v>EPA
EPA-FInal
Human Health Toxicity Values for
Hexafluoropropylene Oxide (HFPO) Dimer Acid and Its
Ammonium Salt (CASRN 13252-13-6 and CASRN
62037-80-3)
Also Known as "GenX Chemicals"
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OCTOBER 2021
Human Health Toxicity Values for Hexafluoropropylene Oxide (HFPO)
Dimer Acid and Its Ammonium Salt (CASRN 13252-13-6 and CASRN 62037-
80-3)
Also Known as "GenX Chemicals"
Prepared by:
U.S. Environmental Protection Agency
Office of Water (4304T)
Health and Ecological Criteria Division
Washington, DC 20460
EPA Document Number: 822R-21-010
October 2021
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Disclaimer
This document has been reviewed in accordance with U.S. Environmental Protection Agency
policy and approved for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
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OCTOBER 2021
Acknowledgments
This document was prepared by the Health and Ecological Criteria Division, Office of Science
and Technology, Office of Water (OW) of the U.S. Environmental Protection Agency (EPA).
OW leads for the assessment include Brittany Jacobs, PhD; Greg Miller, MS; and Jamie Strong,
PhD. OW scientists who provided valuable contributions to the development of this assessment
include Joyce Donohue, PhD, Barbara Soares, PhD; Casey Lindberg, PhD; and Susan Euling,
PhD. The agency gratefully acknowledges the valuable contributions of EPA scientists from the
Office of Research and Development Earl Gray, PhD; Justin Conley, PhD; Beth Owens, PhD;
Jason Lambert, PhD; Samantha Jones, PhD; and Kris Thayer, PhD. The agency gratefully
acknowledges the valuable contributions of scientists from the National Institute of
Environmental Health Sciences Suzanne Fenton, PhD and Bevin Blake, PhD. The agency
gratefully acknowledges the valuable contributions of EPA scientists from the Office of
Pollution Prevention and Toxics, including Catherine Aubee; Amy Babcock, MPH, DABT,
MRSB; Amy Benson, MS, DABT; Tracy Behrsing, PhD; Chris Brinkerhoff, PhD; Tala Henry,
PhD; and Laurence Libelo, PhD.
This document was provided for review by staff in the following EPA Program Offices and
Regions:
• Office of Water
• Office of Chemical Safety and Pollution Prevention, Office of Pesticide Programs
• Office of Chemical Safety and Pollution Prevention, Office of Pollution Prevention and
Toxics
• Office of Chemical Safety and Pollution Prevention, Office of Science Coordination and
Policy
• Office of Land and Emergency Management
• Office of Air and Radiation, Office of Transportation and Air Quality
• Office of Air and Radiation, Office of Air Quality Planning and Standards
• Office of Policy
• Office of Children's Health Protection
• Office of Research and Development
• Regions 1-10
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Contents
1.0 Introduction and Background 1
1.1 History of Assessment of GenX Chemicals 1
1.2 Uses of GenX Chemicals under TSCA 2
1.3 Occurrence 3
1.4 Other Assessments of GenX Chemicals 5
1.4.1 North Carolina Assessment 5
1.4.2 Report by the Netherlands National Institute for Public Health and the
Environment 6
2.0 Nature of the Stressor 6
2.1 Chemical/Physical Properties 6
2.2 Environmental Fate 10
2.2.1 Water 10
2.2.2 Air 10
2.2.3 Sediments and Soils 11
2.2.4 Biodegradation 11
2.2.5 Incineration 11
2.2.6 Bioaccumulation 11
2.3 Toxicokinetics 12
2.3.1 Absorption 12
2.3.2 Distribution 14
2.3.3 Distribution during Gestation and Lactation 16
2.3.4 Metabolism 20
2.3.5 Excretion 20
2.3.6 Clearance and Half-Life Data 21
3.0 Problem Formulation 26
3.1 Conceptual Model 26
3.2 Overall Scientific Objectives 28
3.3 Methods 30
3.3.1 Literature Search Strategy and Results 30
3.3.2 Study Screening and Evaluation 31
3.4 Approach to Deriving Reference Values 33
3.5 Measures of Effect 34
4.0 Study Summaries 35
4.1 Acute Toxicity Studies 35
4.2 Short-Term Toxicity Studies 36
4.3 Subchronic Toxicity Studies 39
4.4 Chronic Toxicity and Carcinogenicity Studies 45
4.5 Reproductive and Developmental Toxicity Studies 49
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4.6 Other Studies 63
4.6.1 Immunotoxicity Studies 63
4.6.2 Mechanistic Studies 63
4.6.3 Genotoxicity Studies 71
5.0 Summary of Hazard 71
5.1 Hepatic 75
5.2 Hematological 77
5.3 Renal 78
5.4 Reproductive/Developmental 79
5.5 Immune System 81
5.6 Cancer 81
6.0 Mode of Action 82
7.0 Dose-Response Assessment 86
7.1 Identification of Studies and Effects for Dose-Response Analysis 86
7.2 Methods of Analysis 90
7.2.1 BMD Modeling 90
7.2.2 Dosimetric Adjustment of the Experimental Animal-Based POD to PODhed 90
7.3 Derivation of Candidate RfD Values 92
7.4 Selection of Overall RfD 98
7.4.1 Subchronic RfD 98
7.4.2 Chronic RfD 99
8.0 Effects Characterization 99
8.1 Uncertainty and Variability 99
8.2 Composition of Test Substance 100
8.3 Use of Data-Derived Extrapolation Factors 101
8.4 Use of Data-Derived Dosimetric Adjustment Factor 101
8.5 Limited Data on Carcinogenicity 103
8.6 Internal Dosimetry Data for GenX Chemicals 103
8.7 Effects on Bilirubin 104
8.8 Susceptible Populations and Life Stages 104
9.0 References 106
Appendix A: Literature Search Strategy A-l
Appendix B: Acute and 7-Day Study Summaries B-l
Appendix C: Genotoxicity Study Summary C-l
Appendix D: NTP PWG Final Report on the Pathology Peer Review of Liver
Findings D-l
Appendix E: Benchmark Dose Modeling E-l
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Figures
Figure 1. Structure of HFPO Dimer Acid and HFPO Dimer Acid Ammonium Salt 6
Figure 2. Conceptual Model for HFPO Dimer Acid and Its Ammonium Salt 27
Figure 3. Evaluation Results for Animal Studies Assessing Effects of GenX Chemicals
Exposure (Click to see interactive data graphic for rating rationales) 32
Tables
Table 1. Chemical and Physical Properties of HFPO Dimer Acid and HFPO Dimer Acid
Ammonium Salt 7
Table 2. Plasma Concentration in Crl:CDl(ICR) Mice at 2 Hours after the First Gavage
Exposure to HFPO Dimer Acid Ammonium Salt 13
Table 3. Concentrations of HFPO Dimer Acid in CD1 Pregnant Mice and Their Embryos
at Embryonic Day 11.5 or 17.5a 16
Table 4. Maternal Serum and Fetal Plasma Concentrations on GD18 in Crl:CD(SD) Rats
Exposed to HFPO Dimer Acid Ammonium Salt from GDI4-18 18
Table 5. Maternal and Offspring HFPO Dimer Acid Anion Concentrations in Serum and
Liver Samples Collected on GD20 or PND2 from Crl:CD(SD) Rats Orally
Exposed to HFPO Dimer Acid Ammonium Salt from GDI6-20 or GD8-PND2 19
Table 6. Clearance Times in Plasma for Male and Female Rats and Mice Following a
Single Oral Dosea 22
Table 7. T1/2 Estimates from Intravenous Injection in Sprague Dawley Rats and
Cynomolgus Monkeys 23
Table 8. T1/2 Estimates from Single Oral Dose in Sprague Dawley Rats and Crl/CD1(ICR)
Mice 24
Table 9. Mean Plasma Concentrations with Standard Deviations of Dosing Crl:CDl(ICR)
Mice with HFPO Dimer Acid Ammonium Salt for at Least 90 Days 25
Table 10. Comparison of Results from 90-Day Mouse Study (DuPont-18405-1307, 2010)
and NTP PWG Reevaluation (NTP, 2019) 44
Table 11. Comparison of Study Results from DuPont-18405-1037 (2010), Thompson et al.
(2019), and NTP PWG Reevaluation of DuPont-18405-1037 (2019) 52
Table 12. Summary of Study NOAELs/LOAELs 72
Table 13. Summary of Determination of PODhed 91
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Table 14. Candidate Subchronic RfD Values
Table 15. Candidate Chronic RfD Values
Table 16. Comparison of PODhed using different allometric scaling methods
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Acronyms and Abbreviations
°c
degree Celsius
3D
three dimensional
A/G
albumin-to-globulin
AAALAC
American Association for
Accreditation of
Laboratory Animal Care
ADME
absorption, distribution,
metabolism, and
excretion
AGD
anogenital distance
AIC
Akaike information
criterion
ALD
approximate lethal dose
ALP
alkaline phosphatase
ALT
alanine aminotransferase
AOP
adverse outcome pathway
AR
androgen receptor
AST
aspartate
aminotransferase
atm-m3/mol
atmosphere cubic meter
per mole
ATP
adenosine triphosphate
BAF
bioaccumulation factor
BBDR
biologically based dose-
response
BCF
bioconcentration factor
BCRP
breast cancer resistance
protein
BMD
benchmark dose
BMDio
dose level corresponding
to the 95% lower
confidence limit for a
10% response level
BMDL
benchmark dose lower
limit
BMDLio
lower bound on the
BMDio
BMDS
Benchmark Dose
Software
BMR
benchmark response
BOD
biochemical oxygen
demand
BUN
blood urea nitrogen
BW
body weight
BWa
animal body weight
BWh
human body weight
CASRN
Chemical Abstracts
Service Registry Number
CFR
Code of Federal
Regulations
cm/hr
centimeter per hour
CoA
coenzyme A
COV
coefficient of variation
Crl:CD(SD)
Sprague Dawley
DAF
dosimetric adjustment
factor
DMEM/F-12
Dulbecco's Modified
Eagle Medium: Nutrient
Mixture F-12
DMSO
dimethyl sulfoxide
DNA
deoxyribonucleic acid
DWEL
drinking water equivalent
level
DWTP
drinking water treatment
plant
E
embryonic day
El
heptafluoropropyl
1,2,2,2-tetrafluoroethyl
ether
E2
estradiol
ELISA
enzyme-linked
immunosorbent assay
EPA
U.S. Environmental
Protection Agency
ERa
estrogen receptor alpha
ERP
estrogen receptor beta
F0
parent generation
Fi
offspring of the Fo
generation
FABP
fatty acid-binding protein
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FIFRA
Federal Insecticide,
Fungicide, and
Rodenticide Act
FRD-902
synonym for HFPO dimer
acid ammonium salt
FRD-903
synonym for HFPO dimer
acid
g
gram
g/L
gram per liter
g/mol
gram per mole
GenX chemicals
hexafluoropropy 1 ene
oxide dimer acid and its
ammonium salt
GD
gestation day
GLP
Good Laboratory
Practices
GWG
gestational weight gain
H30+
hydronium ion
H&E
hematoxylin and eosin
HAWC
Health Assessment
Workspace Collaborative
HDL
high-density lipoprotein
HED
human equivalent dose
HERO
Health & Environmental
Research Online
HFPO
hexafluoropropy 1 ene
oxide
HFPO-DA
HFPO dimer acid
HFPO dimer acid 2,3,3,3-tetrafluoro-2-
(heptafluoropropoxy)
propanoic acid
HFPO-TA
HFPO trimer acid
HFPO-TeA
hexafluoropropy 1 ene
oxide tetramer acid
hL-FABP
human liver fatty acid-
binding protein
HPLC
high-performance liquid
chromatography
HPLC/MS/MS
high-performance liquid
chromatography-tandem
mass spectrometry
IC50
concentration at which
50% inhibition is
observed
ICR
Institute of Cancer
Research
IgM
immunoglobulin M
INHAND
International
Harmonization of
Nomenclature and
Diagnostic Criteria
IUPAC
International Union of
Pure and Applied
Chemistry
i.v.
intravenous
Koc
soil-water partition
coefficient for organic
compounds
Kow
octanol-water partition
coefficient
kPa
kilopascal
L/kg
liter per kilogram
LC50
median lethal
concentration
LD
lactation day
LD50
median lethal dose
LDL
low-density lipoprotein
LLNA
local lymph node assay
LOAEL
lowest-observed-adverse-
effect level
LOD
limit of detection
LOQ
limit of quantification
l-ig/g
microgram per gram
^g/L
microgram per liter
|ig/mL
microgram per milliliter
|iL
microliter
|iM
micromolar
mg
milligram
mg/kg
milligram per kilogram
mg/kg/day
milligram per kilogram
per day
mg/L
milligram per liter
mg/m3
milligram per cubic meter
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mg/mL
milligram per milliliter
mL
milliliter
mM
millimolar
mm Hg
millimeter of mercury
MOA
mode of action
MMAD
mass median
aerodynamic diameter
mRNA
messenger ribonucleic
acid
MRP2
multidrug resistance-
associated protein 2
MTT
3-(4,5-dimethylthiazol-2-
yi)-2,5-
diphenyltetrazolium
bromide
N/A
not applicable
NAM
new approach
methodology
NC DHHS
North Carolina
Department of Health and
Human Services
ND
not detected
ng/g
nanogram per gram
ng/mL
nanogram per milliliter
NHANES
National Health and
Nutrition Examination
Survey
NIEHS
National Institute of
Environmental Health
Sciences
NLM
National Library of
Medicine
nM
nanomolar
nm
nanometer
NOAEL
no-ob served-adverse-
effect level
NQ
not quantified
NR
not rated
NTP
National Toxicology
Program
OECD
Organization for
Economic Cooperation
and Development
OPPT
Office of Pollution
Prevention and Toxics
ORD
Office of Research and
Development
P-gP
P-glycoprotein
PBPK
physiologically based
pharmacokinetic
PBTK
physiologically based
toxicokinetic
PCR
polymerase chain reaction
PECO
population, exposure,
comparator, and outcome
PFAS
per- and polyfluoroalkyl
substances
PFBA
perfluorobutanoic acid
PFBS
perfluorobutanesulfonic
acid
PFHxA
perfluorohexanoic acid
PFHxS
perfluorohexane sulfonic
acid
PF04DA
3,5,7,9-tetraoxadecanoic
perfluoro acid
PFOA
perfluorooctanoic acid
PFOS
perfluorooctane sulfonate
PK
pharmacokinetic
Pka
acid dissociation constant
Pkb
base dissociation constant
pM
picomolar
pmol
picomole
PMN
premanufacture notice
PMOH
ammonium perfluoro(2-
methyl-3 -oxahexanoate)
PMPP
3H-perfluoro-3 -(3 -
methoxypropoxy)
propanoic acid
PND
postnatal day
POD
point of departure
PODhed
point of departure human
equivalent dose
PPAR
peroxisome proliferator-
activated receptor
PPARa
peroxisome proliferator-
activated receptor alpha
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PPAR-p/5
peroxisome proliferator-
activated receptor
UFh
beta/delta
UFl
PPARy
peroxisome proliferator-
activated receptor gamma
ppm
parts per million
UFs
PWG
Pathology Working
Group
RBC
red blood cell
RfD
Reference dose
UFtot
RIVM
National Institute for
VTG
Public Health and the
WOS
Environment
(Rijksinstituut voor
Volksgezondheid en
Milieu)
RNA
ribonucleic acid
rT3
reverse triiodothyronine
SD
standard deviation
SDH
sorbitol dehydrogenase
TEM
transmission electron
microscopy
TG
Test Guideline
TK
toxicokinetic
ToxRTool
Toxicological Data
Reliability Assessment
Tool
TSCA
Toxic Substances Control
Act
TSCATS
Toxic Substances Control
Act Test Submissions
UF
uncertainty factor(s)
UFa
interspecies uncertainty
factor
UFd
database uncertainty
factor
SE
standard error
SM
Standard Model
T1/2
half-life
T3
triiodothyronine
14
thyroxine
TDAR
T cell-dependent antibody
response
intraspecies uncertainty
factor
LOAEL to NOAEL
extrapolation uncertainty
factor
extrapolation from
sub chronic to a chronic
exposure duration
uncertainty factor
total uncertainty factor
vitellogenin
Web of Science
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Executive Summary
The U.S. Environmental Protection Agency (EPA) is issuing final subchronic and chronic oral
toxicity values (i.e., reference doses, or RfDs) for 2,3,3,3-tetrafluoro-2-
(heptafluoropropoxy)propanoic acid (Chemical Abstracts Service Registry Number (CASRN)
13252-13-6)—or hexafluoropropylene oxide (HFPO) dimer acid—and ammonium 2,3,3,3-
tetrafluoro-2-(heptafluoropropoxy)propanoate (CASRN 62037-80-3)—or HFPO dimer acid
ammonium salt. These chemicals are also known as "GenX chemicals" because they are the two
major chemicals associated with GenX processing aid technology. The toxicity assessment for
GenX chemicals is a scientific and technical report that provides an assessment of all available
toxicity and carcinogenicity data and includes toxicity values associated with potential noncancer
health effects following oral exposure (in this case, oral RfDs). This toxicity assessment
evaluates human health hazards. It is not a risk assessment as it does not include an exposure
assessment nor an overall risk characterization. Further, the toxicity assessment does not address
the legal, political, social, economic, or technical considerations involved in risk management.
The GenX chemicals toxicity assessment can be used by EPA, states, tribes, and local
communities, along with specific exposure and other relevant information, to determine, under
the appropriate regulations and statutes, if, and when, it is necessary to take action to address
potential risk associated with human exposures to GenX chemicals.
These GenX chemicals are organic fluorinated ether chemicals that are part of a larger group of
chemicals referred to as "per- and polyfluoroalkyl substances" or PFAS. In 2006, EPA initiated a
stewardship program with the goal of eliminating chemical emissions of perfluorooctanoic acid
(PFOA) and related chemicals by 2015. GenX chemicals are replacements for PFOA.
Specifically, GenX is a trade name for a processing aid technology that enables the creation of
fluoropolymers without the use of PFOA. Information on specific products containing these
chemicals is not available, however, GenX chemicals may be used in the manufacture of the
same or similar commercial fluoropolymer end products that formerly used PFOA.
Fluoropolymers are used in many applications, including the manufacture of nonstick coatings
for cookware, water repellent garments, and other specialty agrochemical and pharmaceutical
applications.
For HFPO dimer acid and its ammonium salt, acute, short-term, subchronic, chronic, and
reproductive and developmental oral animal toxicity studies are available in rats and mice.
Limited information identifying health effects in animals from inhalation of or dermal exposures
to GenX chemicals is available. Repeated-dose toxicity data are available for oral exposure, but
not for the other exposure routes (inhalation and dermal exposures). Thus, this assessment
applies only to the oral route of exposure. These studies report liver toxicity (increased relative
liver weight, hepatocellular hypertrophy, apoptosis, and single-cell/focal necrosis), kidney
toxicity (increased relative kidney weight), immune effects (antibody suppression),
hematological effects (decreased red blood cell count, hemoglobin, and hematocrit),
reproductive/developmental effects (increased early deliveries, placental lesions, changes in
maternal gestational weight gain, and delays in genital development in offspring), and cancer
(liver and pancreatic tumors). Overall, the available toxicity studies demonstrate that the liver is
particularly sensitive to HFPO dimer acid- and HFPO dimer acid ammonium salt-induced
toxicity. Consistent with the Guidelines for Carcinogen Risk Assessment (EPA, 2005a), EPA
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concluded that there is Suggestive Evidence of Carcinogenic Potential of oral exposure to GenX
chemicals in humans, based on the female hepatocellular adenomas and hepatocellular
carcinomas and male combined pancreatic acinar adenomas and carcinomas observed in the
chronic 2-year study in rats.
EPA followed the general guidelines for risk assessment set forth by the National Research
Council (1983) and EPA's Framework for Human Health Risk Assessment to Inform Decision
Making (EPA, 2014a) in determining the point of departure (POD) for the derivation of the RfDs
for these chemicals. Consistent with the recommendations presented in EPA's A Review of the
Reference Dose and Reference Concentration Processes (EPA, 2002), EPA applied uncertainty
factors (UFs) to address intraspecies variability, interspecies variability, and extrapolation from a
subchronic to a chronic exposure duration.
The critical study chosen for determining the subchronic and chronic RfDs for HFPO dimer acid
and/or its ammonium salt was the oral reproductive/developmental toxicity study in mice with a
no-observed-adverse-effect level (NOAEL) of 0.1 milligram per kilogram per day (mg/kg/day)
based on liver effects (a constellation of lesions, including cytoplasmic alteration, hepatocellular
single-cell and focal necrosis, and hepatocellular apoptosis) in females (DuPont-18405-1037,
2010; NTP, 2019). EPA determined that the constellation of liver lesions observed in the rodent
are relevant to human health and not a result of PPARa-induced cell proliferation unique to
rodents. Using EPA's Benchmark Dose Technical Guidance Document (EPA, 2012), EPA
conducted benchmark dose modeling to empirically model the dose-response relationship in the
range of observed data. Additionally, EPA's Recommended Use of Body Weight4 as the Default
Method in Derivation of the Oral Reference Dose (EPA, 201 lb) was used to allometrically scale
a toxicologically equivalent dose of orally administered agents from adult laboratory animals to
adult humans. Allometric scaling addresses some aspects of cross-species extrapolation of
toxicokinetic and toxicodynamic processes (i.e., interspecies UFs). The resulting POD human
equivalent dose is 0.01 mg/kg/day. UFs applied include a 10 for intraspecies variability, 3 for
interspecies differences, and 10 for database deficiencies, including immune effects and
additional developmental studies, to yield a subchronic RfD of 0.00003 mg/kg/day or 0.03
|ig/kg/day. In addition to those above, a UF of 10 was also applied for extrapolation from a
subchronic to a chronic duration in the derivation of the chronic RfD of 0.000003 mg/kg/day or
0.003 |ig/kg/day.
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1.0 Introduction and Background
1.1 History of Assessment of GenX Chemicals
In 2008, DuPont de Nemours, Inc. (hereinafter DuPont) submitted premanufacture notices
(PMNs) to the U.S. Environmental Protection Agency (EPA) under the Toxic Substances
Control Act (TSCA) (Title 15 of the United States Code § 2601 et seq.) for two chemicals—
2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid (Chemical Abstracts Service Registry
Number (CASRN) 13252-13-6)—or hexafluoropropylene oxide (HFPO) dimer acid—and
ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoate (CASRN 62037-80-3)—or
HFPO dimer acid ammonium salt—which are part of the GenX processing aid technology they
developed.
Note: In July 2015, DuPont announced it had separated its Performance Chemicals segment
through the creation of The Chemours Company. As a result, the GenX processing technology
and associated chemicals are now products of The Chemours Company (Chemours, 2018).
Because the submitted studies were conducted prior to the 2015 separation, however, the studies
are referenced with DuPont identifiers.
Upon receipt, EPA assigned these PMNs case numbers P-08-0508 and P-08-0509, and they were
reviewed by the New Chemicals Program in the Office of Pollution Prevention and Toxics
(OPPT) and posted in the Federal Register (73 FR 46263, August 8, 2008) for public comment
(EPA, 2008). A PMN assessment was completed and included a hazard assessment based on
EPA review of test data submitted to the agency with the PMNs (including two 28-day oral
(gavage) toxicity studies in mice (DuPont-24459, 2008) and rats (DuPont-24447, 2008)), as well
as publicly available literature and TSCA confidential business information on other per- and
polyfluoroalkyl substances (PFAS). Submitted test data on HFPO dimer acid and/or its
ammonium salt were available for numerous endpoints such as acute toxicity, metabolism and
toxicokinetics, genotoxicity, and systemic toxicity in mice and rats with dosing durations of up to
28 days.
EPA OPPT evaluated the methods and data submitted and deemed the studies acceptable to the
agency. The studies submitted in 2008 with the PMNs formed the primary basis of EPA's hazard
assessment at that time. The 28-day toxicity study in mice, from which EPA OPPT derived the
point of departure (POD) of 0.1 milligrams per kilogram per day (mg/kg/day), was conducted
according to Organization for Economic Cooperation and Development (OECD) Test Guideline
(TG) 407 (OECD, 2008a) and followed Good Laboratory Practices (GLP) (DuPont-24459, 2008;
OECD, 2008a). The submitted studies were also used, in concert with information on other
PFAS chemicals, to inform the decision to require further testing, as described in the Consent
Order that concluded the PMN review (EPA, 2009).
The Consent Order included, among other things, additional testing pertaining to human health.
The tests were identified in the Consent Order according to OECD TG numbers and/or EPA
health effects TGs for pesticides and toxic substances numbers. Following are the studies
included in the Consent Order relevant to human health and this assessment:
• Repeated dose metabolism and pharmacokinetics studies (OPPTS 870.7485) in mice and
rats (Dupont-18405-1017, 2011)
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• Modified Oral (Gavage) Reproduction/Developmental Toxicity Study in Mice (OECD
TG 421) (Dupont-18405-1037, 2010; OECD, 2016a)
• 90 Day Oral (Gavage) Toxicity Study (OECD, 1998) (species not specified): Both mice
(DuPont-18405-1307, 2010) and rats (Dupont-17751-1026, 2009) were submitted
• Combined Chronic Toxicity/Oncogenicity Study in Rats (OECD, 2009) (Dupont-18405-
1238, 2013)
The OECD TGs are accepted internationally as standard methods for safety testing and:
... are covered by the Mutual Acceptance of Data, implying that data generated in the
testing of chemicals in an OECD member country, or a partner country having adhered to
the Decision, in accordance with OECD Test Guidelines and Principles of GLP, be
accepted in other OECD countries and partner countries having adhered to the Decision,
for the purposes of assessment and other uses relating to the protection of human health
and the environment (OECD, 2018a).
Specifically, for the required oral reproductive/developmental toxicity test, EPA OPPT included
requirements for specific modifications to the test to increase the robustness of the study for this
class of chemicals (DuPont-18405-1037, 2010; OECD, 2016a). These modifications are stated in
the Consent Order (EPA, 2009) and were followed by the testing laboratory as outlined in the
study report (DuPont-18405-1037, 2010). For the required combined chronic
toxicity/oncogenicity study, EPA reviewed and concurred with protocols submitted to the agency
prior to the study being conducted (DuPont-18405-1238, 2013). In addition, the submitter
consulted with EPA on study findings to determine the need for additional data (e.g., further
toxicokinetic testing based on results of the first tier OPPTS 870.7485 study). Finally, while not
specifically required under the Consent Order, DuPont conducted and submitted results for
additional OECD TG studies for Agency review (e.g., the prenatal and developmental toxicity
study in rats (OECD, 2001b) (DuPont-18405-841, 2010).
1.2 Uses of GenX Chemicals under TSCA
GenX is a trade name for a processing aid technology developed by DuPont to make high-
performance fluoropolymers without the use of perfluorooctanoic acid (PFOA) (Chemours,
2018). Transition to GenX processing aid technology began in 2009 as part of the company's
commitment under the 2010/2015 PFOA Stewardship Program to work toward the elimination of
these chemicals from emissions and products by 2015. Although production of most long-chain
PFAS has been phased out in the United States and has been generally replaced by production of
shorter chain PFAS, EPA is aware of ongoing use of long-chain PFAS by companies that did not
participate in the PFOA Stewardship Program and ongoing use of the chemicals available in
existing stocks or being newly introduced via imports.
Fluoropolymers are used in many applications because of their unique physical properties such
as resistance to high and low temperatures, resistance to chemical and environmental
degradation, and nonstick characteristics. Fluoropolymers also have dielectric and fire-resistant
properties that have a wide range of electrical and electronic applications, including architecture,
fabrics, automotive uses, cabling materials, food processing, electronics, pharmaceutical and
biotech manufacturing, and semiconductor manufacturing (Gardiner, 2014).
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One of the two PMNs EPA received in 2008, P-08-0508, was for HFPO dimer acid, a chemical
used as an intermediate to make the polymerization aid HFPO dimer acid ammonium salt. The
PMN for HFPO dimer acid ammonium salt was received by EPA under PMN P-08-0509 and is
used as a replacement for PFOA in the manufacture of fluoropolymers. The GenX resin
manufacturing process includes the thermal transformation of the HFPO dimer acid ammonium
salt processing aid into a hydrophobic hydride. HFPO is used in the manufacture of HFPO dimer
acid, HFPO dimer acid ammonium salt, other HFPO dimer acid derivatives, fluoropolymers
(including polyethers), and other specialty agrochemical and pharmaceutical applications.
Information on specific products containing GenX chemicals is not available, however, GenX
chemicals may be used in the manufacture of the same or similar commercial fluoropolymer end
products that formerly used PFOA. GenX chemicals may also be generated as a byproduct of
fluoromonomer production. When in water, both HFPO dimer acid and HFPO dimer acid
ammonium salt dissociate to form the HFPO dimer acid anion (HFPO") as a common analyte.
HFPO is manufactured from hexafluoropropene. HFPO dimer acid can react with additional
HFPO to form the HFPO trimer acid and longer polymer fluorides. Other PFAS chemicals might
be part of the GenX processing aid technology, but HFPO dimer acid and its ammonium salt are
the major chemicals associated with this technology.
1.3 Occurrence
GenX chemicals were identified in North Carolina's Cape Fear River and its tributaries in the
summer of 2012 (Strynar et al., 2015). Following this discovery, between June and December
2013, Sun et al. (2016) sampled source water at three drinking water treatment plants (DWTPs)
(identified as DWTPs A, B, and C) treating surface water from the Cape Fear River watershed.
The mean concentration of HFPO dimer acid in the finished drinking water treated by DWTP C
was 0.631 microgram per liter (|ig/L) (Sun et al., 2016). In a separate experiment to look at
removal efficiency of DWTP C, water samples were taken during August 2014 from the raw
water intake and after each treatment process step used by DWTP C (i.e., coagulation/
flocculation/sedimentation, raw and settled water ozonation, biological activated carbon
filtration, and disinfection by medium-pressure ultraviolet lamps and free chlorine). GenX
chemicals were found at concentrations of 0.4-0.5 |ig/L at all steps of the treatment process,
indicating that the concentrations of HFPO dimer acid were only slightly decreased by the
conventional and advanced water treatment processes used at this DWTP.
The publication of these data prompted the North Carolina Department of Environmental Quality
to sample sites for GenX chemicals along the Cape Fear River and in private wells close to the
Chemours facility. In certain samples of surface water, groundwater, and finished drinking water,
GenX chemicals were detected above 0.140 |ig/L, which is North Carolina's drinking water
health goal for GenX chemicals (NCDEQ, 2018c). Chemours has indicated that GenX chemicals
have been discharged into the Cape Fear River for several decades as a byproduct of other
manufacturing processes (NCDEQ, 2017). Petre et al. (2021) quantified the mass transfer of
PFAS from contaminated groundwater to five tributaries of the Cape Fear River, including GenX
chemicals. HFPO dimer acid and another fluoroether accounted for 61% of the total quantified
PFAS. The study authors calculated that 32 kg/year of PFAS discharges from the groundwater to
the five tributaries and the movement of these fluoroethers from the groundwater through the
subsurface and into the streams occurred in less than the past 50 years. These data indicate that
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the discharge of contaminated groundwater has led to long-term contamination of surface water
and could lead to subsequent impacts on downstream drinking water (Petre et al, 2021).
Community concern over the detection of GenX chemicals in the Cape Fear Watershed led to the
initiation of the GenX exposure study in Wilmington, North Carolina1. Blood samples from 344
Wilmington residents were collected between November 2017 and May 2018 and repeated blood
samples from 44 of the participants were collected 6 months after the first sample collection. The
blood sampling coincided with source control of GenX chemicals, and it is unknown whether
study participants were drinking tap water at the time of collection. GenX chemicals were not
detected above the analytical reporting limit of 2 ng/mL in any of the blood samples collected
(Kotlarz et al., 2020).
GenX chemicals and other PFAS were also analyzed in 2682 urine samples from 2013-2014
National Health and Nutrition Examination Survey (NHANES) participants > 6 years of age
(Calafat et al., 2019). GenX chemicals were one of the few tested PFAS to be detected in the
urine and was detected in approximately 1.2% of the population. The limit of detection was 0.1
[j,g/L. Importantly, this study demonstrated that the urine does not appear to be a good biomarker
for PFAS. For example, PFOA and PFOS were detected in serum samples for > 98% of this
study population, yet PFOA and PFOS were only detected in paired urine samples for < 0.1% of
the same population.
In a report submitted by The Chemours Company to EPA, 24 human plasma samples were
analyzed for HFPO dimer acid and were found at concentrations ranging from 1.0 ng/mL - 51.2
ng/mL. In seven of the samples, HFPO dimer acid was not detected above the analytical
reporting limit of less than 1.0 ng/mL. No additional information about the study participants
was provided in the report (DuPont- C30031 516655, 2017). GenX chemicals have been
identified in other media, including rainwater and air emissions. North Carolina Department of
Environmental Quality estimates for the Chemours Fayetteville Works plant (in the North
Carolina Cape Fear watershed) indicate that Chemours' annual emissions of GenX chemicals
could have exceeded 2,700 pounds per year during the reporting period (2017-2018) (NCDEQ,
2018a). Additional details on air emissions of GenX chemicals at the Fayetteville Works plant
can be found at
https://files.nc.gov/iicdeq/GeiiX/2018 Apiil6 Letter to Chemours P Vy. signed.pdf.
Rainwater samples were collected between February 28 and March 2, 2018 up to 7 miles from
the North Carolina plant (NCDEQ, 2018b). The highest concentration of GenX chemicals in a
rainwater sample (0.810 |ig/L) was detected 5 miles from the Fayetteville Works facility center.
The three samples collected 7 miles from the plant ranged from 0.045 to 0.060 |ig/L (NCDEQ,
2018b). GenX chemicals also have been detected in three on-site production wells and one on-
site drinking water well at the Chemours Washington Works facility in Parkersburg, WV. EPA
subsequently requested that Chemours test for GenX chemicals in both raw and finished water at
four public drinking water systems and 10 private drinking water wells. Chemours agreed to the
testing and completed sampling during February 2018. The results from these samples are
available at https://www.epa.gov/sites/production/files/2018-
04/documents/hfpo chemours wash works sampling 2018.pdf and range before treatment
from less than 0.010-0.081 |ig/L in the public drinking water systems and less than 0.010-0.052
1 https://senxstudv.iicsu.edu/
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|ig/L in the private drinking water wells (EPA, 2018a). All samples were below the limit of
detection (0.010 |ig/L) after treatment (EPA, 2018a).
Additionally, between the summer of 2016 and March 2018, GenX chemicals were identified in
surface water and some soil samples collected upstream and downwind of a fluoropolymer
production facility in Parkersburg, WV (Galloway et al., 2020). The highest concentrations of
HFPO dimer acid in surface water samples (37-227 ng/L) were found in the direction of
prevailing winds, directly across the Ohio River to the north and upstream to the northeast of the
plant on the East Fork of the Little Hocking River. HFPO dimer acid was found in surface water
samples up to 24 kilometers north of the facility, close to Beverly, OH. HFPO dimer acid was
also detected in soil samples from Drag Strip Road, Veto Lake, and the Little Hocking Water
Association at concentrations ranging from 3.09 nanograms per gram (ng/g) to 8.14 ng/g. These
data reveal the downwind atmospheric transport of HFPO dimer acid.
Low concentrations of HFPO dimer acid (0.003-0.004 |ig/L) were detected in the Delaware
River, as reported in the recent publication by Pan et al. (2018).
The Kentucky Department of Environmental Protection (2019) reported detecting HFPO dimer
acid in 11 samples from DWTPs at concentrations ranging from more than 1.32 ng/L to 29.7
ng/L. The study analyzed DWTPs using both surface water and ground water as sources and
found the most frequent and highest detections of HPFO dimer acid at plants that use the Ohio
River and ground water from the Ohio River alluvial aquifer as sources. For HFPO dimer acid,
10 detections were from surface water DWTPs and one detection was from a ground water
DWTP. The ground water DWTP reported the highest concentration of HFPO dimer acid of all
detections.
Globally, GenX chemical occurrence has been reported in Germany (Heydebreck et al., 2015;
Pan et al., 2018), China (Heydebreck et al., 2015; Pan et al., 2017, 2018; Song et al., 2018), the
Netherlands (Heydebreck et al., 2015; Gebbink et al., 2017; Pan et al., 2018), the United
Kingdom (Pan et al., 2018), South Korea (Pan et al., 2018), and Sweden (Pan et al., 2018).
HFPO dimer acid was also detected with a mean concentration of 30 pg/L in Artie surface water
samples, suggesting long range transport (Joerss et al., 2020).
1.4 Other Assessments of GenX Chemicals
1.4.1 North Carolina Assessment
The North Carolina Department of Health and Human Services (NC DHHS) released a health
assessment and provisional drinking water health goal for GenX chemicals in July 2017, which
was finalized in October 2018 (NCDEQ, 2018c). North Carolina defines "health goal" as a
nonregulatory, non-enforceable level of contamination below which no adverse health effects
would be expected over a lifetime of exposure. The provisional health goal for exposure to GenX
chemicals in drinking water is 0.140 |ig/L, which is intended to protect the most sensitive
population, namely bottle-fed infants. The state selected bottle-fed infants as the most sensitive
population because they drink the largest volume of water per body weight (BW).
North Carolina's provisional health goal is based on a reference dose (RfD) derived from a
NOAEL of 0.1 mg/kg/day for liver effects (single-cell necrosis) in mice (DuPont-24459, 2008;
DuPont-18405-1037, 2010). The total UF applied was 1,000, including individual factors to
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account for interspecies variability (10), intraspecies variability (10), and extrapolation from a
subchronic to a chronic exposure duration (10). This RfD of 0.0001 mg/kg/day was used to
derive a drinking water equivalent level (DWEL), which considers exposure. The DWEL was
calculated using BW and drinking water intake for bottle-fed infants and a relative source
contribution of 20% to account for potential exposure to GenX chemicals from other media and
routes, including air, soil, dust, and food (NCDEQ, 2018c). Additional details are available at
NC DH.H.S.
1.4.2 Report by the Netherlands National Institute for Public Health and the
Environment
The National Institute for Public Health and the Environment (RIVM) in the Netherlands
evaluated the data for GenX chemicals to set a safe limit for air. RIVM's assessment focused on
the precursor 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid (FRD-903) (a synonym
for IIFPO dimer acid), the processing agent ammonium 2,3,3,3-tetrafluoro-2-
(heptafluoropropoxy )propanoate (FRD-902) (a synonym for HFPO dimer acid ammonium salt),
and the transformation product heptafl uoropropyl 1,2,2,2-tetrafluoroethyl ether (El). Overall,
RIVM concluded that there is no health risk expected for people living near plants from
emissions of FRD-902 and FRD-903 at a limit of 73 nanograms per cubic meter (insufficient
data are available to determine the toxicity of E1) (Beekman et al., 2016). RIVM used the oral
carcinogenicity study in rats as the critical study (DuPont-18405-1238, 2013) and concluded that
the study NOAEL was 0.1 mg/kg/day, based on increased albumin and the albumin-to-globulin
(A/G) ratio observed at 12 months in males dosed with 1 mg/kg/day, an effect that indicates the
potential for immunotoxicity. Using route-to-route extrapolation, RIVM converted this NOAEL
to an air concentration to be used as the POD. UFs to account for intraspecies differences (10)
and interspecies differences (1.8), and an additional factor to account for uncertainty in the
human elimination of GenX chemicals (66) were applied to the POD to determine the chronic
inhalation limit.
2.0 Nature of the Stressor
2.1 Chemical/Physical Properties
I IFPO dimer acid and its ammonium salt are fluorinated organic compounds (Figure 1).
F F F
F F F F
OH
-F O
F F F
F F F F
-F O
H
H—N —H
H
I IFPO dimer acid
CASRN 13252-13-6
I IFPO dimer acid ammonium salt
CASRN 62037-80-3
Figure 1. Structure of HFPO Dimer Acid and HFPO Dimer Acid Ammonium Salt
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HFPO dimer acid is a liquid whereas its ammonium salt is a solid at room temperature. Both are
highly soluble in water. Except in very acidic solvents (pH less than 3), the acid will dissolve and
be present as the acid anion with a -1 charge. The associated cation ion will be a hydronium ion
(H30+) in water if other hydrogen ion acceptors are absent. Both compounds can volatilize from
water to air, where they will dissolve in aerosolized water droplets or bind to suspended
particulate matter. In soils, they will migrate with the aqueous phase or bind to the soil particle
surfaces with areas of positive charge. The organic portions of HFPO dimer acid and its
ammonium salt are stable to environmental degradation. Table 1 presents the chemical and
physical properties of HFPO dimer acid and its ammonium salt.
Table 1. Chemical and Physical Properties of HFPO Dimer Acid and HFPO Dimer Acid
Ammonium Salt
Property
HFPO dimer acid
HFPO dimer acid ammonium
salt
Source
CASRN
13252-13-6
62037-80-3
Chemical Abstracts
Service.
CAS Index Name
Propanoic acid, 2,3,3,3-
tetrafluoro-2-(l,l,2,2,3,3,3-
heptafluoropropoxy)
Propanoic acid, 2,3,3,3-
tetrafluoro-2-(l, 1,2,2,3,3,3-
heptafluoropropoxy)-ammonium
salt (1:1)
Chemical Abstracts
Service.
IUPAC Name
2,3,3,3 -tetrafluoro-2-
(1,1,2,2,3,3,3-
heptafluoropropoxy propanoic
acid
azanium;2,3,3,3 -tetrafluoro-2-
(1,1,2,2,3,3,3-
heptafluoropropoxy)propanoate
PubChem.
Synonyms
GenX Acid
FRD 903
H-28307
C3 dimer acid
2,3,3,3 -tetrafluoro-2-
(heptafluoropropoxy)propanoic
acid
GenX salt308
FRD 902
FDR 90208
H-21216
H-27529
H-28072
H-28397
H-28308
H-28548
HFPO dimer ammonium salt
C3 dimer salt
Ammonium, 2,3,3,3-tetrafluoro-2-
(heptafluoropropoxy)propanoate
Ammonium perfluoro(2-methyl-3-
oxahexanoate)
PMOH
DuPont-24637, 2008;
DuPont- 24698, 2008.
Chemical Formula
c6hf„o3
C6H4F11NO3
Molecular Weight
330.06 g/mol
347.08 g/mol
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Property
HFPO dimcr acid
HFPO dimcr acid ammonium
salt
Source
Color/Physical
State
Clear, colorless liquid
(20 °C, 101.3 kPa)
Solid
DuPont-24637, 2008;
DuPont-24698, 2008.
Boiling Point
129 °C
108 °C (as 86% salt solution in
water)
No measurement available for salt
form
DuPont-24637, 2008;
DuPont-24698, 2008.
Melting Point
< -40.0 °C
-21.0 °C (as 86% salt solution in
water)
No measurement available for salt
form
DuPont-24637, 2008;
DuPont-24698, 2008.
Vapor Pressure
306 Pa (2.7 mm Hg)
(20 °C)
No measurement available
DuPont-24128, 2008;
DuPont-24129, 2008.
Henry's Law
Constant
< 2.5 x 10"4 atm-m3/mol
No measurement available
Calculated from
measured vapor
pressure and highest
measured solubility.
Water solubility is
reported to be "infinite"
(DuPont-24128, 2008;
DuPont-24129, 2008),
so the actual Kh is
expected to be much
lower. These values
should not be used to
estimate partitioning
between water and air.
Pka
2.84 (20 °C)
3.82
DuPont-26349, 2008.
Pkb
8.1
8.1
DuPont-24198, 2008
(HFPO dimer acid
ammonium salt).
Koc
Soil-12 L/Kg (log 1.10)
Sludge-12.6 L/Kg (log = 1.08)
DuPont-17568-1675,
2008.
K-ow
Not applicable3
Not applicable3
Solubility in Water
@20 °C
>751 g/L
>739 g/L
Highest tested values.
Actual solubility not
determined but
described as "infinite"
(DuPont-24128, 2008;
DuPont-24129, 2008).
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Property
HFPO dimcr acid
HFPO dimcr acid ammonium
salt
Source
Half-life (T1/2) in
Water
(25 °C)
Stable
Stable
Measured hydrolysis
values for salt. No
degradation in 5 days at
50 °C and pH 4, 7, and
9 (DuPont-24199,
2008).
Half-life (T1/2) in
Air
Stable
Stable
Ultraviolet-visible and
visible
spectrophotometry
spectra for acid showed
little absorption above
240 nm (DuPont-26349,
2008). EPA concurs
withDuPont's
assessment that the salt
is assumed to be
similar. Measured OH-
reaction rate for El
reaction product
indicates T'A >37
years.
Biodegradation
Biodegradation was not
observed in ready
biodegradation and inherent
biodegradation tests
Biodegradation was not observed
in ready biodegradation and
inherent biodegradation tests
DuPont-A080558,
2009;
DuPont-13 88231-
R2009NC03 l(a)-02,
2010; DuPont-1388231-
R2009NC03 l(s)-02,
2010.
Bioconcentration
(Fish BCF)
< 30 (log < 1)
< 30 (log < 1)
Measured BCFb < 30 at
0.02 mg/L and < 3 at
0.2 mg/L in Medaka 28
days (DuPont-A080560,
2009).
Bioaccumulation
(Field BAF)
< 10
< 10
Pan et al., 2017.°
Notes: °C = degrees Celsius; atm-m3/mol = atmosphere cubic meters per mole; BAF = bioaccumulation factor; BCF =
bioconcentration factor; g/L = grams per liter; g/mol = grams per mole; International Union of Pure and Applied Chemistry
(IUPAC); Koc = soil-water partition coefficient for organic compounds; Kow = octanol-water partition coefficient; kPa =
kilopascals; L/kg = liters per kilogram; mg/L = milligrams per liter; mm Hg = millimeters of mercury; nm = nanometer; Pka =
acid dissociation constant; Pkb = base dissociation constant; T1/2 = half-life.
a Surfactants are surface acting agents that lower the interfacial tension between two liquids. Their amphiphilic nature (i.e., they
contain both a hydrophilic part and a hydrophobic part) causes them to accumulate at interfaces such as the water-air interface,
the water-food interface, and glass walls, which hampers the determination of their aqueous concentration. These surfactant
properties present difficulties in applying existing methods for the experimental determination of log Kow and produce
unreliable results.
b The concentration of the propionate ion was not quantified in the BCF study, so the values are limits based on the limit of
quantification for the analytical technique employed in the study.
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c Pan et al. (2017) quantified the propionate ion and found that the concentrations were low in the tissues expected to most likely
accumulate perfluorinated compounds (e.g., muscle, blood, and so forth). The tissue values indicate a BAF less than 10. Tipid
tissue concentrations are not the basis for this BAF as is common for "traditional" organic compounds.
2.2 Environmental Fate
HFPO dimer acid and its ammonium salt are stable to photolysis, hydrolysis, and biodegradation.
The degradation data suggest that the substances will be persistent (i.e., have a half-life (T1/2)
longer than 6 months) in air, water, soil, and sediments. Based on measured physical-chemical
and sorption data, they are expected to run off into surface water and to rapidly leach to ground
water from soil and landfills. As seen with PFOA and chemicals with similar properties, HFPO
dimer acid and its ammonium salt might undergo long-range atmospheric transport in the vapor
phase and associated with particulates. They are not expected to be removed during conventional
wastewater treatment or conventional drinking water treatment.
When released to the freshwater environment, HFPO dimer acid will dissociate to the HFPO
carboxylate anion and H30+. The ammonium salt will dissolve to the HFPO carboxylate anion
and the ammonium cation (NH4+). It is expected that other salts of the HFPO dimer acid (e.g.,
potassium and sodium salts) will behave similarly. Both have high solubilities in water and are
expected to remain in water with low sorption to sediment or soil. Given the vapor pressure, the
acid can partition to air as well as to water. The salt can also be transported in air, although the
mechanism of vapor phase transport is not understood (DuPont CCAS, 2009). In the vapor
phase, the acid and salt are expected to be stable to direct photolysis and will undergo hydroxyl
radical catalyzed indirect photolysis very slowly.
2.2.1 Water
Measured data for HFPO dimer acid and/or ammonium salt show that they are highly soluble in
water (Table 1). The measured base dissociation constants (pKb) indicate that the chemicals will
exist primarily as the propionate ion at most environmental pH levels.
The chemicals are stable to hydrolysis. A hydrolysis study on the ammonium salt found no
degradation at pH 4, 7, and 9 at 50 degrees Celsius (°C) in 5 days, indicating a hydrolysis T1/2 of
more than 1 year at 20 °C (DuPont-24199, 2008). Calculated Henry's Law constants (Table 1)
suggest that partitioning from water to air might occur. Experimental data on the transfer of the
acid and salt from water to air indicate that partitioning from surface water to the vapor phase
might occur and some transfer from surface water to air is expected (DuPont CCAS, 2009).
Water-air transport of these chemicals, however, is not well understood. Their surfactant
properties, equilibrium between chemical forms as a function of pH, and interaction with
dissolved cations make it difficult to accurately predict how the chemicals will behave in the
aquatic environment.
2.2.2 Air
The acid was described as having "a significant vapor pressure" (DuPont CCAS, 2009). As
observed with PFOA and other perfluorochemicals, these chemicals could be transported in the
vapor phase or could associate with particulate material and be transported with the solids when
released or partitioned into air.
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When released to air or volatilized from water, the chemicals are stable and short- and long-
range transport has occurred (D'Ambro et al., 2021; Galloway et al., 2020). For example,
D'Ambro et al. (2021) demonstrated that just 2.5% of the total GenX concentrations (defined as
the HFPO dimer acid and HFPO dimer acid fluoride) emitted from a fluoropolymer
manufacturing facility in North Carolina were deposited within 150 kilometers of the facility.
Removal from air is expected to occur through scavenging by water droplets and attachment to
particulates followed by precipitation and settling. No studies of long-range transport or air
removal rates are available.
2.2.3 Sediments and Soils
Organic carbon normalized sorption coefficients were measured by high-performance liquid
chromatography (HPLC) (following OECD, 2001a). The sorption of the HFPO dimer acid
ammonium salt to soil and sludge were 12.0 liters per kilogram (L/kg) (log = 1.10) and 12.6 L/kg
(log = 1.08), respectively (DuPont-17568-1675, 2008; OECD, 2001a). Their high water
solubility and low sorption potential indicate that the chemicals will tend to remain largely in
water with little partitioning to soil or sediment. If applied or deposited to soil, they will run off
or leach to ground water and, as indicated by the vapor pressure, could volatilize to air.
2.2.4 Biodegradation
GenX chemicals are resistant to biodegradation; no degradation was observed in standardized
internationally recognized test methods for biodegradability. The aerobic aquatic biodegradation
T1/2 is on the order of years based on no measured inherent biodegradation of the acid or
ammonium salt in OECD 302C, modified Ministry of International Trade and Industry studies
(DuPont-1388231-R2009NC031 (a)-02, 2010; DuPont-1388231-R2009NC031(s)-02, 2010;
OECD, 2008b).2 The HFPO dimer acid ammonium salt showed no inhibition of activated sludge
respiration (OECD TG 209) (OECD, 2010a) at up to 1,000 milligrams per liter (mg/L) (DuPont-
25938 RV1, 2008).
2.2.5 Incineration
A preliminary study submitted to EPA by DuPont/Chemours indicates that thermal degradation
occurs (DuPont-PMN Attachment 119, 2008) and the potential for significant removal during
incineration exists. Thermal degradation was reported to be rapid for HFPO dimer acid and/or its
ammonium salt. The acid T1/2 was reported to be about 2,500 seconds (about 42 minutes) at 150
°C and about 1,900 seconds (about 32 minutes) at 200 °C. The salt T1/2 was 500 seconds (8.3
minutes) at 150 °C and 200 seconds (3.3 minutes) at 200 °C (DuPont-PMN Attachment 119,
2008).
2.2.6 Bioaccumulation
Measured steady-state fish BCFs in medaka (Oryzias latipes) exposed to the acid at 0.2 mg/L
and 0.02 mg/L in a flow-through system for 28 days were less than 3 and less than 30,
respectively (DuPont-A080560, 2009). These BCF results were observed—BCFs of less than 3
2 HFPO dimer acid aerobic aquatic biodegradation T1/2 = 0% by biochemical oxygen demand (BOD) and 1.5% by
high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS); HFPO dimer acid
ammonium salt aerobic aquatic biodegradation T1/2 = < 1% by BOD and 0% by HPLC/MS/MS in 28 days (DuPont-
138823 l-R2009NC031(a)-02, 2010; DuPont-1388231-R2009NC031(s)-02, 2010).
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and less than 30 when exposures were 0.2 mg/L and 0.02 mg/L of the acid, respectively—under
the same conditions in common carp (Cyprinus carpio) (Hoke et al., 2016). A field-derived BAF
was determined from a water body impacted by industrial perfluoroether releases. The log BAFs
for specific tissues in the carp were 0.86 for blood, 0.50 for liver, and 0.61 for muscle. The tissue
values indicate a BAF of less than 10 (Pan et al., 2017).
In a 4-day trout hepatocyte bioaccumulation screening test (non-TG) with the HFPO dimer acid
ammonium salt, no metabolism was observed, suggesting that in vivo metabolism does not
significantly affect potential bioaccumulation (DuPont-23459, 2007).
2.3 Toxicokinetics
In rats and mice, HFPO dimer acid and its ammonium salt are both absorbed from the
gastrointestinal tract at levels that are proportional to dose following acute oral exposures.
Transfer from plasma/serum to the liver, but not adipose tissue, was demonstrated in the few
studies that conducted tissue analysis. The potential for maternal transfer to the fetus (Conley et
al., 2019; Blake et al., 2020) during development and to the neonate during lactation (DuPont-
18405-1037, 2010) was noted. Urine is the primary pathway for excretion. Based on data from
studies of acute, single-dose, gavage, and intravenous exposures, T1/2S in the beta (elimination)
phase are longer in male rats and mice than in females. The male rats' T1/2S in the beta
(elimination) phase are relatively comparable to those for the male and female monkeys, whereas
the female rats' T1/2S are shorter.
HFPO dimer acid is a strong acid (acid dissociation constant (pKa) = 2.84) and will be
predominantly ionized in aqueous solutions with pH values higher than 4 and in both plasma and
serum (DuPont-26349, 2008). Once in solution, the cation that counterbalances the HFPO dimer
anion will vary with the salt used or the mineral ion composition of the solvent, plasma, serum,
intercellular, and intracellular fluids. Based on the physical and chemical properties of HFPO
dimer acid and its ammonium salt, once these chemicals enter physiologic compartments with
pH values higher than 4 (e.g., most ambient water, serum, or blood), they will either dissociate
(acid) or dissolve (ammonium salt) to yield the carboxylate anion. Thus, what is being measured
in the studies outlined in this section is the HFPO dimer acid anion concentration regardless of
whether animals are dosed with HFPO dimer acid or its ammonium salt.
2.3.1 Absorption
Oral. Sprague Dawley (Crl:CD(SD)) rats (five of each sex (5/sex)) were administered (via
gavage) a single oral dose of 30 milligrams per kilogram (mg/kg) HFPO dimer acid ammonium
salt in aqueous solution (purity 84%) in a study conducted according to EPA TG OPPTS
870.7485. Two animals of each sex served as controls. Urine and feces were collected at 0-6
hours, 6-12 hours, 12-24 hours, and every 24 hours until 168 hours post-dose. The 0-12-hour
urine collections accounted for a mean of 95% to 97% of the dose, supporting a conclusion that
these GenX chemicals are rapidly absorbed from the GI tract by male and female rats (DuPont-
18405-1017 RV1, 2011).
In a similar guideline study with Crl/CD-1(ICR) mice (5/sex) (OPPTS 870.7485), the animals
were administered a single oral dose of 3 mg/kg HFPO dimer acid ammonium salt (purity 84%)
by gavage in aqueous solution (DuPont-18647-1017 RV1, 2011). Two animals of each sex
served as controls. Urine and feces were collected at 0-6 hours, 6-12 hours, 12-24 hours, and
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every 24 hours until 168 hours post-dose. In the 0-12-hour urine collections, 31% (mean) of the
substance was found for the males and 39% (mean) for the females. By 168 hours post-dosing,
the total accumulated urine values accounted for 90% and 92% of the total dose for male and
female mice, respectively, indicating that both rats and mice extensively absorb the HFPO dimer
acid anion. This study additionally shows mice either incompletely absorb HFPO dimer acid
anions or eliminate it in urine at a slower rate than was seen in the rats (DuPont-18647-1017
RV1, 2011).
A 28-day gavage study by Rushing et al. (2017) indicates a potentially more complex
toxicokinetic profile for HFPO dimer acid when dosing occurs over multiple days. Groups of six
male and six female C57BL/6 mice were given doses of 1, 10, or 100 mg/kg/day of HFPO dimer
acid daily for 28 days. Serum concentrations were measured at intervals of 1, 5, 14, and 28 days,
and urine concentrations were measured on days 1, 2, 3, 5, 10, and 14. At each time point, serum
levels reflected the magnitude of the dose, but not the exposure duration. The peak serum
concentration occurred at day 5 for all but the high-dose males, where it occurred at day 14.
Serum measurements for the 1- and 10-mg/kg/day doses were lower on days 14 and 28 than on
day 5. The differences in serum concentration between days 5, 14, and 28 are not explained by
the study authors, but could possibly indicate changes in absorption, tissue storage, or
elimination after repeated dosing. The males exposed to 10 and 100 mg/kg/day had higher serum
and urine concentrations than the females, as described in section 2.3.5 (Excretion). Based on the
higher serum and urine concentrations, there appeared to be greater absorption in males than in
females.
In a repeated-dose study following OECD TG 408 (OECD, 1998) guidelines, HFPO dimer acid
ammonium salt (purity 84%) was administered to Crl:CDl(ICR) mice for 95 (males) or 96
(females) consecutive days via gavage at doses of 0, 0.1, 0.5, and 5 mg/kg/day (DuPont-18405-
1307, 2010). Ten animals per sex per group (animals/sex/group) were included for toxicity
evaluation, and an additional 15/sex/group were included for quantitation of the test substance
plasma concentration 2 hours after dosing on day 0 (the first day of dosing) (5/sex/dose),
providing a measure of post-dosing absorption (Table 2). Overall, plasma concentrations
increased with increasing dose, indicating that absorption was not saturated, and displayed broad
standard deviations indicative of considerable inter-animal variability in the absorption. The
doses evaluated differ from those used by Rushing et al. (2017), limiting comparisons of the
postexposure serum and plasma data. The sex difference seen by Rushing et al. (2017) (i.e.,
where male uptake to serum for the 1 and 10 mg/kg/day doses at the end of day 1 was greater
than female uptake) is not as apparent at 2 hours post-dosing in this dataset.
Table 2. Plasma Concentration in Crl:CDl(ICR) Mice at 2 Hours after the First Gavage
Exposure to HFPO Dimer Acid Ammonium Salt
Dose
mg/kg/day
Males
Females
Hg/mL
SD
lig/mL
SD
0
Not detected3
N/A
Not detected
N/A
0.1
0.736
0.099
0.824
0.072
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Dose
mjj/kg/dav
Males
Females
0.5
3.806
1.197
3.606
1.308
5
42.58
5.214
35.34
9.262
Source: Dupont-18405-1307, 2010.
Notes'. N/A = not applicable; ng/mL = micrograms per milliliter; SD = standard deviation.
a Detection limit of the method was 0.005 ng/mL in plasma.
Inhalation. There are no studies investigating HFPO dimer acid or its ammonium salt's uptake
following inhalation exposures of aerosols. In a study conducted by Dupont (17751-723, 2009),
one group of 5 male and 5 female Crl:CD(SD) rats were exposed to 5,200 milligrams per cubic
meter (mg/m3) and two groups of male and female Crl:CD(SD) rats (3/sex/group) were exposed
to aerosols containing 0, 13, and 100 mg/m3 of HFPO dimer acid ammonium salt (84% purity)
for a single 4 hour period. One male and one female rat exposed to air only were used as the
control. The rats in the 0, 13, and 100 mg/m3 groups had a 2-day recovery period. The rats in the
5,200 mg/m3 group recovered for 14-days. There were no measurements of the chemical in
serum or plasma, however, to support an estimate of absorption by way of the respiratory tract.
Dermal. Absorption of HFPO dimer acid ammonium salt through the skin was determined in
vitro with rat and human skin specimens (DuPont-25292, 2008). HFPO dimer acid ammonium
salt (86% purity) was diluted with water to a concentration of 124 milligrams per milliliter
(mg/mL). Serial receptor fluid samples were collected at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 12, and 24
hours and analyzed for cumulative HFPO anion concentration.
Steady-state penetration rates were 70.3 ±5.3 and 6.2 ±5.3 micrograms per square centimeter
per hour for rat and human skin, respectively, which yielded dermal permeability coefficients of
5.71E-4 ± 4.3E-5 centimeters per hour (cm/hr) for rats and 5.02E-5 ± 4.3E-5 cm/hr for humans.
These dermal kinetic parameters demonstrate dermal absorption occurs, but at a relatively slower
rate than chemicals that are well absorbed dermally.
2.3.2 Distribution
Crl:CD(SD) rats (3/sex/dose) were administered a single oral dose of 10 or 30 mg/kg by gavage
in aqueous solution of either HFPO dimer acid ammonium salt (purity 84.5%) or HFPO dimer
acid (purity 98%) (DuPont-24281, 2008; DuPont-24286, 2008). Plasma samples were collected
at 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, and 168 hours, as described in section 2.3.6
(Clearance and Half-life Data). Liver and fat samples were presumed to be collected for analysis
after the 168-hour plasma sample collection. In male rats dosed with HFPO dimer acid
ammonium salt, the mean concentration in plasma at 168 hours post-dose was 0.036 ± 0.011
micrograms per milliliter (|ig/mL) (36 ± 11 nanograms per milliliter (ng/mL)) for the low dose
(10 mg/kg) and 0.057 ± 0.036 |ig/mL (57 ± 36 ng/mL) for the high dose (30 mg/kg). In male rats
dosed with HFPO dimer acid, the mean concentration in plasma at 168 hours post-dose was
0.041 ± 0.01 |ig/mL (41 ± 10 ng/mL) for the low dose (10 mg/kg) and 0.128 ± 0.023 |ig/mL (128
± 23 ng/mL) for the high dose (30 mg/kg). In female rats, plasma concentrations of HFPO dimer
acid anion were not above the limit of quantification (LOQ) in any sample at 168 hours post-
dosing. In males dosed with HFPO dimer acid ammonium salt, the mean concentration of HFPO
dimer acid anion in the liver 168 hours post-dose was 73 ± 25 ng/g for the low dose (10 mg/kg)
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and 38 ± 15 ng/g for the high dose (30 mg/kg). In males dosed with HFPO dimer acid, the mean
concentration of HFPO dimer acid anion in the liver 168 hours post-dose was 24 ± 6 ng/g for the
low dose (10 mg/kg) and 89 ± 4 ng/g for the high dose (30 mg/kg). The mean liver tissue-to-
plasma concentration ratio was higher in males for the ammonium salt (2.19) than for the acid
(0.64) at the low dose (10 mg/kg). At the high 30 mg/kg dose, the liver tissue-plasma
concentration ratio values in male rats were similar: 0.78 for the ammonium salt and 0.71 for the
acid. Females at both doses, however, had a lower accumulation of HFPO dimer acid and its
ammonium salt in the liver than in the male did. Overall, 10 out of 12 female rats dosed with
HFPO dimer acid or its ammonium salt had undetectable concentrations of HFPO dimer acid
anion in the liver (LOQ = 20 ng/g). Two females dosed with HFPO dimer acid ammonium salt at
the low dose (10 mg/kg) had liver HFPO dimer acid anion concentrations above the LOQ,
containing 20.6 and 54.1 ng/g of HFPO dimer acid anion. Females dosed with HFPO dimer acid
did not have liver anion concentrations above the LOQ (20 ng/g). HFPO dimer acid anion
concentrations in the fat tissue samples were below the LOQ of 20 ng/g in all the rats given
HFPO dimer acid or HFPO dimer acid ammonium salt (DuPont-24281, 2008; DuPont-24286,
2008).
