^Cm
EPA/635/R-23/027Fa
www.ftpa.nnv/iris
IRIS Toxicological Review of Perfluorohexanoic Acid
[PFHxA, CASRN 307-24-4] and Related Salts
April 2023
Integrated Risk Information System
Center for Public Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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Toxicological Review of PFHxA and Related Salts
DISCLAIMER
This document has been reviewed by the U.S. Environmental Protection Agency, Office of
Research and Development and approved for publication. Any mention of trade names, products, or
services does not imply an endorsement by the U.S. government or the U.S. Environmental
Protection Agency. EPA does not endorse any commercial products, services, or enterprises.
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Toxicological Review of PFHxA and Related Salts
CONTENTS
AUTHORS| CONTRIBUTORS| REVIEWERS xi
EXECUTIVE SUMMARY xiii
1. OVERVIEW OF BACKGROUND INFORMATION AND ASSESSMENT METHODS 1-1
1.1. BACKGROUND INFORMATION ON PFHXA AND RELATED AMMONIUM AND SODIUM
SALTS 1-1
1.1.1. Physical and Chemical Properties 1-1
1.1.2. Sources, Production, and Use 1-3
1.1.3. Environmental Fate and Transport 1-4
1.1.4. Potential for Human Exposure and Populations with Potentially Greater Exposure 1-4
1.2. SUMMARY OF ASSESSMENT METHODS 1-7
1.2.1. Literature Search and Screening 1-7
1.2.2. Evaluation of Individual Studies 1-10
1.2.3. Data Extraction 1-11
1.2.4. Evidence Synthesis and Integration 1-12
1.2.5. Dose-Response Analysis 1-14
2. SUMMARY OF LITERATURE IDENTIFICATION AND STUDY EVALUATION RESULTS 2-1
2.1. LITERATURE SEARCH AND SCREENING RESULTS 2-1
2.2. STUDY EVALUATION RESULTS 2-3
3. PHARMACOKINETICS, EVIDENCE SYNTHESIS, AND EVIDENCE INTEGRATION 3-1
3.1. PHARMACOKINETICS 3-1
3.1.1. Absorption 3-2
3.1.2. Distribution 3-2
3.1.3. Metabolism 3-8
3.1.4. Elimination 3-8
3.1.5. PBPK Models 3-15
3.1.6. Summary 3-15
3.2. NONCANCER EVIDENCE SYNTHESIS AND INTEGRATION 3-19
3.2.1. Hepatic Effects 3-19
3.2.2. Developmental Effects 3-43
3.2.3. Renal Effects 3-54
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Toxicological Review of PFHxA and Related Salts
3.2.4. Hematopoietic Effects 3-65
3.2.5. Endocrine Effects 3-77
3.2.6. Male Reproductive Effects 3-88
3.2.7. Female Reproductive Effects 3-96
3.2.8. Immune Effects 3-104
3.2.9. Nervous System Effects 3-112
3.3. CARCINOGENICITY 3-119
3.3.1. Cancer 3-119
4. SUMMARY OF HAZARD IDENTIFICATION CONCLUSIONS 4-1
4.1. SUMMARY OF CONCLUSIONS FOR NONCANCER HEALTH EFFECTS 4-1
4.2. CONCLUSIONS REGARDING SUSCEPTIBLE POPULATIONS AND LIFESTAGES 4-6
4.3. SUMMARY OF CONCLUSIONS FOR CARCINOGENICITY 4-6
5. DERIVATION OF TOXICITY VALUES 5-1
5.1. HEALTH EFFECT CATEGORIES CONSIDERED (CANCER AND NONCANCER) 5-1
5.2. NONCANCER TOXICITY VALUES 5-1
5.2.1. Oral Reference Dose (RfD) Derivation 5-2
5.2.2. Inhalation Reference Concentration (RfC) 5-37
5.3. CANCER TOXICITY VALUES 5-37
REFERENCES R-l
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Toxicological Review of PFHxA and Related Salts
TABLES
Table ES-1. Evidence integration judgments and derived toxicity values for PFHxA xv
Table 1-1. Physicochemical properties of PFHxA 1-2
Table 1-2. PFHxA levels at 10 military installations and National Priority List sites 1-6
Table 1-3. Populations, exposures, comparators, and outcomes (PECO) criteria 1-8
Table 3-1. Summary of PK evidence for PFHxA 3-17
Table 3-2. Evaluation results for animal studies assessing effects of PFHxA exposure on the
hepatic system 3-21
Table 3-3. Percent increase in relative liver weight due to PFHxA exposure in short-term and
subchronic oral toxicity studies 3-22
Table 3-4. Incidence of hepatocellular hypertrophy findings in adult rats due to PFHxA exposure
in short-term and subchronic oral toxicity studies 3-23
Table 3-5. Percent change in alanine aminotransferase due to PFHxA exposure in short-term,
subchronic, and chronic oral toxicity studies 3-26
Table 3-6. Percent change in aspartate aminotransferase due to PFHxA exposure in short-term,
subchronic, and chronic oral toxicity studies 3-27
Table 3-7. Percent change in alkaline phosphatase due to PFHxA exposure in short-term,
subchronic, and chronic oral toxicity studies 3-28
Table 3-8. Percent change in total protein due to PFHxA exposure in short-term, subchronic, and
chronic oral toxicity studies 3-30
Table 3-9. Percent change in globulins due to PFHxA exposure in short term, subchronic, and
chronic oral toxicity studies 3-31
Table 3-10. Gene targets identified from EPA CompTox Chemicals Dashboard after PFHxA
treatment in human liver cell lines3 3-36
Table 3-11. Evidence profile table for hepatic effects 3-40
Table 3-12. Study design characteristics and outcome-specific study confidence for
developmental endpoints 3-43
Table 3-13. Incidence of perinatal mortality following PFHxA ammonium salt exposure in a
developmental oral toxicity study 3-45
Table 3-14. Percent change relative to control in offspring body weight due to PFHxA sodium or
ammonium salt exposure in developmental oral toxicity studies 3-48
Table 3-15. Percent change relative to control in eye opening due to PFHxA ammonium salt
exposure in a developmental oral toxicity study 3-49
Table 3-16. Evidence profile table for developmental effects 3-52
Table 3-17. Renal endpoints for PFHxA and associated confidence scores from repeated-dose
animal toxicity studies 3-55
Table 3-18. Percent increase in relative and absolute kidney weight due to PFHxA exposure in
short-term, subchronic, and chronic oral toxicity studies 3-56
Table 3-19. Evidence profile table for renal effects 3-62
Table 3-20. Hematopoietic endpoints for PFHxA and associated confidence scores from
repeated-dose animal toxicity studies 3-65
Table 3-21. Percent change in red blood cells due to PFHxA exposure in short-term, subchronic,
and chronic oral toxicity studies 3-67
Table 3-22. Percent change in hematocrit due to PFHxA exposure in short-term, subchronic, and
chronic oral toxicity studies 3-69
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Toxicological Review of PFHxA and Related Salts
Table 3-23. Percent change in hemoglobin due to PFHxA exposure in short-term, subchronic,
and chronic oral toxicity studies 3-70
Table 3-24. Percent change in reticulocytes due to PFHxA exposure in short-term, subchronic,
and chronic oral toxicity studies 3-72
Table 3-25. Evidence profile table for hematopoietic effects 3-75
Table 3-26. Endocrine endpoints for PFHxA and associated confidence scores from
repeated-dose animal toxicity studies 3-78
Table 3-27. Percent change in thyroid hormone levels following PFHxA exposure in a 28-day oral
toxicity study 3-79
Table 3-28. Incidence of thyroid follicular epithelial cell hypertrophy following PFHxA or PFHxA
ammonium salt exposures in rats 3-80
Table 3-29. Evidence profile table for endocrine effects 3-85
Table 3-30. Study design, exposure characteristics, and individual outcome ratings 3-90
Table 3-31. Evidence profile table for male reproductive effects 3-94
Table 3-32. Study design characteristics 3-97
Table 3-33. Evidence profile table for female reproductive effects 3-102
Table 3-34. Study design characteristics and individual outcome ratings for immune endpoints 3-105
Table 3-35. Evidence profile table for immune effects 3-110
Table 3-36. Nervous system endpoints for PFHxA and associated confidence scores from
repeated-dose animal toxicity studies 3-112
Table 3-37. Changes in expression of genes related to neurodevelopment in early life stage
zebrafish 3-114
Table 3-38. Evidence profile table for nervous system effects 3-116
Table 3-39. Summary of PFHxA genotoxicity studies 3-121
Table 4-1. Hazard conclusions across published EPA PFAS human health assessments 4-4
Table 5-1. Endpoints considered for dose-response modeling and derivation of points of
departure 5-4
Table 5-2. Benchmark response levels selected for BMD modeling of PFHxA health outcomes 5-8
Table 5-3. Summary of serum half-lives and estimated clearance for PFHxA 5-14
Table 5-4. Two options for rat, mouse, and human clearance values and data-informed
dosimetric adjustment factor 5-15
Table 5-5. PODs considered for the derivation of the RfD 5-19
Table 5-6. Uncertainty factors for the development of the RfD for PFHxA 5-22
Table 5-7. Candidate values for PFHxA 5-24
Table 5-8. Confidence in the organ/system specific RfDsfor PFHxA 5-26
Table 5-9. Organ/system specific RfD values for PFHxA 5-28
Table 5-10. PODs considered for the derivation of the subchronic RfD 5-29
Table 5-11. Candidate subchronic toxicity values for PFHxA 5-32
Table 5-12. Confidence in the subchronic organ/system specific RfDs for PFHxA 5-33
Table 5-13. Subchronic osRfD values for PFHxA 5-36
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Toxicological Review of PFHxA and Related Salts
FIGURES
Figure 1-1. Linear chemical structures of (from left to right) PFHxA, PFHxA NH4, and PFHxA-Na 1-2
Figure 2-1. Literature search and screening flow diagram for PFHxA and related salts, PFHxA-NH4
and PFHxA-Na 2-2
Figure 2-2. Study evaluation results for human epidemiology studies 2-4
Figure 2-3.Study evaluation results for animal toxicology studies 2-5
Figure 3-1. Study evaluation for human epidemiological studies reporting hepatic system
findings from PFHxA exposures 3-20
Figure 3-2. Liver weights (absolute and relative) after short-term and subchronic PFHxA
exposures 3-22
Figure 3-3. Clinical chemistry findings (serum enzymes) after short-term, subchronic, and chronic
PFHxA exposures 3-26
Figure 3-4. Blood protein findings after short-term, subchronic, and chronic PFHxA exposures 3-29
Figure 3-5. Hepatobiliary findings in rats exposed by gavage to PFHxA or PFHxA sodium salt 3-32
Figure 3-6. Peroxisomal beta oxidation activity in rats exposed by gavage to PFHxA or PFHxA
sodium salt 3-35
Figure 3-7. Developmental effects on offspring viability in mice exposed to PFHxA ammonium
salt 3-45
Figure 3-8. Developmental effects on offspring body weight in mice exposed to PFHxA
ammonium salt and rats exposed to PFHxA sodium salt 3-47
Figure 3-9. Developmental effects on eye opening (percent change relative to control) in mice
exposed to PFHxA ammonium salt 3-49
Figure 3-10. Study evaluation for human epidemiological studies reporting findings from PFHxA
exposures 3-54
Figure 3-11. Animal toxicological renal histopathology after PFHxA exposure 3-58
Figure 3-12. PFHxA Effects on blood and urine biomarkers of renal function 3-60
Figure 3-13. Hematological findings (HCT, HGB, and RBC) in rats exposed by gavage to PFHxA or
PFHxA sodium salt 3-67
Figure 3-14. Hematological findings (MCH, MCHC, and MCV) in rats exposed by gavage to PFHxA
or PFHxA sodium salt 3-69
Figure 3-15. Hematological findings (reticulocytes) in rats exposed by gavage to PFHxA or PFHxA
sodium salt 3-71
Figure 3-16. Hemostasis findings in rats exposed by gavage to PFHxA or PFHxA sodium salt 3-73
Figure 3-17. Study evaluation for human epidemiologic studies reporting toxicity findings from
PFHxA exposures 3-77
Figure 3-18. Thyroid hormone measures from the serum of rats exposed by gavage to PFHxA or
PFHxA sodium salt 3-79
Figure 3-19. Study evaluation for human epidemiological studies reporting male reproductive
findings from PFHxA exposures 3-89
Figure 3-20. Male reproductive effects on sperm parameters in male rats exposed to PFHxA or
sodium salt for 28 or 90 days 3-91
Figure 3-21. Male reproductive effects on epididymis and testis weight in rats exposed to PFHxA
or PFHxA sodium salt 3-92
Figure 3-22. Study evaluation for human epidemiological studies reporting female reproductive
findings from PFHxA exposures 3-96
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Toxicological Review of PFHxA and Related Salts
Figure 3-23. Effects on body weight in female rats and mice exposed to PFHxA or PFHxA
ammonium salt in reproductive studies 3-99
Figure 3-24. Female reproductive effects on uterine horn dilation in rats exposed to PFHxA for
28 days 3-100
Figure 3-25. Study evaluation for human epidemiological studies reporting findings from PFHxA
exposures 3-104
Figure 3-26. Immune organ weights in rats exposed by gavage to PFHxA or PFHxA sodium salt 3-106
Figure 3-27. Immune cell counts in rats exposed by gavage to PFHxA or PFHxA sodium salt 3-108
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Toxicological Review of PFHxA and Related Salts
ABBREVIATIONS AND ACRONYMS
ADME
absorption, distribution, metabolism,
IUR
inhalation unit risk
and excretion
i.v.
intravenous
AFFF
aqueous film-forming foam
LDH
lactate dehydrogenase
A:G
albumin:globulin ratio
LOQ
limit of quantitation
AIC
Akaike's information criterion
LOAEL
lowest-observed-adverse-effect level
ALP
alkaline phosphatase
LOD
limit of detection
ALT
alanine aminotransferase
LOEC
lowest-observed-effect concentration
APTT
activated partial thromboplastin time
MCH
mean cell hemoglobin
AST
aspartate aminotransferase
MCHC
mean cell hemoglobin concentration
atm
atmosphere
MCV
mean cell volume
ATSDR
Agency for Toxic Substances and
MOA
mode of action
Disease Registry
MW
molecular weight
AUC
area under the curve
NCTR
National Center for Toxicological
BMD
benchmark dose
Research
BMDL
benchmark dose lower confidence limit
NOAEL
no-observed-adverse-effect level
BMDS
Benchmark Dose Software
NPL
National Priorities List
BMR
benchmark response
NTP
National Toxicology Program
BUN
blood urea nitrogen
ORD
Office of Research and Development
BW
body weight
OECD
Organisation for Economic
Cmax
maximum concentration
Co-operation and Development
CAR
constitutive androstane receptor
OSF
oral slope factor
CASRN
Chemical Abstracts Service registry
osRfD
organ/system-specific oral reference
number
dose
CBC
complete blood count
PBPK
physiologically based pharmacokinetic
CI
confidence interval
PC
partition coefficient
CL
clearance
PECO
populations, exposures, comparators,
CLa
clearance in animals
and outcomes
CLh
clearance in humans
PFAA
perfluoroalkyl acids
CPHEA
Center for Public Health and
PFAS
per- and polyfluoroalkyl substances
Environmental Assessment
PFBA
perfluorobutanoic acid
CPN
chronic progressive nephropathy
PFBS
perfluorobutane sulfonate
DAF
dosimetric adjustment factor
PFCA
perfluorinated carboxylic acid
DNA
deoxyribonucleic acid
PFDA
perfluorodecanoic acid
DTXSID
DSSTox substance identifier
PFHxA
perfluorohexanoic acid
eGFR
estimated glomerular filtration rate
PFHxS
perfluorohexane sulfonate
EPA
Environmental Protection Agency
PFNA
perfluorononanoic acid
ER
extra risk
PFOA
perfluorooctanoic acid
FTOH
fluorotelomer alcohol
PFOS
perfluorooctane sulfonate
GD
gestation day
PK
pharmacokinetic
GGT
y-glutamyl transferase
PND
postnatal day
HAWC
Health Assessment Workplace
POD
point of departure
Collaborative
PODhed
human equivalent dose POD
HCT
hematocrit
PPAR
peroxisome proliferated activated
HED
human equivalent dose
receptor
HERO
Health and Environmental Research
PQAPP
programmatic quality assurance
Online
project plan
HGB
hemoglobin
PT
prothrombin time
HSA
human serum albumin
QA
quality assurance
IQR
interquartile range
QAPP
quality assurance project plan
IRIS
Integrated Risk Information System
QMP
quality management plan
ISI
Influential Scientific Information
RBC
red blood cells
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RD
RfC
RfD
RNA
ROS
RXR
SD
TP
TRI
TSCATS
TSH
UF
UFa
UFc
UFd
UFh
UFl
UFs
Vi
Vi
Toxicological Review of PFHxA and Related Salts
relative deviation
reference concentration
oral reference dose
ribonucleic acid
reactive oxygen species
retinoid X receptor
standard deviation
total protein
Toxics Release Inventory
Toxic Substances Control Act Test
Submissions
thyroid stimulating hormone
uncertainty factor
interspecies uncertainty factor
composite uncertainty factor
evidence base deficiencies uncertainty
factor
human variation uncertainty factor
LOAEL to NOAEL uncertainty factor
subchronic to chronic uncertainty
factor
volume of distribution of peripheral
compartment (two-compartment PK
model)
volume of distribution
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Toxicological Review of PFHxA and Related Salts
Assessment Team (Lead Authors)
Michelle M. Angrish. Ph.D.
Laura Dishaw. Ph.D.
U.S. EPA/Office of Research and Development/Center
for Public Health and Environmental Assessment
Authors
I. Allen Davis. M.S.P.H.
leffrv L. Dean II. Ph.D.
Andrew Kraft. Ph.D.
Elizabeth G. Radke. Ph.D.
Paul Schlosser. Ph.D.
Yu-Sheng Lin. M.S.
Shana White. Ph.D.
lav Zhao. Ph.D., M.P.H.
Todd Zurlinden. Ph.D.
U.S. EPA/Office of Research and Development/Center
for Public Health and Environmental Assessment
Contributors
Xabier Arzuaga. Ph.D.
Johanna Congleton, M.S.P.H., Ph.D.
Ingrid L. Druwe. Ph.D.
I. Phillip Kaiser. Ph.D., DABT
Elizabeth Oesterling Owens, Ph.D.
Michele M. Taylor. Ph.D.
Andre Weaver, Ph.D.
Amina Wilkins, M.P.H.
Michael Wright, Sc.D.
Belinda Hawkins, Ph.D.
Jason C. Lambert, Ph.D., DABT
Kelly Garcia, B.S.*
Carolyn Gigot, B.A.*
Andrew Greenhalgh, B.S.*
Shahreen Hussain, B.S.*
Brittany Schulz. B.S.
U.S. EPA/Office of Research and Development/Center for
Public Health and Environmental Assessment
U.S. EPA/Office of Research and Development/Office of
Science Advisor, Policy, and Engagement
U.S. EPA/Office of Research and Development/Center for
Computational Toxicology and Exposure
Oak Ridge Associated Universities [ORAU] Contractor
*No longer with U.S. EPA
Production Team
Maureen Johnson
Ryan Jones
Dahnish B. Shams
Vicki M. Soto
Jessica M. Soto Hernandez
Samuel Thacker
Garland Waleko
U.S. EPA/Office of Research and Development/Center for
Public Health and Environmental Assessment
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Toxicological Review of PFHxA and Related Salts
Production Team
Ashlei Williams Former Oak Ridge Associated Universities (ORAU)
Contractor
Executive Direction
Wayne Cascio
V. Kay Holt
Samantha Jones
Kristina A. Thayer
Andrew D. Kraft
Paul D. White
Ravi Subramaniam
Janice S. Lee
Viktor Morozov
Elizabeth G. Radke
Garland Waleko
CPHEA Center Director
CPHEA Deputy Center Director
CPHEA Associate Director
CPAD Division Director
IRIS PFAS Team Lead, CPAD Associate Division Director
CPAD Senior Science Advisor
Acting CPAD Senior Science Advisor
CPHEA/CPAD/Toxic Effects Assessment (RTP) Branch Chief
CPHEA/CPAD/Quantitative Assessment Branch Chief
CPHEA/CPAD/Science Assessment Methods Branch Chief
Acting CPAD Toxic Effects Assessment (DC) Branch Chief
Review
CPAD Executive Review Committee
Janice S. Lee CPHEA/CPAD/Toxic Effects Assessment (RTP) Branch Chief
Kristina A. Thayer CPAD Division Director
Paul D. White CPAD Senior Science Advisor
Alan Stern NJDEP (retired), Contractor
Karen Hogan CPHEA/CPAD/Emeritus
Agency Reviewers
This assessment was provided for review to scientists in EPA's program and regional offices.
Comments were submitted by:
Office of Children's Health Protection
Office of Land and Emergency Management
Office of Pollution Prevention and Toxics
Office of Water
Region 2, New York
Region 8, Colorado
Interagency Reviewers
This assessment was provided for review to other federal agencies and the Executive Office of the
President (EOP). Comments were submitted by:
Agency for Toxic Substances and Disease Registry
Department of Defense
Executive Office of the President
Food and Drug Administration
National Institute for Occupational Safety and Health
National Institutes of Health
National Toxicology Program
United States Department of Agriculture
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EXECUTIVE SUMMARY
Summary of Occurrence and Health Effects
Perfluorohexanoic acid (PFHxA, CASRN 307-24-4]1 and its related salts are members
of the group per and polyfluoroalkyl substances (PFAS). This assessment applies to
PFHxA as well as salts of PFHxA, including ammonium perfluorohexanoate (PFHxA-
NH4, CASRN 21615-47-4), and sodium perfluorohexanoate (PFHxA-NA, CASRN 2923-
26-4], and other nonmetal and alkali metal salts of PFHxA that would be expected to
fully dissociate in aqueous solutions of pH ranging from 4-9 (e.g., in the human body)
and not release other moieties that would cause toxicity independent of PFHxA.
Notably, due to the possibility of PFHxA-independent contributions of toxicity, this
assessment would not necessarily apply to nonalkali metal salts of PFHxA (e.g., silver
perfluorohexanoate; CASRN 336-02-7). The synthesis of evidence and toxicity value
derivation presented in this assessment focuses on the free acid of PFHxA and related
ammonium and sodium salts given the currently available toxicity data.