Crl:CDl(ICR) mice (3/sex/dose) were administered a single oral dose of 10 or 30 mg/kg by
gavage in aqueous solution of HFPO dimer acid ammonium salt (purity 86%) (DuPont-25300,
2008). Unlike the rat studies, HFPO dimer acid was not evaluated in the mice. Plasma samples
were collected at 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, and 168 hours, as described
in section 2.3.6 (Clearance and Half-Life Data). Liver and fat samples were presumed to be
collected for analysis after the 168 hour plasma sample collection. In males, the mean
concentration of HFPO dimer acid anion in the liver was 384 ± 472 ng/g for the low dose
(10 mg/kg) and 457 ± 337 ng/g for the high dose (30 mg/kg). The mean concentration in fat
tissue was 31.6 ng/g in males for the high dose (30 mg/kg) and less than the LOQ (20 ng/g) for
the low dose (10 mg/kg) and for both doses in females.
In male mice, the mean concentration in plasma at 168 hours post-dose was 0.759 ± 0.946
|ig/mL (759 ± 946 ng/mL) for the low dose (10 mg/kg) and 0.83 ± 0.618 |ig/mL (830 ± 618
ng/mL) for the high dose (30 mg/kg). In females, only one of three mice in each dose group had
a plasma concentration above the LOQ at 168 hours post-dose, which was 0.0232 |ig/mL (23.2
ng/mL) for the high dose (30 mg/kg) and 29.2 ng/g for the low dose (10 mg/kg). Based on the
plasma and liver concentrations reported in the study, a liver-to-plasma ratio was calculated for
males, but not for females because the females did not have liver concentrations above the LOQ.
At the low dose (10 mg/kg), the average male liver-to-plasma ratio was 0.52, and at the high
dose (30 mg/kg), it was 0.58.
Because the perfluorinated portion of the HFPO dimer acid ether is similar to that of the
perfluorinated alkane acids (e.g., PFOA), HFPO dimer acid and its ammonium salt are
anticipated to be transported in serum either freely dissolved or bound to serum protein (Gomis
et al., 2018). Additionally, studies have demonstrated that the major serum protein interaction
site for some PFAS, including PFOA and perfluorohexanoic acid (PFHxA), is albumin (D'eon et
al., 2010; Han et al., 2003). Considering these points and that albumin is the major transport
protein in the blood, it is likely that GenX chemicals are also distributed via serum albumin
(Peters, 1995). Indeed, Allendorf et al. (2019) demonstrate that bovine serum albumin binds
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HFPO dimer acid, and that the albumin/water partition coefficient is in the same range as other
PFAS (e.g., perfluorobutanoic acid (PFBA) and perfluorohexane sulfonic acid (PFHxS)).
A study by Sheng et al. (2018) reported that the HFPO dimer acid anion also binds to fatty acid-
binding protein (FABP). FABPs are intracellular lipid carrier proteins for long-chain fatty acids,
phospholipids, and a variety of chemicals that induce peroxisome proliferation (Erol et al.,
2003). They constitute 2%-5% of the cytosolic protein in the liver. FABPs can be synthesized in
the gastrointestinal tract and act as a systemic carrier of long-chain fatty acids in plasma and
serum (Storch and McDermott, 2009). Thus, FABPs likely play a role in the systemic
distribution of HFPO dimer acid in both its neutral and ionized forms.
2.3.3 Distribution during Gestation and Lactation
HFPO dimer acid ammonium salt can be transferred (distributed) from a pregnant animal to the
fetus, as demonstrated in multiple studies. In an OECD TG 421 (OECD, 2016a)
reproduction/developmental toxicity study (DuPont-18405-1037, 2010), pregnant Crl:CDl(ICR)
mice (25/sex/group) were administered, by gavage, 0, 0.1, 0.5, or 5 mg/kg/day HFPO dimer acid
ammonium salt from premating day 14 to lactation day (LD) 20/21. Blood was collected from
the dams 2 hours after dosing on LD/postnatal day (PND) 21 (scheduled termination) and
pooled. The litters were normalized on PND4 to 8 pups per litter (4/sex). Blood was collected
and pooled from the pups not randomly selected on PND4. The HFPO dimer acid anion was
present in the pooled plasma of PND4 pups at concentrations approximately two to four times
lower than the concentrations in the dams on LD21. These results indicate that there is transfer of
HFPO dimer acid anion from maternal serum. The PND/LD21 plasma levels in both male and
female pups, however, were forty- to sixtyfold lower than the maternal LD21 plasma
concentrations, indicating that the majority of fetal transfer occurred during gestation (DuPont-
18405-1037, 2010).
Blake et al. (2020) demonstrated that HFPO dimer acid can be transferred from the pregnant dam
to the embryo during gestation. Pregnant CD-I mouse dams were dosed from embryonic day (E)
1.5 to El 1.5 or E17.5 with either deionized water (vehicle control), 1 or 5 mg/kg/day of PFOA,
or 2 or 10 mg/kg/day of HFPO dimer acid. At El 1.5 and E17.5, serum and a portion of the
hepatic left lateral lobe were collected from pregnant dams after the final dose. Amniotic fluid
was collected by needle aspiration from litters euthanized on El 1.5 and whole embryos were
collected on El 1.5 and E17.5 to determine the concentration of HFPO dimer acid. HFPO dimer
acid was detected in both the amniotic fluid and the whole embryo at 2 and 10 mg/kg/day and at
both time points, demonstrating transfer of HFPO dimer acid from the pregnant dam to the fetus
during gestation (Table 3).
Table 3. Concentrations of HFPO Dimer Acid in CD1 Pregnant Mice and Their Embryos
at Embryonic Day 11.5 or 17.5a
Measurementb
Embryonic
day
HFPO dimer acid
2 mg/kg/day
10 mg/kg/day
Maternal Serum (|ig/mL)
11.5
33.5 ± 15.7
118.1 ± 10.4
17.5c
22.9 ± 17.1
58.5 ±34.5
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Measurementb
Embrvonie
day
HFPO dimer aeid
2 mg/kg/day
10 mg/kg/day
Amniotic Fluid (|ig/mL)
11.5
3.6 ±2.2
9.3 ±2.0
17.5
NQ
NQ
Maternal Liver (|ig/g)
11.5
5.45 ±3.43
19.9 ±4.2
17.5
4.56 ±2.80
14.2 ±7.6
Whole Embryo (|ig/g)
11.5
0.91 ±0.22
3.21 ±0.51
17.5
3.23 ± 1.28
7.69 ±2.92
Source: Blake et al., 2020.
Notes'. ng/mL = micrograms per milliliter; (ig/g = micrograms per gram embryo weight; SD = standard deviation; NQ = not
quantified due to limited volume.
a For each reported measurement in this table, N = 6-8 per group.
b Limit of detection was 0.010 ng/mL; note all vehicle control samples were below the limit of detection.
c HFPO dimer acid was detected in the serum of vehicle control mice in the E17.5 group (0.211 ± 0.55 (ig/mL).
HFPO dimer acid concentrations increased with increasing dose in all samples. The
concentration of HFPO dimer acid in the whole embryo increased from El 1.5 to E17.5 in both
dose groups, indicating bioaccumulation in the embryo over the gestational period. Conversely,
the concentration of HFPO dimer acid in the maternal serum decreases from El 1.5 to E17.5 in
both dose groups. The authors note that the decrease in maternal serum HFPO dimer acid could
be the result of increased transfer to embryos over time or to dilution effect from blood volume
expansion over the course of gestation.
In the DuPont-18405-1037 (2010) study, generally, the standard deviations were large in all dose
groups, especially as compared to PND21. The male pups tended to have slightly higher plasma
concentrations of HFPO dimer acid anion than the female pups at PND40. For example, at the
0.1 mg/kg/day-dose group, the concentration of HFPO dimer acid anion was 1.352 and 0.946
|ig/mL (1,352 and 946 ng/mL) in male and female pups, respectively. Similarly, at the 0.5
mg/kg/day-dose group, the concentration of HFPO dimer acid anion was 6.282 and 4.074 |ag/m L
(6,282 and 4,074 ng/mL) in male and female pups, respectively, and it was 51.34 |ig/mL (51,340
ng/mL) in male pups and 43.34 |ag/mL (43,340 ng/mL) in female pups at 5 mg/kg/day (DuPont-
18405-1037, 2010).
Transfer of HFPO dimer acid anion to the fetus was also demonstrated in groups of five
Crl:CD(SD) rats exposed to doses of 0, 5, 10, 100, or 1,000 mg/kg/day from gestation day (GD)6
to GD20 (Dupont-18405-849 RV1, 2011). On GD20, blood was collected from individual dams
2 hours after dosing and trunk blood was collected from the fetuses and pooled by litter for
analysis. The plasma concentration in the blood samples from the dams was three times higher
than the plasma concentration in the pooled blood of their fetuses. The detection of HFPO dimer
acid anion in the pooled fetus plasma demonstrates gestational transfer from dam to fetus.
Similarly, Conley et al. (2019) demonstrated transfer of HFPO dimer acid anion to the fetus by
measuring serum concentrations of pregnant Crl:CD(SD) rats exposed to 0, 1, 3, 10, 30, 62.5,
125, 250, and 500 mg/kg/day HFPO dimer acid ammonium salt from GD14 through GD18.
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Serum was collected from the dams in all dose groups and plasma was collected from the fetuses
in the 0, 1, 3, 10 and 30 mg/kg/day groups. On GD18, trunk blood was collected from individual
dams 2 hours after dosing and blood was collected from the fetuses' jugular vein and pooled per
litter for analysis. HFPO dimer acid anion was detected in the pooled fetal plasma at all doses
and the concentration increased with increasing maternal dose (Table 4). The study authors noted
that, while the increases in maternal serum and fetal plasma were linear in the lower dose range
(0-30 mg/kg/day), the maternal slope was significantly greater than the fetal slope. The maternal
serum concentration of HFPO dimer acid anion increased 0.46 |ag/m L (460 ng/mL) per 1 mg/kg
increase in maternal dose while the fetal plasma concentration increased 0.12 jag/m L (120
ng/mL) per 1 mg/kg increase in maternal dose. Additionally, the study authors modeled uptake
over the full maternal dose range (1-500 mg/kg) (Table 4) using exponential one-phase
association and determined that a plateau was reached at 112 ± 15 |ig/mL (112,000 ± 15,000
ng/mL), indicative of uptake saturation (Conley et al., 2019).
Table 4. Maternal Serum and Fetal Plasma Concentrations on GD18 in Crl:CD(SD) Rats
Exposed to HFPO Dimer Acid Ammonium Salt from GD14-18
Oral dose
mjj/kjj/day
Pregnant dam scrum
Fetal plasma
jig/mL
SE
jig/mL
SE
0
0.027
0.008
0.018
0.01
1
0.68
0.08
0.13
0.06
3
1.2
0.3
0.49
0.04
10
4.6
1.1
1.9
0.2
30
13.9
3.1
3.5
0.4
62.5
30.7
2.9
N/A
N/A
125
46.0
10.3
N/A
N/A
250
81.8
21.6
N/A
N/A
500
100.7
26.4
N/A
N/A
Source: Conley et al., 2019, Table S10.
Notes'. ng/mL = micrograms per milliliter; N/A = not applicable because no sample collected at that dose; SE = standard error.
Conley et al. (2021) also demonstrated transfer of HFPO dimer acid anion to the fetus and pup
by measuring serum concentrations of pregnant Crl:CD(SD) rats exposed to 0, 1, 3, 10, 30, 62.5,
or 125 mg/kg/day HFPO dimer acid ammonium salt from GD16 through GD20 or to 0, 10, 30,
62.5, 125, or 250 mg/kg/day from GD8 through PND2. Serum was collected from the dams and
fetuses in all dose groups on GD20 in the GDI6-20 experiment and from the dams on PND2 in
the neonatal experiment. In the GDI6-20 experiment, trunk blood and liver samples were
collected from both dams and fetuses 2 to 4 hours after the final oral dose on GD20. Fetal serum
was pooled per litter for analysis. On PND2 in the neonatal experiment, trunk blood and liver
samples were collected from the dams 2 to 5.5 hours after the final oral dose and liver samples
were collected from the pups. Maternal serum and liver HFPO dimer acid anion concentrations
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increased as a function of dose during both experiments (Table 5). The study authors noted that
there was no statistically significant difference in serum or liver concentration within a given
dose group between the two experiments indicating that bioaccumulation did not occur after
longer exposure. HFPO dimer acid anion was detected in the pooled fetal serum at all doses and
the concentration generally increased with increasing maternal dose. Regression analyses
showed that fetal and maternal serum concentrations increased log-linearly as a function of
maternal oral dose, and maternal serum concentrations were approximately 2- to 3-fold greater
than fetal serum concentrations. Liver concentrations of HFPO dimer acid anion in dams,
fetuses, and pups also increased log-linearly. The fetal and maternal liver concentrations on
GD20 were nearly identical for the 30-125 mg/kg/day dose levels. On PND2, male pup liver
concentrations were significantly greater than female pup liver concentrations, which was most
prominent at the 125 mg/kg/day dose level. PND2 liver concentrations for both sexes were
approximately 10-fold lower than concentrations observed in GD20 fetal livers.
Table 5. Maternal and Offspring HFPO Dimer Acid Anion Concentrations in Serum and
Liver Samples Collected on GD20 or PND2 from Crl:CD(SD) Rats Orally Exposed to
HFPO Dimer Acid Ammonium Salt from GD16-20 or GD8-PND2
Oral dose
mg/kg/day
Maternal
serum GD20
(W/mL)
Fetal serum
GD20
WmL)
Maternal
serum PND2
(M«/mL)
Maternal
liver GD20
(Mg/g)
Fetal liver
GD20 (nfi/g)
Maternal
liver PND 2
(Mg/g)
Female pup
liver PND 2
(Mg/g)
Male pup
liver PND 2
(Mg/g)
0
0.016 ±0.014
0.014 ±0.008
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2.3.4 Metabolism
In two in vitro studies, hepatocytes (1 x 106 cells/mL for clearance incubations and 5 x 106
cells/mL for biotransformation incubations) prepared from male and female Crl:CD(SD) rats
were incubated with 2 micromolar (|iM) (clearance) or 200 |iM (biotransformation) solutions of
HFPO dimer acid ammonium salt for a total of 120 minutes (DuPont-23460, 2007). Samples
were analyzed for HFPO dimer acid ammonium salt and potential metabolites at 5, 15, 30, 45,
60, 90, and 120 minutes. Heat-inactivated hepatocytes were used as negative controls and 4-
nonylphenol in live hepatocytes were used as a positive control. There was no difference in the
concentration of HFPO dimer acid between the viable and heat-inactivated hepatocytes,
indicating that HFPO dimer acid ammonium salt is not metabolized by rat hepatocytes.
Additionally, no metabolites were detected in the biotransformation incubation samples
(DuPont-23460, 2007). Similar in vitro studies were conducted in rat hepatocytes in Gannon et
al. (2016). Hepatocytes (1 x 106 cells/mL for clearance incubations and 5 x 106 cells/mL for
biotransformation incubations) prepared from male and female Crl:CD(SD)rats were incubated
with 5 |iM (clearance) or 50 |iM (biotransformation) solutions of HFPO dimer acid ammonium
salt for a total of 120 minutes. Heat-inactivated hepatocytes were used as negative controls and
samples were collected at 0, 30, 45, 60, 90, and 120 minutes. Gannon et al. (2016) concluded that
the test substance was not metabolized by rat hepatocytes because there was no difference in
clearance rate between live and heat-inactivated hepatocytes and no metabolites were identified.
In the single oral (gavage) study of rats described in section 2.3.1 (Absorption), the total
accumulated amount of HFPO dimer acid ammonium salt at 168 hours post-dosing in the
collected urine accounted for 103% + 2.73% and 99.8% + 6.41% of the administered dose for
male and female rats, respectively, and there was no detection of metabolites (DuPont-18405-
1017 RV1, 2011).
Similarly, in the single oral (gavage) study of mice described in section 2.3.1 (Absorption), the
total accumulated amount of HFPO dimer acid ammonium salt accounted for 89.5% ± 6.91%
and 91.5% ± 6.04% of the total dose for male and female mice, respectively, and there was no
detection of metabolites in the urine (DuPont-18647-1017 RV1, 2011).
2.3.5 Excretion
Urine. Studies in rats, mice, and monkeys indicate that urine is the primary excretory pathway
for GenX chemicals. In the DuPont-18405-1017 RV1 (2011) study, Crl:CD(SD)rats (5/sex)
administered a single oral (gavage) dose of 30 mg/kg HFPO dimer acid ammonium salt excreted
95% to 97%) of the dose in urine within 12 hours. The pooled urine collections accounted for
virtually all the substance administered with no evidence of metabolic alteration. Study authors
calculated the elimination T1/2 in the urine for male and female rats to be 3 hours and 8 hours,
respectively. In a companion study, Crl/CD1(ICR) mice (5/sex) were administered a single oral
(gavage) dose of 3 mg/kg HFPO dimer acid ammonium salt (purity 84%) (DuPont-18647-1017
RV1, 2011). Urinary elimination in mice appeared to be less efficient than in the rats given that
only 31%) (mean) and 39% (mean) of the dose material was found in the 12-hour pooled urine for
the male and female mice, respectively. At 168 hours post-dosing, the mean values for the
pooled urine samples accounted for 90% and 92% of the total dose for the male and female mice,
respectively (DuPont-18647-1017 RV1, 2011). Study authors calculated the elimination T1/2 in
the urine for male and female mice to be 21 hours and 18 hours, respectively. Based on the
amounts in urine and the clearance from blood (see section 2.3.6), mice appear to have less of an
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ability than rats to clear the HFPO dimer acid anion by transferring it to urine in the early
postexposure period. The differences in the results of these studies might have been influenced
by the different doses given to the rats (30 mg/kg) and the mice (3 mg/kg) (DuPont-18647-1017
RV1, 2011; DuPont-18405-1017 RV1, 2011).
The dynamic relationship across dose and exposure duration observed in serum measurements
from the Rushing et al. (2017) study is also reflected in their data on urinary excretion. Urine
concentrations were monitored on exposure days 1, 2, 3, 5, 10, and 14. For the 1- and 10-
mg/kg/day doses, urinary concentration peaked on day 3 and, thereafter, declined monotonically.
Males had higher urine concentrations than females at each time point, consistent with their
higher serum concentrations. For the 100-mg/kg/day-dose group, the concentrations in urine
peaked at day 2 and again at day 14 in males while in females they appeared to peak at 5 days
followed by a decline at 10 and 14 days.
Feces. Fecal elimination of HFPO dimer acid appears to be minor in rats and mice in the
available single-dose studies (DuPont-18405-1017 RV1, 2011; DuPont-18647-1017 RV1, 2011).
Specifically, feces + cage wash (dried fecal matter) from male and female rats had 2% and 6% of
recovered compound, respectively, while feces + cage wash from male and female mice had 12%
and 8% of recovered compound, respectively. The data for combined fecal matter and cage wash
suggest that mice might lose slightly more HFPO dimer acid through fecal matter than rats. Low
fecal excretion could reflect low levels of hepatic loss via biliary excretion.
2.3.6 Clearance and Half-Life Data
Clearance time. In multiple study reports, the study authors did not calculate pharmacokinetic
parameters such as Ty2 or area under the curve and instead defined the metric "clearance time" as
the time when 98.4% of the anion from the HFPO dimer acid ammonium salt was cleared from
the plasma.
A total of 12 Crl:CD(SD) rats, 3/sex/dose, received a single oral dose of 10 or 30 mg/kg/day
HFPO dimer acid ammonium salt (84.5% purity) by gavage (Dupont-24281, 2008). Plasma
samples were collected from animals serially at 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120,
144, and 168 hours. In males, plasma levels peaked within the first 1-2 hours after dosing for the
low dose, and within the first 30 minutes to 1 hour for the high dose. By days 4 to 5, plasma
concentrations were less than 1% of the peak level, although still above the LOQ (0.02 jag/m L
(20 ng/mL)). In females, the plasma levels peaked at 1 hour for the low dose and had usually
declined to the LOQ (0.02 |ig/mL (20 ng/mL)) by 24 hours. At the 30-mg/kg dose, the plasma
levels of female rats peaked at 30 minutes to 1-hour post-dosing and declined to the LOQ (0.02
|ig/mL (20 ng/mL)) by 24 or 48 hours. In male rats, the authors identified 12 hours as the
clearance time at the low dose and 22 hours at the high dose (Table 6). In female rats, the
clearance values were 4 hours and 8 hours for the low dose and high dose, respectively.
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Table 6. Clearance Times in Plasma for Male and Female Rats and Mice Following a Single
Oral Dosea
Chemical
Male rat
Male mouse
Female rat
Female mouse
10 mg/kg
HFPO dimer acid ammonium salt
12 hr
143 hr
4 hr
57 hr
HFPO dimer acid
28 hr
ND
8 hr
no data
30 mg/kg
HFPO dimer acid ammonium salt
22 hr
139 hr
8 hr
62 hr
HFPO dimer acid
22 hr
ND
4 hr
no data
Sources: Dupont-24281, 2008; Dupont-24286,2008; Dupont-25300, 2008.
Notes', hr = hour
a "Clearance time" is defined as the time when 98.4% of the HFPO dimer acid ammonium salt was cleared from the plasma.
The same protocol was followed using HFPO dimer acid (98% purity) (Dupont-24286, 2008). At
the low dose, plasma concentrations peaked within 1 hour in both male and female rats, while at
the high dose, the peak plasma concentrations occurred in males at 1 or 2 hours and in females at
15 minutes. The clearance times in males were 28 hours and 22 hours for the low dose and high
dose, respectively. The clearance times in females were 8 hours and 4 hours for the low dose and
high dose, respectively (Table 6).
The protocol outlined in this section was also followed for mice with a total of 12 Crl:CD(ICR)
mice, 3/sex/dose, receiving a single oral dose of 10 or 30 mg/kg/day HFPO dimer acid
ammonium salt (86% purity) by gavage (Dupont-25300, 2008). Plasma samples were collected
from animals serially at 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, and 168 hours post-
dosing. Peak plasma HFPO dimer acid anion concentrations were reached within 8 hours for the
males and 4 hours for the females at the 10-mg/kg dose. At the 30-mg/kg dose, the peak HFPO
dimer acid anion concentrations were reached within 2 hours for both males and females. The
mean clearance time was slower in the males (143 hours and 139 hours at the low dose and high
dose, respectively) than in the females (57 hours and 62 hours at the low dose and high dose,
respectively) (Table 6).
In the oral toxicokinetic studies, the clearance times were shorter in rats than in mice and were
shortest in female rats compared to male rats for both anions from HFPO dimer acid and its
ammonium salt. In rats at the 10-mg/kg dose, HFPO dimer acid took longer to clear than its
ammonium salt in both male and female rats. At the 30-mg/kg dose, however, both HFPO dimer
acid and its ammonium salt had the same clearance times in male rats, but the HFPO dimer acid
ammonium salt took longer to clear in female rats.
In a cross-species pharmacokinetic study, Crl:CD(SD) rats (3/sex) were administered a single
intravenous bolus of 10 or 50 mg/kg of HFPO dimer acid ammonium salt and Cynomolgus
monkeys (3/sex) were administered a single intravenous bolus of the HFPO dimer acid
ammonium salt (10 mg/kg) (DuPont-17751-1579 RV1, 2009). Plasma samples were collected at
intervals over the first 24 hours post-dosing and once per day for the subsequent 7 days in the
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rats and 21 days in the monkeys. In the rats, the plasma concentrations were consistently higher
for the males than for females by approximately one to two orders of magnitude, consistent with
the indication that female rats have more rapid elimination. The clearance times for male rats
were 22 hours and 17 hours in the 10- and 50-mg/kg dose groups, respectively. The clearance
times for female rats were 3 hours and 4 hours in the 10- and 50-mg/kg dose groups,
respectively. Notably, the calculated clearance time in the male rats was longer for the 10-mg/kg
dose group (22 hours) than the clearance time calculated in Dupont-24281 (2008) for male rats in
the 10-mg/kg dose group (12 hours). Female rats had similar clearance times. Additionally, the
standard deviations on each serum mean were broad for the rats in the 50-mg/kg dose group,
indicative of wide differences between the three males and three females evaluated at that dose.
In the monkeys, the standard deviations on each serum mean were broad, especially for the
female monkeys over the first 2 hours, which is indicative of wide differences between the three
males and three females evaluated. The plasma levels were generally higher in females over the
first 2 hours, were nearly identical at 4 hours, and were slightly higher in the males from 4 to 336
hours. The levels of the anion from HFPO dimer acid ammonium salt were very low at 168 hours
in male (0.004 |ig/mL (4 ng/mL)) and female (0.001 |ig/mL (1 ng/mL)) monkeys. For 408 hours
and beyond, concentrations were below the LOQ of 0.001 |ag/m L (1 ng/mL). The clearance
times calculated for the male and female monkeys were 11 hours and 10 hours, respectively.
Half-lives. In Gannon et al. (2016), the goodness of fit was calculated for the plasma
concentrations after oral and intravenous dosing (DuPont studies outlined above) using one- and
two-compartment models, and the two-compartment model had a better fit. Pharmacokinetic
parameters identified by Gannon et al. (2016) are presented for the intravenous studies in Table 7
and for the oral studies in Table 8. The alpha phase T1/2 represents the plasma concentration in
the early post-injection period and is considered to reflect the plasma distribution phase
(Klaassen, 1996). The beta phase T1/2 represents the period during which the chemical in the
plasma has established an equilibrium with the levels in the body tissues and represents the
elimination phase. The two-compartment model is a refinement of the prior pharmacokinetic
analysis in which the clearance time was calculated. The two-compartment model better fits the
data and separates distribution and elimination phases; therefore, generally for comparisons
across the datasets, the T1/2S are preferred.
Table 7. T1/2 Estimates from Intravenous Injection in Sprague Dawley Rats and
Cynomolgus Monkeys
Tib
Intravenous Exposures (in hours)
Male rat
Male monkey
Female rat
Female monkey
Alpha (Plasma Distribution) Phase
3.6
2.3
0.4
1.9
Beta (Plasma Elimination) Phase
89.1
64.1
22.6
79.6
Source: Gannon et al., 2016.
In the intravenous injection studies, the T1/2 of the alpha phase of distribution is similar (about 2
hours) for male and female monkeys, but the T1/2 of the beta (elimination) phase is longer in
female monkeys. The T1/2 of the beta (elimination) phase in female monkeys is longer than it is
in the female rats, which could be a result of female monkeys having higher tissue stores than
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female rats or clearance of HFPO dimer acid anion from their tissues might be slower. There are
no studies, however, to distinguish these explanations such as a study of tissue concentrations
over time. In rats, both the alpha and beta phases are shorter in females than in males; the beta
phase T1/2 is about four times longer in males, suggesting higher levels in tissues of males or
slower clearance of HFPO dimer acid anion from their tissues (Gannon et al., 2016).
Gannon et al. (2016) also used the data from the single oral dose studies in rats and mice to
derive estimates of alpha and beta phase T1/2S to represent the distribution and elimination
phases. The oral exposure data are not ideal for this calculation because the chemical is not
directly injected into the blood. However, because intestinal uptake of HFPO dimer acid anion
from the ammonium salt is believed to be rapid and there appears to be no metabolism, the
estimates are reasonable for a two-compartment model.
In rats, following oral exposure, the alpha (distribution) T1/2 phase is shorter in females than in
males and the beta (elimination) phase T1/2 is comparable for both sexes (Table 8). In mice, the
T1/2 estimates for the alpha phase are similar for both sexes and the T1/2 estimates for the beta
phase are shorter for females than for males (Table 8). The T1/2 estimated for the beta phase in
female rats is shorter from the intravenous data (22.6 hours) than from the oral gavage data (67.4
hours), while the other estimates of T1/2 from the intravenous and oral gavage data for males and
females are similar.
Table 8. T1/2 Estimates from Single Oral Dose in Sprague Dawley Rats and Crl/CD1(ICR)
Mice
Tl/2
Oral Exposures (in hours)
Male rat
Female rat
Male mouse
Female mouse
Alpha (Plasma Distribution) Phase
2.8
0.2
5.8
4.6
Beta (Plasma Elimination) Phase
72.2
67.4
36.9
24.2
Source: Gannon et al., 2016.
The time it takes to achieve a balance between gastrointestinal uptake and excretion (i.e., steady
state) following daily gavage exposures to the HFPO dimer acid anion is dependent on the T1/2S
of the alpha and beta phases. When the data are well described by a multicompartmental model,
the steady state is a function of the multiple T1/2S for the intercompartmental distribution (alpha
phase) and elimination (beta phase); however, at later times, the elimination T1/2 is expected to
dominate the time to steady state and to be reached approximately within four T1/2S, or 6.15 days,
for male mice (Ito, 2011). This was calculated by multiplying the oral gavage beta phase T1/2
(36.9 hours) for male mice by 4 and dividing that product by 24 hours. The data from Rushing et
al. (2017) for male mice clearly demonstrate a lack of serum steady state for male mice after
receiving doses of 1, 10, and 100 mg/kg/day for 28 days because the serum concentrations do not
remain constant after the expected 6 days. In fact, HFPO dimer acid concentrations continue to
change between 5 and 14 days and 14 and 28 days. These continual changes in plasma
concentration after 6 days indicate dynamics over multiple days that are not represented by
typical multicompartment models and, therefore, are not appropriate for modeling the
complexity of the pharmacokinetics of HFPO dimer acid and its ammonium salt.
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Repeated-dose study. In a repeated-dose study with Crl:CDl(ICR) mice dosed with 0, 0.1, 0.5,
or 5 mg/kg/day for at least 90 days, plasma measurements were determined 2 hours post-dosing
on days 0, 28, and 95 (Dupont 18405-1307, 2010). Plasma concentrations increased less than
twofold between the 2 hour and the 28-day measurements for both the males and females in all
dose groups (Table 9). Unfortunately, the study provides no measurements between the 2-hour
and 28-day time points to allow for a determination regarding steady state. As mentioned above,
however, the Rushing et al. (2017) study in mice provides measurements in serum at 1, 5, 14,
and 28 days following daily gavage dosing of C57BL/6 mice that clearly establish the lack of
steady-state conditions, which supports development of a more complex model to represent these
data.
Table 9. Mean Plasma Concentrations with Standard Deviations of Dosing Crl:CDl(ICR)
Mice with HFPO Dimer Acid Ammonium Salt for at Least 90 Days
Dose
mg/kg/day
Day 0
Day 28
Day 95
jig/m L
SD
COV
jig/mL
SD
COV
jig/mL
SD
COV
Males
0
NDa
N/A
N/A
ND
N/A
N/A
ND
N/A
N/A
0.1
0.736
0.099
13%
1.124
0.238
21%
1.276
0.309
24%
0.5
3.806
1.197
31%
7.182
3.055
43%
7.068
2.398
34%
5
42.58
5.214
12%
52.240
16.725
32%
67.98
13.717
20%
Females
0
ND
N/A
N/A
N/D
N/A
N/A
ND
N/A
N/A
0.1
0.824
0.072
9%
0.704
0.35
50%
0.74
0.282
38%
0.5
3.606
1.308
36%
4.198
1.239
30%
5.438
1.696
31%
5
35.34
9.262
26%
46.58
16.842
36%
45.58
5.741
13%
Source: Dupont 18405-1307, 2010.
Notes'. COV = coefficient of variation (SD / mean); ng/mL = micrograms per milliliter; N/A = not applicable; ND = not detected;
SD = standard deviation.
a Limit of detection = 0.005 ng/mL
Plasma concentrations remained relatively constant between 28 days and 95 days for male and
female mice administered the 0.1-mg/kg/day dose in the Dupont 18405-1307 (2010) study
(Table 9). At the 0.5-mg/kg/day dose, plasma concentrations were relatively constant from day
28 to 95 days for the males, but the females' plasma concentrations increased from 4.198 to
5.438 |ig/mL (4,198 ng/mL to 5,438 ng/mL) (a 30% increase). This indicates that the HFPO
dimer acid anion does not appear to accumulate at 0.1 mg/kg/day; however, it might have
accumulation potential at 0.5 mg/kg/day. Interestingly, this increase in female plasma
concentrations from 28 days to 95 days is equal to the coefficient of variation (COV) in the 28-
day measurement, thus the difference between days 28 and 95 could be the result of inter-animal
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differences in response to the same dose. Also interesting is that, at the 5-mg/kg/day dose, female
plasma levels returned to approximately the same levels at 28 and 95 days (46.58 and 45.58
|ig/mL (46,580 and 45,580 ng/mL), respectively) (Table 9). In the males, the plasma levels at 28
days increased from 52.24 to 67.98 |ig/mL (52,240 ng/mL to 67,980 ng/mL) at 95 days (a 30%
increase), again equaling the COV in the 28-day measurement. Thus, the difference between
days 28 and 95 could be the result of variability in these measurements as a result of inter-animal
differences and might not necessarily reflect accumulation of HFPO dimer acid anion.
3.0 Problem Formulation
3.1 Conceptual Model
The conceptual model provides useful, publicly available information to characterize and
communicate the potential health hazards related to oral exposure to HFPO dimer acid and its
ammonium salt. Figure 2 depicts in a conceptual diagram the sources of these GenX chemicals,
the routes of exposure to biological receptors of concern (e.g., human activities related to
ingested tap water such as drinking, food preparation, and consumption), the potential
assessment endpoints (e.g., effects such as liver toxicity), and populations at risk of exposure to
HFPO dimer acid and its ammonium salt. As outlined in the legend for Figure 2, the green boxes
indicate where there are limited data available for these GenX chemicals. This includes
quantitative data for oral exposure to HFPO dimer acid and its ammonium salt, as well as the
limited data available for some of the potential sources of exposure to these chemicals. The
quantitative data for oral exposure to HFPO dimer acid and its ammonium salt includes animal
toxicity and toxicokinetic studies; no epidemiological studies on health effects in humans are
available. The white boxes indicate that no data are publicly available to allow for determining if
GenX chemicals are found in certain sources and that no human toxicity data exist.
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STRESSORS
POTENTIAL
SOURCES OF
EXPOSURE
EXPOSURE
ROUTES
ORGANS/
SYSTEMS
AFFECTED
HFPO Dimer Acid and its
Ammonium Salt
Drin king
Water
POTENTIAL
RECEPTORS IN
GENERAL
POPULATION
Ambient
Ground and
Surface Water
Industrial
Uses
Ail-
Soil
Dust
Consumer
Products
Food
Oral
Dermal
Inhalation
Liver Effects
Hematological
Effects
Reproductive/
Developmental
Effects
Kidnev Effects
Immune
Effects
Cancer
Adults
Children (including
breastfed/formula fed
infants)
Pregnant Women
and the Developing
Embrvos/Fetuses
Lactating Women
Fire
Fighting
Foams
LEGEND
Data Selected for
Assessment
(Quantitative Data)
Limited Data
Undetermined
Figure 2. Conceptual Model for HFPO Dimer Acid and Its Ammonium Salt
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3.2 Overall Scientific Objectives
This document provides the health effects basis for the development of oral RfDs for subchronic
and chronic durations for GenX chemicals, including the science-based decisions providing the
basis for estimating the POD. This section discusses the factors EPA considers in the process of
developing a POD (depicted in Figure 2).
Stressors: This assessment addresses only HFPO dimer acid and its ammonium salt. It does not
address any other chemicals used in the GenX processing technology or any other precursors,
metabolites, or degradate of HFPO dimer acid and its ammonium salt. Uses of GenX chemicals
include as intermediates and as polymerization aids in the production of fluoropolymers. These
chemicals are two of several replacements for PFOA and its ammonium salt and could have
many applications in consumer products (e.g., stain- and water-repellant textiles) and industrial
processes (e.g., pharmaceutical and semiconductor manufacturing). Information on specific
products containing GenX chemicals is not available, however, GenX chemicals may be used in
the manufacture of the same or similar commercial fluoropolymer end products that formerly
used PFOA. GenX chemicals may also be generated as a byproduct of fluoromonomer
production. Publicly available data, although limited, indicate that sources of exposure to GenX
chemicals include both ground and surface waters used for drinking. Many other potentially
important sources of exposure to GenX chemicals exist given their use as a replacement for
PFOA, including foods; indoor dust in a home or work environment; indoor and outdoor air; soil;
biosolids; and consumer products within the home, workplace, children's schools, and daycare
centers. Very little quantitative information on these sources of exposure, however, is available.
Routes of Exposure: Nonoccupational exposure to GenX chemicals in water can occur through
oral exposure (i.e., drinking water, cooking with water, and incidental ingestion from showering)
and is expected to occur by dermal exposure (i.e., contact of exposed parts of the body with
water containing GenX chemicals during bathing or showering, and dishwashing) and inhalation
exposure (e.g., volatilization of the GenX chemicals from the water during bathing or showering,
or while using a humidifier or vaporizer). There is limited information identifying health effects
from inhalation or dermal exposures to GenX chemicals in animals. Specifically, two acute
dermal toxicity tests (one in rats and one in rabbits), one dermal irritation study in rabbits, and
one acute inhalation toxicity test in rats (see section 4.1) have been conducted. Repeated-dose
toxicity data are available for oral exposure, but not for inhalation and dermal exposures. Since
the only quantitative data available for HFPO dimer acid and its ammonium salt are for oral
exposure, this assessment applies only to that route.
Receptors: The receptors are those in the general population who could be exposed to GenX
chemicals in tap water through ingestion (i.e., adults, the elderly, women of childbearing age,
pregnant women, fetuses, infants, and children). In the conceptual model in Figure 2, the box for
adults includes sensitive life stages (e.g., women of childbearing age and the elderly). In this
toxicity assessment, the first two steps (Step 1. Hazard Identification and Step 2. Dose Response)
of the four-step risk assessment process developed by the National Academy of Sciences are
addressed. This toxicity assessment summarizes potential health effects associated with exposure
to GenX chemicals and identifies levels at which those health effects might occur. Potential
exposure to receptors is not determined. Toxicity values from this assessment can be combined
with specific exposure information (Step 3. Exposure Assessment) to help characterize the
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potential public health risks associated with exposure to these chemicals (Step 4. Risk
Characterization) to the receptors outlined here.
Endpoints: No human epidemiological studies for GenX chemicals are available. Oral exposure
studies of acute, subchronic, and chronic duration are available in rodent species, including rats
and mice. The recommended definitions of study duration were applied as outlined in A Review
of the Reference Dose and Reference Concentration Processes (EPA, 2002). Using this
approach, the employed study durations are as follows:
• Acute: Exposure by the oral, dermal, or inhalation route for 24 hours or less.
• Short-term: Repeated exposure by the oral, dermal, or inhalation route for more than 24
hours, up to 30 days.
• Subchronic: Repeated exposure by the oral, dermal, or inhalation route for more than
30 days, up to approximately 10% of the life span in humans (more than 30 days up to
approximately 90 days in typically used laboratory animal species).
• Chronic: Repeated exposure by the oral, dermal, or inhalation route for more than
approximately 10% of the life span in humans (more than approximately 90 days to
2 years in typically used laboratory animal species).
Adverse effects observed following exposure to HFPO dimer acid and/or its ammonium salt
include liver toxicity (e.g., hypertrophy, single-cell necrosis, focal necrosis and apoptosis),
hematological effects (e.g., decreased red blood cell (RBC) count, hemoglobin, and hematocrit),
kidney toxicity (e.g., increased kidney weight, necrosis, and hyperplasia), reproductive and
developmental effects (e.g., placental lesions, changes in maternal gestational weight gain
(GWG), and BW changes), immune effects (e.g., T cell-dependent antibody response (TDAR)
suppression and lymphocyte increases), and Suggestive Evidence of Carcinogenic Potential of
oral exposure to GenX chemicals in humans (e.g., liver and pancreatic acinar cell tumors).
In most of the available animal studies, hepatocellular hypertrophy and necrosis of the liver
appear to be the most sensitive effects observed. The increases in relative liver weight,
hepatocellular hypertrophy, and peroxisome activity (e.g., peroxisomal beta-oxidation induction)
can be associated with activation of cellular peroxisome proliferator-activated receptor alpha
(PPARa) receptors, making it difficult to determine if this change is a reflection of PPARa
activation or an indication of GenX chemical toxicity. This is important because the PPARa
response could be more relevant to rodents than humans. EPA evaluated liver effects resulting
from exposure to GenX chemicals in the context of the Hall criteria (Hall et al., 2012), through
which changes in liver weight or hepatocellular hypertrophy can be considered adverse when
they are accompanied by histologic or clinical pathology indicative of liver toxicity such as
necrosis, inflammation, and/or fibrosis. In this assessment, EPA listed hepatocellular
hypertrophy or changes in serum liver enzymes as adverse only when they were accompanied by
histologic pathology indicative of liver toxicity such as necrosis, inflammation, and/or fibrosis.
The observance of liver necrosis indicates that cytotoxicity also could be a mode of action
(MOA) for liver damage.
No physiologically based pharmacokinetic (PBPK) models are available that address the
relationship between external exposure and internal dose for GenX chemicals; however,
allometric scaling methodology is available to calculate a toxicologically equivalent dose of
orally administered agents from adult laboratory animals to adult humans (EPA, 201 lb). The use
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of allometric scaling addresses some aspects of the cross-species extrapolation of toxicokinetic
and toxicodynamic processes.
The toxicity values for this assessment include a chronic oral RfD (chronic RfD) and a
subchronic oral RfD (subchronic RfD) for HFPO dimer acid and its ammonium salt. An RfD is
an estimate of the concentration or dose of a substance (with uncertainty spanning perhaps an
order of magnitude) to which a human population (including sensitive subgroups) can be
exposed that is likely to be without an appreciable risk of deleterious effects during a lifetime. In
addition to chronic RfDs, other durations of exposure can be considered, including subchronic
exposures. RfDs are derived for noncarcinogenic toxicological endpoints of concern.
3.3 Methods
3.3.1 Literature Search Strategy and Results
EPA assembled and evaluated available information on toxicokinetics; acute, short-term,
subchronic, and chronic toxicity; developmental and reproductive toxicity; neurotoxicity;
immunotoxicity; genotoxicity; and cancer in animals. Most of the available data for HFPO dimer
acid and its ammonium salt were submitted with PMNs to EPA by DuPont/Chemours, the
manufacturer of GenX chemicals, under TSCA, as required pursuant to a consent order (EPA,
2009) or as required under TSCA reporting requirements (15 U.S.C. § 2607.8(e)). Submitted test
data on HFPO dimer acid and its ammonium salt were available for numerous endpoints such as
acute toxicity, metabolism and toxicokinetics, genotoxicity, and systemic toxicity in mice and
rats with dosing durations of up to 2 years. Most of these submitted studies were conducted
according to OECD TGs and/or EPA health effects TGs for pesticides and toxic substances,
which:
... are generally intended to meet testing requirements for human health impacts of
chemical substances under the Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA) and TSCA (EPA, 2021b).
All available studies were considered for inclusion. Most of the studies considered for dose-
response analysis in this assessment adhered to the principles of GLP, and full study reports were
submitted for Agency review. As noted by OECD,3 the OECD TGs are accepted internationally
as standard methods for safety testing and:
... are covered by the Mutual Acceptance of Data, implying that data generated in the
testing of chemicals in an OECD member country, or a partner country having adhered to
the Decision, in accordance with OECD Test Guidelines and Principles of GLP, be
accepted in other OECD countries and partner countries having adhered to the Decision,
for the purposes of assessment and other uses relating to the protection of human health
and the environment.
To identify public literature available for HFPO dimer acid and its ammonium salt, literature
searches were conducted of four databases (PubMed, Toxline, Web of Science (WOS), and
Toxic Substances Control Act Test Submissions (TSCATS)) using CASRN, synonyms, and
additional relevant search strings (see Table A-2 in appendix A for a full list). Because the
results of this core search were so limited, additional databases were searched for
3 http://www.oecd.ors/chemicaJsafetv/testins/oecdgniclelinesfortlietestinsofchemicaJs.litm.
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physicochemical property information, health effects, toxicokinetics, and mechanistic
information. A list of the additional databases searched is provided in Table A-3 and Table A-4
in appendix A. The initial searches of these databases specific to HFPO dimer acid were
conducted in July 2017 and specific to the HFPO dimer acid ammonium salt in January and
February 2018. They returned 27 studies for HFPO dimer acid and its ammonium salt, after
accounting for duplicates. Additional updates to the literature search were completed in February
2019, October 2019, and March 3, 2020 using the same search strategy as described in appendix
A. These searches returned an additional 48 studies.
The submitted studies from DuPont/Chemours and the literature identified by the search of
publicly available sources are available through EPA's Health & Environmental Research Online
website at https://hero.epa.gov/hero/iiidex.cfm/proiect/page/proiect id/2627.
3.3.2 Study Screening and Evaluation
In accordance with EPA's Office of Research and Development (ORD) systematic review
practices, relevancy screenings were conducted on all the studies submitted from
DuPont/Chemours and the publicly available, peer-reviewed literature resulting from the
literature searches mentioned above (EPA, 2020). These studies were subjected to title and
abstract screening to determine relevancy according to the PECO criteria statement/inclusion and
exclusion criteria outlined in Table A-6 in appendix A. The title and abstract of each study were
independently screened by two screeners using Distiller SR4. The studies that met the PECO
criteria were tagged as having relevant human data, animal data in a mammalian model, or a
PBPK model. A study was included as relevant if it was unclear from the title and abstract
whether it met the inclusion or exclusion criteria. Studies that did not meet the inclusion criteria
but provide supporting information were categorized as supplemental, relative to the type of
supporting information they provided. These supplemental categories are outlined in Table A-7
in appendix A. When two screeners did not agree if a study should be included, excluded, or
tagged as supplemental, a third reviewer made the final decision. The title and abstract screening
resulted in 12 studies tagged as relevant (i.e., containing dose-response information). The
relevancy of these studies was confirmed by a full-text review.
The twelve studies providing dose-response information were then evaluated for study quality
using an approach consistent with the draft ORD Handbook for developing IRIS assessments
(DuPont-24447, 2008; DuPont-24459, 2008; DuPont-17751-1026, 2009; DuPont-18405-1307,
2010; DuPont-18405-1037, 2010; DuPont-18405-841, 2010; DuPont-18405-1238, 2013;
Rushing et al., 2017, Conley et al., 2019, 2021; Thompson et al., 2019; Blake et al., 2020; EPA,
2020). Study quality was determined by two independent reviewers who assessed risk of bias
and sensitivity for the following domains: reporting quality, risk of bias (selection or
performance bias, confounding/variable control, and reporting or attrition bias), and study
sensitivity (exposure methods sensitivity, and outcome measures and results display) using
EPA's version of HAWC5. A third reviewer made the final decision on the quality ratings based
on the primary ratings. The results of the study quality evaluation are provided in Figure 3 and an
interactive version of the heatmap can be found here:
4 Distiller SR is a fee-based, multi-user, web-based platform that manages, tracks, and streamlines the screening of
literature reviews.
5 HAWC is a free and open-source web-based software application that enables multiple users to synthesize multiple
data sources into an overall human health assessment of chemicals.
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https://hawcprd.epa.gov/summarv/visual/assessment/100500273/GenX-SQE-Heatmap/. All
twelve studies were rated as medium or high-quality studies and were summarized in section 4
and considered for dose response in section 7.
Reporting -
ooi0 . -dP vjO1 9)^
.. *• v .rft->¦ ..rft-* . rv
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3.4 Approach to Deriving Reference Values
Development of the hazard identification and dose-response assessment for HFPO dimer acid
and its ammonium salt has followed the general guidelines for risk assessment published by the
National Research Council (1983) and EPA's Framework for Human Health Risk Assessment to
Inform Decision Making (EPA, 2014a). Additional EPA guidelines and other Agency reports
used in developing this assessment include the following:
• Guidelines for Developmental Toxicity Risk Assessment (EPA, 1991)
• Guidelines for Reproductive Toxicity Risk Assessment (EPA, 1996)
• Guidelines for Neurotoxicity Risk Assessment (EPA, 1998)
• A Review of the Reference Dose and Reference Concentration Processes (EPA, 2002)
• Guidelines for Carcinogen Risk Assessment (EPA, 2005a)
• Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to
Carcinogens (EPA, 2005b)
• A Framework for Assessing Health Risks of Environmental Exposures to Children (EPA,
2006a)
• Exposure Factors Handbook (EPA, 2011a)
• Recommended Use of Body Weight4 as the Default Method in Derivation of the Oral
Reference Dose (EPA, 201 lb)
• Benchmark Dose Technical Guidance Document (EPA, 2012)
• Child-Specific Exposure Scenarios Examples (EPA, 2014b)
• Guidance for Applying Quantitative Data to Develop Data-Derived Extrapolation
Factors for Interspecies and Intraspecies Extrapolation (EPA, 2014c)
EPA's A Review of the Reference Dose and Reference Concentration Processes describes a
multistep approach to dose-response assessment, including analysis in the range of observation
followed by extrapolation to lower levels (EPA, 2002). EPA conducted a dose-response
assessment to define a POD and extrapolated from the POD to an RfD. For HFPO dimer acid
and its ammonium salt, EPA used benchmark dose (BMD) modeling to refine the critical effect
POD in deriving the RfD.
The steps for deriving an RfD are summarized below.
Step 1: Evaluate the data to identify and characterize endpoints related to exposure to
GenX chemicals. This step involves determining the relevant studies and adverse effects to be
considered for BMD modeling. Once the appropriate data are collected, evaluated for study
quality, and characterized for adverse outcomes, the risk assessor selects endpoints judged to be
relevant and the most sensitive (typically defined by the NOAEL value). Considerations that
might influence selection of endpoints include data with dose response, percent change from
controls, adversity of effect, and consistency across studies.
Step 2: Conduct BMD Modeling. Using EPA's Benchmark Dose Technical Guidance
Document (EPA, 2012), a benchmark response (BMR) is selected and BMD modeling is applied
to the endpoints selected as most relevant. The BMR is a predetermined change in the response
rate of an adverse effect. It serves as the basis for obtaining the benchmark dose lower limit
(BMDL), which is the 95% lower bound of the BMD. A family of BMD models are fit to the
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dose-response data that describe the dataset of the identified adverse effect. From the family of
models, either a best fitting model with the corresponding BMD and BMDL is derived or, if no
adequate models are found, the NOAEL or lowest-observed-adverse-effect level (LOAEL)
identified in step 1 is used as the POD.
Step 3: Convert the POD to a human equivalent dose (HED) or point of departure human
equivalent dose (PODhed). The POD (either a BMDL, NOAEL, or LOAEL) is then converted
to an HED following the method described in EPA's Recommended Use of Body Weight4 as the
Default Method in Derivation of the Oral Reference Dose (EPA, 201 lb).
Step 4: Provide rationale for selecting UFs. UFs are selected in accordance with EPA
guidelines considering variations in sensitivity among humans, differences between animals and
humans, the duration of exposure in the critical study compared to the lifetime of the species
studied, and the completeness of the toxicology database.
Step 5: Calculate the chronic and subchronic RfDs. The RfDs are calculated by dividing
PODhed by the selected UF.
RfD = PODhed
Total UF
where:
• PODhed = calculated from the BMDL or NOAEL/LOAEL using a BW3 4 allometric
scaling approach consistent with EPA guidance (EPA, 201 lb).
• UF = Total UF established in accordance with EPA guidelines considering variations in
sensitivity among humans, differences between animals and humans, duration of
exposure in the critical study compared to the lifetime of the species studied, and
completeness of the toxicology database.
3.5 Measures of Effect
The available dataset regarding the toxicity of these GenX chemicals includes in vivo and in vitro
studies. The in vivo studies were considered in the dose-response assessment for HFPO dimer
acid and its ammonium salt. The available data indicate that the liver, kidney, RBCs,
immunological responses, and reproductive and developmental effects (BW and fetal
development) are adversely impacted by exposure to GenX chemicals. Tumors were also
observed following oral exposure to GenX chemicals (DuPont-18405-1238, 2013). In this
analysis, all reported changes in relative organ weights were presented as relative to BW (data
relative to brain weight were not included). The endpoints presented in this assessment represent
potentially adverse effects that were statistically significantly different (p < 0.05 or 0.01) from
control unless otherwise noted. Additionally, statistically significant changes from the control are
presented as the percent change from control, unless otherwise noted.
The animal studies demonstrated dose-related effects on the liver in rodent species (rats and
mice) following exposure to HFPO dimer acid and/or its ammonium salt for durations of 28 days
to 104 weeks. The studies and endpoints reviewed as possible critical studies and effects for
determination of the POD were evaluated for experimental design, data quality, and dose
response identified through the range of experimental NOAELs/LOAELs. A route-to-route
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extrapolation of oral toxicity data from which to derive an inhalation reference concentration was
not conducted because of data limitations. For example, no toxicokinetic data are available
characterizing the uptake of GenX chemicals through the lung for systemic distribution, and only
one acute inhalation toxicity study is available (DuPont-17751-723, 2009). This study identifies
the portal of entry effects, albeit at a high dose.
4.0 Study Summaries
4.1 Acute Toxicity Studies
There are over 10 studies available detailing the acute toxicity and irritation effects of HFPO
dimer acid and its ammonium salt. This section summarizes the available acute oral, dermal, and
inhalation toxicity studies as well as dermal and eye irritation studies for HFPO dimer acid and
its ammonium salt. Appendix B provides additional details on each of the studies.
Oral Toxicity. Several studies have evaluated oral toxicity in rats and mice from single doses of
the HFPO dimer acid ammonium salt at doses ranging from 1.5 mg/kg to 17,000 mg/kg
(DuPont-22932, 2007; DuPont-24126, 2007; DuPont-25438 RV1, 2008; DuPont-2-63, 1963;
DuPont-770-95, 1996). Also, male and female rats were evaluated with doses of 175-5,000
mg/kg HFPO dimer acid (DuPont-25875, 2008). The rats and mice in these studies received a
single dose of the compound and were observed for clinical effects of toxicity for 14 days.
Four studies were conducted according to OECD TG 425 (OPPTS 870.1100) (OECD, 2008c)
using the Up-and-Down Procedure (DuPont-22932, 2007; DuPont-25438 RV1, 2008; DuPont-
25875, 2008; DuPont-24126, 2007). Two studies that estimated approximate lethal doses (ALDs)
did not have identified TGs (DuPont-2-63, 1963; DuPont-770-95, 1996). For HFPO dimer acid,
the oral median lethal doses (LDsos) were 1,730 mg/kg and 1,750 mg/kg in male rats and female
rats, respectively (DuPont-25875, 2008). For the HFPO dimer acid ammonium salt, the LD50 was
3,129 mg/kg for female rats (DuPont-22932, 2007); 1,030 mg/kg for female mice (DuPont-
24126, 2007); and 1,750 mg/kg for male rats (DuPont-25438 RV1, 2008). The estimated ALD
for male rats for the ammonium salt ranged from 5,000 mg/kg to 7,500 mg/kg (DuPont-2-63,
1963; DuPont-770-95, 1996).
The more common clinical signs observed across studies included wet fur, fur/skin stain or
discoloration, altered posture, and lethargy; changes in BW were also seen (DuPont-770-95,
1996; DuPont-22932, 2007; DuPont-24126, 2007; Dupont-25438 RV1, 2008; DuPont-25875,
2008). Effects in mice were observed after exposure to HFPO dimer acid ammonium salt (86%
purity) doses at 550 mg/kg and higher. Effects in rats were observed after exposure to either
HFPO dimer acid (98% purity) or its ammonium salt (82.6% to 99% purity) at doses of 175
mg/kg and higher (DuPont-22932, 2007; DuPont-25875, 2008).
Gross evidence of organ or tissue damage included discoloration of lungs, stomach, skin, lymph
nodes, liver, and/or esophagus (DuPont-22932, 2007; DuPont-25438 RV1, 2008; DuPont-25875,
2008). Enlarged livers and enlarged hepatocytes were observed in young male rats following
single doses of 2,250, 3,400, or 5,000 mg/kg for HFPO dimer acid ammonium salt (DuPont-2-
63, 1963).
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Dermal Toxicity. Two studies reported acute dermal toxicity of HFPO dimer acid ammonium
salt in rats or rabbits following acute dermal exposure (DuPont-24113, 2007; DuPont-839-95,
1996). In an OECD TG 402 (OPPTS 870.1200) (OECD, 2017) study, 5,000 mg/kg HFPO dimer
acid ammonium salt (86% purity) was applied to shaved, intact skin of male and female rats
under a semi-occlusive dressing for 24 hours. The dermal LD50 was more than 5,000 mg/kg
(both sexes). Erythema was observed only in females, whereas hyperkeratosis and ulceration
were observed in rats of both sexes. All dermal effects cleared by 13 days posttreatment
(DuPont-24113, 2007). In another study (in which no guideline is cited), HFPO dimer acid
ammonium salt (99% purity) was applied to shaved, intact skin of New Zealand white rabbits for
24 hours. The ALD was determined to be more than 5,000 mg/kg. In this study, erythema
persisted for 13 days post application and was accompanied by scaling and sloughing of skin.
One of the rabbits also exhibited necrosis for 2-6 days post application (DuPont-839-95, 1996).
Inhalation Toxicity. One study (conducted using the GLP Compliance Statement in compliance
with Title 40 of the Code of Federal Regulations (CFR) part 792) evaluated acute inhalation
toxicity of HFPO dimer acid ammonium salt (84% purity) in male and female rats following a
single 4-hour nose-only exposure to aerosol concentrations of 0, 13, 100, and 5,200 mg/m3. The
median lethal concentration (LC50) was more than 5,200 mg/m3. Red discharge from the nose,
eyes, and mouth was observed in rats at doses of 100 and 5,200 mg/m3 for up to 2 days
postexposure. No gross lesions were observed. Microscopic evaluation of respiratory tract tissue
(lung, larynx/pharynx, trachea, and nose) from rats exposed to concentrations of 0, 13, and 100
mg/m3 detected no substance-related effects (DuPont-17751-723, 2009).
Dermal Irritation. In an OECD TG 404 (OPPTS 870.2500) (OECD, 2002) dermal irritation
study, very slight-to-well-defined erythema was observed in three male New Zealand white
rabbits following a single application of a 0.5-mL aliquot of HFPO dimer acid ammonium salt
(86%) purity) in an area of shaved skin for a period of 4 hours on the day of application.
Erythema cleared by 24 hours postexposure (DuPont-24030, 2007).
Eye Irritation. New Zealand white rabbits were administered a single application of a 0.1 mL
aliquot of HFPO dimer acid ammonium salt (86%> purity) to the lower conjunctival sac in an eye
irritation study conducted according to OECD TG 405 (OPPTS 870.2400) (OECD, 2020a). At
28 hours after instillation of the compound, necrosis, corneal opacity, iritis, conjunctival
chemosis (swelling), discharge, and corneal injury were observed (DuPont-24114, 2007).
Short-Term Toxicity Studies
Seven-Day Toxicity Studies. Hepatic effects were observed in 6-week-old mice and rats of both
sexes in four 7-day studies (in which no TG is cited) evaluating repeated-dose oral toxicity of
HFPO dimer acid and its ammonium salt (DuPont-24010, 2008; DuPont-25281, 2008; DuPont-
24116, 2008; DuPont-24009, 2008). Water was used as the vehicle control in all studies. Two 7-
day studies evaluated the toxicity of HFPO dimer acid ammonium salt (86.6%> purity) and HFPO
dimer acid (99% purity) at doses of 30 mg/kg/day in male mice and rats, respectively. In both
studies, a twofold increase in liver weight relative to control, cell necrosis of hepatocytes, and
hepatocellular hypertrophy were observed in all exposed animals (DuPont-24010, 2008; DuPont-
25281, 2008). A third 7-day study evaluating toxicity of HFPO dimer acid (99% purity) also
detected increased liver weight in male rats (at 30, 100, and 300 mg/kg/day) and in female rats
(at 300 mg/kg/day). Hepatocellular hypertrophy was present in both sexes at all doses (DuPont-
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24116, 2008). Hypertrophy and increased liver weight were observed in another similar 7-day
gavage study evaluating effects of HFPO dimer acid ammonium salt (86.6% purity). Males
appeared to be more sensitive to hepatic effects because increases in liver weight were observed
at 30, 300, and 1,000 mg/kg/day, whereas increased liver weight was observed in females only at
1,000 mg/kg/day. These effects were accompanied by increases in P-oxidation and increases in
cytochrome P450 enzyme activity, biomarkers for activation of PPARa nuclear receptors.
Mild-to-minimal hepatocellular hypertrophy was observed in both sexes at 1,000 mg/kg/day
(DuPont-24009, 2008).
Twenty-Eight-Day Toxicity Studies. Two 28-day studies evaluating systemic toxicity in rats
and mice are available for HFPO dimer acid ammonium salt.
DuPont-24447 (2008)
In a study with 7-week-old Crl:CD(SD) rats (10/sex/group) conducted according to OECD TG
407 (OECD, 2008a), HFPO dimer acid ammonium salt (purity 88%) was administered on 28
consecutive days via gavage (vehicle was deionized water) (OECD, 2008a; DuPont-24447,
2008). Male rats received doses of 0, 0.3, 3, or 30 mg/kg/day while females received 0, 3, 30, or
300 mg/kg/day. In this study, there were no mortalities and clinical signs were confined to high-
dose females (e.g., urogenital staining).
Hematological evaluation revealed statistically significantly decreased RBC count, hemoglobin,
and hematocrit at greater than or equal to 3 mg/kg/day in males. The maximum decreases
compared to control at 4 weeks were observed at the highest dose (30 mg/kg/day) and were 6%,
7%>, and 8% for RBC count, hemoglobin, and hematocrit, respectively. Increases in absolute
reticulocyte counts were also observed in males at all dose levels, but this increase was only
statistically significant from control at the highest dose {21%) at 4 weeks. No statistically
significant hematological effects were observed in the females (DuPont-24447, 2008).
Alterations in serum clinical chemistry parameters were seen in both sexes, but most of the
significant effects were observed in the male rats. Decreases in total globulin and increases in the
A/G ratio were observed in males and females. In males, total serum albumin increased (15% at
30 mg/kg/day) while total globulin decreased 13% and 22% compared to control at 3 mg/kg/day
and 30 mg/kg/day, respectively. This resulted in an increase in the A/G ratio to 16% and 41% in
the 3 mg/kg/day and 30 mg/kg/day males, respectively, most likely the result of underproduction
of globulin. Females exhibited a 9% decrease in total globulin and a 20% increase in the A/G
ratio compared to control at 300 mg/kg/day. Males also showed statistically significant decreases
in serum cholesterol at all doses, with the largest decrease compared to control (28%) in the
30-mg/kg/day group. Triglyceride levels were decreased at all doses but were significantly
decreased (22%) only at 3 mg/kg/day. Males also exhibited increases in blood urea nitrogen
(BUN) (24%) and glucose (15%) at 30 mg/kg/day when compared to controls (DuPont-24447,
2008).
In males, relative kidney weight was significantly increased (15% compared to control) only at
the highest dose tested. Minimal mineralization of the kidneys was also observed in 1/10 male
rats in the high-dose group. There were no statistically significant changes in kidney weight in
the females; however, there was minimal basophilic staining of cells in the tubules for 3/10
female mice in the 300-mg/kg/day group, while none were observed in the control group. Dose
response could not be determined for basophilic tubules because no rats were examined in the 3-
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mg/kg/day-dose group and only one rat was examined in the 30-mg/kg/day-dose group. No
statistical analyses were completed on these microscopic observations.
Relative liver weights were statistically increased in a dose-response manner in males, 19%
and 56% compared to control at 3 mg/kg/day and 30 mg/kg/day, respectively. These increases
were accompanied by decreases compared to control in sorbitol dehydrogenase (SDH) at 0.3
mg/kg/day (-36%) and 30 mg/kg/day (-21%) in males. In females, the only statistically
significant change in liver weight was a 12% increase compared to control at the highest dose
(300 mg/kg/day). Microscopically, 4/10 and 7/10 male rats exhibited hepatocellular hypertrophy
at 3-mg/kg/day and 30-mg/kg/day doses, respectively. In female rats, hepatocellular hypertrophy
was observed in 4/10 rats in the high-dose group. Hepatocellular necrosis (3/10) and single-cell
necrosis (1/10) were observed in males at 30 mg/kg/day. No statistical analyses were completed
on these histological observations. The authors note that hepatic peroxisomal P-oxidation activity
was induced in both sexes at the middle and high doses. Specifically, P-oxidation activity was
determined using [14C] palmitoyl-coenzyme A (CoA) as the substrate and total cytochrome
P450 content as markers of peroxisome proliferation. In the males, P-oxidation activity was
significantly increased compared to control at dosages of 0.3 mg/kg/day, 3 mg/kg/day, and 30
mg/kg/day by 42%, 274%), and 772%), respectively, and total cytochrome P450 content was
significantly increased by 23% at 30 mg/kg/day (DuPont-24447, 2008). In female rats dosed
with 30 mg/kg/day and 300 mg/kg/day, P-oxidation activity was significantly increased
compared to control by 49% and 198%, respectively, while total cytochrome P450 content
remained unaltered (DuPont-24447, 2008). EPA identified the NOAEL to be 0.3 mg/kg/day and
the LOAEL to be 3 mg/kg/day based on hematological (decreased hemoglobin, RBC count, and
hematocrit) and immune (decreased globulin levels) findings in males (DuPont-24447, 2008).
These findings were also accompanied by liver effects, including an increase in relative liver
weight and hepatocellular hypertrophy; however, necrosis was observed only at the high dose
(30 mg/kg/day).
DuPont-24459 (2008)
In another repeated-dose study conducted according to OECD TG 407 (OECD, 2008a), 7-week-
old Crl:CD-l mice (10/sex/group) were administered 0, 0.1, 3, or 30 mg/kg/day HFPO dimer
acid ammonium salt (purity 88%) for 28 consecutive days via gavage (vehicle was deionized
water) (DuPont-24459, 2008). Increases in mean BW gain were observed at 30 mg/kg/day in
both males and females. In males, increases in mean cumulative BWs were reported as
statistically different from the control group in the 30-mg/kg/day group during study weeks 1, 2,
3, and 4. In females, mean cumulative BW gains were significantly increased in the 30-
mg/kg/day group during study weeks 2, 3, and 4.
Similar to the findings observed in the 28-day toxicity study in Crl:CD(SD) rats (DuPont-24447,
2008), decreases of 5.0% in hemoglobin and hematocrit were reported at greater than or equal to
3 mg/kg/day, and RBC count was significantly decreased by 7.6% in the Crl:CD-l male mice at
30 mg/kg/day. In both males and females, the A/G ratio was statistically increased compared to
control at greater than or equal to 3 mg/kg/day. Albumin alone was significantly increased by
31.3%) compared to controls in males at 30 mg/kg/day, and globulin alone was decreased in
females at greater than or equal to 3 mg/kg/day by 15.8% and 21.1% at 3 mg/kg/day and
30 mg/kg/day, respectively. Finally, in males, the serum liver enzymes aspartate
aminotransferase (AST) (478%), alanine aminotransferase (ALT) (1,254%), alkaline
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phosphatase (ALP) (1,222%), and SDH (1,800%) were significantly increased from control at
the 30-mg/kg/day dose. Note that the hematology measures in female mice were inexplicably
underpowered. Though a sample size of 9-10 mice per dose group was expected, only 2, 6, 3,
and 5 female mice had hematology measurements in the 0, 0.1, 3 and 30 mg/kg/day dose groups,
respectively.
In male mice, no statistically significant effect was observed on kidney weight. Female kidney
weight findings were equivocal with the mean relative kidney weight showing statistically
significant increases compared to control only at the low dose (8%) and high dose (17%).
Minimal increases in basophilic tubular cells and tubular dilatation were observed in females at
30 mg/kg/day (3 of 10 animals for both effects) (DuPont-24459, 2008).
Macroscopic and microscopic tissue pathology evaluations were conducted for all dose groups.
The inspection of male adrenal cortex at the highest dose found minimal hypertrophy in 8 of 10
tissue samples examined, while females showed mild or minimal adrenal cortex congestion at
only the highest dose (DuPont-24459, 2008). No statistical analyses were completed on these
microscopic observations.
Liver effects were also reported in both males and females in this study. In males, relative liver
weights were significantly increased compared to control at 3 mg/kg/day and 30 mg/kg/day by
78%) and 163%, respectively. In females, relative liver weights were increased at 3 mg/kg/day
and 30 mg/kg/day by 32% and 103%), respectively, compared to controls. Absolute liver weights
also increased at these doses in both sexes and to similar extents. Increases in liver weight
correlated with microscopic liver findings (including single-cell necrosis, increased mitosis, and
hepatocellular hypertrophy). Single-cell necrosis was observed in 40% (4/10) and 100% (10/10)
of the male mice at 3 mg/kg/day and 30 mg/kg/day, respectively, while no liver necrosis was
observed in the control mice. As noted above, serum liver enzymes were significantly increased
from control at the 30 mg/kg/day dose: AST (478%), ALT (1,254%), ALP (1,222%), and SDH
(1,800%)). Single-cell necrosis was also detected in 40% (4/10) of female mice at 30 mg/kg/day
compared to zero in the control. This was associated with an increase in serum SDH (186%) at
30 mg/kg/day. Hepatic peroxisomal P-oxidation activity was induced in both sexes. Specifically,
P-oxidation activity was determined using [14C] palmitoyl-CoA as the substrate and total
cytochrome P450 content as markers of peroxisome proliferation. In the male mice, P-oxidation
activity significantly increased compared to control at doses of 0.1 mg/kg/day, 3 mg/kg/day, and
30 mg/kg/day HFPO dimer acid ammonium salt by 57%, 744%), and 648%), respectively, yet
total cytochrome P450 content significantly decreased at 3 mg/kg/day and 30 mg/kg/day by 26%
and 53%), respectively (DuPont-24459, 2008). P-oxidation activity significantly increased
relative to control in female mice at 3 mg/kg/day and 30 mg/kg/day by 495%) and 823%),
respectively, with no alterations in total cytochrome P450 content. EPA identified the NOAEL
for this study as 0.1 mg/kg/day and the LOAEL as 3 mg/kg/day based on increase in single-cell
necrosis in males, which was accompanied by increased relative liver weight and hepatocellular
hypertrophy, hematological, and immune effects.
4.3 Subchronic Toxicity Studies
DuPont-17751-1026 (2009)
In a repeated-dose study with rats, HFPO dimer acid ammonium salt (purity 84%) was
administered to 8-week-old Crl:CD(SD) rats (10-20/sex/dose) on 90 consecutive days via oral
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gavage (vehicle was deionized water) in accordance with OECD TG 408 (DuPont-17751-1026,
2009; OECD, 1998). Male rats were administered the test substance at doses of 0, 0.1, 10, or 100
mg/kg/day while females received 0, 10, 100, or 1,000 mg/kg/day. In this study, three high-dose
females died before dosing was complete (two deaths considered as treatment-related; one death
of undetermined cause).
Hematological evaluations revealed decreased hemoglobin, erythrocyte counts, and hematocrit in
males administered greater than or equal to 10 mg/kg/day. The decreases in all three parameters
for males were significantly different from control at 10 and 100 mg/kg/day and decreased in a
dose-dependent manner at 90 days (study week 13). The maximum decreases from control in
males were observed at the highest dose and were 11%, 13%, and 12% for RBC count,
hemoglobin, and hematocrit, respectively. Likewise, female rats exhibited significant and dose-
dependent decreases in RBC count (28%), hemoglobin (21%), and hematocrit (18%), but only at
the 1,000 mg/kg/day dose. In males, absolute (52%) and percent {61%) reticulocytes and platelet
count (17%>) were significantly increased from control at the highest dose and exhibited a dose
response. Additionally, both the absolute and percent of basophils (a type of white blood cell)
were significantly decreased relative to control at 10 mg/kg/day (25%) and 100 mg/kg/day (50%)
in males. Finally, female rats saw significant increases from control in mean corpuscular volume
(15%>), mean corpuscular hemoglobin (11%), mean corpuscular hemoglobin concentration (4%),
platelet count (30%), and absolute (212%) and percent (392%) reticulocytes and a decrease
relative to control in the percent of basophils (33%) at the high dose (1,000 mg/kg/day) (DuPont-
17751-1026, 2009).
There were alterations in the clinical chemistry values in both sexes. Males exhibited a dose-
dependent increase in total albumin and the A/G ratio and a decrease in total globulin compared
to control. These changes were statistically significant at 10 mg/kg/day and 100 mg/kg/day. The
maximum increases compared to control observed at the highest dose in total albumin, total
globulin, and A/G ratio were 12%, 15%, and 35%, respectively. As in the 28-day study, females
exhibited a dose-dependent decrease in globulin (33%) and an increase in A/G ratio (58%) that
was significantly different from control for both effects at the highest dose only. Males and
females also showed dose-dependent decreases in serum cholesterol that were statistically
significantly different from control at 100 mg/kg/day (31%) in males and at both 100 mg/kg/day
(20%) and 1,000 mg/kg/day (31%) in females. BUN was significantly increased relative to
control in males at 100 mg/kg/day (38%). The trend for BUN was dose-related and positive in
both sexes. ALP levels were significantly increased from control in a dose-dependent manner at
10 mg/kg/day (48%) and 100 mg/kg/day (106%) in the males and at 1,000 mg/kg/day (66%) in
the females. Serum phosphorus levels increased dose-dependently in males and females and
were significantly different from control at 10 mg/kg/day (10%) and 100 mg/kg/day (11%) in
males and at 1,000 mg/kg/day (18%) in females. Total bilirubin was significantly decreased from
control in a dose-dependent manner at the mid-dose (25%) and high dose (50%) only in females.
Total protein and y-glutamyl transferase decreased 10% and 69%, respectively, at the high dose
in females. Finally, a slight but significant and dose-dependent decrease compared to controls in
urine pH (8%) and a large increase in total urine volume (252%) were observed in female rats at
1,000 mg/kg/day (DuPont-17751-1026, 2009).
Kidney weight relative to BW was significantly and dose-dependently increased from control at
10 mg/kg/day (13%) and 100 mg/kg/day (16%) in male rats. Likewise, kidney weight relative to
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BW was significantly increased at all dose levels in females and reached a maximum increase of
23% from control; however, microscopic damage of the kidney (tubular and papillary necrosis)
was observed in only one of the rats at the highest dose. Additionally, one of the females that
died prior to study termination exhibited tubular and papillary necrosis of the kidney.
Transitional cell hyperplasia and mild acute inflammation were observed in the kidney of 1/10
male rats at the 100-mg/kg/day dose. Statistical analyses were not completed for the microscopic
renal findings.
Liver weight relative to BW was significantly and dose-dependently increased from control at
10 mg/kg/day (31%) and 100 mg/kg/day {61%) in male rats. Females exhibited an 85% increase
from control in liver weight at the high dose (1,000 mg/kg/day). Hepatocellular hypertrophy was
observed in 3/10 and 10/10 males at the 10-mg/kg/day dose and 100-mg/kg/day dose,
respectively, and in 10/10 females at the 1,000-mg/kg/day dose. Statistical analyses were not
conducted for hepatocellular hypertrophy. Furthermore, it is not documented in the data tables
whether other histological effects such as liver necrosis were detected in the 90-day study,
although the pathology report states that the hypertrophy was not associated with microscopic
changes indicative of liver injury such as necrosis (DuPont-17751-1026, 2009). EPA has
determined the study NOAEL to be 0.1 mg/kg/day and the LOAEL to be 10 mg/kg/day based on
blood effects (i.e., decreased RBC count, hemoglobin, and hematocrit) in males.
DuPont-18405-1307 (2010)
DuPont-18405-1307 (2010) was submitted to EPA under a TSCA Consent Order (see section 1.1
for more detail). Subsequently, in comments submitted to regulations.gov (Docket EPA-HQ-
OW-2018-0614) by ToxStrategies LLC (2019a,b) a reevaluation of the study results for DuPont-
18405-1307 (2010) and DuPont-18405-1037 (2010) was submitted. The reevaluation of DuPont-
18405-1037 (2010) was published as Thompson et al. (2019) (discussed in section 4.5); however,
the results of the reevaluation of DuPont-18405-1307 (2010) were not included in this
publication. In response to these comments and the publication, EPA requested an independent
review of DuPont-18405-1307 (2010) by the National Toxicology Program (NTP, 2019)
Pathology Working Group (PWG) (appendix D). The results of the DuPont-18405-1307 (2010)
and the NTP PWG review are described next.
In a repeated-dose, subchronic study with 7-week-old Crl:CDl(ICR) mice, the HFPO dimer acid
ammonium salt (purity 84%) was administered to 10/sex/group for 95 days (males) or 96 days
(females) via gavage (vehicle was deionized water) at doses of 0, 0.1, 0.5, and 5 mg/kg/day in
accordance with OECD TG408 (DuPont-18405-1307, 2010; OECD, 1998). A statistically
significant increase in male BW and overall BW gain was observed at the high dose only. Mean
daily food consumption was statistically increased in males between days 0 and 91 in a dose-
related manner. The study authors reported that there were no treatment-related deaths. Two
female mice (one at 0.5 mg/kg/day on day 6 and one at 5 mg/kg/day on day 20) died during the
study. The authors reported that these animals displayed signs indicative of injury from gavage
misdosing. The mice that died prematurely were included in the study results presented in the
report.
A small decrease compared to control in mean corpuscular hemoglobin concentration (3%) in
males and increased bilirubin (14%) in females was reported at 5 mg/kg/day. Clinical chemistry
changes were more evident among male mice than female mice. Specifically, AST, ALT, and
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ALP were statistically increased from control 106%, 420%, and 1,134%, respectively, at the 5-
mg/kg/day dose in males. Comparatively, female mice saw significant increases relative to
control in ALT (42%) and ALP (143%). SDH levels significantly increase compared to control
in both males (308%) and females (32%) at 5 mg/kg/day. Albumin levels were increased relative
to control in the 5-mg/kg/day-dose group in both males (14%) and females (4%), but total serum
protein was significantly increased (14%) only in males at this dose (DuPont-18405-1307, 2010).
Macroscopic and microscopic tissue pathology evaluations were conducted for all dose groups.
Male mice exhibited kidney tubular epithelial hypertrophy (9/10 treated mice compared to 0 in
control) while females exhibited dilated kidney tubules (4/10 in treated compared to 2/10 in
control) in the 5-mg/kg/day-dose group. Both effects were classified as minimal by the study
authors. Female mice exhibited a decrease in relative spleen weight (10%, 21%, and 18% at 0.1
mg/kg/day, 0.5 mg/kg/day, and 5 mg/kg/day, respectively). No effects on the spleen were
observed in male mice in any dose group. The study authors reported that changes in female
spleen weight did not occur in a dose-related manner and were not associated with changes in
absolute spleen weights or histological abnormalities in the spleen (DuPont-18405-1307, 2010).
Increased relative liver weights compared to control in both male mice (130%) and female mice
(69%) were accompanied by minimal-to-mild hepatocellular hypertrophy at 5 mg/kg/day in all
dosed mice. Minimal hepatocellular hypertrophy was also observed at the 0.5-mg/kg/day dose as
well in males (8/10 mice). No hepatocellular hypertrophy was observed in the control group.
Large and discolored livers were observed at doses greater than or equal to 0.5 mg/kg/day in
males, but only in the 5-mg/kg/day-dose group in females. Key treatment-related findings
considered as adverse at 5 mg/kg/day included increased enzymes indicative of liver injury
(i.e., AST, ALT, ALP, and SDH) and increased total bile acids that co-occurred with
histopathological findings in the liver. Histopathological findings in male mice included an
increase in the incidence of single-cell necrosis (10/10 treated mice versus 0 in control), Kupffer
cell pigments (10/10 treated mice versus 0 in control), and mitotic figures (9/10 treated mice
versus 0 in control). Females also exhibited histopathological liver findings, but to a lesser
degree. For example, 3/10 female mice exhibited focal necrosis and only 1/10 mice presented
single-cell necrosis at 5 mg/kg/day (DuPont-18405-1307, 2010).
EPA concluded that the NOAEL in this study is 0.5 mg/kg/day and the LOAEL is 5 mg/kg/day
based on the histological findings for the liver (i.e., necrosis and mitotic figures) accompanied by
the clinical chemistry changes (i.e., AST, ALT, ALP, and SDH).
Reanalysis ofDuPont 18405-1307 (2010) by National Toxicology Program Pathology Working
Group (NTP, 2019)
The National Institute of Environmental Health Sciences (NIEHS), NTP in Research Triangle
Park, NC convened a pathology working group (PWG) to provide an independent review of
slides from the 90-day mouse study (DuPont-18405-1307, 2010) and the
reproductive/developmental study (DuPont-18405-1037, 2010). All pathology slides provided by
DuPont/Chemours were reviewed by the NTP PWG, including those of animals that died on
study. The data and slides were reviewed per NTP standards (Sills et al., 2019).
As part of this PWG, one pathologist reviewed slides from the two studies and classified liver
effects according to the International Harmonization of Nomenclature and Diagnostic Criteria
(INHAND) Organ Working Group's diagnostic criteria which describes how pathologists can
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distinguish between apoptosis and single-cell necrosis in standard hematoxylin and eosin- (H&E)
stained tissue sections (Elmore et al., 2016). The PWG coordinator then confirmed the
classifications and selected example slides representative of the observed liver effects for review
by the full, eight-member PWG. The selected slides included three examples each of normal
liver, hepatocellular apoptosis, hepatocellular single-cell necrosis, and hepatocellular
cytoplasmic alteration; two examples each of focal necrosis, pigment, increased mitoses, mixed-
cell infiltrates, and cytoplasmic vacuolation; and one example of oval cell hyperplasia. The
PWG's description of cytoplasmic alteration indicates that this endpoint includes hepatocellular
hypertrophy occurrence along with eosinophilic change to the hepatocytes. There was a majority
consensus for all reviewed lesions. The PWG consensus opinion for each slide, including any
additional diagnoses made by the PWG panel, was recorded and presented in the final PWG
report (appendix D of this revised assessment).
The PWG's classification of liver lesions included, but was not limited to, the following:
apoptosis, single-cell necrosis, cytoplasmic alteration, and focal necrosis. Single-cell necrosis
was observed in the high-dose group for male and female mice (DuPont-18405-1307, 2010). The
PWG agreed that the observed single-cell necrosis was often accompanied with inflammation.
Findings of apoptosis were also observed in the high-dose groups in both sexes.
Additionally, the PWG offered general observations about the histopathology reported in the
original study. The NTP pathologists identified hepatocellular hypertrophy, including
morphological changes such as eosinophilic stippling. The pathologists agreed that hypertrophy
was present, but often less severe than reported in the original study. In addition, the pathologists
recommended adding the diagnosis of cytoplasmic alteration to account for the eosinophilic,
granular appearance of the cytoplasm of the hepatocytes. The pathologists recommended using
this term to account for hypertrophy and eosinophilic changes as they are considered part of the
same process. Cytoplasmic alteration was noted in the mid- and high-dose groups in males.
The PWG majority consensus opinion for each slide was recorded in review worksheets in a
final report to EPA (see appendix D). Overall, the PWG review confirmed the results of the
original study. Specifically, the PWG confirmed that single-cell necrosis was observed and is a
treatment-related, adverse effect. The PWG concluded that the dose response and constellation of
lesions (i.e., cytoplasmic alteration, apoptosis, single-cell necrosis, and focal necrosis) rather
than one lesion individually, represents adversity within these studies (appendix D). EPA
interpreted the NTP PWG's definition that the constellation of liver lesions is adverse applies to
the dose group level instead of the individual animal level since the histopathological evaluation
represents a snapshot in time of a biological process within one portion of the liver that can vary
across animals. Table 10 presents a comparison of the incidence data for the 90-day mouse study
(DuPont-18405-1307, 2010) and the NTP (2019) PWG reevaluation. Because the PWG analysis
reflects more recent histopathological criteria for the grading of liver lesions, the incidence data
as reported by NTP (see appendix D) were considered the more appropriate measure of response
in the liver from the 90-day mouse study (DuPont-18405-1307, 2010). The NTP PWG reported
that 10 out of 10 male mice exhibited cytoplasmic alteration, compared to 0 in control at the 0.5-
mg/kg/day dose in this study. Although NTP classified cytoplasmic alteration as part of the
constellation of liver lesions considered adverse, no other liver lesions indicative of liver damage
(i.e., single-cell or focal necrosis or apoptosis) were observed at the 0.5-mg/kg/day dose level in
males. Consistent with the Hall criteria, EPA did not consider the cytoplasmic alteration findings
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alone as an adverse effect in the 0.5 mg/kg/day dose group but considered the constellation of
liver lesions observed across the male mice in the high-dose group as adverse. Additionally, the
female mice in this study did not exhibit a dose response for the constellation of liver lesions.
Based on EPA's interpretation of the NTP PWG results, EPA derived the study NOAEL for
DuPont-18405-1307 (2010) of 0.5 mg/kg/day and the LOAEL is 5 mg/kg/day based on the
histological findings for the liver (i.e., cytoplasmic alteration, apoptosis, single cell necrosis, and
focal necrosis) in male and female mice.
Table 10. Comparison of Results from 90-Day Mouse Study (DuPont-18405-1307, 2010)
and NTP PWG Reevaluation (NTP, 2019)
Reference
Results
Doses mg/kg/day)
0
0.1
0.5
5
Single-cell necrosis [incidence (%)]
Male
0/10 [0]
0/10 [0]
0/10 [0]
10/10 [100]
Female
0/10 [0]
0/10 [0]
0/10 [0]
9/10 [90]
Hepatocellular hypertrophy [incidence (%)]
Male
0/10 [0]
0/10 [0]
8/10 [80]
9/10 [90]
DuPont-18405-1307
(2010)
Female
0/10 [0]
0/10 [0]
0/10 [0]
10/10 [100]
Mitotic figures [incidence (%)]
Male
0/10 [0]
0/10 [0]
0/10 [0]
9/10 [90]
Female
0/10 [0]
0/10 [0]
0/10 [0]
0/10 [0]
Pigment increased, Kupffer cells [incidence (%)]
Male
0/10 [0]
0/10 [0]
0/10 [0]
10/10 [100]
Female
0/10 [0]
0/10 [0]
0/10 [0]
2/10 [20]
Doses mg/kg/day)
0
0.1
0.5
5
Single-cell necrosis [incidence (%)]
Male
0/10 [0]
1/10 [10]
0/10 [0]
9/10 [90]
NTP (2019) PWG
Reevaluation of DuPont-
18405-1307 (2010)
Female
0/10 [0]
0/9a [0]
0/9b [0]
3/9b [33]
Cytoplasmic alteration [incidence (%)]
Male
0/10 [0]
0/10 [0]
10/10 [100]
10/10 [100]
Female
0/10 [0]
0/9a [0]
0/9b [0]
9/9b [100]
Focal necrosis [incidence (%)]
Male
0/10 [0]
0/10 [0]
0/10 [0]
1/10 [10]
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Reference
Results
Female
1/10 [10]
0/9a [0]
2/9b [22]
3/9b [33]
Apoptosis [incidence (%)]
Male
0/10 [0]
0/10 [0]
0/10 [0]
10/10 [100]
Female
0/10 [0]
0/9a [0]
0/9b [0]
3/9b [33]
Combined Necrosis (single cell and focal necrosis) [incidence (%)]
Male
0/10 [0]
1/10 [10]
0/10 [0]
9/10 [90]
Female
1/10 [10]
0/9a [0]
2/9b [22]
4/9b [44]
Constellation of lesions (cytoplasmic alteration, focal necrosis, single-cell necrosis,
apoptosis) [incidence (%)]
Male
0/10 [0]
1/10 [10]
10/10 [100]
10/10 [100]
Female
1/10 [10]
0/9b [0]
2/9b [22]
9/9b [100]
Mitotic figures increased [incidence (%)]
Male
0/10 [0]
0/10 [0]
0/10 [0]
7/10 [70]
Female
0/10 [0]
0/9b [0]
0/9b [0]
0/9b [0]
Pigment increased [incidence (%)]
Male
0/10 [0]
0/10 [0]
0/10 [0]
10/10 [100]
Female
0/10 [0]
0/9b [0]
0/9b [0]
4/9b [44]
Notes:
a Slides for animal number 251 were not provided for analysis.
b EPA did not include animals that died due to gavage misdosing in the presentation of incidence data from the NTP PWG.
4.4 Chronic Toxicity and Carcinogenicity Studies
DuPont-18405-1238 (20J3)
In a combined chronic toxicity/carcinogenicity study in 7-week-old Crl:CD(SD) rats, HFPO
dimer acid ammonium salt (purity 84%) was administered by oral gavage (vehicle was deionized
water) for up to 104 weeks (80/sex/group, of which 10/sex/group were designated for a 12-
month interim necropsy in accordance with OECD TG453) (DuPont-18405-1238, 2013; OECD,
2009, 2018b; Caverly Rae et al., 2015). Dose levels administered were 0, 0.1, 1, and 50
mg/kg/day for males and 0, 1, 50, and 500 mg/kg/day for females. Numerous animals in all dose
groups (both male and female) were found dead or euthanized in extremis over the course of the
study. Across all dosing groups in both male and female rats, 25.4% of the test animals survived
to their planned terminal necropsy while 74.6% of the animals experienced unscheduled death/
moribundity prior to the scheduled study termination at 104 weeks. The authors state that mean
survival in males and females was unaffected by treatment; however, all females were sacrificed
before study termination at 101 weeks because of decreased survival across all groups, including
the control. There were no statistically significant differences in survival across the female
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dosing groups and female survival was comparable across all dosing groups. Among the animals
that experienced unscheduled death/moribundity on study due to effects determined to be
unrelated to treatment, DuPont stated the males most commonly died from pituitary tumors and
undetermined causes while the females most commonly died from pituitary tumors and
mammary tumors.
The females in the high-dose group were observed to have papillary necrosis and inflammation
of the kidneys deemed by the authors to be related to treatment. BW and BW gain were
unaffected in males but reduced compared to control (13% and 20%, respectively) in high-dose
females at 52 weeks. The incidence of alopecia and hypotrichosis (abnormal patterns of hair
growth) was statistically significantly increased in females at 500 mg/kg/day.
Statistically significant hematological effects were observed in this study, primarily in female
rats. Blood samples were taken at 3, 6, and 12 months. At 3 months, RBC count, hemoglobin,
and hematocrit were significantly decreased at the highest dose in males and females, although
these decreases did not occur in a dose-dependent manner. Similarly, at 6 months, hemoglobin
and hematocrit were significantly decreased at the highest dose in males, yet these decreases did
not occur in a dose-dependent manner. There were no significant differences in any of these
parameters in male rats at the 12-month time point. At 6 and 12 months, female rats exhibited a
significant decrease in RBC count, hemoglobin, and hematocrit at 500 mg/kg/day and in a dose-
dependent manner. The RBC count was also significantly decreased at 50 mg/kg/day in females
at the 12-month time point; however, hemoglobin and hematocrit were not. The largest decreases
compared to control in RBC count, hemoglobin, and hematocrit in female rats were 28%, 24%,
and 20%, respectively, which were observed at 12 months. Additionally, the percent change
from control of these effects increased over time (i.e., 3 months < 6 months < 12 months). At
12 months, serum albumin levels increased in males at 1 mg/kg/day and 50 mg/kg/day by 8%
and 16%) from control, respectively, which led to a concomitant increase in the A/G ratio by 16%
and 28%), respectively.
Statistically significant changes from control were observed in the kidneys of females, but only
at the highest dose (500 mg/kg/day). For example, there were increased incidences of tubular
dilatation (increased by 34% compared to control), edema of the renal papilla (increased by 56%
compared to control), transitional cell hyperplasia (increased by 39% compared to control),
tubular and pelvic mineralization (increased by 15% and 24% compared to control, respectively),
renal papillary necrosis (increased by 23% compared to control), and chronic progressive
nephropathy (increased by 36% compared to control), all statistically significant from control.
These microscopic indications of kidney damage were also associated with a 15% increase in
relative kidney weight compared to control in females administered 500 mg/kg/day of HFPO
dimer acid ammonium salt.
Liver enzyme levels also were affected by exposure to HFPO dimer acid ammonium salt at
12 months in the chronic study. In males, statistically significant increases in ALP (180%), ALT
(228%>), and SDH (141%) were observed at 50 mg/kg/day. These enzyme changes were
correlated with microscopic findings in the liver, including focal necrosis. Relative liver weights
were increased in high-dose males (16% compared to controls) and females (69% compared to
controls) at the 12-month sacrifice. The change in liver weight in females corresponded to
centrilobular hypertrophy in the high-dose females at the interim sacrifice. Females exposed to
500 mg/kg/day of HFPO dimer acid ammonium salt for 2 years also had significantly increased
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relative liver weights (43% compared to control) at terminal sacrifice. There was no difference in
organ weights in males at any dose at terminal sacrifice despite the changes observed at 12
months. Male and female rats exposed to 50 mg/kg/day and 500 mg/kg/day, respectively, had
statistically significantly increased centrilobular hepatocellular hypertrophy compared to control
rats (7/70 in treated males compared to 0/70 in control; 65/70 in treated females compared to
0/70 in control) and centrilobular hepatocellular necrosis (5/70 in treated males compared to 1/70
in control; 7/70 in treated females compared to 1/70 in control). Male rats also saw a decrease in
incidence from control of 16% and 10% in focal and periportal vacuolization, respectively, at
50 mg/kg/day, and female rats had a 4% decrease from control in centrilobular vacuolation at
500 mg/kg/day. Finally, in females, panlobular hepatocellular hypertrophy (increase in incidence
compared to control of 4%), individual cell hepatocellular necrosis (increase in incidence
compared to control of 4%), and angiectasis (i.e., dilation of a blood or lymphatic vessel)
(increase in incidence compared to control of 6%) were reported at the high dose.
Nonneoplastic effects also were observed in the stomach and tongue of females exposed to the
high dose. Specifically, there were increased incidences of hyperplasia of the limiting ridge of
the nonglandular stomach (increased by 13% compared to control; incidence was 9/70 for treated
females and 0/70 in control) and of the squamous cell in the tongue (increased 16% from control;
incidence was 13/70 in treated females and 2/70 in control). The tongue also exhibited an
increased incidence of inflammation (increased 14% from control; incidence was 13/70 in treated
and 3/70 in control). EPA concluded that the NOAEL for chronic toxicity in this study was 1
mg/kg/day and the LOAEL was 50 mg/kg/day for the liver effects in males.
Statistically significant increases in the incidence of liver tumors in females at 500 mg/kg/day
and pancreatic acinar cell tumors in males at 50 mg/kg/day were reported. An increase in
testicular interstitial (Leydig) cell tumors was noted at the high dose but was not statistically
significant. Because of the observed early deaths in both control and treated male rats, EPA
recommended that the submitter (a) reexamine their test data, (b) identify the animals that died
without Ley dig cell tumor within the first year, (c) exclude the animals identified in the previous
step (i.e., those that died within the first year and had no tumors) from consideration for cancer
data analysis, (d) recalculate tumor incidences, and (e) perform statistical analyses. Because the
initial results indicated that the increased incidences of liver tumors in female rats (500 mg/kg/d)
and combined pancreatic acinar tumors in male rats (50 mg/kg/d) were significantly increased
from control despite the inclusion of early deaths, EPA decided to limit the reanalysis to
testicular hyperplasia and tumors in male rats only. Additional discussion of tumor findings for
the liver, pancreas, and testes is presented below.
Females. There were increases in the incidence of liver tumors at the high dose only (500
mg/kg/day), where degenerative and necrotic changes were also observed. The tumor incidences
were 0/70 (0%), 0/70 (0%), 0/70 (0%), and 11/70 (15.7%) for hepatocellular adenomas and 0/70
(0%), 0/70 (0%), 0/70 (0%), and 4/70 {5.1%) for hepatocellular carcinomas at the doses of 0, 1,
50, and 500 mg/kg/day, respectively. The increased incidences of hepatocellular adenomas were
statistically significant by the Cochran-Armitage trend test, the Peto test, and the pairwise Fisher
Exact test and the increased incidences of hepatocellular carcinomas were statistically significant
by the Cochran-Armitage trend test and the Peto test. The incidences of adenomas and
carcinomas observed at 500 mg/kg/day also exceeded the test laboratory historical control ranges
of 0%—5% and 0%—1.7%, respectively.
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Males: A statistically significant increase was reported in the incidence of pancreatic acinar cell
adenomas/carcinomas combined (but not adenomas or carcinomas alone) at 50 mg/kg/day.
Incidences of pancreatic acinar cell adenomas were 0/70 (0%), 1/70 (1.4%), 0/70 (0%), and 3/70
(4.3%) at 0 mg/kg/day, 0.1 mg/kg/day, 1 mg/kg/day, and 50 mg/kg/day, respectively. The
increased incidence at the high dose was not statistically significant and was within the test
laboratory historical control range (0%—5%). The incidence of pancreatic acinar cell carcinomas
was 0/70 (0%) in all groups other than the high-dose group, in which 2/70 (2.9%) were observed.
The incidence of carcinomas at 50 mg/kg/day was not statistically significant but was slightly
higher than the upper end of the laboratory's historical control range (0%—1.7%). When these
two types of tumor were combined, the incidences of adenoma/carcinoma were 0/70 (0%), 1/70
(1.4%>), 0/70 (0%>), and 5/70 (7.1%>) at 0 mg/kg/day, 0.1 mg/kg/day, 1 mg/kg/day, and
50 mg/kg/day, respectively, with the increased incidence at the high dose significant by the
Cochran-Armitage trend test and the Peto test. For reference, the incidences of pancreatic acinar
cell hyperplasia were 16/70 (22.9%), 18/70 (25.7%), 7/70 (10%), and 21/70 (30%) at
0 mg/kg/day, 0.1 mg/kg/day, 1 mg/kg/day, and 50 mg/kg/day, respectively, indicating a lack of
dose-response relationship for this finding. Furthermore, the increased incidence of hyperplasia
at the high dose was not statistically significant (compared to control).
In the testes, the incidences of interstitial cell adenomas were 4/70 (5.7%), 4/70 (5.7%), 1/70
(1.4%>), and 8/70 (11.4%) at 0 mg/kg/day, 0.1 mg/kg/day, 1 mg/kg/day, and 50 mg/kg/day,
respectively at 2 years. An interstitial cell adenoma was also present in 1/10 high-dose males at
the interim sacrifice (12 months). The increased adenoma incidence at 50 mg/kg/day (11.4%)
was not statistically significant but was slightly higher than the upper end of the testing
laboratory's historical control range (0%—8.3%). For reference, the incidences of interstitial cell
hyperplasia were 7/70 (10%), 7/70 (10%), 3/70 (4.3%), and 15/70 (21.4%) at 0 mg/kg/day,
0.1 mg/kg/day, 1 mg/kg/day, and 50 mg/kg/day, respectively. The increased incidence of
hyperplasia at the high dose was not statistically significant (compared to control), although the
incidence of hyperplasia at 50 mg/kg/day exceeded the historical control range (0%—8.3%). The
observed incidences in the control and low-dose groups (both 10%) were also slightly above the
upper end of historical controls. DuPont's reanalysis of these findings in the testes indicated that
the number of male rats that died before 1 year was 4, 9, 8, and 3 in the 0 mg/kg/day (control),
0.1 mg/kg/day, 1 mg/kg/day, and 50 mg/kg/day groups, respectively. The causes of death were
generally dosing injury or undetermined causes, and there were no testicular lesions or tumors in
the testicular tissues of these animals. Excluding these early deaths, the incidences of testicular
interstitial cell hyperplasia were 7/66 (10.6%), 7/61 (11.5%), 3/62 (4.8%), and 15/67 (22.4%) in
the 0 mg/kg/day (control), 0.1 mg/kg/day, 1 mg/kg/day, and 50 mg/kg/day groups, respectively.
The corresponding incidences of testicular interstitial cell adenomas were 4/66 (6.0%), 4/61
(6.6%>), 1/62 (1.6%), and 8/67 (11.9%). Thus, there were no statistically significant differences
for either hyperplasia or adenoma, consistent with results from the original report in which all
early deaths were included. Although the incidence of testicular interstitial cell adenomas was
not statistically significant compared to controls, the authors of the study conclude that "a
relationship to treatment for these findings in the 50 mg/kg/day group cannot be ruled out" while
also suggesting that Ley dig cell tumor induction in rodents might have low relevance to humans
(Caverly Rae et al., 2015).
Based upon EPA's review of the study, the increased incidence of liver tumors in females at
500 mg/kg/day and combined pancreatic acinar adenomas and carcinomas in males at
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50 mg/kg/day are treatment related. The increased incidence of testicular interstitial cell
adenoma was not statistically significant, and EPA accepted the results of the reanalysis that
excluded the early deaths. EPA concluded that the NOAEL is 1 mg/kg/day and the LOAEL is
50 mg/kg/day based on the reported liver effects (i.e., centrilobular necrosis in both sexes;
increased ALP, ALT, and SDH in males; and increased centrilobular hepatocellular hypertrophy
and cystic focal degeneration in males).
4.5 Reproductive and Developmental Toxicity Studies
DuPont-l8405-1037 (2010)
DuPont-18405-1037 (2010) was submitted to EPA under a TSCA Consent Order (see section 1.1
for more detail). Subsequently, Thompson et al. (2019), a contractor to Chemours (previously
DuPont), performed a reevaluation of the study results for DuPont-18405-1037 (2010). In
response to this publication, EPA requested an independent review of DuPont-18405-1037
(2010) by the NTP PWG (appendix D). The results of the original DuPont study, and these two
reanalyses are described next.
In a combined oral gavage reproductive/developmental toxicity study in mice with HFPO dimer
acid ammonium salt, the test compound (purity 84%) was administered by oral gavage (vehicle
was deionized water) to Crl:CDl(ICR) mice (25/sex/group) at doses of 0, 0.1, 0.5, or 5
mg/kg/day, according to a modified OECD TG421 (DuPont-18405-1037, 2010; OECD, 2016a).
The male mice were approximately 6 weeks old and the female mice were approximately 10
weeks old. Parental (Fo) males were dosed 70 days prior to mating and throughout mating
through 1 day prior to scheduled termination, for a total of 84 to 85 total doses. Parental Fo
females were dosed for 2 weeks prior to pairing and were dosed through LD20 for a total of 53
to 65 doses (exceptions include females with no evidence of mating or those that failed to deliver
yet were administered a total of 37 to 50 doses). Fi animals (offspring) were dosed daily
beginning on PND21 through PND40.
In this study, increases in BWs and food consumption were observed at 5 mg/kg/day in Fo
animals. In Fo males, increased mean BW gains were reported in the 5-mg/kg/day group during
study days 0-49; differences from the control group achieved significance during study days 0-
7, 14-21, and 21-28. Significantly higher mean BW gains were observed in this high-dose male
group when the overall premating period (study days 0-69) and treatment period (study days 0-
84) were evaluated. Mean BW gains were statistically significantly increased in females during
both the premating period and throughout gestation at 0.5 and 5 mg/kg/day. Specifically, during
the pre-mating period, BW gain increased by 100% and 70% in the 0.5- and 5-mg/kg/day-dose
groups, respectively. Mean maternal GWG, calculated from individual differences, also
significantly increased over the gestational period (0-18 days) by 18% and 22% in the 0.5- and
5-mg/kg/day-dose groups, respectively. At the high dose, mean BW gains were increased (5.1%-
14.0%) compared to controls throughout lactation; the differences were significant on LD1, LD4,
and LD21. BWs were unaffected at 0.1 and 0.5 mg/kg/day during lactation. Overall, final BW
was significantly increased from control by 9% and 14% in males and females, respectively,
administered 5 mg/kg/day.
The authors reported no treatment-related deaths in the Fo mice. However, three males (one in
each of the dose groups) and six females (one in the control, three in the low-dose group, and one
each in the mid- and high-dose group) did not survive until scheduled sacrifice. The cause of
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death was undetermined in all cases except the male in the mid-dose group, which appeared to
have ulcerative dermatitis. Due to the lack of dose response, the study authors concluded that
these deaths were not related to treatment. The study authors did not include the mice with
premature deaths in the study results (e.g., histopathological incidence counts).
An increase in relative kidney weight compared to control by 6.5% was observed only in Fo
females at the 5-mg/kg/day dose. Mild increases in tubular cell hypertrophy were observed in the
kidneys of males at greater than or equal to 0.5 mg/kg/day-6/24 mice or 25% and 18/24 mice or
75%) of male mice at 0.5 mg/kg/day and 5 mg/kg/day, respectively, compared to 1/25 mice or 4%
in the control. Chronic progressive nephropathy was also noted in males at 0.5 mg/kg/day (4/24
mice or 17%) and 5 mg/kg/day (5/24 mice or 21%). This effect was not associated with any
evidence of tubular cell degeneration.
Liver effects also were reported in both males and females in this study. In males, mean absolute
liver weights were increased 26% and 142% at 0.5 mg/kg/day and 5 mg/kg/day, respectively, as
compared to control values. Mean relative liver weights were increased by 26% and 121%,
respectively, at the 0.5-mg/kg/day and 5-mg/kg/day doses. In females, mean absolute liver
weights were increased by 26% and 101% at 0.5 mg/kg/day and 5 mg/kg/day, respectively, as
compared to control values. Mean relative (% BW) liver weights were increased by 17% and
80%), respectively. Microscopic findings observed in the liver of Fo males and females
administered 0.5-5 mg/kg/day included increases in hepatocellular hypertrophy, single-cell
necrosis, mitotic figures, and lipofuscin pigment. Fo females exhibited an increase in the
incidence of gross white areas in the liver at 5 mg/kg/day, which correlated with microscopic
focal and single-cell necrosis. At doses greater than or equal to 0.5 mg/kg/day, minimal-to-
moderate hepatocellular hypertrophy was observed in both sexes, along with the corresponding
increases in relative liver weight outlined above. Specifically, male mice exhibited a 50% and
100%) increase in the incidence of hepatocellular hypertrophy compared to control at 0.5
mg/kg/day and 5 mg/kg/day, respectively, and similar increases in incidence was also observed
in female mice (58% and 100% at 0.5 mg/kg/day and 5 mg/kg/day, respectively, compared to
control). At greater than or equal to 0.5 mg/kg/day, single-cell necrosis of hepatocytes was
observed in males. Specifically, single-cell necrosis was observed in 5/24 mice at 0.5 mg/kg/day
and 24/24 mice at 5 mg/kg/day compared to 1/25 mice in the control. Female mice exhibited an
increase compared to control in both focal/multifocal necrosis and single-cell necrosis
at 5mg/kg/day. Specifically, 5/24 mice had focal/multifocal necrosis compared to 1/24 in the
control and 21/24 mice had single-cell necrosis compared to 1/24 mice in the control. Finally, the
incidence of mitotic figures increased in males and females administered 5 mg/kg/day by 75%
and 21%) compared to control, respectively, while the incidence of lipofuscin pigment increased
by 88%) and 21% compared to control, respectively.
No treatment-related effects were identified for reproductive parameters (mating, fertility, and
copulation indices; mean days between pairing and coitus), although male epidydimal weight
relative to final BW was statistically decreased at 5 mg/kg/day in both the left and right testes
(12%) decrease relative to control). No treatment-related effects were observed for mean
gestation length, mean numbers of implantation sites, mean numbers of pups born, live litter size,
percentage of males at birth, postnatal survival, or general condition of pups. At 5 mg/kg/day,
however, Fi male and female pups exhibited lower mean BWs at PND4, PND7, PND14, PND21,
and PND28. Fi male pups continued to exhibit lower mean BWs at PND35 and PND40.
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Although values for the attainment of balanopreputial separation and vaginal patency (markers of
pubertal onset) were within the range of historical control values, the pups showed statistically
significant delays in these endpoints at 5 mg/kg/day (a finding that might be related to the
observed effects on BW during the preweaning period). Additionally, the day for attainment of
vaginal patency did not exhibit a dose response. The NOAEL (Fo) is 0.1 mg/kg/day, and the
LOAEL is 0.5 mg/kg/day based on liver effects (single-cell necrosis in males). The NOAEL (Fi)
is 0.5 mg/kg/day based on decreased pup BW and delays in attainment of balanopreputial
separation and vaginal patency at the high dose.
Reanalysis ofDuPont 18405-1037 (2010) published by Thompson etal. (2019)
In a publication presenting alternative approaches to deriving toxicity values and subsequent
drinking water concentrations for GenX chemicals, Thompson et al. (2019) present a
reevaluation of slides of liver sections in the reproductive/developmental toxicity study in mice
(DuPont-18405-1037, 2010). Thompson et al. (2019) presents the reevaluation of the liver
sections from the reproductive/developmental toxicity study in mice in the supplemental file,
Table S3.
Thompson et al. (2019) reevaluated these slides using more current diagnostic criteria (Elmore et
al., 2016) than those used in the original study (DuPont-18405-1037, 2010) to distinguish
between apoptosis and single-cell necrosis in standard H&E-stained tissue sections. Cell death
was classified as apoptosis and necrosis based on the proposed nomenclature from the
Terminology Recommendations from the INHAND Apoptosis/Necrosis Working Group
described by Elmore et al. (2016). The INHAND Nomenclature for Non-neoplastic Findings of
the Rodent Liver was also consulted for final diagnostic nomenclature (Thoolen et al., 2010).
The samples were specifically evaluated for the presence and type of individual hepatocyte
necrosis. The veterinary pathologist who reviewed the slides concluded that apoptosis was the
primary adverse effect of note at 5 mg/kg/day. Thompson et al. (2019) also reported increased
mitosis at doses with apparent increased apoptosis; the study authors concluded that it is well
established that peroxisome proliferator-activated receptor (PPAR) activators can increase
mitosis and apoptosis in vivo. Therefore, the authors conclude that this effect is likely a part of
PPARa signaling pathways that are specific to rodents. EPA identified the NOAEL for this study
as 0.5 mg/kg/day and the LOAEL as 5 mg/kg/day based on increased apoptosis in male mice.
Reanalysis ofDuPont 18405-1037 (2010) by National Toxicology Program Pathology Working
Group (2019)
As described in section 4.3, slides from the 90-day mouse study (DuPont-18405-1307, 2010) and
the reproductive/developmental study (DuPont-18405-1037, 2010) were reevaluated by anNTP
PWG (see appendix D). The same protocol was used by the PWG in their analysis of each of
these studies (see section 4.3 for protocol details). The NTP PWG consensus opinion for each
slide was recorded on a review worksheet. Worksheets for the slides were provided as
appendices A and B in the final PWG report to EPA (see the full report provided in appendix D
of this assessment). Similar to the Thompson et al. (2019) publication, the NTP used the
terminology of the INHAND document containing standardized terminology of the liver
(Thoolen et al., 2010) except where it would be superseded by the terminology published by the
INHAND committee with reference to cell death/necrosis/apoptosis (Elmore et al., 2016). For
the reproductive/developmental study (DuPont-18405-1037, 2010), the PWG confirmed single-
cell necrosis and focal necrosis in the mid- and high-dose groups of both sexes. Single-cell
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necrosis alone and single-cell and focal necrosis combined exhibited a dose-response
relationship in both sexes. The PWG agreed that the observed single-cell necrosis was often
accompanied with inflammation in this study. Findings of apoptosis were observed but were
limited to the highest dose groups in both sexes. Additionally, cytoplasmic alteration (which
includes hepatocellular hypertrophy occurrence along with eosinophilic change to the
hepatocytes) was noted in the mid- and high-dose groups in both males and females.
The PWG review confirmed the results of the original DuPont study and did not agree with the
conclusion of the reanalysis published by Thompson et al. (2019). Specifically, the PWG
concluded that the dose response and constellation of lesions (i.e., cytoplasmic alteration,
apoptosis, single-cell necrosis, and focal necrosis) rather than one lesion by itself, represents
adversity within the confines of the study. Table 11 presents a comparison the incidence data for
the reproductive/developmental toxicity study (DuPont-18405-1037, 2010), Thompson et al.
(2019), and the NTP PWG reevaluation (NTP, 2019) of DuPont-18405-1037 (2010). The
incidence data as reported by NTP (see appendix D) were considered the more appropriate
measure of response in the liver from the reproductive/developmental study (DuPont-18405-
1037, 2010) because the PWG analysis reflects the more recent scientific histopathological
criteria developed for the grading of liver lesions and the PWG results were the consensus of
eight pathologists. The NTP PWG confirmed that the study NOAEL for DuPont-18405-1037
(2010) is 0.1 mg/kg/day and the LOAEL is 0.5 mg/kg/day based on the constellation of liver
effects (i.e., cytoplasmic alteration, apoptosis, single-cell necrosis, and focal necrosis) in male
and female mice.
Table 11. Comparison of Study Results from DuPont-18405-1037 (2010), Thompson et al.
(2019), and NTP PWG Reevaluation of DuPont-18405-1037 (NTP, 2019)
Reference
Results
Doses (mg/kg/day)
0
0.1
0.5
5
Single-cell necrosis [incidence (%)]
Male
1/25 [4]
1/24 [4]
5/24 [21]
24/24 [100]
Female
1/24 [4]
3/22 [14]
2/24 [8]
21/24 [88]
Focal /multifocal necrosis [incidence (%)]
DuPont-18405-
Male
0/25 [0]
0/24 [0]
1/24 [4]
1/24 [4]
1037 (2010)
Female
1/24 [4]
0/22 [0]
0/24 [0]
5/24 [21]
Hepatocellular hypertrophy [incidence (%)]
Male
0/25 [0]
0/24 [0]
12/24 [50]
24/24 [100]
Female
0/24 [0]
0/22 [0]
14/24 [58]
24/24 [100]
Mitotic figures [incidence (%)]
Male
0/25 [0]
0/24 [0]
0/24 [0]
18/24 [75]
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OCTOBER 2021
Reference
Results
Female
0/24 [0]
0/22 [0]
0/24 [0]
5/24 [21]
Pigment increased [incidence (%)]
Male
0/25 [0]
0/24 [0]
0/24 [0]
21/24
Female
0/24 [0]
0/22 [0]
0/24 [0]
5/24
Doses (mg/kg/day)
0
0.1
0.5
5
Apoptosis [incidence (%)]
Male
2/25 [8]
1/25 [0]
0/25 [0]
23/25 [92]
Female
N/A
N/A
N/A
N/A
Thompson et al.
Necrosis3 [incidence (%)]
(2019)
Male
2/25 [8]
0/25 [0]
1/25 [4]
1/25 [4]
Female
N/A
N/A
N/A
N/A
Mitosis [incidence (%)]
Male
0/25 [0]
0/25 [0]
0/25 [0]
15/25 [60]
Female
N/A
N/A
N/A
N/A
Doses (mg/kg/day)
0
0.1
0.5
5
Single-cell necrosis [incidence (%)]
Male
1/25 [4]
l/24b [4]
2/24b [8]
23/24b [96]
Female
0/24b [0]
2/22b [9]
3/24b [13]
19/24b [79]
Cytoplasmic alteration [incidence (%)]
Male
0/25 [0]
0/24b [0]
10/24b [42]
24/24b [100]
NTP (2019) PWG
Reevaluation of
Female
0/24b [0]
l/22b [5]
16/24b [67]
24/24b [100]
DuPont-18405-
1037 (2010)
Focal necrosis [incidence (%)]
Male
0/25 [0]
0/24b [0]
4/24b [17]
3/24b [13]
Female
2/24b 8]
l/22b [5]
4/24b [17]
5/24b [21]
Apoptosis [incidence (%)]
Male
0/25 [0]
0/24b [0]
0/24b [0]
21/24b [88]
Female
0/24b [0]
0/22b [0]
0/24b [0]
10/24b [42]
Combined Necrosis (single cell and focal necrosis) [incidence (%)]
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Reference
Results
Male
1/25 [4]
l/24b [4]
6/24b [25]
24/24b [100]
Female
2/24b [8]
3/22b [14]
6/24b [25]
20/24b [83]
Constellation of lesions (cytoplasmic alteration, focal necrosis, single-cell necrosis,
apoptosis) [incidence (%)]
Male
1/25 [4]
l/24b [4]
13/24b [54]
24/24b [100]
Female
2/24b [8]
3/22b [14]
17/24b [71]
24/24b [100]
Mitotic figures increased [incidence (%)]
Male
0/25 [0]
0/24b [0]
0/24b [0]
17/24b [71]
Female
0/24b [0]
0/22b [0]
0/24b [0]
2/24b [8]
Pigment increased [incidence (%)]
Male
0/25 [0]
0/24b [0]
0/24b [0]
20/24b [83]
Female
0/24b [0]
0/22b [0]
0/24b [0]
2/23b [9]
Notes'. N/A = not applicable
a Thompson et al. (2019) stated that "Emphasis was placed on evaluating the samples for the presence and type of individual
hepatocyte necrosis. The two terms recommended for hepatocyte death were apoptosis and necrosis based on the proposed
nomenclature from the Terminology Recommendations from the INHAND Apoptosis/Necrosis Working Group."
b EPA did not include animals that died due to gavage misdoing in the presentation of incidence data from the NTP PWG.
DuPont-18405-841 (2010)
In a prenatal and developmental toxicity study in 12-week-old female Crl:CD(SD) rats, HFPO
dimer acid ammonium salt (purity 84%) was administered via oral gavage (vehicle was
deionized water) once daily from GD6 through GD20 at doses of 0, 10, 100, and 1,000
mg/kg/day (22 females/group), according to OECD TG 414 (DuPont-18405-841, 2010; OECD,
2001b). The parental males and females were not dosed prior to or during mating and dosing for
the dams was not initiated until GD6. Lack of dosing for males and females prior to and during
mating and failure to dose the dams during the GD0 to GD6 period are limitations when
evaluating this study to fully reflect the ability of the HFPO dimer acid ammonium salt to cause
reproductive/developmental toxicity.
The dams' BW decreased at all doses, but significantly decreased (-22% compared to control) at
1,000 mg/kg/day. This decrease in BW also resulted in a decrease (-25%) in maternal GWG
compared to control at 1,000 mg/kg/day. Moreover, gravid uterine weight was significantly
decreased by 10% and 25% compared to control at 100 mg/kg/day and 1,000 mg/kg/day,
respectively. Food consumption in the dams was significantly decreased by 9% over the dosing
period (GD6-GD21) at the highest dose. Early delivery on GD21 was observed in 18% and 41%
of the dams at 100 mg/kg/day and 1,000 mg/kg/day, respectively. Importantly, the authors noted
that, in the available historical controls data for early deliveries in this rat strain (17 datasets), no
females showed early deliveries (i.e., before GD21).
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Statistically significant increases relative to control in absolute liver weight (12% and 34%) were
observed at 100 mg/kg/day and 1,000 mg/kg/day, respectively. Changes in liver weight relative
to BW were not documented. This increase in liver weight was associated with hepatocellular
hypertrophy at the high dose (19/22 rats, or 86%) and focal necrosis was observed in 9% and
23% of the dams dosed with 100 mg/kg/day and 1,000 mg/kg/day, respectively. Additionally,
absolute kidney weight increased dose-dependently in the dams and was significantly increased
compared to control (10%) at the highest dose. Changes in kidney weight relative to BW were
not documented, and there were no notable microscopic changes in the kidney tissue of the
dams. Of note is that a 1,000-mg/kg/day dam that died on GD20 had moderate multifocal/focal
necrosis of the liver and disseminated intravascular coagulation in the kidney glomerular
capillaries.
The pups experienced a 9% and 28% decrease compared to control in fetal weight at doses of
100 mg/kg/day and 1,000 mg/kg/day, respectively. The percentage of male (47%) and female
(53%>) pups born were significantly altered from control (55% male; 45% female) at 1,000
mg/kg/day. Additionally, a 14th rudimentary rib developed in 9% of the control fetuses, 10% of
fetuses in the 10-mg/kg/day-dose group, 12% of fetuses in the 100-mg/kg/day-dose group, and
27% of the fetuses in the 1,000-mg/kg/day-dose group. Statistical analyses were not completed
for the development of the 14th rudimentary rib in individual pups, but a statistically significant
increase in the number of litters developing a 14th rudimentary rib was observed for those
receiving the high dose.
The NOAEL for this prenatal and developmental toxicity study is 10 mg/kg/day based on an
increase in early deliveries, decreases in gravid uterine weight, and decreased fetal weights for
both sexes, all occurring at the LOAEL of 100 mg/kg/day.
Conley et al. (2019)
Conley et al. (2019) reported on two experiments evaluating the effects of oral gestational
exposures to HFPO dimer acid ammonium salt. In the first experiment, pregnant Crl:CD(SD)
rats were dosed from GD14 to GD18 with either water (control), or 1, 3, 10, 30, 62.5, 125, 250,
or 500 mg/kg/day of HFPO dimer acid ammonium salt. HFPO dimer acid purity was 100% as
determined by the supplier via perchloric acid titration. Dams were dosed during GDI4 to GDI8
because this window is identified as the critical period for masculinization of the male
reproductive tract. The study authors stated that the experiment was completed in three separate
"blocks" of animals (15 animals/block). There was a total of nine control animals (three control
animals/block), three animals each for the 62.5-, 125-, 250-, and 500-mg/kg/day doses (first
block) and six animals each for the 1-, 3-, 10-, or 30-mg/kg/day doses (second and third blocks).
Across all three blocks, GWG, reproductive output (number of fetuses and absorptions),
maternal sera, and maternal liver weight were measured. In the first two blocks, fetal testis gene
expression and testosterone production, fetal BW, fetal and maternal liver gene expression, and
maternal serum thyroid hormone and lipid concentrations were also evaluated. In the third block,
fetal plasma was collected for determining HFPO dimer acid ammonium salt concentrations (see
section 2.3.3 for detail).
A variety of effects were observed in the dams at doses greater than or equal to 30 mg/kg/day.
Serum total triiodothyronine (T3) levels were decreased at doses greater than or equal to 30
mg/kg/day and total thyroxine (T4) levels decreased at doses greater than or equal to 125
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mg/kg/day. Liver weight was increased on GDI8 at doses greater than or equal to 62.5
mg/kg/day. Decreases in serum low-density lipoprotein (LDL) were observed at doses greater
than or equal to 125 mg/kg/day and in serum high-density lipoprotein (HDL) and total
cholesterol at doses greater than or equal to 250 mg/kg/day. Additionally, serum triglycerides
were decreased at the highest dose tested. GWG was also decreased at doses greater than or
equal to 250 mg/kg/day.
No significant effects from control were observed on the number of fetuses or resorptions.
In a second pilot experiment evaluating postnatal development, five Crl:CD(SD) dams were
dosed from GD14 through GD18 with either water (control; n=2 pregnant dams) or 125
mg/kg/day of HFPO dimer acid ammonium salt (n=3 pregnant dams) (Conley et al., 2019). The
single dose of 125 mg/kg/day was selected because it was the highest dose evaluated that did not
cause a significant decrease in GWG during the study described above. Pup delivery began on
GD22 and the following schedule was followed for postnatal monitoring:
• PND2: Pups were weighed and sexed and anogenital distance (AGD) was measured.
• PND13: Pups were weighed, sexed, and evaluated for retention of female-like
nipples/areolae.
• PND27: Dams were euthanized, and uterine implantation sites scored. Pups were weaned
to 2/sex/treatment group.
• PND31-PND37: Fi female offspring were examined daily for vaginal opening (a marker
of pubertal onset).
• PND41-PND45: Fi male offspring were evaluated daily for balanopreputial separation (a
marker of pubertal onset).
• PND128: Fi females were weighed, euthanized, and examined for reproductive tract
malformations. Tissue weights were recorded for the uterus, paired ovaries, liver, paired
kidneys, and visceral adipose tissues.
• PND146: Fi males were weighed, euthanized, and examined for reproductive track
abnormalities. Tissue weights were collected for glans penis, ventral prostate, paired
seminal vesicles, paired testes, paired epididymis, levator ani-bulbocavernosus, paired
bulbourethral (Cowper's) glands, paired kidneys, and visceral and epididymal adipose
tissues. Total sperm counts were measured in epididymal sections.
Viable pup number was not affected by treatment. The only significant effect in the treated Fi
generation was a decrease in right epididymis weight on a litter mean basis compared to control.
However, multiple significant effects were observed on an individual pup basis. For example, Fi
female BW was significantly decreased compared to control on PND2, PND27, and at the time
of vaginal opening (PND31-PND37). Additionally, AGD and liver weight were significantly
decreased in Fi female offspring on an individual pup basis. For Fi males, paired testes, paired
epididymides, right testis, right corpus/caput, right epididymis, left testis, and epididymal
adipose tissue were significantly decreased compared to control on an individual pup basis.
Conley et al. (2019) conducted gene expression analyses to determine if HFPO dimer acid
ammonium salt activates PPAR signaling pathways. Maternal and fetal livers and fetal testes
were collected on GDI8 for gene expression analyses. Gene expression was assessed using
reverse transcriptase real-time polymerase chain reaction (PCR) of complementary
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deoxyribonucleic acid (DNA). Maternal and fetal livers were assessed for 84 target genes
relevant to PPARa, PPAR beta/delta (PPAR-p/S), and -y signaling pathways in the rat.
Maternal and fetal livers shared upregulation of 16 genes and most of these shared genes were
associated with fatty acid metabolism. Enoyl-CoA, hydratase/3-hydroxyacyl-CoA
dehydrogenase (Ehhadh) was the most highly upregulated gene in both the maternal (fifty-five-
fold at 500 mg/kg/day) and fetal (321-fold at 500 mg/kg/day) livers. Other shared upregulated
genes were associated with adipogenesis (e.g., Echl), PPAR transcription factors (e.g., Rxrg),
and PPAR ligand transporters (e.g., Slc27a5). Generally, the fetal liver tended to display a
greater sensitivity to HFPO dimer acid ammonium salt exposure with respect to the number of
genes upregulated and the magnitude of upregulation. For example, the fetal liver exhibited
upregulation of 12 genes that were not affected in the maternal liver (e.g., Pckl, Aqp7, Gk
(gluconeogenesis) and AngptU (lipid transport)). Additionally, all but one of the upregulated
genes shared by maternal and fetal livers (i.e., Echl) was upregulated to a greater extent in the
fetal liver. In maternal livers, the genes most sensitive to HFPO dimer acid ammonium salt
exposure were Echl and Rxrg and, in the fetal livers, Cptlb (mitochondrial fatty acid
metabolism), Acoxl (fatty acid metabolism) and AngptU were the most sensitive. These genes
were significantly increased at 1 mg/kg/day of HFPO dimer acid ammonium salt.
Overall, Conley et al. (2019) concluded that HFPO dimer acid ammonium salt activated PPAR
signaling pathways in maternal and fetal livers, but the effects observed in this study are not
exclusive to PPARa or even general PPAR signaling.
Conley et al. (2019) also measured fetal testis testosterone production and gene expression to
understand if HFPO dimer acid ammonium salt exposure produces effects similar to those of
some phthalate ester metabolites. Fetal testes were collected from male pups on GDI8, with a
single testis from the first three male pups used for the ex vivo testosterone production assay and
the remaining testes for gene expression analysis. Unlike some phthalate ester metabolites, there
was no effect of HFPO dimer acid ammonium salt exposure on fetal testis testosterone
production or on the expression of genes that are typically changed in the fetal testis by exposure
to phthalates (e.g., steroidogenic enzymes).