Concerns about PFHxA and other PFAS stem from the resistance of these compounds
to hydrolysis, photolysis, and biodegradation, which leads to their persistence in the
environment. PFAS are not naturally occurring in the environment; they are
manmade compounds that have been used widely over the past several decades in
industrial applications and consumer products because of their resistance to heat, oil,
stains, grease, and water. PFAS in the environment are linked to industrial sites,
military fire training areas, wastewater treatment plants, and commercial products
(Appendix A, Section 2.1.2)
The Integrated Risk Information System (IRIS) Program is developing a series of five
PFAS assessments (i.e., perfluorobutanoic acid [PFBA], perfluorohexanoic acid
[PFHxA], perfluorohexane sulfonate [PFHxS], perfluorononanoic acid [PFNA],
perfluorodecanoic acid [PFDA], and their associated salts) at the request of EPA
National Programs and Regions. Specifically, the development of human health
toxicity assessments for exposure to these individual PFAS represents only one
component of the broader PFAS strategic roadmap at the EPA
f https://www.epa.gov/pfas/pfas-strategic-roadmap-epas-commitments-action-
2021-2024). The systematic review protocol (see Appendix A) for these five PFAS
assessments outlines the related scoping and problem formulation efforts, including
a summary of other federal and state assessments of PFHxA. The protocol also lays
out the systematic review and dose-response methods used to conduct this review
(see also Section 1.2). The systematic review protocol was released for public
comment in November 2019 and was updated based on those public comments.
Appendix A links to the updated version of the protocol and summary of revisions.
1 The CASRN given is for linear PFHxA; the source PFHxA used in toxicity studies was reported to be >93%
pure. No explicit statement that only the linear form was used was available from the studies. Therefore,
there is the possibility that a minor proportion of the PFHxA used in the studies were branched isomers and
thus observed health effects may apply to the total linear and branched isomers in a given exposure source.
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Toxicological Review of PFHxA and Related Salts
Human epidemiological studies have examined possible associations between PFHxA
exposure and health outcomes, such as liver enzymes, thyroid hormones, blood lipids,
blood pressure, insulin resistance, body mass index, semen parameters, reproductive
hormones, and asthma. The ability to draw conclusions regarding these associations
is limited by the overall conduct of the studies (studies were generally low
confidence); the few studies per health outcome; and, in some studies, the lack of a
quantifiable measure of exposure. No studies were identified that evaluated the
association between PFHxA exposure and carcinogenicity in humans.
Animal studies of PFHxA exposure exclusively examined the oral exposure route, and
therefore, no inhalation assessment was conducted nor was an RfC derived (see
Section 5.2.2). The available animal studies of oral PFHxA exposure examined a
variety of noncancer and cancer endpoints, including those relevant to hepatic,
developmental, renal, hematopoietic, endocrine, reproductive, immune, and nervous
system effects.
Overall, the available evidence indicates that PFHxA likely causes hepatic,
developmental, hematopoietic, and endocrine (see Sections 3.2.1, 3.2.2, 3.2.4, and
3.2.5, respectively) effects in humans given sufficient exposure conditions.
Specifically, for hepatic effects, the primary support for this hazard conclusion
included evidence of increased relative liver weights and increased incidence of
hepatocellular hypertrophy in adult rats. These hepatic findings correlated with
changes in clinical chemistry (e.g., serum enzymes, blood proteins) and necrosis. For
hematopoietic effects, the primary supporting evidence included decreased red blood
cell counts, decreased hematocrit values, and increased reticulocyte counts in adult
rats. Developmental effects were identified as a hazard based on evidence of
decreased offspring body weight and increased perinatal mortality in exposed rats
and mice. A short-term (28-day) study in rats showed a strong dose dependent effect
on serum thyroid hormones in males. Selected quantitative data from these identified
hazards were used to derive toxicity values (see Table ES-1).
Although some human and animal evidence was also identified for renal, male, and
female reproductive, immune, and nervous system effects, the currently available
evidence is inadequate to assess whether PFHxA may cause these health effects in
humans (see Sections 3.2.3, 3.2.6, 3.2.7, 3.2.8, and 3.2.9 respectively) and were not
used to derive toxicity values.
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Toxicological Review of PFHxA and Related Salts
Table ES-1. Evidence integration judgments and derived toxicity values for PFHxA
Health system
Evidence
integration
judgment
Toxicity
value type
Value for
PFHxA
(mg/kg-d)
Value for
PFHxA-Naa
(mg/kg-d)
Value for
PFHxA-NH4a
mg/kg-d)
Confidence
in toxicity
valueb
UFcc'd,e
Basis
Hepatic
Evidence indicates
(likely)
osRfD
4 x 10"4
4 x 10"4
4 x 10"4
Medium
300
Increased hepatocellular hypertrophy in
adult rats (Loveless et al., 2009)
Subchronic
osRfD
1 x 10"3
1 x 10"3
1 x 10"3
Medium
100
Increased hepatocellular hypertrophy in
adult rats (Loveless et al., 2009)
Hematopoietic
Evidence indicates
(likely)
osRfD
5 x 10"3
6 x 10"3
5 x 10"3
Medium
100
Decreased red blood cells in adult rats
(Klaunig et al., 2015)
Subchronic
osRfD
8 x 10"4
8 x 10"4
8 x 10"4
Medium-Low
100
Decreased red blood cells in adult rats
(Chengelis et al., 2009b)
Developmental
Evidence indicates
(likely)
osRfD
5 x 10"4
5 x 10"4
5 x 10"4
Medium
100
Decreased Fi body weight at PND 0 in rats
(Loveless et al., 2009)
Subchronic
osRfD
5 x 10"4
5 x 10"4
5 x 10"4
Medium
100
Decreased Fi body weight at PND 0 in rats
(Loveless et al., 2009)
Endocrine
Evidence indicates
(likely)
osRfD
NA
NA
NA
NA
NA
Not derived due to high degree of
uncertainty with deriving a lifetime value
from a short-term study.
Subchronic
osRfD
1 x 10"3
1 x 10"3
1 x 10"3
Medium
300
Decreased Free T4 in adult male rats
(NTP, 2018)
RfDd
5 x 10"4
5 x 10"4
5 x 10"4
Medium
100
Decreased Fi body weight at PND 0 in rats
(Loveless et al., 2009)
Subchronic RfD8
5 x 10"4
5 x 10"4
5 x 10"4
Medium
100
Decreased Fi body weight at PND 0 in rats
(Loveless et al., 2009)
See Section 5.2.1 for full details on study and dataset selection, modeling approaches (including BMR selection), uncertainty factor application, candidate value selection, and
characterization of confidence in the osRfDs and RfDs.
RfD = reference dose (in mg/kg-day) for lifetime exposure; subchronic RfD = reference dose (in mg/kg-d) for less-than-lifetime exposure; osRfD = organ/system specific oral
reference dose (in mg/kg-d); UFC = composite uncertainty factor which is the product of the interspecies uncertainty factor (UFA), interindividual human variability uncertainty
factor (UFh), subchronic-to-chronic uncertainty factor (UFS), LOAEL-to-NOAEL uncertainty factor (UFL), and database uncertainty factor (UFD); NA = not applicable.
aSee Tables 5-7 and 5-11 for details on how to calculate candidate values for salts of PFHxA. The osRfDs presented in this table have been rounded to 1 significant digit from the
candidate values presented in Tables 5-7 and 5-11.
bThe overall confidence in the derived toxicity values is synthesized from confidence judgments regarding confidence in the study used to derive the toxicity value, confidence in
the evidence base supporting the hazard, and confidence in the quantification of the point of departure; see Table 5-8 for full details regarding the confidence judgments.
cSee Table 5-6 for an explanation of the uncertainty factors applied to derive the osRfD and subchronic osRfD values.
developmental and hematopoietic UFC = 100 based on UFA = 3, UFH = 10, UFS = 1, UFL = 1, and UFD = 3; hepatic UFC = 300 based on UFA = 3, UFH = 10, UFS = 3, UFL = 1, and
UFd = 3.
eHepatic, developmental, and hematopoietic UFC = 100 based on UFA = 3, UFH = 10, UFS = 1, UFL = 1, and UFD = 3; endocrine UFC = 300 based on UFA = 3, UFH = 10, UFS = 3, UFL = 1,
and UFd = 3.
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ES.1 CHRONIC ORAL REFERENCE DOSE (RFD) FOR NONCANCER EFFECTS
From the identified hazards of potential concern (i.e., endocrine, hepatic, hematopoietic,
and developmental toxicity), decreased offspring body weight in neonatal rats fLoveless etal..
20091 was selected as the basis for the RfD of 5 x 10 4 mg/kg-day. A BMDLsrd of 10.62 mg/kg-day
was identified for this endpoint and was used as the point of departure (POD). The human
equivalent dose POD (PODhed) of 0.048 mg/kg-day was derived by applying the ratio of the
clearance between female rats and humans and a normalization from the sodium salt to the free
acid using a molecular weight conversion. The overall RfD for PFHxA was calculated by dividing the
PODhed by a composite uncertainty factor of 100 to account for pharmacodynamic uncertainty in
the extrapolation from rats to humans (UFa = 3), inter individual differences in human susceptibility
(UFh = 10), and deficiencies in the toxicity evidence base (UFd = 3).
ES.2 CONFIDENCE IN THE ORAL REFERENCE DOSE (RFD)
The study conducted by Loveless etal. (2009) reported developmental effects following
administration of PFHxA sodium salt to pregnant Sprague-Dawley rats dosed by gavage for
approximately 70 days prior to cohabitation through gestation and lactation, for a total of 126 days
daily gavage with 0, 20,100, or 500 mg/kg-day sodium PFHxA. The overall confidence in the osRfD
is medium and is primarily driven by medium confidence in the overall evidence base for
developmental effects, high confidence in the study (click the HAWC link for full study evaluation
details), and medium confidence in quantitation of the POD (see Table 5-8). High confidence in the
study was not interpreted to warrant changing the overall confidence in the RfD from medium.
ES.3 SUBCHRONIC ORAL REFERENCE DOSE (RFD) FOR NONCANCER EFFECTS
In addition to providing RfDs for chronic oral exposures in multiple systems, a less-than-
lifetime subchronic RfD was derived for PFHxA. The same study and endpoint fLoveless etal..
20091 and decreased Fi body weight and value was selected as the basis for the subchronic RfD of
5 x 10 4 mg/kg-day (see Table ES-1). Details are provided in Section 5.2.1.
ES.4 NONCANCER EFFECTS FOLLOWING INHALATION EXPOSURE
No studies that examine toxicity in humans or experimental animals following inhalation
exposure and no physiologically based pharmacokinetic (PBPK) models are available to support
route-to-route extrapolation; therefore, no RfC was derived.
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ES.5 EVIDENCE FOR CARCINOGENICITY
Under EPA's Guidelines for Carcinogen Risk Assessment fU.S. EPA. 20051. EPA concluded
there is inadequate information to assess carcinogenic potential for PFHxA by all routes of exposure.
The lack of data on the carcinogenicity of PFHxA precludes the derivation of quantitative estimates
for either oral (oral slope factor [OSF]) or inhalation (inhalation unit risk [IUR]) exposure (see
Section 3.3).
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1.OVERVIEW OF BACKGROUND INFORMATION
AND ASSESSMENT METHODS
A series of five PFAS assessments (perfluorobutanoic acid [PFBA], perfluorohexanoic acid
[PFHxA], perfluorohexane sulfonate [PFHxS], perfluorononanoic acid [PFNA], perfluorodecanoic
acid [PFDA], and their associated salts) are being developed by the Integrated Risk Information
System (IRIS) Program at the request of the U.S. Environmental Protection Agency (EPA) National
Programs and Regions. Appendix A is the systematic review protocol for these five PFAS
assessments. The protocol outlines the scoping and problem formulation efforts relating to these
assessments, including a summary of other federal and state reference values for PFHxA. The
protocol also lays out the systematic review and dose-response methods used to conduct this
review (see also Section 1.2). This systematic review protocol was released for public comment in
November 2019 and was subsequently updated based on those public comments. Appendix A
includes the updated version of the protocol, including a summary of the updates in the protocol
history section (see Appendix A, Section 12).
1.1. BACKGROUND INFORMATION ON PFHxA AND RELATED AMMONIUM
AND SODIUM SALTS
This section provides a brief overview of aspects of the physiochemical properties, human
exposure, and environmental fate characteristics of perfluorohexanoic acid (PFHxA, CASRN
307-24-4), ammonium perfluorohexanoate (PFHXA-NH4, CASRN 21615-47-4), and sodium
perfluorohexanoate (PFHxA-Na, CASRN 2923-26-4). This overview is not intended to provide a
comprehensive description of the available information on these topics and is not recommended
for use in decision making. The reader is encouraged to refer to source materials cited below, more
recent publications on these topics, and the assessment systematic review protocol (see
Appendix A).
1.1.1. Physical and Chemical Properties
PFHxA and related sodium and ammonium PFHxA salts covered in this assessment are
members of the group of per- and polyfluoroalkyl substances (PFAS). Concerns about PFHxA and
other PFAS stem from the resistance of these compounds to hydrolysis, photolysis, and
biodegradation, which leads to their persistence in the environment fNLM. 2017. 2016. 20131.
PFHxA and related salts are classified as a perfluorinated carboxylic acids (PFCAs) fOECD. 20151.
PFHxA and its associated salts are considered short-chain PFAS fATSDR. 20211. The linear chemical
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structures 2 of these chemicals are presented in Figure 1-1, and select physiochemical properties
are provided in Table 1-1. When available, experimental values are provided in the table but
predicted values that may be less reliable are included in the absence of experimental data.
NKj o
Na
CASRN
DTXSID
PFHxA
307-24-4
3031862
PFHxA
ammonium salt
21615-47-4
90880232
PFHxA
sodium salt
2923-26-4
3052856
Figure 1-1. Linear chemical structures of (from left to right) PFHxA,
PFHxA NH4, and PFHxA-Na.
PFHxA = perfluorohexanoic acid; PFHxA NH4 = ammonium perfluorohexanoate; PFHxA-Na = sodium
perfluorohexanoate.
Source: EPA CompTox Chemicals Dashboard.
Table 1-1. Physicochemical properties of PFHxA
Property (unit)
PFHxA value
PFHxA-NH4 value
PFHxA-Na value
Formula
CsHFiiChCsHFuCh
C6H4F11NO2
CsFnNaCh
Molecular weight (g/mol)
314
331
336
Melting point (°C)
12.2a
39.2b
70.2b
Boiling point (°C)
157a
156b
216b
Density (g/cm3)
1.69b
1.72b
1.69b
Vapor pressure (mm Hg)
0.908a
2.00b
1.63b
Henry's law constant (atm-m3/mole)
2.35 x I0~10
2.35 x I0"10
2.35 x I0"10
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Toxicological Review of PFHxA and Related Salts
Property (unit)
PFHxA value
PFHxA-NH4 value
PFHxA-Na value
PKa
-0.16°
-
-
LogP Octanol-Water^
2.85a
3.97b
0.70a
Soil adsorption coefficient (L/kg)
l,070b
l,070b
l,070b
Bioconcentration factor
49.3b
5.47b
49.3b
= data not available.
a(U.S. EPA, 2018a). CompTox Chemicals Dashboard; access date 2/18/2021. Median or average experimental
values.
bAverage or median predicted values are, in general, less reliable than experimental values.
Predicted value reported by Steinle-Darling and Reinhard (2008).
Experimental measure of PFAS octanol/water partition coefficient are difficult due to the tendency for alkyl acids
to aggregate at the interface between octanol and water (Kim et al., 2015).
1.1.2. Sources, Production, and Use
PFAS have been used widely over the past several decades in consumer products and
industrial applications because of their resistance to heat, oil, stains, grease, and water. fATSDR.
2021: U.S. EPA. 2020. 2019c. 2013. 2007. 2002b). Fluorinated compounds have also been used in
consumer products including stain-resistant fabrics for clothing, carpets, and furniture; nonstick
cookware; ski wax; certain leather products; and personal care products (e.g., dental floss,
cosmetics, and sunscreen) fATSDR. 2021: U.S. EPA. 2020. 2019c. 2013. 2007. 2002b). PFAS also
have been detected from foam used in firefighting and in industrial surfactants, emulsifiers, wetting
agents, additives, and coatings; they are also used in aerospace, automotive, building, and
construction industries to reduce friction fU.S. EPA. 2020. 2019c: ATSDR. 2018: U.S. EPA. 2013.
2007. 2002b). PFHxA has been detected as a breakdown product of PFAS used in water- and stain-
protective coatings for carpets, paper, and textiles including textiles used in some protective
clothing fKlaunig et al.. 2 0151. PFAS have been found at private and federal facilities associated
with various material or processes involving aqueous film-forming foam (AFFF), chrome plating,
and are associated with other industries using PFAS (e.g., textiles, carpets) fATSDR. 2021: U.S. EPA.
2020. 2019c. 2013. 2007. 2002b). In AFFF, PFHxA has been detected at concentrations ranging
from 0.1 to 0.3 g/L fBaduel etal.. 2015: Houtz etal.. 20131. In the occupational sectors and/or
products PFAS occurances have been found in wood particle board, rubber insulation,
electroplating, metal treatments, paints, varnishes, and flame retardants fOECD. 2022: Gliige etal..
2020).
No quantitative PFHxA information on production volume is available (U.S. EPA. 2019a).
and EPA's Toxics Release Inventory (TRI) contains no information on releases to the environment
from facilities manufacturing, processing, or otherwise using PFHxA fATSDR. 2021: U.S. EPA.
2018dl.
Wang etal. f20141 estimates global emissions of 39 to 1,691 tons of PFHxA from direct and
indirect (i.e., degradation of precursors) sources between 1951 and 2030. The lower estimate
assumes manufacturers cease production and use of long-chain PFCAs and that their precursors
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stay consistent with global transition trends. The higher estimate assumes the 2015 emission
scenario remains constant until 2030.
1.1.3. Environmental Fate and Transport
PFAS are highly stable and persistent worldwide, and many are found in environmental
media (e.g., soils, water, the atmosphere, foods, wildlife, and humans) fU.S. EPA. 2019cl
(Appendix A).
Uptake of soil PFAS to plants can occur (ATSDR. 20211. and estimates are available of PFAS
accumulation in vegetation when plants are grown in PFAS-contaminated soil. Yoo etal. (20111
estimated grass-soil accumulation factors of 3.4 (grass concentration divided by soil concentration)
for PFHxA using samples collected from a site with biosolids-amended soil. Venkatesan and Halden
f20141 analyzed archived samples from outdoor mesocosms to investigate the fate over 3 years of
PFAS in agricultural soils amended with biosolids. The mean half-life for PFHxA was estimated to be
417 days. Volatilization of PFHxA from moist soil is not expected to be an important fate process
(NLM. 20161. PFHxAbioaccumulates in foods grown on PFAS-containing soils. Blaine etal. (20131
conducted a series of greenhouse and field experiments to investigate the potential for PFAS uptake
by lettuce, tomatoes, and corn when grown in industrially impacted and biosolids-amended soils.
Blaine etal. f20131 calculated PFHxA bioaccumulation factors of 9.9-11.7 for lettuce and 2.9-6.8 for
tomatoes (no bioaccumulation factor was reported for corn).
1.1.4. Potential for Human Exposure and Populations with Potentially Greater Exposure
The general population can be exposed to PFAS via inhalation of air or dust, ingestion of
drinking water and food, and dermal contact with PFAS-containing products and during susceptible
lifestages (see Appendix A). The oral route of exposure is considered the dominant exposure
pathway for the general population fSunderland etal.. 20191. for which contaminated drinking
water is likely a significant source of exposure to PFAS, including PFHxA. Due to the high water
solubility and mobility of PFAS in groundwater (and potential lack of remediation at some water
treatment facilities), populations consuming drinking water from any contaminated watershed
could be exposed to PFAS (Shao etal.. 20161.
Infants potentially have higher exposure due to greater ingestion of food per body weight.
PFHxA has been detected in human breast milk from many nations, including U.S. fZheng etal..
20211. French, Korean, and Spanish populations (summarized in Table 5 of Anderson etal. f201911.
Exposure can also occur through hand-to-mouth transfer of materials containing these compounds
(ATSDR. 20211 or in infants through ingestion of formula reconstituted with contaminated drinking
water.
Air and Dust
PFHxA has not been evaluated under the National Air Toxics Assessment program and no
additional information on atmospheric concentration was identified. PFAS, including PFHxA, have
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been measured in indoor air and dust and might be associated with the indoor use of consumer
products such as PFAS-treated carpets or other textiles (ATSDR. 20211. For example, Kato et al.
f20091 detected PFHxA in 46.2% of the dust samples collected from 39 homes in the United States,
United Kingdom, Germany, and Australia. Karaskova et al. f20161 detected PFHxA in all 56 dust
samples collected from 41 homes in the Czech Republic, Canada, and the United States at mean
concentrations of 12.8,14.5, and 20.9 ng/g, respectively. Strvnar and Lindstrom f20081 analyzed
dust samples from 110 homes and 10 daycare centers in North Carolina and Ohio and detected
PFHxA in 92.9% of the samples. Knobeloch etal. (20121 detected PFHxA in 20% of samples of
vacuum cleaner dust collected from 39 homes in Wisconsin. PFHxA concentrations ranged from
below the reporting limit (1 ng/g) to 180 ng/g. Fraser etal. T20131 analyzed dust samples collected
from offices [n = 31), homes [n = 30), and vehicles [n = 13) in Boston, Massachusetts. PFHxA was
detected in 68% of the office samples at concentrations ranging from 5.1 to 102 ng/g, 57% of the
home samples at concentrations ranging from 4.9 to 1,380 ng/g, and 54% of the vehicle samples at
concentrations ranging from 5.0 to 18.2 ng/g.
Water
EPA conducted monitoring for several PFAS in drinking water as part of the third and fifth
Unregulated Contaminant Monitoring Rules (UCMR3 and UCMR5) fU.S. EPA. 2019b. 2016dl. PFHxA
was added to UCMR5 for public water system monitoring, which applies to 2022-2026 with sample
collection occurring between 2023 and 2025. Some drinking water PFHxA data are available from
other publications. For example, samples from seven municipal wells in Oakdale, Minnesota were
analyzed for PFHxA where the concentrations ranged from <0.025 to 0.235 |ig/L (U.S. EPA. 2016dl.
PFHxA was also detected in 23% of raw water samples collected from public water systems in New
Jersey at concentrations ranging from nondetectable to 0.017 |ig/L fPostetal.. 20121. In a more
recent study of surface waters sampled from 11 waterways in New Jersey, PFHxA was detected in
10 samples, ranging from 0.0015 to 0.026 |ig/L fGoodrow et al.. 20201.
AFFF Training Sites
PFHxA was detected at an Australian training ground where AFFFs had been used. Baduel et
al. (20151 and Braunigetal. (20171 observed mean concentrations of PFHxA of 0.6 |ig/L in water,
8.4 |ig/kg dry weight in soil, and 3.0 |J.g/kg wet weight in grass at an Australian town where the
groundwater had been impacted by PFAS from a nearby firefighting training facility. Houtz et al.
f20131 analyzed samples of groundwater, soil, and aquifer solids collected at an Air Force
firefighting training facility in South Dakota where AFFF had been used. PFAS concentrations in
groundwater decreased with increased distance from the burn pit, and PFHxA was detected at a
median concentration of 36 |ig/L. PFHxA was detected in surficial soil at a median concentration of
11 Hg/kg and in aquifer solids at a median concentration of 45 |J.g/kg.
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Military and National Priorities List (NPL) Sites
PFHxA levels in environmental samples collected in 2014 have been measured at military
and National Priorities List (NPL) sites in the United States. Table 1-2 provides the concentrations
at these sites fATSDR. 2021: Anderson etal.. 20161.
Table 1-2. PFHxA levels at 10 military installations and National Priority
List sites
Media
PFHxA value
Site
Source
Surface soil
Frequency of detection (%)
Median (ppb)
Maximum (ppb)
70.33
1.75
51.0
Military3
Anderson et al. (2016)
Subsurface soil
Frequency of detection (%)
Median (ppb)
Maximum (ppb)
65.38
1.04
140
Military3
Anderson et al. (2016)
Sediment
Frequency of detection (%)
Median (ppb)
Maximum (ppb)
63.64
1.70
710
Military3
Anderson et al. (2016)
Surface Water
Frequency of detection (%)
Median (ppb)
Maximum (ppb)
96.00
0.320
292
Military3
Anderson et al. (2016)
Groundwater
Frequency of detection (%)
Median (ppb)
Maximum (ppb)
94.20
0.820
120
Military3
Anderson et al. (2016)
Water (ppb)
Median
Geometric mean
0.25
0.10
NPLb
ATSDR (2021)
Soil (ppb)
Median
Geometric mean
1,175
1,175
NPLb
ATSDR (2021)
Air (ppbv)
Median
Geometric mean
ND
ND
NPLb
ATSDR (2021)
aSamples collected between March and September 2014 from 10 active U.S. Air Force installations located
throughout the United States, including Alaska, with a historic use of AFFFs; data originally reported as ng/kg.
Concentrations found in ATSDR site documents; water and soil values represent data from two NPL sites.
Other Exposures
Schecter et al. f20121 collected 31 food samples from 5 grocery stores in Texas and
analyzed them for persistent organic pollutants, including PFHxA. PFHxA was not detected in the
samples. Chen etal. (20181 analyzed PFAS in a wide range of foods in Taiwan and detected PFHxA
at geometric mean concentrations ranging from 0.03 ng/mL in milk to 1.58 ng/g in pork liver. Heo
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etal. (20141 analyzed a variety of foods and beverages in Korea for PFAS. PFHxA was detected in
8.1% of the fish and shellfish samples at a mean concentration of 0.037 ng/g; 8.1% of the dairy
samples at a mean concentration of 0.051 ng/g; 9.5% of the beverage samples at a concentration of
0.187 ng/L; 20.5% of the fruit and vegetable samples at a mean concentration of 0.039 ng/g; and
51.3% of the meat samples at a mean concentration of 0.515 ng/g. Heo etal. f20141 also detected
PFHxA in tap water in Korea at a mean concentration of 11.7 ng/L; PFHxA was not detected in
bottled water. Perez etal. (20141 analyzed PFAS in 283 food items (38 from Brazil, 35 from Saudi
Arabia, 36 from Serbia, and 174 from Spain). PFHxA was detected in 6.0%, 21.3%, and 13.3% of the
samples from Brazil, Saudi Arabia, and Spain, respectively. The mean concentrations of PFHxA were
270, 931, and 418 pg/g sample, respectively. The study did not find PFHxA in any of the Serbian
samples. PFHxA was detected in microwave popcorn packaging materials at a range of 3.4 to 497
ng/g but was not detected in the corn or popcorn fMoreta and Tena. 20141.
Stahl etal. (20141 characterized PFAS in freshwater fish from 164 U.S. urban river sites and
157 near-shore Great Lakes sites. PFHxA was not detected in the fish from U.S. urban rivers but was
detected in fish from 15% of the Great Lakes sites at a maximum concentration of 0.80 ng/g.
1.2. SUMMARY OF ASSESSMENT METHODS
This section summarizes the methods used for developing this assessment. A detailed
description of these methods is provided in the PFAS Systematic Review Protocol for the PFDA,
PFNA, PFHxA, PFHxS, and PFBA IRIS Assessments (see Appendix A and online 1. The protocol
includes additional problem formulation details, including the specific aims and key science issues
identified for this assessment
1.2.1. Literature Search and Screening
The detailed search approach, including the query strings and populations, exposures,
comparators, and outcomes (PECO) criteria, are provided in Appendix A, Table 3-1. The results of
the current literature search and screening efforts are documented in Section 2.1. Briefly, a
literature search was first conducted in 2017 and regular yearly updates have been performed (the
literature fully considered in the assessment was until April 2021). As described in the protocol,
studies identified after peer review begins will only be considered for inclusion if they meet the
PECO criteria and are expected to fundamentally alter the assessment's conclusions (see Appendix
A, Section 4.1).
The literature search queries the following databases (no literature was restricted by
language):
• PubMed fNational Library of Medicine 1
• Web of Science fThomson Reutersl
• Toxline (moved to PubMed)
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• TSCATS fToxic Substances Control Act Test Submissions!
In addition, relevant literature not found through evidence base searching was identified
by:
• Review of studies cited in any PFHxA PECO-relevant studies and published journal reviews;
finalized or draft U.S. state, U.S. federal, and international assessments (e.g., the draft
Agency for Toxic Substances and Disease Registry [ATSDR] assessment released publicly in
2018). In addition, studies included in ongoing IRIS PFAS assessments (PFHxS, PFNA, PFDA)
were also scanned for any studies that met PFHxA PECO criteria.
• Review of studies submitted to federal regulatory agencies and brought to EPA's attention.
For example, studies submitted to EPA by the manufacturers in support of requirements
under the Toxic Substances Control Act (TSCA).
• Identification of studies during screening for other PFAS. For example, epidemiological
studies relevant to PFHxA sometimes were identified by searches focused on one of the
other four PFAS currently being assessed by the IRIS Program.
• Other gray literature (i.e., primary studies not indexed in typical evidence bases, such as
technical reports from government agencies or scientific research groups; unpublished
laboratory studies conducted by industry; or working reports/white papers from research
groups or committees) brought to EPA's attention.
All literature, including literature search updates, is tracked in the EPA Health and
Environmental Research Online (HERO) database..3 The PECO criteria identify the evidence that
addresses the specific aims of the assessment and focuses the literature screening, including study
inclusion/exclusion (see Table 1-3).
Table 1-3. Populations, exposures, comparators, and outcomes (PECO) criteria
PECO element
Evidence
Populations
Human: Any population and lifestage (occupational or general population, including children and
other sensitive populations). The following study designs will be included: controlled exposure,
cohort, case-control, and cross-sectional. (Note: Case reports and case series will be tracked as
potential supplemental material.)
Animal: Nonhuman mammalian animal species (whole organism) of any lifestage (including
preconception, in utero, lactation, peripubertal, and adult stages).
Other: In vitro, in silico, or nonmammalian models of genotoxicity. (Note: Other in vitro, in silico,
or nonmammalian models will be tracked as potential supplemental material.)
Exposures
Human: Studies providing quantitative estimates of PFAS exposure based on administered dose
or concentration, biomonitoring data (e.g., urine, blood, or other specimens), environmental or
occupational-setting measures (e.g., water levels or air concentrations, residential location and/or
3EPA's Health and Environmental Research Online (HERO) database provides access to the scientific
literature behind EPA science assessments. The database includes more than 3,000,000 scientific references
and data from the peer-reviewed literature EPA uses to develop its risk assessments and related regulatory
decisions.
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PECO element
Evidence
duration, job title, or work title). (Note: Studies that provide qualitative, but not quantitative,
estimates of exposure will be tracked as supplemental material.)
Animal: Oral or Inhalation studies including quantified exposure to a PFAS of interest based on
administered dose, dietary level, or concentration. (Note: Nonoral and noninhalation studies will
be tracked as potential supplemental material.) PFAS mixture studies are included if they employ
an experimental arm that involves exposure to a single PFAS of interest. (Note: Other PFAS
mixture studies are tracked as potential supplemental material.)
Studies must address exposure to one or more of the following: PFHxA (CASRN 307-24-4), PFHxA
sodium salt (CASRN 2923-26-4), PFHxA ammonium salt (CASRN 21615-47-4). [Note: although
while these PFHxA is not metabolized or transformed in the body, there are precursor compounds
known to be biotransformed to a PFAS of interest; for example, 6:2 fluorotelomer alcohol is
metabolized to PFHxA and PFBA (Russell et al., 2015). Thus, studies of precursor PFAS that
identify and quantify a PFAS of interest will be tracked as potential supplemental material
(e.g., for ADME analyses or interpretations).]
Comparators
Human: A comparison or reference population exposed to lower levels (or no exposure/exposure
below detection levels) or for shorter periods of time.
Animal: Includes comparisons to historical controls or a concurrent control group that is
unexposed, exposed to vehicle-only or air-only exposures. (Note: Experiments including exposure
to PFAS across different durations or exposure levels without including one of these control
groups will be tracked as potential supplemental material [e.g., for evaluating key science issues;
Section 2.4].)
Outcomes
All cancer and noncancer health outcomes. (Note: Other than genotoxicity studies, studies
including only molecular endpoints [e.g., gene or protein changes; receptor binding or activation]
or other nonphenotypic endpoints addressing the potential biological or chemical progression of
events contributing toward toxic effects will be tracked as potential supplemental material
[e.g., for evaluating key science issues; Section 2.4].)
PBPK models
Studies describing physiologically based pharmacokinetic (PBPK) and other PK models for PFDA
(CASRN 335-76-2), PFDA ammonia salt (CASRN 3108-42-7), PFDA sodium salt (CASRN 3830-45-3),
PFNA (CASRN 375-95-1), PFNA ammonium salt (CASRN 4149-60-4), PFNA sodium salt
(CASRN 21049-39-8), PFHxA (CASRN 307-24-4), PFHxS (CASRN 355-46-4), PFHxS potassium salt
(CASRN 3871-99-6), PFBA (CASRN 375-22-4), or PFBA ammonium salt (CASRN 10495-86-0).
ADME = absorption, distribution, metabolism, and excretion; PK = pharmacokinetic.
In addition to those studies meeting the PECO criteria and studies excluded as not relevant
to the assessment, studies containing supplemental material potentially relevant to the specific
aims of the assessment were inventoried during the literature screening process. Although these
studies did not meet PECO criteria, they were not excluded. Rather, they were considered for use in
addressing the identified key science issues (see Appendix A, Section 2.4) and other potential
scientific uncertainties identified during assessment development but unanticipated at the time of
protocol posting. Studies categorized as "potentially relevant supplemental material" included the
following:
• In vivo mechanistic or mode-of-action studies, including non-PECO routes of exposure
(e.g., intraperitoneal injection) and populations (e.g., nonmammalian models);
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• In vitro and in silico models;
• Absorption, distribution, metabolism, and excretion (ADME) and pharmacokinetic (PK)
studies (excluding models);4
• Exposure assessment or characterization (no health outcome) studies;
• Human case reports or case-series studies; and
The literature was screened by two independent reviewers with a process for conflict
resolution, first at the title and abstract level and subsequently the full-text level, using structured
forms in DistillerSR fEvidence Partners! Literature inventories for studies meeting PECO criteria
and studies tagged as "potentially relevant supplemental material" during screening were created
to facilitate subsequent review of individual studies or sets of studies by topic-specific experts.
1.2.2. Evaluation of Individual Studies
The detailed approaches used for the evaluation of epidemiological and animal toxicological
studies used in the PFHxA assessment are provided in the systematic review protocol (see
Appendix A, Section 6). The general approach for evaluating health effect studies meeting PECO
criteria is the same for epidemiological and animal toxicological studies although the specifics of
applying the approach differ. Approaches for evaluating mechanistic evidence are described in
detail in Appendix A, Section 6.5. The key concerns during the review of epidemiological and animal
toxicological studies are potential bias (factors that affect the magnitude or direction of an effect in
either direction) and insensitivity (factors that limit the ability of a study to detect a true effect; low
sensitivity is a bias toward the null when an effect exists). Briefly, for epidemiology studies,
evaluation of risk of bias and study sensitivity are conducted for the following domains: exposure
measurement, outcome ascertainment, participant selection, potential confounding, analysis, study
sensitivity, and selective reporting. The principles and framework used for evaluating epidemiology
studies are based on the Cochrane Risk of Bias in Nonrandomized Studies of Interventions
(ROBINS-I) but have been modified to address environmental and occupational exposures. For
animal studies, risk of bias and study sensitivity are evaluated for the following domains: reporting
quality, allocation, observational bias/blinding, confounding, selective reporting and attrition,
chemical administration and characterization, exposure timing, frequency and duration, endpoint
sensitivity and specificity, and results presentation. Details of the human epidemiology and animal
toxicology methodology, including core and prompting questions as well as PFAS-specific criteria,
are described in greater detail in Appendix A (see Appendix A, Sections 6.2 and 6.3, respectively).
In evaluating individual studies, two or more reviewers independently arrived at judgments
about the reliability of the study results (reflected as study confidence determinations; see below)
4Given the known importance of ADME data, this supplemental tagging was used as the starting point for a
separate screening and review of PK data (see Appendix A, Section9.2 for details).
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with regard to each outcome or outcome grouping of interest; thus, different judgments were
possible for different outcomes within the same study. The results of these reviews were tracked
within EPA's version of the Health Assessment Workplace Collaborative (HAWC). To develop these
judgments, each reviewer assigned a rating of good, adequate, deficient (or not reported, which
generally carried the same functional interpretation as deficient), or critically deficient (see
Appendix A, Section 6.1 for definitions) related to each evaluation domain representing the
different characteristics of the study methods that were evaluated based on the criteria outlined in
HAWC. Once all domains were evaluated, the identified strengths and limitations were collectively
considered by the reviewers to reach a final study confidence classification:
• High confidence: No notable deficiencies or concerns were identified; the potential for bias
is unlikely or minimal, and the study used sensitive methodology.
• Medium confidence: Possible deficiencies or concerns were noted, but the limitations are
unlikely have a significant impact on the results.
• Low confidence: Deficiencies or concerns were noted, and the potential for bias or
inadequate sensitivity could have a significant impact on the study results or their
interpretation. Low confidence results were given less weight compared to high or medium
confidence results during evidence synthesis and integration (see Section 1.2.4).
• Uninformative: Serious flaw(s) were identified that make the study results unusable.
Uninformative studies were not considered further, except to highlight possible research
gaps.
Using the HAWC platform (and conflict resolution by an additional reviewer, as needed), the
reviewers reached a consensus judgment regarding each evaluation domain and overall
(confidence) determination. The specific limitations identified during study evaluation were carried
forward to inform the synthesis (see Section 1.2.4) within each body of evidence for a given health
effect (i.e., study confidence determinations were not used to inform judgments in isolation).
1.2.3. Data Extraction
The detailed data extraction approach is provided in Appendix A, Section 8, and data
extraction and content management is carried out using HAWC (see Appendix C). Data extraction
elements that may be collected from epidemiological, controlled human exposure, animal
toxicological, and in vitro studies are available in HAWC. As described in the systematic review
protocol (see Appendix A), not all studies that meet the PECO criteria go through data extraction:
For example, studies evaluated as being uninformative are not used to inform assessment
judgments and, therefore, do not undergo full data extraction.
All findings from informative studies are considered for extraction, regardless of statistical
significance. The level of extraction for specific outcomes within a study might differ (e.g., ranging
from a qualitative description to full extraction of dose response effect size information). For
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quality control, data extraction is performed by one member of the evaluation team and
independently verified by at least one other member. Discrepancies in data extraction are resolved
by discussion or consultation with a third member of the evaluation team.
1.2.4. Evidence Synthesis and Integration
For the purposes of this assessment, evidence synthesis and integration are considered
distinct but related processes (see Appendix A, Sections 9 and 10 for full details). For each assessed
health effect, the evidence syntheses provide a summary discussion of each body of evidence
considered in the review that directly informs the integration across evidence that was used to
draw an overall judgment for each health effect. The available human and animal evidence
pertaining to the potential health effects were synthesized separately, with each synthesis resulting
in a summary discussion of the available evidence that addresses considerations regarding
causation adapted from Hill T1965I Briefly, the following aspects are considered for the available
evidence: study confidence, consistency, strength (effect magnitude) and precision, biological
gradient/dose response, coherence, mechanistic evidence related to biological plausibility, and
natural experiments (applicable to human studies only). Detailed descriptions and application of
these considerations are described in Appendix A, Section 9. Mechanistic evidence is also
synthesized as necessary to help inform key decisions regarding the human and animal evidence;
processes for synthesizing mechanistic information are covered in detail in Appendix A, Section 9.2.
The syntheses of the human and animal health effects evidence focus on describing aspects
of the evidence that best inform causal interpretations, including the exposure context examined in
the sets of available studies. The evidence synthesis is based primarily on studies of high and
medium confidence. The systematic review protocol (see Appendix A) describes that, in certain
instances (i.e., few or no studies with higher confidence are available), low confidence studies might
be used to help evaluate consistency, or if the study designs of the low confidence studies address
notable uncertainties in the set of high or medium confidence studies on a given health effect. If low
confidence studies are used, a careful examination of the study evaluation and sensitivity with
potential effects on the evidence synthesis conclusions will be included in the narrative. For the
current assessment all studies meeting PECO criteria were used for evidence synthesis and
included in the narrative. When possible, results across studies are compared using graphs and
charts or other data visualization strategies. The synthesis of mechanistic information informs the
integration of health effects evidence for both hazard identification (e.g., biological plausibility or
coherence of the available human or animal evidence; inferences regarding human relevance, or the
identification of susceptible populations and lifestages across the human and animal evidence) and
dose-response evaluation (e.g., selection of benchmark response levels, selection of uncertainty
factors). Evaluations of mechanistic information typically differ from evaluations of phenotypic
evidence (e.g., from routine toxicological studies). This is primarily because mechanistic data
evaluations consider the support for and involvement of specific events or sets of events within the
context of a broader research question (e.g., support for a hypothesized mode of action; consistency
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with known biological processes), rather than evaluations of individual apical endpoints considered
in relative isolation.
Following the synthesis of human and animal health effects data, and mechanistic data,
integrated judgments are drawn across all lines of evidence for each assessed health effect. During
evidence integration, a structured and documented process was used, as follows:
• Building from the separate syntheses of the human and animal evidence, the strength of the
evidence from the available human and animal health effect studies was summarized in
parallel, but separately, using a structured evaluation of an adapted set of considerations
first introduced by Bradford Hill fHill. 19651. This process is similar to that used by the
Grading of Recommendations Assessment, Development, and Evaluation (GRADE) (Morgan
etal.. 2016: Guvatt etal.. 2011: Schiinemann etal.. 2011). which arrives at an overall
integration conclusion based on consideration of the body of evidence. These summaries
incorporate the relevant mechanistic evidence (or mode of action [MOA] understanding)
that informs the biological plausibility and coherence within the available human or animal
health effect studies. The terms associated with the different strength of evidence
judgments within evidence streams are robust, moderate, slight, indeterminate, and
compelling evidence of no effect.
• The animal, human, and mechanistic evidence judgments are then combined to draw an
overall judgment that incorporates inferences across evidence streams. Specifically, the
inferences considered during this integration include the human relevance of the animal
and mechanistic evidence, coherence across the separate bodies of evidence, and other
important information (e.g., judgments regarding susceptibility). Note that without
evidence to the contrary, the human relevance of animal findings is assumed. The final
output is a summary judgment of the evidence base for each potential human health effect
across evidence streams. The terms associated with these summary judgments are evidence
demonstrates, evidence indicates (likely), evidence suggests, evidence inadequate, and strong
evidence of no effect. The decision points within the structured evidence integration process
are summarized in an evidence profile table for each considered health effect
• In some instances (i.e., key science questions, coherence within and across biologically
related outcomes, areas of uncertainty) the supplemental mechanistic information was
reviewed and prioritized based on potential impact on assessment conclusions. For
example, interpreting a pattern of changes or collection of findings from various health
outcomes is strengthened by biological understanding (e.g., disruption of thyroid
homeostasis through increased clearance of thyroid hormones during gestation may affect
fetal growth and nervous system development) and progression to adverse outcomes and
applicability in humans. Biological understanding, including strong mechanistic support for
the chemical molecular interactions and conservation of those reactions and subsequent
biological responses between species can increase certainty in strength of the evidence.
As discussed in the protocol (see Appendix A), the methods for evaluating the potential
carcinogenicity of PFAS follow processes laid out in the EPA cancer guidelines (U.S. EPA. 2005) and
that the judgments described here for different cancer types are used to inform the evidence
integration narrative for carcinogenicity and selection of one of EPA's standardized cancer
descriptions. These are: (1) carcinogenic to humans, (2) likely to be carcinogenic to humans, (3)
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suggestive evidence of carcinogenic potential, (4) inadequate information to assess carcinogenic
potential, or (5) not likely to be carcinogenic to humans. However, for PFHxA, data relevant to cancer
were sparse and did not allow for such an evaluation (see Section 3.3).
1.2.5. Dose-Response Analysis
The details for the dose-response analysis completed for this assessment are in Appendix A,
Section 11. Briefly, although procedures for dose-response assessments were developed for both
noncancer and cancer health hazards, and for the oral route of exposure following exposure to
PFHxA, the existing data for PFHxA only supported derivation of an oral reference dose (RfD) for
noncancer hazards (see Appendix A, Section 11 for the health hazard conclusions necessary for
deriving other values). An RfD is an estimate, with uncertainty spanning perhaps an order of
magnitude, of an exposure to the human population (including susceptible subgroups) that is likely
without an appreciable risk of deleterious health effects over a lifetime fU.S. EPA. 2002cl The
derivation of a reference value like the RfD depends on the nature of the health hazard conclusions
drawn during evidence integration. For noncancer outcomes, dose-response assessments were
conducted for evidence integration judgments of evidence demonstrates and evidence indicates
(likely). In general, toxicity values are not developed for noncancer hazards with evidence suggests
conclusions (see Appendix A, Section 10.2 for exceptions).
Consistent with EPA practice, the PFHxA assessment applied a two-step approach for
dose-response assessment that distinguishes analysis of the dose-response data in the range of
observation from any inferences about responses at lower, environmentally relevant exposure
levels (TJ.S. EPA. 2012a. 20051.
• Within the observed dose range, the preferred approach is to use dose-response modeling
to incorporate as much of the data set as possible into the analysis, and considering
guidance on modeling dose-response data, assessing model fit, selecting suitable models,
and reporting modeling results [see the EPA Benchmark Dose Technical Guidance (U.S. EPA.
2012a)] as elaborated in Appendix A, Section 11. Thus, modeling to derive a POD attempted
to include an exposure level near the lower end of the range of observation, without
significant extrapolation to lower exposure levels.
• As derivation of cancer risk estimates and reference values nearly always involves
extrapolation to exposures lower than the POD; the approaches to be applied in these
assessments are described in more detail in Appendix A, Section 11.2.
When sufficient and appropriate human and laboratory animal data are available for the
same outcome, human data are generally preferred for the dose-response assessment because use
of human data eliminates the need to perform interspecies extrapolations. For reference values,
this assessment will derive a candidate value from each suitable data set. Evaluation of these
candidate values grouped within a given organ/system were used to derive a single
organ/system-specific RfD (osRfD) for each organ/system under consideration from which a single
overall reference value will be selected to cover all health outcomes across all organs/systems.