HFPO dimer acid ammonium salt was also assessed for in vitro agonism and antagonism of
transcriptional activation for estrogen (100 picomolar (pM) to 10 |iM), androgen (100 pM to 100
|iM), and glucocorticoid (100 pM to 100 |iM) receptors (Conley et al., 2019). HFPO dimer acid
ammonium salt displayed no agonism of any of the receptors. At 100 |iM, the study authors
classified HFPO dimer acid ammonium salt antagonism as slight for the glucocorticoid receptor
(28% reduction in luciferase expression) and as moderate for the androgen receptor (AR) (42%
reduction in luciferase expression). The study authors noted that the 100 |iM dose was
approaching the cytotoxic dose of 300 |iM.
EPA concluded that the study NOAEL is 62.5 mg/kg/day and the LOAEL is 125 mg/kg/day
based on the indications of reduced BW in Fi females and tissue weights in Fi animals,
decreased maternal GWG, and decreased maternal serum total T4 levels. Although maternal
serum total T3 levels were significantly decreased compared to control at 30 mg/kg/day, EPA
selected the LOAEL at 125 mg/kg/day because the deiodination of free T4 results in the
formation of T3 (Forhead and Fowden, 2014), and T4 is the thyroid hormone that preferentially
crosses the placenta of humans and rodents during early gestation (Calvo et al., 2002).
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Blake et al. (2020)
Blake et al. (2020) evaluated the effects of gestational PFOA and HFPO dimer acid exposure on
maternal and embryonic endpoints in mice. Pregnant CD-I dams were dosed from El.5 to El 1.5
or E17.5 with either deionized water (vehicle control), 1 or 5 mg/kg/day PFOA, or 2 or 10
mg/kg/day HFPO dimer acid. PFOA results, as they compare to HFPO dimer acid, are presented
in section 2.3.3. These time points were selected because the placenta had not fully matured at
El 1.5 and this time point overlaps with critical periods of placental development, including
vascularization with the uterine wall and chorioallantoic branching of vessels. The El 7.5 time
point was selected to capture treatment-related effects on embryo weight and because the
placenta is fully mature at E17.5.
Blake et al. (2020) evaluated albumin, ALP, ALT, AST, BUN, total cholesterol, creatine,
glucose, HDL, LDL, SDH, total bile acid, total protein, triglycerides, and urinary creatine in
maternal serum at El 1.5 and E17.5. Total cholesterol and HDL were significantly increased 66%
and 56%, respectively, compared to vehicle control in the 2 mg/kg/day HFPO dimer acid-dose
group at El 1.5, but these effects did not reach statistical significance at E17.5. Additionally,
serum triglyceride levels were significantly decreased at 2 mg/kg/day (-43%) and 10 mg/kg/day
(-61%) of HFPO dimer acid at El 1.5 and remained significantly decreased in the 10-mg/kg/day
(-74%) dose group at E17.5. Finally, serum ALP was significantly increased (53%) compared to
vehicle control at El7.5 in the 10-mg/kg/day HFPO dimer acid-dose group.
Absolute and relative maternal liver weights significantly increased compared to vehicle control
at both time points and in both HFPO dimer acid dose groups. Specifically, absolute liver weight
increased by 41% and 91% and relative liver weights increased 37% and 73% compared to
vehicle control at 2 and 10 mg/kg/day, respectively, at El 1.5. At E17.5, absolute liver weight
increased by 30% and 70% and relative liver weights increased 31% and 69% compared to
vehicle control at 2 and 10 mg/kg/day, respectively. A variety of hepatocellular lesions were
observed to increase as compared to vehicle control, including cytoplasmic alteration, mitotic
figures, cell death (included both apoptosis and single-cell necrosis), and vacuolation. At El 1.5,
all dosed livers presented with cytoplasmic alteration, which increased in severity at the 10
mg/kg/day HFPO dimer acid dose. Mitotic figures and cell death increased in both dose groups
and vacuolation rated as minimal was observed in 100% of the 10 mg/kg/day-dose group livers.
At E17.5, all dosed livers presented with more severe cytoplasmic alteration than at El 1.5, and
this cytoplasmic alteration was most severe in the 10-mg/kg/day HFPO dimer acid-dose group.
Mitotic figures were no longer increased at E17.5 and increased cell death was only observed in
the 10-mg/kg/day-dose group. Vacuolation rated as minimal and mild was observed in the 10-
mg/kg/day-dose group. Additionally, a portion of El 7.5 livers from all dose groups were
processed for transmission electron microscopy (TEM). As compared to vehicle control, the
livers from the 2- and 10-mg/kg/day HFPO dimer acid-dose groups exhibited "abnormal
ultrastructure with enlarged hepatocytes containing more abundant cytoplasmic organelles
consistent with mitochondria and peroxisomes and vacuolation" (Blake et al., 2020).
Additionally, the livers in the 10-mg/kg/day HFPO dimer acid-dose group presented
"vacuolation often with remnant membrane material as myelin figures, abundant rough
endoplasmic reticulum with few ribosomes present, and unevenly dispersed glycogen appearing
as clustered clumps" (Blake et al., 2020).
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Absolute and relative kidney weights were unchanged at El 1.5. Absolute (19%) and relative
(16%) kidney weight was increased compared to vehicle control in the 10-mg/kg/day HFPO
dimer acid-dose group at E17.5. No histopathologic changes in kidneys were noted in any dose
groups.
GWG was significantly increased (30%) relative to vehicle control at El 1.5 in the 10-mg/kg/day
HFPO dimer acid-dose group. When controlling for litter size, GWG was significantly greater in
the 10-mg/kg/day HFPO dimer acid-dose group than in vehicle control at both El 1.5 (7.1%) and
E17.5 (19.1%)). Finally, GWG was significantly increased compared to vehicle control at 2
mg/kg/day and 10 mg/kg/day at El7.5 using effect estimates from mixed effect models adjusting
for repeated measures of relative GWG, litter size, and embryonic day.
Implantation sites, viable embryos, nonviable embryos, and resorptions were not significantly
different than vehicle control in any dose group. Placental weight was significantly increased by
-15.5 milligrams (mg) and the embryo:placental weight ratio significantly decreased by 15% in
the 10-mg/kg/day HFPO dimer acid-dose group relative to vehicle control at El7.5.
Additionally, placentas from litters (an average of seven individual placentas per litter) per
treatment group and sacrifice time point were evaluated for histopathology. There were no
significant histopathological changes at El 1.5 between vehicle control and the HFPO dimer acid
dose groups, with nearly all the placentas evaluated within normal limits. However, 58% and
83%) of placentas evaluated at E17.5 were classified as abnormal in the 2 and 10 mg/kg/day
HFPO dimer acid dose groups, respectively, compared to 2% in the vehicle control group. The
number of abnormal placentas in the 10 mg/kg/day HFPO dimer acid dose group was
significantly different than vehicle control. The most frequent lesion detected was labyrinth
atrophy, which was observed in 0/41 (0%), 15/31 (48%), and 16/35 (46%) placentas in 0-, 2-,
and 10-mg/kg/day-dose groups, respectively. Labyrinth congestion and early fibrin clots
increased with increasing HFPO dimer acid dose. Specifically, labyrinth congestion was
observed in 0/41 (0%), 1/31 (3%), and 8/35 (23%) placentas in 0-, 2-, and 10-mg/kg/day-dose
groups, respectively, and early fibrin clot was observed in 0/41 (0%), 1/31 (3%), and 4/35 (11%)
placentas in 0-, 2-, and 10-mg/kg/day-dose groups, respectively. Placental lesions were evaluated
against the proportion of placentas within a litter within normal limits to account for litter effects,
and the proportion of abnormal placentas was significantly higher at the 2- and 10-mg/kg/day
HFPO dimer acid-dose groups relative to vehicle control. Finally, placental thyroid hormones
(reverse triiodothyronine (rT3), T3, and T4) were quantified at E17.5 from 2-3 pooled placental
tissues of same-sex embryos. Each pooled sample was considered as one biological replicate and
three replicates were used for each sex and treatment group. There was no significant effect of
sex or treatment on rT3, T3, T3:T4 ratio, or rT3:T4 ratio. A significant increase (60%) in T4
relative to vehicle control was reported for the 10-mg/kg/day HFPO dimer acid-dose group.
The authors noted that, in some HFPO dimer acid-exposed dams, gross anomalies were apparent,
including excess abdominal fluid, edematous tissues, and clotted placentas.
EPA concluded that there is no NOAEL for this study because the study LOAEL is 2 mg/kg/day,
which is the lowest dose tested. The LOAEL is based on increased incidence of placental lesions
within a litter and increased GWG using effect estimates from mixed-effect models adjusting for
repeated measures of relative GWG, litter size, and embryonic day in maternal mice.
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Conley et al. (2021)
In a follow-up to their 2019 study, Conley et al. (2021) reported on two experiments evaluating
the effects of oral gestational exposures to HFPO dimer acid ammonium salt. In one experiment,
pregnant Crl:CD(SD) rats were dosed once daily by gavage from GDI6 to GD20 with either
water (control) or 1, 3, 10, 30, 62.5, or 125 mg/kg/day of HFPO dimer acid ammonium salt.
HFPO dimer acid purity was 100% as determined by the supplier via perchloric acid titration.
The study authors stated that the experiment was completed in two separate "blocks" of animals
(15 animals/block). There were three control animals/block (total of six control animals) and two
animals/treatment group/block (total of four treated animals/group). In both blocks, dams and
fetuses were euthanized on GD20 and maternal and fetal sera were collected for determining
HFPO dimer acid concentrations (see section 2.3.3 for detail). Maternal serum was also analyzed
for thyroid hormone concentrations (total T3 and total T4) and clinical chemistry parameters
(ALT, AST, triglycerides, cholesterol, albumin, and glucose (non-fasting)). Maternal weight
gain, reproductive output (number of fetuses and resorptions), and maternal liver weight were
measured. Maternal liver samples were collected for determining HFPO dimer acid
concentrations and gene expression analyses. In the first block, two male and two female fetuses
were randomly selected from each litter for measurements of body and liver weight and HFPO
dimer acid concentration in liver samples. The individual body weights of the remaining fetuses
were recorded irrespective of sex. Because there was no indication of an effect of sex on fetal
body weight in the first block, body weights in the second block were recorded for three
randomly selected fetuses per litter (irrespective of sex); out of those fetuses, one was randomly
selected per litter to determine liver weight and HFPO dimer acid concentration in the liver and
for gene expression analyses.
There were no significant differences observed for fetal body weight or liver weight (broken out
by sex or combined), maternal body weight gain, or maternal terminal body weight in any dose
groups compared with controls. Maternal liver weight was increased at 62.5 and 125 mg/kg/day.
Maternal serum T3 and T4 levels were decreased at doses >62.5 mg/kg/day. Albumin was
decreased at 3, 62.5, and 125 mg/kg/day. Triglycerides were decreased at doses >10 mg/kg/day
and cholesterol was decreased at doses >30 mg/kg/day. There were no significant effects on the
numbers of viable fetuses or resorptions in any dose groups compared with controls.
In another experiment reported in Conley et al. (2021), pregnant Crl:CD(SD) rats (five per
group) were dosed once daily by gavage from GD8 to PND2 with either water (control) or 10,
30, 62.5, 125, or 250 mg/kg/day of HFPO dimer acid ammonium salt. Dams gave birth naturally
and were checked for parturition beginning on GD22. Once delivery was complete, pups were
counted and the litter weight was recorded. All pups were returned to the nest except for two
randomly selected pups per litter that were sacrificed. Trunk blood was collected, and serum was
analyzed for HFPO dimer acid concentration and clinical chemistry parameters. Livers were
collected for histopathological examination and gene expression analyses. The carcasses of three
deceased newborn pups (one each from 30, 125, and 250 mg/kg/day dose groups) were sent for
histopathological examination. On PND2, dams received their final dose in the morning and
were weighed and euthanized 2-5.5 hours later. Maternal trunk blood was collected, and serum
was analyzed for thyroid hormone concentrations (total T3 and total T4), clinical chemistry
parameters (ALT, AST, triglycerides, cholesterol, albumin, and glucose (non-fasting)), and
HFPO dimer acid concentration. Maternal liver weight was recorded, and liver samples were
collected for gene expression analyses and HFPO dimer acid determination. Uterine implantation
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sites were scored. The pups were sexed and weighed, anogenital distance was measured, trunk
blood was collected, liver weight was recorded (one male and one female per litter), and liver
samples were analyzed for HFPO dimer acid concentration. PND2 pup serum was analyzed for
clinical chemistry parameters (ALT, AST, triglycerides, cholesterol, albumin, and glucose (non-
fasting)) and HFPO dimer acid concentration.
All pups were alive at birth with no remarkable gross external malformations. Dams displayed
typical nesting behaviors; however, shortly after delivery, pups in the higher dose groups began
displaying lethargy, morbidity, or were found dead. Pups continued to die or require euthanasia
throughout PNDO and PND1. Pup survival was reduced on PND1 and PND2 at doses >62.5
mg/kg/day. Many of the pups that died had visible milk bands indicating they had nursed. Pup
survival scores on PND2 were 100 ± 0, 96 ± 2, 97 ± 2, 87 ± 5, 38 ± 13, and 5 ± 5% in the 0, 10,
30, 62.5, 125, 250 mg/kg/day groups, respectively. Pup survival scores were significantly
decreased at doses >62.5 mg/kg/day Pup body weight gain (birth to PND2) and PND2 body
weight in the surviving pups were both reduced at doses >30 mg/kg/day. Anogenital distance
was not affected in male or female pups, but relative liver weight was increased in all dose
groups. No remarkable histopathological lesions were observed in pup livers, but glycogen
accumulation scores in the liver were significantly lower in all dose groups compared with
control pups. Significant changes were observed in some pup serum clinical chemistry
parameters. Glucose was decreased at doses >62.5 mg/kg/day in newborn pups and at doses
>125 mg/kg/day in PND2 pups. Albumin was decreased at 62.5 and 250 mg/kg/day only in
newborn pups. Cholesterol was increased at doses >125 mg/kg/day in newborn pups and at doses
>62.5 mg/kg/day in PND2 pups. Triglycerides were increased at doses >125 mg/kg/day in
newborn pups, and AST was increased at doses >30 mg/kg/day in PND2 pups.
Purple discoloration of the entire right hind limb was observed in one pup each from the 30, 125,
and 250 mg/kg/day dose groups beginning on PND1 and those pups were examined for
histopathology. All three had milk protein in the stomach lumen, vascular thrombi in various
vessels, and small dense basophilic cells throughout liver lobes. The two from the higher dose
groups also had moderate subcutaneous hemorrhage in the area of the umbilical artery and vein.
Subcutaneous edema or vascular congestion of the lower limb was observed in the pups from the
30 and 250 mg/kg/day dose groups.
A variety of significant adverse effects were observed in the dams. Maternal body weight (on
GD22 and PND2) and gestational weight gain were reduced at doses >125 mg/kg/day. At
necropsy on PND2, maternal absolute liver weight was increased at doses >30 mg/kg/day, and
relative liver weight was increased at all dose levels. There was no significant effect on the
number of uterine implants. Maternal serum total T3 and T4 levels were decreased at doses
>62.5 mg/kg/day (with the exception of T3 at 250 mg/kg/day). Albumin was decreased at 250
mg/kg/day, and triglycerides were increased at 125 and 250 mg/kg/day. Serum AST was
increased at all dose levels. The study authors noted that, even though maternal serum and levels
of HFPO dimer acid ammonium salt did not increase when dosing was extended from 4 days in
the fetal study to 16 days in the postnatal study (see section 2.3.3), maternal liver weight was
more affected in the postnatal study and at lower dose levels.
Conley et al. (2021) also conducted gene expression analyses using liver samples from both
experiments (GDI 6-20 and GD8-PND2) to determine if HFPO dimer acid ammonium salt
activates PPAR signaling pathways or alters genes related to glucose and glycogen metabolism.
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Gene expression was assessed using reverse transcriptase real-time polymerase chain reaction
(PCR) of complementary deoxyribonucleic acid (DNA) synthesized from ribonucleic acid
(RNA) extracted from sample homogenates. Maternal (GD20), fetal (GD20), and neonatal
(PNDO) livers were assessed for 84 target genes relevant to PPARa, PPARp/S, and PPAR-y
signaling pathways in the rat. Fetal and neonatal livers were also assessed for 84 genes involved
in the regulation and enzymatic pathways of glucose and glycogen metabolism.
Expression of five genes related to glucose metabolism were affected in the GD20 fetal livers.
Four genes {Pckl, Pdk4, G6pc, Pdp2) were significantly upregulated compared with controls and
one (Ugp2, critical to glycogen synthesis) was significantly downregulated. All genes were
significantly different at doses >10 mg/kg/day, except for G6pc (critical to gluconeogenesis)
which was significantly different at doses >3 mg/kg/day. Pckl (critical to gluconeogenesis) was
the most highly upregulated gene (37.5-fold compared with control at the highest dose). No
genes were significantly affected at 1 mg/kg/day.
Conley et al. (2021) compared the gene alterations observed in PPAR signaling pathways for
fetal livers exposed GD16 to 20 (this study) with those exposed GD14 to 18 (reported in Conley
et al., 2019). All 28 genes involved in PPAR signaling that were significantly upregulated on
GDI 8 were also upregulated on GD20, and 16 of these genes had a highly significant interval
effect with greater upregulation on GD20 than on GDI8. The remaining upregulated genes did
not differ significantly between GDI8 and GD20. There were no significantly downregulated
PPAR signaling genes. Overall, Conley et al. (2021) concluded that greater gene expression
effects were observed later in gestation on genes that code for proteins critical to mitochondrial
(Acaa2, Acadm, Cptla) or peroxisomal (Acoxl, Echl, Ehhadh) fatty acid P-oxidation or both
(Mlycd), gluconeogenesis (Pckl), glycerol metabolism (Gk), fatty acyl-CoA conversion (Acsll,
Acs/3), mediation of triglyceride clearance (Angptl4), triglyceride biosynthesis (Dgatl), fatty
acid biosynthesis (Fads2, Scdl), and PPAR coactivation (Rxrg).
Analysis of maternal livers showed that the 19 PPAR signaling genes that were upregulated on
GD18 (Conley et al., 2019) were also upregulated on GD20, and seven of those showed greater
upregulation on GD20. The upregulated genes code for proteins critical to mitochondrial and
peroxisomal fatty acid P-oxidation, ketogenesis, fatty acid transport, fatty acyl-CoA conversion,
triglyceride turnover, carnitine transport, mitochondrial protein import, accumulation of reactive
oxygen species, and transcriptional coactivation. Conley et al. (2021) concluded that the data
from this study provide evidence for PPARa activation in the maternal, fetal, and neonatal livers
following exposure to HFPO dimer acid ammonium salt.
Gene expression analyses of newborn pup livers showed that 13 glucose metabolism genes were
upregulated and 15 were downregulated, 11 of which were significantly different from controls
in all dose groups. Pdk4 was upregulated and Ugp2 was downregulated, similar to fetal livers,
but Pckl and G6pc were unaffected. The most highly affected upregulated genes were Fbp2
(gluconeogenesis) and Ldha (anaerobic glycolysis); the most highly affected downregulated
genes included Aldob (glycolysis), Agl (glycogen degradation), Ugp2 (glycogen synthesis), and
Gsk3a (glycogen synthesis). There were 21 upregulated and 8 downregulated PPAR signaling
pathway genes, 21 of which were significantly different from controls in all dose groups. Several
gene expression changes were unique to PNDO livers including Fabp2 (downregulated, a lipid
sensor and high affinity long-chain fatty acid binding protein), Slc27a5 (downregulated, activates
very long-chain fatty acids and bile acids), Apoc3 (downregulated, associated with metabolism of
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triglyceride-rich lipoproteins), Ppara (downregulated, codes for the PPAR alpha nuclear
receptor), and Cd36 (upregulated, has pleiotropic effects associated with angiogenesis,
inflammation, and fatty acid metabolism). Overall, Conley et al. (2021) concluded that many
genes associated with carbohydrate and lipid metabolism were affected at multiple stages of
development by HFPO dimer acid ammonium salt exposure.
Conley et al. (2021) observed multiple significant adverse effects when dams were dosed with
HFPO dimer acid ammonium salt from GD8 to PND2. Significant pup mortality was observed at
doses >62.5 mg/kg/day and growth rates were significantly lower at doses >30 mg/kg/day.
Newborns displayed hypoglycemia at doses >62.5 mg/kg/day, elevated serum lipid levels at
doses >125 mg/kg/day, and significantly lower glycogen accumulation scores in the PND2 livers
of all dose groups. Maternal body weights were decreased at doses >125 mg/kg/day, and
maternal relative liver weight was increased in all dose groups. Maternal serum total T3 and T4
levels were decreased at doses >62.5 mg/kg/day (with the exception of T3 at 250 mg/kg/day).
The authors noted that disruption of carbohydrate and lipid metabolism across the maternal-
placental-fetal unit (beginning in the 1 mg/kg/day dosing group) were likely key events in the
observed adverse effects, including decreases in pup body weight and survival. EPA concluded
that the study NOAEL is 10 mg/kg/day and the LOAEL is 30 mg/kg/day based on reduced BW
in Fi pups at PND0 and PND2.
4.6 Other Studies
4.6.1 Immunotoxicity Studies
Rushing et al. (2017)
Male and female C57BL/6 mice (6-12/sex/group) were administered HFPO dimer acid by
gavage at doses of 0, 1, 10, or 100 mg/kg/day for 28 days (Rushing et al., 2017). The animals
were immunized with sheep RBC antigen on day 24 and, 5 days later, were evaluated for TDARs
and splenic lymphocyte subpopulations. Organs were collected 1 day after the final gavage
exposure.
T lymphocyte numbers were significantly increased (the average increase of CD8+, CD4+/CD8+,
and CD4VCD8" T cells was 74%) in males at 100 mg/kg/day, yet suppression of TDAR was
observed in female mice only at 100 mg/kg/day. TDAR suppression was measured through
immunoglobulin M (IgM) antibody production, which decreased by 7.3% in females at the high
dose. Liver weight relative to BW significantly increased (40%-160%) in both sexes at 10
mg/kg/day in a dose-dependent manner. Relative spleen weights significantly decreased by 11%
in females treated with 100 mg/kg/day, and there were no significant changes in thymus weight.
Peroxisomal fatty acid oxidation was measured using hepatic acyl-CoA oxidase activity as a
readout. In male mice, hepatic acyl-CoA oxidase activity increased 122% and 222% at 10
mg/kg/day and 100 mg/kg/day, respectively. Female mice had a 100% increase in acyl-CoA
oxidase activity at the highest dose tested. The NOAEL for immune effects that include TDAR
suppression in females and increased T cells in males is 10 mg/kg/day.
4.6.2 Mechanistic Studies
The studies in this section provide mechanistic insight into the effects of HFPO dimer acid
and/or its ammonium salt. Available studies address biological mechanisms applicable to liver
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effects, serum lipids and lipoproteins, thyroid hormones, and developmental effects. Of note,
many of the studies outlined here report using dimethyl sulfoxide (DMSO) to prepare HFPO
dimer acid or its ammonium salt. This is important because a 2020 publication (Gaballah et al.,
2020) demonstrates that HFPO dimer acid is unstable in DMSO but is stable in deionized water.
Where reported, EPA has listed the vehicle that the study authors used to dissolve these
chemicals and the vehicle control.
Wang et al. (2017)
In one study investigating changes in gene expression, male ICR mice (//= 12/group) were
administered either (control) or 1 mg/kg/day HFPO dimer acid ammonium salt prepared in 0.5%
Tween-20 via oral gavage for 28 days (Wang et al., 2017). Although the authors state that HFPO
dimer acid was tested and its chemical structure is presented, the CASRN is listed as 62037-80-
3, which is the HFPO dimer acid ammonium salt. Nevertheless, whether the chemical evaluated
was the acid or the ammonium salt does not impact the form dissolved in serum or plasma. In
both cases, the HFPO dimer anion is present in solution.
At the end of 28 days, blood samples were collected and analyzed. After sacrifice the liver was
recovered for measurement of organ weight and histological examination. High-throughput
ribonucleic acid (RNA)-sequencing was conducted to gain mechanistic insights into the observed
liver effects. Liver tissue samples from three controls and three treated animals were frozen for
RNA isolation, library preparation, and sequencing.
Statistically significant treatment-related findings reported include increased absolute liver
weight (31%) and relative liver weight (28%), ALP (51%), LDL cholesterol (50%), decreased
total bilirubin (-37%), and decreased direct bilirubin (-45%) when compared to control.
Qualitative hepatic histopathological findings documented abnormalities from the treated
animals, including lipid droplet accumulation, hepatocellular hypertrophy, mild steatosis, and
karyolysis.
High-throughput RNA-sequencing of liver tissues resulted in the identification of 146 transcripts
(101 upregulated and 45 downregulated) with altered differential gene expression due to
treatment with the HFPO dimer. Pathway analyses (using the National Center for Biotechnology
Information, Ensemble, gene ontology, and Kyoto Encyclopedia of Genes and Genomes
databases) revealed four enriched pathways from these altered hepatic transcripts: the PPAR
signaling pathway, arachidonic acid (an essential polyunsaturated fatty acid) metabolism, retinol
metabolism, and fatty acid degradation. All four of these pathways are associated with lipid
metabolism. Gene ontology analyses of the 146 altered transcripts identified several other
enriched processes, cellular components, and molecular functions related to immune system
function, lipid metabolism, membrane parameters, and others that were altered by HFPO dimer
acid treatment.
Behr et al. (2018)
H295R, MDA-kb2, HEK293T, LNCaP, and MCF-7 cell lines were cultured and incubated with
various individual PFAS in a variety of experiments to investigate effects on cytotoxicity,
estrogen and AR activity, and steroidogenesis (Behr et al., 2018). The study authors do not report
how the HFPO dimer acid ammonium salt was prepared but report 99% purity.
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The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was employed
to assess cell viability in HEK293T, H295R, MCF-7, and LNCaP cell lines following exposure
to various concentrations of each PFAS. HEK293T and LNCaP cells were exposed for 24 hours,
H295R cells for 48 hours, and MCF-7 cells for 6 days. The WST-1 assay was used to determine
viability of MDA-kb2 cells following 24 hours of exposure to each PFAS. Although the study
authors do not report how the HFPO dimer acid ammonium salt was prepared, 0.1% DMSO was
used as the vehicle control in this assy. HFPO dimer acid ammonium salt (referred to as
"PMOH" (ammonium perfluoro(2-methyl-3-oxahexanoate)) in this study) was not cytotoxic in
HEK293T, MDA-kb2, H295R, or LNCaP cell lines up to concentrations of 500 |iM and caused
cytotoxicity in the MCF-7 cell line at 500 |iM.
HEK293T cells were assayed for agonistic and antagonistic estrogen receptor (ER) alpha and
beta (ERa and ERP) transactivation. None of the tested PFAS were found to affect either ERa or
ERP at the highest tested concentrations (500 |iM for HFPO dimer acid ammonium salt).
Estrogen co-exposure with 500 |iM HFPO dimer acid ammonium salt was found to co-stimulate
ERP activity. Additionally, HFPO dimer acid ammonium salt was found to enhance estrogen-
mediated ERP activation.
The agonistic and antagonistic AR reporter gene assay was performed in MDA-kb2 cells. HFPO
dimer acid ammonium salt was found to be negative for AR transactivation and inhibition up to
concentrations of 100 |iM. HFPO dimer acid ammonium salt enhanced dihydrotestosterone-
stimulated AR activity in a dose-responsive fashion at concentrations above 50 |iM.
A steroidogenesis assay was performed in which H295R cells were exposed for 48 hours and
enzyme-linked immunosorbent assay (ELISA) kits were used to quantify estradiol (E2), estrone,
testosterone, and progesterone levels. HFPO dimer acid ammonium salt significantly decreased
testosterone at 100 |iM.
An E-screen assay was used to evaluate proliferation of MCF-7 cells following 6 days of
exposure to various PFAS. HFPO dimer acid ammonium salt did not significantly affect cell
proliferation compared to estrogen. Exposure to high concentrations of HFPO dimer acid
ammonium salt (100 |iM) in combination with estrogen slightly diminished cell proliferation, but
the effect was not statistically significant.
MCF-7 and LNCaP cells were cultured with HFPO dimer acid ammonium salt for 24 hours, and
H295R cells were cultured for 48 hours prior to RNA extraction followed by quantitative reverse
transcription PCR. HFPO dimer acid ammonium salt did not stimulate estrogenic responsive
gene expression of TFF1, GREB1, PGR, ESR1, ESR2, or CTSD in MCF-7 cells, or AR, PSA,
NKX3-1, TMPRSS2, or CDKN1A in LNCaP cells at concentrations up to 100 |iM. Additionally,
HFPO dimer acid ammonium salt did not affect expression of CYP19A1, CYP17A1, CYP21A2,
CYP11A1, STAR, or HSD3B at concentrations up to 100 |iM.
Shengetal. (2018)
Sheng et al. (2018) used in vitro experiments to investigate perfluoroalkyl cytotoxicity and
binding to proteins for HFPO dimer acid ammonium salt (referred to as "HFPO-DA" in this
study), HFPO dimer acid trimer, HFPO dimer acid tetramer, PFOA, and perfluorooctane
sulfonate (PFOS) in a human liver HL-7702 cell line. The study authors assessed cell viability to
determine the cytotoxicity of the various perfluoroalkyl substances and used flow cytometry to
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investigate effects on cell proliferation. The authors noted, however, that no effects of HFPO
dimer acid ammonium salt on cytotoxicity and cell proliferation could be determined through
these assays because of the chemical's low boiling point and high volatility. The study authors
do not report how the HFPO dimer acid ammonium salt was prepared.
Data quantifying the HFPO dimer acid anion's ability to bind to human liver fatty acid-binding
protein (hL-FABP) was also generated. Binding affinity was explored because other PFAS
compounds have exhibited effective binding to hL-FABP and such binding might explain how
PFAS can enter into hepatocytes, a potential target cell for HFPO dimer acid and/or its
ammonium salt (Luebker et al., 2002; Sheng et al., 2016; Zhang et al., 2013). Binding affinity
was measured in a fluorescence competitive binding assay and found that HFPO dimer acid
anion exhibited a weaker binding affinity than PFOA or PFOS. However, the study found that
the HFPO dimer acid anion fit well in the hL-FABP binding pocket with a docking energy in
between PFOS and PFOA. This indicates direct interaction between the HFPO dimer acid anion
and hL-FABP. Additionally, the HFPO dimer acid anion bound differently to hL-FABP than
PFOA and PFOS (Sheng et al., 2018). These results were replicated using a predictive model of
binding affinity to hL-FABP (Cheng and Ng, 2018).
Li et al. (2019)
Li et al. (2019) investigated the binding affinity of HFPO dimer acid (referred to as "HFPO-DA"
in the paper), HFPO trimer acid (HFPO-TA), and PFOA to human and mouse PPAR gamma
(PPARy) ligand binding domains. PPARy is a second member of the PPAR family of nuclear
receptors. It functions as a regulator of cell proliferation and differentiation in addition to
impacting lipid metabolism. The study authors report that HFPO dimer acid was dissolved in
DMSO to make stock solutions and was reported as 97% pure. Binding affinity was measured in
a fluorescence competitive binding assay. The study authors observed a higher affinity for the
human PPARy ligand binding domain for HFPO-TA and PFOA, while HFPO dimer acid bound
with greater affinity for the mouse PPARy ligand binding domain. Among the three PFAS tested,
a binding potency order of HFPO-TA> PFOA>HFPO dimer acid was identified for both human
and mouse ligand binding. Li et al. (2019) also assessed the activity of HFPO dimer acid, HFPO-
TA, and PFOA using HEK293 cells transfected with a luciferin-tagged PPARy vector. After
exposure to HFPO dimer acid, HFPO-TA, and PFOA, the luciferase activity of the cells was
quantified as an indicator of the PFAS's ability to impact PPARy transcription. The authors
conclude that HFPO dimer acid, HFPO-TA, and PFOA acted as transcriptional agonists,
resulting in enhanced PPARy transcriptional activity in a dose-dependent manner.
Because PPARy activation is involved in the modulation of adipogenesis (Tontonoz et al., 1994),
Li et al. (2019) also exposed cultured human (HPA-s) and mouse (3T3-L1) preadipocytes to the
three compounds for ten days during a period of cellular differentiation into adipocytes. To
quantify adipogenic activity, an Oil Red O staining assay was performed to quantify lipid
accumulation using the dosed human and mouse adipocytes. HFPO dimer acid significantly
increased lipid accumulation at 6 |iM and 25 |iM for the human HPA-s cells and mouse 3T3-L1
cells, respectively. HFPO dimer acid showed comparable or weaker adipogenesis activity than
PFOA and HFPO-TA. Relative PPARy messenger RNA (mRNA) levels were statistically
significantly increased in human HPA-s cells exposed to 25 |iM HFPO dimer acid and at 50 |iM
HFPO dimer acid in mouse 3T3-L1 cells.
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Sun etal. (2019)
Three dimensional (3D) spheroids were used to evaluate the cytotoxicity of PFOA, HFPO dimer
acid (referred to as "HFPO-DA" in this study) and PF04DA (3,5,7,9-tetraoxadecanoic perfluoro
acid) (Sun et al., 2019). HFPO dimer acid was diluted with serum-free DMEM/F-12 (Dulbecco's
Modified Eagle Medium: Nutrient Mixture F-12) medium and was reported as 95% pure. 3D
spheroids were exposed to 100 |iM of each of these three substances for 28 days. No changes in
adenosine triphosphate (ATP) content or albumin secretion were observed. Lactic dehydrogenase
and reactive oxygen species levels were significantly increased (compared with controls) after
PFOA, HFPO dimer acid, or PF04DA exposure. A 1.5- to twofold increase in Scdl expression
(compared with control) was observed in PFOA, HFPO dimer acid, and PF04DA exposure
groups. PFOA exposure, but not HFPO dimer acid exposure, significantly increased PPARa
expression compared to the control. For the apoptosis-related genes, PFOA exposure
significantly increased expression of caspase3, p53, and p21 compared to control, whereas
HFPO dimer acid exposure produced no changes. For the oxidative stress genes, only PFOA
significantly increased Nqol expression, and PFOA, HFPO-dimer acid, and PF04DA all
significantly induced expression of Gsta2 and Ho-1.
Xin et al. (2019)
Estrogenic effects of PFOA and HFPO dimer acid (referred to as "HFPO-DA" in this study)
were evaluated in a series of in vitro assays (Xin et al., 2019). All tests were also performed on
additional HFPO homologs (HFPO-TA and hexafluoropropylene oxide tetramer acid (HFPO-
TeA)). HFPO dimer acid was prepared in DMSO at a concentration of 20 millimolar (mM) and
was reported as 97% pure.
HFPO dimer acid binding affinity to the human ERa and ERP ligand binding domains were
compared to that of PFOA using a fluorescence polarization-based competitive fluorescence
binding assay. HFPO dimer acid did not bind either ERa or ERp (not detected; no IC50
(concentration at which 50% inhibition is observed) could be derived). PFOA displaced estrogen
in a concentration-dependent manner, with IC50 values of 469.5 |iM for ERa and 384.4 |iM for
ERp.
The cytotoxic effects of HFPO dimer acid at concentrations ranging from 0.8 to 1,600 |iM were
determined in MVLN cells using the WST-1 assay. HFPO dimer acid produced no cytotoxicity,
whereas PFOA inhibited cell viability at concentrations above 800 |iM. MVLN cells were also
exposed to PFOA or HFPO dimer acid at concentrations ranging from 1.6 to 800 |iM, with or
without E2 for 12 or 24 hours, and estrogenic/anti-estrogenic activity was assessed. Exposure to
HFPO dimer acid did not result in any effects on ERs. PFOA exposure resulted in concentration-
dependent antagonism of ERs and PFOA was also found to compete with E2 to activate ERs.
ELISA kits were used to measure E2, testosterone, and vitellogenin (VTG) in wildtype zebrafish
larvae exposed to 0.4 or 1.6 |iM PFOA or HFPO dimer acid for 168 hours post-fertilization.
HFPO dimer acid and PFOA significantly increased E2, testosterone, and VTG compared to
controls in all dose groups, except for VTG levels at 0.4 |iM HFPO dimer acid.
Molecular docking and molecular dynamics simulations were performed using AutoDock 4.2 to
compare binding interactions of HFPO dimer acid and PFOA with ERa and ERp. The
simulations illustrated that both HFPO dimer acid and PFOA fit into the binding cavity of ERa
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and ERp. The calculated energies from the simulations indicated that the order of the binding
affinity for these compounds is HFPO-TeA > HFPO-TA > PFOA > HFPO dimer acid.
Behr et al. (2020)
The cytotoxicity, human nuclear receptor activation, and gene expression changes induced by
HFPO dimer acid ammonium salt (referred to as "PMOH" (ammonium perfluoro(2-methyl-3-
oxahexanoate)) in this study) was investigated in vitro using HEK293T and HepG2 cells. Seven
other PFAS (PFOA, PFOS, PFHxA, perfluorobutanesulfonic acid (PFBS), PFBA, PFHxS, and
3H-perfluoro-3-((3-methoxypropoxy) propanoic acid (PMPP) were also analyzed in this study
(Behr et al., 2020). The study authors do not report how the HFPO dimer acid ammonium salt
was prepared.
The cytotoxicity of HFPO dimer acid ammonium salt was assessed in HepG2 cells. The cells
were exposed to 50-500 |iM of HFPO dimer acid ammonium salt for 24 hours, and cellular
viability was determined using the MTT assay. Cell viability was not significantly decreased at
any concentration.
Luciferase-based reporter gene assays were used to determine the ability of HFPO dimer acid
ammonium salt to activate various human nuclear receptors that function in the regulation of
lipid or xenobiotic metabolism. HEK293T cells were transfected with expression plasmids for
hCAR, hFXR, hLXRa, hPPARa, hPPARS, hPPARy, hPXR, hRARa, or hRXRa. The cells were
co-transfected with a luciferase reporter plasmid and the i?e«z7/a-luciferase construct pcDNA3-
Rluc for normalization. Positive controls were included. Receptor activity was measured after 24
hours of exposure to 25, 50, or 100 |iM of each chemical. Values were normalized to Renilla
reniformis luciferase activities and compared to untreated cells. HFPO dimer acid ammonium
salt significantly induced PPARa activation (sevenfold) at 25 |iM and higher. HFPO dimer acid
ammonium salt also significantly induced activation of PPARy (2.4-fold) at 100 |iM, The other
human nuclear receptors were not significantly affected. Reporter gene assays for PPARa were
repeated for PFOA and PMOH using concentrations up to 250 |iM, Concentration-response
curves were calculated and ECio values were determined relative to a positive control
(GW7647). HFPO dimer acid ammonium salt activated PPARa to a level of 10% at 5 [xM, and a
comparable activation was induced by PFOA at 50 [xM.
HepG2 cells were exposed to concentrations up to 250 |iM HFPO dimer acid ammonium salt for
24 hours, and the RNA was extracted for analysis of PPARa-dependent target gene expression.
Untreated cells served as control. At 250 [xM, PMOH significantly induced expression of CPT1A
(1.7-fold), HMGCS2 (2.8-fold), and PLIN2 (1.4-fold). Compared to PFOA, the effects of PMOH
were not as substantial. PFOA produced similar effects on the target genes as PPARa agonists
GW7647 and WY14,643.
Although HFPO dimer acid ammonium salt was a more potent PPARa agonist than PFOA under
the conditions of this study, it produced weaker effects on PPARa-dependent target gene
expression.
Wen et al. (2020)
The epigenetic toxicities of HFPO dimer acid (referred to as "GenX" in this study) and PFOA
were explored and compared in vitro using a liver hepatocellular carcinoma cell line (Wen et al.,
2020). HepG2 cells were exposed to concentration gradients of the ammonium salt form of
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PFOA (20-600 |iM) or HFPO dimer acid (20-1,000 |iM) for 48 hours. The test chemicals were
first dissolved in DMSO (< 0.4% volume/volume), and vehicle controls were included. HFPO
dimer acid was reported as 97% pure.
The MTT and neutral red assays were used to assess cell metabolism rates and viability.
Following HFPO dimer acid exposure, cell metabolic activity was only slightly increased at all
concentrations compared to control. Cell viability was increased from 20 to 200 [xM, and then
decreased linearly from 200 to 1,000 [xM.
Following PFOA exposure, cell metabolic activity increased in a concentration-dependent
fashion from 0 to 100 [xM, peaked at 100 [xM, and decreased in a concentration-dependent
fashion from 100 to 400 [xM. Cell viability decreased with increasing PFOA concentration
(decreased by 87% at 600 (xM).
Gene expression analysis was performed for 22 genes related to cell cycle, proliferation,
apoptosis, and lipid metabolism. HepG2 cells were cultured in flasks and treated with 100-600
[xM HFPO dimer acid for 48 hours, and the RNA was extracted for gene expression analysis.
Overall, HFPO dimer acid did not have a strong impact on the genes examined; expression of
most lipid metabolism and transport-related genes was either decreased or not significantly
affected by HFPO dimer acid. In contrast, expression of lipid synthesis-related genes was mostly
elevated, and expression of lipid transport genes was mostly decreased by PFOA.
Global methylation assays were performed using genomic DNA from HepG2 cells extracted
immediately after the treatment period. Expression profiles of 10-11 translocation
methylcytosine dioxygenases (TETs) and DNA methyltransferases (DNMTs) were also
evaluated. In HFPO dimer acid-treated cells, global methylation (5mc) levels significantly
decreased from 100 to 400 [xM, and then increased from 600 to 800 [xM. GenX caused decreased
expression of DNMTs but had no clear effect on TETs. PFOA caused a significant,
concentration-dependent decrease in global methylation (5-mC) levels from 20 to 400 [xM, and
significant concentration-dependent changes in TETs (TET1 decreased whereas TET2 and TET3
increased with increasing PFOA concentration), but no significant trends in the expression of
DNMTs.
Cannon et al. (2020)
The effects of HFPO dimer acid ammonium salt (referred to as "GenX" in this paper) on
expression and activity of three ATP binding cassette (ABC) transporters at the blood-brain
barrier were studied using rat brain capillaries exposed ex vivo to low nanomolar (nM)
concentrations (Cannon et al., 2020). Rats were also exposed to 97% pure HFPO dimer acid
ammonium salt in vivo followed by ex vivo measurement of transport activity. ATPase levels
were measured in vitro, and protein levels were measured with Western blotting. The
cytotoxicity of HFPO dimer acid ammonium salt was assessed using two human cell lines.
HFPO dimer acid ammonium salt was prepared in fresh DMSO (0.1% volume/volume) prior to
each experiment.
The brains from 4-6 male or female Hsd:Sprague Dawley rats (age 12-15 weeks) were harvested
and capillaries were isolated from cortical gray matter. Capillaries were exposed to varying
concentrations of HFPO dimer acid ammonium salt (0.01-1,000 nM) for 3 hours and P-
glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-
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associated protein 2 (MRP2) transporter activities were measured with a confocal microscopy-
based method. Hourly changes in transport activities were also measured during a 4-hour
exposure to 100 nM HFPO dimer acid ammonium salt. In addition, reversibility assays were
performed using 100 nM HFPO dimer acid ammonium salt exposure for 1 hour for P-gp and 2
hours for BCRP. After transport activities were measured, HFPO dimer acid ammonium salt was
removed from the assay media and transport activities were measured at 0.5, 1, 2, and 3 hours
after removal. In the main assays, P-gp transport activity was significantly lower in male
capillaries exposed to 0.1-100 nM HFPO dimer acid ammonium salt and in female capillaries
exposed to 1.0-100 nM HFPO dimer acid ammonium salt compared with controls. P-gp
transport activity was significantly decreased beginning at 15 or 30 minutes of treatment with
100 nM and persisted to the 4-hour mark. BCRP transport activity was significantly lower in
male capillaries exposed to 1.0-1,000 nM HFPO dimer acid ammonium salt and in female
capillaries exposed to 0.1-1,000 nM, and a significant decrease in activity during a 4-hour
exposure to 100 nM was observed beginning at the 1-hour time point for both sexes. MRP2
transport activity was not significantly affected by HFPO dimer acid ammonium salt. In the
reversibility assays, P-gp transport activity in capillaries from both sexes was restored to control
levels within 1 hour of HFPO dimer acid ammonium salt removal, but BCRP transport activity
remained lowered for 2 hours after removal for both sexes.
A reconstituted transport assay system containing vesicle membranes and transport proteins was
used to determine the effects of HFPO dimer acid ammonium salt on transport-associated
ATPase activity in vitro. Purified P-gp and BRCP transport proteins were exposed to 0.001-1.0
|iM HFPO dimer acid ammonium salt for 20 minutes and the enzymatic hydrolysis of ATP to
inorganic phosphate was measured. The substrate was stimulated with paclitaxel for P-gp and
sulfasalazine for BCRP. HFPO dimer acid ammonium salt did not alter ATPase activity
associated with P-gp or BCRP transport either when the substrate was stimulated or when no
substrate was added, indicating that HFPO dimer acid ammonium salt was not a substrate for
either transporter using this particular in vitro reconstituted transport assay system.
Isolated brain capillaries pooled from male or female rats (n=6 rats/sex) were exposed to 100 nM
HFPO dimer acid ammonium salt for 4 hours, and P-gp and BCRP protein levels were measured
by Western blotting. No significant differences in P-gp or BCRP protein levels were identified
for treated capillaries compared with control (vehicle-treated) capillaries for either sex.
Isolated brain capillaries from male or female rats (n=6 rats/sex) were also treated with HFPO
dimer acid ammonium salt (1.0 or 100 nM) for 4 hours with or without the PPARy inhibitor
GW9662. HFPO dimer acid ammonium salt decreased P-gp transport activity at both
concentrations for both sexes compared with controls. The addition of GW9662 blocked the
reduction in activity in male capillaries at both concentrations, but only at 1.0 nM HFPO dimer
acid ammonium salt in female capillaries. BCRP transport activity was lowered by treatment at
both concentrations in both sexes, and co-treatment with GW9662 had no effect on the reduced
BCRP transport activities for either sex.
Male and female Hsd:Sprague Dawley rats (5/sex/group) were administered a single oral gavage
dose of 0, 10, 100, or 1,000 ng/kg (30 picomole (pmol)/kg, 300 pmol/kg, or 3 nanomole/kg) of
HFPO dimer acid ammonium salt and sacrificed 5 hours later. Brains from each dose group were
pooled, and capillaries were isolated for measurement of P-gp and BCRP transport activities. All
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dose levels produced significant decreases in P-gp and BCRP transport activities in both sexes
compared with controls.
Cell survival following HFPO dimer acid ammonium salt exposure was determined using the
human ovarian cell line NCI/ADR-RES (with high P-gp expression) and human mammary
epithelial cell line MX-MCF-7 (with high BCRP expression). The cells were first exposed to
HFPO dimer acid ammonium salt alone at increasing concentrations of 10 9—10 4 M for 72
hours, and the remaining cells were counted. No significant differences in survival were
observed. Next, to determine if HFPO dimer acid ammonium salt affected the toxicity of known
cytotoxic substrates for P-gp or BCRP, each cell line was exposed to 100 nM HFPO dimer acid
ammonium salt for 72 hours in the presence of Adriamycin (for NCI/ADR-RES cells) or
mitoxantrone (for MX-MCF-7 cells). Co-treatment of 100 nM HFPO dimer acid ammonium salt
and 10 |iM Adriamycin significantly reduced NCI/ADR-RES cell survival from 85% to 45%,
and co-treatment of 100 nM HFPO dimer acid ammonium salt and 100 [xM mitoxantrone
significantly reduced MX-MCF-7 cell survival from 63% to 37%.
The results of these assays show that both ex vivo and in vivo exposure to low nM levels of
HFPO dimer acid ammonium salt can inhibit P-gp and BCRP transport in rat brain capillaries.
The effect of HFPO dimer acid ammonium salt on P-gp transport was shown to involve PPARy.
4.6.3 Genotoxicity Studies
HFPO dimer acid ammonium salt was not observed to induce genetic mutations both with and
without metabolic activation of the test substance by rat liver S9 fraction in two species of
prokaryotes: Escherichia coli (strain WP2uvrA) and Salmonella typhimurium (strains TA98,
TA100, TA1535, and TA 1537) (DuPont-19713 RV1, 2008; DuPont-22734 RV1, 2008). An in
vitro gene mutation test of the HFPO dimer acid ammonium salt in mouse lymphoma cells
(strain L5178Y/TK+/-) was negative in the presence and absence of rat liver S9 fraction
(DuPont-26129, 2008). HFPO dimer acid ammonium salt was observed to induce chromosomal
aberrations in Chinese hamster ovary cells in vitro in the presence and absence of S9 activation
(DuPont-19714 RV1, 2008; DuPont-22620 RV1, 2009). In in vivo mammalian studies, exposure
to HFPO dimer acid ammonium salt by the oral route did not induce chromosomal mutations in
the form of structural aberrations, numerical aberrations, or micronuclei nor DNA effects in the
form of unscheduled DNA synthesis (DuPont-23219, 2007; DuPont-23220, 2007). A table
summarizing the findings of the available genotoxicity studies is provided in appendix C.
5.0 Summary of Hazard
The available studies indicate adverse effects including liver, developmental, hematological, and
immune effects occur following exposures in the range of 0.5-1,000 mg/kg/day GenX
chemicals. Table 12 presents the available studies and their NOAELs and LOAELs. Discussion
of the weight of evidence for hazard is presented following the table.
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Table 12. Summary of Study NOAELs/LOAELs
Study
Overall study
quality
Doses (mg/kg/day)
NOAEL or LOAEL
(mg/kg/day)
Effects at the LOAEL
28 Day Oral (Gavage)
Toxicity Study in Rats
(OECD, 2008a)
DuPont-24447 (2008)
Medium
Males: 0, 0.3, 3,
and 30
Females: 0, 3, 30,
and 300
NOAEL = 0.3
LOAEL = 3
Hematological effects (j RBC count, hemoglobin, and hematocrit in
males)
Immune effects (j globulin, and t A/G ratio in males)
28 Day Oral (Gavage)
Toxicity Study in Mice
(OECD, 2008a)
DuPont-24459 (2008)
Medium/Low
Males and
Females: 0, 0.1, 3,
and 30
NOAEL = 0.1
LOAEL = 3
Liver effects (single-cell necrosis in males, t relative liver weight in
in males, and t hepatocellular hypertrophy in males)
Hematological effects (J, hemoglobin and hematocrit in males)
Immune effects (J, globulin in females, and t A/G ratio in both
sexes)
28 Day Oral (Gavage)
Immunotoxicity Study in
Mice
Rushing et al. (2017)
Medium
Males and
Females: 0, 1, 10,
and 100
Note: HFPO dimer
acid
NOAEL = 10
LOAEL = 100
Immune effects (TDAR suppression in females, and t lymphocytes
in males)
90 Day Oral (Gavage)
Toxicity Study in Rats
(OECD, 1998)
DuPont-17751-1026
(2009)
High
Males: 0,0.1, 10,
and 100
Females: 0, 10,
100, and 1,000
NOAEL = 0.1
LOAEL = 10
Hematological effects (J, RBC count, hemoglobin, and hematocrit in
males)
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Study
Overall study
quality
Doses (mg/kg/day)
NOAEL or LOAEL
(mg/kg/day)
Effects at the LOAEL
90 Day Oral (Gavage)
Toxicity Study in Mice
(OECD, 1998)
DuPont-18405-1307
(2010); Reevaluation by
NTP PWG Pathology
(NTP, 2019)
High
Males and
Females: 0, 0.1,
0.5, and 5
NOAEL = 0.5
LOAEL = 5
Liver effects (t AST. ALT, and ALP in males; t relative liver weight
in males and females; and t in constellation of liver lesions:
cytoplasmic alteration, single-cell necrosis, focal necrosis, and
hepatocellular apoptosis in males and females)
Combined Chronic
Toxicity/ Oncogenicity
Study in Rats
(OECD, 2009)
DuPont-18405-1238
(2013)
Medium
Males: 0,0.1, 1,
and 50
Females: 0, 1, 50,
and 500
NOAEL = 1
LOAEL = 50
Liver effects (centrilobular necrosis in both sexes; t ALP, ALT, and
SDH in males; and t centrilobular hepatocellular hypertrophy and
cystic focal degeneration in males)
Oral (Gavage)
Reproduction/
Developmental Toxicity
Study in Mice (OECD,
2016a; modified
according to the Consent
Order)
DuPont-18405-1037
(2010); Reevaluation by
NTP PWG Pathology
(NTP, 2019)
High
Males and
Females: 0, 0.1,
0.5, and 5
NOAEL (Fo) = 0.1
LOAEL (F0) = 0.5
NOAEL (Fi) = 0.5
LOAEL (Fi) = 5
Liver effects ((single-cell necrosis, focal necrosis, and cytoplasmic
alteration), and t relative liver weight in males and females);
reproductive/developmental effects (t maternal GWG from GD0
through GDI8)
Developmental effects (J, pup weights, and delays in the attainment
of balanopreputial separation and vaginal patency)
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Study
Overall study
quality
Doses (mg/kg/day)
NOAEL or LOAEL
(mg/kg/day)
Effects at the LOAEL
Prenatal and
Developmental Toxicity
Study in Rats (OECD,
2001b)
DuPont-18405-841
(2010)
Medium
Females: 0, 10,
100, and 1,000
NOAEL (F0 and Fi) =
10
LOAEL (F0 and Fi) =
100
Developmental effects (t early deliveries, j fetal weights in both
sexes, i gravid uterine weight, and focal liver necrosis)
Reproductive and
Developmental Toxicity
in Rats
Conley etal. (2019)
Medium
Females: 0, 1, 3,
10, 30, 62.5, 125,
250, and 500
NOAEL (F0 and Fi) =
62.5
LOAEL (F0 and Fi) =
125
Reproduction/developmental effects (j maternal GWG,, and
indications of reduced body (females) and reproductive and non-
reproductive organ weights inFi animals)
Thyroid effects (|maternal serum total T3 and T4 levels)
Reproductive and
Developmental Toxicity
in Mice
Blake et al. (2020)
Medium
Females: 0, 2, and
10
NOAEL = NA
LOAEL = 2
Reproductive/developmental effects (t mean abnormal placental
lesions (including labyrinth atrophy, labyrinth congestion, labyrinth
necrosis, early fibrin clot, and placental nodule), and t maternal
GWG)
Reproductive and
Developmental Toxicity
in Rats
Conley et al. (2021)
High
Females: 0, 10, 30,
62.5, 125, or 250
NOAEL (F0) = 30
LOAEL (F0) = 62.5
NOAEL (Fi) = 10
LOAEL (Fi) = 30
Thyroid effects (jmaternal serum total T3 and T4 levels)
Reproductive/developmental effects (j BW in FI pups at PND0 and
PND2)
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5.1 Hepatic
The liver is a target organ for toxicity from oral exposure to HFPO dimer acid and its ammonium
salt. Liver effects are observed in both male and female mice and rats at varying durations of
exposures and doses of GenX chemicals. Liver effects are also the endpoints that are observed at
the lowest doses for these chemicals. Hepatocellular hypertrophy and an increased liver-to-BW
ratio are common findings in rodents but are considered nonadverse and less relevant to humans
when there is evidence that PPARa activation is the only MOA. The increased relative liver
weight and hepatocellular hypertrophy were only considered adverse when accompanied by
effects such as necrosis, fibrosis, inflammation, and significantly increased serum levels for
enzymes indicative of liver tissue damage (Hall et al., 2012).
Significant increases in liver weight relative to BW were observed in male and female
Crl:CD(SD) rats and several strains of male and female mice treated with 0.5 mg/kg/day-1,000
mg/kg/day of HFPO dimer acid ammonium salt for 28-90 days (DuPont-17751-1026, 2009;
DuPont-18405-1037, 2010; DuPont-18405-1307, 2010; DuPont-24447, 2008; DuPont-24459,
2008; Rushing et al., 2017; Wang et al., 2017). These increases were observed in doses as low as
0.5 mg/kg/day in male Crl:CD-l mice (26% increase) over 84-85 days (DuPont-18405-1037,
2010), and the greatest increases were observed when male (163%) and female (102.7%)
Crl:CD-l mice were administered 30 mg/kg/day for 28 days. Likewise, male Crl:CD(SD) rats
exhibited increased relative liver weights of 19%-61% compared to control when administered
3 mg/kg/day-100 mg/kg/day for 28-90 days, while female rats' relative liver weights compared
to control did not increase until much higher doses (12% at 300 mg/kg/day for 28 days and 85%
at 1,000 mg/kg/day for 90 days) were administered. Comparatively, the one available chronic
study in rats indicates that liver weight may increase and return to control levels after a time. For
example, relative liver weights in male rats increased only 15% when administered 50 mg/kg/day
for 1 year and did not exhibit a significant increase from control at 2 years. Likewise, female rat
relative liver weights increased 67% and 42% after administration of 500 mg/kg/day for 1 and 2
years, respectively (DuPont-18405-1238, 2013).
Indications of liver damage were also reflected through increases in serum liver enzymes of
Crl:CD-l mice, particularly males, and Crl:CD(SD) rats administered HFPO dimer acid
ammonium salt. For example, significant increases in ALT (420%—1,254%), AST (106%-
478%>), ALP (1,134%-1,221%), and SDH (1,134%—1,221%) were observed in male mice
administered the ammonium salt at 5-30 mg/kg/day for 28-90 days. Female mice saw smaller
increases in ALP (140%—143%) and SDH (32%—186%) compared to male mice administered the
same dose. Overall, rats exhibited far fewer and smaller increases in serum liver enzyme levels
following subchronic exposure than the mouse, with increases in AST (106%) and ALP (52%) at
100 mg/kg/day in male rats and AST (66%) in female rats at 1,000 mg/kg/day. In the chronic
study, however, ALT (228%), ALP (180%), and SDH (140%) significantly increased in male
rats only when administered 50 mg/kg/day for 1 year (DuPont-18405-1238, 2013).
Liver damage was confirmed microscopically in male and female mice and rats in several less-
than-chronic studies (15-90 days) and one chronic study (DuPont-17751-1026, 2009; DuPont-
18405-841, 2010; DuPont-18405-1037, 2010; DuPont-18405-1238, 2013; DuPont-18405-1307,
2010; DuPont-24447, 2008; DuPont-24459, 2008; Wang et al., 2017; Thompson et al., 2019;
NTP, 2019). The most prevalent liver effects following both subchronic and chronic exposure to
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HFPO dimer acid and/or its ammonium salt were hepatocellular hypertrophy (also referenced
here as cytoplasmic alteration per NTP PWG's review) and single-cell and/or focal necrosis.
In both sexes of mice exposed either short term (28 days) or subchronically (30-90 days),
hepatocellular hypertrophy was observed at 0.5 mg/kg/day, while male and female rats showed
this effect at 3 mg/kg/day and 30 mg/kg/day, respectively. Interestingly, in the chronic study,
male rats did not show any significant increases in hepatocellular hypertrophy when
administered 0.1-50 mg/kg/day of HFPO dimer acid ammonium salt for 1 year, and only 10% of
the rats exhibited minimal hypertrophy with 50 mg/kg/day administered for 2 years (DuPont-
18405-1238, 2013). Conversely, female rats had significant hepatocellular hypertrophy at 500
mg/kg/day after 1 year (100%) and 2 years (93%).
Single-cell and focal necrosis were detected in all the available studies. The reanalysis of the
liver pathology slides from DuPont 18405-1037 (2010) by Thompson et al. (2019) did not report
necrosis in mice. This interpretation conflicts with the results from the original pathology
conducted in DuPont 18405-1037 (2010) and the 2019 NTP PWG reanalysis (NTP, 2019) of
DuPont 18405-1037 (2010). The incidence data as reported by NTP (see appendix D) were
considered the appropriate measure of response in the liver from the reproductive/developmental
study (DuPont-18405-1037, 2010) because the PWG analysis reflects more recent
histopathological criteria for the grading of liver lesions and the PWG results were the consensus
of eight pathologists.
In the subchronic toxicity studies in mice, males and females presented with single-cell and focal
necrosis in doses as low as 0.5 mg/kg/day, which significantly increased at 5 mg/kg/day.
Specifically, the incidence rates for single-cell and focal necrosis at 5 mg/kg/day were 100% and
83%) in males and females, respectively, in DuPont 18405-1037 (2010) and 90% and 44% in
males and females, respectively, in DuPont 18405-1307 (2010) (NTP, 2019). Apoptosis was
observed in the 5 mg/kg/day-dose groups in these studies as well, but not in the 0.5 mg/kg/day -
dose group (NTP, 2019). As noted in section 4.0 and appendix D in this assessment, the NTP
PWG agreed that the dose response and constellation of liver lesions (i.e., hepatocellular
hypertrophy, single-cell and focal necrosis and apoptosis) observed in DuPont 18405-1037
(2010) and DuPont 18405-1307 (2010) should be considered as adverse (NTP, 2019). Male and
female rats exhibited hepatocellular necrosis at much higher doses in the available short-term
study, with males exhibiting what was classified as general necrosis (30%) at 30 mg/kg/day and
females presenting focal liver necrosis at 100 mg/kg/day (9%) and 1,000 mg/kg/day (23%).
Interestingly, no liver necrosis was reported for either sex in the subchronic rat study (DuPont-
17751-1026, 2009). It is possible that apoptosis could have been present in the other DuPont
studies, but these studies might not have separated apoptotic lesions from other liver lesions
reported (i.e., single-cell necrosis) since they were conducted prior to the histopathological
guidance on separating apoptosis from single-cell necrosis (i.e., Elmore et al., 2016) and were
not reanalyzed by the 2019 NTP PWG.
These findings suggest that mice are more sensitive to liver necrosis than rats in short-term and
subchronic exposure scenarios. In the 2-year chronic rat study, centrilobular necrosis increased at
50 mg/kg/day and 500 mg/kg/day for males (7%) and females (4%), respectively, while single-
cell necrosis was observed only in females (4%) at 500 mg/kg/day. Taken together, the male rat
liver necrosis data appear to be inconsistent. Specifically, 30% of male rats have necrotic liver
cells after 28 days of dosing with 30 mg/kg/day of HFPO dimer acid ammonium salt, yet no
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necrosis is reported in male rats after 90 days of dosing with 0.1-100 mg/kg/day. However,
necrosis returns in 50% of male rats after 1 year of dosing with 50 mg/kg/day to then be reduced
to 7-13% incidence after 2 years of dosing.
Similarly, these data suggest that the pregnant rodent might be more susceptible than
nonpregnant rodents to liver effects following exposures to GenX chemicals. Liver effects were
reported in the pregnant dams in the available reproductive/developmental studies dosing during
gestation (DuPont-18405-841, 2010; Conley et al., 2019; DuPont 18405-1037, 2010; Blake et al.,
2020). All the studies reported increases in liver weight ranging from 12% to 34% in rats and
26%) to 101%) in mice over the gestational period. Conley et al. (2019) did not conduct liver
histopathology, but both DuPont-18405-841 (2010) and Blake et al. (2020) reported
hepatocellular hypertrophy and increased cell death as compared to control with increasing
HFPO dimer acid ammonium salt concentration. Specifically, focal necrosis was observed in
2/22 (9%>) and 5/22 (23%>) pregnant rats after 15 days (GD6-GD20) of 10 mg/kg/day or 100
mg/kg/day of HFPO dimer acid ammonium salt, respectively, compared to 0 in the control
group. Comparatively, nonpregnant female rats dosed from 28 to 90 days did not exhibit necrosis
when treated with doses up to 1,000 mg/kg/day of HFPO dimer acid ammonium salt. Necrosis
was observed in female rats only after 2 years of dosing with 500 mg/kg/day of HFPO dimer
acid ammonium salt. Increased cell death (including both apoptosis and single-cell necrosis) or
focal necrosis was observed in pregnant mice after 11 and 17 days (GD1.5-GD11.5 or 17.5) of 2
mg/kg/day or 10 mg/kg/day of HFPO dimer acid ammonium salt. Similarly, and as noted above,
female mice dosed 14 days prior to mating and throughout gestation/lactation exhibited
cytoplasmic alteration, apoptosis, single-cell necrosis, and focal necrosis after 53-64 days of
dosing (NTP, 2019 reanalysis of DuPont 18405-1037, 2010). The incidence of single-cell and
focal necrosis in the Fo females was 6/24 (25%>) and 20/24 (83%>) in the 0.5- and 5-mg/kg/day-
dose groups, respectively (NTP, 2019).