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Although this overall RfD represents the focus of the dose-response assessment, the osRfDs can be
useful for subsequent cumulative risk assessments that consider the combined effect of multiple
PFAS (or other agents) acting at a common organ/system. For noncancer toxicity values,
uncertainties in these estimates are characterized and discussed. In addition, a less-than-lifetime,
"subchronic" RfD was similarly estimated. Uncertainties in these toxicity values are transparently
characterized and discussed. For dose-response purposes, EPA has developed a standard set of
models (http://www.epa.gov/bmds) that can be applied to typical data sets, including those that
are nonlinear. In situations where alternative models with significant biological support are
available (e.g., pharmacodynamic models), those models are included as alternatives in the
assessment(s) along with a discussion of the models' strengths and uncertainties. EPA has
developed guidance on modeling dose-response data, assessing model fit, selecting suitable models,
and reporting modeling results [see the EPA Benchmark Dose Technical Guidance fU.S. EPA.
2012a)]. Additional judgment or alternative analyses are used if the procedure fails to yield reliable
results; for example, if the fit is poor, modeling might be restricted to the lower doses, especially if
competing toxicity at higher doses occurs. When alternative approaches fail or are not applicable,
the NOAEL/LOAEL approach is used for POD estimation. For each modeled response, a POD from
the observed data was estimated to mark the beginning of extrapolation to lower doses. The POD is
an estimated dose (expressed in human-equivalent terms) near the lower end of the observed
range without significant extrapolation to lower doses. The POD is used as the starting point for
subsequent extrapolations and analyses. For noncancer effects, the POD is used in calculating the
RfD.
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2.SUMMARY OF LITERATURE IDENTIFICATION AND
STUDY EVALUATION RESULTS
2.1. LITERATURE SEARCH AND SCREENING RESULTS
The evidence base searches yielded 975 unique records identified from core database
searches (WOS, Toxline, PubMed, Toxnet) and other sources (see Figure 2-1). Of the 975 studies
identified, 74 were excluded at the title and abstract level on the basis that they did not meet the
PECO and did not contain potentially relevant supplemental information. At the full-text level, 57
studies met PECO criteria including 24 human health effect studies, 23 in vivo animal studies,
7 genotoxicity study, and 5 PBPK/PK studies (see Table 1-3). An interactive summary of the
literature screening results is available as a literature tag-tree accessible via the link: PFHxA
Literature Tagtree. Additional information, including high-throughput screening data on PFHxA, are
available from EPA's CompTox Chemicals Dashboard fU.S. EPA. 2018a!
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PFHxA Literature Searches (through April 2022)
s
Database searches(713)
\
\
PubMed (n = 416)
WOS (n = 434)
Toxline (n = 16)
Toxnet(23)
Other(326)
Additional Strategies (n = 23)
PFAS SEMs(n = 131)
Other(186)
\
Scopus(618)
)
V
)
1
TITLE AND ABSTRACT
Title & Abstract Screening
(records after duplicate removal)
(n = 975)
I
FULL TEXT SCREENING
Full-Text Screening
(n = 59)
I
Studies Meeting PECO (n = 57)
• Human health effects studies (n = 24)
• Animal health effect studies (n = 23)
• Genotoxicity studies (n = 7)
• PBPK/PK models (n = 5)
1
Studies Considered for Dose Response
(n = 24)
• Human health effects studies (n = 17)
• Animal health effect studies (n = 7)
Excluded
Not relevant to PECO (n = 74)
Tagged as Supplemental (n= 845)*
• Human MOA non-cancer (n= 3)
• In vivo mechanistic or MOA (n= 41)
• In vitro or in silico studies
(nongenotoxicity) (n= 72)
• ADME/toxicokinetic (excluding models)
(n= 77)
• Exposure assessment or characterization
(no health outcome) (n= 121)
• PFAS Mixture Study (no individual PFAS
comparison (n = 4)
• Ecotoxicity (n = 51)
• Environmental fate or occurrence
(including food) (n = 288)
• Manufacture, engineering, use,
treatment, remediation, or laboratory
methods (n = 160)
• Other assessments or records with no
original data (e.g., reviews, editorials,
commentaries; abstract-only) (n = 148)
• Susceptible populations (n = 11)
• Non-oral/non-inhalation route (n = 2)
• OtherPFAS(n=13)
"Some studies were assigned multiple
tags
Figure 2-1. Literature search and screening flow diagram for PFHxA and
related salts, PFHxA-NH4 and PFHxA-Na. Literature HAWC tree are available in
HAWC and all studies are available in PFHxA HERO.
PFHxA-NhU = ammonium perfiuorohexanoate; PFHxA-Na = sodium perfluorohexanoate.
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2.2. STUDY EVALUATION RESULTS
Human and animal studies evaluated potential hepatic, developmental, hematopoietic,
endocrine, cardiometabolic, renal, reproductive, immune, and nervous system effects following
exposure to PFHxA. The evidence informing these potential health effects is presented and assessed
in Sections 3.2.1-3.2.9. Seventeen epidemiological studies were identified that report on the
potential association between PFHxA and human health effects. Of these, five were considered
uninformative due to critical deficiencies in one or more domains, including participant selection,
exposure measurement, confounding, or analysis (Zhang etal.. 2019: Seo etal.. 2018: Kim etal..
2016a: Tiang etal.. 20141. The remaining 12 studies were rated medium fLiu etal.. 2022: Nian etal..
2019: Bao etal.. 2017: Zeng etal.. 2015: Dong etal.. 20131 or low confidence fVelarde etal.. 2022:
Tian etal.. 2019: Wang etal.. 2019: Song etal.. 2018: Li etal.. 2017: Zhou etal.. 2016: Fu etal..
20141.
Seven unique reports of animal studies met PECO criteria. The available evidence base of
animal toxicity studies on PFHxA and the related ammonium and sodium salts primarily consists of
five reports in rats and mice including short-term fNTP. 20181. subchronic fChengelis etal.. 2009b:
Loveless etal.. 20091. chronic fKlaunig etal.. 20151. and reproductive/developmental flwai and
Hoberman. 2014: Loveless etal.. 20091 experiments. These studies were generally well conducted
and judged high or medium confidence. In cases where a study was rated low confidence for one or
more of the evaluated outcomes, the specific limitations identified during evaluation are discussed
in the applicable synthesis section(s). An acute (<24 hours) oral toxicity study was also available
(Riker Labs. 19791 but not considered informative as other longer duration (>30 day) studies were
available. A reproductive study by Kirkpatrick f2005al was initially considered uninformative due
to reporting deficiencies (i.e., all summary and individual animal data [pages 110-1,334] were
missing). During public comment, the study sponsor submitted the missing data tables fKirkpatrick.
2005a. b, c, d, e, £ g). Consistent with the protocol and the study was reconsidered for incorporation
in the assessment based on the potential impact on assessment conclusions. The study was
considered medium confidence for most outcomes. Immune findings from this study were
incorporated into the evidence synthesis on the basis that, although they do not change the
assessment conclusions, they may help to address critical data gaps for this potential health effect.
For all other health effects it was determined that the data would not impact assessment
conclusions based on the following rationales: for reproductive, developmental and renal effects,
the findings were weak or null and did not address key data gaps; for hepatic and hematopoietic
effects, the results were similar to effects observed in other studies and supported hematopoietic
and hepatic conclusions (i.e., decreased hemoglobin, MCH, MCHC, total protein, globulin; increased
relative liver weight, incidence of hepatocellular hypertrophy), but largely limited to the high dose
that was associated with overt toxicity, including high mortality (approx. 33%) which may impact
the reliability of the results. Thus, these findings were not incorporated into the evidence synthesis
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and integration sections for hepatic, hematopoietic, developmental, renal, and reproductive health
effects.
Detailed rationales for each domain and overall confidence rating are available in HAWC.
Results shown below in Figure 2-2 for human studies are (interactive version available: link) and
Figure 2-3 for animal studies (interactive version available: link! Graphical representations of the
outcome-specific ratings are also presented in the organ/system-specific integration sections (in
Section 3.2). All outcomes rated low confidence or higher were used for evidence synthesis and
integration.
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* Multiple judgments exist
Figure 2-2. Study evaluation results for human epidemiology studies.
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* T*1 V ^ ^
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*%9
Reporting quality
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NR Not reported
N/A Not applicable
* Multiple judgments exist
T Bias towards null
Figure 2-3.Study evaluation results for animal toxicology studies.
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3.PHARMACOKINETICS, EVIDENCE SYNTHESIS, AND
EVIDENCE INTEGRATION
3.1. PHARMACOKINETICS
Only a few human PK studies on PFHxA are available, but the studies provide sufficient data
to estimate PFHxA half-life, a dependent variable for the estimation of clearance (along with volume
of distribution). Several studies such as Ericsonetal. f20071 reported PFHxA in blood or serum of
human populations (e.g., in relation to age and sex) but, because exposure levels are not known for
the subjects and the concentrations are not measured over time in specific subjects for whom the
exposure level is known to be zero, such observations cannot be used to obtain ADME information.
Several other studies that investigate specific aspects of PFHxA ADME in humans are discussed
briefly below but were not used in the derivation of toxicity values. One analysis provides an
estimate of PFHxA elimination in humans fRussell etal.. 20131 using data from an observational
study by Nilssonetal. f20131. Luz etal. f 20191 describes a reanalysis of these data but based only
on the three participants with the most rapid clearance. While EPA considers the data reported by
Nilsson etal. (2013) to be sufficient for the estimation of a half-life in humans, the approaches used
by Russell etal. (2013) and Luz etal. (2019) were not considered adequate. Therefore, the data of
Nilsson etal. (2013) have been re-analyzed as described in Approach for Animal-Human
Extrapolation of PFHxA Dosimetry (see Section 5.2.1).
Animal experiments in rats, mice, and monkeys have provided valuable information on PK
processes of PFHxA. In brief, PFHxA and other perfluoroalkyl acids (PFAA) have similar PK aspects:
They are well absorbed following oral exposure and quickly distribute throughout the body
(Iwabuchi etal.. 2017). particularly to blood, liver, skin, and kidney (Gannon etal.. 2011).
Dzierlenga etal. (2019) noted that following intravenous (i.v.) administration of 40 mg/kg PFHxA,
the PK profiles were generally similar between sexes, but a lower dose-normalized area under the
curve (AUC, 3.05 mM-h/mmol/kg), a faster clearance (CL, 327 mL/h-kg), and a lower volume of
distribution of peripheral compartment (V2 = 59.6 mL/kg) was observed in female Sprague-Dawley
rats, as compared to their male counterparts (dose-normalized AUC = 7.38 mM-h/(mmol/kg),
CL = 136 mL/h-kg, and V2 = 271 mL/kg, respectively). Likewise, kinetic parameters (e.g., the
maximum concentration [Cmax]) were comparable between sexes following an oral dose of
40 mg/kg exceptthat female rats exhibited a lower dose-adjusted AUC/dose and a faster CL. A PK
study in mice similarly showed an AUC/dose in male animals 2-3 times higher than in females,
indicating slower elimination in males fGannon et al.. 2 0111. Thus, apparent sex-related
quantitative differences in PFHxA PK occur in rats and mice. On the other hand, the AUC in monkeys
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Toxicological Review of PFHxA and Related Salts
given a 10 mg/kg i.v. dose of PFHxA was only slightly lower in females than in males (75 vs. 84 mg-
h/L), suggesting no significant sex difference in nonhuman primates.
PFHxA is resistant to metabolic transformation, and urinary excretion is the main
elimination route, followed by feces fGannon etal.. 2011: Iwai. 2011: Chengelis etal.. 2009al.
3.1.1. Absorption
Absorption is rapid in rodents and monkeys f I wabuchi etal.. 2017: Gannon etal.. 2011:
Chengelis etal.. 2009a). PFHxA was extensively absorbed with an average time to reach maximum
concentration (Tmax) of 1 hour in Sprague-Dawley rats given 26-day repeated gavage doses of 50,
150 or 300 mg PFHxA/kg fChengelis etal.. 2009al. After gavage at 2 or 100 mg [l-14C]PFHxA/kg
using a single dose or 14 daily consecutive doses, Gannon etal. f20111 also observed a short Tmax of
30 and 15 minutes, respectively, in male and female Sprague-Dawley rats. Similarly, rapid
absorption was also observed in CD-I mice f Gannon etal.. 20111. For female rats and male and
female mice, PFHxA absorption does not appear to be saturated between 2 and 100 mg/kg as
suggested by dose normalized AUCo^i68 hour, but the data in male rats indicate either a 25%
reduction in absorption or a corresponding increase in PFHxA clearance between these two dose
levels f Gannon et al.. 2011: Chengelis etal.. 2009al.
In a recent PK study by Dzierlenga etal. f20191. Sprague-Dawley rats were given PFHxA, by
i.v. injection (40 mg/kg) or gavage (40, 80, and 160 mg/kg). Besides collection of blood samples to
evaluate the time course of plasma PFHxA for each dose and route, liver, kidney, and brain samples
were collected to determine the distributions of PFHxA in tissues following 80 mg/kg gavage dose.
A two-compartmental model was used to evaluate the PK profiles. The estimated oral
bioavailability for PFHxA was >100% (Dzierlenga etal.. 2019): this result simply could reflect
experimental and analytical uncertainty in estimating the serum concentration AUC from
intravenous versus oral exposure, but also might be due to increased reabsorption from the
intestinal lumen by intestinal transporters of material excreted in the bile. The data indicate that
Tmax increased slightly but not significantly with increasing oral PFHxA dose levels for both sexes.
For instance, Tmax increased from 0.668 ± 0.154 to 0.890 ± 0.134 hour (mean ± standard error) and
from 0.529 ± 0.184 to 0.695 ± 0.14 hour with increased gavage doses of PFHxA for male and female
rats, respectively (Dzierlenga etal.. 2019).
3.1.2. Distribution
PFHxA has an aqueous solubility of 15.7 g/L fZhou etal.. 20101. Computational chemistry
predictions conclude that PFHxA and its salts have a pKa < 0 (Ravne and Forest. 2010). so it likely
exists exclusively in anionic form at physiological pH (Russell etal.. 2013). Therefore, it is relatively
water soluble, but limited data are available to examine its distribution to various organs and
tissues upon exposure in mammalian systems (Russell etal.. 2013: Gannon etal.. 2011). The largest
concentrations were found in liver, skin, heart, lung, and kidney and concentrations peaked within
hours flwabuchi etal.. 2017: Gannon etal.. 20111. For example, Gannon etal. f20111 reported
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heart, kidneys, liver, and lungs had detectable but not quantifiable concentrations of PFHxA at 24
hours in rats dosed with 100 mg/kg (Gannon etal.. 20111. Similarly, the highest uptake
concentrations occurred in the liver and femur (10 ± 2% and 5 ± 1% of the injected dose,
respectively), in male CD-I mice fBurkemper et al.. 20171. As described in detail below, the volume
of distribution (Vd) was generally similar (within a factor of three) among male and female mice,
rats, and monkeys fRussell etal.. 20131.
Distribution in Animal (Rats, Mice, and Monkeys) and In Vitro Studies
Chengelis etal. (2009a) gave both Sprague Dawley rats and cynomolgus monkeys (3/sex)
PFHxA (10 mg/kg) via a single i.v. injection to determine PFHxA PK using noncompartmental
analysis. In monkeys they observed a distribution phase of 8 hours and an apparent Vd of 0.77 and
0.35 L/kg in males and females, respectively. In male and female rats, Vd was reported as 0.18 and
0.47 L/kg, respectively, and the distribution phase after gavage dosing was about 1-2 hours in both
sexes. Serum concentrations of PFHxA were up to 17-fold higher for male than female rats after i.v.
dosing. In a separate experiment male and female Sprague-Dawley rats were given oral gavage
doses of 50,150, or 300 mg/kg/d PFHxA for 25 days (6 rats/sex/dose) and the PK evaluated on the
first and last day of dosing. The AUC after oral dosing was approximately 4fold higher in males than
females given a 50 mg/kg gavage dose on both day 1 and day 25. The half-life in males, however,
was only 2.5 times greater than females after i.v. dosing and was similar to that in females after oral
dosing. Together these lead to the conclusion of higher Vd for females than for males.
Using a one-compartment model, Iwabuchi etal. (2017) evaluated the distribution of PFHxA
and other PFAAs (PFOA, PFOS and perfluorononanoic acid, [PFNA]) in multiple tissues (brain,
heart, liver, spleen, kidney, whole blood, and serum) in 6-week-old male Wistar rats. The rats were
given a single oral dose or 1- and 3-month exposures in drinking water. For the single oral dose,
rats were given drinking water containing a mixture of PFAAs by gavage (PFHxA, PFOA, PFOS:
100 M-g/kg body weight [BW], PFNA: 50 M-g/kg BW). Although the estimated Tmax for PFHxA was 1
hour for all tissues, the Tmax for other PFAAs was 12 hours in the tissues except the brain (72 h) and
whole blood (24 h), indicating PFHxA was distributed rapidly throughout the body. Peak
concentrations occurred between 15 minutes and 1 hour after dosing, depending on the tissue. Of
examined tissues, the highest concentrations of PFHxA were found in the serum and kidney,
equivalent to 7.9% and 7.1% of the administered PFHxA, respectively. Note that the peak
concentrations measured in liver and brain were roughly 40% (at 15 minutes) and 1.5% (at 1 hour)
of the corresponding peak serum levels (4.6% and 0.027% of administered PFHxA dose),
respectively. The earlier peak in liver concentration is likely due to initial delivery there from oral
absorption, although the results show low delivery to the brain.
Dzierlenga etal. (2019) measured levels of PFHxA in rat liver, kidney and brain over 12
hours following an 80 mg/kg oral gavage dose. In general tissue distribution was rapid, with peak
concentration occurring at 0.5 hours (first time-point) in male rat liver and kidney or 1 hour
(second time-point) in male rat brain and in female rat liver, kidney, and brain. The concentrations
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declined exponentially after the peak, with tissue: plasma ratios mostly remaining in a limited
range. For example, in male and female rat kidney and female rat liver the tissue: plasma ratio only
varied between 0.5 and 0.75, though the liver: plasma ratio varied between 1 and 0.5 in male rats,
though without a clear pattern. However, the kidney: plasma ratio in female rats showed a steady
increase from around 0.8 at 0.5 hours to around 1.7 at 3 hours, after which it slowly declined to
around 1.4 at 12 hours fDzierlenga etal.. 20191. Since tissue: plasma ratios are generally less than
1, this result in the female rat kidney indicates a mechanism that was not active in the liver or male
rats, perhaps involving active transport into the tissue.
For the 1- or 3-month exposures, rats were given a mixture of four PFAA dose levels: 0,1, 5
and 25 ng/L in drinking water with similar intake rate across dose groups
(0.072-0.077 L/kg BW-day) flwabuchi etal.. 20171. In general, the long-term tissue concentrations
of PFHxA predicted based on the data from the single-exposure studies were comparable to that
measured after the 1- and 3-month exposures, suggesting that steady-state tissue levels were
achieved rather quickly and the tissue distribution of PFHxA remained relatively constant over time
flwabuchi etal.. 20171.
An in vitro study using lung epithelial cells (NCI-H292) and adipocytes (3T3-L1K) made
similar observations of no appreciable cellular accumulation and retention of PFHxA (Sanchez
Garcia etal.. 20181.
Distribution in Humans
The tissue distribution of PFHxA and other PFAAs were analyzed in 99 human autopsy
samples (brain, liver, lung, bone, and kidney) (Perez etal.. 20131. Perez etal. (20131 used the term
"accumulation," which in PK terminology describes a steady increase in the amount of a substance
in the body tissues over an extended time while exposure continues at a relatively constant level.
So, to demonstrate accumulation, one must have repeated measures of the blood or tissue
concentration in an individual over a significant period of time. If the body quickly reaches a
constant level (with ongoing exposure), that would not be called "accumulation." Because the study
data were collected from cadavers, they show only the tissue levels in the individuals at time of
death, and thus do not actually demonstrate accumulation but simply that exposure, absorption,
and distribution have occurred. These tissue concentrations could represent approximate steady-
state concentrations that were achieved quickly after the start of exposure, without accumulation.
More generally, these data cannot inform the specific exposure scenarios that might have occurred
before the time of death, in particular the duration of exposure that was required to reach the
observed concentrations.
Perez etal. (20131 found PFHxA to be the main PFAA compound in the brain
(mean = 180 ng/g tissue weight, median = 141 ng/g). PFHxA was detected in all collected tissue
types at levels ranging from below the detection limit to an observed concentration of 569 ng/g in
the lung. These observations generally demonstrate the distribution of short-chain PFAAs like
PFHxA, for which the mean (or median) concentration ranged from 5.6 ng/g (2.7 ng/g) tissue in the
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kidney to 180 ng/g (141 ng/g) in the brain. The liver and lung had tissue levels somewhat below
that in the brain but within the same range, with mean (or median) levels of 115 ng/g (68.3 ng/g)
and 50.1 ng/g (207 ng/g), respectively.
Considering the relatively rapid elimination of PFHxA in humans, the high levels reported
by Perez etal. f20131 for brain, liver and lung are surprising. Abraham etal. f20211 found much
lower levels of PFBA in human tissues than Perez etal. f20131 and attributed the discrepancy to
issues in the analytic chemistry. Sanan and Magnuson (2020) describe analytic issues that can
impact a broad range of PFAS, including PFHxA. Multiple PFAS may co-elute from chromatographic
separation, so the measurements attributed to PFHxA by Perez etal. (2013) may reflect multiple
PFAS, with PFHxA being only a small fraction of the total. Resolution of this issue is beyond the
scope of this review. Perez etal. f20131 describes what appear to be good quality control methods
and their results are reported here for completeness but are not used in the quantitative dosimetric
analyses presented later.
Because blood plasma concentrations could not be evaluated in the cadavers, the data of
Perez etal. (2013) lack this component of total PFHxA body burden. Plasma is a small fraction of
total body mass (~ 4% in humans), but due to PFHxA's substantial binding to serum proteins
(>99% bound to serum albumin fBischel etal.. 201111 it will carry a disproportionate amount of the
PFHxA. For example, if the overall volume of distribution in humans is 0.5 L/kg, plasma will then
contain about 8% of the PFHxA.
Fabrega etal. (2015) attempted to estimate tissue: blood partition coefficients (PCs) for
PFHxA using the data of Perez etal. (2013). Because Perez etal. (2013) did not measure or report
blood concentrations, Fabrega etal. (2015) used the mean blood concentration reported 4 years
earlier for residents of the same county (Ericson etal.. 2007). The resulting set of PCs ranged from 6
(unitless ratio) in the kidney to 202 in the brain, indicating a Vd in the human body around 40 L/kg
or higher.
Zhang etal. f2013al evaluated the distribution of several PFAS including PFHxA in matched
samples of maternal blood, cord blood, placenta, and amniotic fluid among Chinese women. Only
45% of maternal blood samples were above the limit of quantitation (LOQ), with a mean
concentration of 0.07 ng/mL, although 87% of cord blood samples were above the LOQ, with a
mean of 0.21 ng/mL PFHxA. Only 17% of placenta samples were above the LOQ (mean
concentration 0.04 ng/mL) and 45% of amniotic fluid samples (mean concentration 0.19 ng/mL).