5.2 Hematological
The hematologic system could be a target of HFPO dimer acid ammonium salt toxicity as effects
have been observed across studies of varying durations of oral exposure to the chemical. The
primary effects observed are decreases in RBC number, hemoglobin, and percentage of RBCs in
the blood, indicating that oral exposure to HFPO dimer acid ammonium salt might promote
anemic conditions. In male mice and rats, the percent change in these effects from the controls
was relatively small. For example, male Crl:CD-l mice and Crl:CD(SD) rats treated with 3
mg/kg/day-100 mg/kg/day of HFPO dimer acid ammonium salt for 28-180 days had maximum
decreases of 12%>, 11%>, and 12%> in hemoglobin, erythrocyte count, and hematocrit, respectively
(DuPont-17751-1026, 2009; DuPont-18405-1238, 2013; DuPont-18405-1307, 2010; DuPont-
24447, 2008; DuPont-24459, 2008). Interestingly, in the available chronic study, no
hematological effects were observed at the 12-month time point in male rats (DuPont-18405-
1238, 2013). Female Crl:CD-l mice and Crl:CD(SD) rats presented hematological effects at
greater than 90 days and typically at higher doses than males, with one exception. Hemoglobin
significantly decreased by 4%> when female Crl:CD(SD) rats were administered 1 mg/kg/day of
HFPO dimer acid ammonium salt for 90 days (DuPont-18405-1238, 2013). Otherwise,
hematological effects occurred at doses greater than or equal to 50 mg/kg/day and the maximum
decreases from control were 24%>, 28%>, and 20%> for hemoglobin, erythrocyte count, and
hematocrit, respectively (DuPont-18405-1238, 2013; DuPont-24447, 2008).
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5.3 Renal
The kidney could also be a target organ for toxicity from oral exposure to HFPO dimer acid
and/or ammonium salt; however, kidney effects typically presented at higher doses than the liver
effects.
Significant increases in kidney weight relative to BW were observed in several less-than-chronic
studies in Crl:CD-l mice and Crl:CD(SD) rats treated with 0.1 mg/kg/day-1,000 mg/kg/day
(DuPont-17751-1026, 2009; DuPont-18405-1037, 2010; DuPont-24459, 2008; DuPont-24447,
2008). The maximum increase in kidney weight for male rodents was an increase of 16%
compared to control in male rats treated with 100 mg/kg/day of HFPO dimer acid ammonium
salt over 90 days. Likewise, the maximum kidney weight relative to BW increase in female
rodents was 23% in female rats administered 1,000 mg/kg/day over 90 days (DuPont-17751-
1026, 2009). Interestingly, increases in relative kidney weights were not observed in the same
type of male rat when administered HFPO dimer acid ammonium salt for 1 or 2 years (DuPont-
18405-1238, 2013). Relative kidney weight did increase in female Crl:CD(SD) rats by 25% and
14% when administered 500 mg/kg/day of HFPO dimer acid ammonium salt for 1 and 2 years,
respectively (DuPont-18405-1238, 2013).
These increases in kidney weight were often associated with increases in BUN, which can be
used as an indicator of renal damage. In several studies, urea nitrogen levels were significantly
increased (16%-38%) in male mice and rats administered doses greater than or equal to 30
mg/kg/day of HFPO dimer acid ammonium salt for 28-180 days (DuPont-17751-1026, 2009;
DuPont-18405-1238, 2013; DuPont-24447, 2008; DuPont-24459, 2008). Female rats exhibited
an increase in urea nitrogen levels (35%) only when administered 500 mg/kg/day of HFPO dimer
acid ammonium salt for 1 year (DuPont-18405-1238, 2013). Kidney damage was equivocal
microscopically in the less-than-chronic studies (28-90 days), and typically presented as
increases in basophilic tubular cells and tubular epithelial hypertrophy or dilation without tubular
degeneration and/or necrosis (DuPont-17751-1026, 2009; DuPont-18405-1037, 2010; DuPont-
24459, 2008; DuPont-24447, 2008).
In the chronic study, the increases in BUN and relative kidney weight noted above for female
rats were associated with multiple microscopic observations of kidney damage when female rats
were treated with HFPO dimer acid ammonium salt for 2 years. For example, at 50 mg/kg/day-
500 mg/kg/day, female rats exhibited transitional cell hyperplasia, tubular dilation, pelvic and
tubular mineralization, and papillary edema, which ultimately resulted in papillary necrosis at
500 mg/kg/day (DuPont-18405-1238, 2013).
To summarize, significant and dose-dependent increases in relative kidney weight occurred in
rats at lower doses (e.g., 10 mg/kg/day) in a subchronic study (DuPont-18405-1307, 2010).
Kidney hypertrophy, however, was not associated with microscopic damage of the kidney such
as necrosis in this study. Additionally, there are instances in which kidney hypertrophy occurred
at low doses in female mice (e.g., 0.1 mg/kg/day (DuPont-24459, 2008) or 5 mg/kg/day
(DuPont-18405-1037, 2010)), but there was not a dose response in these datasets, and
microscopic damage to the kidney tissues was not reported. Of the available studies, kidney
hypertrophy was associated with significant microscopic damage only in female rats treated with
500 mg/kg/day of HFPO dimer acid ammonium salt for 2 years (DuPont-18405-1238, 2013).
Thus, the observed kidney effects are potentially of concern. The biological significance,
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however, of the observed hypertrophy and increases in BUN without microscopic evidence of
kidney damage is not clear.
5.4 Reproductive/Developmental
Evidence in animals suggests HFPO dimer acid and/or ammonium salt could target the
reproductive system and the developing fetus.
In a reproduction/developmental toxicity mouse study, there were no effects on mating, fertility,
or copulation indices; mean days between pairing and coitus; mean gestation length; mean
numbers of implantation sites; mean numbers of pups born; live litter size; percentage of males
at birth; postnatal survival; or the general condition of pups (DuPont-18405-1037, 2010).
Similarly, implantation sites, viable embryos, nonviable embryos, and resorptions were not
significantly different than control when pregnant mice were dosed with 2 or 10 mg/kg/day of
HFPO dimer acid from El.5 to E17.5 (Blake et al., 2020). In the rat developmental toxicity
study, however, early delivery on GD21 was observed in 18% and 41% of the dams at 100
mg/kg/day and 1,000 mg/kg/day, respectively, and the percentage of male (47%) and female
(53%>) pups born was significantly altered from control at 1,000 mg/kg/day (DuPont-18405-841,
2010). Conley et al. (2019) reported no significant effects from control on the number of fetuses
or resorptions in pregnant rats dosed with 1-500 mg/kg/day HFPO dimer acid from GD14
through GD18. Conley et al. (2021) also reported no significant effects on the numbers of viable
fetuses or resorptions in pregnant rats dosed with 1-125 mg/kg/day HFPO dimer acid from
GDI6 through GD20. However, pup survival was significantly reduced on PND1 and PND2 at
doses >62.5 mg/kg/day in pups born to dams dosed from GD8 to PND2. Specifically, pup
survival percentages on PND2 were 100 ± 0, 96 ± 2, 97 ± 2, 87 ± 5, 38 ± 13, and 5 ± 5% in the
0-250 mg/kg/day groups, respectively.
Changes in maternal GWG were a consistently observed effect. In pregnant rats dosed during
gestation and through PND2, maternal GWG significantly decreased 25%>-70%> compared to
control at doses greater than or equal to 125 mg/kg/day of HFPO dimer acid ammonium salt
(DuPont-18405-841, 2010; Conley et al., 2019, 2021). Conversely, pregnant mice dosed during
gestation saw increases in maternal GWG ranging from 7% to 22% at doses as low as 0.5
mg/kg/day (DuPont-18405-1037, 2010; Blake et al, 2020). It is unclear why this response is
different for mice and rats, but in Blake et al. (2020), the study authors hypothesize that it could
be the result of differences in the exposure window or interspecies toxicokinetic differences in
elimination rates. Specifically, the available rat studies dosed from GD6 through GD20 (DuPont-
18405-841, 2010), GD14-GD18 (Conley et al., 2019), GD16-GD20 (Conley et al., 2021), or
GD8-PND2 (Conley et al., 2021), while the mice were dosed earlier in gestation (GDI.5-
GD17.5) in Blake et al. (2020) and 14 days prior to mating through LD21 in DuPont-18405-
1037. Additionally, the elimination T1/2 in urine (see section 2.3.5) for female mice (18 hours) is
much longer than for female rats (8 hours) and there are also differences in the alpha and beta
phase T1/2 for female rats and mice (see Table 8).
Blake et al. (2020) presented data indicating that the placenta might be a target of GenX
chemical exposure. Placental lesions were detected in 58% and 83% of mouse placentas
evaluated after dosing with 2 and 10 mg/kg/day of HFPO dimer acid from El. 5 to El 7.5,
respectively, compared to 2% in the control group (Blake et al., 2020). The most frequent lesion
detected was labyrinth atrophy, which was observed in 0/41 (0%), 15/31 (48%), and 16/35 (46%)
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placentas in 0-, 2-, and 10-mg/kg/day-dose groups, respectively. Labyrinth congestion and early
fibrin clots increased with increasing HFPO dimer acid doses. These placental lesions are
indicative of a placental insufficiency phenotype (Blake et al., 2020). Additionally, placental
weights increased in the 10-mg/kg/day-dose group and large placentas are associated with
adverse health outcomes in neonates and adult offspring (Hutcheon et al., 2012; Risnes et al.,
2009). It is unclear how these effects might impact reproductive and developmental outcomes.
In some of the available developmental studies, there was also a decrease in rodent pup weight
that ranged from 9% to 24% when the pups were exposed to 5 mg/kg/day-1,000 mg/kg/day in
utero (DuPont-18405-841, 2010; DuPont-18405-1037, 2010; Conley et al., 2019, 2021). The
mouse pups showed delays in attaining balanopreputial separation and vaginal patency at 5
mg/kg/day of 2.6 days and 3.4 days, respectively, which could be related to the observed effects
on BW during the preweaning period (DuPont-18405-1037, 2010). Additionally, the attainment
of vaginal patency did not exhibit a dose-response relationship. The decrease in pup weight was
associated with a decrease in gravid uterine weight by 10% and 25% at 100 mg/kg/day and
1,000 mg/kg/day, respectively, in the rat prenatal developmental toxicity study (DuPont-18405-
841, 2010). Moreover, in a rat prenatal developmental study, a 14th rudimentary rib developed in
9% of the control fetuses, 10% of fetuses in the 10-mg/kg/day dose, 12% of fetuses in the 100-
mg/kg/day dose, and 27% of the fetuses in the 1,000-mg/kg/day dose (DuPont-18405-841,
2010). Statistical analyses were not completed on the development of the 14th rudimentary rib in
individual fetuses, but a statistically significant increase in the number of litters developing a
14th rudimentary rib was observed at the high dose. Conley et al. (2019) reported significant
effects for the Fi generation in their postnatal pilot study where Fo pregnant rats were dosed with
125 mg/kg/day of HFPO dimer acid ammonium salt from GD14 through GD18. Fi male pups
had a decrease in right epididymis weight on a litter mean basis compared to control. Multiple
significant effects were observed on an individual pup basis, including AGD and liver weight
decreases in female Fi offspring and paired testes, paired epididymides, right testis, right
corpus/caput, right epididymis, left testis, and epididymal adipose tissue decreases in Fi male
mice. Similarly, Fi male mice in the 5 mg/kg/day-dose group exhibited a decrease of 12% in the
relative epididymis weight in a reproduction/developmental toxicity mouse study (DuPont-
18405-1037, 2010).
Changes in thyroid hormones, which are important for neurodevelopment, were reported in
Conley et al. (2019), Conley et al. (2021) and Blake et al. (2020). In pregnant rats (n=3) dosed
with 0-500 mg/kg/day of HFPO dimer acid ammonium salt from GD14 through GD18 (Conley
et al., 2019), maternal serum total T3 levels were decreased at greater than or equal to 30
mg/kg/day and total T4 levels at greater than or equal to 125 mg/kg/day. The decreases in
maternal serum total T4 levels compared to control were -50%, -63%, and -76% in the 125-,
250-, and 500-mg/kg/day-dose groups, respectively. The decreases in maternal serum total T3
levels compared to control were -21%, -39%, and -48% in the 30-, 62.5-, and 125-mg/kg/day-
dose groups, respectively. Maternal total T3 levels in the 250 and 500 mg/kg/day-dose groups
were below the detection limit. Similar findings were reported in Conley et al. (2021) for
pregnant rats (n=4-5) dosed with 0-250 mg/kg/day of HFPO dimer acid ammonium salt from
GDI6 through GD20 and GD8 through PND2. Notably, significant decreases in maternal total
T4 (-35%) for GD16-GD20 and -51% for GD8-PND2) were also observed in the 62.5 mg/kg/day
dose groups in Conley et al. (2021). In Blake et al. (2020), placental thyroid hormones (rT3, T3,
and T4) were quantified at GDI7.5 from 2-3 pooled placental tissues of same-sex embryos. A
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significant increase (60%) in T4 relative to control was reported for the 10-mg/kg/day HFPO
dimer acid-dose group.
5.5 Immune System
In the one available study specifically addressing immunotoxicity, suppression of TDARs was
measured through IgM antibody production in mice (Rushing et al., 2017). IgM antibody
production was decreased by 7.3% in female C57BL/6 mice treated with 100 mg/kg/day of
HFPO dimer acid. In male mice treated with the same dose of HFPO dimer acid, significant
increases in the number of T lymphocytes were observed, but no suppression of TDARs.
In two studies of less-than-chronic duration (28-90 days), decreases in spleen weight relative to
BW were observed in female mice and rats (DuPont-18405-1307, 2010; Rushing et al., 2017).
For example, in C57BL/6 mice, relative spleen weights significantly decreased by 11% in
females treated with 100 mg/kg/day of HFPO dimer acid for 28 days (Rushing et al., 2017).
Changes in early markers of potential immunotoxic effects were observed in multiple studies
examining the oral toxicity of HFPO dimer acid and/or ammonium salt. The most prevalent
indications were statistically significant decreases from control in serum globulin levels (6%-
22%), which resulted in an increase in the serum A/G ratio (7%-58%) from the controls when
both sexes of Crl:CD-l mice and Crl:CD(SD) rats were treated with 1 mg/kg/day-500
mg/kg/day of HFPO dimer acid ammonium salt for 12 months or less (DuPont-17751-1026,
2009; DuPont-18405-1238, 2013; DuPont-18405-1307, 2010; DuPont-24447, 2008; DuPont-
24459, 2008). Alterations in the serum levels of globulin can be associated with decreases in
antibody production (FDA, 2002). To determine the biological significance of the apparent
decrease in globulin production, however, immune function tests (such as TDAR) need to be
conducted. Finally, female Crl:CD-l mice exhibited a 21% and 18% decrease in spleen weight
relative to BW when administered 0.5 mg/kg/day and 5 mg/kg/day of HFPO dimer acid
ammonium salt for 90 days, respectively (DuPont-18405-1307, 2010). For HFPO dimer acid
and/or ammonium salt, there were also two local lymph node assays (LLNAs) conducted in mice
that showed equivocal results (DuPont-19897, 2006; DuPont-22616 RV1, 2007).
In summation, the results of the Rushing et al. (2017) TDAR assay in combination with the
supportive findings of decreased globulin levels and spleen weight provide evidence that GenX
chemicals can induce immune suppression in female mice.
5.6 Cancer
The single cancer bioassay for HFPO dimer acid ammonium salt showed increased incidence of
liver tumors (females) and combined pancreatic acinar adenomas and carcinomas (males) in rats
at the high doses only. Additionally, a statistically insignificant increase in the incidence of
testicular interstitial cell adenoma was noted at the high dose. Although that result was not
statistically significant compared to controls, the authors of the study conclude that "a
relationship to treatment for these findings in the 50 mg/kg/day group cannot be ruled out," while
also suggesting that Ley dig cell tumor induction in rodents might have low relevance to humans
(Caverly Rae et al., 2015). Given these uncertainties and the large number of early deaths in the
study (see section 4.4), the existing evidence from this single chronic study is considered
inadequate to justify a quantitative assessment. Further, the available data for HFPO dimer acid
ammonium salt suggest that mice might be more sensitive to exposure to GenX chemicals than
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rats. The available study (DuPont-18405-1238, 2013) only evaluated rats; there are no studies
measuring cancer endpoints in mice. Given the evidence that the liver is the target organ for
toxicity and primary organ for tumor development, the lack of data evaluating cancer in mice is a
database deficiency. Thus, under EPA's Guidelines for Carcinogen Risk Assessment (EPA,
2005a), there is Suggestive Evidence of Carcinogenic Potential of oral exposure to GenX
chemicals in humans, based on the female hepatocellular adenomas and hepatocellular
carcinomas and male combined pancreatic acinar adenomas and carcinomas. No data are
available to evaluate cancer risk via dermal or inhalation exposure.
6.0 Mode of Action
The available data indicate that multiple MO As could be involved in the liver effects observed
after GenX chemical exposure. The available studies provide support for a role for PPARa,
cytotoxicity, mitochondrial dysfunction, and PPARy. The potential MOA(s) for the observed
reproductive and developmental effects (e.g., changes in GWG and placental lesions) are
unknown. Additionally, no data support identification of a potential carcinogenic MOA for
tumors in the pancreas and testes as being related to any of the proposed MO As for the tumor
development in either organ.
For some PFAS (e.g., PFOA), PPARa activation has been proposed as a potential MOA for
some of the effects in the liver (i.e., liver tumors) (Klaunig et al., 2003, 2012; Maloney and
Waxman, 1999). PPARa is primarily expressed in the liver, but also is present in the kidney,
intestines, heart, and brown adipose tissue (Hall et al., 2012). Klaunig et al. (2003) describes the
causal key events of the PPARa MOA for liver tumors as activation of PPARa, perturbation of
cell proliferation and apoptosis, and selective clonal expansion. There are multiple effects
associated with the PPARa MOA such as hepatocellular hypertrophy, peroxisome proliferation,
expression of peroxisomal genes, Kupffer cell-mediated events, and increased liver weight.
However, these associative effects might not be specific to the PPARa MOA (e.g., hepatocellular
hypertrophy) or might not be causal to the development of liver tumors (e.g., peroxisome
proliferation) (Klaunig et al., 2003). According to Klaunig et al. (2003), demonstration of
PPARa agonism combined with microscopic evidence for peroxisome proliferation or increases
in liver weight and one or more of the specific in vivo markers of peroxisome proliferation (e.g.,
induction of acyl-CoA oxidase or cytochrome P450 4A) are sufficient to establish a PPARa
MOA.
For HFPO dimer acid and/or ammonium salt, there are data that demonstrate peroxisome
proliferation in the liver. Activation of PPARa was measured in multiple 28-day studies in
rodents (DuPont-24447, 2008; DuPont-24459, 2008; Rushing et al., 2017; Wang et al., 2017).
Using acyl-CoA oxidase activity as a measure, Rushing et al. (2017) showed increased activity
compared to control in male C57BL/6 mice administered 10 mg/kg/day and 100 mg/kg/day of
HFPO dimer acid (122% and 222%, respectively) and a 100% increase compared to control in
C57BL/6 female mice at 100 mg/kg/day. Notably, there were no significant increases in acyl-
CoA oxidase activity at 1 mg/kg/day, indicating that it might be a high dose effect.
The DuPont studies used P-oxidation activity and total cytochrome P450 content as markers of
peroxisome proliferation in the livers of rats and mice (DuPont-24447, 2008; DuPont-24459,
2008). In Crl:CD-l male mice, P-oxidation activity significantly increased compared to control at
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doses of 0.1 mg/kg/day, 3 mg/kg/day, and 30 mg/kg/day of HFPO dimer acid ammonium salt by
57%, 744%, and 648%), respectively, and total cytochrome P450 content significantly decreased
at 3 mg/kg/day and 30 mg/kg/day by 26% and 53%, respectively (DuPont-24459, 2008). P-
oxidation activity significantly increased compared to control in female Crl:CD-l mice at 3
mg/kg/day and 30 mg/kg/day by 495%) and 823%), respectively, with no alterations in total
cytochrome P450 content (DuPont-24459, 2008). In male Crl:CD(SD) rats, P-oxidation activity
was significantly increased relative to control at dosages of 0.3 mg/kg/day, 3 mg/kg/day, and 30
mg/kg/day by 42%, 274%), and 772%), respectively, and total cytochrome P450 content was
significantly increased by 23% at 30 mg/kg/day (DuPont-24447, 2008). In female rats dosed
with 30 mg/kg/day and 300 mg/kg/day, P-oxidation activity was significantly increased
compared to control to 49% and 198%, respectively, while total cytochrome P450 content
remained unaltered (DuPont-24447, 2008).
Induction of genes associated with peroxisome proliferation in the liver was also demonstrated
(Wang et al., 2017; Conley et al., 2019). Wang et al. (2017) demonstrates significant increases in
hepatic mRNA levels of many PPAR targets (e.g., CD36 antigen, acyl-CoA oxidase 1, and
cytochrome P450 family members) after administration of 1 mg/kg/day of HFPO dimer acid
ammonium salt for 28 days. Relatedly, Conley et al. (2019) found upregulation of gene
expression associated with PPARa signaling in maternal and fetal livers following in vivo
exposure during GD14-GD18.
Additionally, significant increases in liver weight relative to BW were observed in male and
female Crl:CD(SD) rats and several strains of male and female mice treated with 0.5 mg/kg/day-
1,000 mg/kg/day of HFPO dimer acid ammonium salt for 28-90 days (DuPont-17751-1026,
2009; DuPont-18405-1037, 2010; DuPont-18405-1307, 2010; DuPont-24447, 2008; DuPont-
24459, 2008; Rushing et al., 2017; Wang et al., 2017; NTP, 2019). Increases in liver weight were
also reported in the pregnant dams in the available reproductive/developmental studies dosing
during gestation (Blake et al., 2020; Conley et al., 2019; DuPont-18405-841, 2010; DuPont
18405-1037, 2010). Additionally, hepatocellular hypertrophy was observed at 0.5 mg/kg/day in
both sexes of mice, while male and female rats showed these effects at 3 mg/kg/day and 30
mg/kg/day, respectively, in subchronic studies. Interestingly, in the chronic study, male rats
showed only a 10%) incidence of hepatocellular hypertrophy with dosing at 50 mg/kg/day for 2
years (DuPont-18405-1238, 2013). Conversely, female rats had significant hepatocellular
hypertrophy at 500 mg/kg/day after 1 year (100%>) and 2 years (93%>).
There is evidence of perturbations to cell proliferation and apoptosis in the liver following short-
term and subchronic exposure to HFPO dimer acid ammonium salt, particularly in the high-dose
groups. In the 28-day mouse study, increased mitosis was observed in male (9/10) and female
(5/10) mice in the high-dose groups only (30 mg/kg/day) and apoptosis was not reported
(DuPont-24459, 2008). In the 90-day mouse study, increases in mitotic figures and apoptosis
were reported in 7/10 and 10/10 male mice in the high-dose (5 mg/kg/day) group, respectively
(NTP, 2019). No mitotic figures were detected in female mice, but an increase in apoptosis was
observed in 3/9 mice (NTP, 2019). In the reproductive/developmental mouse study, mitotic
figures were observed in 17/24 males and 2/24 females in the 5-mg/kg/day-dose group, but in no
other dose groups (NTP, 2019). Similarly, apoptosis was reported in 21/24 males and 10/24
females in the 5-mg/kg/day high-dose group (NTP, 2019). Notably, decreases in the rates of
apoptosis are typically observed with PPARa agonists, with Klaunig et al. (2003) describing
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decreased rates of apoptosis as a "hallmark of liver growth seen in the early stages of treatment
with PPARa agonists." Interestingly, increases in mitoses/mitotic figures and apoptosis are
consistently restricted to the high-dose group in all available mouse studies; however, necrosis is
observed in both the mid- and high-dose groups. These data suggest that PPARa's role in the
observed liver effects may be dose dependent. In the 28-day rat study, mitosis/mitotic figures,
hyperplasia, and apoptosis were not reported (DuPont-24447, 2008). In the 90-day rat study,
mitosis/mitotic figures, hyperplasia, and apoptosis were not reported (DuPont-17751-1026,
2009). In the chronic rat study, mitotic figures and apoptosis were not reported, and hyperplasia
was no different than control in the male and female rats in any dose group (DuPont-18405-
1238, 2013). It is possible that the rat studies might not have separated apoptotic lesions from
other liver lesions reported (i.e., single-cell necrosis) since these studies were conducted prior to
the guidelines outlined in Elmore et al. (2016) and were not reanalyzed by the NTP PWG.
Although there is evidence for a PPARa MOA in the liver, particularly in the high-dose groups
in the available studies, data indicate that liver toxicity extends beyond a single PPARa-based
MOA. For example, liver necrosis was consistently observed in rodent toxicity studies with
HFPO dimer acid ammonium salt and was reaffirmed by the NTP PWG's review of the 90-day
subchronic study in mice and the reproductive and developmental toxicity study in mice
(appendix D), which suggests that cytotoxicity is also a possible MOA. Felter et al. (2018)
identified the following key events for establishing a cytotoxicity MOA:
1.) The chemical is not DNA reactive.
2.) Clear evidence of cytotoxicity by histopathology such as the presence of necrosis and/or
increased apoptosis.
3.) Evidence of toxicity by increased serum enzymes that are relevant to humans.
4.) Presence of increased cell proliferation as evidenced by increased labeling index and/or
increased number of hepatocytes.
5.) Demonstration of a parallel dose response for cytotoxicity and formation of tumors.
6.) Reversibility (ideally).
The available data for HFPO dimer acid support cytotoxicity as a potential MOA. For example,
HFPO dimer acid does not appear to be DNA reactive in vivo (see section 4.6.3 and appendix C).
It did not induce chromosomal mutations in the form of structural aberrations, numerical
aberrations, or micronuclei or DNA effects in the form of unscheduled DNA synthesis (DuPont-
23219, 2007; DuPont-23220, 2007). Secondly, clear evidence of cytotoxicity in the form of
increased liver necrosis and apoptosis was confirmed microscopically in male and female mice
and rats in several less-than-chronic studies (15-90 day) and one 2-year chronic study (DuPont-
17751-1026, 2009; DuPont-18405-841, 2010; DuPont-18405-1037, 2010; DuPont-18405-1238,
2013; DuPont-18405-1307, 2010; DuPont-24447, 2008; DuPont-24459, 2008; Wang et al., 2017;
NTP, 2019). There is also evidence of increased serum liver enzymes. Hall et al. (2012)
identifies significant increases in ALT/AST, ALP, and bilirubin/bile acids as potentially
clinically relevant. Additionally, other enzymes such as SDH might reflect alterations in liver
function (Hall et al., 2012). For HFPO dimer acid, significant increases in ALT (420%-l,254%),
AST (106%-478%), ALP (1,134%-1,221%), and SDH (1,134%-1,221%) were observed in male
mice administered the ammonium salt at 5-30 mg/kg/day for 28-90 days. Female mice had
smaller increases in ALP (140%-143%) and SDH (32%-186%) as compared to male mice
administered the same dose over the same duration. Overall, rats exhibited far fewer and smaller
increases in serum liver enzyme levels following subchronic exposure compared to the mouse,
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with increases in AST (106%) and ALP (52%) at 100 mg/kg/day in male rats and AST (66%) in
female rats at 1,000 mg/kg/day. In the chronic study, however, ALT (228%), ALP (180%), and
SDH (140%>) significantly increased in male rats only when administered 50 mg/kg/day for 1
year. Typically, an increase in bilirubin, when accompanied with increased bile acids, is a
reliable index of liver toxicity (Hall et al., 2012). For HFPO dimer acid, however, a decrease in
serum bilirubin is a consistent effect observed across multiple studies, especially in female
rodents (DuPont-17751-1026, 2009; DuPont-18405-1238, 2013; DuPont-18405-1307, 2010;
Wang etal., 2017).
Data gaps exist for the other key events related to a cytotoxic MOA. Studies investigating if
exposure to HFPO dimer acid result in increased labeling index and/or increased number of
hepatocytes are unavailable. A 2-year chronic study in rats reported centrilobular and single cell
necrosis in females in the 500-mg/kg/day high-dose group only (DuPont-18405-1238, 2013).
Additionally, treatment-related liver tumors were also observed in the 500-mg/kg/day dose group
(0/70 in control versus 11/70 in 500 mg/kg/day), which suggests a parallel response for
cytotoxicity and formation of tumors. However, these effects were observed only in the high-
dose group and dose selection in this study resulted in a large gap between the mid-dose (50
mg/kg/day) and high-dose (500 mg/kg/day). Therefore, the potential for a parallel dose response
is unclear. Additionally, while liver necrosis exhibits a dose response in the 84/85 day modified
reproductive developmental study (DuPont-18405-1037, 2010; NTP, 2019), there are no chronic
studies in the mouse to determine if liver tumors form. The available data indicate that the mouse
is the more sensitive to the liver effects resulting from HFPO dimer acid exposure.
Additionally, Blake et al. (2020) reports an increase in subcellular organelles consistent with
peroxisomes and mitochondria in pregnant dam livers exposed to 2 or 10 mg/kg/day of HFPO
dimer acid from El.5 to El 1.5 or E17.5 using TEM. This increase in mitochondria is not typical
of PPARa activation and suggests an alternate MOA such as mitochondrial alteration could also
be operative for the liver effects resulting from exposure to HFPO dimer acid and/or ammonium
salt. Further supporting this alternate MOA, a number of genes upregulated in maternal and fetal
livers exposed to 1-500 mg/kg/day of HFPO dimer acid ammonium salt from GD14 to GD18 are
specific to mitochondrial beta oxidation (iCptla, Cptlb, Cpt2, Acaa2, Acadl, Acadm),
mitochondrial ketogenesis (Hmgcs2), and mitochondrial electron transfer (Etfdh) (Conley et al.,
2019).
Finally, a study of HFPO dimer acid in HEK293 embryonal kidney cells found activation of
genes associated with the PPARy signaling pathway (Li et al., 2019). Further supporting a role
for the PPARy signaling pathway, Conley et al. (2019) reports upregulation of genes in maternal
and fetal livers exposed to 1-500 mg/kg/day of HFPO dimer acid ammonium salt from GD14 to
GDI8, which are associated with PPARy signaling, including Pckl, Aqp7, and Ok. Additionally,
Rosen et al. (2017) concluded that 11 %>-24%> of the PF AS-induced increase in transcriptional
activity is PPARa independent, depending on the PFAS. This study identified 67 genes that were
similarly upregulated in wild type (129Sl/SvlmJ) and PPARa-null (129S4/SvJa.e-!)paratm 1 (,"n7J)
mouse livers exposed to either 3 or 10 mg/kg/day of PFHxS, 1 or 3 mg/kg/day of
perfluorononanoic acid, 3 mg/kg/day of PFOA, or 10 mg/kg/day PFOS for 7 days, indicative of
PPARa independence. The authors note that genes typically associated with the activation of
PPARa such as Acoxl were similarly upregulated in wild type and PPARa-null mice livers,
suggesting that these genes might not be specific indicators of PPARa activation. Interestingly,
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Conley et al. (2019) found that five of the 67 genes identified as PPARa independent in the
Rosen et al. (2017) study are also significantly upregulated in the liver of pregnant rats and their
fetuses exposed to HFPO dimer acid at doses greater than or equal to 1 mg/kg/day (i.e., Ehhadh,
Slc22a5, Echl, Cpl2, and Acoxl). Slc22a5 and Cpt2 are associated with mitochondrial fatty acid
oxidation.
Taken together, the available data indicate that a PPARa MOA is plausible in the liver in
response to GenX chemical exposure, especially at doses greater than 0.5 mg/kg/day; however,
there are not yet enough data to conclude that PPARa activation is the sole mechanism
underlying the liver effects associated with exposure to GenX chemicals. For example, there are
no studies investigating GenX chemical exposure in PPARa-null mice. It is worth noting that
exposure to PFOA has been demonstrated to induce liver effects in PPARa-null mice, including
hepatocellular hypertrophy (Minata et al., 2010). Additionally, available studies indicate that
other MO As (e.g., PPARy, mitochondrial dysfunction, and cytotoxicity) are also plausible. The
data are not adequate to conclude that any of the MO As described here are the sole toxicologic
MOA for HFPO dimer acid and/or ammonium salt in the liver and especially in other organ
systems. For example, the potential MOA(s) for the observed reproductive and developmental
effects (e.g., changes in GWG, placental lesions, reduced pup body weight, and reduced pup
survival) are unknown, though Conley et al. (2021) provides mechanistic evidence that
dysregulation of carbohydrate and lipid metabolism in the mother and developing offspring may
be contributing to some of these effects. Of note, glycogen accumulation scores in pup livers
were significantly lower compared to control in pups exposed to doses as low as 10 mg/kg/day of
HFPO dimer acid ammonium salt from GD8-PND2. Additionally, no data support identification
of a potential carcinogenic MOA for tumors in the pancreas or testes as being related to PPARa
or any of the proposed alternative MO As for the tumor development in either organ.
7.0 Dose-Response Assessment
7.1 Identification of Studies and Effects for Dose-Response Analysis
Several studies were evaluated further for identification of specific endpoints to carry forward
for dose-response (BMD) modeling. EPA evaluated studies based on identification of adverse
effects, duration of exposure, use of a control and two or more doses, and provision of NOAEL
and/or LOAEL values. Data from available studies indicate that the liver is the most sensitive
target of toxicity from exposure to GenX chemicals. Liver effects were observed in both male
and female mice and rats at varying durations of exposures and doses. These effects occurred at
the lowest doses and shortest durations of exposure to GenX chemicals.
Because liver effects such as increases in liver weight and hepatocellular hypertrophy (also
referenced here as cytoplasmic alteration per NTP PWG's review) can be associated with
activation of cellular PPARa receptors, EPA evaluated observed liver effects resulting from
HFPO dimer acid ammonium salt exposure against the Hall criteria (Hall et al., 2012). These
criteria indicate that increased liver weight and hepatocellular hypertrophy must be accompanied
by histologic or clinical pathology indicative of liver toxicity to be considered adverse.
Histologic or clinical pathology indicative of liver toxicity can include changes in liver enzyme
concentrations in the serum, necrosis, inflammation, and degeneration. With these criteria in
mind, EPA concluded that some of the observed liver effects such as single-cell and focal
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necrosis, increased apoptosis, and increases in serum liver enzymes indicate toxicity of relevance
to humans as opposed to PPARa-induced cell proliferation unique to rodents.
For the GenX chemicals database, many studies identified the mouse as the most sensitive
species and the liver as a target organ for toxicity. Liver effects at low doses (e.g., less than or
equal to 5 mg/kg/day) were identified in the 28 day oral (gavage) toxicity study in mice (DuPont-
24459, 2008), the 90 day oral (gavage) toxicity study in mice (DuPont-18405-1307, 2010), and
the oral (gavage) reproduction/developmental toxicity study in mice (DuPont-18405-1037,
2010). In these studies, increases in relative liver weight were accompanied by increases in
hepatocellular hypertrophy, single-cell/focal necrosis and apoptosis.
EPA requested that NIEHS, NTP convene a PWG to provide independent, expert review of the
liver tissues from the oral (gavage) reproduction/ developmental toxicity study in mice (DuPont-
18405-1037, 2010) and the 90 day oral (gavage) toxicity study in mice (DuPont-18405-1307,
2010). Given the availability of longer duration studies demonstrating effects at low doses, the
28-day study in mice was not included in this review. The NTP PWG classified cell death
according to the INHAND Organ Working Group's diagnostic criteria that describes how
pathologists can distinguish between apoptosis and single-cell necrosis in standard H&E-stained
tissue sections (Elmore et al., 2016). These criteria were unavailable at the time the DuPont
studies were conducted and submitted to EPA.
The liver effects noted in the 28 day oral (gavage) toxicity study in mice (DuPont-24459, 2008)
were not considered as a potential POD in support of the derivation of the RfD. The 28 day study
did not use a dose range optimized for the identification of low-dose effects compared to the 90
day and reproduction/developmental toxicity studies (0, 0.1, 3, and 30 mg/kg/day-dose groups in
the 28 day study versus 0, 0.1, 0.5 and 5 mg/kg/day in the 90 day and
reproduction/developmental studies). For example, in DuPont-18405-1037 (2010), the LOAEL
(i.e., the lowest dose at which an adverse effect is observed) of 0.5 mg/kg/day falls between the
low and mid-doses of the dosing design used in DuPont-24459 (2008). Additionally, as
described above, this short-term study was not reviewed by the NTP PWG because there were
two longer duration studies in the most sensitive species.
The liver effects noted in the 90 day and reproduction/developmental toxicity studies (DuPont-
18405-1307, 2010 and DuPont-18405-1037, 2010) were considered for determination of PODs
in support of the derivation of RfDs. The NTP PWG concluded, that the dose response and
constellation of lesions (i.e., cytoplasmic alteration (including hepatocellular hypertrophy),
single-cell necrosis, focal necrosis, and apoptosis), rather than each lesion individually, represent
adversity in these studies (appendix D). EPA interpreted the NTP PWG's definition that the
constellation of liver lesions is adverse to apply to the dose group level, as opposed to individual
animal level, given that the histopathology assessment represents a snapshot in time of a
biological process within one portion of the liver that can vary across animals. Therefore, if
multiple liver lesion types and progression of adverse liver effects (e.g., necrosis or apoptosis)
were observed within a dose group, all animals in that dose group were included in the dose-
response modeling. The constellation of liver lesions in the reproduction/developmental toxicity
study in mice (DuPont-18405-1037, 2010) was selected for BMD modeling based on the
incidence data as reported by the NTP PWG. Multiple liver lesions, including cytoplasmic
alteration, single-cell, and focal necrosis, exhibited a dose response in both male and female
mice in this study. These effects were observed at doses as low as 0.5 mg/kg/day. A constellation
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of liver lesions observed in the 90-day toxicity study in mice (DuPont-18405-1307, 2010) were
observed at higher doses (5 mg/kg/day) than in the reproduction/developmental toxicity study in
mice (DuPont-18405-1037, 2010). The NTP PWG reported that 10 out of 10 male mice
exhibited cytoplasmic alteration, compared to 0 in control at the 0.5-mg/kg/day dose in the 90-
day toxicity study in mice (DuPont-18405-1307, 2010). Although NTP classified cytoplasmic
alteration as part of the constellation of liver lesions considered adverse, no other liver lesions
indicative of liver damage (i.e., single-cell or focal necrosis or apoptosis) were observed at the
0.5-mg/kg/day dose group in males. Consistent with the Hall criteria, EPA did not consider the
cytoplasmic alteration findings alone as an adverse effect in the 0.5 mg/kg/day dose group but
considered the constellation of liver lesions observed across the male mice in the high-dose
group as adverse. Additionally, the female mice in this study did not exhibit a dose response for
the constellation of liver lesions. For these reasons, the constellation of liver lesions observed in
the 90-day toxicity study in mice were not selected for BMD modeling.
Additionally, the chronic rat 2-year cancer bioassay (DuPont-18405-1238, 2013) was not
selected for the derivation of candidate RfDs for several reasons. Across all dosing groups in
both male and female rats, just 25.4% of the test animals survived to their planned terminal
necropsy with most of the animals experiencing unscheduled death/moribundity prior to the
scheduled study termination at 104 weeks. Effects observed at low doses in this study include
changes in serum albumin levels and the A/G ratio in male rats. For males, an increase in A/G
ratio at 1 mg/kg/day at the 3-month time point and increases in both albumin and A/G ratio at the
12-month time point were observed, but these changes were not seen at 6 months. These
changes, while indicative of an immune system effect, were deemed of unclear biological
significance especially given these temporal inconsistencies. For these reasons, the changes in
albumin and A/G ratio observed in DuPont-18405-1238 (2013) were not considered for
determination of PODs in support of the derivation of the RfD. Liver effects were also observed
in this study but did not occur at comparable doses to the oral reproductive/developmental
toxicity study in mice. Also, the available chronic study evaluated only rats, and the data indicate
that mice appear to be more sensitive. For example, mice presented with single-cell necrosis in
doses as low as 0.5 mg/kg/day, with a large increase in response at 5 mg/kg/day in the oral
reproductive/developmental toxicity study in mice (DuPont-18405-1037, 2010; NTP, 2019).
Female mice also had a large increase in incidence compared to control at 5 mg/kg/day for both
focal/multifocal and single-cell necrosis (DuPont-18405-1037, 2010; NTP, 2019). Conversely,
the study authors did not report subchronic hepatocellular necrosis in the 90-day study of male
and female rats. (DuPont-17751-1026, 2009). Hepatocellular necrosis is observed in the 2-year
chronic rat study, but at higher doses (50 mg/kg/day for male rats and 500 mg/kg/day for female
rats) as compared to the developmental/reproductive mouse study (0.5 mg/kg/day for male and
female mice) (DuPont-18405-1238, 2013; NTP, 2019 reread of DuPont-18405-1037, 2010).
While a chronic study is typically the preferred duration for development of lifetime RfD, in this
case, the oral reproductive/developmental toxicity study indicates that adverse effects in the liver
are observed in the parental mice at lower doses than those reported in the chronic study in rats.
For these reasons, the adverse liver effects observed in DuPont-18405-1238 (2013) were not
selected for determination of PODs in support of the derivation of the RfD.
Adverse health outcomes resulting from exposure from HFPO dimer acid or its ammonium salt
are not limited to the liver. Studies in both rats and mice indicate that exposure to GenX
chemicals during pregnancy and gestation results in adverse effects at low doses. Specifically,
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Blake et al. (2020) determined that 58% and 83% of placentas evaluated at E17.5 were classified
as abnormal in the 2- and 10-mg/kg/day HFPO dimer acid dose groups, respectively, with the
number of abnormal placentas in the 10-mg/kg/day HFPO dimer acid dose group reaching
statistical significance. Different placental lesions were recorded in the study, including labyrinth
atrophy, labyrinth congestion, labyrinth necrosis, early fibrin clot, and the presence of placental
nodules. Placental lesions were also evaluated against the proportion of placentas within a litter
that were within normal limits to account for litter effects. The proportion of abnormal placentas
was significantly higher at the 2- and 10-mg/kg/day dose groups relative to vehicle control. The
placental lesions observed in Blake et al. (2020) exhibited a dose response; however, only two
dose groups were used in this study, and the study LOAEL (2 mg/kg/day) is much higher than
the LOAELs observed for liver effects (0.5 mg/kg/day). It is possible that the placental lesions
occur at lower doses, especially given that 58% of placentas were classified as abnormal at the
lowest dose tested, but these data are lacking. While the placental lesions observed are
considered adverse, additional research is needed to understand if they would be seen at lower
doses. Additionally, further research should evaluate the impact of GenX chemicals-induced
placental lesions on development after gestation, including latent health outcomes. Blake et al.
(2020) reported that these lesions did not impact some measured reproductive and developmental
outcomes such as implantation sites, viable embryos, nonviable embryos, and resorptions.
Because, however, a full two-generation reproductive toxicity study is not available for mice, the
impact of placental lesions on development after gestation, including latent health outcomes, is
unclear.
An increase in maternal GWG ranging from 13 to 22% was reported by DuPont-18405-1037
(2010) at doses as low as 0.5 mg/kg/day. Similarly, an increase in maternal GWG in mice at
E17.5 at doses greater than or equal to 2 mg/kg/day (i.e., the lowest tested dose) was also
reported by Blake et al. (2020) using a mixed-effect modeling approach that adjusts for repeated
measures of relative maternal GWG, litter size, and embryonic day. Furthermore, Conley et al.
(2019) evaluated maternal GWG in rats and observed a decrease in GWG following exposure to
dosing greater or less than 250 mg/kg/day of HFPO dimer acid. A decrease in maternal GWG in
rats was also reported in DuPont-18405-841 (2010), which suggests that the shift in maternal
GWG might be species specific. Given the lack of mechanistic clarity for the maternal GWG
endpoints in two similar species, the endpoint was not considered for determination of PODs in
support of the RfD derivation. According to Blake et al. (2020), the inconsistency in maternal
GWG response between rats (Conley et al., 2019; DuPont-18405-841, 2010) and mice (Blake et
al., 2020; DuPont-18405-1037, 2010) might be due to differing statistical methods, interspecies
elimination rates, and/or developmental exposure windows. All other reproductive and
developmental effects reported as a result of gestational exposure to GenX chemicals (see Table
12 for a summary) were observed at higher doses than the placental lesions and changes in GWG
and were not selected for determination of PODs in support of the RfD derivation.
Immune and hematological effects were also observed at low doses; however, these endpoints
are not as consistently observed as the liver effects. Additionally, there is some uncertainty
regarding the biological significance of both the hematological and immune endpoints. For
example, the observed changes in albumin and A/G ratio at dosing of 3 mg/kg/day (DuPont-
24447, 2008; DuPont-24459, 2008) are considered early markers of potential immunotoxic
effects. Evaluation of additional immune function assays, histopathology, and immune endpoints
such as antibody levels, however, are not available. Currently little or no data exist on the
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potential for GenX chemicals to impact aspects of immune function beyond the
immunosuppression (e.g., allergic responses and autoimmunity). Furthermore, while considered
adverse, the hematological effects were inconsistently observed, especially as study duration
increased. For example, the hematological effects observed in the 28-day mouse study at
3 mg/kg/day were not observed in the 90-day subchronic study in mice, except for a 3% decrease
in hemoglobin concentration at 5 mg/kg/day. No hematological changes were observed at the
0.1- or 0.5-mg/kg/day dose in the subchronic mouse study (DuPont-18405-1307, 2010).
Likewise, the hematological effects observed in the subchronic rat study at low doses are not
observed in the chronic rat study (DuPont-17751-1026, 2009; DuPont-18405-1238, 2013).
Specifically, decreases in hemoglobin, hematocrit, and RBC count that are observed at 10
mg/kg/day in the subchronic study are not observed after 12 months of dosing, which adds
additional uncertainty to the significance of these effects (DuPont-18405-1238, 2013). For these
reasons, hematological and immune endpoints from these studies were not considered further for
determination of PODs in support of the derivation of the RfD.
7.2 Methods of Analysis
7.2.1 BMD Modeling
There are no biologically based dose-response (BBDR) models available for HFPO dimer acid
and its ammonium salt. Thus, using the most current version of its Benchmark Dose Software at
the time data were modeled, EPA evaluated a range of dose-response models thought to be
consistent with underlying biological processes to determine how best to empirically model the
dose-response relationship in the range of observed data (appendix E).
Consistent with EPA's Benchmark Dose Technical Guidance (EPA, 2012), the BMD and the
BMDL were estimated using a BMR of 10% extra risk for dichotomous data, in the absence of
information regarding the level of change considered biologically important, and to facilitate a
consistent basis of comparison across endpoints, studies, and assessments. Using the pathology
analysis from the NTP PWG, candidate PODs were estimated from all three doses (plus control)
for DuPont-18405-1037 (2010) (Table 13).
Further details, including the BMD modeling output and graphical results for the selected
models, are provided in appendix E of this assessment.
7.2.2 Dosimetric Adjustment of the Experimental Animal-Based POD to POD hid
EPA guidance was followed to calculate a candidate PODhed from the animal-based POD using
a BW3/4 allometric scaling approach (EPA, 201 lb), which is derived from the relationship
between body surface area and basal metabolic rate in adults. With infants and children, surface
area and basal metabolic rates are very different than for adults with a slower metabolic rate.
While this BW3/4 allometric scaling is not appropriate for infants and children because of the
limited toxicokinetic data available, the critical effect of liver single-cell necrosis observed in
adult mice is not a developmental endpoint nor is it specific to early life stages. However, the
exposure for the parental females in DuPont-18405-1037 (2010) took place during pregnancy.
EPA indicates that:
.. .exposure and internal dosimetry of pregnant, nursing, and growing animals may vary
compared to adult animals, so use of the administered dose for toxicity studies involving
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these periods is associated with relatively greater uncertainty, absent life stage-specific
information (EPA, 2011b).
In this case, however, BW3/4 allometric scaling relied on life stage-specific BW data from the
pregnant or lactating dams as appropriate. The HFPO dimer acid ammonium salt PODheds from
the experimental animal studies (DuPont-18405-1037, 2010) were adjusted via the dosimetric
adjustment factor (DAF) equation below:
DAF = (BWa1/4/BWh1/4),
where:
• BWa = animal BW
• BWh = human BW
For the chronic reproductive/developmental toxicity study (DuPont-18405-1037, 2010), aBWa
value of 0.0372 kg was identified as the mean BW of the Fo male mouse controls on study day
84 (the final day of animal dosing). The mean BWa for the Fo females in this study was 0.0349
kg taken from the controls upon sacrifice on LD21.
A BWh of 80 kg for humans was selected based on National Health and Nutrition Examination
Survey (NHANES) sampling data (EPA, 201 la). For adults more than 21 years of age, EPA
updated the default BW assumption from 70 kg to 80 kg based on NHANES data from 1999 to
2006 as reported in Table 8.1 of EPA's Exposure Factors Handbook (EPA, 201 la). The updated
BW represents the mean weight for adults ages 21 and older. The resulting DAF for the
allometric scaling of doses from male mice to humans is 0.15 for DuPont-18405-1037 (2010).
For the female mice, the DAF is 0.14 for DuPont-18405-1037 (2010). Applying the DAF to the
identified PODs identified for liver effects in adult mice yields a PODhed as follows:
PODhed = POD animal dose (mg/kg/day) x DAF
Table 13. Summary of Determination of PODhed
Endpoint and
reference
Species/
Sex
Model
BMR
BMDio
(mg/kg/day)
POD
(mg/kg/day)
POD
Type
DAF
PODiiicd"
(mg/kg/day)
HEPATIC
Constellation of
liver lesions in
parental males
(DuPont-18405-
1037, 2010)b
Crl:CDl(ICR)
mice
Fo parental
male
Benchmark
dose (ver.
3.1.2)
Probit
10%
0.19
0.14
BMDLio
0.15
0.02
Constellation of
liver lesions in
parental females
(DuPont-18405-
1037, 2010)b
Crl:CDl(ICR)
mice
F0 parental
female
Benchmark
dose (ver.
3.1.2)
Probit
10%
0.12
0.09
BMDLio
0.14
0.01
Notes'. N/A = not applicable.
a Calculated using BW3/4 scaling (EPA, 201 lb).
bCalculations for DuPont 18405-1037 (2010) rely on pathology conclusions of the NTP PWG (Appendix D)
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7.3 Derivation of Candidate RfD Values
To calculate the candidate RfD values, EPA applied UFs to the PODheds from the oral
reproduction/developmental toxicity study in mice as described in this section. UFs were applied
according to guidance in EPA's Review of the Reference Dose and Reference Concentration
Processes (EPA, 2002).
An interspecies uncertainty factor (UFa) of 3 (101/2 =3.16, rounded to 3) was applied to account
for uncertainty in extrapolating from laboratory animals to humans. The UFa is generally
presumed to include both toxicokinetic (i.e., absorption, distribution, metabolism, and
elimination) and toxicodynamic (i.e., MO A) aspects. A PODhed was derived from the BMDL
using EPA's Recommended Use of Body Weight4 as the Default Method in Derivation of the
Oral Reference Dose (EPA, 201 lb). This guidance describes approaches for deriving PODheds
from data from laboratory animals, with the preferred approach being PBPK modeling. For
HFPO dimer acid and ammonium salt, no PBPK models have been developed or published.
Other approaches described by the guidance include the use of chemical-specific data to inform
the derivation of human equivalent oral exposures. In the absence of either PBPK models or
chemical-specific information, a BW scaling to the 3/4 power approach is applied to extrapolate
toxicologically equivalent doses of orally administered agents from adult laboratory animals to
adult humans. Although this scaling addresses most aspects of cross-species extrapolation of
toxicokinetic processes, there is some residual uncertainty for toxicokinetics and uncertainty
around toxicodynamic processes (EPA, 201 lb). Thus, in the absence of chemical-specific data to
quantify this uncertainty, a UF of 3 was applied.
An intraspecies uncertainty factor (UFh) of 10 is applied to account for variability in the
responses within the human populations because of both intrinsic (toxicokinetic, toxicodynamic,
genetic, life stage, and health status) and extrinsic (lifestyle) factors that can influence the
response to dose. No information to support a UFh other than 10 was available to characterize
interindividual and age-related variability in the toxicokinetics or toxicodynamics.
A LOAEL-to-NOAEL extrapolation uncertainty factor (UFl) of 1 is applied because a BMDL is
used as the basis for the PODhed derivation. When the POD type is a BMDL, the current
approach is to address this factor as one of the considerations in selecting a BMR for BMD
modeling. In this case, the BMR of a 10% change for the modeled liver endpoints was selected
under the assumption that it represents a minimal, but biologically significant, change for these
effects.
A UF for extrapolation from a subchronic to a chronic exposure duration (UFs) of 10 was
applied for the derivation of the chronic RfD, but not of the subchronic RfD. The reproduction/
developmental study (DuPont-18405-1037, 2010) considered for dose-response analysis is
shorter than the duration of a chronic study. Chronic studies typically employ repeated dosing for
longer than 90 days or for more than 10% of the human life span (EPA, 2002). In DuPont-
18405-1037 (2010), Fo females that delivered were dosed daily starting 14 days prior to pairing
and were dosed through LD20 for a total of 53 to 64 days of exposure, depending on delivery
date. By contrast, Fo males in this study were dosed 70 days prior to mating and throughout
mating through 1 day prior to scheduled termination, for a total of 84 to 85 days of exposure.
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Because a 2-year chronic mouse study is unavailable and since female mice were dosed well
below the 90-day exposure window typically employed in a subchronic study, the impact of a
longer dosing duration on both the incidence and severity of liver effects in mice is unknown.
This is important because duration of exposure appears to play a role in the progression and
severity of liver effects resulting from GenX chemical exposure, as evidenced in female rats.
Specifically, necrosis in female rats was not reported in the 28- or 90-day rat studies or the
interim 1-year time point in the 2-year chronic rat study, which dosed the rats from 3 to 1,000
mg/kg/day. However, at the completion of the 2-year chronic rat study, centrilobular and single-
cell necrosis are reported in the 500-mg/kg/day-dose group. Moreover, treatment-related liver
tumors were observed in the 500-mg/kg/day rat dose group (0/70 in control versus 11/70 in the
500-mg/kg/day group). These data demonstrate progression of liver effects over the 2-year
dosing period. Additionally, Blake et al. (2020) did not find clear evidence of changes in
maternal liver serum enzymes (i.e., ALP, ALT or AST) or increases in liver necrosis as
compared to control after 10-16 days of dosing at 2 mg/kg/day. Similarly, DuPont-24459 (2008)
did not report single cell necrosis in female mice treated with 0.1 or 3 mg/kg/day after 28 days of
dosing, though 4/10 mice displayed single cell necrosis in the 30 mg/kg/day dose group.
However, DuPont-18405-1037 (2010) found liver necrosis in mice after 53-85 days of dosing at
0.5 mg/kg/day, indicating progression of liver effects as the duration of dosing increases.
Because the mouse presents with liver necrosis at much lower doses and shorter durations (0.5
mg/kg/day at 53-85 days) than the rat and because the mode of action for these liver effects is
uncertain (see section 6), it is critical to have a 2-year chronic study in the mouse to understand
the progression of these liver effects. Specifically, a longer duration study would likely result in
an increased frequency and/or magnitude of response and could also reveal additional adverse
effects at lower doses than currently observed in the existing less-than-chronic mouse studies
(DuPont-24459, 2008; DuPont-18405-1307, 2010; DuPont-18405-1037, 2010). For these
reasons, EPA applied a UF of 10 to account for duration of exposure for the chronic RfD. For the
subchronic RfD, a UF was not applied to account for duration as the study is of subchronic
duration.
A database uncertainty factor (UFd) of 10 was applied to account for database deficiencies. The
database uncertainty factor is applied to account for a potentially lower reference value as a
result of an incomplete characterization of a chemical's toxicity (EPA, 2002). The GenX
chemicals database contains a number of toxicological studies including acute studies in both
mice and rats, subchronic studies in mice and rats, a chronic study in rats, a one generation
reproductive and developmental study in mice and gestational reproductive and developmental
toxicity studies in mice and rats, as well as a single immunotoxicity study; however, when
evaluating the available endpoints and studies to ensure comprehensive characterization of the
potential toxicity, there are important deficiencies that need to be considered, particularly for
understanding developmental toxicity. If data from the available toxicology studies raise
suspicions of developmental toxicity and signal the need for developmental data on specific
organ systems (e.g., detailed nervous system, immune system, carcinogenesis, or endocrine
system), then the database factor should take into account whether or not these data are available
and used in the assessment (EPA, 2002). For GenX chemicals, there are reproductive or
developmental effects of concern in mice occurring at similar dose levels to the liver effects
(changes in maternal GWG and placental lesions indicative of placental insufficiency) or
ongoing research related to these and other endpoints or effects that have not been studied yet
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(skeletal ossification, mammary gland development, altered metabolism in offspring, changes in
thyroid hormones in the mouse).
For example, increases in maternal gestational weight gain and placental lesions were observed
in mice at doses similar to the observed liver effects. In DuPont-18405-1037 (2010) mean
maternal GWG, calculated from individual differences, significantly increased over the
gestational period (0-18 days) by 18% and 22% in the 0.5- and 5-mg/kg/day-dose groups,
respectively. The NOAEL for this effect would be 0.1 mg/kg/day which is the same as the liver
effects. Additionally, Blake et al., 2020 found that maternal GWG was significantly increased
compared to vehicle control at 2 mg/kg/day and 10 mg/kg/day at gestational day 17.5 using
effect estimates from mixed effect models adjusting for repeated measures of relative GWG,
litter size, and embryonic day. This is a consistent effect observed in two studies conducted by
two different groups. Conley et al. (2019, 2021) also evaluated GWG in rats and observed a
decrease following exposure to dosing greater than 125 mg/kg/day of HFPO dimer acid. A
decrease in GWG in rats was also reported in DuPont-18405-841 (2010), which suggests that the
shift in GWG might be species specific. Blake et al. (2020) also suggested that differing
statistical methods, interspecies elimination rates, and exposure windows could explain these
disparate results. In humans, altered GWG has been shown to adversely impact both mothers and
infants. Effects including pregnancy-induced hypertension, gestational diabetes, postpartum
weight retention, difficulty breast feeding, increased risk of stillbirth and infant mortality, and
preterm birth have been associated with increased GWG (Rasmussen and Yaktine, 2009).
Secondly, Blake et al. (2020) reports a statistically significant increase (58%) in placental lesions
over control at 2 mg/kg/day, the lowest dose used in this study. The placenta is critical to the
transfer of nutrients, oxygen, and waste between mother and baby. Because of its role in
maintaining pregnancy and programming latent health outcomes it is a relevant endpoint to
evaluate maternal and embryo health. Placental insufficiency, as evidenced by effects such as
those observed by Blake can result in reduced transfer of vital oxygen and nutrients.
Additionally, deficiencies in placental development or function can result in hypertensive
disorders of pregnancy which increases the risk of post-pregnancy hypertension, heart disease,
and stroke in affected women, as well as increased risk for adverse cardiometabolic outcomes in
adult offspring (Pinheiro et al., 2016). EPA notes that it is unclear how the placental lesions
might impact reproductive and developmental outcomes. For example, implantation sites, viable
embryos, nonviable embryos, and resorptions were not significantly different than control in
Blake et al. (2020). Because, however, a full two-generation reproductive toxicity study is not
available for mice, the impact of placental lesions on development after gestation or latent effects
resulting from a placental insufficiency phenotype are unclear. Notably, Blake et al. (2020) also
reported placental lesions for PFOA and, studies in humans have shown associations between
PFOA exposure and health outcomes resulting from placental insufficiency such as pregnancy-
induced hypertension or preeclampsia (EPA, 2016a).
As mentioned above, other database deficiencies include the absence of a full two-generation
reproductive and developmental toxicity study to understand if latent effects occur as a result of
exposure to GenX chemicals during development (e.g., adverse cardiometabolic outcomes in
adult offspring associated with placental insufficiency). Additionally, Conley et al. (2021)
reported that survival of pups born to dams dosed from GD8-PND2 was significantly reduced on
PND1 and PND2 at doses >62.5 mg/kg/day. Pup body weight gain (birth to PND2) and PND2
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body weight in the surviving pups were both reduced at doses >30 mg/kg/day. These effects
were attributed to the hypoglycemia and elevated serum lipid levels newborns displayed,
consistent with impaired fetal glycogen storage. Metabolic disturbance during fetal development
is likely to lead to long-term negative metabolic outcomes in the offspring. These effects are
among the most sensitive effects observed in the rat resulting from exposure to GenX chemicals
and highlight the importance of having a full two-generation reproductive and developmental
toxicity study.
Additionally, the evaluation of particular developmental endpoints during early organogenesis
(i.e., GDO to GD6) such as delayed skeletal ossification and mammary gland development in the
mouse that have been observed following exposure to other PFAS like PFOA (EPA, 2016a,b) are
lacking. For PFOA, the LOAEL for mammary gland developmental effects was 0.01 mg/kg/day,
with no study NOAEL. There are no published studies looking at mammary gland development
for GenX chemicals at this time. Similarly, studies that evaluate skeletal ossification in the more
sensitive species, mice, do not exist for GenX chemicals. The LOAEL for reduced skeletal
ossification was 1 mg/kg/day for PFOA (no study NOAEL) and studies looking at lower dose
ranges were not available. These studies are especially important considering that Blake et al.
(2020) demonstrated accumulation of HFPO dimer acid in whole mouse embryos from El.5 to
El 1.5 to El.5 to E17.5. The lack of studies evaluating these endpoints at or below doses included
in the critical study identifies this as a significant gap in the understanding of the developmental
toxicity of GenX chemicals.
In addition to the gaps in the database concerning reproductive and developmental toxicity, other
database gaps are noted for GenX chemicals with respect to potential immune, hematological
and neurological effects, which are outlined below. Additionally, there are no human toxicity
data from epidemiological studies in the general population or worker cohorts evaluating the
health effects of exposure to these GenX chemicals.
The immunotoxicity of GenX chemicals has not yet been fully elucidated. PFAS chemicals,
including PFOS and PFOA, interact with the immune system in studies of both humans and
animals (NTP, 2016; EPA, 2016a,b). The GenX chemical immunotoxicity database is less robust
than PFOA and PFOS, but does include two LLNAs (DuPont-19897, 2006; DuPont-22616 RV1,
2007) and a 28-day immunotoxicity study (Rushing et al., 2017). Rushing et al. (2017) identified
suppression of TDAR by a reduction in antigen-specific IgM antibody production in females and
increased T cell numbers in males at the high dose only (100 mg/kg/day). The LLNA is typically
used to identify potential skin-sensitizing chemicals through their ability to induce allergic
immune response (OECD, 2010b). The LLNAs were conducted with HFPO dimer acid
ammonium salt preparations of varied purity and yielded equivocal results (one positive
(DuPont-19897, 2006) and one negative (DuPont-22616 RV1, 2007). Evaluations of additional
immune function assays, histopathology, and immune endpoints such as antibody levels are not
available. The combined GenX chemicals immunotoxicity dataset was found to be incomplete as
it did not include sufficient measures of immunopathology, humoral immunity, cell-mediated
immunity, nonspecific immunity, or host resistance, but the available studies are suggestive of a
potential immune hazard. Data on the potential for these GenX chemicals to impact aspects of
immune function beyond immunosuppression are lacking. Additional studies, therefore, would
be useful to support a more conclusive determination of immunotoxic potential.