The authors urge caution in interpreting their results because recovery of PFHxA from test samples
was more variable than for most other PFAAs. These data do show, however, that PFHxA
distributes into the fetus during pregnancy.
More recently, Li etal. (2020) evaluated the transplacental transfer of multiple PFAS,
including PFHxA, in preterm versus full-term births, and evaluated for correlation with the
expression of nine placental transporters. The transplacental transfer efficiency (TTE) was
calculated as the ratio of PFAS concentration in cord serum, collected at the time of birth, to the
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concentration in maternal serum collected within 1 week of (prior to) birth. The median TTE for
preterm births was 0.8, with first and third quartiles of 0.5 and 1.17, respectively (n = 27), hence
the distribution in the preterm fetus was slightly less than but not significantly different from 1.
This contrasts with PFDA, for example, for which the median TTE was 0.23 and the third quartile
0.29, indicating that the placenta limited distribution of PFDA to the fetus much more so than
PFHxA. In full-term births the median TTE for PFHxA was 2.26 (Q1 = 0.88, Q3 = 5.30, n = 88), which
is exceptionally high compared to most other PFAS for which the median TTE ranged between 0.35
(PFDA) and 1.32. Only PFTeDAhad a median full-term TTE nearly as high as PFHxA, i.e., 1.84. The
authors conjectured that the general shift to higher TTE in full-term pregnancies may be due to loss
of placental integrity as a barrier fLi etal.. 20201. but for most PFAS this is a shift from a median
value below one to a value that is higher but still less than or close to one. In the case of PFHxA
these results do not indicate a significant placental barrier for the preterm fetus and an elevated
transfer at full term. The TTE was not significantly correlated with any of the transporters analyzed.
The p-value for association with equilibrative nucleoside transporter (ENT1) was 0.054, indicating
some possibility for its role, but with a negative correlation coefficient There was a non-significant
but positive correlation between the TTE and p-glycoprotein (MDR1), which had a significant
positive association with other PFAS fLi etal.. 20201. So, there is no clear evidence or explanation
for why PFHxA would be elevated in full-term cord blood compared to maternal blood, but it seems
possible that active transport could play a role in some individuals.
The partitioning of PFHxA and 15 perfluoroalkyl substances (C6-C11) between plasma and
blood cells was investigated using blood samples collected from human subjects (n = 60) (Tin etal..
20161. The results showed that although the estimated mass fraction in plasma generally increased
with the carbon chain length, PFHxA appeared to have lowest mass fraction in plasma (0.24) as
compared with other PFAA chemicals (0.49 to 0.95). In a study population of 61 adults in Norway,
Poothong et al. f20171 also found that although PFHxA was detected in 100% of the whole blood
samples, it was not detected in serum or plasma. Given the strong partitioning to whole blood
(perhaps due to partitioning into blood cells), the whole blood, rather than serum or plasma, was
suggested as a better blood matrix for assessing PFHxA exposure (Poothongetal.. 20171.
Synthesis of Distribution Across Species
In contrast to the estimated PCs of Fabregaetal. f20151. Chengelis etal. f2009al estimated
Vd of 0.18 and 0.47 L/kg, respectively, in male and in female rats. For monkeys, the individual
estimates of Vh Chengelis etal. f2009al reported varied widely for each sex; for example, the
coefficient of variation among the three females was 74%. Therefore, EPA recalculated male and
female values for this analysis from the mean values of AUCo-oo and the beta-phase elimination
constant, Kei:
Vd = dose/[mean(AUCo-oo) x mean(Kei)]. (3-1)
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The resulting values of Vd were 0.77 L/kg and 0.35 L/kg for male and female monkeys,
respectively. Although the reported values for rats and these re-estimated values for monkeys were
within similar ranges, spanning less than a factor of five, the difference between males and females
of each species is larger than expected. The underlying data indicate significant PK differences
between males and females of each species.
The average Vd for rats (0.33 L/kg) is only 40% lower than the average for monkeys
(0.56 L/kg), a modest species difference that could occur due to differences in the relative
concentration of binding proteins and phospholipids in blood (e.g., albumin) versus the rest of body
(Sanchez Garcia etal.. 2018). Partitioning or distribution is primarily a function of the
physicochemical properties of a tissue versus blood (binding site content and phospholipid
concentration being significant components for PFAS) and are typically similar across mammalian
species, not differing by orders of magnitude as suggested by the difference between the results of
Fabrega etal. (2015) for humans and the animal PC data. This raises a significant question about
reasons for the apparent disparity. EPA is unaware of a specific mechanism that could explain this
discrepancy, particularly one that differs between monkeys and humans to such a large extent but
not between monkeys and rats.
Therefore, the most likely explanation for the differences in the PCs estimated by Fabrega et
al. f20151 are an artifact of combining data from nonmatched human samples Perez etal. f20131
whereas Ericson etal. f20071 collected data over several years (e.g., due to a change in PFHxA
exposure in that population across those times). Thus, these results are considered too uncertain
for further analysis of human pharmacokinetics. Instead, the Vd estimated for male and female
monkeys by Chengelis etal. (2009a) is assumed to provide the best estimates for men and women,
respectively, given the biochemical properties of tissues that determine the relative affinity for
PFHxA in tissue versus blood are more similar between humans and a nonhuman primate than
between humans and rats or mice. Because the Vd in monkeys is similar to that in rats (see details
above, Distribution in Animals) and an assumption of similar partitioning in humans versus other
mammals has been successfully used for many PBPK models, this assumption is considered modest
with minimal associated uncertainty.
A generally accepted assumption in pharmacokinetics is that renal clearance (via
glomerular filtration) is limited to the fraction unbound in plasma (Tanku. 1993). PFAS
accumulation in tissues appears to correlate with phospholipid binding and content and like lipids
the relative distribution of phospholipids, albumin, and other binding sites is not expected to differ
by orders of magnitude between humans and other animals f Sanchez Garcia etal.. 20181. Some
evidence suggests plasma protein binding (e.g., serum albumin) could also play a role in PFHxA
pharmacokinetics. A study by D'eon etal. (2010) evaluated the molecular interactions of PFHxA and
PFOA with human serum albumin (HSA) using nuclear magnetic resonance spectroscopy. They
found the interaction of both PFHxA and PFOA with HSA—assessed based on data for selected HSA
ligands including oleic acid, phenylbutazone, and ibuprofen—could affect its pharmacokinetics.
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Organic anion transporters, a family of transmembrane proteins, had been suggested to
play a role in the renal reabsorption of PFAAs (Kudo. 2015: Weaver etal.. 20101 (see further
discussion below for rat studies). Weaver etal. f 20101 found that renal transport of PFAAs with
different chain lengths (C2-C18) could occur via specific transporters (Oatl, Oat2, Oat3, Uratl, and
Oatplal) that were differentially located in the basolateral membrane and apical membrane in rats
(Chinese hamster ovary cell line and kidney RNA from Sprague-Dawley rats). Although PFHxA was
capable of inhibiting Oatl-mediated transport of p-aminohippurate, the model substrate used for
PFAA transport tests, the quantitative role of organic anion transporters in PFHxA PK remains
uncertain due to the rapid elimination kinetics of PFHxA (Weaver etal.. 20101. The role of Oatplal
and its regulation by sex hormones is discussed at further length below (Rat Studies).
On the other hand, although Bischel etal. f20111 measured the binding of PFHxA to bovine
serum albumin (BSA) in vitro, the measured fraction bound is 99%, which appears quantitatively
inconsistent with the empirical observation that the elimination half-life is on the order of 2-3
hours in rats, for example. Renal elimination is generally predicted to be proportional to the
fraction of a compound unbound in plasma (e.g., Tanku and Zvara (199311. Transporter-mediated
renal resorption would only reduce elimination to a greater extent. If the binding of PFHxA to BSA
is indicative of its overall fraction bound in serum and glomerular filtration could remove only 1%
(i.e., the free faction) of PFHxA carried in the corresponding serum flow, the elimination half-life
should be much longer than is observed. Thus, although plasma protein binding could play some
role in PFHxA distribution and elimination, one must be careful in quantitatively interpreting such
results. Because it is reversible, protein binding could have a limited impact on distribution and
elimination, despite a relatively high fraction of plasma protein binding at equilibrium. Therefore,
the empirically determined distribution and elimination rates for PFHxA in various species and
sexes are used rather than the rate one might predict based on albumin binding.
3.1.3. Metabolism
Similar to other PFAA compounds, PFHxA is not readily metabolized as evidenced by the
findings that no metabolites were recovered from either the liver or urine following oral dosing of
mice or rats (Gannon et al.. 2011: Chengelis etal.. 2009a). Although PFHxA is resistant to
metabolism, fluorotelomer-alcohols and sulfonates can undergo biotransformation to form PFHxA
or its glucuronide and sulfate conjugates in rodents and humans fKabadi etal.. 2018: Russell etal..
201 SI.
3.1.4. Elimination
Existing evidence has consistently suggested PFHxA has a shorter half-life than those of
other longer chained PFAAs (e.g., PFOA or PFOS). For instance, approximately 80% of the
administered dose of PFHxA appeared in the urine of rats 24 hours post-dosing regardless of sex
followingi.v. injection fChengelis etal.. 2009al. Daikin Industries recovered approximately 90% of
an oral dose of 50 mg/kg PFHxA, either as a single dose or on the 14th day of dosing by 24 hours
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after the single or last dose in male and female rats and mice (Daikin Industries. 2009a. b). Likewise
Dzierlenga etal. (20191 reported that liver and kidney concentrations peaked by 30 min in male
rats and by 1 hour in female rats after gavage and decreased steadily thereafter (observations at
0.5,1, 3, 6, 9 and 12 hours). The tissues concentrations of PFHxA tended to be very low or not
quantifiable 24 hours after dosing in both sexes of mice and rats flwabuchi etal.. 2017: Gannon et
al.. 20111.
The comparable weight-normalized blood elimination half-life of PFHxA across mammalian
species further implies the lack of species-specific roles for renal tissue transporters, either in
facilitating elimination or impeding elimination through renal resorption for PFHxA, unlike the
situation for some long-chain PFAAs. Gomis etal. f 20181 concluded PFHxA had a relatively short
elimination half-life and the lowest bioaccumulation among the six PFAAs they evaluated based on
applying a one-compartment PK model combined with PK data compiled from previous studies on
male rats. In particular, the beta- or elimination-phase half-life (ty2,p) values estimated were:
PFHxA = 2.4 hours, perfluorobutane sulfonate (PFBS) = 4.7 hours, pentafluorobenzoic acid
(PFBA) = 9.2 hours, ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate
(GenX) = 72 hours, PFOA = 136 hours, and PFOS = 644 hours (Gomis etal.. 20181. PK model
simulations from a 10-day oral experiment with a dose of 1 mg/kg-day predicted that, as compared
to other PFAAs, PFHxA had the lowest serum and liver AUC levels. Likewise, Chengelis etal. f2009al
compared PFHxA dosimetry in naive male and female rats to results after 25 days of dosing (50-
300 mg/kg-day) and found no significant difference in the parameters evaluated, with the serum
half-life remaining in the range of 2-3 hours.
Rat Studies
Iwai f20111 evaluated PFHxA excretion in Sprague-Dawley rats and CD-I mice treated with
single and multiple (4 days) oral dose(s) at 50 mg/kg of [14C] ammonium perfluorohexanoate
(APFHx). Urine and feces samples were collected for 0-6 hours (urine only) and 6-24 hours and
then followed 24-hour intervals until 72 hours after dosing. Expired air was collected over 0-24
and 24-48 hours following oral exposure. For the single dose administration in rats, 97%-100% of
administered PFHxA dose was recovered within 24 hours with urine as the major route of
elimination (73.0%-90.2%), followed by feces (7.0%-15.5% of the administered dose). No
appreciable PFHxA was found in expired air. Two percent of the dose remained in the
gastrointestinal tract and carcass. Comparable findings were observed with the multiple oral dose
administration (14 daily doses) scenarios flwai. 20111.
Chengelis etal. (2009a) reported the terminal half-life of PFHxA in serum was about
2.4-fold shorter for female Sprague-Dawley rats than for male rats (0.42 hours compared to
1.0 hour) with a single dose of 10 mg/kg i.v. injection. Likewise, Gannon etal. (2011) reported
elimination half-lives for PFHxA of 1.7 and 1.5 hours in male rats and 0.5 and 0.7 hours in female
rats for doses of 2 and 100 mg/kg, respectively. On the other hand, after repeated oral
administration (50-300 mg/kg-day) of PFHxA, Chengelis etal. f2009al found the serum terminal
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half-life of PFHxA was generally in the range of 2-3 hours regardless of sex. Comparable urinary
elimination half-lives following single 10 mg/kg i.v. were also observed (males: 2.1 hours; females
2.5 hours) fChengelis etal.. 2009al It is unclear why Chengelis etal. f2009al obtained different
half-lives for males versus females from some of their results, but not in others. Evaluation of the
half-life from any PK data set depends on the study design, especially the number and spacing of
data points relative to the half-life, the type of PK analysis done, and analytic sensitivity. EPA
analyzed PFHxA half-lives that combined data across studies to obtain sex-specific values,
described in Section 5.2.1 (Approach for Animal-Human Extrapolation of PFHxA Dosimetry).
As noted above, Daikin Industries evaluated urinary and fecal excretion in Sprague-Dawley
rats after 50 mg/kg oral doses for 1 or 14 days fDaikin Industries. 2009a. b). The elimination
pattern is consistent with other studies described here, with approximately 90% of the dose
recovered in feces and urine by 24 hours. Because excretion was only evaluated at 6 hours (urine
only), 24 hours, and multiple days thereafter, these specific studies are not considered
quantitatively informative for evaluation of half-life or clearance.
Russell etal. (2015) conducted PK modeling analysis of 3,3,4,4,5,5,6,6,7,7,8,8,8-
Tridecafluorooctanol (6:2 FTOH) inhalation (0.5 or 5 ppm) in rats, including its metabolite PFHxA,
as described above. The estimated PFHxA half-lives were 1.3 and 0.5 hours in male and female rats,
respectively, from single-day exposures, with the estimated yield of PFHxA ranging from 0.5 mol%
to 1.9 mol%. The model assumes, however, that the yield of PFHxA from 6:2 FTOH is independent
of time. This apparent time-dependence in the half-life could be an artifact of that assumption if
induction of metabolism during the dosing period leads to a higher yield with later times. A more
comprehensive multiday PK analysis would be needed to demonstrate time-dependent PFHxA
clearance unequivocally. Using a noncompartmental PK analysis Kabadi etal. (2018) reanalyzed the
1-day data of Russell etal. f20151 and obtained the same half-life values (1.3 and 0.5 hours in males
and females).
A recent study by Dzierlenga etal. f20191 and NTP f20171 showed no apparent pattern in
ty2, p among the i.v. (40 mg/kg) and two lower oral doses (40 and 80 mg/kg) for each sex (ranges
5.74-9.3 hours for male rats and 2.3-7.3 hours for female rats), which likely reflects experimental
variability. The ty2, p for the 160 mg/kg oral dose appeared higher than the other three
measurements (13.7 ± 14.2 and 12.2 ± 23.6 hours [mean ± standard error of the mean] for males
and females, respectively), but a loss of dose-concordance occurred among the PK data starting at
6 hours (i.e., the serum concentrations were similar for all dose levels at 6 hours and beyond). Also,
the data at the last time point (24 hours) varied considerably, resulting in large uncertainty in the
estimated terminal half-lives (Dzierlenga etal.. 2019).
Similar to the elimination half-life in male Sprague-Dawley rats, the estimated serum
elimination half-life of PFHxA in male Wistar rats (6 weeks old) was about 2.6 hours for a single
dose of 100 M-g/kg BW or 2.9 hours for exposures in drinking water of 1 or 3 months flwabuchi et
al.. 20171. Using a single-compartment PK model with an elimination constant defined as
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ke = ln(2)/ti/2 and obtained from a single-day exposure, the predicted serum concentration after 1
and 3 months of exposure was only 10% higher and 15% lower than the measured concentrations
at these time points, respectively. Thus, a systematic change in the half-life or clearance with
repeated dosing is not apparent
In support of the empirical estimates of half-lives described above indicating sex-specific
differences in the elimination of PFHxA, the differences can be explained (at least in part) based on
available mechanistic information. Specifically, sex hormone-dependent differences occur in
expression of transporter proteins in the rat kidney. In rats, kidney Oatplal is expressed at the
apical membrane of the proximal tubule (Bergwerk etal.. 19961 and mediates sodium-independent
transport of thyroid hormones, cholesterol-derived molecules fHata etal.. 2003: Shitara etal..
20021. and PFAS fHan etal.. 2012: Yang etal.. 20101. In male rats, Oatplal mRNA expression was
2.5-fold greater than in females, undetectable in castrated rats, and inducible in male rats by
treatment with estradiol (Kudo etal.. 20021.
A separate study (Lu etal.. 19961 reported the same sex hormone-dependent effect on
Oatplal mRNA expression in castrated males or ovariectomized females treated with testosterone
or estradiol. Further, Gotoh etal. (20021 confirmed that Oatplal protein levels were undetectable
from female rat kidney and highly expressed in male rat kidney. Because these hormone-dependent
transporters are predicted to increase renal resorption of PFHxA in male rats, the implication is
that PFAS elimination in female rats should be more rapid compared with male rats. Not all the
results above match this expectation, which could reflect a limited activity of the renal transporters
toward PFHxA, or simply aspects of experimental design and sampling that measure the PK
parameters better in some studies than others. The empirical results of Chengelis etal. (2009a) and
Dzierlenga etal. (20191. however, are consistent with this prediction: higher clearance and shorter
half-lives in female rats compared to male rats.
Some evidence also suggests the affinity for Oatplal depends on PFAS chain length.
Specifically, Yang etal. f20091 examined the role of PFAS (C4-C12) in inhibiting the uptake of
estrone-3-sulfate (E3S3S) using Oatplal-expressing Chinese hamster ovary cells. They showed the
level of inhibition of E3S uptake increased as the chain length increased; for example, PFHxA
inhibited E3SS uptake with an inhibition constant (Ki) of 1,858 |iM, as compared with 84 |iM for
PFOA. This high Ki for PFHxA (i.e., the concentration required to inhibit one-half the Oatplal
activity, 584 [ig/mL] indicates a low affinity of PFHxA for the transporter and thus leads to
predictions of a low impact of Oatplal expression on PFHxA elimination kinetics, contrary to the
empirical PK data discussed above. Chengelis etal. f2009al clearly showed more rapid elimination
in female rats versus male rats at serum concentrations below 40 |ig/mL, that is, an order of
magnitude or more below the Ki. As most of the water is resorbed from the renal filtrate, however,
the concentration of PFHxA in the remaining fluid will increase proportionately. Thus, the PFHxA
concentrations in the proximal tubule of these rats (where Oatplal is expressed) could be high
enough for significant transporter activity, but below the level of saturation.
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Toxicological Review of PFHxA and Related Salts
Collectively, the evidence provides a biologically plausible explanation for the observed sex-
specific PFHxA elimination in rats (i.e., the 2- to 3-fold longer half-life in male versus female rats),
although uncertainties remain fHan etal.. 2012: Gannon etal.. 2011: Chengelis etal.. 2009bl. Most
notably, whether this apparent sex difference in re-uptake exists in humans or in species other than
rats is unclear. Organic-anion transporters are known to be under hormonal regulation in rat and
mouse kidney, with gender-specific differences in their expression likely regulated by sex-hormone
receptors. Some evidence suggests similar sex-related differences in humans (Sabolic etal.. 20071.
Kudo etal. (20011 demonstrated that the sex-related difference in PFOA elimination in rats was
abolished when male rats were castrated, increasing to match that in females, and that its
elimination was reduced in both females and castrated males treated with testosterone. This
demonstration of hormone-related elimination for PFOA and observations of sex differences in the
elimination of other PFAS such as PFNA, PFOA, and PFBS fChengelis etal.. 2009a: Kudo etal.. 20011
suggest this is a common underlying mechanism for PFAS elimination.
Mouse Studies
As stated above (Elimination, Rat Studies), Iwai (20111 evaluated PFHxA excretion in CD-I
mice after single and 14-day oral exposures. Results were similar for single and multiple dose
administrations. After multiple doses, >95% of the administered PFHxA was recovered within 24
hours with urine as the major route of elimination (77.8%-83.4%), followed by feces (9.6%-12.9%
of the administered dose). Only 0.6%-0.9% remained in the gastrointestinal tract and carcass.
Gannon etal. (20111 also evaluated PFHxA PK in mice but state they did not report half-lives in
mice because the data showed a biphasic clearance pattern that precluded use of the standard
noncompartmental modeling.
As noted above, Daikin Industries evaluated urinary and fecal excretion in CD-I mice after
50 mg/kg oral doses for 1 or 14 days fDaikin Industries. 2009a. b). The elimination pattern is
consistent with Iwai f20111. with approximately 90% of the dose recovered in the urine and feces
(total) after 24 hours. Because excretion was only evaluated at 6 hours (urine only), 24 hours, and
multiple days after the PFHxA dosing ended, however, the studies cited are not considered
quantitatively informative for evaluation of half-life or clearance.
Daikin Industries (20101 evaluated the time-course of PFHxA in female Crl:CD(lCR) mouse
plasma after single oral gavage doses of 35,175, and 350 mg/kg, with concentrations measured at
0.5, 2, 4, 6, 8, and 24 hours. The estimated half-life was between 0.9 and 1.2 hours for the three dose
groups but lacked a dose-dependent pattern. However, the Cmax/dose was 2.76,1.88, and 1.30 kg/L
for the 35,175, and 350 mg/kg doses, respectively, indicating saturation of absorption with higher
doses. The AUCo-oo/dose was not dose-dependent, although it varied between 5.1 and 6.5 kg-h/L,
indicating that clearance was not dose-dependent
The plasma time-course data from Gannon etal. (20111 and Daikin Industries (20101 were
reevaluated by EPA as described with the derivation of the HED in Section 5.2.1 (Approach for
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Toxicological Review of PFHxA and Related Salts
Animal-Human Extrapolation of PFHxA Dosimetry) and Appendix C to obtain overall
pharmacokinetic parameters.
Monkey Studies
In the study on cynomolgus monkeys by Chengelis etal. f2009al. three males and three
females received 10 mg/kg PFHxA by i.v. injection. The mean clearance was nearly the same in both
sexes (0.122 L/h-kg in males and 0.136 L/h-kg in females), but the estimated half-life appeared
longer in males (5.3 ± 2.5 hours) than in females (2.4 ± 1.7 hours) with a corresponding apparent
difference in Vd (0.989 L/kg in males and 0.474 L/kg in females). The similarity of the clearance
values and the nearly identical serum values for males and females after the first 4 hours suggest no
striking sex differences in the pharmacokinetics of PFHxA in monkeys.