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Finally, additional research is needed to help determine if the inconsistent hematological effects
observed in many of the studies are adverse and to investigate potential neural effects of GenX
chemical exposure. As mentioned above, the hematological effects observed in the 28-day mouse
study at 3 mg/kg/day were not observed in the 90-day subchronic study in mice, except for a 3%
decrease in hemoglobin concentration at 5 mg/kg/day. No hematological changes were observed
at the 0.1- or 0.5-mg/kg/day dose in the subchronic mouse study (DuPont-18405-1307, 2010).
Likewise, the hematological effects observed in the subchronic rat study at low doses are not
observed in the chronic rat study (DuPont-17751-1026, 2009; DuPont-18405-1238, 2013).
Cannon et al. (2020) demonstrated that HFPO dimer acid can modify the activity of transporters
at the blood-brain barrier. Specifically, HFPO dimer acid inhibited P-gp and BCRP transport in
rat brain capillaries. The potential neural effects that might result from inhibition of transport
activity are unknown and require additional investigation.
Furthermore, Conley et al. (2019), Conley et al. (2021) and Blake et al. (2020) observed
alterations in thyroid hormones in the pregnant dam after gestational exposure to GenX
chemicals. Specifically, Conley et al. (2019, 2021) demonstrated significant decreases in
maternal serum total T3 and T4 levels in the pregnant rat (e.g., a 51% decrease in total T4 in
pregnant dams dosed with 62.5 mg/kg/day from GD8-PND2) while Blake et al. (2020) reported a
significant increase in mouse placental total T4 levels relative to control. In the Blake et al
(2020) study, there was a trend towards a significant effect of higher T4 in placentas exposed to
2 mg/kg/day GenX (38% increase) though not statistically significant. Maternal serum thyroid
hormones could not be measured due to volume constraints in the study. The potential
neurodevelopmental effects that might result from the disruption of these thyroid hormones are
unknown and require additional investigation at lower doses.
Given the residual concerns for potentially more sensitive effects outlined above, a database
uncertainty factor is considered necessary to account for the possibility that the currently
available database for GenX chemicals may result in an under-protective point of departure.
Specifically, a value of 10 was selected for the UFd to account for the uncertainty surrounding
reproductive or developmental effects of concern occurring at similar dose levels to the liver
effects (maternal GWG, placental lesions indicative of placental insufficiency, changes in
thyroid hormones) or effects that observed to occur with exposure to other PFAS (e.g., PFOA)
but have not been studied or do not have published studies currently for GenX chemicals
(skeletal ossification, changes in thyroid hormones, mammary gland development, and altered
metabolism in the mouse).
The UFs described above were applied to the PODheds from section 7.2.2 to derive a candidate
RfDs applicable to both subchronic and chronic exposures. Table 14 summarizes the results of
this quantification for the subchronic scenario. The subchronic candidate RfDs range from
0.00003 mg/kg/day to 0.00007 mg/kg/day. Likewise, Table 15 summarizes the results of this
quantification for the chronic scenario. The chronic candidate RfDs range from 0.000003
mg/kg/day to 0.000007 mg/kg/day. Each PODhed is impacted by the doses used in the subject
study, the endpoints monitored, and the animal species/gender studied. Thus, the array of
outcomes, combined with knowledge of the individual study characteristics, helps to inform
selection of a subchronic and chronic RfDs that will be protective for humans.
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Table 14. Candidate Subchronic RfD Values
Endpoint and reference
POD hud"
(nig/kg/day)
POD Type
UFl
UFs
UFa
UFh
UFd
UFtot
Candidate RfD value
(nig/kg/day)
HEPATIC
Liver constellation of lesions in parental male mice
(DuPont-18405-1037, 2010)
0.02
BMDLio
1
1
3
10
10
300
7 x 10"5
Liver constellation of lesions in parental female
mice (DuPont-18405-1037, 2010)
0.01
BMDLio
1
1
3
10
10
300
3 x 10"5
Note:
a Calculated using BW3/4 scaling (EPA, 201 lb).
Table 15. Candidate Chronic RfD Values
Endpoint and reference
POD hud"
(nig/kg/day)
POD Type
UFl
UFs
UFa
UFh
UFd
UFtot
Candidate RfD value
(nig/kg/day)
HEPATIC
Liver constellation of lesions in parental male mice
(DuPont-18405-1037, 2010)
0.02
BMDLio
1
10
3
10
10
3000
7 x 10"6
Liver constellation of lesions in parental female
mice (DuPont-18405-1037, 2010)
0.01
BMDLio
1
10
3
10
10
3000
3 x 10"6
Note:
a Calculated using BW3/4 scaling (EPA, 201 lb).
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7.4 Selection of Overall RfD
The oral reproductive/developmental toxicity mouse study (DuPont-18405-1037, 2010) and its
pathologic demonstration of liver effects in females (constellation of lesions including
cytoplasmic alteration, hepatocellular single-cell and focal necrosis, and hepatocellular
apoptosis) were selected as the critical study and effect, respectively, for deriving the subchronic
and chronic RfDs for HFPO dimer acid and its ammonium salt. The RfD based on this grouping
of effects occurred at the lowest dose and therefore provides the most health-protective RfD
among the modeled endpoints based on the available data. The selection of the constellation of
lesions is supported by the NTP PWG conclusion that the dose response and constellation of
lesions (i.e., cytoplasmic alteration, apoptosis, single-cell necrosis, and focal necrosis) rather
than one lesion by itself, represents adversity within the confines of the study. Because there is a
negligible difference between the molecular weight of the HFPO dimer acid ammonium salt
(347.08 grams per mol (g/mol)) and the free HFPO dimer acid (330.06 g/mol), the subchronic
and chronic RfDs presented here are applicable for both chemicals.
Several of the other studies provide support for the selection of the DuPont-18405-1037 (2010)
study as the critical analysis and the constellation of liver lesions as the critical effect (DuPont-
24447, 2008; DuPont-24459, 2008; DuPont-18405-841, 2010; DuPont-18405-1307, 2010;
DuPont-18405-1238, 2013) on which to base the subchronic and chronic RfDs. The liver is the
primary target organ for toxicity from oral exposure to HFPO dimer acid and its ammonium salt.
Liver effects are observed in both male and female mice and rats at varying durations of
exposures and doses of GenX chemicals. Specifically, changes in liver enzyme levels,
histopathological lesions, and tumors are observed in both male and female mice and rats at
varying durations of exposures (15 days to 2 years) and doses of these GenX chemicals (0.5-
1,000 mg/kg/day).
7.4.1 Subchronic RfD
This section provides the calculation for the subchronic RfD. The values and rationale describing
the input parameters for the RfD calculation can be found in sections 7.2 and 7.3, and appendix
E.
Subchronic RfD = P0Dhed
Total UF
JM^day
300
= 3 x 10~5 mg/kg/day or 0.03 jug/kg/day
where:
• PODhed = 0.01 mg/kg/day, the HED based on the BMDLio for liver effects (constellation
of liver lesions as defined by the NTP PWG) in parental female mice exposed to HFPO
dimer acid ammonium salt by gavage for 53-64 days (DuPont-18405-1037, 2010).
• Total UF = 300, including 10 for UFh, 3 for UFa, and 10 for UFd.
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7.4.2 Chronic RfD
This section provides the calculation for the chronic RfD. The values and rationale describing the
input parameters for the RfD calculation can be found in sections 7.2 and 7.3, and appendix E.
Chronic RfD = ^£SS£.
Total UF
_ 0.01 ^/day
3000
= 3 x lO 6 mg/kg/day or 0.003 jig/kg/day
where:
• PODhed = 0.01 mg/kg/day, the HED based on the BMDLio for liver effects (constellation
of liver lesions as defined by the NTP PWG) in parental female mice exposed to HFPO
dimer acid ammonium salt by gavage for 53-64 days (DuPont-18405-1037, 2010).
• Total UF = 3000, including 10 for UFh, 3 for UFa, 10 for UFs, and 10 for UFd.
8.0 Effects Characterization
8.1 Uncertainty and Variability
The uncertainty and variability in an RfD are a function of both intrinsic and extrinsic factors.
EPA has identified multiple short-term subchronic and chronic studies that provide dose-
response information and were considered during the quantitative assessment of risk. The range
of external dose NOAELs among these studies is 0.1 mg/kg/day-10 mg/kg/day. The LOAELs
range from 0.5 mg/kg/day to 100 mg/kg/day.
The intrinsic uncertainties in the assessment reflect the fact that the NOAELs and LOAELs are
derived using central tendency estimates for variables such as BW, food and drinking water
intakes, and dose. The central tendency estimates are derived from small numbers of relatively
genetically similar animals representing one or more strains of rats or mice living in controlled
environments. The animals lack the heterogeneous genetic complexity, behavioral diversity, and
complex habitats experienced by humans. These differences, to some extent, have been
minimized using the modeled outcomes and use of allometric scaling to help inform the
application of the UF.
While EPA has routinely used BW to allometrically scale toxicity data from animal test species
to HEDs during the development of human health risk assessments, the applied methodology is
not without limitation (EPA, 201 lb). Allometric scaling using BW scaled to the 3/4 power
primarily addresses uncertainty associated with toxicokinetics, although the exact amount of
uncertainty addressed by this method for any specific chemical is often not quantifiable. In
following the recommended method to apply BW3/4 scaling, it remains possible that the
toxicokinetic uncertainty associated with GenX chemicals might be more or less than what is
accounted for using this scaling methodology.
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For all selected candidate studies, BW3/4 scaling was found to be appropriate because GenX
chemicals are not metabolized and have relatively short clearance times, especially compared to
other longer chain PFAS chemicals such as PFOA (DuPont-18405-1017 RV1, 2011; EPA,
201 lb; Gannon et al., 2016). The BW3/4 scaling methodology is not appropriate, however, when
using children's BWs. This limitation exists due to the absence of quantitative information
describing the toxicokinetic and toxicodynamic differences between test animals and early life-
stage humans (EPA, 201 lb). Because the liver effects observed following exposure to GenX
chemicals were in adult animals, the allometric scaling methodology was scaled to the average
adult human BW.
Variability in the study outcomes is extrinsically a function of study design and the endpoints
monitored. Studies of systemic toxicity monitor an array of endpoints that are not evaluated in
studies of reproductive, developmental, neurological, and immunological toxicity. The reverse is
true for the other types of toxicity studies compared to standard short-term and long-term
systemic studies. Studies of systemic toxicity do not often examine neurological or
immunological endpoints. Increases in liver weight were seen in many of the studies with dose-
response information, and the histological evaluation of the liver supported a determination that
the increase in liver weight when it is accompanied by necrosis can be considered as adverse
rather than adaptive, according to the Hall et al. (2012) criteria. Increases in relative liver weight
with confirmed liver necrosis were observed in DuPont-24447 (2008), DuPont-24459 (2008),
DuPont-18405-1037 (2010), DuPont-18405-1307 (2010), and DuPont-18405-1238 (2013).
The subchronic and chronic RfDs are based on the PODhed derived from the parental females
from the oral reproductive/developmental toxicity study in mice with application of UFs to
account for variability in the human population, database uncertainties, and possible differences
in the ways in which humans and rodents respond to HFPO dimer acid and/or its ammonium salt
that reaches their tissues (DuPont-18405-1037, 2010). Uncertainty associated with relying on a
less-than-chronic study to derive a chronic RfD is addressed with a UF applied only for the
chronic RfD calculation. The selected RfDs are based on the adverse liver effects observed in the
parental female animals. Selection of this endpoint is expected to provide protection to both the
sensitive life stages and the general population. The RfDs are supported by the outcomes from
other studies based on different endpoints, including hematological, immune, and developmental
effects (DuPont-24459, 2008; DuPont-17751-1026, 2009; DuPont-18405-1037, 2010). These
supporting data from the HFPO dimer acid and its ammonium salt database increase confidence
in the RfD.
8.2 Composition of Test Substance
Most of the available data for HFPO dimer acid and its ammonium salt with PMNs were
submitted to EPA by DuPont, the manufacturer of GenX chemicals, under TSCA, as required
pursuant to a consent order for these chemicals (EPA, 2009) or as required under TSCA
reporting requirements (e.g., section 8(e) 15 U.S.C. § 2607.8(e)). In these submissions, DuPont
provided information on the purity of the test substance used in each of the studies. Purity ranged
from 84% to 88% across the toxicity studies considered in this assessment. DuPont provided a
certificate of these analyses and noted that they were conducted under EPA GLP standards (40
CFR part 792). The major impurity identified is water (12.7%-13.3%). Trace amounts of PFOA
were also identified in the test substance (3.4-150 parts per million). DuPont noted that test
results were adjusted for purity based on the reported test article formulations. Based on the
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information provided, administered doses of PFOA present as a contaminant in the formulations
used by DuPont are low. For example, in the critical study chosen for the derivation of the RfDs,
the dose of administered PFOA is 0.000075 mg/kg/day at the GenX chemicals NOAEL (0.1
mg/kg/day) (DuPont-18405-1037, 2010). For PFOA, NOAELs ranging from 0.01 mg/kg/day to
30 mg/kg/day have been identified for effects including developmental, liver, and immune
endpoints (EPA, 2016a). Despite trace amounts of PFOA that might be present as an impurity,
EPA recognizes the potential for this impurity to contribute to the observed toxicity at very high
doses of GenX chemicals. At present, however, discerning the contribution of this low level of
PFOA to observed toxicity is not possible. Thus, EPA concluded that the presence of PFOA at
these low levels is not the primary driver of toxicity observed in the studies. Of note is that the
same test substance (Lot H-28548) was used in the 90-day mouse and rat studies, the chronic rat
study, and the oral reproductive and developmental toxicity and prenatal developmental toxicity
studies (DuPont-17751-1026, 2009; DuPont-18405-841, 2010; DuPont-18405-1307, 2010;
DuPont-18405-1238, 2013). Additionally, the same test substance (Lot H-28397) was used in
both the mouse and the rat 28-day studies (DuPont-24447, 2008; DuPont-24459, 2008). Despite
differences in test substance purity, adverse effects were observed consistently across the DuPont
studies. Many of the peer-reviewed studies did not report purity in their methods or formulations
of HFPO dimer acid and ammonium salt (Behr et al., 2020; Blake et al., 2020; Rushing et al.,
2017; Sheng et al., 2018; Wang et al., 2017).
Given the database for GenX chemicals, the quality of these studies—including adequacy of
reporting of methods and results—and the weight of evidence for effects on the liver,
hematological and immune systems, and reproductive and developmental endpoints, EPA
concluded that the DuPont studies demonstrated adverse effects as a result of exposure to the
HFPO dimer acid ammonium salt formulations and were appropriate for derivation of toxicity
values for these chemicals.
8.3 Use of Data-Derived Extrapolation Factors
For HFPO dimer acid and/or ammonium salt, there are limited human half-life data (see section
8.4) and no BBDR or PBPK models available to evaluate toxicokinetic and toxicodynamic
differences between humans and animals. Additionally, only a few repeat-dose studies are
available on rats and mice that evaluate toxicokinetics. These studies indicate that there is little-
to-no metabolism and that clearance is relatively rapid compared to other longer chain PFAS.
MOA (both in vivo and in vitro) data are also inadequate. EPA considered the 2014 Guidance for
Applying Quantitative Data to Develop Data-Derived Extrapolation Factors for Interspecies and
Intraspecies Extrapolation in determining UFa and UFh (EPA, 2014c). Using the decision
process described in Figure 2, EPA concluded that data are not adequate to support derivation of
data-derived extrapolation factors. Specifically, given the lack of available models and data to
address external dose and clearance in humans, default approaches to the application of UFa and
UFh were employed, including BW scaling for oral exposure (EPA, 201 lb). These approaches
are described further in section 7.3.
8.4 Use of Data-Derived Dosimetric Adjustment Factor
EPA guidance recommends a hierarchical approach to deriving human equivalent oral exposures
from animal studies, with the preferred approach being physiologically based toxicokinetic
modeling. There are no such toxicokinetic models available for GenX chemicals. The next
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preferred approach is to use chemical-specific information to derive a data-informed dosimetric
adjustment factor (DAF). For GenX chemicals there are limited human data (outlined below) and
a few repeat-dose studies available on rats and mice that evaluate toxicokinetics (see section
2.3.6).
In the one available human half-life study, twenty-five workers from a Chemours facility in the
Netherlands volunteered blood samples before an off-work weekend and twenty-two workers
provided a second sample at the start of the next shift (72-96 hours between sample collections)
(Clark, 2021). Samples were sent to two independent laboratories. HFPO dimer acid
concentrations ranged from below the level of detection (less than 0.5 |ig/L) to 25 |ig/L (Arbo
Unie, 2020). Samples containing measurable amounts of HFPO dimer acid at both time points
were used to calculate an average approximate half-life of 81 ± 55 hours, assuming an
exponential rate of decay (Clark, 2021). The range was 42 to 333 hours with a median of 66
hours. Serum from eighteen of the twenty-two workers contained HFPO dimer acid at detectable
levels (i.e., at or above the limit of detection) at both time points.
A letter summarizing the data and briefly outlining the methods used to calculate the human half-
life was provided to TSCA in 2021 by Chemours (similar information can also be found on
EC HA). However, EPA has not received the full study report and these data have not been peer
reviewed. The dataset used by Chemours to calculate the half-life is limited to only 18
individuals. Chemours also provided EPA with an unpublished report containing the raw data
(Arbo Unie, 2020); however, this report did not stratify the data based on sex or provide any
additional details on the test subjects (including sex). Sex-stratification of the human worker data
is potentially important because the critical effect in mice is more severe in females (DuPont-
18405-1037, 2010). Because the information provided are insufficient, EPA did not use the
human half-life data to estimate a data-informed DAF. Instead, EPA employed the default
procedure of body weight scaling to the 3/4 power (i.e., BW3/4) to derive human equivalent oral
exposures from animal studies in concordance with EPA guidance (EPA, 201 lb; outlined in
section 7.2.2).
Although the Chemours human half-life data are insufficient for use in the allometric scaling of
animal to human dose for toxicity and risk assessment purposes, EPA conducted an exploratory
analysis to determine the magnitude of the impact on the resulting PODhed if this information
was used to calculate a PODhed in place of the default BW3/4DAF (which, as outlined above, is
the agency's standard approach where acceptable data are not available) (Table 16).
Table 16. Comparison of PODhed using different allometric scaling methods
Endpoint and reference
PODnni) (mg/kg/day) calculated using...
BW3 4 DAF
Data Derived Human DAF
Liver constellation of lesions in parental males (DuPont-
18405-1037, 2010)
0.02
0.06
Liver constellation of lesions in parental females (DuPont-
18405-1037, 2010)
0.01
0.03
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The method used to calculate the data-derived DAF is outlined in Section 6.1.1.2 of the Human
Health Toxicity Values for Perfluorobutane Sulfonic Acid (CASRN 375-73-5) and Related
Compound Potassium Perfluorobutane Sulfonate (CASRN 29420-49-3) (EPA, 2021a). Briefly,
the ratio of elimination half-life in animals from which the POD is obtained (ti/2A) to that in
humans (ti/2H) can be used to calculate the DAF, and the human equivalent dose (HED) can be
calculated as follows:
PODhed = POD x ^
tl/2H
For the comparison exercise in Table 16, the ti/2AUsed for GenX chemicals were the male and
female mouse data from the beta elimination phase outlined in Table 8 and the ti/2Hwas the 81
hours calculated from the data outlined above (Arbo Unie, 2020; Clark, 2021). Although the
Chemours human half-life data were found to be insufficient for this purpose (Chemours, 2021)
describes the dataset as "limited"), this comparison demonstrates that the PODhed calculated
using either the BW34 DAF or the Data Derived Human DAB are similar. This comparison
exercise illustrates a degree of consistency between the BW3/4 approach and the use of the only
available human half-life dataset for deriving human equivalent oral doses for GenX chemicals.
8.5 Limited Data on Carcinogenicity
One study is available on evaluating carcinogenicity of HFPO dimer acid and its ammonium salt
in rats (DuPont-18405-1238, 2013). In this study, liver and pancreatic tumors were noted at the
highest doses tested. Although the incidence of testicular interstitial cell adenomas was not
statistically significant compared to controls, the authors of the study conclude that "a
relationship to treatment for these findings in the 50 mg/kg/day group cannot be ruled out" while
also suggesting that Bey dig cell tumor induction in rodents might have low relevance to humans
(Caverly Rae et al., 2015).The available data for HFPO dimer acid ammonium salt suggest that
mice might be more sensitive than rats to exposure to these GenX chemicals. Given the evidence
that the liver is the target organ for toxicity and the primary organ for tumor development,
additional research is needed using chronic duration exposures in mice. This uncertainty was not
considered in the application of the UBd because a noncancer toxicity value was developed for
this assessment.
8.6 Internal Dosimetry Data for GenX Chemicals
EPA recognizes that there are similarities in the health effects observed across various PBAS.
Specifically, GenX chemicals are linked to adverse effects on the liver, kidney, immune system,
development, and cancer and these health effects have also been associated with PBOA exposure
(EPA, 2016a,b). There are data available that demonstrate that the toxicokinetic profile for GenX
chemicals is different than PBOA in that GenX chemicals are more rapidly excreted than PBOA
and appear not to bioaccumulate like PBOA. These data lead one to question whether
administering the same dose of these chemicals could result in a much lower internal dose for
GenX chemicals than PBOA or PBOS and thus differences in potency between the two
chemicals.
There are currently two studies evaluating the internal dose of the HFPO dimer acid and
comparing it to the internal dose of either PBOA (Blake et al., 2020) or PBOS (Conley et al.,
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2021). Specifically, Blake et al. (2020) evaluated internal dose of both chemicals in pregnant
mice and their embryos. Concentrations of PFOA and HFPO dimer acid were measured in the
maternal serum, maternal liver, amniotic fluid, and whole embryo dosed with 0, 1, or 5
mg/kg/day of PFOA or 2 or 10 mg/kg/day of HFPO dimer acid from El.5 to El 1.5 or E17.5.
Although concentrations in the maternal serum were relatively similar, the total concentration of
HFPO dimer acid is an order of magnitude less than PFOA in the maternal liver. Additionally,
PFOA appears to accumulate in the liver from El 1.5 to E17.5 in mice exposed to 1 mg/kg/day
PFOA (48.3 ± 12.5 ug/mL to 181.1 ± 46.0 ug/mL); however, the concentration of HFPO dimer
acid at 2 mg/kg/day is similar at both time points (5.45 ± 3.43 ug/mL to 4.56 ± 2.80 ug/mL).
These differences are noteworthy because PFOA and HFPO dimer acid affected the maternal
liver similarly in this study (e.g., increased liver weight and increased incidence of liver lesions)
despite the concentration of HFPO dimer acid being an order of magnitude lower than PFOA and
displaying no apparent accumulation between El 1.5 and E17.5. The concentrations of PFOA and
HFPO dimer acid are similar in the amniotic fluid and whole embryo in the 1- and 2-mg/kg/day -
dose groups, respectively. These data suggest that a lower internal dose of HFPO dimer acid
elicits the same effects on the liver as a higher internal dose of PFOA in the pregnant mouse.
Additional research is needed to further elicit whether internal dosimetry is in fact different
between these chemicals and to determine if these results are specific to the pregnant mouse.
Conley et al. (2021) compared the maternal serum levels of HFPO dimer acid and PFOS with
respect to neonatal mortality. Conley et al. (2021) concluded that based on maternal serum
concentrations, HFPO dimer acid (ECso = 35.4 ug/mL) was ~2-fold more potent than PFOS
(ECso = 74.5 ug/mL). However, given that the molecular weight of PFOS (500 g/mol) is 34%
greater than HFPO-DA (330 g/mol), the potency of PFOS (EC50 =148.9 uM) and HFPO dimer
acid (EC50 = 107.1 uM) are very similar when correcting for molecular weight differences.
8.7 Effects on Bilirubin
A decrease in serum bilirubin is a consistent effect observed across multiple studies, especially in
female rodents (DuPont-17751-1026, 2009; DuPont-18405-1307, 2010; DuPont-18405-1238,
2013; Wang et al., 2017). This finding was surprising given that increased rather than decreased
levels of serum bilirubin are typically indicative of liver damage, and multiple studies outlined
above have confirmed microscopic liver damage (DuPont-18405-841, 2010; DuPont-18405-
1037, 2010; DuPont-18405-1307, 2010; DuPont-18405-1238, 2013; Tietze, 2012). In female
mice and rats, however, serum bilirubin levels were significantly decreased by 14%-50%
relative to controls when the females were administered 5 mg/kg/day-1,000 mg/kg/day of HFPO
dimer acid ammonium salt for 3-12 months (DuPont-17751-1026, 2009; DuPont-18405-1307,
2010; DuPont-18405-1238, 2013). Additionally, male ICR mice treated with 1 mg/kg/day of
HFPO dimer acid ammonium salt exhibited a significant 37% and 45% decrease in total and
direct bilirubin, respectively, when compared to controls (Wang et al., 2017); this finding was
not replicated in the other 28-day studies (DuPont-24447, 2008; DuPont-24459, 2008). The
biological or mechanistic significance of this effect is unknown, yet its consistency across
multiple studies is noteworthy.
8.8 Susceptible Populations and Life Stages
Data for the elucidation of differential susceptibility dependent on life stage (e.g., developing
embryo/fetus, women of reproductive age, or pregnant women) are not available. Children are
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frequently more vulnerable to pollutants than the average adult because of the differences in their
behaviors and biology. These differences can result in greater exposure and/or unique windows
of developmental susceptibility during the prenatal and postnatal periods for both the pregnant
mother and the developing fetus. No human toxicity or epidemiological studies are available in
the literature that address early developmental or reproductive life stages. Peer-reviewed
literature and DuPont submitted data examining reproductive and developmental endpoints in
both mice and rats (Blake et al., 2020; Conley et al., 2019, 2021; DuPont-18405-841, 2010;
DuPont-18405-1037, 2010) and summaries of these studies can be found in section 5.4
(Reproductive/Developmental). HFPO dimer acid ammonium salt can be transferred from a
pregnant animal to the fetus during gestation and lactation (Blake et al., 2020; Conley et al.,
2019, 2021; DuPont-18405-1037, 2010; Dupont-18405-849 RV1, 2011). When present,
developmental and reproductive effects were found at doses similar to and higher than those
associated with the selected critical effect: liver effects in females (constellation of lesions as
defined by the NTP PWG to include cytoplasmic alteration, hepatocellular single-cell and focal
necrosis, and hepatocellular apoptosis). The UFh of 10 accounts for variability in the responses
within human populations because of both intrinsic (including life stage) and extrinsic (lifestyle)
factors that can influence the response to dose. No information to characterize interindividual
and age-related variability in the toxicokinetics or toxicodynamics is available. Thus, the RfDs
provided in sections 7.4.1 and 7.4.2 (Subchronic RfD and Chronic RfD) are applicable to all life
stages. When reviewing data pertinent to the hazard potential of GenX chemicals, EPA adhered
to the requirements of its 2013 reaffirmation of the Policy on Evaluating Health Risks to
Children (EPA, 2013).
There is some sex-specific variation in the toxicokinetics of these two GenX chemicals in
rodents. Toxicokinetic data from DuPont calculate clearance times from the urine and plasma,
which is defined by DuPont as the time when 98.4% of the anion from the HFPO dimer acid
ammonium salt was cleared from the urine or plasma. These data show the HFPO dimer acid and
its ammonium salt clearance time in the plasma to be considerably faster for female rodents than
for male rodents (see the summary in section 2.3.6 (Clearance and Half-Life Data). For example,
Dupont-25300 (2008) identified 143 hours as the clearance time for HFPO dimer acid
ammonium salt in male mice at 10 mg/kg and 139 hours for 30 mg/kg. In female mice, the
clearance values were 57 and 62 hours for the low dose and the high dose, respectively.
However, this difference was not as pronounced in mice in the T1/2 estimates. Specifically, the
alpha (distribution) phase T1/2S were 5.8 and 4.6 hours for male and female mice, respectively,
and the beta (elimination) phase T1/2S were 36.9 hours and 24.2 hours for male and female mice,
respectively. It is unknown if or how these observed sex-specific toxicokinetic differences in
rodents contribute to the toxic response.
The available data suggest that the pregnant rodent might be more susceptible to liver effects
following exposure to GenX chemicals during gestation. Liver effects were reported in the
pregnant dams in the available reproductive/developmental studies dosed during gestation (Blake
et al., 2020; Conley et al., 2019; DuPont-18405-841, 2010; DuPont 18405-1037, 2010). All the
studies reported increases in liver weight ranging from 12% to 34% in rats and 26% to 101% in
mice over the gestational period. Conley et al. (2019) did not conduct liver histopathology, but
both DuPont-18405-841 (2010) and Blake et al. (2020) reported hepatocellular hypertrophy and
increased cell death as compared to controls with increasing HFPO dimer acid ammonium salt
concentration. Specifically, focal necrosis was observed in 2/22 (9%) and 5/22 (23%) pregnant
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rats after just 15 days (GD6-GD20) of 10 mg/kg/day or 100 mg/kg/day of HFPO dimer acid
ammonium salt, respectively, compared to 0 in the control group. Comparatively, nonpregnant
female rats dosed from 28 to 90 days did not exhibit necrosis when treated with doses up to
1,000 mg/kg/day of HFPO dimer acid ammonium salt. Necrosis was observed in nonpregnant
female rats only after 2 years of dosing with 500 mg/kg/day of HFPO dimer acid ammonium
salt. Increased cell death (including both apoptosis and single-cell or focal necrosis) was
observed in pregnant mice after 11 and 17 days (GD1.5-GD11.5 or GD17.5) of 2 mg/kg/day or
10 mg/kg/day of HFPO dimer acid ammonium salt. Similarly, and as noted above, female mice
dosed 14 days prior to mating and throughout gestation and lactation exhibited cytoplasmic
alteration, apoptosis, single-cell necrosis, and focal necrosis after 53-64 days of dosing (NTP,
2019 reread of DuPont 18405-1037, 2010). The incidence of single-cell and focal necrosis in the
Fo females was 6/24 (25%) and 20/24 (83%) in the 0.5- and 5-mg/kg/day-dose groups,
respectively (NTP, 2019). A chronic study in mice is not available to compare to the gestational
exposures in female pregnant mice, and comparisons to the 90-day subchronic study in mice is
potentially limited by sample size (n = 9) in the 0.1 and 0.5 mg/kg/day-dose groups.
Susceptible populations include groups who have relatively high exposure to GenX chemicals.
While data are currently unavailable, there is the potential for highly exposed populations. For
example, formula fed infants, who have high daily water ingestion relative to body weight, have
the potential for relatively high exposure to GenX chemicals when GenX chemicals are present
in tap water and this tap water is used to reconstitute formula. As a second example, workers and
their families who work at and/or live near facilities that use the GenX processing aid technology
have the potential for greater exposure levels and duration of exposure. Finally, communities
living in close proximity to facilities using the GenX processing aid technology have the
potential for increased exposure as evidenced by the detection of GenX chemicals in drinking
water, surface water, soil and rainwater samples collected close to the facility (see section 1.3).
9.0 References
Allendorf, F., U. Berger, K. Goss, and N. Ulrich. 2019. Partition coefficients of four
perfluoroalkyl acid alternatives between bovine serum albumin (BSA) and water in
comparison to ten classical perfluoroalkyl acid. Environmental Science: Processes &
Impacts 21:1852-1863. doi: 10.1039/C9EM00290A.
Arbo Unie. 2020. Study Half-Time HFPO-DA. Arbo Unie, Utrecht, the Netherlands.
https://heronet.epa.gov/heronet/iiidex.cfin/reference/details/reference id/8631852.
Beekman, M., P. Zweers, A. Muller, W. de Vries, P. Janssen, and M. Zeilmaker. 2016.
Evaluation of Substances Used in the GenX Technology by Chemours, Dordrecht. RIVM
Letter Report 2016-0174. National Institute for Public Health and the Environment,
Ministry of Health, Welfare and Sport, Netherlands. Accessed May 2018.
https://www.rivm.nl/bibliotheek/rapporten/2016-
f#:~:text=Evaluation%20of%20substances%20used%20in%20the%20GenX%20
technology. people%201iving%20in%20the%20vicinitv%20of%20the%20plant.
106
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Behr, A.C., D. Lichtenstein, A. Braeuning, A. Lampen, and T. Buhrke. 2018. Perfluoroalkylated
substances (PFAS) affect neither estrogen and androgen receptor activity nor
steroidogenesis in human cells in vitro. Toxicology Letters 291:51-60.
doi /i .toxtet.2018.03.029.
Behr, A.C., C. Plinsch, A. Braeuning, and T. Buhrke. 2020. Activation of human nuclear
receptors by perfluoroalkylated substances (PFAS). Toxicology in Vitro 62:104700.
doi: 10.1016/j.tiv.2019.104700.
Blake, B E., H.A. Cope, S.M. Hall, R.D. Keys, B.W. Mahler, J. McCord, B. Scott, H.M.
Stapleton, M.J. Strynar, S.A. Elmore, and S.E. Fenton. 2020. Evaluation of maternal,
embryo, and placental effects in CD-I mice following gestational exposure to
perfluorooctanoic acid (PFOA) or hexafluoropropylene oxide dimer acid (HFPO-DA or
GenX). Environmental Health Perspectives 128(2):027006. doi:10.1289/EHP6233.
Calafat, A.M., K. Kato, K. Hubbard, T. Jia, J.C. Botelho, and L. Wong. 2019. Legacy and
alternative per- and polyfluoroalkyl substances in the U.S. general population: paired
serum-urine data from the 2013-2014 National Health and Nutrition Examination
Survey. Environment International 131:10548. doi: 10.1016/j.envint.2019.105048.
Calvo, R.M., E. Jauniaux, B. Gublis, M. Asuncion, C. Gervy, B. Contempre, and G.M. de
Escobar. 2002. Fetal tissues are exposed to biologically relevant free thyroxine
concentrations during early phases of development. The Journal of Clinical
Endocrinology & Metabolism 87(4): 1768-1777. doi:10.1210/jcem.87.4.8434.
Cannon, R.E., A.C. Richards, A.W. Trexler, C.T. Juberg, B. Sinhra, G.A. Knudsen, and L.S.
Birnbaum. 2020. Effect of GenX on p-glycoprotein, breast cancer resistance protein, and
multidrug resistance-associated protein 2 at the blood-brain barrier. Environmental
Health Perspectives 128(3):037002. doi:10.1289/EHP5884.
Caverly Rae, J.M., L. Craig, T.W. Slone, S.R. Frame, L. Buxton, and G.L. Kennedy. 2015.
Evaluation of chronic toxicity and carcinogenicity of ammonium2,3,3,3-tetrafluoro-2-
(heptafluoropropoxy)-propanoate in Sprague-Dawley rats. Toxicology Reports 2:939-
949. doi: 10.1016/j.toxrep.2015.06.001.
Chemours. 2018. Sustainability-GenX. The Chemours Company, Wilmington, DE.
Cheng, W., and C.A. Ng. 2018. Predicting relative protein affinity of novel per- and
polyfluoroalkyl substances (PFAS) by an efficient molecular dynamics approach.
Environmental Science Technology 52:7972-7980. doi:10.1021/acs.est.8b01268.
Clark, Dawn, The Chemours Company. 2021, March 17. Letter to EPA, Office of Pollution
Prevention and Toxics regarding propanoic acid, 2,3,3,3-tetrafluoro-2-(l, 1,2,2,3,3,3-
heptafluoropropoxy)-CAS RN 13252-13-6 (also known as HFPO-DA).
https://heroiiet.epa.gov/heroiiet/iiidex.cfm/reference/details/refereiice id/8631852.
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Conley, J.M., C.S. Lambright, N. Evans, J. McCord, M.J. Strynar, D Hill, E. Medlock-Kakaley,
V.S. Wilson, and L.E. Gray, Jr. 2021. Hexafluoropropylene oxide-dimer acid (HFPO-DA
or GenX) alters maternal and fetal glucose and lipid metabolism and produces neonatal
mortality, low birthweight, and hepatomegaly in the Sprague-Dawley rat. Environmental
International 146:106204. doi: 10.1016/j.envint.2020.106204.
Conley, J.M., C.S. Lambright, N. Evans, M.J. Strynar, J. McCord, B.S. Mclntyre, G.S. Travlos,
M.C. Cardon, E. Medlock-Kakaley, P.C. Hartig, V.S. Wilson, and L.E. Gray, Jr. 2019.
Adverse maternal, fetal, and postnatal effects of hexafluoropropylene oxide dimer acid
(GenX) from oral gestational exposure in Sprague-Dawley rats. Environmental Health
Perspectives 127(3):037008. doi:10.1289/EHP4372.
D'Ambro, E.L., H.O.T. Pye, J.O. Bash, J. Bowyer, C. Allen, C. Efstathiou, R.C. Gilliam, L.
Reynolds, K. Talgo, and B.N. Murphy. Characterizing the air emissions, transport, and
deposition of per- and polyfluoroalkyl substances from a fluoropolymer manufacturing
facility. Environmental Science & Technology 55(2):862-870.
doi: 10.1021/acs.est.0c06580.
D'eon, J.C., A.J. Simpson, R. Kumar, A.J. Baer, and S.A. Mabury. 2010. Determining the
molecular interactions of perfluorinated carboxylic acids with human sera and isolated
human serum albumin using nuclear magnetic resonance spectroscopy. Environmental
Toxicology and Chemistry 29(8): 1678-1688. doi: 10.1002/etc.204.
DuPont CCAS (DuPont Corporate Center for Analytical Sciences). 2009. Sublimation of
Processing Aids FRD-903K and PRD-902, ed. A.D. English. CCAS, Wilmington, DE.
DuPont-2-63: E.I. du Pont de Nemours and Company. 1963. Acute Oral Test. Test guideline not
identified. Study conducted by Haskell Laboratory for Toxicology and Industrial
Medicine (Study Completion Date: January 3, 1963). Testing laboratory location not
identified.
DuPont-770-95: E.I. du Pont de Nemours and Company. 1996. Approximate Lethal Dose (ALD)
ofH-21216 in Rats. Test guideline not identified. Study conducted by E.I. du Pont de
Nemours and Company (Study Completion Date: February 26, 1996), Newark, DE.
DuPont-839-95: E.I. du Pont de Nemours and Company. 1996. Approximate Lethal Dose (ALD)
by Skin Absorption ofH-21216 in Rabbits. Test guideline not identified. Study conducted
by E.I. du Pont de Nemours and Company (Study Completion Date: April 1, 1996),
Newark, DE.
DuPont-17568-1675: E.I. du Pont de Nemours and Company. 2008. Estimation of the Adsorption
Coefficient (Koc) of the HFPO Dimer Acid Ammonium Salt on Soil and Sludge. OECD
Test Guideline 121. Study conducted by DuPont Haskell Global Centers for Health and
Environmental Sciences (Study Completion Date: September 11, 2008), Newark, DE.
DuPont-17751-723: E.I. du Pont de Nemours and Company. 2009. H-28548: Inhalation Acute
Exposure with Anatomic Pathology Evaluation in Rats. Test guideline not identified.
Study conducted by E.I. du Pont de Nemours and Company (Study Completion Date:
May 11, 2009), Newark, DE.
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DuPont-17751-1026: E.I. du Pont de Nemours and Company. 2009. A 90-Day Oral (Gavage)
Toxicity Study of H-28548 in Rats with a 28-Day Recovery. OECD Test Guideline 408.
Study conducted by WIL Research Laboratories, LLC (Study Completion Date: October
5, 2009), Ashland, OH.
DuPont-17751-1579 RV1: E.I. duPont de Nemours and Company. 2009. Cross-species
Comparison of FRD-902 Plasma Pharmacokinetics in the Rat and Primate Following
Intravenous Dosing. Test guideline not identified. Study conducted by E.I. du Pont de
Nemours and Company (Original Report Completed: December 8, 2008; Report Revision
1 Completed: February 2, 2009), Newark, DE.
DuPont-18405-841: E.I. du Pont de Nemours and Company. 2010. An Oral (Gavage) Prenatal
Developmental Toxicity Study of H-28548 in Rats. U.S. EPA OPPTS 850.3700; OECD
Test Guideline 414. Study conducted by WIL Research Laboratories, LLC (Study
Completion Date: July 2, 2010), Ashland, OH.
DuPont-18405-849 RV1: E.I. du Pont de Nemours and Company. 2011. H-28548: Toxicokinetic
Study in Pregnant Rats. Test guideline not identified. Study conducted by E.I. du Pont de
Nemours and Company (Original Report Completed: March 29, 2011; Report Revision 1
Completed: April 11, 2011), Newark, DE.
DuPont-18405-1017 RV1: E.I. duPont de Nemours and Company. 2011. H-28548: Absorption,
Distribution, Metabolism, and Elimination in the Rat. U.S. EPA OPPTS 870.7485. Study
conducted by E.I. du Pont de Nemours and Company (Original Report Completed:
November 3, 2010; Report Revision 1 Completed: April 21, 2011), Newark, DE, and
Wilmington, DE.
DuPont-18405-1037: E.I. du Pont de Nemours and Company. 2010. An Oral (Gavage)
Reproduction/Developmental Toxicity Screening Study of H-28548 in Mice. U.S. EPA
OPPTS 870.3550; OECD Test Guideline 421. Study conducted by WIL Research
Laboratories, LLC (Study Completion Date: December 29, 2010), Ashland, OH.
DuPont-18405-1238: E.I. duPont de Nemours and Company. 2013. H-28548: Combined
Chronic Toxicity/Oncogenicity Study 2-Year Oral Gavage Study in Rats. U.S. EPA
OPPTS 870.4300; OECD Test Guideline 453. Study conducted by MPI Research, Inc.
(Study Completion Date: March 28, 2013), Mattawan, MI.
DuPont-18405-1307: E.I. duPont de Nemours and Company. 2010. H-28548: Subchronic
Toxicity 90-Day Gavage Study in Mice. OECD Test Guideline 408. Study conducted by
E.I. du Pont de Nemours and Company (Study Completion Date: February 19, 2010),
Newark, DE.
DuPont-18647-1017 RV1: E.I. duPont de Nemours and Company. 2011. H-28548: Absorption,
Distribution, Metabolism, and Elimination in the Mouse. U.S. EPA OPPTS 870.7485.
Study conducted by E.I. du Pont de Nemours and Company (Original Report Completed:
November 3, 2010; Report Revision 1 Completed: April 21, 2011), Newark, DE, and
Wilmington, DE.
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DuPont-19713 RV1: E.I. du Pont de Nemours and Company. 2008. H-27529: Bacterial Reverse
Mutation Test. U.S. EPA OPPTS 870.5100; OECD Test Guideline 471. Study conducted
by E.I. du Pont de Nemours and Company (Original Report Completed: May 31, 2006;
Report Revision 1 Completed: February 22, 2008), Newark, DE.
DuPont-19714 RV1: E.I. du Pont de Nemours and Company. 2008. H-27529: In Vitro
Mammalian Chromosome Aberration Test in Chinese Hamster Ovary Cells. U.S. EPA
OPPTS 870.5375; OECD Test Guideline 473. Study conducted by E.I. du Pont de
Nemours and Company (Original Report Completed: June 27, 2006; Report Revision 1
Completed: February 25, 2008), Newark, DE.
DuPont-19897: E.I. du Pont de Nemours and Company. 2006. H-27529: Local Lymph Node
Assay (LLNA) in Mice. U.S. EPA OPPTS 870.2600; OECD Test Guideline 429. Study
conducted by E.I. du Pont de Nemours and Company (Study Completion Date: June 9,
2006), Newark, DE.
DuPont-22616 RV1: E.I. du Pont de Nemours and Company. 2007. H-28072: Local Lymph
Node Assay (LLNA) in Mice. U.S. EPA OPPTS 870.2600; OECD Test Guideline 429.
Study conducted by E.I. du Pont de Nemours and Company (Original Report Completed:
July 2, 2007; Report Revision 1 Completed: October 1, 2007), Newark, DE.
DuPont-22620 RV1: E.I. du Pont de Nemours and Company. 2009. H-28072: In Vitro
Mammalian Chromosome Aberration Test in Chinese Hamster Ovary Cells. U.S. EPA
OPPTS 870.5375; OECD Test Guideline 473. Study conducted by E.I. du Pont de
Nemours and Company (Original Report Completed: July 25, 2007; Report Revision 1
Completed: September 23, 2009), Newark, DE.
DuPont-22734 RV1: E.I. du Pont de Nemours and Company. 2008. H-28072: Bacterial Reverse
Mutation Test. U.S. EPA OPPTS 870.5100; OECD Test Guideline 471. Study conducted
by E.I. du Pont de Nemours and Company (Original Report Completed: July 26, 2007;
Report Revision 1 Completed: August 13, 2008), Newark, DE.
DuPont-22932: E.I. du Pont de Nemours and Company. 2007. H-28072: Acute Oral Toxicity
Study in Rats—Up-and-Down Procedure. U.S. EPA OPPTS 870.1100; OECD Test
Guideline 425. Study conducted by E.I. du Pont de Nemours and Company (Study
Completion Date: July 25, 2007), Newark, DE.
DuPont-23219: E.I. du Pont de Nemours and Company. 2007. H-28072: UnscheduledDNA
Synthesis (USD) Test with Mammalian Cells in Vivo. OECD Test Guideline 486. Study
conducted by BioReliance (Study Completion Date: August 14, 2007), Rockville, MD.
DuPont-23220: E.I. du Pont de Nemours and Company. 2007. H-28072: In VivoMicronucleus
and Chromosome Aberration Assay in Mouse Bone Marrow Cells. U.S. EPA OPPTS
870.5395; OECD Test Guidelines 474 and 475. Study conducted by BioReliance (Study
Completion Date: October 10, 2007), Rockville, MD.
110
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OCTOBER 2021
DuPont-23459: Study Sponsor not identified. 2007. In Vitro Trout Hepatocyte Bioaccumulation
Screen. Test guideline not identified. Study conducted by Haskell Laboratory Discovery
Toxicology Group (Study Completion Date: June 15, 2007). Testing laboratory location
not identified.
DuPont-23460: Haskell Laboratory Discovery Toxicology Group. 2007. In Vitro Rat Hepatocyte
Screen. Test guideline not identified. (Study Completion Date: June 12, 2007). Testing
laboratory location not identified.
DuPont-24009: Dupont Haskell Global Centers for Health and Environmental Sciences. 2008.
Repeated Dose Oral Toxicity 7-Day Gavage Study in Rats. Test guideline not identified.
(Report Issue Date: February 14, 2008). Testing laboratory location not identified.
DuPont-24010: Dupont Haskell Global Centers for Health and Environmental Sciences. 2008.
Repeated Dose Oral Toxicity 7-Day Gavage Study in Mice. Test guideline not identified.
(Report Issue Date: February 14, 2008). Testing laboratory location not identified.
DuPont-24030: E.I. du Pont de Nemours and Company. 2007. FRD-902: Acute Dermal
Irritation Study in Rabbits. U.S. EPA OPPTS 870.2500; OECD Test Guideline 404.
Study conducted by E.I. du Pont de Nemours and Company (Study Completion Date:
November 21, 2007), Newark, DE.
DuPont-24113: E.I. du Pont de Nemours and Company. 2007. FRD-902: Acute Dermal Toxicity
Study in Rats. U.S. EPA OPPTS 870.1200; OECD Test Guideline 402. Study conducted
by E.I. du Pont de Nemours and Company (Study Completion Date: November 28,
2007), Newark, DE.
DuPont-24114: E.I. du Pont de Nemours and Company. 2007. FRD-902: Acute Eye Irritation
Study in Rabbits. U.S. EPA OPPTS 870.2400; OECD Test Guideline 405. Study
conducted by E.I. du Pont de Nemours and Company (Study Completion Date: December
14, 2007), Newark, DE.
DuPont-24116: Dupont Haskell Global Centers for Health and Environmental Sciences. 2008.
Repeated Dose Oral Toxicity 7-Day Gavage Study in Rats. Test guideline not identified.
(Report Issue Date: February 14, 2008). Testing laboratory location not identified.
DuPont-24126: E.I. du Pont de Nemours and Company. 2007. FRD-902: Acute Oral Toxicity
Study inMice—Up-and-DownProcedure. U.S. EPA OPPTS 870.1100; OECD Test
Guideline 425. Study conducted by E.I. du Pont de Nemours and Company (Study
Completion Date: November 29, 2007), Newark, DE.
DuPont-24128: E.I. du Pont de Nemours and Company. 2008. Determination of the Water
Solubility and Vapor Pressure o/H-28307. U.S. EPA OPPTS 830.7840 and 830.7950;
OECD Test Guidelines 104 and 105. Study conducted by Wildlife International, Ltd.
(Study Completion Date: March 27, 2008), Easton, MD.
Ill
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OCTOBER 2021
DuPont-24129: E.I. du Pont de Nemours and Company. 2008. Determination of the Water
Solubility and Vapor Pressure of H-28308. U.S. EPA OPPTS 830.7840 and 830.7950;
OECD Test Guidelines 104 and 105. Study conducted by Wildlife International, Ltd.
(Study Completion Date: March 27, 2008), Easton, MD.
DuPont-24198: E.I. du Pont de Nemours and Company. 2008. Determination of Dissociation
Constant of H-28308: Revision 1. U.S. EPA OPPTS 830.7370; OECD Test Guideline
112. Study conducted by Wildlife International, Ltd. (Study Completion Date: April 1,
2008), Easton, MD.
DuPont-24199: E.I. du Pont de Nemours and Company. 2008. H-28308: An Evaluation of
Hydrolysis as a Function ofpH. U.S. EPA OPPTS 835.2110; OECD Test Guideline 111.
Wildlife International, Ltd. (Study Completion Date: March 27, 2008), Easton, MD.
DuPont-24281: Dupont Haskell Global Centers for Health and Environmental Sciences. 2008.
Biopersistence and Pharmacokinetic Screen in the Rat. Test guideline not identified.
(Report Issue Date: February 13, 2008). Testing laboratory location not identified.
DuPont-24286: Dupont Haskell Global Centers for Health and Environmental. 2008.
Biopersistence and Pharmacokinetic Screen in the Rat. Test guideline not identified.
Study conducted by Critical Path Services Sciences (Study Completion Date: October 10,
2007). Testing laboratory location not identified.
DuPont-24447: E.I. du Pont de Nemours and Company. 2008. A 28-Day Oral (Gavage) Toxicity
Study ofH-28397 in Rats with a 28-Day Recovery. OECD Test Guideline 407. Study
conducted by WIL Research Laboratories, LLC (Study Completion Date: August 22,
2008), Ashland, OH.
DuPont-24459: E.I. du Pont de Nemours and Company. 2008. A 28-Day Oral (Gavage) Toxicity
Study ofH-28397 in Mice with a 28-Day Recovery. OECD Test Guideline 407. Study
conducted by WIL Research Laboratories, LLC (Study Completion Date: August 29,
2008), Ashland, OH.
DuPont-24637: DuPont Haskell Global Centers for Health and Environmental Sciences. 2008.
Physical and Chemical Characteristics ofFRD-902: State of the Substance,
Melting/Freezing Point, Boiling Point, Relative Density, Surface Tension, Flash Point,
Auto-Ignition Temperature and Viscosity. OECD Test Guidelines 102 and 115; ASTM
Methods D 92, 445, 446, 891, and 1120; ASTM Method E 659-78. Study conducted by
Case Consulting Laboratories, Inc. (Study Completion Date: May 5, 2008), Whippany,
NJ.
DuPont-24698: DuPont Haskell Global Centers for Health and Environmental Sciences. 2008.
Physical and Chemical Characteristics ofFRD-903: State of the Substance,
Melting/Freezing Point, Boiling Point, Relative Density, Surface Tension, Flash Point,
Auto-Ignition Temperature and Viscosity. OECD Test Guidelines 102 and 115; ASTM
Methods D 92, 445, 446, 891, and 1120; ASTM Method E 659-78. Study conducted by
Case Consulting Laboratories, Inc. (Study Completion Date: May 5, 2008), Whippany,
NJ.
112
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OCTOBER 2021
DuPont-25281: Dupont Haskell Global Centers for Health and Environmental Sciences. 2008.
Repeated Dose Oral Toxicity 7-Day Gavage Study in Male Mice. Test guideline not
identified. (Report Issue Date: February 14, 2008). Testing laboratory location not
identified.
DuPont-25292: E.I. du Pont de Nemours and Company. 2008. Determination of a Permeability
Coefficient (Kp) for H-28308 Using Human and Rat Skin Mounted in an in Vitro Static
Diffusion Cell. Test guideline not identified. Testing laboratory not identified (Study
Completion Date: February 27, 2008). Testing laboratory location not identified.
DuPont-25300: Dupont Haskell Global Centers for Health and Environmental Sciences. 2008.
Biopersistence and Pharmacokinetic Screen in the Mouse. Test guideline not identified.
(Report Issue Date: July 31, 2008). Testing laboratory location not identified.
DuPont-25438 RV1: E.I. du Pont de Nemours and Company. 2008. H-28308: Acute Oral
Toxicity Study in Rats—Up-and-Down Procedure. U.S. EPA OPPTS 870.1100; OECD
Test Guideline 425. Study conducted by E.I. du Pont de Nemours and Company (Original
Report Completed: May 28, 2008; Report Revision 1 Completed: July 23, 2008),
Newark, DE.
DuPont-25875: E.I. du Pont de Nemours and Company. 2008. FRD-903: Acute Oral Toxicity
Study in Rats—Up-and-Down Procedure. U.S. EPA OPPTS 870.1100; OECD Test
Guideline 425. Study conducted by E.I du Pont de Nemours and Company (Study
Completion Date: October 13, 2008), Newark, DE.
DuPont-25938 RV1: E.I. du Pont de Nemours and Company. 2008. H-28397: Activated Sludge
Respiration Inhibition Test. OECD Test Guideline 209. Study conducted by DuPont
Haskell Global Centers for Health and Environmental Sciences (Study Completion Date:
September 5, 2008; Revision Date: October 21, 2008), Newark, DE.
DuPont-26129: E.I. du Pont de Nemours and Company. 2008. H-28548: In Vitro Mammalian
Cell Gene Mutation Test (L5178Y/TK+/-Mouse Lymphoma Assay). U.S. EPA OPPTS
870.5300; OECD Test Guideline 476. Study conducted by BioReliance (Study
Completion Date: June 25, 2008), Rockville, MD.
DuPont-26349: E.I. du Pont de Nemours and Company. 2008. Determination of the Dissociation
Constant and UV-VIS Absorption Spectra ofH-28307. U.S. EPA OPPTS 830.7370;
OECD Test Guidelines 101 and 112. Study conducted by Wildlife International, Ltd.
(Study Completion Date: September 17, 2008), Easton, MD.
DuPont-1388231-R2009NC03 l(a)-02: E.I. du Pont de Nemours and Company. 2010. Report for
Inherent Biodegradation ofFRD903 (ModifiedMITI (II) Test). OECD Test Guideline
302C. Study conducted by Key Lab of Pesticide Environmental Assessment and
Pollution Control, MEP (Study Completion Date: January 20, 2009), Nanjing, China.
DuPont-1388231-R2009NC03 l(s)-02: E.I. du Pont de Nemours and Company. 2010. Report for
Inherent Biodegradation ofFRD902 (ModifiedMITI (II) Test). OECD Test Guideline
302C. Study conducted by Key Lab of Pesticide Environmental Assessment and
Pollution Control, MEP (Study Completion Date: January 20, 2009), Nanjing, China.
113
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OCTOBER 2021
DuPont-A080558: Du Pont-Mitsui Fluorochemicals Company, Ltd. 2009. Ready
Biodegradability Test ofFRD903. Test guideline not identified. Study conducted by
Mitsubishi Chemical Medience Corporation, Yokohama Laboratory (Study Completion
Date: May 25, 2009), Yokohama, Japan.
DuPont-A080560: Du Pont-Mitsui Fluorochemicals Company, Ltd. 2009. Bioconcentration
Study ofFRD903 with Carp. Test guideline not identified. Study conducted by Mitsubishi
Chemical Medience Corporation, Yokohama Laboratory (Study Completion Date: June
26, 2009), Yokohama, Japan.
DuPont-C30031_516655: The Chemours Company. 2017. Determination ofHFPO-DA in EDTA
human plasma samples. Test Site Study No. 516655. Study conducted by Charles River
Laboratories Den Bosch BV (Study Completion Date: October 27, 2017), Schaijk, The
Netherlands.
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Creasy. 2016. Recommendations from the INHAND apoptosis/necrosis working group.
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Appendix A: Literature Search Strategy
This appendix presents the details of the literature search strategy U.S. Environmental Protection
Agency (EPA) used to identify primary, peer-reviewed literature pertaining to
hexafluoropropylene oxide (HFPO) dimer acid (Chemical Abstracts Service Registry Number
(CASRN) 13252-13-6) and its ammonium salt (CASRN 62037-80-3). The literature searches
were conducted using the databases listed in Table A-l.
The initial literature searches for these GenX chemicals were conducted in July 2017 (acid) and
January/February 2018 (ammonium salt). Subsequent literature searches were conducted from
2018 to March 2020. The searches were conducted using CASRN, synonyms, and additional
relevant search strings (see Table A-2). Because the results of this core search were so limited,
additional databases were identified and searched for physiochemical property information,
health effects, toxicokinetics, and mechanistic information (see Table A-3 and Table A-4).
Combined, these initial literature searches returned 27 studies for HFPO dimer acid and HFPO
dimer acid ammonium salt after duplicates across the two chemicals were deleted. The literature
searches conducted after publication of the public comment draft in November 2018 resulted in
48 additional studies for HFPO dimer acid and HFPO dimer acid ammonium salt after duplicates
were deleted.
As previously stated, the available data for GenX chemicals come primarily from studies
submitted under the Toxic Substances Control Act (TSCA). Those studies were combined with
the results of the search of the publicly available peer-reviewed literature for evaluation for
relevance to the assessment. The submitted studies and literature identified by the search of
publicly available sources are available through EPA's Health & Environmental Research Online
(HERO) website at https://hero.epa.gov/hero/iiidex.cfm/proiect/page/proiect id/2627. Potential
relevance was based primarily on a title and abstract screen. Table A-5 presents the
inclusion/exclusion criteria applied to conducting the literature searches. An additional 48 studies
from peer-reviewed literature were identified during the updated literature searches conducted in
February 2019, October 2019, and March 3, 2020. These studies were subjected to title and
abstract screening to determine relevancy according to the inclusion/exclusion criteria outlined in
Table A-6. Relevancy was confirmed by review of the full text of studies included in the title
abstract screen. Studies that did not meet the inclusion criteria but provide supporting
information were categorized as supplemental, relative to the type of supporting information they
provided. These supplemental categories are outlined in Table A-7.
A-l
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OCTOBER 2021
Table A-l. Summary of Core Database Search Results
Search date
Pub Med
WOS
Toxline
TSCATS via
Toxlinc/NLM
Other sou rccs
Combined datasct after
duplicate removal
HFPO dimer acid (CASRN 13252-13-6)
7/24/17
3
12
0
0
3
16
7/17-2/19
6
11
0
0
0
11
2/19-10/19
9
8
0
0
9
16
10/19-3/20
7
4
N/Aa
0
1
9
HFPO dimer acid ammonium salt (CASRN 62037-80-3)
1/18 and 2/18
9
12
0
0
3
18
2/18-2/19
8
13
0
0
1
15
2/19-10/19
15
11
0
0
2
20
10/19-3/20
7
3
N/Aa
0
1
8
Note: N/A = not applicable; NLM = National Library of Medicine; TSCATS = Toxic Substances Control Act Test Submissions;
WOS = Web of Science.
a Toxline was no longer available in March 2020.