Human Studies
No controlled exposure PK studies of PFHxA elimination in humans are available but
Russell etal. (2013) applied PK analysis to biomonitoring data from Nilssonetal. (2013) to
estimate the half-life of PFHxA in humans. Specifically, Russell etal. (2013) estimated the apparent
half-life of PFHxA in humans by analyzing biomonitoring data collected from professional ski wax
technicians and then compared the human estimates of PFHxA elimination to that for mice, rats,
and monkeys. For the human monitoring study, blood samples (n = 94) were collected from male
professional ski wax technicians (n = 11) and analyzed for PFHxA in plasma and serum. (Individual
data for eight of the technicians are shown in Appendix C.2; complete data are available as the
supplemental information for Nilssonetal. (2013)). Personal and area air concentration monitoring
of the ski wax subjects and facilities demonstrated both the metabolic precursor, 6:2 FTOH, and
PFHxA were present in all locations, but the arithmetic mean concentration of 6:2 FTOH ranged
from over 100 times higher than PFHxA to almost 100 times lower, across the monitoring locations.
A one-compartment model with first-order kinetics was used for PK analyses. The estimated
geometric mean half-life of PFHxA was 32 days with a range of 14-49 days in the studied
population (Russell etal.. 2013). PFHxA plasma concentrations declined below the plasma
detection limit of 0.05 ng/mL within a period of 2-4 months after exposure ceased, reflecting the
relatively rapid elimination rate of PFHxA. In contrast, the half-life of PFHxS in humans was
estimated to range from 5 to 9 years fOlsen etal.. 20071.
Analysis by Luz et al. f20191 found no significant species- or sex-related differences in the
elimination kinetics of PFHxA. The PK analysis, however, is attributed to a meeting abstract (Buck
and Gannon. 2017) and provides no details of the methods the authors used. The text of Luz et al.
(2019) indicates the analysis of Buck and Gannon (2017) used data from only 3 of the 11 subjects of
Nilsson etal. (2013). specifically the 3 with the most rapid elimination, reducing the extent to which
the conclusion can be reliably extrapolated to the population as a whole. Luz etal. (2019) state
slower apparent elimination could occur in some subjects because of ongoing exposure. Although
ongoing exposure could cause this effect, it is also possible that elimination in some individuals is
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Toxicological Review of PFHxA and Related Salts
slower than others due to interindividual variability. In the absence of independent evidence that
ongoing exposure occurred in other human subjects of Nilsson etal. (20131 who were excluded in
this later analysis, EPA does not consider basing conclusions on human elimination on only the
three individuals who had the most rapid elimination appropriate.
EPA examined the data of Nilsson etal. f20131. and the observed seasonal variation appears
to show a longer systemic period of exposure (when blood levels are elevated versus. Declining) for
some individuals than others. Also, the data set includes samples with concentrations below the
limit of detection (LOD) that should be treated with an appropriate statistical model to account for
the censoring of these data. Finally, only the data collected encompassing the 2007-2008 ski
season, during which only 8 of the 11 technicians were sampled, includes post-exposure samples
needed to quantify elimination. A detailed description of EPA's analysis of the eight technicians
sampled during the 2007-2008 season is provided in Appendix C.2. Briefly, each ski-wax technician
in the study was presumed to have a constant rate of exposure up to a date that is different for each
individual when exposure stopped, and elimination began. Specifically, we used a one-compartment
i.v.-infusion model to fit the data:
Where A = dose/Va, tinf is the time period of exposure (treated as an infusion), and ke is the
elimination rate. The model is analyzed through hierarchical Bayesian analysis, with A and tinf
estimated independently for each individual technician although the technician-level ke is drawn
from a population-level distribution. Note blood concentrations were measured only once a month
and no other data on exposure is available. Thus, although the model clearly simplifies the exposure
estimation, estimating a larger number of parameters reliably would not be possible. As such, the
model allows for estimating variation among individuals without subjectively selecting a subset of
the technicians for analysis. The resulting distribution of ke had a mean (90% confidence interval,
CI) of 0.00252 (0.00136-0.00477) h"1. Using an average Fd of 0.7315 L/kg (731.5 mL/kg) for male
and female monkeys from Chengelis etal. f2009al. the resulting mean for human clearance is
CL = Vd-ke = 1.84 mL/kg-h. Given the expected similarity of Vd across mammalian species, EPA
considers the average value estimated for rats (0.33 L/kg) to be a reasonable lower bound for
humans and the highest value reported by Chengelis etal. (2009a) for an individual (male) monkey
(1.54 L/kg) to be a reasonable upper bound. Combining these with the 90% CI for ke (0.00136-
0.00477 h"1) yields a possible range for human clearance of 0.45-7.35 mL/kg-h, a range of 16-fold
from 4-fold above to 4-fold below the estimated mean.
Xiao etal. f20111 measured the serum concentrations of 10 PFAA chemicals in 227
nonoccupational exposed individuals aged 0.3-90 years (133 males and 94 females) in China.
Significant positive correlations were observed between age and serum levels of PFAA chemicals
(3-2)
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Toxicological Review of PFHxA and Related Salts
except for PFBS, PFHpA, and PFHxA. Spearman correlation coefficients between age and serum
PFHxA were 0.20, -0.02, and 0.08 for males, females, and the combined data, respectively.
Collectively, the findings indicated no age-related accumulation of PFHxA in human bodies, which is
consistent with the relatively short half-life.
3.1.5. PBPK Models
No PBPK model is available for PFHxA in rats, mice, or monkeys. Fabrega etal. (20151
described a PBPK model for multiple PFAS in humans, including PFHxA. However, Fabrega et al.
(20151 state two key parameters that determine the rate of resorption from glomerular filtrate in
the kidney were identified using the data from the Ericsonetal. f20071 epidemiological survey of
PFAS exposure in residents of Catalonia, Spain. Because PFHxA was not detected in any individuals
sampled by Ericson et al. f20071. EPA does not consider it possible to reliably identify elimination
parameters from that data set. Further, the individual exposure or elimination data needed to
associate the blood concentrations of Ericson etal. (20071 with urinary clearance rates are not
reported in either paper. Thus, uniquely identifying two parameters with a single combination of
average PFHxA exposure and average blood concentration is impossible. Finally, as described above
(Distribution, Distribution in Humans), the tissue: blood partition coefficients Fabrega etal. f20151
estimated are not considered suitable for the purposes of this assessment due to the 4+-year lag in
measurements between collection of the blood samples and the tissue samples and because they
are inconsistent with data on PFHxA distribution in other species, including monkeys. Thus, the
PBPK model of Fabrega etal. (20151 is not considered sufficiently suitable for use in this
assessment.
3.1.6. Summary
The PFHxA elimination half-lives and clearance values reported in studies are important for
interpreting and quantifying health outcomes potentially associated with PFHxA exposure. The
most notable finding was the apparent sex-specific PK differences between male and female mice
and rats where female rodents eliminate PFHxA 2-3 times faster than males (see Table 3-1).
Although monkeys have half-lives and clearance values in the same range as mice and rats, the
clearance in female monkeys is only 11% faster than in males. This indicates that the significant sex
differences observed in rodents does not appear to apply to primates. Humans have a much longer
serum elimination half-life (EPA estimate: 275 hours) than rodents and monkeys (2-7 hours). The
difference could be a consequence of species differences in the expression or activity of the renal
transporters that reabsorb PFAS, but this has not been demonstrated. All available PK evidence is
summarized below in Table 3-1.
According to EPA's BW°75 guidelines (U.S. EPA. 20111. use of chemical-specific data for
dosimetric extrapolation such as the PFHxA-specific data described above is preferable to the
default method of BW°75 scaling. That is the case here. For example, using the standard species BWs
of 0.25 kg in rats and 80 kg in humans, the half-life in humans is predicted to be 4.2 times greater
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Toxicological Review of PFHxA and Related Salts
than rats. Given half-lives in the range of 0.4-14 hours among male and female rats (see Table 3-1),
one would then predict half-lives of 1.6-57 hours in humans, 20-200 times lower than the range
estimated by Russell etal. f20131 and 10-100 times lower than the range estimated by EPA (see
Table 3-1). Thus, based on the PFHxA-specific PK data, use of BW0 75 for dosimetric extrapolation
could lead to an underprediction of human elimination by 1-2 orders of magnitude. Therefore, use
of BW°75 as an alternative means of extrapolation is not considered further for PFHxA, and the
preferred, data-driven approach will be used for the dosimetric extrapolation.
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Table 3-1. Summary of PK evidence for PFHxA
Species/sex
Study design (dose)
Elimination half-
life (beta)(h)
AUC/dose
(kg-h/L)
Clearance
(mL/h-kg)
Volume of
distribution (mL/kg)
Reference
Rats
Male
Single i.v. dose (10 mg/kg)
1.0
8.7
116
175
Chengelis et al. (2009a)
Single oral dose (50 mg/kg)
2.2
10.0
NR
NR
Single oral dose (150 mg/kg)
2.4
6.1
NR
NR
Single oral dose (300 mg/kg)
2.5
8.4
NR
NR
Female
Single i.v. dose (10 mg/kg)
0.42
1.3
775
466
Single oral dose (50 mg/kg)
2.6
2.4
NR
NR
Single oral dose (150 mg/kg)
2.2
2.2
NR
NR
Single oral dose (300 mg/kg)
2.1
3.5
NR
NR
Male
Single i.v. dose (40 mg/kg)
8.0 ±2.2
7.4 ±0.7
136 ± 13
430 ±112
Dzierlenga et al. (2019)
Single oral dose (40 mg/kg)
9.3 ±20.8
9.7 ± 1.3
103 ± 13
601± 470
NTP (2017)
Single oral dose (80 mg/kg)
5.7 ±4.6
6.6 ±0.5
153 ± 11
496 ± 81
Single oral dose (160 mg/kg)
14 ± 14
6.8 ±0.6
147 ± 14
615 ± 367
Female
Single i.v. dose (40 mg/kg)
7.3 ±2.0
3.1 ±0.3
327 ± 33
223 ± 45
Single oral dose (40 mg/kg)
2.3 ±213
6.1 ± 1.1
164 ± 29
327± 149
Single oral dose (80 mg/kg)
5.5 ±2.6
3.2 ±0.4
314 ± 39
560 ±113
Single oral dose (160 mg/kg)
12 ±24
3.7 ±0.5
274 ± 37
473 ±158
Male
Single oral dose (2 mg/kg)
1.7 ±0.6
8± 1.5
NR
NR
Gannon et al. (2011)
Single oral dose (100 mg/kg)
1.5 ±0.2
6.5 ± 1.4
NR
NR
Female
Single oral dose (2 mg/kg)
0.5 ±0.1
2.5 ±0.5
NR
NR
Single oral dose (100 mg/kg)
0.7 ±0.3
2.5 ±0.7
NR
NR
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Species/sex
Study design (dose)
Elimination half-
life (beta)(h)
AUC/dose
(kg-h/L)
Clearance
(mL/h-kg)
Volume of
distribution (mL/kg)
Reference
Male
Single i.v. dose (0.1 mg/kg)
2.7
9.8
NR
400
Iwabuchi et al. (2017)
Male
Single inhalation3 (0.5 ppm)
1.3
NDb
107
NR
Kabadi et al. (2018)
Single inhalation3 (5.0 ppm)
1.3
NDb
277
NR
Female
Single inhalation3 (0.5 ppm)
0.5
NDb
107
NR
Single inhalation3 (5.0 ppm)
0.5
NDb
277
NR
Mice
Male
Single oral dose (2 mg/kg)
ND
12
NR
NR
Gannon et al. (2011)
Single oral dose (100 mg/kg)
ND
12
NR
NR
Female
Single oral dose (2 mg/kg)
ND
4
NR
NR
Single oral dose (100 mg/kg)
ND
6.4
NR
NR
Monkeys
Male
Single i.v. dose (10 mg/kg)
5.3 ±2.5
8.4 ± 1.8
122 ± 24
989 ± 579
Chengelis et al. (2009a)
Female
Single i.v. dose (10 mg/kg)
2.4 ± 1.7
7.5 ± 1.3
136 ± 22
474 ± 349
Humans
Males and females
Post-exposure observation in
male ski wax technicians
768 (336-1,176)
275 (145-509)
ND
ND
1.84 (0.45-7.35)
ND
Russell et al. (2013)
Current analysis
i.v. = intravenous; ND = not determined; NR = not reported.
a6-hour inhalation exposure to 6:2 FTOH.
bDose of PFHxA unknown.
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3.2. NONCANCER EVIDENCE SYNTHESIS AND INTEGRATION
For each potential health effect discussed below, the synthesis describes the evidence base
of available human and animal studies. The PFHxA animal literature inventory summarizes the
evidence base on potential health effects (organized by organ or system) from the available high
and medium confidence short-term, developmental, subchronic, and chronic studies in mice and
rats (NTP. 2018: Klaunig etal.. 2015: Iwai and Hoberman. 2014: Chengelis etal.. 2009b: Loveless et
al.. 2009: Kirkpatrick. 2005a). These animal studies represent the primary evidence available for
this PFAS, and a more detailed summarization of study methods and findings is provided in HAWC.
Some organs/systems for which animal data were available are summarized in the animal
literature inventory, but these data were not synthesized due to insufficient evidence to draw
hazard judgments (i.e., evidence is inadequate). Specifically, for these health effects there were
either few studies with null results (i.e., dermal, musculoskeletal, sensory, ocular) or few studies
with sporadic findings of unclear toxicological significance (i.e., respiratory, gastrointestinal system,
cardiovascular, and metabolic effects), including small changes in indirect outcome measures and
other effects of unclear biological significance in isolation (e.g., decreases in cholesterol). Similarly,
one low confidence cross-sectional study in general population adults in China examined the
association between PFHxA exposure and adiposity fTian etal.. 20191: however, due to concern for
exposure misclassification resulting from reverse causation, no judgment could be drawn (i.e.,
evidence is inadequate) and these data were not synthesized. Effects on body weights and
survival, which had no effect or lowest effect levels at the highest administered dose in animal
studies, were also not separately synthesized but were used to aid the interpretation of other
potential health effects of PFHxA exposure.
3.2.1. Hepatic Effects
Human
Three epidemiological studies report on the relationship between PFHxA exposure and liver
enzymes. Of these, one (Tiang etal.. 2014). a cross-sectional study of pregnant women in China, was
critically deficient in the confounding domain and was considered overall uninformative. There was
no consideration of potential confounding in the study design and analysis. Most notably, there was
no adjustment for age, which is a highly relevant potential confounder of the association. Based on
these deficiencies, the study was excluded from further analysis (see Figure 3-1). The remaining
studies (Liu etal.. 2022: Nian etal.. 2019) were cross-sectional studies in general population adults
in China and were classified as medium confidence (see Figure 3-1). Liu etal. (2022) reported
positive associations (i.e., higher liver enzyme levels with higher PFHxA exposure) for serum
albumin (p < 0.05) and alanine aminotransferase (ALT) and alkaline phosphatase (ALP) (not
statistically significant), but no association with aspartate aminotransferase (AST), total protein, or
y-glutamyl transferase (GGT). Nian etal. f20191 did not observe an association between PFHxA
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levels and ALT, AST, total protein, ALP, GGT, total bilirubin, or cholinesterase. Sensitivity was a
concern in both studies due to limited exposure contrast (i.e., likely insufficient variability in
exposure levels to detect an association), which may explain the limited observed associations.
Participant selection ¦
Exposure measurement^
Outcome ascertainment-
Confounding ¦
Analysis ¦
Sensitivity -
Selective Reporting •
Overall confidence ¦
W
Legend
Good (metric) or High confidence (overall)
Adequate (metric) or Medium confidence (overall)
Deficient (metric) or Low confidence (overall)
Critically deficient (metric) or Uninformative (overall)
Figure 3-1. Study evaluation for human epidemiological studies reporting
hepatic system findings from PFHxA exposures (full details available by
clicking the HAWC link). Note that for N/A, critical deficiencies in confounding
domains were identified and the study was judged as uninformative; thus, the
remaining domains were not evaluated.
Animal
Hepatic outcomes were evaluated in multiple short-term, subchronic, or chronic studies in
rats and mice fNTP. 2018: Klaunig etal.. 2015: Iwai and Hoberman. 2014: Chengelis etal.. 2009b:
Loveless etal.. 20091. Generally, studies were rated as medium or high confidence for the hepatic
outcomes, but some outcome-specific considerations for study evaluation were influential on the
overall confidence ratings for hepatic effects. Histopathology for Chengelis etal. f2009bl was rated
low confidence because of issues related to observational bias, endpoint sensitivity and specificity,
and results presentation. Results of the outcome-specific confidence evaluations are presented in
Table 3-2 below, and details are available by clicking the HAWC link.
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Toxicological Review of PFHxA and Related Salts
Table 3-2. Evaluation results for animal studies assessing effects of PFHxA
exposure on the hepatic system
Author (year)
Species, strain
(sex)
Exposure
design
Exposure route and
dose range
Organ weight
Histopathology
Clinical chemistry
Peroxisomal beta
oxidation
NTP (2018)
Rat, Harlan
Sprague-Dawley
(male and female)
Short term
(28 d)
Gavage3
Male and female: 0,
62.5, 125, 250, 500,
1,000 mg/kg-d
+ +
+ +
++
NM
Chengelis et al.
(2009b)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and female)
Subchronic
(90 d)
Gavage3
Male and female: 0,10,
50, 200 mg/kg-d
+ +
"
++
"
Loveless et al.
(2009)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and female)
Subchronic
(90 d)
Gavageb
Male and female: 0, 20,
100, 500 mg/kg-d
+ +
+ +
++
+ +
Klaunig et al.
(2015)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and female)
2-yr cancer
bioassay
Gavage3
Male: 0, 2.5,15,
100 mg/kg-d
Female: 0, 5, 30,
200 mg/kg-d
NM
+ +
++
NM
++ Outcome rating of high confidence; + outcome rating of medium confidence; - outcome rating of low
confidence; - outcome rating of uninformative; NM, outcome not measured.
a bStudy evaluation for animal toxicological hepatic endpoints reported from studies with male and female rats
receiving by gavage PFHxA3 or PFHxA sodium salt.b Study evaluation details for all outcomes are available by
clicking the HAWC link.
Organ Weight
Overall, findings of increased liver weights after oral PFHxA or PFHxA sodium salt
exposures in rats were consistent (see Figure 3-2; exposure response array link). Relative liver
weights (see Table 3-3) are generally considered more reliable than absolute liver weights because
they consider large variations in body weight that could skew organ weight interpretation fHall et
al.. 20121. Large variations in body weights were not observed after PFHxA exposures in male and
female adult rats, and changes in both relative and absolute liver weights were similarly increased
and dose responsive. Increases in relative and absolute liver weights were dose-dependently
increased in all three short-term and subchronic studies. Statistically significant increases in male
rat relative liver weights were observed with oral doses of >200-250 mg/kg-day, whereas
statistically significant increases in female rats were observed only at >500 mg/kg-day. Specifically,
in the 28-day study, relative liver weight was increased (14%) in male rats at 250 mg/kg-day,
where a similar increase (15%) was observed in female rats at 500 mg/kg-day fNTP. 20181. In the
subchronic studies, relative liver weights were increased (22%) at 200 mg/kg-day in males (with
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Toxicological Review of PFHxA and Related Salts
no change in females) in one study (Chengelis etal.. 2009b). and the other study observed increases
of 63% and 37% at 500 mg/kg-day in males and females, respectively (Loveless etal.. 20091. Note
that PFHxA effects on relative liver weights had resolved by 30 days in the recovery group
fChengelis etal.. 2009bl. Liver weights were not evaluated in the chronic study fKlaunig etal..
201 SI.
Endpoint
Study
Experiment
Animal Description
Observation Time
PFHxA Hepatic Effects: Liver Weight
Liver Weight, Absolute
NTP, 2018, 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley (
S) Day 29
A a
A
A
V *
Rat, Harlan Sprague-Dawley (
0) Day 29
A a
A
A
V *
Chengelis, 2009, 2850404
90-Day Oral
Rat, Crl:CD(SD) (c?)
Day 90
• •
a
Rat, Crl:CD(SD) (?)
Day 90
• •
-a
Loveless, 2009, 2850369
90-Day Oral
Rat. Crl:CD(SD) (S)
Day 92
A
*
aX
Rat, Crl:CD(SD) (?)
Day 93
A
Liver Weight, Absolute, Recovery
Chengelis, 2009, 2850404
90-Day Oral
Rat, Crl:CD(SD) (t?)
Day 118
Rat. Crl:CD(SD) (?)
Day 118
*
*
Liver Weight, Relative
NTP, 2018, 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley (
3) Day 29
a
A
A
A
V *
Rat, Harlan Sprague-Dawley (
0) Day 29
A a
A
v •
Chengelis. 2009, 2850404
90-Day Oral
Rat. Crl:CD(SD) (c?)
Day 90
9#
A
Rat. Crl:CD(SD) (?)
Day 90
• •
-a
Loveless, 2009, 2850369
90-Day Oral
Rat. Crl:CD(SD) ($)
Day 92
A
m
Rat, Crl:CD(SD) (?)
Day 93
A
u
Liver Weight, Relative, Recovery
Chengelis, 2009, 2850404
90-Day Oral
Rat, Crl:CD(SD) (c?)
Day 118
•
w
a
1 •
No significant changeA
Significant increase v Significant decrease
^ Significant Trend |
100 0
100
200 300
400 500 600 700 800
900 1,0001,100
Dose (mg/kg-day)
Figure 3-2. Liver weights (absolute and relative) after short-term and
subchronic PFHxA exposures (full details available by clicking the HAWC link).
Table 3-3. Percent increase in relative liver weight due to PFHxA exposure in
short-term and subchronic oral toxicity studies
Study design and
reference
Dose (mg/kg-d)
in
-------
Toxicological Review of PFHxA and Related Salts
Histopathology
Treatment-related increases in liver weight can result from various changes in hepatic
morphology including hyperplasia of any resident liver cell type, cellular hypertrophy
inflammation, fibrosis, increase in hepatocyte size, neoplasia, congestion, or metabolic enzyme
induction fHall etal.. 2012: Thoolen etal.. 2010: U.S. EPA. 2002al. As shown in Table 3-4 and
summarized in the HAWC link, four studies evaluated liver histopathology in rats. One observed
effect of PFHxA exposure was hepatocellular hypertrophy that was consistent across the short term
and subchronic studies. Hepatic hypertrophy can refer to an increase in liver weight and size; an
increase in hepatocyte size caused by abnormal storage of water, glycogen, lipids, or organelle
proliferation; and an increase in hepatic enzyme induction fHall etal.. 2012: Thoolen etal.. 2010:
U.S. EPA. 2002al. Coherent with findings on liver weight, the observations of hepatocellular
hypertrophy were dose-dependent and male rats were more sensitive than females. Specifically,
increased hepatocellular hypertrophy was observed in adult male and female rats in the high
confidence short-term (NTP. 20181 and high confidence subchronic (Loveless etal.. 20091 studies
at doses >100-500 mg/kg-day. In the subchronic study, hypertrophy persisted 30 and 90 days after
recovery in males, and 30 days after recovery in females fLoveless etal.. 20091. In the low
confidence (for histopathology outcomes) subchronic study, centrilobular hepatocellular
hypertrophy was observed in male rats only (incidence 7/10, 200 mg/kg-day) and resolved after
28-day recovery (Chengelis etal.. 2009b). In the chronic study (Klaunig etal.. 2015). hepatocellular
hypertrophy findings were null consistent with null observations at similar doses in the short-term
and subchronic studies.