A-2
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OCTOBER 2021
Table A-2. Database Search Strings
Database
HFPO dimcr acid (CASRN 13252-13-6)
HFPO dimcr acid ammonium salt (CASRN 62037-80-3)
PubMed
13252-13-6[rn] OR "2,3,3,3-Tetrafluoro-2-
(heptafluoropropoxy)propionic acid"[tw] OR "2,3,3,3-tetrafluoro-2-
(l,l,2,2,3,3,3-heptafluoropropoxy)-Propanoic acid"[tw] OR
"Perfluoro(2-methyl-3-oxahexanoate) "[tw] OR "Propanoic acid,
2,3,3,3-tetrafluoro-2-(l,l,2,2,3,3,3-heptafluoropropoxy)- "[tw] OR
"Perfluorinated aliphatic carboxylic acid"[tw] OR "Perfluoro(2-
methyl-3-oxahexanoic) acid"[tw] OR "2,3,3,3-tetrafluoro-2-
(l,l,2,2,3,3,3-heptafluoropropoxy)propanoic acid"[tw] OR "2,3,3,3-
tetrafluoro-2-(heptafluoropropoxy)propanoic acid"[tw] OR
"perfluoro-2-(propyloxy)propionic acid"[tw] OR "perfluoro-2-methyl-
3-oxahexanoic acid"[tw] OR "perfluoro-2-propoxypropanoic
acid"[tw] OR "perfluoro-2-propoxypropionic acid"[tw] OR
"perfluoro-a-propoxypropionic acid"[tw] OR "propanoic acid,
2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-"[tw] OR "propionic acid,
2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-"[tw] OR (GenX AND
(fluorocarbon*[tw] ORfluorotelomer*[tw] ORpolyfluoro*[tw] OR
perfluoro-*[tw] ORperfluoroa*[tw] ORperfluorob*[tw] OR
perfluoroc*[tw] ORperfluorod*[tw] ORperfluoroe*[tw] OR
perfluoroh*[tw] ORperfluoron*[tw] ORperfluoroo*[tw] OR
perfluorop*[tw] ORperfluoros*[tw] ORperfluorou*[tw] OR
perfluorinated[tw] OR fluorinated[tw])) OR (("2,3,3,3-Tetrafluoro-2-
(heptafluoropropoxy)propionic"[tw] OR "2,3,3,3-tetrafluoro-2-
(1,1,2,2,3,3,3 -heptafluoropropoxy)-Propanoic" [tw] "Perfluorinated
aliphatic carboxylic"[tw] OR "Perfluoro(2-methyl-3-
oxahexanoic)"[tw] OR "2,3,3,3-tetrafluoro-2-(l,1,2,2,3,3,3-
heptafluoropropoxypropanoic" [tw] "2,3,3,3 -tetrafluoro-2-
(heptafluoropropoxy)propanoic"[tw] OR "perfluoro-2-
(propyloxy)propionic"[tw] OR "perfluoro-2-methyl-3-
oxahexanoic"[tw] OR "perfluoro-2-propoxypropanoic"[tw] OR
"perfluoro-2-propoxypropionic"[tw] OR "perfluoro-a-
propoxypropionic "[tw]) AND (acid[tw] OR acids[tw]))
(62037-80-3[rn] OR "62037-80-3"[tw] OR "Ammonium 2,3,3,3-
tetrafluoro-2-(heptafluoropropoxy)propanoate"[tw] OR "Propanoic
acid, 2,3,3,3-tetrafluoro-2-(l,l,2,2,3,3,3-heptafluoropropoxy)-,
ammonium salt"[tw] OR "Perfluorinated aliphatic carboxylic acid,
ammonium salt"[tw] OR "2,3,3,3-Tetrafluoro-2-(l,l,2,2,3,3,3-
heptafluoropropoxy)propanoic acid, ammonium salt"[tw] OR
"Ammonium 2-(perfluoropropoxy)perfluoropropionate"[tw] OR
"Ammonium Perfluoro(2-methyl-3-oxahexanoate)"[tw] OR
"Ammonium perfluoro(2-methy 1-3-oxahexanoic) acid"[tw] OR
"Ammonium perfluoro-2-methy 1-3-oxahexanoate"[tw] OR "FRD-
902"[tw] OR "GenX-H3N"[tw] OR "HFPO-DA"[tw] OR "Propanoic
acid, 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-, ammonium salt"[tw]
OR "Undecafluoro-2-methy 1-3-oxahexanoic acid"[tw] OR ((GenX[tw]
AND (fluorocarbon*[tw] ORfluorotelomer*[tw] ORpolyfluoro*[tw]
ORperfluoro-*[tw] ORperfluoroa*[tw] ORperfluorob*[tw] OR
perfluoroc*[tw] ORperfluorod*[tw] ORperfluoroe*[tw] OR
perfluoroh*[tw] ORperfluoron*[tw] ORperfluoroo*[tw] OR
perfluorop*[tw] ORperfluoros*[tw] ORperfluorou*[tw] OR
perfluorinated[tw] OR fluorinated[tw])) OR (("Undecafluoro-2-
methyl-3-oxahexanoic" [tw] OR "Ammonium perfluoro(2-methy 1-3-
oxahexanoic)"[tw] OR "2,3,3,3-Tetrafluoro-2-(l,l,2,2,3,3,3-
heptafluoropropoxy)"[tw] OR "Perfluorinated aliphatic
carboxylic "[tw]) AND (salt[tw] OR salts[tw] OR acid[tw] OR
acids[tw])))) OR (((Undecafluoro AND oxahexanoic) OR
(Ammonium AND perfluoro AND oxahexanoic) OR (Tetrafluoro
AND heptafluoropropoxy) OR "Perfluorinated aliphatic
carboxylic"[tw] OR "Perfluorinated aliphatic carboxylic"[tw]) AND
(salt[tw] OR salts[tw] OR acid[tw] OR acids[tw]))
A-3
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OCTOBER 2021
Database
HFPO dimcr acid (CASRN 13252-13-6)
HFPO dimcr acid ammonium salt (CASRN 62037-80-3)
wos
TS="2,3,3,3-Tetrafluoro-2-(heptafluoropropoxy)propionic acid" OR
TS="2,3,3,3-tetrafluoro-2-(l,l,2,2,3,3,3-heptafluoropropoxy)-
Propanoic acid" OR TS="Perfluoro(2-methyl-3-oxahexanoate)" OR
TS="Propanoic acid, 2,3,3,3-tetrafluoro-2-(l,l,2,2,3,3,3-
heptafluoropropoxy)-" OR TS="Perfluorinated aliphatic carboxylic
acid" OR TS="Perfluoro(2-methyl-3-oxahexanoic) acid" OR
TS="2,3,3,3-tetrafluoro-2-(l,l,2,2,3,3,3-
heptafluoropropoxy)propanoic acid" OR TS="2,3,3,3-tetrafluoro-2-
(heptafluoropropoxy)propanoic acid" OR TS="perfluoro-2-
(propyloxy)propionic acid" OR TS="perfluoro-2-methyl-3-
oxahexanoic acid" OR TS="perfluoro-2-propoxypropanoic acid" OR
TS="perfluoro-2-propoxypropionic acid" OR TS="perfluoro-a-
propoxypropionic acid" OR TS="propanoic acid, 2,3,3,3-tetrafluoro-
2-(heptafluoropropoxy)-" OR TS="propionic acid, 2,3,3,3-tetrafluoro-
2-(heptafluoropropoxy)-" OR (TS="GenX" AND TS=(fluorocarbon*
OR fluorotelomer* OR polyfluoro* OR perfluoro-* OR perfluoroa*
OR perfluorob* OR perfluoroc* OR perfluorod* OR perfluoroe* OR
perfluoroh* OR perfluoron* OR perfluoroo* OR perfluorop* OR
perfluoros* OR perfluorou* OR perfluorinated OR fluorinated OR
PFAS ORPFOS OR PFOA)) OR ((TS="2,3,3,3-Tetrafluoro-2-
(heptafluoropropoxy)propionic" OR TS="2,3,3,3-tetrafluoro-2-
(l,l,2,2,3,3,3-heptafluoropropoxy)-Propanoic" OR
TS="Perfluorinated aliphatic carboxylic" OR TS="Perfluoro(2-
methyl-3-oxahexanoic)" OR TS="2,3,3,3-tetrafluoro-2-(l, 1,2,2,3,3,3-
heptafluoropropoxy)propanoic" OR TS="2,3,3,3-tetrafluoro-2-
(heptafluoropropoxy)propanoic" OR TS="perfluoro-2-
(propyloxy)propionic" OR TS="perfluoro-2-methyl-3-oxahexanoic"
OR TS="perfluoro-2-propoxypropanoic" OR TS="perfluoro-2-
propoxypropionic" OR TS="perfluoro-a-propoxypropionic") AND
TS=(acid OR acids))
TS=("Ammonium 2,3,3,3-tetrafluoro-2-
(heptafluoropropoxy)propanoate" OR "Propanoic acid, 2,3,3,3-
tetrafluoro-2-(l,l,2,2,3,3,3-heptafluoropropoxy)-, ammonium salt"
OR "Perfluorinated aliphatic carboxylic acid, ammonium salt" OR
"2,3,3,3-Tetrafluoro-2-( 1,1,2,2,3,3,3-heptafluoropropoxy)propanoic
acid, ammonium salt" OR "Ammonium 2-
(perfluoropropoxy)perfluoropropionate" OR "Ammonium
Perfluoro(2-methyl-3-oxahexanoate)" OR "Ammonium perfluoro(2-
methyl-3-oxahexanoic) acid" OR "Ammonium perfluoro-2-methyl-3-
oxahexanoate" OR "FRD-902" OR "GenX-H3N" OR "HFPO-DA"
OR "Propanoic acid, 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-,
ammonium salt" OR "Undecafluoro-2-methyl-3-oxahexanoic acid")
OR ((TS=GenX AND (TS=(fluorocarbon* OR fluorotelomer* OR
polyfluoro* OR perfluoro-* OR perfluoroa* OR perfluorob* OR
perfluoroc* OR perfluorod* OR perfluoroe* OR perfluoroh* OR
perfluoron* OR perfluoroo* OR perfluorop* OR perfluoros* OR
perfluorou* OR perfluorinated OR fluorinated)))) OR
((TS=("Undecafluoro-2-methyl-3-oxahexanoic" OR "Ammonium
perfluoro(2-methyl-3-oxahexanoic)" OR "2,3,3,3-Tetrafluoro-2-
(1,1,2,2,3,3,3-heptafluoropropoxy)" OR "Perfluorinated aliphatic
carboxylic" OR "Perfluorinated aliphatic carboxylic")) AND (TS=(salt
OR salts OR acid OR acids)))
Timespan: All years. Indexes: SCI-EXPANDED, CPCI-S,
CPCI-SSH, BKCI-S, BKCI-SSH, CCR-EXPANDED, IC.
A-4
-------
OCTOBER 2021
Database
HFPO dimcr acid (CASRN 13252-13-6)
HFPO dimcr acid ammonium salt (CASRN 62037-80-3)
Toxline
(13252-13-6[rn] OR "2,3,3,3-Tetrafluoro-2-
(heptafluoropropoxy)propionic acid" OR "2,3,3,3-tetrafluoro-2-
(l,l,2,2,3,3,3-heptafluoropropoxy)-Propanoic acid" OR "Perfluoro(2-
methyl-3-oxahexanoate)" OR "Propanoic acid, 2,3,3,3-tetrafluoro-2-
(1,1,2,2,3,3,3-heptafluoropropoxy)-" OR "Perfluorinated aliphatic
carboxylic acid" OR "Perfluoro(2-methyl-3-oxahexanoic) acid" OR
"2,3,3,3 -tetrafluoro-2-(l, 1,2,2,3,3,3 -heptafluoropropoxy propanoic
acid" OR "2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid"
OR "perfluoro-2-(propyloxypropionic acid" OR "perfluoro-2-methyl-
3-oxahexanoic acid" OR "perfluoro-2-propoxypropanoic acid" OR
"perfluoro-2-propoxypropionic acid" OR "perfluoro-a-
propoxypropionic acid" OR "propanoic acid, 2,3,3,3-tetrafluoro-2-
(heptafluoropropoxy)-" OR "propionic acid, 2,3,3,3-tetrafluoro-2-
(heptafluoropropoxy)-" OR (GenX AND (fluorocarbon* OR
fluorotelomer* OR polyfluoro* OR perfluoro* OR perfluorinated OR
fluorinated OR PFAS OR PFOS OR PFOA)) OR (("2,3,3,3-
Tetrafluoro-2-(heptafluoropropoxy)propionic" OR "2,3,3,3-
tetrafluoro-2-(l, 1,2,2,3,3,3-heptafluoropropoxy)-Propanoic" OR
"Perfluorinated aliphatic carboxylic" OR "Perfluoro(2-methyl-3-
oxahexanoic)" OR "2,3,3,3-tetrafluoro-2-(l,l,2,2,3,3,3-
heptafluoropropoxy)propanoic" OR "2,3,3,3-tetrafluoro-2-
(heptafluoropropoxy)propanoic" OR "perfluoro-2-
(propyloxy)propionic" OR "perfluoro-2-methyl-3-oxahexanoic" OR
"perfluoro-2-propoxypropanoic" OR "perfluoro-2-propoxypropionic"
OR "perfluoro-a-propoxypropionic") AND (acid OR acids))) AND ((
aneupl [org] OR biosis [org] OR cis [org] OR dart [org] OR pubdart
[org] OR emic [org] OR epidem [org] OR fedrip [org] OR heep [org]
OR hmtc [org] OR ipa [org] OR riskline [org] OR mtgabs [org] OR
niosh [org] OR ntis [org] OR pestab [org] OR ppbib [org]) AND
NOT pubmed [org] AND NOT pubdart [org])
(62037-80-3[rn] OR "Ammonium 2,3,3,3-tetrafluoro-2-
(heptafluoropropoxy)propanoate" OR "Propanoic acid, 2,3,3,3-
tetrafluoro-2-(l,l,2,2,3,3,3-heptafluoropropoxy)-, ammonium salt"
OR "Perfluorinated aliphatic carboxylic acid, ammonium salt" OR
"2,3,3,3 -Tetrafluoro-2-( 1,1,2,2,3,3,3 -heptafluoropropoxy propanoic
acid, ammonium salt" OR "Ammonium 2-
(perfluoropropoxy)perfluoropropionate" OR "Ammonium
Perfluoro(2-methyl-3-oxahexanoate)" OR "Ammonium perfluoro(2-
methyl-3-oxahexanoic) acid" OR "Ammonium perfluoro-2-methy 1-3-
oxahexanoate" OR "FRD-902" OR "GenX-H3N" OR "HFPO-DA"
OR "Propanoic acid, 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-,
ammonium salt" OR "Undecafluoro-2-methyl-3-oxahexanoic acid"
OR "GenX" OR (("Undecafluoro-2-methyl-3-oxahexanoic" OR
"Ammonium perfluoro(2-methyl-3-oxahexanoic)" OR "2,3,3,3-
Tetrafluoro-2-(l,l,2,2,3,3,3-heptafluoropropoxy)" OR "Perfluorinated
aliphatic carboxylic" OR "Perfluorinated aliphatic carboxylic") AND
(salt OR salts OR acid OR acids))) AND ((aneupl [org] OR biosis
[org] OR cis [org] OR dart [org] OR pubdart [org] OR emic [org] OR
epidem [org] OR fedrip [org] OR heep [org] OR hmtc [org] OR ipa
[org] OR riskline [org] OR mtgabs [org] OR niosh [org] OR ntis [org]
OR pestab [org] OR ppbib [org]) AND NOT pubmed [org] AND
NOT pubdart [org])
TSCATS 1
13252-13-6[rn] AND (TSCATS [org])
62037-80-3 [rn] AND (TSCATS [org])
Notes'. PFAS = per- and polyfluoroalkyl substances; PFOA = perfluorooctanoic acid; PFOS = perfluorooctane sulfonate; TSCATS = Toxic Substances Control Act Test
Submissions; WOS = Web of Science.
A-5
-------
OCTOBER 2021
Table A-3. Processes Used to Augment the Search of Core Databases for HFPO Dimer Acid (CASRN 13252-13-6)
System used
Selected key rcfcrcncc(s) or sources
TSCATS3
TSCA Test Submissions 2.0; website now retired dittDs://vosemite.era.gov7oDDts/eratscat8.iisf/ReDortSearch?ODenForni)
Chemical Data Access Tool (CDAT); website now retired dittDs://iava.eDa.gov/oDDt chemical search/)
ChemView dittos://iava .eoa. gov/chemv iew~)
Resources searched for
physiochemical property
information
Agencv for Toxic Substances and Disease Registry (ATSDR) dittDs://www.atsdr.cdc.gov/)
Australian National Industrial Chemicals Notification and Assessment Scheme CNICNAS) dittos://www.nicnas.gov.au/chemical-
information)
CAMEO Chemicals (httDs://cameochemicaIs.noaa.gov/)
Canada DSL List (httD://webnet.oecd.org/CCRWEB/Search.asDx)
Chemical Risk Information Platform (CHRIP) dittD://www.nite.eo.io/eii/clieiii/clirio/clirio search/svstemToDN)
ChemlDolus dittos://cliem.tiliri.nili.sov/cliemidoliis/N)
ChemSmder (httD://www.chemsDider.com/)
CRC Handbook of Chemistry and Physics
(httD://hbc do nl ine.com/face s/contents/ContentsSearch.xhtml;isessionid=9408875156F724E0E945D3A6D0454891")
ECHA Information on Chemicals (httDs://echa.euroDa.eu/)
eChemPortal dittos://www.echemtx)rtal.org/echemtx)rtal/index.action)
Hazardous Substances Data Bank (HSDB) httDs://toxiiet.nlnuuh.gov/cgi-bin/sis/htnilgen?HSDBN)
HSNO Chemical Classification and Information Database (CCID) updated linkb
rtittDs://www.eDa.govt.nz/database-search/chemical-classification-and-information-database-ccid/)
IARC Monographs dittD://www.inchem.org/Daees/iarc.litiiiB
Integrated Risk Information Svstem (IRIS) dittos://www.era.gov/iris)
J-Check dittD://www.safe.nite.go.iD/icheck/search.action?reauest locale=en)
Kirk-Othmer Encyclopedia of Chemical Technology updated linkb
dittDs://onlinelibrarv.wilev.com/doi/book/l 0.1002/0471238961)
NIEHS (httDs://www.niehs.nih.gov/)
OSHA Occupational Chemical Database dittDs://www.osha.gov/chemicaldata/)
PubChem dittos ://Diibchein. iicbi.iiliii.iiih.gov/searcli/iiidex.htmn
SRC Fate Pointers dittD://esc.sv rres.com/fatetx> inter/search, aso)
Ullmann's Encvclopedia updated link' dittDs://onlinelibrarv.wilev.com/doi/book/l 0.1002/14356007")
EPA ACToR dittDs://actor.eDa.gov/actor/home.xhtmB
EPA CDAT; website now retired ditti)s://iava.eDa.gov/oDDt chemical search/)
EPA Chemistry Dashboard dittos ://co iriDtox. era. gov/dashboard/")
EPA ChemView dittos://iava.era.gov/chemview")
EPA Substance Registry Services (SRS)
dittos://ofniDub.era.gov/sor interne t/registrv/substreg/searchandretrieve/substaneesearch/search.do")
Web-based search for chemical manufacturer documents
A-6
-------
OCTOBER 2021
System used
Selected key rcfcrcncc(s) or sources
Resources searched for
health effects,
toxicokinetics, and
mechanistic information
ATSDR (httD://www.atsdr.cdc.20v/substarices/index.asDN)
CalEPA OEHHA dittD://www.oeMia.ca.eov/risk.litniB
CPSC ChttD://www.cDsc.eovN)
ECHA dittD://eclia.eiiroDa.eii/iiifoniiatioii-oii-clieiiiicalsN)
eChemPortal0 ditto://www.echemportal.org/echeniTX)rtalA
EFSA Europe ditto://www.efsa.eurooa.eu/)
Environment Canada (IittD://www.ec.gc.ca/default.asD?lang=En&n=ECD35C36N)
European Union Risk Assessment Reports dittDs://ec.euroDa.eu/irc/eii/Dublicatioiis-listN)
Federal Docket (lit tp://www. regulations. gov)
Health Canada (httDs://www.canada.ca/en/health-catiada.htmn
IARC dittD://iiioiioeraDlis.iarc.fr/ENG/Classification/iiidex.DliDN)
ITER ditto ://www. tera. o rg/iterA
Japan Existing Chemical Data Base ChttD://dra4.nihs.go.iD/nihlw data/iso/SearchPageENG.isd)
NICNAS ditto://www. nicnas. gov.au/chemical-infomiatioirt
NIEHS ditto://www.iiielis.iiili.eov/N)
NTP ditto://iitDsearcli.iiielis.nili.gov/hoirieN)
OEHHA Toxicity Criteria Database (httD://www.oehha.ca.gov/tcdb/index.asDN)
EPA NSCEP dittDs://www.eDa.gov/nsceDN)
FDA ChttD://www.fda.govA
WHO dittD://www.wlio.iiit/iDcs/assessiiieiit/eii/N)
Notes'. TSCATS = Toxic Substances Control Act Test Submissions
a Only relevant TSCATS studies from these interfaces were added to the HERO project page.
b The URL has been updated (as listed here) since the literature search; during the search, a previous URL was used.
c eChemPortal includes the following databases: ACToR, AGRITOX, CCR, CCR DATA, CESAR, CHRIP, ECHA CHEM, EnviChem, EPA-IRIS, EPA-SRS, ESIS, GHS-J,
LIP VIS, HSDB, HSNO CCID, INCHEM, J-CHECK, JECDB, NICNAS PEC, OECD-HPV, OECD SIDS IUCLID, SIDS UNEP, and UK CCRMP Outputs.
A-7
-------
OCTOBER 2021
Table A-4. Processes Used to Augment the Search of Core Databases for HFPO Dimer Acid Ammonium Salt (CASRN 62037-
80-3)
System used
Scleetcd key rcfcrcncc(s) or sources
TSCATS3
Chemical Data Access Tool (CDAT); website now retired (Imps.//iava.era.gov/oppi ulienuuai searuli/)
ChemView dittos://iava.era. eov/chemvlew)
Resources searched for
physiochemical property
information
ATSDR dittDs://www.atsdr.cdc. gov/)
CAMEO Chemicals (httDs://cameochemicaIs.noaa.gov/)
Canada DSL List (httD://webnet.oecd.org/CCRWEB/Search.asDx)
ChemlDolus ClittDs://cheiii.iiliii.iiih.eov/chemidi>liisA
CRC Handbook of Chemistry and Physics
(httD://hbc do nline.com/face s/contents/ContentsSearch.xhtml;isessiomd=9408875l56F724E0E945D3A6D0454891)
ECHA Information on Chemicals (httDs://echa.euroDa.eu/)
eChemPortal dittos://www.echeniDortal.org/echemtx)rtal/index.action)
Hazardous Substances Data Bank (HSDB) (httDs://toxnet.nIm.nih.gov/cgi-bin/sis/htmlgen?HSDB)
HSNO Chemical Classification and Information Database (CCID) updated linkb
rtittDs://www.eDa.govt.nz/database-search/chemical-classification-and-information-database-ccid/)
IARC Monographs (httD://www.inchem.org/Dages/iarc.html)
Integrated Risk Information Svstem (IRIS) dittos://www.era.gov/iris)
J-Check dittD://www.safe.nite.go.iD/icheck/search.action?reauest locale=en)
Kirk-Othmer Encyclopedia of Chemical Technology updated linkb
ChttDs://onlinelibrarv.wilev.co m/doi/book/10.1002/0471238961
NICNAS (httDs://www.rucnas. gov.au/chemical-information)
NIEHS (httDs://www.niehs.nih.gov/)
OSHA Occupational Chemical Database (httDs://www.osha.gov/chemicaldata/)
PubChem dittos ://Diibchein. iicbi.iiliTi.iiih.gov/searcli/index.litiiin
SRC Fate Pointers dittD://esc.sv rres.com/fateDointer/search.asD)
Ullmann's Encyclopedia updated link' dittDs://onlinelibrarv.wilev.com/doi/book/l 0.1002/14356007)
EPA ACToR dittDs://actor.eDa.gov/actor/home.xhtml)
EPA CD AT; website now retired ditti)s://iava.eDa.gov/oDDt chemical search/)
EPA Chemistry Dashboard dittos ://co iriDtox. era. gov/dashboard/)
EPA ChemView (httDs://iava.era.gov/chemview)
EPA Substance Registry Services (SRS)
ChttDs://ofmDub.era.gov/sor internet/registrv/substreg/searchandretrieve/substancesearch/search.do)
Web-based search for chemical manufacturer documents
A-8
-------
OCTOBER 2021
System used
Scleetcd key rcfcrcncc(s) or sources
Resources searched for health
effects, toxicokinetics, and
mechanistic information
ATSDR (httD://www.atsdr.cdc.20v/substaiices/index.asDN)
CalEPA - OEHHA (httD://www.oehha.ca.gov/risk.htnik littD://www.oelilia.ca.sov/tcdb/iiidex.asDN)
CPSC ChttD://www.cDsc.eovN)
ECHA ('httD://echfl.euroKl.ell/info^nation-on-chemicals,)
eChemPortal0 (littPl//www;ecJienii)o^
EFSA Europe ditto://www.efsa.eurooa.eu/)
Environment Canada (IittD://www.ec.gc.ca/default.asD?lang=En&n=ECD35C36N)
EPA-NSCEP ChttDs://www.eDa.gov/nsceD)
European Union Risk Assessment Reports (httDs://ec.eurora.eu/irc/en/Dublicatioiis-lisO
Federal Docket (lit to://www. regulations. gov)
Google (Quick search onlv www.google.com)
Health Canada (httDs://www.canada.ca/en/health-canada.html)
IARC dittD://iiioiioeraDlis.iarc.fr/ENG/Classification/iiidex.DliDN)
ITER (TERA database) dittD://www. tera.o rg/iter/)
Japan Existing Chemical Data Base (JECDB) ChttD://dra4.nihs.go.io/irihlw data/isD/SearchPageENG.isD)
NICNAS ditto ://www. nicnas. gov. an/chemical -info nnatio n)
NIEHS dittD://www.niehs.nih.gov/)
NTP ditto://iitDsearcli.iiielis.nili.gov/home)
FDA (httD://www.fda.gov)
WHO (httD://www.who.int/iDcs/assessment/en/)
Notes'. TSCATS = Toxic Substances Control Act Test Submissions
a Only relevant TSCATS studies from these interfaces were added to the HERO project page.
b The URL has been updated (as listed here) since the literature search; during the search, a previous URL was used.
c eChemPortal includes the following databases: ACToR, AGRITOX, CCR, CCR DATA, CESAR, CHRIP, ECHA CHEM, EnviChem, EPA-IRIS, EPA-SRS, ESIS, GHS-J,
LIP VIS, HSDB, HSNO CCID, INCHEM, J-CHECK, JECDB, NICNAS PEC, OECD-HPV, OECD SIDS IUCLID, SIDS UNEP, and UK CCRMP Outputs.
A-9
-------
OCTOBER 2021
Table A-5. Inclusion-Exclusion Criteria for HFPO Dimer Acid and HFPO Dimer Acid Ammonium Salt Studies
PECO
Parameter
Inelusion eriteria
Exelusion eriteria
Population
• Humans
• Standard mammalian animal models, including rat, mouse, rabbit,
guinea pig, hamster, monkey, dog
• Alternative animal models in standard laboratory conditions (e.g.,
Xenopus, zebrafish, minipig)
• Human or animal cells, tissues, or organs (not whole animals); bacteria;
nonmammalian eukaryotes; other nonmammalian laboratory species
• Ecological species
Exposure
• Exposure is to HFPO dimer acid and/or its ammonium salt
• Exposure via oral, inhalation, dermal, intraperitoneal, or intravenous
injection routes
• Exposure is measured in air, dust, drinking water, diet, gavage, or
injection vehicle, or via a biomarker of exposure (PFAS levels in whole
blood, serum, plasma, or breast milk)
• Exposure is via cells in culture or subcellular matrices
• Study population is not exposed to HFPO dimer acid and/or
its ammonium salt
• Exposure is to a mixture only without evaluating HFPO
dimer acid and/or its ammonium salt individually
Outcome
• Studies that include a measure of one or more health effect endpoints,
including effects on reproduction, development, developmental
neurotoxicity, liver, thyroid, immune system, nervous system,
genotoxicity, and cancer
• In vivo and/or in vitro studies related to toxicity mechanisms or
physiological effects/adverse outcomes, and studies useful for
elucidating toxic modes of action
• Qualitative or quantitative description of absorption, distribution,
metabolism, elimination, and toxicokinetic and/or toxicodynamic
models (e.g., PBPK, PBTK, PBTK/TD)
• Studies addressing risks to infants, children, pregnant women,
occupational workers, the elderly, and any other susceptible or
differentially exposed populations
A-10
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OCTOBER 2021
PECO
Parameter
Inelusion eriteria
Exelusion eriteria
Other
• Structure and physiochemical properties
• Reviews and regulatory documents
Not on topic, including:3
• Abstract only, inadequately reported abstract, or no abstract
and not considered further because study was not potentially
relevant
• Bioremediation, biodegradation, or chemical or physical
treatment of HFPO dimer acid and/or its ammonium salt,
including evaluation of wastewater treatment technologies
and methods for remediation or contaminated water and soil
• Ecosystem effects, studies in ecological species that are not
relevant to health effects in humans
• Studies of environmental fate and transport of HFPO dimer
acid and/or its ammonium salt compounds in environmental
media
• Analytical methods for detecting/measuring HFPO dimer
acid and/or its ammonium salt compounds in environmental
media and use in sample preparations and assays
• Studies describing the manufacture and use of HFPO dimer
acid and/or its ammonium salt compounds
• Not chemical-specific (studies that do not involve testing of
HFPO dimer acid and/or its ammonium salt compounds)
• Studies that describe measures of exposure to HFPO dimer
acid and/or its ammonium salt compounds without data on
associated health effects
Notes'. PBPK = physiologically based pharmacokinetic; PBTK = physiologically based toxicokinetic; PBTK/TD = physiologically based toxicokinetic and toxicodynamic; PFAS =
pre- and polyfluoroalkyl substances.
a Although these criteria were used for the peer-reviewed literature, the current document describes environmental fate data submitted by DuPont (now the Chemours Company). A
subsequent targeted search for bioconcentration and bioaccumulation data was also conducted. In addition, a summary of occurrence data is also provided in the current document
to give context to the toxicity values.
A-ll
-------
OCTOBER 2021
Table A-6. Inclusion-Exclusion Criteria for HFPO Dimer Acid and HFPO Dimer Acid Ammonium Salt Studies after the
Public Comment Draft
PECO
Parameter
Inelusion eriteria
Exelusion eriteria
Population
• Humans
• Standard mammalian animal models, including rat, mouse,
rabbit, guinea pig, hamster, monkey, dog
• Ecological species (supplemental tag —non-PECO model)
• Alternative animal models in standard laboratory conditions
(e.g,,Xenopus, zebrafish, minipig) (supplemental tag—non-PECO
model)
• Human or animal cells, tissues, or organs (not whole animals);
bacteria; nonmammalian eukaryotes; other nonmammalian laboratory
species (supplemental tag—mechanistic)
Exposure
• Exposure is to HFPO dimer acid and/or its ammonium salt
• Must include 2 or more levels of exposure to HFPO dimer acid
and/or its ammonium salt (if not stated, include at title/abstract
screening)
• Humans: Exposure is measured in air. dust, drinkine water,
diet, or gavage or injection vehicle, or via a biomarker of
exposure (PFAS levels in whole blood, serum, plasma, or
breast milk)
• Any exposure length is acceptable
• Animals: Exposure via oral route onlv
• Any exposure length for an animal study is acceptable for
reproductive or developmental exposures
• Exposure duration for all other animal study designs require an
exposure duration of 28 days or more (if not stated, include at
title/abstract screening)
• Study population is not exposed to HFPO dimer acid and/or its
ammonium salt
• There is only 1 exposure group (supplemental tag—single-dose group
in study)
• Exposure is to a mixture only without evaluating HFPO dimer acid
and/or its ammonium salt individually (supplemental tag—mixture
study)
• Exposure via inhalation, dermal, intraperitoneal, or intravenous
injection routes (supplemental tag—non-oral route of administration)
• Exposure is via cells in culture or subcellular matrices (supplemental
tag—mechanistic)
• Acute exposures (< 28 days) in animal studies (supplemental tag—
acute/short-term duration exposures)
Comparator
• A concurrent control group exposed to vehicle-only treatment
or an untreated control
• A comparison or referent population exposed to HFPO dimer
acid and/or its ammonium salt at lower levels (or no
exposure/exposure below detection limits) or for shorter
periods of time
• Biological monitoring (e.g., whole blood, serum, plasma, or
breast milk) that can be used to establish a range of exposure
• Case reports and case series (supplemental tag—case report or case
series)
A-12
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OCTOBER 2021
PECO
Parameter
Inelusion eriteria
Exelusion eriteria
Outcome
• Studies that include a measure of one or more health effect
endpoints, including effects on reproduction, development,
developmental neurotoxicity, liver, thyroid, immune system,
nervous system, genotoxicity, and cancer
• Qualitative or quantitative description of absorption,
distribution, metabolism, elimination, and toxicokinetic and/or
toxicodynamic models (e.g., PBPK, PBTK, PBTK/TD)
• In vivo and/or in vitro studies related to toxicity mechanisms,
physiological effects/adverse outcomes, and studies useful for
elucidating toxic modes of action (supplemental tag—mechanistic)
• Studies addressing risks to infants, children, pregnant women,
occupational workers, the elderly, and any other susceptible or
differentially exposed populations (supplemental tag—susceptible
population)
Other Exclusion
Criteria
• Not on topic, including:
• Structure and physiochemical properties (supplemental tag—
structure and physiochemical properties)
• Reviews and regulatory documents (supplemental tag—other
assessments or records with no original data)
• Abstract only, inadequately reported abstract, or no abstract and not
considered further because study was not potentially relevant
{supplemental tag—conference abstract)
• Ecosystem effects, studies in ecological species that are not relevant
to health effects in humans (supplemental tag—non-PECO model)
• Bioaccumulation of the target chemical in fish (supplemental tag—
bioaccumulation data in fish)
• Studies of environmental fate and transport of HFPO dimer acid
and/or its ammonium salt compounds in environmental media or food
{supplemental tag—environmental fate or occurrence)
• Studies that describe measures of exposure to HFPO dimer acid
and/or its ammonium salt compounds without data on associated
health effects {supplemental tag—exposure characteristics)
• Bioremediation, biodegradation, or chemical or physical treatment of
HFPO dimer acid and/or its ammonium salt, including evaluation of
wastewater treatment technologies and methods for remediation or
contaminated water and soil
• Analytical methods for detecting/measuring HFPO dimer acid and/or
its ammonium salt compounds in environmental media and use in
sample preparations and assays
• Studies describing the manufacture and use of HFPO dimer acid
and/or its ammonium salt compounds
• Not chemical specific (studies that do not involve testing of HFPO
dimer acid and/or its ammonium salt compounds)
Notes: PBPK = physiologically based pharmacokinetic; PBTK = physiologically based toxicokinetic; PBTK/TD = physiologically based toxicokinetic and
toxicodynamic; PFAS = per- and polyfluoroalkyl substances.
A-13
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OCTOBER 2021
Table A-7. Supplemental Tags for the GenX Chemicals Literature Search
Category
Evidence
Mechanistic studies
Studies reporting measurements related to a health outcome that inform the biological or chemical events associated with
phenotypic effects, in both mammalian and non-mammalian model systems, including in vitro, in vivo (by various routes of
exposure), ex vivo, and in silico studies. When possible, mechanistic studies will be sub-tagged as pertinent to cancer, non-cancer,
or unclear/unknown.
Non-mammalian model
systems
Studies in non-mammalian model systems (e.g., fish, birds, C. elegans).
ADME and toxicokinetic
Studies designed to capture information regarding absorption, distribution, metabolism, and excretion, including toxicokinetic
studies. Such information might be helpful in updating or revising the parameters used in existing PBPK models.
Acute/short-term duration
exposures
Animal studies of less than 28 days.
Single-dose group
Studies that used only a single-dose group were tagged as supplemental due to the GenX chemicals database having several multi-
dose group studies.
Exposure characteristics
Studies that include data unrelated to toxicological endpoints, but which provide information on exposure sources or measurement
properties of the environmental agent (e.g., demonstrate a biomarker of exposure).
Susceptible populations
Studies that identify potentially susceptible subgroups (e.g., studies that focus on a specific demographic, life stage, or genotype).
Mixture studies
Studies not considered PECO-relevant because they do not contain an exposure or treatment group assessing only the chemical of
interest.
Non-oral routes of exposure
Studies not addressing routes of exposure that fall outside the PECO scope, and include inhalation and dermal exposure routes
Case studies or case series
Case reports and case series will be tracked as potentially relevant supplemental information.
Records with no original
data
Records that do not contain original data such as other agency assessments, informative scientific literature reviews, editorials, or
commentaries.
Conference abstracts
Records that contain insufficient documentation to support study evaluation and data extraction.
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Category
Bioaccumulation in fish
BAFs were mentioned in the public comment draft assessment.
Notes'. ADME = absorption, distribution, metabolism, and excretion; BAFs = bioaccumulation factors; PBPK = physiologically based pharmacokinetic; and PECO = population,
exposure, comparator, and outcome.
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Appendix B: Acute and 7-Day Study Summaries
This appendix summarizes studies evaluating acute exposure to hexafluoropropylene oxide
(HFPO) dimer acid or HFPO dimer acid ammonium salt by the oral, dermal, and inhalation
routes of exposure and investigating dermal and eye irritation.
Oral Toxicity. In a study of the HFPO dimer acid ammonium salt (no Test Guideline (TG)
cited), a single dose of 1.5, 12, 130, 1,000, 2,250, 3,400, 5,000, 7,500, 11,000, 12,963, or 17,000
milligrams per kilogram (mg/kg) of HFPO dimer acid ammonium salt was administered by
stomach tube to young male rats. The approximate lethal dose (ALD) was determined to be
7,500 mg/kg. Discomfort, gasping, and tonic convulsions were observed before death at lethal
doses (7,500 mg/kg and higher). Discomfort, increased water intake, inactivity, polyuria, and
initial weight loss were observed in rats at the three highest sublethal doses (2,250 mg/kg, 3,400
mg/kg, and 5,000 mg/kg). Slightly enlarged livers with enlarged hepatocytes and pronounced
cell membranes were also observed in rats at the three highest sublethal doses. Slight-to-
moderate degenerative changes in the pancreas were also observed in doses at 2,250 mg/kg and
higher. No effects were observed at doses of less than or equal to 1,000 mg/kg (DuPont-2-63,
1963).
In another study evaluating toxicity of HFPO dimer acid ammonium salt by the oral route of
exposure (no TG identified), a single dose of 670, 2,300, 3,400, 5,000, 7,500, or 11,000 mg/kg of
HFPO dimer acid ammonium salt (purity > 99%) was administered to 7-week-old male rats
(1/dose group). Rats were evaluated for clinical signs of toxicity over a 14-day observation
period. No clinical signs of toxicity were observed in the rat dosed at 670 mg/kg. Rats dosed at
2,300 and 3,400 mg/kg exhibited weight loss (17% and 14%, respectively); ruffled fur; and a
wet, yellow-stained perineum at 1 day postexposure. The rats dosed at 2,300 and 3,400 mg/kg no
longer exhibited these effects at 2 days and 4 days postexposure, respectively. Rats dosed with
greater than or equal to 5,000 mg/kg died by 1 day after dosing. The rat dosed with 11,000
mg/kg exhibited lethargy, low carriage, and low posture before its death. The ALD was
determined to be 5,000 mg/kg (DuPont-770-95, 1996).
A single dose of HFPO dimer acid ammonium salt (82.6% purity) was administered by oral
gavage to 10- to 11-week-old female rats at a dose of 175, 550, 1750, or 5,000 mg/kg (1-3
rats/group) in a study conducted according to Organization of Economic Cooperation and
Development (OECD) TG 425 (Up-and-Down Procedure) (OECD, 2008c). Rats were then
evaluated for clinical signs of toxicity over a 14-day observation period. All rats exhibited
clinical signs of toxicity such as hair loss, lethargy, high posture, stained fur/skin, clear ocular
discharge, prostrate posture, partially closed eyes, or salivation. With the exception of hair loss,
clinical signs disappeared by 2 days postexposure. All three rats dosed at 5,000 mg/kg died
within 2 days after dosing. Grossly observable evidence of organ or tissue damage in these rats
included discoloration of lungs (rat #1651), discoloration of lungs and mandibular lymph nodes
(rat #1746), and discoloration of lungs and liver (rat #1975). No visible lesions were observed in
females dosed at 175 mg/kg, 550 mg/kg, or 1,750 mg/kg. With the exception of rats dosed at
5,000 mg/kg, increases in body weight (BW) were observed in all rats over the course of the
study. The oral median lethal dose (LD50) was estimated to be 3,129 mg/kg for female rats
(DuPont-22932, 2007).
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Oral toxicity of HFPO dimer acid ammonium salt was also evaluated in male rats in a study
conducted according to OECD TG 425 (OECD, 2008c). A single dose of HFPO dimer acid
ammonium salt (86% purity) was administered by oral gavage to 9- to 11-week-old male rats at a
dose of 175, 550, 1,750, or 5,000 mg/kg (three rats). Rats were then evaluated for clinical signs
of toxicity over a 14-day observation period. All rats exhibited clinical signs of toxicity such as
lethargy, wet fur, stained fur/skin, decreased muscle tone, low posture, or lung noise. One rat
dosed at 1,750 mg/kg and all three rats dosed at 5,000 mg/kg died either the day dosed or by the
day after dosing. Grossly observable evidence of organ or tissue damage in rats dosed at 5,000
mg/kg included expanded lungs and discolored stomach, discoloration and cloudiness of eyes,
and stained skin. With the exception of rats dosed at 5,000 mg/kg, increases in BW were
observed in all rats over the course of the study. The oral LD50 was determined to be 1,750
mg/kg for male rats (DuPont-25438 RV1, 2008).
Another study evaluated oral toxicity of HFPO dimer acid in both male and female rats in a
study conducted according to OECD TG 425 (OECD, 2008c). A single dose of HFPO dimer acid
(98% purity) was administered to 9- to 11-week-old rats. Males were dosed at 175, 550, 1,750,
or 5,000 mg/kg (2-6 rats/group). Female rats were also dosed at 175, 550, 1,750, or 5,000 mg/kg
(1-4 rats/group). Clinical signs were not observed in rats dosed at 175 mg/kg or in one male rat
dosed at 550 mg/kg. The rest of the rats in this study exhibited clinical signs of toxicity. Clinical
signs of toxicity in male rats observed up to 5 days after dosing included lung noise, absent
feces, lethargy, not eating, stained fur/skin, wet fur, labored breathing, decreased muscle tone,
prostrate posture, tremors, clear oral discharge, diarrhea, ataxia, and/or high posture. Clinical
signs in female rats were observed for up to 3 days after dosing and included wet fur, stained
fur/skin, ataxia, labored breathing, cold to touch, clear ocular or oral discharge, lethargy, lung
noise, absent feces, not eating, and/or rubbing face on the bottom of the cage (DuPont-25875,
2008).
All rats dosed at 5,000 mg/kg died by the day after dosing. Among rats dosed at 1,750 mg/kg,
two males and three females died by the day after dosing. One male rat dosed at 550 mg/kg (rat
#274) was sacrificed in extremis on the fourth day after dosing following a 23% reduction in
BW. Gross findings were detected in three male rats dosed at 5,000 mg/kg, in four rats dosed at
1,750 mg/kg, and in one rat dosed at 550 mg/kg. Small testes and epididymis were observed in
rat #274. A discolored, glandular stomach was observed in two of the male rats dosed at
1,750 mg/kg. Gross findings for male rats dosed at 5,000 mg/kg included a glandular stomach; a
glandular, discolored stomach (rats #640, #796, and #821); and discolored skin (rat #796). Gross
findings for female rats dosed at 1,750 mg/kg included a glandular, discolored stomach (rats
#478, #527, and #626); discolored lymph nodes (rat #527); and discolored skin (#527). The
female rat dosed at 5,000 mg/kg exhibited wet skin; a discolored esophagus with foamy fluid;
and a thick, discolored stomach. Increases in BW were observed in animals that survived until
the end of the study. The oral LD50 was estimated to be 1,730 mg/kg for male rats and 1,750
mg/kg for female rats (DuPont-25875, 2008).
Another study conducted according to OECD TG 425 (OECD, 2008c) evaluated toxicity of
HFPO dimer acid by the oral route of exposure in female mice. A single dose of HFPO dimer
acid ammonium salt (86% purity) was administered to 8- to 9-week-old female mice at a dose of
175, 550, or 1,750 mg/kg (1-3 mice). No clinical signs of toxicity were observed in mice dosed
at 175 mg/kg or in two mice dosed at 550 mg/kg. One mouse dosed at 550 mg/kg, however,
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exhibited wet fur on the day of dosing. All three mice dosed at 1,750 mg/kg died on the day of
dosing. Discoloration of lungs and an ovarian cyst were observed in a mouse dosed at 550
mg/kg. Skin stain was also observed in two mice dosed at 1,750 mg/kg. These observations were
considered by study authors to be nonspecific and not indicative of test substance related. With
the exception of mice dosed at 1,750 mg/kg, increases in BW were observed in all mice over the
course of the study. The oral LD50 was estimated to be 1,030 mg/kg for female mice (DuPont-
24126, 2007).
Dermal Toxicity. In a study evaluating toxicity through dermal absorption (no TG identified),
5,000 mg/kg of HFPO dimer acid ammonium salt (purity > 99%) was applied directly onto the
shaved, intact skin of two young adult male New Zealand white rabbits for a period of 24 hours.
One rabbit exhibited necrosis from days 2-6 post-application in a small area of treated skin. The
necrotic area sloughed off by day 7, and alopecia was then observed in this area until the study
was completed. Moderate erythema was observed in both rabbits at 1 day post-application and
was still observed up to 3 days post-application. Erythema persisted until 13 days post-
application, with the degree of severity decreasing over time. Both rabbits exhibited scaling and
sloughing of skin 6-13 days after application. Increases in BW were observed for both rabbits at
the conclusion (day 14) of the study. The ALD was determined to be higher than 5,000 mg/kg
(DuPont-839-95, 1996).
The dermal toxicity of HFPO dimer acid ammonium salt (86% purity) was also evaluated in rats
in a study conducted according to OECD TG 402 (OPPTS 870.1200) (OECD, 2017). A single
dose of 5,000 mg/kg (five males and five females) was applied directly onto the shaved, intact
skin for 24 hours. Rats were then observed daily for 14 days posttreatment. All female rats
exhibited mild erythema on the test site 1 day post-application. Erythema was no longer
detectable by the second day after application. Erythema was not observed in male rats.
Hyperkeratosis was observed in four male and four female rats. Ulceration was observed in one
male and two female rats. All dermal effects cleared up by 13 days posttreatment. Increases in
BW were observed for male and female rats by the conclusion (day 14) of the study. The LD50 of
the compound was determined to be higher than 5,000 mg/kg (DuPont-24113, 2007).
Inhalation Toxicity. The toxicity of HFPO dimer acid ammonium salt by the inhalation route of
exposure was evaluated in 8-week-old male and female rats (no TG identified) (DuPont-17751-
723, 2009). One group of five male and five female rats were exposed to an aerosol atmosphere
containing 5,200 milligrams per cubic meter (mg/m3) of HFPO dimer acid ammonium salt (84%
purity) to determine the inhalation median lethal concentration (LC50). Two other groups of three
male and three female rats were exposed to HFPO dimer acid ammonium salt at concentrations
of 0, 13, and 100 mg/m3 in air to evaluate respiratory tract pathology. All rats were exposed
nose-only for a single 4-hour period. Rats exposed to 0, 13, and 100 mg/m3 of HFPO dimer acid
ammonium salt in air were evaluated for clinical signs of toxicity for 2 days following exposure
and rats exposed to 5,200 mg/m3 were evaluated for a period of 14 days following exposure.
Respiratory tract tissues (lung, larynx/pharynx, trachea, and nose) of the 0-, 13-, and 100-mg/m3
exposure groups were also evaluated microscopically. According to study authors, no clinical
signs of toxicity were observed for any animals at any exposure in this study. However,
following the 100 mg/m3 exposure, all rats displayed a red nasal discharge immediately after
exposure. Rats exposed to 5,200 mg/m3 exhibited red discharge from eyes, nose, and mouth as
well as red stains on skin/fur immediately after exposure. Red discharge and staining were absent
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within 1 or 2 days after exposure. Rats in the 5,200-mg/m3 exposure group lost 2.5% to 6.8% of
their original BW for 1 or 2 days after exposure but exhibited normal weight gain for the
remainder of the experiment. The LCso was determined to be greater than 5,200 mg/m3 (DuPont-
17751-723, 2009).
Dermal Irritation. The dermal irritation of HFPO dimer acid ammonium salt (86% purity) was
evaluated in three male New Zealand white rabbits in a study conducted according to OECD TG
404 (OPPTS 870.2500) (OECD, 2002). A 0.5-mL aliquot of the compound was applied to an
area of shaved skin for a period of 4 hours. Very slight erythema was observed in one rabbit
following removal of the compound. At 60 minutes post-application, very slight erythema was
observed in one rabbit and well-defined erythema was observed in the other two rabbits.
Erythema had cleared by 24 hours postexposure (DuPont-24030, 2007).
Eye Irritation. In an OECD TG 405 (OPPTS 870.2400) (OECD, 2020a) study evaluating eye
irritation of HFPO dimer acid ammonium salt (86% purity), a 0.1 -mL aliquot of compound was
administered to one eye of a young adult male New Zealand white rabbit. Necrosis, characterized
by brown and white discoloration of the conjunctival membrane of the treated eye, was observed
at 1, 24, and 28 hours after application. Corneal opacity, iritis, conjunctival chemosis, and
discharge were also observed. Fluorescein stain examination of the treated eye indicated corneal
injury (DuPont-24114, 2007).
Seven-Day Toxicity Studies. Four 7-day studies are available for HFPO dimer acid or
ammonium salt in rats or mice. The toxicity of HFPO dimer acid ammonium salt (86.6% purity)
by the oral route of exposure was evaluated in 6-week-old male and female rats (DuPont-24009,
2008). Five rats of each sex were exposed to 0, 30, 300, or 1,000 mg/kg HFPO by oral gavage
for 7 days. No clinical signs of toxicity were observed in either sex at any dose level tested. A
significant decrease in BW was observed on test day 7 in males exposed to 1,000 mg/kg versus
control. Significant decreases in red blood cells (RBCs), hemoglobin, and hematocrit were
observed in male rats at 300 milligrams per kilogram per day (mg/kg/day) and in both male and
female rats at 1,000 mg/kg/day. A significant increase in red cell distribution width,
reticulocytes, and neutrophils was also observed in female rats exposed to 1,000 mg/kg/day.
Decreases in serum lipids and globulins were observed in males at all dosage groups as well as in
females at 300 and 1,000 mg/kg/day. Increased alanine aminotransferase, urea nitrogen, and
glucose as well as decreased sorbitol dehydrogenase, creatinine, and calcium were observed at
doses of 300 and/or 1,000 mg/kg/day. Increases in liver weight were observed in males at all
doses and in females at 1,000 mg/kg/day and corresponded with increases in B-oxidation and/or
increases in P450 enzyme activity. Mild-to-minimal hepatocellular hypertrophy was also
observed in both sexes at 1,000 mg/kg/day. Decreases in heart weight were observed in males at
1,000 mg/kg and increases in kidney weight were observed in females at 1,000 mg/kg/day: No
microscopic changes were observed in these organs.
In another study evaluating toxicity of HFPO dimer acid (99% purity) by the oral route of
exposure, 6-week-old male and female rats (5/sex) were exposed to 0, 30, 100, and 300 mg/kg of
HFPO dimer acid by gavage over a period of 7 days (DuPont-24116, 2008). No clinical signs of
toxicity were observed. Significant decrease in RBC count and a significant increase in red cell
distribution width were observed in females at 300 mg/kg/day. Significant decreases in
hemoglobin and hematocrit were observed in male rats at 300 mg/kg/day. A significant increase
in mean corpuscular cell volume was observed in males at 30 mg/kg/day. Decreases in serum
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lipids were detected in all dosed male groups versus control. Increased alkaline phosphatase and
urea nitrogen and decreased bilirubin, creatinine, total protein, globulin, and calcium were
observed at 30 and/or 300 mg/kg/day. Increased liver weight was observed in males at all doses
and in females at 300 mg/kg/day. Microscopic examination of livers detected hepatocellular
hypertrophy in all treated males and females. Lesions observed in males and females were mild
and minimal, respectively. A statistically significant increase in P-oxidation was detected in
females exposed to 300 mg/kg/day versus control.
A 7-day study was conducted in 6-week-old male mice to evaluate toxicity of HFPO dimer acid
ammonium salt (86.6% purity) by the oral route of exposure (DuPont-24010, 2008). Doses of
0 or 30 mg/kg/day were administered over a period of 7 days. By test day 7, BWs were
significantly higher in exposed males versus controls. A twofold increase in liver weight relative
to control was detected in exposed males. No grossly observable lesions in the liver were
observed. Microscopic changes in the liver observed at 30 mg/kg/day included minimal single-
cell necrosis of hepatocytes, moderate hepatocellular hypertrophy, and moderate increases in
mitotic figures. Minimal vacuolation of hepatocytes was also observed in one treated mouse.
Another 7-day gavage study was conducted in 6-week-old male mice to evaluate toxicity of
HFPO dimer acid (99% purity) by the oral route of exposure (DuPont-25281, 2008). Doses of
0 or 30 mg/kg/day were administered over a period of 7 days. By test day 7, BWs were
significantly higher in exposed males versus controls. A twofold increase in liver weight was
detected in exposed males versus control. Microscopic changes to the liver of exposed animals
included minimal single-cell necrosis of hepatocytes, moderate hepatocellular hypertrophy, and
moderate increases in mitotic figures. Minimal vacuolization was also observed in 2/5 treated
mice.
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Appendix C: Genotoxicity Study Summary
Table C-l provides a summary of the available genotoxicity data for hexafluoropropylene oxide (HFPO) dimer acid and/or its
ammonium salt.
Table C-l. Genotoxicity Study Summary
Study
Assay
Strain/Spccics
Dosing
Activation
Results
DuPont-
19713
RV1
(2008)
In vitro
Bacterial
Reverse
Mutation Test
(OECD TG
471) (OECD,
2020b)
Salmonella
typhimurium (strains
TA98, TA100,
TA1535, and
TA1537) and
Escherichia coli
(strain WP2uvrA)
HFPO dimer acid ammonium salt (85% purity)
With S9
Negative.
33.3, 66.7, 100, 333, 667, 1,000, 3,333, and 5,000
Hg/plate for preliminary toxicity test
333, 667, 1,000, 3,333, and 5,000 ng/plate for
toxicity-mutation test
Negative control (sterile water) and positive control
(benzo[a]pyrene, 2-nitrofluorine, 2-aminoanthracene,
sodium azide, acridine mutagen Institute of Cancer
Research (ICR)-191, or 4-nistroquinoline-N-oxide)
also included in study
Without S9
Negative.
DuPont-
22620
RV1
(2009)
In vitro
Mammalian
Chromosome
Aberration Test
(OECD TG
473) (OECD,
1997a)
Chinese hamster
ovary cells
HFPO dimer acid ammonium salt (83% purity)
49, 98, 244, 489, 977, 1954, and 3391 ng/mL for
preliminary toxicity test*
977, 1954, and 3391 |ig/mL for the 4-hour
nonactivated and activated test conditions*
With S9
Positive at 3,391 (ig/mL* in 4-
hour activated test conditions.
(CHO-Ki line)
Without S9
Negative.
489, 977, and 1954 |ig/mL for the 20-hour
nonactivated test condition*
Negative control (sterile water) and positive control
(mitomycin C or cyclophosphamide) also included in
study
* Doses have been corrected to account for 83%
HFPO dimer acid ammonium salt purity.
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Study
Assay
Strain/Spccics
Dosing
Activation
Results
DuPont-
23219
(2007)
In vivo
Unscheduled
DNA Synthesis
Test in
Mammalian
Cells (OECD
TG 486)
(OECD, 1997b)
Primary hepatocytes
harvested from male
rats (5/dose group)
HFPO dimer acid ammonium salt (83% purity)
1, 10, 100, 1,000, and 2,000 mg/kg for preliminary
toxicity test
500, 1,000, and 2,000 mg/kg/day for Unscheduled
DNA Synthesis Test
Negative control (distilled water) and positive control
(dimethylnitrosamine) also included in study
Negative-No significant increase in the mean number
of net nuclear grain counts in hepatocytes at 2-4 or
12-16 hours after dosing.
Dupont-
26129
(2008)
In vitro
Mammalian
Cell Gene
Mutation Test
(OECD TG
476) (OECD,
1997c)
L5178Y/TK+'"
Mouse lymphoma
cells
HFPO dimer acid ammonium salt (87% purity)
0.5, 1.5, 5, 15, 50, 150, 500, 1,500, and 3,500 ng/mL
for both non-activated and S9-activated cultures at
both 4-hour and 24-hour exposures for preliminary
toxicity assay
500, 750, 1,000, 1,500, and 2,000 ng/mL for
nonactivated cultures with a 4-hour exposure
150, 250, 500, 600, and 750 ng/mL for S9-activated
cultures with a 4-hour exposure
250, 500, 600, 750, and 1,000 ng/mL for
nonactivated cultures with a 24-hour exposure
Negative control (sterile, distilled water) and positive
control (methyl methanesulfonate or 7,12-
dimethylbenz(a)anthracene) also included in study
With S9
Negative.
Without S9
Negative.
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Study
Assay
Strain/Spccics
Dosing
Activation
Results
Dupont-
19714
RV1
(2008)
In vitro
Mammalian
Chromosome
Aberration Test
(OECD TG
473) (OECD,
1997a)
Chinese hamster
ovary cells
(CHO-Ki line)
HFPO dimer acid ammonium salt (85% purity)
0.3, 1, 3, 10, 30, 100, 300, 1,000, and 3,471 ng/mL
for preliminary toxicity test
100, 500, 1,000, 2500, and 3,471 (ig/mL for the
chromosome aberration assay for the 4-hour
nonactivated, 4-hour S9-activated, and 20-hour
nonactivated test conditions
Cytogenetic evaluations were conducted at 1,000,
2,500, and 3,471 (ig/mL for the 4-hour nonactivated
and 4-hour S9-activated test conditions and at 100,
500, and 1,000 (ig/mL for the 20-hour nonactivated
test condition
With S9
The percentage of cells with
structural aberrations in the test
substance-treated groups was not
increased above that of the
vehicle control at any
concentration.
The percentage of cells with
numerical chromosome
aberrations at 2,500 and 3,471
(ig/mL in the 4-hour S9-
activated test conditions was
increased in a dose-dependent
manner above that of the vehicle
control. The change was outside
the historical control range and
considered biologically relevant.
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Study
Assay
Strain/Spccics
Dosing
Activation
Results
Negative control (sterile water) and positive control
(mitomycin-C or cyclophosphamide) also included in
study
Without S9
In the 20-hour nonactivated test
condition, substantial toxicity
was observed at 3,471 (ig/mL
and a substantial reduction in
mitotic index relative to vehicle
control was observed in the
mitotic index relative to vehicle
control.
The percentage of cells with
structural aberrations in the test
substance-treated groups was not
increased above that of the
vehicle control at any
concentration.
An increase in the percentage of
cells with numerical
chromosome aberrations was
observed at 3,471 (ig/mL in the
4-hour nonactivated condition
relative to vehicle control.
DuPont-
22734
RV1
(2008)
In vitro
Bacterial
Reverse
Mutation Test
(OECD TG
471) (OECD,
2020b)
Salmonella
typhimurium (strains
TA98, TA100,
TA1535, and
TA1537) and
Escherichia coli
(strain WP2uvrA)
HFPO dimer acid ammonium salt (82.6% purity)
32.5, 65.2, 97.7, 325, 652, 977, 3,256, and 4,885
(ig/plate for the toxicity-mutation assay*
325, 652, 977, 3256, and 4885 (ig/plate for the
mutagenicity test*
* Doses have been correct to account for 82.6%
HFPO dimer acid ammonium salt purity.
With S9
Negative.
Without S9
Negative.
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Study
Assay
Strain/Spccics
Dosing
Activation
Results
DuPont-
23220
(2007)
In vivo
Micronucleus
and
Chromosome
Aberration
Assay (OECD
TGs 474 and
475) (OECD,
2014, 2016b)
Primary bone
marrow cells
harvested from male
and female ICR
mice
(2 males or 5 of
each sex/dose for
preliminary toxicity
study)
(5 of each sex/dose
for toxicity study)
(5 of each sex/dose
for Micronucleus
and Chromosome
Aberration Assay)
HFPO dimer acid ammonium salt (82.6% purity)
1, 10, 98, 975, and 1,950 mg/kg by oral gavage for
preliminary toxicity study*
1170, 1365, 1560, and 1,755 mg/kg by oral gavage
for toxicity study*
317, 634, and 1,268 mg/kg by oral gavage for
Micronucleus and Chromosome Aberration Assay*
Positive control (colchicine) and negative control
(sterile water) also included in the study
* Doses have been corrected to account for 82.6%
HFPO dimer acid ammonium salt purity.
Negative-No statistically significant increases in the
incidence of micronucleated polychromatic
erythrocytes or structural or numerical chromosomal
aberrations in bone marrow of male and female ICR
mice at doses up to and including the maximum
tolerated dose (1,268 mg/kg).
Notes'. DNA = deoxyribonucleic acid; |xg/mL = micrograms per milliliter; (ig/plate = micrograms per plate; mg/kg = milligrams per kilogram; mg/kg/day = milligrams per
kilogram per day; OECD = Organization for Economic Cooperation and Development; TG = test guideline.
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Appendix D: NIP PWG Final Report on the Pathology Peer Review of
Liver Findings
FINAL REPORT
December 4, 2019
PATHOLOGY PEER REVIEW
OF LIVER FINDINGS
H-28548: SUBCHRONIC TOXICITY 90 DAY GAVAGE STUDY IN MICE
(PROJECT ID: DUPONT-18405-1307)
&
AN ORAL (GAVAGE) REPRODUCTION/DEVELOPMENTAL TOXICITY SCREENING
STUDY OF H-28548 IN MICE
(STUDY NUMBER WIL-189225)
(STUDY SPONSOR NUMBER: DUPONT-18405-1037)
Prepared by:
Susan A. Elmore, MS, DVM, DACVP, DABT, FIATP
Amy Brix, DVM, PhD, DACVP
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OCTOBER 2021
INTRODUCTION
The study report, summary tables and individual animal findings, along with hematoxylin and
eosin stained microscope slides used in the Subchronic Toxicity 90 Day Gavage Study In Mice
(Project ID: DuPont-18405-1307) and An Oral (Gavage) Reproduction/Developmental Toxicity
Screening Study of H-28548 In Mice (Study Number WIL-180225) (Project ID: DuPont-18405-
1037) were received by the NTP reviewing pathologist. The slides for review each contained two
liver lobes presumed to be the left and median lobes. The data and slides of the liver were
reviewed per NTP standards (Sills et al, 2017), and the results are summarized in this report. The
experimental design for this study is as follows:
DUPONT-l8405-1307
SUBCHRONIC TOXICITY STUDY
DOSAGE
(nig/kg/day)
MICE
MALE
FEMALE
0
10
10
0.1
10
10
0.5
10
10
5
10
10
DUPONT-l8405-103 7
REPRODUCTIVE/DEVELOPMENTAL TOXICITY STUDY
DOSAGE
(mjj/kjj/day)
Fo MICE
MALE
FEMALE
0
25
25
0.1
25
25
0.5
25
25
5
25
25
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OCTOBER 2021
SUMMARY OF ORIGINAL STUDY RESULTS
DUPONT-18405-1307 (Subchronic Toxicity 90 Day Gavage Study)
The following information is excerpted from the Final Report entitled "H-28548: Subchronic
Toxicity 90 Day Gavage Study in Mice," dated February 19, 2010:
In 5 mg/kg/day male and female dose groups, increases were observed in the
incidence of single cell necrosis, mitotic figures, and/or pigment. The liver effects at 5
mg/kg/day correlated with clinical chemistry effects and were considered test
substance related and adverse. Other test substance-related effects were observed in
the livers of 0.5 and 5 mg/kg/day males and 5 mg/kg/day females, including increases
in absolute and/or relative liver weight, enlarged and/or discolored livers, and
centrilobular hepatocellular hypertrophy. The liver effects observed in 0.5 mg/kg/day
males were considered to be non-adverse adaptive responses as they were not
correlated with clinical or microscopic pathology evidence of liver toxicity.
DUPONT-18405-1037 (Reproduction/Developmental Toxicity Screening Study)
The following information is excerpted from the Final Report entitled "An Oral (Gavage)
Reproduction/Developmental Toxicity Screening Study of H-28548 in Mice," dated December
29, 2010:
In male and female mice given 5 mg/kg/day, mild to moderate hepatocellular
hypertrophy was observed microscopically. The hepatocellular hypertrophy was
characterized by cytoplasmic eosinophilic stippling that is consistent with peroxisome
proliferation and was associated with correlative increases in liver weights. Other
microscopic changes in the liver at 5 mg/kg/day included increases in single cell
necrosis, mitotic figures, pigment, and focal necrosis (females only). In male and
female mice given 0.5 mg/kg/day, the incidence and severity of hepatocellular
hypertrophy, as well as the correlative liver weight changes was reduced. Other
lesions at the 0.5 mg/kg/day dose level were limited to minimal single cell necrosis in
5 of 24 males.
SLIDE REVIEW WORK SHEETS (SRWS)
The Slide Review Work Sheets are presented in appendix A (Dupont-18405-1307 Subchronic
Toxicity Study) and appendix B (Dupont-18405-1037 Reproduction/ developmental Toxicity
Screening Study). These work sheets list, in animal ID number order, the original study
pathologist's findings, along with the reviewing pathologist's comments. Entries other than
"Agree" under the reviewing pathologist's comments indicate a disagreement with the study
pathologist's (SP's) diagnosis. In each instance, space is provided to record remarks made during
the Slide Review.
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OCTOBER 2021
FINDINGS OF THE SLIDE REVIEW
DUPONT-18405-1307 (Subchronic Toxicity 90 Day Gavage Study)
The slides reviewed during this quality assessment were of adequate quality and had no artifacts
that interfered with making diagnoses. The liver was reviewed from all animals for all lesions. It
was requested by the NTP pathologist that the reviewing pathologist use the terminology of the
UNHAND document containing standardized terminology of the liver (Thoolen et al, 2010)
except where it would be superseded by the terminology published by the UNHAND committee
with reference to cell death/necrosis/apoptosis (Elmore et al, 2016). The study pathologist
diagnosed hepatocellular hypertrophy which included the morphologic change of eosinophilic
stippling commonly observed with peroxisome proliferators. The reviewing pathologist agreed
that there was hypertrophy of the hepatocytes, but often regarded the severity to be less than
recorded by the study pathologist. In addition, the reviewing pathologist recommended adding
the diagnosis cytoplasmic alteration to account for the brightly eosinophilic, frequently granular,
appearance of the cytoplasm of hepatocytes. After reviewing these lesions with the NTP
pathologist, the NTP pathologist recommended using the term cytoplasmic alteration to
encompass both hypertrophy and eosinophilic change to the hepatocytes, as she considered them
part of the same process. The reviewing pathologist agreed with most occurrences and severities
of single cell necrosis. However, the reviewing pathologist also observed apoptosis, and
recommended adding the diagnosis of "apoptosis, hepatocellular" when present. Descriptions of
individual lesions recorded during this review are listed below. The summary incidences are
found in Table 1.
DUPONT-18405-1037 (Reproduction/Developmental Toxicity Screening Study)
The slides reviewed during this quality assessment were of adequate quality and had no artifacts
that interfered with making diagnoses. The liver was reviewed from all animals for all lesions.
For the most part, the reviewing pathologist agreed with the study pathologist's diagnoses and
severities. It was requested by the NTP pathologist that the reviewing pathologist use the
terminology of the INHAND document containing standardized terminology of the liver
(Thoolen et al, 2010) except where it would be superseded by the terminology published by the
INHAND committee with reference to cell death/necrosis/apoptosis (Elmore et al, 2016). The
study pathologist diagnosed hepatocellular hypertrophy which included the morphologic change
of eosinophilic stippling commonly observed with peroxisome proliferators. The reviewing
pathologist agreed that there was hypertrophy of the hepatocytes, but used the terminology
"cytoplasmic alteration" at the request of the NTP pathologist, based upon review of the slides
from the 18405-1307 subchronic study. The reviewing pathologist agreed with most occurrences
and severities of single cell necrosis. However, the reviewing pathologist also observed
apoptosis, and recommended adding the diagnosis of "apoptosis, hepatocellular" when present.
The reviewing pathologist recorded additional occurrences of mixed cell infiltrates in most
groups of animals. Descriptions of individual lesions recorded during this review are listed
below. The summary incidences are found in Table 2.
DESCRIPTIONS OF LESIONS
Single cell necrosis (Figures 1 & 2) consisted of individual hepatocytes that had pale, granular,
vacuolated or eosinophilic cytoplasm; nuclei were either swollen or pyknotic and karyorrhectic.
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OCTOBER 2021
The cells frequently appeared fragmented and were often surrounded by degenerative
inflammatory cells. Inflammatory cells were not documented separately as they were considered
a response to the necrosis. All of the lesi ons were considered minimal in severity, which was
recorded when 1-10 cells were observed in ten 20X fields. Single cell necrosis was not recorded
unless at least two affected cells were observed in the entirety of the liver sections examined; if 2
or more necrotic cells were observed, counting of ten 20X fields was done to determine severity.
This lesion was observed in both the 18405-1307 subchronic & the 18405-1037
reproduction/developmental studi es.
Figure J. Single cell necrosis in the liver of a Group 4 male mouse (animal 410) from the
18405-1307 subchronic study. The necrotic cell (arrow) is fragmented and surrounded by
inflammatory cells.
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OCTOBER 2021
Figure 2. Single cell necrosis in the liver of a Group 4 male mouse (animal 405) from the
18405-1307 subchronic study. The necrotic cell (arrow) is swollen, and has brightly
eosinophilic cytoplasm and a karyorrhectic nucleus.
Apoptosis; hepatocellular was recorded when individual hepatocytes were observed that had
characteristics, as described in the article by Elmore (Elmore et al, 2016), of apoptosis. Briefly,
affected cells were typically shrunken, with hyper-eosinophilic cytoplasm and condensed,
pyknotic or karyorrhectic nuclei. The cells were round and often small; occasionally they were
phagocytosed by surrounding cells. There was a lack of associated inflammatory cells with
apoptotic hepatocytes. Grading was done based upon the Thompson article (Thompson et al,
2018) to be consistent with the reviewing pathologist's grading criteria. All of the lesions were
considered minimal to mild in severity, which was recorded when 1-10 cells, or 11-40 cells,
respectively, were observed in ten 20X fields. Apoptosis was not recorded unless at least two
affected cells were observed in the entirety of the liver sections examined; if 2 or more apoptotic
cells were observed, counting of ten 20X fields was done to determine severity. This lesion was
observed in both the 18405-1307 subchronic & the 18405-1037 reproduction/developmental
studies.
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OCTOBER 2021
Figure 3. Apoptosis in the liver of a Group 4 male mouse (animal 410) from the 18405-1307
subchronic study. The apoptotic hepatocytes are small, rounded, and brightly eosinophilic
(arrows).
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OCTOBER 2021
Figure 4. Apoptosis in the liver of a Group 4 male mouse (animal 410) from the 18405-1307
subchronic study. Evidence of apoptosis is provided by small round eosinophilic remnants
of hepatocytes (arrows).
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OCTOBER 2021
Figure 5. Apoptosis (long arrows) and single cell necrosis (short arrow) in the liver of a
Group 4 male mouse (animal 405) from the 18405-1307 subchronic study.
Focal necrosis consisted of a localized area of coagulative necrosis. Generally, there was a loss
of cellular detail of the affected hepatocytes; rarely there was a small amount of mineral
(dystrophic) associated with the areas of necrosis. Inflammatory cell infiltrates typically ringed
the region of necrotic hepatocytes. This lesion was observed in both the 18405-1307 subchronic
& the 18405-1037 reproduction/developmental studies.
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OCTOBER 2021
Figure 6. Focal necrosis (outlined) in the liver of a Group 4 male mouse (animal 7744) from
the 18405-1037 reproduction/developmental study. There is a small area of contiguous
hepatocytes that are necrotic. Many of the cell borders are indistinct.
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OCTOBER 2021
Figure 7. Focal necrosis (area delineated by arrows) in the liver of a Group 4 male mouse
(animal 456) from the 18405-1307 subchronic study. This example of focal necrosis is more
extensive than that shown in figure 6.
Cytoplasmic alteration was characterized by a bright eosinophilia to the cytoplasm of
hepatocytes, usually accompanied by a slight increase in cell, and sometimes nuclear, size. The
cytoplasm usually had a granular appearance to it, although with greater severities, the cytoplasm
lost its granular appearance, and was just filled with smooth, homogeneous, brightly eosinophilic
material. Severity grading was subjectively based on the number of hepatocytes involved and the
amount of material within the affected hepatocytes. Minimal (+1) cytoplasmic alteration was
recorded when there was an eosinophilic granular appearance to the hepatocytes in the
centrilobular region of most hepatic lobules. With mild (+2) cytoplasmic alteration, more of each
hepatic lobule was involved, so that many of the hepatic lobules appeared to be completely
affected, rather than having alteration limited to the centrilobular area. All the hepatocytes
appeared to be affected with moderate (+3) cytoplasmic alteration and those in the centrilobular
area usually had lost the granular appearance to the cytoplasm and instead had a more solid,
brightly eosinophilic appearance to it. Many of the hepatocytes with moderate cytoplasmic
alteration were also larger than normal, and some also had larger than normal nuclei. These latter
changes were not recorded separately, but were considered part of the cytoplasmic alteration.
Marked (+4) cytoplasmic alteration was a diffuse change, with most of the hepatocytes distended
by increased amounts of brightly eosinophilic cytoplasm that lacked granularity or definition,
D-ll
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OCTOBER 2021
similar to what was seen with moderate cytoplasmic alteration, but affected the entire hepatic
lobule rather than just the centrilobular part. This lesion was observed in both the 18405-1307
subchronic & the 18405-1037 reproduction/developmental studies.
Figure 8. Control liver from a Group 1 mouse (animal 101) on the left; cytoplasmic
alteration in the liver of a Group 4 male mouse (animal 401) on the right; from the 18405-
1307 subchronic study. At this low magnification, the Group 4 mouse liver appears brightly
eosinophilic when compared to the Group 1 (control) liver. The nuclei are also spaced
further apart from each other, consistent with hypertrophied cells. CV=central vein.
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OCTOBER 2021
Figure 9. Control liver from a Group 1 mouse (animal 105) on the left; cytoplasmic
alteration in the liver of a Group 4 male mouse (animal 401) on the right; from the 18405-
1307 subchronic study. This higher magnification photo reveals the eosinophilic granular
nature of the cytoplasm in the Group 4 mouse. CV=central vein.
Mixed cell infiltrate was characterized by the presence clusters of inflammatory cells within the
hepatic parenchyma. They were often found randomly scattered throughout the liver, and less
commonly in the periportal or centrilobular areas. The infiltrates were composed primarily of
lymphocytes with fewer macrophages and plasma cells. Neutrophils were a small component of
many of the foci, and were a major component of a few of them. Occasionally, a necrotic
hepatocyte could be found within the focus of inflammatory cells. Mixed cell infiltrate was used
as a diagnostic term as it is the term preferred in INHAND (Thoolen et al, 2010). This lesion was
observed in both the 18405-1307 subchronic & the 18405-1037 reproduction/developmental
studies. Mixed cell infiltrates are common background lesions in mice, although they may be
exacerbated with treatment.
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OCTOBER 2021
Figure 10. Mixed cell infiltrates (circled) in the liver of a Group 4 male mouse (animal
7744) from the 18405-1037 reproduction/developmental study. Different types of
inflammatory cells, including macrophages (some containing pigment consistent with cell
breakdown product), lymphocytes, plasma cells, and neutrophils are present in a focal
area. Some of these areas also contained an occasional necrotic hepatocyte.
Mitotic figures were considered to be present when there were an increased number of mitotic
figures observed in the sections of liver examined. Typically, if 3 or more mitotic figures were
observed, ten 20X fields were counted for the number of mitotic figures, and severity scores
were based upon how many mitotic figures were counted: Minimal if 1-10 cells were observed
in ten 20X fields; mild if 11-40 cells were observed in ten 20X fields. All the occurrences of
mitotic figures were considered of minimal or mild severity. This lesion was observed in both the
18405-1307 subchronic & the 18405-1037 reproduction/developmental studies.
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OCTOBER 2021
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from the 18405-1037 reproduction/developmental study. Although mitotic figures can be
found in the livers of normal mice, there were increased numbers observed in the livers of
some animals in this study.
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OCTOBER 2021
Figure 12. Mitotic figures (arrows) in the liver of a Group 4 male mouse (animal 409) from
the 18405-1307 subchronic study. Increased mitotic figures were observed in both the
18405-1307 subchronic and the 18405-1037 reproduction/developmental studies.
Pigment increased; was characterized by golden brown pigment that was found primarily in bile
canaliculi, Kupffer cells, but occasionally in hepatocytes as well. All the occurrences were of
minimal severity. This lesion was observed in both the 18405-1307 subchronic & the 18405-
1037 reproduction/developmental studies.
D-16
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OCTOBER 2021
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Figure 13. Pigment (arrows) in the liver of a Group 4 male mouse (animal 410) from the
18405-1307 subchronic study. Special stains were not performed to identify the pigment,
but the appearance and location were consistent with either inspissated bile in canaliculi
(arrows) or byproducts from cell breakdown in Kupffer cells (see figure 14).
D-17
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OCTOBER 2021
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Figure 14. Pigment in the liver of a Group 4 male mouse (animal 410) from the 18405-1307
subchronic study. The pigment was found primarily in bile canaliculi or macrophages
(Kupffer cells) but also in hepatocytes on occasion and was consistent with either
inspissated bile in canaliculi or byproducts from cell breakdown (e.g. hemosiderin) (arrow).
Extramedullary hematopoiesis was recorded in one animal (a Group 4 male) in the 18405-1307
subchronic, and in 4 animals (a Group 3 male; two Group 4 males; & a Group 4 female) in the
18405-1037 reproduction/developmental study. It consisted of tight clusters of hematopoietic
cells in varying degrees of maturity; most of the cells appeared to be myeloid cells.
Cytoplasmic vacuolation was recorded in several male mice in Group 3 and several female mice
in Group 4 in the 18405-1037 reproduction/developmental study. It was characterized by very
small vacuoles within the cytoplasm of hepatocytes, consistent with microvesicular fatty change.
In most of the animals, it primarily involved the centrilobular hepatocytes, although in some
animals the change was also present in midzonal hepatocytes.
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OCTOBER 2021
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Figure 15. Cytoplasmic vacuolation in the liver of a Group 4 female mouse (animal 5073)
from the 18405-1037 reproduction/developmental study. The vacuoles were small and
slightly less regular than those observed in inacrovesicular fatty change, and found
primarily in the centrilobular region (area delineated by arrows), around the central vein.
CV=central vein.
Oval cell hyperplasia was recorded in six Group 4 male mice in the 18405-1037
reproduction/developmental study. Only minimal oval cell hyperplasia was recorded, and it was
characterized by an increase in oval cells in several periportal regions, with some oval cells
present in the surrounding hepatic parenchyma
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OCTOBER 2021
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Figure 16. Oval cell hyperplasia in the liver of a Group 4 male mouse (animal 7730) from
the 18405-1037 reproduction/developmental study. Oval cells (arrows), small cells with
round to oval nuclei, appear to originate in the portal region and branch out from there.
Bile duct hyperplasia was recorded in one Group 4 male mouse in the 18405-1307 subchronic
study, and was characterized by increased profiles of bile ducts in the periportal region; only
minimal bile duct hyperplasia was recorded.
Table 1. Study 18405-1307 Subchronic Toxicity 90-Day Gavage Study
Summary incidences of lesions observed in the liver during slide review
Group
Group 1
Male
Group 2
Male
Group 3
Male
Group 4
Male
Group 1
Female
Group 2
Female
Group 3
Female
Group 4
Female
Number evaluated
10
10
10
10
10
9
10
10
Within normal limits
4
4
0
0
4
6
7
0
Mixed cell infiltrate
6
6
4
6
5
3
3
7
Single cell necrosis;
hepatocellular
0
1
0
9
0
0
0
3
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OCTOBER 2021
Group
Group 1
Male
Group 2
Male
Group 3
Male
Group 4
Male
Group 1
Female
Group 2
Female
Group 3
Female
Group 4
Female
Number evaluated
10
10
10
10
10
9
10
10
Cytoplasmic
alteration
0
0
10
10
0
0
0
10
Focal necrosis
0
0
0
1
1
0
2
4
Cytoplasmic
vacuolation
0
0
0
0
0
0
0
0
Extramedullary
hematopoiesis,
0
0
0
1
0
0
0
0
Pigment, increased
0
0
0
10
0
0
0
4
Apoptosis;
hepatocellular
0
0
0
10
0
0
0
3
Mitotic figures
increased
0
0
0
7
0
0
0
0
Bile duct hyperplasia
0
0
0
1
0
0
0
0
Table 2. Study 18405-1037 Reproduction/developmental Toxicity Screening Study
Summary incidences of lesions observed in the liver during slide review
Group
Group 1
Male
Group 2
Male
Group 3
Male
Group 4
Male
Group 1
Female
Group 2
Female
Group 3
Female
Group 4
Female
Number evaluated
25
25
25
25
25
25
25
25
Within normal limits
18
21
5
0
11
14
3
0
Mixed cell infiltrate
6
3
11
8
12
7
17
15
Single cell necrosis;
hepatocellular
1
1
2
24
0
2
3
19
Cytoplasmic
alteration
0
0
10
25
0
1
16
25
Focal necrosis
0
0
4
3
2
2
4
5
Cytoplasmic
vacuolation
0
0
3
0
0
0
0
1
Extramedullary
hematopoiesis
0
0
1
2
0
0
0
1
Pigment, increased
0
0
0
21
0
0
0
3
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OCTOBER 2021
Group
Group 1
Male
Group 2
Male
Group 3
Male
Group 4
Male
Group 1
Female
Group 2
Female
Group 3
Female
Group 4
Female
Number evaluated
25
25
25
25
25
25
25
25
Apoptosis;
hepatocellular
0
0
0
22
0
0
0
10
Mitotic figures
increased
0
0
0
17
0
0
0
2
Oval cell hyperplasia
0
0
0
4
0
0
0
0
Inflammation,
granulomatous
0
0
0
0
0
0
1
0
Polyarteritis nodosa
0
0
0
0
0
0
0
1
PATHOLOGY WORKING GROUP
A PWG was convened on October 15, 2019 at the National Institute of Environmental Health
Sciences (NIEHS), Research Triangle Park (RTP), NC to histologically evaluate selected tissues
from this study. The participants were Drs. Susan A. Elmore, MS, DVM, DACVP, DABT
(NTP/NIEHS - PWG Coordinator), Amy Brix, DVM, PhD, DACVP (EPL - Reviewing
Pathologist), David Malarkey, DVM, PhD, DACVP (NTP/NIEHS), Arun Pandiri, BVSc&AH,
PhD, DACVP, DABT (NTP/NIEHS), Robert Sills, DVM, PhD, DACVP (NTP/NIEHS), Brian
Berridge, DVM, PhD, DACVP (NTP/NIEHS), Robert Maronpot, DVM, MS, MPH (Maronpot
Consulting, LLC) and Michael Elwell, DVM, PhD (Apex ToxPath, LLC).
The PWG Coordinator selected slides for review by the PWG that included 3 examples each of
normal liver, hepatocellular apoptosis, hepatocellular single cell necrosis and hepatocellular
cytoplasmic alteration, as well as 2 examples each of focal necrosis, pigment, increased mitoses,
mixed cell infiltrates, cytoplasmic vacuolation and 1 example of oval cell hyperplasia. There was
a majority consensus for all reviewed lesions. The PWG consensus opinion for each slide,
including any additional diagnoses made by the PWG panel, was recorded on the slide review
worksheet attached to the end of this report.
After review of all lesions, there was discussion about potential adversity. Adversity is a term
indicating "harm" to the test animal within the constraints of a given study design (dose,
duration, etc.). Assessment of adversity should represent empirical measurements (i.e. objective
data) integrated with well-informed subjective judgements to determine whether or not a
response is considered harmful to an organism (Kerlin et al. 2016). After discussion, the PWG
members agreed that the dose response and constellation of lesions (i.e. cytoplasmic alteration,
apoptosis, single cell necrosis, and focal necrosis) rather than one lesion by itself, represents
adversity within the confines of this study.
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OCTOBER 2021
SUMMARY
This review generally supported the study pathologist's findings. When appropriate, the
diagnosis of apoptosis, hepatocellular was added to distinguish cells with morphological
characteristics of apoptosis from those with morphologic characteristics of single cell necrosis.
The diagnostic term of "cytoplasmic alteration" was used to indicate hepatocyte hypertrophy,
frequently coupled with a brightly eosinophilic, often granular appearance of the cytoplasm of
hepatocytes. Other changes were recommended based upon using terminology preferred by the
NTP. The dose response and constellation of lesions were together considered to be indicators of
adversity within the confines of this study.
CONFLICT OF INTEREST STATEMENT
This statement is to certify that the reviewing pathologist, Dr. Brix, an on-site NTP Pathologist
employed by Experimental Pathology Laboratories, Inc. (EPL®), participated in the pathology
peer review of the liver of the Subchronic Toxicity 90 Day Gavage Study In Mice (Project ID:
DuPont-18405-1307) and An Oral (Gavage) Reproduction/Developmental Toxicity Screening
Study of H-28548 In Mice (Study Number WIL-180225) (Project ID: DuPont-18405-1037). She
has not been involved in any aspect of the study for any organization other than NTP which
conducted the study nor the generation and/or evaluation of materials or data which were
reviewed prior to the receipt of materials from the study lab. Hence, her participation in the
review poses no apparent or actual conflict of interest.
REFERENCES
Elmore SA, Dixon D, Hailey JR, Harada T, Herbert RA, Maronpot RR, Nolte T, Rehg JE,
Rittinghausen S, Rosol TJ, Satoh H, Vidal JD, Willard-Mack CL, Creasy DM.
Recommendations from the INHAND Apoptosis/Necrosis Working Group. Toxicol
Pathol. 2016 Feb;44(2): 173-88. Epub 2016 Feb 14. PubMed PMID: 26879688; PubMed
Central PMCID: PMC4785073.
https ://i ournal s. sagepub. com/doi/10.1177/0192623315625859
Kerlin, R., Bolon, B., Burkhardt, J., Francke, S., Greaves, P., Meador, V., & Popp, J. (2016).
Scientific and Regulatory Policy Committee: Recommended ("Best") Practices for
Determining, Communicating, and Using Adverse Effect Data from Nonclinical
Studies. Toxicologic Pathology, 44(2), 147-162.
https://doi.org/10.1177/0192623315623265
Sills, R. C., Cesta, M. F., Willson, C. J., Brix, A. E., Berridge, B. R. (2019). National Toxicology
Program Position Statement on Informed ("Nonblinded") Analysis in Toxicologic
Pathology Evaluation. Toxicologic Pathology 47(7): 887-890.
https://doi.org/10.1177/0192623319873974
Thompson, CM, Fitch, SE, Ring, C, Rish, W, Cullen, JM, Haws, LC. Development of an oral
reference dose for the perfluorinated compound GenX. J Appl Toxicol.
2019; 1- 16. https://doi.org/10.1002/jat.3812
D-23
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OCTOBER 2021
Thoolen, B., Maronpot, R. R., Harada, T., Nyska, A., Rousseaux, C., Nolte, T., ... Ward, J. M.
(2010). Proliferative and Nonproliferative Lesions of the Rat and Mouse Hepatobiliary
System. Toxicologic Pathology, 38(7_suppl), 5S-81S.
https://doi.org/10.1177/0192623310386499
PWG SLIDE REVIEW WORKSHEET
Flat/#
Animal #
Study
Lesion in question
Other/
Comments
1-1
101
1307
Within normal limits
7 agreed, 0 disagreed
1-2
7722
1037
Within normal limits
7 agreed, 0 disagreed
1-3
7718
1037
Within normal limits
7 agreed, 0 disagreed
1-4
410
1307
Apoptosis
7 agreed, 0 disagreed
1-5
401
1307
Apoptosis
7 agreed, 0 disagreed
1-6
7770
1037
Apoptosis
7 agreed, 0 disagreed
1-7
405
1307
Single cell necrosis
7 agreed, 0 disagreed
1-8
7730
1037
Single cell necrosis
7 agreed, 0 disagreed
1-9
7804
1037
Single cell necrosis
7 agreed, 0 disagreed
1-10
406
1307
Cytoplasmic alteration
7 agreed, 0 disagreed
1-11
404
1307
Cytoplasmic alteration
7 agreed, 0 disagreed
1-12
7759
1037
Cytoplasmic alteration
7 agreed, 0 disagreed
1-13
7744
1037
Focal necrosis
7 agreed, 0 disagreed
1-14
456
1307
Focal necrosis
7 agreed, 0 disagreed
1-15
403
1307
Pigment
7 agreed, 0 disagreed
1-16
7780
1037
Pigment
7 agreed, 0 disagreed
1-17
408
1307
Increased mitoses
7 agreed, 0 disagreed
1-18
409
1307
Increased mitoses
7 agreed, 0 disagreed
1-19
407
1307
Mixed cell infiltrates
5 agreed, 2 voted for "inflammation"
1-20
7723
1037
Mixed cell infiltrates
5 agreed, 2 voted for "inflammation"
2-1
5073
1037
Cytoplasmic Vacuolation
7 agreed, 0 disagreed
2-2
7799
1037
Cytoplasmic Vacuolation
7 agreed, 0 disagreed
2-3
7778
1037
Oval cell hyperplasia
6 agreed, 1 voted for biliary hyperplasia
D-24
-------
OCTOBER 2021
Appendix A. Slide Review Worksheets
Project 18405-1307 Males
Animal #
Organ
SP Diagnosis
NTP Diagnosis
101
Liver
Within normal limits
Agree with SP
102
Liver
Within normal limits
Agree with SP
103
Liver
Mononuclear cell infiltrate; minimal
Mixed cell infiltrate; minimal
104
Liver
Within normal limits
Mixed cell infiltrate; minimal
105
Liver
Within normal limits
Agree with SP
106
Liver
Mononuclear cell infiltrate; minimal
Mixed cell infiltrate; minimal
107
Liver
Within normal limits
Agree with SP
108
Liver
Mononuclear cell infiltrate; minimal
Mixed cell infiltrate; minimal
109
Liver
Within normal limits
Mixed cell infiltrate; minimal
110
Liver
Mononuclear cell infiltrate; minimal
Mixed cell infiltrate; minimal
201
Liver
Within normal limits
Agree with SP
202
Liver
Within normal limits
Agree with SP
203
Liver
Within normal limits
Agree with SP
204
Liver
Within normal limits
Agree with SP
205
Liver
Mononuclear cell infiltrate; minimal
Mixed cell infiltrate; minimal
Single cell necrosis; hepatocellular;
minimal
206
Liver
Mononuclear cell infiltrate; minimal
Mixed cell infiltrate; minimal
207
Liver
Mononuclear cell infiltrate; minimal
Mixed cell infiltrate; minimal
208
Liver
Mononuclear cell infiltrate; minimal
Mixed cell infiltrate; minimal
209
Liver
Mononuclear cell infiltrate; minimal
Mixed cell infiltrate; minimal
210
Liver
Mononuclear cell infiltrate; minimal
Mixed cell infiltrate; minimal
301
Liver
Within normal limits
Cytoplasmic alteration; mild
302
Liver
Hepatocellular hypertrophy; minimal
Cytoplasmic alteration; mild
303
Liver
Hepatocellular hypertrophy; minimal
Cytoplasmic alteration; minimal
304
Liver
Hepatocellular hypertrophy; minimal
Mononuclear cell infiltrate; minimal
Cytoplasmic alteration; minimal
Mixed cell infiltrate; minimal
D-25
-------
OCTOBER 2021
Animal #
Organ
SP Diagnosis
NTP Diagnosis
305
Liver
Hepatocellular hypertrophy; minimal
Cytoplasmic alteration; minimal
306
Liver
Hepatocellular hypertrophy; minimal
Cytoplasmic alteration; minimal
307
Liver
Hepatocellular hypertrophy; minimal
Cytoplasmic alteration; minimal
308
Liver
Hepatocellular hypertrophy; minimal
Mononuclear cell infiltrate; minimal
Cytoplasmic alteration; minimal
Mixed cell infiltrate; minimal
309
Liver
Hepatocellular hypertrophy; minimal
Mononuclear cell infiltrate; minimal
Cytoplasmic alteration; minimal
Mixed cell infiltrate; minimal
310
Liver
Mononuclear cell infiltrate; minimal
Mixed cell infiltrate; minimal
Cytoplasmic alteration; minimal
401
Liver
Single cell necrosis; hepatocellular;
minimal
Pigment increased; Kupffer cells; minimal
Hepatocellular hypertrophy; mild
Mitotic figures; minimal
Agree with SP
Pigment, increased; minimal
Cytoplasmic alteration; marked
Not present in section
Apoptosis; hepatocellular; minimal
402
Liver
Single cell necrosis; hepatocellular;
minimal
Pigment increased; Kupffer cells; minimal
Hyperplasia; bile duct; minimal
Hepatocellular hypertrophy; mild
Mitotic figures; minimal
Agree with SP
Pigment, increased; minimal
Agree with SP
Cytoplasmic alteration; marked
Agree with SP
Apoptosis; hepatocellular; minimal
403
Liver
Single cell necrosis; hepatocellular;
minimal
Pigment increased; Kupffer cells; minimal
Hepatocellular hypertrophy; mild
Mitotic figures; minimal
Mononuclear cell infiltrate; minimal
Agree with SP
Pigment, increased; minimal
Cytoplasmic alteration; marked
Agree with SP
Mixed cell infiltrate; minimal
Apoptosis; hepatocellular; minimal
404
Liver
Single cell necrosis; hepatocellular;
minimal
Pigment increased; Kupffer cells; minimal
Hepatocellular hypertrophy; mild
Mitotic figures; mild
Mononuclear cell infiltrate; minimal
Agree with SP
Pigment, increased; minimal
Cytoplasmic alteration; marked
Agree with SP
Mixed cell infiltrate; minimal
Apoptosis; hepatocellular; minimal
Focal necrosis; minimal
D-26
-------
OCTOBER 2021
Animal #
Organ
SP Diagnosis
NTP Diagnosis
405
Liver
Single cell necrosis; hepatocellular;
minimal
Pigment increased; Kupffer cells; minimal
Hepatocellular hypertrophy; mild
Mitotic figures; mild
Agree with SP
Pigment, increased; minimal
Cytoplasmic alteration; marked
Agree with SP
Apoptosis; hepatocellular; minimal
406
Liver
Single cell necrosis; hepatocellular;
minimal
Pigment increased; Kupffer cells; minimal
Hepatocellular hypertrophy; minimal
Mononuclear cell infiltrate; minimal
Not present in section
Pigment, increased; minimal
Cytoplasmic alteration; marked
Mixed cell infiltrate; minimal
Apoptosis; hepatocellular; minimal
407
Liver
Single cell necrosis; hepatocellular;
minimal
Pigment increased; Kupffer cells; minimal
Hepatocellular hypertrophy; mild
Mitotic figures; minimal
Mononuclear cell infiltrate; minimal
Agree with SP
Pigment, increased; minimal
Cytoplasmic alteration; marked
Not present in section
Mixed cell infiltrate; minimal
Apoptosis; hepatocellular; minimal
408
Liver
Single cell necrosis; hepatocellular;
minimal
Pigment increased; Kupffer cells; minimal
Hepatocellular hypertrophy; mild
Mitotic figures; minimal
Mononuclear cell infiltrate; minimal
Agree with SP
Pigment, increased; minimal
Cytoplasmic alteration; marked
Mitotic figures; mild
Mixed cell infiltrate; minimal
Apoptosis; hepatocellular; minimal
409
Liver
Single cell necrosis; hepatocellular;
minimal
Pigment increased; Kupffer cells; minimal
Hepatocellular hypertrophy; mild
Mitotic figures; mild
Mononuclear cell infiltrate; minimal
Agree with SP
Pigment, increased; minimal
Cytoplasmic alteration; marked
Agree with SP
Not present in section
Apoptosis; hepatocellular; minimal
Extramedullary hematopoiesis; minimal
410
Liver
Single cell necrosis; hepatocellular;
minimal
Pigment increased; Kupffer cells; minimal
Hepatocellular hypertrophy; mild
Mitotic figures; minimal
Mononuclear cell infiltrate; minimal
Agree with SP
Pigment, increased; minimal
Cytoplasmic alteration; marked
Agree with SP
Mixed cell infiltrate; minimal
Apoptosis; hepatocellular; mild
D-27
-------
OCTOBER 2021
Project 18405-1307 Females
Animal #
Organ
SP Diagnosis
NTP Diagnosis
151
Liver
Within normal limits
Mixed cell infiltrate; minimal
152
Liver
Within normal limits
Mixed cell infiltrate; minimal
153
Liver
Within normal limits
Agree with SP
154
Liver
Mononuclear cell infiltrate; minimal
Mixed cell infiltrate; minimal
155
Liver
Mononuclear cell infiltrate; minimal
Mixed cell infiltrate; minimal
156
Liver
Within normal limits
Agree with SP
157
Liver
Focal necrosis, moderate: diffuse and
restricted to one lobe (likely due to
lobular torsion)
Agree with SP
158
Liver
Within normal limits
Agree with SP
159
Liver
Mononuclear cell infiltrate; minimal
Mixed cell infiltrate; minimal
160
Liver
Within normal limits
Agree with SP
251
Liver
Within normal limits
Slide missing
252
Liver
Within normal limits
Agree with SP
253
Liver
Within normal limits
Agree with SP
254
Liver
Within normal limits
Agree with SP
255
Liver
Within normal limits
Agree with SP
256
Liver
Within normal limits
Mixed cell infiltrate; minimal
257
Liver
Within normal limits
Agree with SP
258
Liver
Within normal limits
Agree with SP
259
Liver
Mononuclear cell infiltrate; minimal
Mixed cell infiltrate; minimal
260
Liver
Mononuclear cell infiltrate; minimal
Mixed cell infiltrate; minimal
351
Liver
Mononuclear cell infiltrate; minimal
Mixed cell infiltrate; minimal
352
Liver
Within normal limits
Agree with SP
353
Liver
Within normal limits
Agree with SP
354
Liver
Within normal limits
Agree with SP
355
Liver
Within normal limits
Agree with SP
356
Liver
Within normal limits
Agree with SP
D-28
-------
OCTOBER 2021
Animal #
Organ
SP Diagnosis
NTP Diagnosis
357
Liver
Focal necrosis; minimal
Mononuclear cell infiltrate; minimal
Agree with SP
Mixed cell infiltrate; minimal
358
Liver
Focal necrosis; minimal
Mononuclear cell infiltrate; minimal
Agree with SP
Mixed cell infiltrate; minimal
359
Liver
Within normal limits
Agree with SP
360
Liver
Within normal limits
Agree with SP
451
Liver
Hepatocellular hypertrophy; minimal
Cytoplasmic alteration; mild
452
Liver
Pigment increased; Kupffer cells; minimal
Hepatocellular hypertrophy; mild
Mononuclear cell infiltrate; minimal
Pigment, increased; minimal
Cytoplasmic alteration; moderate
Mixed cell infiltrate; minimal
Single cell necrosis; hepatocellular; minimal
453
Liver
Hepatocellular hypertrophy; minimal
Mononuclear cell infiltrate; minimal
Cytoplasmic alteration; mild
Mixed cell infiltrate; minimal
454
Liver
Hepatocellular hypertrophy; mild
Mononuclear cell infiltrate; minimal
Cytoplasmic alteration; moderate
Mixed cell infiltrate; minimal
Focal necrosis; minimal
Singe cell necrosis; minimal
Pigment, increased; minimal
Apoptosis; hepatocellular; minimal
455
Liver
Hepatocellular hypertrophy; minimal
Cytoplasmic alteration; minimal
Pigment, increased; minimal
456
Liver
Focal necrosis; mild: sub-capsular
Hepatocellular hypertrophy; minimal
Mononuclear cell infiltrate; minimal
Focal necrosis; mild
Cytoplasmic alteration; minimal
Mixed cell infiltrate; minimal
457
Liver
Hepatocellular hypertrophy; mild
Cytoplasmic alteration; moderate
Mixed cell infiltrate; minimal
458
Liver
Hepatocellular hypertrophy; minimal
Cytoplasmic alteration; mild
Apoptosis; hepatocellular; minimal
459
Liver
Single cell necrosis; hepatocellular;
minimal
Pigment increased; Kupffer cells; minimal
Focal necrosis; mild
Hepatocellular hypertrophy; mild
Mononuclear cell infiltrate; mild
Agree with SP
Pigment, increased; minimal
Focal necrosis; minimal
Cytoplasmic alteration; mild
Mixed cell infiltrate; mild
Apoptosis; hepatocellular; minimal
D-29
-------
OCTOBER 2021
Animal #
Organ
SP Diagnosis
NTP Diagnosis
460
Liver
Focal necrosis; mild
Hepatocellular hypertrophy; minimal
Mononuclear cell infiltrate; minimal
Focal necrosis; minimal
Cytoplasmic alteration; mild
Mixed cell infiltrate; minimal
D-30
-------
OCTOBER 2021
Appendix B. Slide Review Worksheets
Project 18405-1037 Males
Animal #
Organ
SP Diagnosis
NTP Diagnosis
7714
Liver
Within normal limits
Agree with SP
7717
Liver
Within normal limits
Agree with SP
7718
Liver
Within normal limits
Agree with SP
7722
Liver
Within normal limits
Agree with SP
7723
Liver
Hematopoiesis, extramedullary; minimal
Mixed cell infiltrate; mild
7732
Liver
Within normal limits
Agree with SP
7734
Liver
Within normal limits
Agree with SP
7742
Liver
Within normal limits
Agree with SP
7750
Liver
Necrosis, single cell; minimal
Single cell necrosis; hepatocellular; minimal
7752
Liver
Within normal limits
Mixed cell infiltrate; minimal
7758
Liver
Within normal limits
Agree with SP
7763
Liver
Within normal limits
Agree with SP
7765
Liver
Within normal limits
Agree with SP
7769
Liver
Within normal limits
Agree with SP
7772
Liver
Within normal limits
Agree with SP
7775
Liver
Within normal limits
Agree with SP
7788
Liver
Within normal limits
Agree with SP
7792
Liver
Within normal limits
Agree with SP
7798
Liver
Within normal limits
Mixed cell infiltrate; minimal
7800
Liver
Within normal limits
Mixed cell infiltrate; minimal
7803
Liver
Within normal limits
Mixed cell infiltrate; minimal
7810
Liver
Within normal limits
Agree with SP
7813
Liver
Within normal limits
Mixed cell infiltrate; minimal
7823
Liver
Within normal limits
Agree with SP
7825
Liver
Within normal limits
Agree with SP
7710
Liver
Within normal limits
Agree with SP
D-31
-------
OCTOBER 2021
Animal #
Organ
SP Diagnosis
NTP Diagnosis
7728
Liver
Within normal limits
Agree with SP
7731
Liver
Within normal limits
Mixed cell infiltrate; minimal
7737
Liver
Necrosis, single cell; minimal
Single cell necrosis; hepatocellular; minimal
7743
Liver
Within normal limits
Agree with SP
7748
Liver
Within normal limits
Agree with SP
7749
Liver
Within normal limits
Agree with SP
7754
Liver
Within normal limits
Agree with SP
7768
Liver
Within normal limits
Agree with SP
7776
Liver
Within normal limits
Mixed cell infiltrate; minimal
7777
Liver
Within normal limits
Agree with SP
7779
Liver
Within normal limits
Agree with SP
7783
Liver
Within normal limits
Agree with SP
7784
Liver
Within normal limits
Agree with SP
7786
Liver
Within normal limits
Agree with SP
7787
Liver
Within normal limits
Mixed cell infiltrate; minimal
7794
Liver
Within normal limits
Agree with SP
7797
Liver
Within normal limits
Agree with SP
7805
Liver
Within normal limits
Agree with SP
7807
Liver
Within normal limits
Agree with SP
7808
Liver
Within normal limits
Agree with SP
7809
Liver
Within normal limits
Agree with SP
7811
Liver
Within normal limits
Agree with SP
7817
Liver
Within normal limits
Agree with SP
7826
Liver
Within normal limits
Agree with SP
7711
Liver
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse;
minimal (with eosinophilic cytoplasmic
stippling)
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; minimal
7720
Liver
Within normal limits
Mixed cell infiltrate; minimal
D-32
-------
OCTOBER 2021
Animal #
Organ
SP Diagnosis
NTP Diagnosis
7721
Liver
Infiltrate, mononuclear cell,
focal/multifocal; minimal
Mixed cell infiltrate; minimal
7729
Liver
Hypertrophy, hepatocellular, diffuse;
minimal (with eosinophilic cytoplasmic
stippling)
Cytoplasmic alteration; minimal
7740
Liver
Within normal limits
Agree with SP
7741
Liver
Within normal limits
Agree with SP
7745
Liver
Infiltrate, mononuclear cell,
focal/multifocal; minimal
Necrosis, single cell; minimal
No remarkable lesion
Focal necrosis; minimal
Hepatocyte; cytoplasmic vacuolation;
minimal
7746
Liver
Hypertrophy, hepatocellular, diffuse;
minimal (with eosinophilic cytoplasmic
stippling)
Cytoplasmic alteration; minimal
Mixed cell infiltrate; minimal
7756
Liver
Within normal limits
Mixed cell infiltrate; minimal
Hepatocyte; cytoplasmic vacuolation;
minimal
7760
Liver
Infiltrate, mononuclear cell,
focal/multifocal; minimal
Hematopoiesis, extramedullary; mild
Mixed cell infiltrate; minimal
Agree with SP
7761
Liver
Within normal limits
Agree with SP
7762
Liver
Hypertrophy, hepatocellular, diffuse; mild
(with eosinophilic cytoplasmic stippling)
Cytoplasmic alteration; mild
7767
Liver
Hypertrophy, hepatocellular, diffuse; mild
(with eosinophilic cytoplasmic stippling)
Cytoplasmic alteration; mild
Mixed cell infiltrate; minimal
7774
Liver
Hypertrophy, hepatocellular, diffuse; mild
(with eosinophilic cytoplasmic stippling)
Cytoplasmic alteration; mild
7789
Liver
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse; mild
(with eosinophilic cytoplasmic stippling)
Focal necrosis; minimal
Cytoplasmic alteration; mild
7790
Liver
Necrosis, single cell; minimal
Agree with SP
7793
Liver
Within normal limits
Mixed cell infiltrate; minimal
7796
Liver
Within normal limits
Agree with SP
7799
Liver
Fatty change, centrilobular; minimal
Hepatocyte; cytoplasmic vacuolation;
minimal
D-33
-------
OCTOBER 2021
Animal #
Organ
SP Diagnosis
NTP Diagnosis
7802
Liver
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse;
minimal (with eosinophilic cytoplasmic
stippling)
Focal necrosis; minimal
Cytoplasmic alteration; minimal
7814
Liver
Hypertrophy, hepatocellular, diffuse;
minimal (with eosinophilic cytoplasmic
stippling)
Not present in section (within normal limits)
7820
Liver
Infiltrate, mononuclear cell,
focal/multifocal; minimal
Necrosis, focal/multifocal; minimal
Mixed cell infiltrate; minimal
Focal necrosis; minimal
7822
Liver
Infiltrate, mononuclear cell,
focal/multifocal; minimal
Hypertrophy, hepatocellular, diffuse;
minimal (with eosinophilic cytoplasmic
stippling)
Mixed cell infiltrate; minimal
Not present in section
7827
Liver
Hypertrophy, hepatocellular, diffuse;
minimal (with eosinophilic cytoplasmic
stippling)
Cytoplasmic alteration; minimal
Mixed cell infiltrate; minimal
7828
Liver
Hypertrophy, hepatocellular, diffuse; mild
(with eosinophilic cytoplasmic stippling)
Cytoplasmic alteration; minimal
Mixed cell infiltrate; minimal
7709
Liver
Pigment, increased; minimal
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Agree with SP
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
7712
Liver
Infiltrate, mononuclear cell,
focal/multifocal; minimal
Mitotic figures increased; minimal
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse; mild
(with eosinophilic cytoplasmic stippling)
Mixed cell infiltrate; minimal
No remarkable lesions
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; mild
7715
Liver
Pigment, increased; minimal
Mitotic figures increased; mild
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Agree with SP
Mitotic figures increased; minimal
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; mild
D-34
-------
OCTOBER 2021
Animal #
Organ
SP Diagnosis
NTP Diagnosis
7716
Liver
Pigment, increased; minimal
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse; mild
(with eosinophilic cytoplasmic stippling)
Agree with SP
Single cell necrosis; hepatocellular; mild
Cytoplasmic alteration; mild
Apoptosis; hepatocellular; minimal
7724
Liver
Pigment, increased; minimal
Hematopoiesis, extramedullary; minimal
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Agree with SP
Agree with SP
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; mild
7726
Liver
Pigment, increased; minimal
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Agree with SP
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
7730
Liver
Pigment, increased; minimal
Mitotic figures increased; mild
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Agree with SP
Mitotic figures increased; minimal
Single cell necrosis; hepatocellular; mild
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
Mixed cell infiltrate; minimal
Oval cell hyperplasia; minimal
7735
Liver
Pigment, increased; minimal
Mitotic figures increased; mild
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate
Agree with SP
Agree with SP
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
Extramedullary hematopoiesis; minimal
7736
Liver
Pigment, increased; minimal
Mitotic figures increased; minimal
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate
Agree with SP
Agree with SP
Single cell necrosis; hepatocellular; mild
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
Mixed cell infiltrate; minimal
D-35
-------
OCTOBER 2021
Animal #
Organ
SP Diagnosis
NTP Diagnosis
7738
Liver
Pigment, increased; minimal
Mitotic figures increased; mild
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Agree with SP
Mitotic figures increased; minimal
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
7739
Liver
Pigment, increased; minimal
Mitotic figures increased; mild
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Agree with SP
Mitotic figures increased; minimal
Single cell necrosis; hepatocellular; mild
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
7744
Liver
Pigment, increased; minimal
Mitotic figures increased; mild
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate
Agree with SP
Mitotic figures increased; minimal
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
Mixed cell infiltrate; minimal
Focal necrosis; minimal
7747
Liver
Pigment, increased; minimal
Mitotic figures increased; minimal
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Agree with SP
Agree with SP
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
7751
Liver
Mitotic figures increased; mild
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Mitotic figures increased; minimal
Single cell necrosis; hepatocellular; mild
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
Mixed cell infiltrate; minimal
7759
Liver
Pigment, increased; minimal
Mitotic figures increased; mild
Necrosis, single cell; moderate
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Agree with SP
Mitotic figures increased; minimal
Single cell necrosis; hepatocellular; mild
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; mild
Oval cell hyperplasia; minimal
D-36
-------
OCTOBER 2021
Animal #
Organ
SP Diagnosis
NTP Diagnosis
7764
Liver
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
7770
Liver
Pigment, increased; minimal
Mitotic figures increased; minimal
Necrosis, single cell; moderate
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Agree with SP
Agree with SP
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; moderate
7778
Liver
Pigment, increased; minimal
Mitotic figures increased; mild
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Agree with SP
Mitotic figures increased; minimal
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; mild
Oval cell hyperplasia; minimal
7780
Liver
Pigment, increased; minimal
Mitotic figures increased; mild
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Agree with SP
Mitotic figures increased; minimal
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
Oval cell hyperplasia; minimal
7781
Liver
Pigment, increased; minimal
Mitotic figures increased; mild
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate
Agree with SP
Mitotic figures increased; minimal
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
7782
Liver
Pigment, increased; minimal
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Agree with SP
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
Mixed cell infiltrate; minimal
D-37
-------
OCTOBER 2021
Animal #
Organ
SP Diagnosis
NTP Diagnosis
7785
Liver
Infiltrate, mononuclear cell,
focal/multifocal; minimal
Pigment, increased; minimal
Mitotic figures increased; mild
Necrosis, single cell; moderate
Hypertrophy, hepatocellular, diffuse;
moderate
Mixed cell infiltrate; minimal
Agree with SP
Mitotic figures increased; minimal
Single cell necrosis; hepatocellular; mild
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
7801
Liver
Pigment, increased; minimal
Mitotic figures increased; mild
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Agree with SP
Mitotic figures increased; minimal
Single cell necrosis; hepatocellular; mild
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
7804
Liver
Pigment, increased; minimal
Mitotic figures increased; mild
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Agree with SP
Mitotic figures increased; minimal
Single cell necrosis; hepatocellular; mild
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; mild
Focal necrosis; minimal
7815
Liver
Pigment, increased; minimal
Necrosis, focal/multifocal; minimal
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse; mild
(with eosinophilic cytoplasmic stippling)
Not present in section
Focal necrosis; minimal
Apoptosis; hepatocellular; minimal
Cytoplasmic alteration; mild
Mixed cell infiltrate; minimal
D-38
-------
OCTOBER 2021
Project 18405-1037 Females
Animal #
Organ
SP Diagnosis
NTP Diagnosis
4956
Liver
Within normal limits
Mixed cell infiltrate; minimal
4958
Liver
Within normal limits
Mixed cell infiltrate; minimal
4962
Liver
Within normal limits
Agree with SP
4966
Liver
Within normal limits
Mixed cell infiltrate; minimal
4967
Liver
Within normal limits
Mixed cell infiltrate; minimal
4968
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Mixed cell infiltrate; minimal
4978
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Mixed cell infiltrate; minimal
4985
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Mixed cell infiltrate; minimal
4986
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Mixed cell infiltrate; minimal
4987
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Mixed cell infiltrate; minimal
4991
Liver
Within normal limits
Agree with SP
4999
Liver
Within normal limits
Agree with SP
5001
Liver
Within normal limits
Agree with SP
5003
Liver
Within normal limits
Agree with SP
5013
Liver
Within normal limits
Agree with SP
5018
Liver
Within normal limits
Agree with SP
5021
Liver
Within normal limits
Agree with SP
5030
Liver
Necrosis, single cell; minimal
Focal necrosis; minimal
5045
Liver
Within normal limits
Agree with SP
5058
Liver
Necrosis, focal/multifocal; minimal
Focal necrosis; minimal
5059
Liver
Within normal limits
Agree with SP
5060
Liver
Within normal limits
Agree with SP
5064
Liver
Within normal limits
Mixed cell infiltrate; minimal
5066
Liver
Within normal limits
Mixed cell infiltrate; minimal
5071
Liver
Within normal limits
Mixed cell infiltrate; minimal
D-39
-------
OCTOBER 2021
Animal #
Organ
SP Diagnosis
NTP Diagnosis
4954
Liver
Within normal limits
Agree with SP
4957
Liver
Within normal limits
Agree with SP
4961
Liver
Within normal limits
Agree with SP
4973
Liver
Within normal limits
Agree with SP
4979
Liver
Necrosis, single cell; minimal
Single cell necrosis; hepatocellular; minimal
4981
Liver
Within normal limits
Agree with SP
4988
Liver
Within normal limits
Focal necrosis; minimal
4989
Liver
Infiltrate, neutrophil, focal/multifocal;
minimal
Mixed cell infiltrate; minimal
4990
Liver
Within normal limits
Mixed cell infiltrate; minimal
4997
Liver
Within normal limits
Mixed cell infiltrate; minimal
5000
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Mixed cell infiltrate; minimal
5004
Liver
Within normal limits
Agree with SP
5005
Liver
Within normal limits
Agree with SP
5010
Liver
Within normal limits
Agree with SP
5015
Liver
Within normal limits
Agree with SP
5025
Liver
Within normal limits
Agree with SP
5036
Liver
Within normal limits
Mixed cell infiltrate; minimal
5040
Liver
Within normal limits
Agree with SP
5041
Liver
Within normal limits
Agree with SP
5046
Liver
Necrosis, single cell; minimal
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; minimal
5047
Liver
Within normal limits
Mixed cell infiltrate; minimal
5049
Liver
Within normal limits
Agree with SP
5061
Liver
Within normal limits
Agree with SP
5063
Liver
Within normal limits
Mixed cell infiltrate; minimal
5072
Liver
Necrosis, single cell; minimal
Focal necrosis; minimal
D-40
-------
OCTOBER 2021
Animal #
Organ
SP Diagnosis
NTP Diagnosis
4960
Liver
Hypertrophy, hepatocellular, diffuse;
minimal (with eosinophilic cytoplasmic
stippling)
Cytoplasmic alteration; minimal
Mixed cell infiltrate; minimal
4963
Liver
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse;
minimal (with eosinophilic cytoplasmic
stippling)
Focal necrosis; minimal
Cytoplasmic alteration; minimal
4969
Liver
Within normal limits
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; minimal
4974
Liver
Within normal limits
Single cell necrosis; hepatocellular; minimal
4975
Liver
Hypertrophy, hepatocellular, diffuse;
minimal (with eosinophilic cytoplasmic
stippling)
Cytoplasmic alteration; minimal
4976
Liver
Within normal limits
Mixed cell infiltrate; minimal
4977
Liver
Hypertrophy, hepatocellular, diffuse;
minimal
Cytoplasmic alteration; minimal
Mixed cell infiltrate; minimal
4980
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Hypertrophy, hepatocellular, diffuse;
minimal
Mixed cell infiltrate; minimal
Cytoplasmic alteration; minimal
4993
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Hypertrophy, hepatocellular, diffuse;
minimal (with eosinophilic cytoplasmic
stippling)
Mixed cell infiltrate; minimal
Cytoplasmic alteration; minimal
5007
Liver
Hypertrophy, hepatocellular, diffuse;
minimal
Cytoplasmic alteration; minimal
Mixed cell infiltrate; minimal
5011
Liver
Within normal limits
Agree with SP
5014
Liver
Necrosis, focal/multifocal; minimal
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse;
minimal
Focal necrosis; minimal
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; minimal
5022
Liver
Hypertrophy, hepatocellular, diffuse;
minimal
Cytoplasmic alteration; minimal
Mixed cell infiltrate; minimal
5023
Liver
Hypertrophy, hepatocellular, diffuse;
minimal
Cytoplasmic alteration; minimal
Mixed cell infiltrate; minimal
5031
Liver
Within normal limits
Mixed cell infiltrate; minimal
D-41
-------
OCTOBER 2021
Animal #
Organ
SP Diagnosis
NTP Diagnosis
5034
Liver
Hypertrophy, hepatocellular, diffuse;
minimal
Within normal limits
5037
Liver
Within normal limits
Mixed cell infiltrate; minimal
5043
Liver
Within normal limits
Mixed cell infiltrate; minimal
5048
Liver
Within normal limits
Agree with SP
5050
Liver
Necrosis, focal/multifocal; minimal
Focal necrosis; minimal
Cytoplasmic alteration; minimal
Mixed cell infiltrate; minimal
5052
Liver
Within normal limits
Mixed cell infiltrate; minimal
5056
Liver
Hypertrophy, hepatocellular, diffuse;
minimal (with eosinophilic stippling)
Cytoplasmic alteration; minimal
Mixed cell infiltrate; minimal
5057
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Necrosis, focal/multifocal; minimal
Hypertrophy, hepatocellular, diffuse;
minimal (with eosinophilic cytoplasmic
stippling)
Mixed cell infiltrate; minimal
Focal necrosis; minimal
Cytoplasmic alteration; minimal
Inflammation; granulomatous; focal;
minimal
5065
Liver
Hypertrophy, hepatocellular, diffuse;
minimal (with eosinophilic cytoplasmic
stippling)
Cytoplasmic alteration; minimal
Mixed cell infiltrate; minimal
5070
Liver
Within normal limits
Cytoplasmic alteration; minimal
Mixed cell infiltrate; minimal
4955
Liver
Mitotic figures increased; minimal
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Not present in section
Apoptosis; hepatocellular; minimal
Cytoplasmic alteration; mild
Polyarteritis nodosa; moderate
4959
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse;
moderate
Mixed cell infiltrate; minimal
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; mild
D-42
-------
OCTOBER 2021
Animal #
Organ
SP Diagnosis
NTP Diagnosis
4972
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Mitotic figures increased; minimal
Necrosis, focal/multifocal; minimal
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Mixed cell infiltrate; minimal
Not present in section
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
4982
Liver
Pigment, increased; minimal
Mitotic figures increased; minimal
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Agree with SP
Not present in section
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
4984
Liver
Pigment, increased; minimal
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Agree with SP
Single cell necrosis; hepatocellular; mild
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
4998
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse; mild
Mixed cell infiltrate; minimal
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; minimal
Apoptosis; hepatocellular; minimal
5002
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Necrosis, focal/multifocal; minimal
Mitotic figures increased; minimal
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Mixed cell infiltrate; minimal
Not present in section
Agree with SP
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
5006
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Necrosis, focal/multifocal; minimal
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Mixed cell infiltrate; minimal
Focal necrosis; minimal
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
5008
Liver
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse; mild
(with eosinophilic cytoplasmic stippling)
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; mild
D-43
-------
OCTOBER 2021
Animal #
Organ
SP Diagnosis
NTP Diagnosis
5009
Liver
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
Mixed cell infiltrate; minimal
5017
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Hematopoiesis, extramedullary; minimal
Necrosis, focal/multifocal; minimal
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse; mild
(with eosinophilic cytoplasmic stippling)
Mixed cell infiltrate; minimal
Agree with SP
Focal necrosis; minimal
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; mild
5020
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Necrosis, single cell; mild
Hypertrophy, hepatocellular, diffuse;
moderate
Mixed cell infiltrate; minimal
Single cell necrosis; hepatocellular; mild
Cytoplasmic alteration; mild
Apoptosis; hepatocellular; minimal
5027
Liver
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Cytoplasmic alteration; mild
5028
Liver
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
Mixed cell infiltrate; minimal
5029
Liver
Necrosis, focal/multifocal; minimal
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse; mild
(with eosinophilic cytoplasmic stippling)
Focal necrosis; minimal
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; mild
Mixed cell infiltrate; minimal
5033
Liver
Hypertrophy, hepatocellular, diffuse; mild
(with eosinophilic cytoplasmic stippling)
Cytoplasmic alteration; mild
5035
Liver
Hypertrophy, hepatocellular, diffuse; mild
Cytoplasmic alteration; mild
5051
Liver
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse;
minimal (with eosinophilic cytoplasmic
stippling)
Not present in section
Cytoplasmic alteration; minimal
Mitotic figures increased; minimal
Mixed cell infiltrate; minimal
Focal necrosis; minimal
D-44
-------
OCTOBER 2021
Animal #
Organ
SP Diagnosis
NTP Diagnosis
5062
Liver
Pigment, increased; minimal
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse; mild
Agree with SP
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; mild
Apoptosis; hepatocellular; minimal
Mixed cell infiltrate; minimal
5068
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse;
moderate (with eosinophilic cytoplasmic
stippling)
Mixed cell infiltrate; minimal
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
5069
Liver
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse; mild
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; mild
Apoptosis; hepatocellular; minimal
5073
Liver
Fatty change, centrilobular; mild
Hypertrophy, hepatocellular, diffuse; mild
(with eosinophilic cytoplasmic stippling)
Hepatocyte; cytoplasmic vacuolation; mild
Cytoplasmic alteration; mild
5074
Liver
Pigment, increased; minimal
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse; mild
(with eosinophilic cytoplasmic stippling)
Not present in section
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; mild
5075
Liver
Infiltrate, mononuclear cell, focal/multifocal;
minimal
Pigment, increased; minimal
Mitotic figures increased; minimal
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse;
moderate
Mixed cell infiltrate; minimal
Not present in section
Not present in section
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; moderate
Apoptosis; hepatocellular; minimal
5077
Liver
Necrosis, single cell; minimal
Hypertrophy, hepatocellular, diffuse; mild
Single cell necrosis; hepatocellular; minimal
Cytoplasmic alteration; mild
Focal necrosis; minimal
Mixed cell infiltrate; minimal
D-45
-------
OCTOBER 2021
Appendix E: Benchmark Dose Modeling
E.l Oral Reproduction/Developmental Toxicity Study in Mice (DuPont-18405-1037
2010)
U.S. Environmental Protection Agency (EPA) Center for Public Health and Environmental
Assessment conducted dose response modeling of this study using the Benchmark Dose
Software (BMDS) 3.1.2. program. This work used data from the reevaluation of the DuPont oral
reproductive/ developmental toxicity study slides by the National Toxicology Program (NTP)
Pathology Working Group (see section 4.5 for a description) and addresses the constellation of
liver lesions the NTP defined as adverse (i.e., cytoplasmic alteration, single-cell and focal
necrosis, and apoptosis) in parental male and parental female mice.
E.l.l Constellation of Lesions (Cytoplasmic Alteration, Apoptosis, Single-Cell Necrosis,
and Focal Necrosis) in the Liver, Parental Males
Increased incidence of a constellation of lesions in the liver was observed in the parental males.
Dichotomous models were used to fit dose-response data (DuPont-18405-1037, 2010). A
benchmark response (BMR) of 10% extra risk was chosen per EP A's Benchmark Dose
Technical Guidance (EPA, 2012). The doses and response data used for the modeling are listed
in Table E-l.
Table E-l. Constellation of Lesions in the Male Liver Selected for
Dose-Response Modeling
Dose
(nig/kg/day)
Number of rniee
(males)
Constellation of Liver
Lesions
0
25
1
0.1
24
1
0.5
24
13
5
24
24
Note: mg/kg/day = milligrams per kilogram per day.
The benchmark dose (BMD) modeling results for the constellation of lesions are summarized in
Table E-2 and Figure E-l. The best fitting model was the Probit model based on adequate
/(-values (greater than 0.1), the benchmark dose lower limits (BMDLs) were sufficiently close
(less than threefold difference) among adequately fitted models, and the Probit model had the
lowest Akaike information criterion (AIC). The lower bound on the dose level corresponding to
the 95% lower confidence limit for a 10% response level (BMDLio) from the selected Probit
model is 0.14 milligram per kilogram per day (mg/kg/day).
E-l
-------
OCTOBER 2021
Table E-2. Summary of BMD Modeling Results for Constellation of Lesions in Male Mice
Model3
Goodness of fit
Scaled residual for:
BMDioPct
(mg/kg/day)
BMDLioPct
(mg/kg/day)
Basis for model
selection
/>-value
AIC
Dose group
near BMD
Dose group
near BMDL
Dichotomous
Hill
N/Ab
57.818
0.007
-0.007
0.29
0.11
EPA ORD selected
the Weibull model.
All models, except
Dichotomous Hill,
had adequate fit (re-
values >0.1), the
BMDLs were
sufficiently close
(<3-fold
difference), and the
Probit model had
the lowest AIC.
Gamma
0.994
55.815
-0.005
0.005
0.26
0.09
Log-Logistic
0.977
55.816
0.000
-0.020
0.34
0.11
Multistage
Degree 3
0.997
53.820
-0.053
0.047
0.26
0.08
Multistage
Degree 2
0.905
54.026
-0.368
0.248
0.19
0.08
Multistage
Degree 1
0.279
57.026
-1.402
0.452
0.08
0.05
Weibull
0.937
53.951
-0.290
0.205
0.20
0.08
Logistic
0.888
54.048
-0.327
0.359
0.22
0.15
Log-Probit
0.990
55.816
0.001
-0.001
0.24
0.10
Probit
0.907
52.444
-0.635
0.093
0.19
0.14
Notes: ORD = Office of Research and Development.
a Selected model in bold.
b degrees of freedom=0, saturated model (Goodness of fit test cannot be calculated).
Frequentist Probit Model with BMR of 10% Extra Risk for the
BMD and 0.95 Lower Confidence Limit for the BMDL
Q
Estimated Probability
^^Response at BMD
O Data
BMD
BMDL
Dose
Figure E-l. Plot of Incidence Rate by Dose with Fitted Curve for the Selected Probit Model
for Constellation of Lesions in Male Mice (dose shown in mg/kg/day)
E-2
-------
OCTOBER 2021
E.1.2 Constellation of Lesions (Cytoplasmic Alteration, Apoptosis, Single-Cell Necrosis,
and Focal Necrosis) in the Liver, Parental Females
Increased incidence of the constellation of lesions in the liver was observed in the parental
females. Dichotomous models were used to fit dose-response data (DuPont-18405-1037, 2010).
A BMR of 10% extra risk was chosen per EPA's Benchmark Dose Technical Guidance (EPA,
2012). The doses and response data used for the modeling are listed in Table E-3.
Table E-3. Constellation of Lesions in the Female Liver Selected for
Dose-Response Modeling
Dose
(nig/kg/day)
Number of rniee
(females)
Constellation of Liver
Lesions
0
24
2
0.1
22
3
0.5
24
17
5
24
24
The BMD modeling results for constellation of lesions are summarized in Table E-4 and Figure
E-2. The best fitting model was the Probit model based on adequate ^-values greater than 0.1),
the BMDLs were sufficiently close (less than threefold difference) among adequately fitted
models, and the Probit model had the lowest AIC. The BMDLio from the selected Probit model
is 0.09 mg/kg/day.
E-3
-------
OCTOBER 2021
Table E-4. Summary of BMD Modeling Results for Constellation of Lesions in Female
Mice
Model"
Goodness of fit
Scaled residual for:
BMDioPct
(mg/kg/day)
BMDLioPct
(mg/kg/day)
Basis for model
selection
/>-value
AIC
Dose group
near BMD
Dose group
near BMDL
Dichotomous
Hill
N/Ab
68.371
0.108
-0.077
0.14
0.05
EPA ORD selected
the Probit model.
All models, except
Dichotomous Hill
and Multistage
Degree 3, had
adequate fit (re-
values > 0.1), the
BMDLs were
sufficiently close
(<3-fold
difference), and the
Probit model had
the lowest AIC.
Gamma
1.000
66.268
0.000
0.000
0.13
0.04
Log-Logistic
0.804
66.371
0.108
-0.077
0.14
0.05
Multistage
Degree 3
N/Ab
68.268
0.003
-0.002
0.15
0.04
Multistage
Degree 2
0.998
66.268
-0.002
0.001
0.14
0.04
Multistage
Degree 1
0.448
66.021
-1.087
0.393
0.05
0.04
Weibull
1.000
66.268
0.000
0.000
0.14
0.04
Logistic
0.993
64.283
-0.086
0.085
0.13
0.09
Log-Probit
0.932
66.282
0.024
-0.015
0.13
0.05
Probit
0.971
62.514
-0.328
-0.101
0.12
0.09
Notes: ORD = Office of Research and Development.
a Selected model in bold.
b degrees of freedom=0, saturated model (Goodness of fit test cannot be calculated).
Frequentist Probit Model with BMR of 10% Extra Risk for the
BMD and 0.95 Lower Confidence Limit for the BMDL
-©
Estimated Probabi
^^Response at BMD
O Data
BMD
BMDL
Dose
lity
Figure E-2. Plot of Incidence Rate by Dose with Fitted Curve for the Selected Probit Model
for Constellation of Lesions in Female Mice; (dose shown in mg/kg/day)
E-4
------- |