Table 3-4. Incidence of hepatocellular hypertrophy findings in adult rats due
to PFHxA exposure in short-term and subchronic oral toxicity studies
Study design and reference
Dose (mg/kg-d)
o
*—1
o
fM
o
m
62.5
o
o
*—i
lO
fsj
*—1
o
o
fM
o
m
fM
o
o
m
OOO'I
28-d, female rat (NTP, 2018)
0/10
0/10
0/10
0/10
9/10*
28-d, male rat (NTP, 2018)
0/10
0/10
0/10
9/10*
10/10*
90-d, female rat
(Chengelis et al., 2009b)
0/10
0/10
0/10
90-d, male rat
(Chengelis et al., 2009b)
0/10
0/10
7/10*
90-d, female rat
(Loveless et al., 2009)
0/10
0/10
5/10*
90-d, male rat
(Loveless et al., 2009)
0/10
4/10*
10/10
*
90-d, female rat, 30-d recovery
(Loveless et al., 2009)
4/10*
3-23
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Toxicological Review of PFHxA and Related Salts
Study design and reference
Dose (mg/kg-d)
o
*—1
o
fsj
O
lO
LO
rsi
ID
o
o
*—1
lO
fsj
*—1
o
o
fsj
O
LO
fsj
O
O
lO
OOO'I
90-d, female rat, 90-d recovery
(Loveless et al., 2009)
0/10
90-d, male rat, 30-d recovery
(Loveless et al., 2009)
9/10*
90-d, male rat, 90-d recovery
(Loveless et al., 2009)
6/10*
* Indicates instances where statistical significance (p < 0.05) compared to controls was reported by study authors;
shaded cells represent doses not included in the individual studies.
Other pathological findings of PFHxA-mediated hepatic effects included increased
hepatocellular necrosis in rats, with a significant increase in female rats [n = 99/10 vs. 0/10 in
controls, p < 0.05) reported in a short-term study at 1,000 mg/kg-day PFHxA, whereas there were
no significant findings of hepatocellular necrosis across male dose groups fNTP. 20181. One
subchronic study reported necrosis in male rats [n = 1/10 vs. 0/10 in controls, not statistically
significant) (Chengelis etal.. 2009b) whereas necrosis was not observed in the other subchronic
study (Loveless etal.. 2009). In the high confidence chronic study, Klaunigetal. (2015) reported
hepatocellular necrosis [n = 12/70 vs. 2/60 in controls, p < 0.05 in the 200 mg/kg-day female dose
group (the highest dose tested). The authors noted most necrosis findings were in animals that died
or were euthanized prior to scheduled necropsy and the increased mortality was not treatment
related, but was due to mechanical injury, gavage trauma, reflux injury, or spontaneous disease
processes (Klaunig et al.. 2015). The authors reported no treatment-related increases in
hepatocellular necrosis (n = 6/70 vs. 4/60 in controls) or necrosis in the centrilobular regions of
the liver lobule (n = 1/70 vs. 2/60 in controls) in male rats up to the highest dose for that sex,
100 mg/kg-day. Other findings included nonsignificant congestion in males [n = 23/70 vs. 15/60 in
controls) and females (n = 8/70 vs. 6/60 in controls) fKlaunig etal.. 20151. Incidence of necrosis
were not observed in the short-term study fNTP. 20181. and the subchronic study by Loveless et al.
(2009) did not report histological findings other than hepatocellular hypertrophy (no data on
necrosis were available).
Other histopathological findings included observations of hepatocellular cytoplasmic
alterations (p < 0.05) in adult male and female rats at the highest dose [1,000 mg/kg-day in the
short-term study fNTP. 20181], All results reported above can be viewed using the HAWC link.
Clinical Chemistry
A clinical chemistry panel measures the proteins, enzymes, chemicals, and waste products
in the blood. These measures, when evaluated together and with other biomarkers are informative
3-24
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Toxicological Review of PFHxA and Related Salts
diagnostic tests of organ function and when interpreted together with histopathology are useful for
the assessment of adverse liver effects.
Serum Enzymes
Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are often useful
indicators of hepatic enzyme induction or hepatocellular damage as increased serum levels are
thought to be due to hepatocyte damage resulting in release into the blood, whereas ALP is
localized to the bile canalicular membrane and more indicative of hepatobiliary damage (Hall etal..
2012: Amacher et al.. 1998). PFHxA effects on the serum enzymes ALT, AST, and ALP included
<2-fold increases in serum enzyme across the three short-term and subchronic studies, except for
one 2.4-fold increase in male rats at 200 mg/kg-day in the high confidence subchronic study
f Chengelis etal.. 2009bl. No clear pattern of effects on the serum enzymes were reported in the
chronic study fKlaunig etal.. 20151. but the highest dose was 100 or 200 mg/kg-day PFHxA in male
or female rats, respectively. Full study details are available in Figure 3-3 and by clicking the HAWC
link. Percent changes in treated relative to controls are provided in Table 3-5, Table 3-6, and
Table 3-7.
Specifically, in the short-term study, ALT, AST, and ALP were increased in a dose-response
gradient in adult male rats at doses as low as 500 mg/kg-day fNTP. 20181. In female rats, ALT and
AST measures were increased in a dose-response gradient at doses as low as 500 mg/kg-day,
whereas ALP was increased only in the highest (1,000 mg/kg-day) dose group (NTP. 2018).
ALT increases were observed only in male rats at PFHxA sodium salt exposures as low as 20
mg/kg-day in one subchronic study (Loveless etal.. 2009) and in the highest PFHxA dose group
(200 mg/kg-day) in the other subchronic study (Chengelis etal.. 2009b). AST was increased in only
one subchronic study in males at >20 mg/kg-day fLoveless etal.. 20091. Chengelis etal. f2009bl
reported increased AST in males only in the 200 mg/kg-day dose group that resolved after the
30-day recovery (see Table 3-6).
ALP was increased in both subchronic studies with significant increases observed in the
highest exposure groups [200 (Loveless etal.. 2009) and 500 mg/kg-day (Chengelis etal.. 2009b)]
that resolved by the 30-day recovery (see Table 3-7). The chronic study did not include a 13-week
endpoint that would have been useful for group mean comparisons with the test measures in the
subchronic studies (as clinical pathology test values often change with animal age) fAACC. 19921.
3-25
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Toxicological Review ofPFHxA and Related Salts
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Study design and reference
Dose (mg/kg-d)
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Toxicological Review of PFHxA and Related Salts
Table 3-7. Percent change in alkaline phosphatase due to PFHxA exposure in
short-term, subchronic, and chronic oral toxicity studies
Study design and
reference
Dose (mg/kg-d
in
-------
Toxicological Review ofPFHxA and Related Salts
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20181. A dose-responsive decrease (6%-14%, >100 mg/kg-day) in TP also was observed in male
rats fChengelis et al.. 2009b: Loveless etal.. 20091 with decreased levels observed in males (-6%,
200 mg/kg-day) atthe 30-day recovery fChengelis etal.. 2009b). Albumin is a major blood protein
that binds fatty acids, cations, bilirubin, thyroxine (T4), and other compounds. Decreased albumin
levels are associated with decreased synthesis in the liver, increased catabolism, severe diffuse liver
disease, subacute hepatitis, hepatocellular damage, ascites, cirrhosis, and chronic alcoholism
fWhalan. 20151. Slight decreases (p < 0.05) in albumin were reported only in males exposed for
3-29
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Toxicological Review of PFHxA and Related Salts
28 days to 125 mg/kg-day (6% decrease) and 1,000 mg/kg-day (7% decrease) PFHxA (NTP. 2018).
The biological significance of this magnitude of change is unclear. No effects on albumin were
identified in the subchronic or chronic studies.
Globulin, a collection of blood proteins larger than albumin made by both the liver and
immune system, were decreased in all but the chronic study (see Table 3-9). Globulin decreases
were observed in both male and female rats treated with PFHxA in the short-term study at
>125 mg/kg-day and 1,000 mg/kg-day, respectively fNTP. 2018). Consistent with the short-term
study, decreases were also observed in both males and females in the highest dose groups
[200 (Chengelis etal.. 2009b) and 100 mg/kg-day (Loveless etal.. 2009)]. Notably, globulin
decreases (10%) persisted at the 30-day recovery in males (200 mg/kg-day) and returned to
normal in females fChengelis etal.. 2009bl.
The decrease in globulin was consistent with increases in A: G, a routine blood test used to
screen for liver, kidney, immune, and gastrointestinal function. The A:G was increased in males and
females (113%-160% at >250 mg/kg-day and 142% at 1,000 mg/kg-day) with significant trends in
both sexes (NTP. 2018). Chengelis etal. (2009b) observed an increase (10%) at the 30-day
recovery in rats receiving an oral dose of 200 mg/kg-day.
Table 3-8. Percent change in total protein due to PFHxA exposure in
short-term, subchronic, and chronic oral toxicity studies
Study design and
reference
Dose (mg/kg-d)
in
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Toxicological Review of PFHxA and Related Salts
Dose (mg/kg-d)
Study design and
reference
in
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Toxicological Review of PFHxA and Related Salts
Hepatobiliary Components
Other indicators of potential liver dysfunction or injury included impacts on bile
components essential for normal lipid metabolism and red blood cell breakdown. ALP (discussed
with serum enzymes and in Table 3-7, see above) is an indicator of bile duct obstruction and was
consistently increased in male and female rats in the short-term study fNTP. 2018] and subchronic
studies (Chengelis etal.. 2009b: Loveless etal.. 2009). In the short-term study fNTP. 2018). bile
acids were increased at the highest dose (1,000 mg/kg-day) with a significant trend (a possible
indication of cholestatic liver injury), and bilirubin was decreased in a dose-response gradient
across both the short-term and subchronic fLoveless etal.. 20091 studies (see Figure 3-5). Lower
than normal bilirubin levels are usually not a concern and can be reduced in response to increased
conjugation rates after hepatic enzyme induction and excretion into bile fHall etal.. 20121.
Endpoint
Study
Study Type
Animal Description
Observation Time
PFHxA Hepatic Effects: Hepatobiliary Components
Bile Salt/Acids
NTP, 2018, 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley ($)
Day 29
4> * « ' • A
Rat, Harlan Sprague-Dawley (?)
Day 29
0 • • ¦ • A
Direct Bilirubin
NTP, 2018, 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley (3)
Day 29
• • •—•—
•
-•
Rat, Harlan Sprague-Dawley ($)
Day 29
• • »—•—
~
•
Loveless, 2009, 2850369
90-Day Oral
Rat, Crl:CD(SD) ( » « . . ~
• No significant changeA Significant increase V Significant decrease Significant Trend
100 0 100 200 300
400 500 600 700 800 900
Dose (mg/kg-day)
1,0001,10
0
Figure 3-5. Hepatobiliary findings in rats exposed by gavage to PFHxA or
PFHxA sodium salt (full details available by clicking the HAWC link).
Mechanistic Evidence and Supplemental Information
The available evidence base reports increased liver weight, hepatocellular hypertrophy,
hepatocellular necrosis, increased (1.5-2.5-fold) serum enzymes, increased peroxisomal beta
oxidation, decreased total protein (driven by decreased globulin), and decreased bilirubin levels in
rats exposed to PFHxA. This collection of findings was considered for their potential adversity and
relevance in humans using Hall criteria (Hall etal.. 2012) along with other available supplemental
mechanistic evidence. The mechanistic findings (i.e., -omics data, transactivation assays, oxidative
stress assays, knockout mouse models) were coherent with animal findings of PFHxA mediated
liver injury. The supplemental mechanistic evidence from other short-chain PFAS (e.g., PFBA)
supported both PPARa independent and dependent pathways that are relevant in humans,
increasing the plausibility that these PFAS-mediated responses also apply to PFHxA. Additional
evidence from non-mammalian models is available and summarized in the PFHxA Literature
Tagtree.
3-32
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Toxicological Review of PFHxA and Related Salts
Considerations for Potentially Adaptive Versus Adverse Responses
Considering that the hepatic effects of PFHxA exposure (increased liver weight and
hepatocyte hypertrophy) observed in rodents could represent adaptive responses, the potential
adversity of these effects was a key consideration and analyzed. In the absence of a known
mechanism leading to increased liver weight, hepatocellular hypertrophy, and necrosis, the
evidence on PFHxA-mediated hepatotoxicity was evaluated to inform interpretations regarding
adversity utilizing guidance from Hall etal. (20121. Increased liver weight and hepatocellular
hypertrophy can be associated with changes that are adaptive in nature (Hall etal.. 20121 and not
necessarily indicative of adverse effects unless observed in concordance with other clinical,
pathological markers of overt liver toxicity (see Appendix A). The IRIS PFAS Assessment Protocol
(which applies to PFHxA) states the panel recommendations from Hall etal. f 20121 can be used to
judge whether observed hepatic effects are adverse or adaptive in nature. Given that Hall et al.
(20121 was focused on framing noncancer liver effects in the context of progression to liver tumors,
however, the protocol further indicates that "...consultation of additional relevant information will
be considered to interpret the adversity of noncancer liver effects over a lifetime exposure, taking
into account that effects perceived as adaptive can progress into more severe responses and lead to
cell injury." Hall etal. f20121 indicates that additional evidence must be considered when
determining adverse versus adaptive response in rodents in the context of hepatocellular
hypertrophy that include:
1) Is there histological evidence of the following structural degenerative or necrotic changes?
• Hepatocyte necrosis, fibrosis, inflammation, and steatotic vacuolar degeneration
• Biliary/oval cell proliferation, degeneration, fibrosis, and cholestasis
• Necrosis and degeneration of other resident cells within the liver
2) In the absence of histological changes, using a weight-of-evidence approach, is there clinical
pathology evidence of hepatocyte damage characterized by a dose dependent and
biologically significant and consistent increase in at least two of the following liver
parameters?
• At least x 2 to x3 increase in ALT
• A biologically significant change in other biomarkers of hepatobiliary change (ALP, AST,
yGT, GLDH, etc.)
• A biologically significant change in another clinical pathology marker indicating liver
dysfunction (albumin, bilirubin, bile acids, coagulation factors, cholesterol, triglycerides,
etc.)
With regard to Step 1 above, histological evidence of structural change included necrosis in
females rats only (incidence of 12/70) receiving 200 mg/kg-day in the chronic study (note the
3-33
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Toxicological Review of PFHxA and Related Salts
highest dose in male rats was half the female dose, 100 mg/kg-dlfKlaunig et al.. 20151 and 2.5-5
times lower than the highest dose in the short term (NTP. 20181 or the subchronic studies
f Chengelis etal.. 2009b: Loveless etal.. 20091. There was one incidence of necrosis male rats only
[n = 1/10 vs. 0/10 in controls, not significant) from the short-term study fNTP. 20181. and no
findings of necrosis from either subchronic study fChengelis etal.. 2009b: Loveless etal.. 20091.
Histological findings did include increased incidence of hepatocellular hypertrophy from the short
term and both subchronic studies. Notably, hypertrophy findings persisted in both male and female
rats 90-day after recovery (Loveless etal.. 20091. Although some uncertainties remain, the necrosis
observed in the female rats in the chronic study (and at a highest dose in a short-term study)
support the adversity of the hepatic effects of PFHxA regarding Step 1.
Regarding Step 2 above, other liver parameter effects were observed after PFHxA exposure
and included increased peroxisomal beta oxidation in both subchronic studies fChengelis etal..
2009b: Loveless etal.. 20091 that persisted at 30 days recovery in both male and female rats
(Loveless etal.. 20091. The serum enzymes ALT was increased 2-3-fold in the short term and
subchronic studies. AST and ALP were also significantly increased, although the magnitude of the
response was <2-fold, at the same or lower doses than the observed increases in hepatocellular
hypertrophy. Other parameters characterized by a dose-dependent PFHxA-mediated effect
included decreased globulin, decreased total protein, and decreased bilirubin. While the collection
of findings was generally observed across both sexes, the magnitude of the change was greater in
males than females. This sex-specific difference is possibly explained by the increased clearance
rate in females compared with males. Although some uncertainties remain, the observed liver
parameter changes in the female and male rats in the subchronic studies (e.g., increased liver
enzymes, increased peroxisomal beta oxidation, decreased blood proteins) support the adversity of
the hepatic effects of PFHxA regarding Step 1.
Considering the Hall etal. f20121 criteria above, the observed increase in relative liver
weight and hepatocellular hypertrophy in rats exposed to PFHxA are interpreted as adverse, human
relevant, and potentially leading to increasingly severe outcomes such as necrosis.
Peroxisomal Beta Oxidation
Peroxisomal proliferation can be induced within the peroxisomes to perform beta oxidation
of lipids into acetyl CoA and hydrogen peroxide (H2O2) fReddv. 20041. Hydrogen peroxide is a
reactive metabolite and can cause oxidative damage to the surrounding tissue. Two subchronic
studies measured PFHxA induction of peroxisomal beta oxidation activity in male and female rats
(Chengelis etal.. 2009b: Loveless etal.. 20091 (see Figure 3-6) and both were considered medium or
high confidence for this outcome. Chengelis etal. (2009b) reported an increase (p < 0.05,1.37-fold)
in males treated with 200 mg/kg-day at 13 weeks. Loveless etal. (2009) found increased
peroxisomal beta oxidation activity in both sexes gavaged with 500 mg/kg-day for 10 and 90 days
(males, 3.1- and 4.36-fold, respectively; females, 1.45- and 2.67-fold, respectively). Notably,
increased activity persisted after the 30-day recovery and male rats were more sensitive than
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Toxicological Review of PFHxA and Related Salts
females, with males in the 100 mg/kg-day group also showing increased peroxisomal beta
oxidation (Loveless etal.. 20091.
F.ndpoint
Study
Experiment
Animal Description
Observation Time
Peroxisomal Beta Oxidation
Perosixomal Beta Oxidation
Loveless, 2009, 2850369
90-Day Oral
Rat, Crl:CD(SD) (cf)
Day 10
«• • A
Rat, Crl:CD(SD) (9)
Day 10
» . A
Rat. Crl:CD(SD) 250 mg/kg-day PFHxA (not measured in females)
were observed.
The hepatic effects of PFHxA exposure observed in the rat studies discussed above were
also evaluated in pathogen-free ICR mice receiving 0, 50, or 200 mg/kg PFHxA by oral gavage daily
for 60 days (Tiang etal.. 20211. The observed hepatotoxicity in ICR mice was similar to the SD rats
and included histopathological findings of hepatocellular hypertrophy, inflammatory cell
infiltration, and degeneration. These findings were coherent with results from whole liver RNA-seq
and proteomics analysis that identified pathways enriched with differentially regulated genes and
proteins involved in PPAR signaling pathways fliang etal.. 20211.
The available evidence also included evidence for mechanistic information supporting
biologically plausible pathways (including those mediated by PPARa activation) leading to the
observed PFHxA-mediated hepatoxicity in rodents and considered relevant in humans. For
example, PFHxA activation of both rodent and human PPARa in HepG2 cells at concentrations
between 1-30 |jM; 12.2 |jM PFHxA Buhrke etal. (20131. The activation of human PPARa by short
and long chain PFAS was also observed by Behr etal. (20201 in human HepG2 cells. In another
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Toxicological Review of PFHxA and Related Salts
study, Wolf etal. (20081 examined, PPARa activation by PFHxA in COS1 cells transfected with
reporter gene constructs containing either the mouse or human PPARa ligand binding domain
fused to a Gal4 DNA binding domain under control of an SV-40 promoter in a luciferase reporter
plasmid. These assays indicated that both mouse and human PPARa are activated by PFHxA in a
treatment-related manner with PFHxA being a more potent activator of the human (lowest-
observed-effect concentration, LOEC = 10 |j.M) compared to the mouse (LOEC = 20 |j.M) receptor
(Wolf etal.. 20081. While the transactivation studies of Wolf etal. (20081 indicated PFHxA
activation of both the mouse and human PPARa, significant effects were reported only for treated
vs. control within a species. In sharp contrast to all other studies Buhrke etal. (20131. Wolf etal.
f20081. Wolf etal. f20081. and liang etal. f20211. Biork and Wallace f20091 reported PFAS
activation of PPARa target gene expression in rat, but not human immortalized and primary
hepatocytes exposed. However, the outcome was considered low confidence due to notable concern
that variables were unaccounted or uncontrolled for (i.e., no positive control, no baseline
characterization of gene expression changes or PPAR activity).
Additional in vitro evidence for PFHxA effects in human cell lines (including HepG2 and
HepaRG cells) is available from EPA's CompTox Chemicals Dashboard (U.S. EPA. 2018a).
Transactivation assays in immortalized human HepG2 cells indicated PFHxA treatment effects that
included activation of the transcription factors PPARa and hypoxia inducible factor 1 subunit alpha
(HIFla, a transcriptional regulator of genes involved in the hypoxia response). Similarly, gene
expression assays in human-derived HepaRG cells identified the induction of 16 genes including
several cytochrome P450 family members, transporters, kinases, and oxidase/oxidoreductase
related activities that are primarily involved in oxidation/reduction and/or lipid metabolism (see
Table 3-10), consistent with evidence of short and long chain PFAS (including PFHxA) activation of
both human and rodent PPAR-dependent and independent pathways. Altogether, the mostly
consistent activation across species of PPARa by PFHxA provides further support for the human
relevance of the PFHxA-induced liver changes observed in rodents.
Table 3-10. Gene targets identified from EPA CompTox Chemicals Dashboard
after PFHxA treatment in human liver cell lines3
Gene Symbol
Gene Name
AC50b
LOGAC50
BMADC
ABCG2*
ATP-binding cassette, sub-family G (WHITE), member 2
(Junior blood group)
9.49
0.977
0.201
ACOX1*
acyl-CoA oxidase 1, palmitoyl
9.47
0.976
0.135
ADK
adenosine kinase
2.88
0.459
0.166
CYP2B6*
cytochrome P450, family 2, subfamily B, polypeptide 6
19.1
1.28
0.251
CYP2C19
cytochrome P450, family 2, subfamily C, polypeptide 19
5.21
0.717
0.187
CYP2C8*
cytochrome P450, family 2, subfamily C, polypeptide 8
7.88
0.896
0.216
CYP2C9
cytochrome P450, family 2, subfamily C, polypeptide 9
6.53
0.815
0.314
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Toxicological Review of PFHxA and Related Salts
Gene Symbol
Gene Name
AC50b
LOGAC50
BMADC
CYP3A7
cytochrome P450, family 3, subfamily A, polypeptide 7
10.3
1.01
0.259
CYP4A11
cytochrome P450, family 4, subfamily A, polypeptide 11
45.3
1.66
0.213
CYP4A22
cytochrome P450, family 4, subfamily A, polypeptide 22
57.6
1.76
0.269
FABP1*
fatty acid binding protein 1, liver
22.3
1.35
0.161
FM03
flavin containing monooxygenase 3
13.3
1.12
0.145
HMGCS2*
3-hydroxy-3-methylglutaryl-CoA synthase 2 (mitochondrial)
4.13
0.616
0.175
PDK4*
pyruvate dehydrogenase kinase, isozyme 4
20.9
1.32
0.161
SLCOIBI
solute carrier organic anion transporter family, member 1B1
9.82
0.992
0.181
UGT1A1
UDP glucuronosyltransferase 1 family, polypeptide Al
15.1
1.18
0.221
* PPARa target gene (http://www.ppargene.org/index.php).
aAfter filtering out results flagged in the Dashboard as uncertain (e.g., high degree of variability) or occurring at
high concentrations associated with cytotoxicity (U.S. EPA, 2018c; Filer et al., 2016; Filer, 2015), 19 assay targets
remained from human liver cell-based assays (at up to 200 nM PFHxA).
bAC50 - active concentration (nM) that elicited half maximal response.
CBMAD - baseline median absolute deviation.
The data discussed above suggest similar PPARa activation occurs in both rodents and
humans (at least in vitro). Potential pathways such as PPARa and CAR activation can contribute to
some of the hepatic changes caused by PFHxA exposure, including hypertrophy. Studies of the
prototypical PPARa agonist, WY-14643, indicate an increased sensitivity of rodents as compared to
humans; however, the PFHxA-specific data do not demonstrate such clear differences with this
structurally different compound.
Overall, although the PFHxA-specific data informing possible biological pathways leading to
the observed hepatic effects are sparse and uncertainties remain, collectively the available in vivo
and in vitro evidence for PFHxA support the potential for the responses observed in rodents to be
relevant to humans.
Evidence from Other PFAS
Although no direct in vivo evidence is available for PFHxA effects in PPARa null rodent
models, evidence from other PFAS, as well as PFAS exposures in PPARa null and humanized mouse
models provide evidence that PPARa independent and dependent pathways from other PFAS are
consistent for other short-chain PFAS (e.g., PFBA) (U.S. EPA. 2022b). and possibly long-chain PFAS
as well (e.g., PFNA, PFOA, PFOS) (U.S. EPA. 2023a. b), supporting the plausibility that these PFAS-
mediated responses also apply to PFHxA. Specifically. Specifically, Foreman etal. (20091 also
observed increased liver weight, hepatic lipid accumulation, ALT increases >2-fold, and
pathologically similar (severity and incidence) hepatocellular hypertrophy in male SV129 wild-type
SV and humanized PPARa mice exposed to PFBA. Although there is no evidence specifically
challenging the role of PPARa in PFHxA-mediated hepatotoxicity, based on PFHxA structural
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Toxicological Review of PFHxA and Related Salts
similarity with other PFAS, most notably PFBA, it is reasonable to infer that PFHxA exposure in
genetic mouse model systems would elicit similar effects.
Evidence Integration
The human evidence base is limited to a single medium confidence study reporting null
associations between serum biomarker levels and PFHxA exposure. Based on these data, there is
indeterminate human evidence of hepatic effects.
The hepatic findings in rodents exposed to PFHxA included increased relative liver weight
observed with increased hepatocellular hypertrophy at doses as low as 100 mg/kg-day (Loveless et
al.. 20091 and 200 mg/kg-day fChengelis etal.. 2009bl in male rats that persisted after 30- and 90-
day recovery. Corroborative evidence for adverse hepatotoxicity included increased serum
enzymes, (e.g., ALT increased >2-fold) in the subchronic studies, although a dose-responsive
relationship was observed in the short term, but not the subchronic, studies. Serum enzyme
changes were not observed in the chronic study (Klaunig et al.. 20151. Hepatocellular necrosis was
observed in male rats in a high confidence short term study (NTP. 20181 at 1,000 mg/kg-day, low
confidence subchronic study (Chengelis etal.. 2009b) and in the high confidence chronic study
(female rats) f Klaunig etal.. 20151 at 200 mg/kg-day. Other clinical findings altered by PFHxA
exposure included decreased bilirubin and decreased total protein mainly driven by decreases in
globulins (see Clinical Chemistry section above). These findings (i.e., increased liver weight with
concurrent hepatocellular hypertrophy, increases in ALT, and decreased protein levels) were
considered adverse as they might lead to the necrosis observed in females at 200 mg/kg-day in the
chronic study. In general, the pattern of findings suggests a generally increased sensitivity in males
as compared to females. Overall, there is robust animal evidence of hepatic effects. This judgment is
based on four studies in Sprague-Dawley rats that were generally rated high confidence on the
outcome-specific evaluations.
Although multiple biological pathways could lead to the histopathological findings
mentioned above, the PFHxA database for molecular evidence was predominantly limited to PPARa
pathways and included in vitro assays measuring PFHxA induction of PPARa activity (Wolf etal..
2014: Wolf etal.. 20081. peroxisomal beta oxidation activity (NTP. 2018: Chengelis etal.. 2009b:
Loveless etal.. 20091. changes in gene expression for CAR and PPARa cytochrome P450 gene
expression fNTP. 20181. and in vivo PPARa knockout and humanized genetic mouse models
exposed to PFAS structurally similar to PFHxA fDas etal.. 2017: Rosen etal.. 2017: Foreman etal..
20091. Wolf etal. f20081 and Wolf etal. f20141 found evidence that PFHxA activated the
human PPARa receptor concentrations that in rodent. Dose-responsive increases in peroxisomal
beta oxidation activity were observed in two subchronic studies (Chengelis etal.. 2009b: Loveless
etal.. 20091 at a dose as low as 100 mg/kg-day and this effect persisted after the 30-day recovery
(Loveless etal.. 20091. Evidence for pathways other than PPARa and CAR were available from
genetic PPARa knockout mouse studies evaluating the effects of PFAS exposure fDas etal.. 2017:
Rosen etal.. 2017: Foreman et al.. 20091 that found similar levels of increased liver weight and
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Toxicological Review of PFHxA and Related Salts
incidence of hepatocellular hypertrophy when comparing between PPARa knockout, humanized,
and wild-type mouse models, suggesting PPARa dependent and independent pathways are
activated by other PFAS, most notably PFBA fU.S. EPA. 2022bl. a short-chain PFAS like PFHxA.
Further supplemental mechanistic evidence indicated that human PPARa and related gene
expression is activated by PFHxA treatment in vitro. Thus, taken together, the findings observed in
rodent models are interpreted as relevant to humans.
Overall, the currently available evidence indicates that PFHxA likely causes hepatic effects
in humans given sufficient exposure conditions (see Table 3-ll)..5This conclusion is based on
studies of animals showing increased liver weight, hepatocellular hypertrophy, increased serum
enzymes (including >2-fold ALT) and decreased serum globulins generally occurring at
>100 mg/kg-day within the evidence base of four primarily high confidence studies of short-term,
subchronic, and chronic PFHxA exposure in (primarily male) rats. The findings in rats were
determined to be adverse and relevant to humans, with the likely involvement of both PPARa-
dependent and -independent pathways and consistent with hepatic effects identified for other PFAS
(U.S. Environmental Protection Agency: (U.S. EPA. 2022b: ATSDR. 2021: U.S. EPA. 2021a. 2018b.
2016a. b)) (summarized in Section 4, see Table 4-2).
5 The "sufficient exposure conditions" are more fully evaluated and defined for the identified health effects through
dose-response analysis in Section 5.
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Toxicological Review of PFHxA and Related Salts
Table 3-11. Evidence profile table for hepatic effects
Evidence stream summary and interpretation
Evidence integration summary
judgment
Evidence from studies of exposed humans
Studies and
confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key
findings
Evidence stream
judgment
®©o
Evidence indicates (likely)
Serum
Biomarkers
2 low confidence
studies
• No factors noted
• Low confidence
studies (low
sensitivity)
• No association
of PFHxA with
serum
biomarkers
ooo
Indeterminate
Primary basis:
Four generally high confidence
studies in rats ranging from short-
term to chronic exposure,
Evidence from animal studies
generally in males at >100 mg/kg-
d PFHxA
Human relevance:
Given the induction of human
PPARa by PFHxA, as well as
support for involvement of both
PPARa-dependent and
independent pathways for PFHxA
and related PFAS, effects in rats
are considered relevant to humans
Cross-stream coherence:
N/A (human evidence
indeterminate)
Studies and
confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key
findings
Evidence stream
judgment
Organ Weight
3 hiqh
confidence:
28-d
90-d (2 studies)
• Consistent increases, all
studies, and sexes
• Dose-response observed
in all studies
• Coherence with
histopathology
• Large magnitude of
effect, up to 63%
• High confidence studies
• No factors noted
• Increased liver
weight at
>200 mg/kg-d;
stronger in
males
®©o
Moderate
Findings considered
adverse based on
observed liver parameter
changes in the female
and male rats in the
subchronic studies (e.g.,
increased liver enzymes,
increased peroxisomal
beta oxidation, decreased
HistoDathologv
3 hiqh confidence
studies in adult
rats:
28-d
90-d
2-yr
• Consistent cellular
hypertrophy across
studies and sexes
• Coherence with liver
weight
• No factors noted
• Cellular
hypertrophy at
>100 mg/kg-d;
stronger in
males
• Necrosis in
females at
blood proteins) at similar
doses, but lower than
doses at which necrosis
was observed
(200 mg/kg-d) consistent
with the progressive
change to necrosis
support the adversity of
Susceptible populations and
lifestages:
No evidence to inform
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Toxicological Review of PFHxA and Related Salts
Evidence stream summary and interpretation
Evidence integration summary
judgment
1 low confidence
study in adult
rats:
90-d
• Dose-response for
hypertrophy
• Concerning severity of
effect—necrosis (with
short term, subchronic,
and chronic exposure)
• High confidence studies
200 mg/kg-d
the hepatic effects with
potential for progression
for more severe
phenotypes, including
necrosis with longer-term
exposure.
Serum
Biomarkers of
Hepatic Iniurv
4 hiqh confidence
studies in adult
rats:
28-d
90-d (2 studies)
2-yr
• Consistent increases in
ALT, AST, and ALP, and
decreases in bilirubin,
across studies
• Magnitude of effect,
>2-fold ALT
• Dose-response for total
protein
• Coherence of ALP and
bilirubin
• High confidence studies
• No factors noted
• Increased ALT,
AST, ALP, and
bile salts/acids
at >20, >100,
>200, and
500 mg/kg-d,
respectively;
stronger in
males
• Decreased total
protein at
>100 mg/kg-d;
stronger in
males
Mechanistic evidence and supplemental information
Biological events
or pathways
Primary evidence evaluated
Key findings, interpretation, and limitations
Evidence stream
summary
Molecular
Initiating
Events—PPARa
Key findings and interpretation:
• Consistent studies demonstrating In vitro induction of human and
rodent PPARa activity in trans-activation experiments. Reporter gene
activation at lower effective concentrations in human versus mouse
constructs.
• Biologically plausible
support for PPARa-
dependent and
independent pathways
contributing to hepatic
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Toxicological Review of PFHxA and Related Salts
Evidence stream summary and interpretation
Evidence integration summary
judgment
• Induction of PPARa in association with hepatic effects in a short-term
oral exposure study.
• PFHxA binding to and activation of human PPARa.
• Activation of PPARa target gene expression in rodents.
• Findings coherent with animal findings of increased peroxisomal beta
oxidation activity.
effects of PFHxA that
are relevant in
humans.
Molecular
Initiating
Events—Other
Pathways
Key findings and interpretation:
• Indirect evidence supporting activation of PPARa-independent
pathways contributing to hepatic effects like those observed for PFHxA
in PPARa knockout and humanized mice after short-term oral
exposure to PFAS other than PFHxA, including the related PFAS, PFBA.
• -omics evidence supporting differential regulation of genes regulated
by PPARa dependent and independent pathways
Limitations: Small evidence base with no experiments specifically
challenging the role of PPARa in PFHxA-induced hepatic injury.
Organ Level
Effects
Key findings and interpretation:
• Increased peroxisomal beta oxidation activity that persisted 30 d post-
exposure (likely not a transient, adaptive response) in short-term and
subchronic rat studies of oral PFHxA exposure.
• Indirect evidence of fatty liver, hepatocellular hypertrophy, and
hepatomegaly in PPARa KO mice after short-term oral exposure to
PFAS other than PFHxA.
Limitations: Small evidence base and the most compelling in vivo
evidence for PPARa-independent pathways with hepatic effects did not
specifically test PFHxA.
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Toxicological Review of PFHxA and Related Salts
3.2.2. Developmental Effects
Human
A single study evaluated associations between PFHxA and developmental effects,
specifically birth size and postnatal growth, in a study in China with follow-up to 5 months after
birth (Tin etal.. 20201. This study was considered uninformative and not considered further due to
lack of consideration of potential confounding, including parity, socioeconomic status and maternal
age.
Animal
Three studies described in two publications examined developmental outcomes, including
offspring viability, body weight, eye opening, and malformations and variations. Rats were exposed
to PFHxA sodium salt during gestation (gestation day [GD] 6-20; developmental study) or
continuously exposed throughout gestation and lactation (reproductive study) (Loveless etal..
2009). Mice were exposed to PFHxA ammonium salt from GD 6-18 (Iwai and Hoberman. 2014).
These studies were rated high confidence. The results from outcome-specific, confidence
evaluations for all individual reproductive outcomes are presented in Table 3-12, and details are
available by clicking the HAWC link. Effects on male and female reproductive system development
following developmental exposure are discussed in the male and female reproductive effects
sections, respectively (see Sections 3.2.6 and 3.2.7).
Table 3-12. Study design characteristics and outcome-specific study
Study
Species,
strain (sex)
Exposure design
Exposure route and
dose
Offspring
viability
Offspring body
weight
Eye Opening
Malformations
and Variations
Loveless et
Rat,
Reproductive study: Po females
Gavage3
++
++
+ +
+ +
al. (2009)
Crl:CD(SD)
dosed 70 d prior to cohabitation,
Female: 0, 20,100,
Sprague-
through gestation and lactation
500 mg/kg-d
Dawley
(126 d); Po males dosed for 110 d
(male and
female)
Developmental study: GD 6-20
Iwai and
Hoberman
(2014)°
Mouse,
Crl:CDl(ICR)
(male and
female)
Developmental: GD 6-18
Gavageb
Phase 1: 0,100,
350, 500 mg/kg-d
Phase 2: 0, 7, 35,
175 mg/kg-d
++
++
+ +
NM
++ Outcome rating of high confidence; NM, outcome not measured.
a bStudy evaluation for animal toxicological developmental endpoints reported from studies with rats receiving
PFHxA sodium salt3 or PFHxA ammonium saltb by gavage. Study evaluation details for all outcomes are available
by clicking the HAWC link.
cPhase 1 was a range finding study used to determine the appropriate dose range for identification of a NOAEL in
Phase 2.
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Toxicological Review of PFHxA and Related Salts
Offspring Mortality
Potential effects of PFHxA exposure on offspring viability were evaluated in a
developmental study flwai and Hoberman. 20141 and a one-generation reproductive study
fLoveless etal.. 20091. Mice exposed to PFHxA ammonium salts during gestation (GD 6-18) showed
a dose-dependent increase in the incidence of offspring mortalities occurring both pre- and
postnatally flwai and Hoberman. 20141. Most deaths occurred between postnatal day (PND) 0-7,
with a statistically significant increase observed in the 350 and 500 mg/kg-day dose groups on PND
1-4. These effects were observed across two experimental cohorts with different but overlapping
dose ranges (cohort 1: 0,100, 350, and 500 mg/kg-day; cohort 2: 0, 7, 35, and 175 mg/kg-day).
Early postnatal losses are reflected in treatment-related effects on several measures of offspring
viability for the 500 mg/kg-day dose group. Specifically, statistically significant changes were
observed in the following related outcomes: decreased viability index for PND 0-4 and PND 0-7,
fewer surviving pups per litter on PND 20, and increased incidence of total litter loss between PND
0-3 (5 of 17 for the 500 mg/kg-day group compared to 1 of 19 dams for concurrent controls). A
dose-dependent increase in the number of stillbirths, a measure of prenatal mortality, was also
reported across the two phases of the experiment (incidence of 3/241, 5/245, and 19/177 for the
175, 350, and 500 mg/kg-d dose groups, respectively). Results are summarized in Figure 3-7 and
Table 3-13.
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Toxicological Review ofPFHxA and Related Salts
Endpoint
Offspring Survival
Study
Iwai, 2014, 2821611
Experiment
1-generation reproductive (GD 6-18)
Animal Description
F1 Mouse. CD-1 ( ":)
Iwai, 2014,2821611 1-generation reproductive (GD 6-18) F1 Mouse. CD-1 ( '.)
Loveless, 2009, 2850369
reproductive (56 d)
F1 Rat, Cri:CD(SD)(_\)
Observation Time
PND0
PND0
PND 4
PND 4
PND 7
PND 7
PND 14
PND 14
PND 20
PND 20
PND 0-4
PND 0-4
PND 0-7
PND 0-7
PND 4-20
PND 4-20
PND 0
PND 0-4
PND 4-21
PFHxA Developmental Effects: Offspring Mortality
No. of Pups, Stillborn Iwai, 2014,2821611
Viability. Litters with Stillborn Pups Iwai, 2014. 2821611
Pups Found Dead/Presumed Cannibalized Iwai, 2014. 2821611
1-generation reproductive (GD 6-18) F1 Mouse. CD-1 ( ' _)
1-generation reproductive (GD 6-18) F1 Mouse. CD-1 (.-'£)
1-generation reproductive (GD 6-18) F1 Mouse. CD-1 (;' ¦')
Total Litter Loss
Iwai, 2014, 2821611 1-generation reproductive (GD 6-18) P0 Mouse, CD-1 ( ')
PND 0
PND 0
PND0
PND 0
PND 0
PND 0
PND 1-4
PND 1-4
PND 5-7
PND 5-7
PND 8-14
PND 8-14
PND 15-20
PND 15-20
PND 0
PND 0
PND 0-3
PND 0-3
PND 4-20
PND 4-20
I No significant change A Significant increase V Significant decrease l
50 100 150 200 250 300
0 400 450 500 550
" ¦ • ¦' '
Figure 3-7. Developmental effects on offspring viability in mice exposed to
PFHxA ammonium salt (HAWC: PFHxA - Animal Toxicity Developmental
Effects linkl.
The Iwai and Hoberman (2014) study was conducted in two phases. Phase 1 was a range-finding study (100, 350,
or 500 mg/kg-d) used to determine the appropriate doses (7, 35,175 mg/kg-d) to identify a NOAEL in Phase 2,
Table 3-13. Incidence of perinatal mortality following PFHxA ammonium salt
exposure in a developmental oral toxicity study
Dose (mg/kg-d)
Study design and reference
0
(Cohort 1)
0
(Cohort 2)
i-»
Ln
m
o
o
*—1
lO
*—i
o
LD
CO
o
o
lO
Stillbirths, male and female (combined) mice (Iwai
and Hoberman,2014)
4
0
oa
0
0
3*
5a
iga*
Mortalities, PND 0, male and female (combined)
mice (Iwai and Hoberman, 2014)
0
0
0
0
0
4
3a
21a*
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Toxicological Review of PFHxA and Related Salts
Study design and reference
Dose (mg/kg-d)
0
(Cohort 1)
0
(Cohort 2)
r-.
CO
o
o
rH
r-.
rH
o
m
CO
o
o
m
Mortalities, PNDs 1-4, male and female
(combined) mice (Iwai and Hoberman, 2014)
2
r
3a
2
2a
oa
13a*
15a*
Mortalities, PNDs 5-7, male and female
(combined) mice (Iwai and Hoberman, 2014)
0a
i
0a
0
0b
3a
2a
0a
Mortalities, PNDs 8-14, male and female
(combined) mice (Iwai and Hoberman, 2014)
0
0
0
0
0a'b
0a
0a
0a
Mortalities, PNDs 15-20, male and female
(combined) mice (Iwai and Hoberman, 2014)
0
0
0
0
2b
1
0
0
Total pups delivered, male and female (combined)
mice (Iwai and Hoberman, 2014)
221
249
211
232
250
241
245
177
Cumulative perinatal mortality/total pups
delivered, male and female (combined) mice (Iwai
and Hoberman,2014)
6/
221
2/
249
3/
211
2/
232
4/
250
11/
241
23/
245
55/
177
The Iwai and Hoberman (2014) study was conducted in two phases. Phase 1 was a range-finding study (100, 350,
or 500 mg/kg-d) used to determine the appropriate doses (7, 35,175 mg/kg-d) to identify a NOAEL in Phase 2.
* Indicates instances where statistical significance (p < 0.05) compared to controls was reported by study authors
aExcludes animals that were missing and presumed cannibalized or where vital status at birth was uncertain due to
maternal cannibalization or autolysis.
bExcludes offspring mortalities that occurred following death of the dam; deaths assumed not treatment related.
Offspring Body Weight
Offspring body weights were available from two developmental studies flwai and
Hoberman. 2014: Loveless etal.. 20091 and a one-generation reproductive study (Loveless etal..
20091. In mice, offspring body weights were statistically significantly decreased at PND 0-7 in
animals exposed gestationally (GD 6-18) to >100 mg/kg-day PFHxA ammonium salt These effects
were observed across two experimental cohorts with different but overlapping dose ranges (cohort
1 = 0,100, 350, and 500 mg/kg-day; cohort 1 = 0, 7, 35, and 175 mg/kg-day). Although there is
some variability in the magnitude of the dose response, consistent body weight deficits of >5%
relative to control, a level of change that may be biologically significant during early development
(U.S. EPA. 2012a. 19911. generally persisted to the end of lactation (Table 3-14). Some of this
variability may be explained by differences in control body weights across the two cohorts or
higher mortality of low body weight pups in the high dose groups. After weaning, some body weight
deficits persisted, with females with the 350 mg/kg-day dose group showing a statistically
significant reduction through the end of the experiment (PND 41) flwai and Hoberman. 20141.
Similar results were reported in two experiments with rats exposed to PFHxA sodium salt
(Loveless etal.. 20091. In the developmental study, fetal body weights (GD 21) of animals exposed
gestationally (GD 6-20) to 500 mg/kg-day were decreased by 9% relative to controls, although this
3-46
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Toxicological Review ofPFHxA and Related Salts
change was not statistically significant, but no effects were observed at the lower doses. In the
one-generation reproductive study, a dose-related decrease (4%, 11%, and 18% decrease relative
to controls for 20,100, and 500 mg/kg-day, respectively, reaching statistical significance atthe
highest dose) was found in pup body weights across all dose groups at PND 0, Similar to results in
the mouse study flwai and Hoberman. 20141. body weight deficits >5% relative to control were
observed through the end of lactation (PND 21) in the 100 and 500 mg/kg-d dose groups, but
resolved after weaning (Loveless et al.. 20091.
Neither study reported treatment-related effects on body weight change (i.e., gains or
losses) between weaning and the end of testing (PND 21-41 for mice; PND 21-60 for rats) flwai
and Hoberman. 2014: Loveless etal.. 20091. Results are presented in Figure 3-8 and Table 3-14.
Endpoint
Body Weight, Absolute
Study
Iwai. 2014. 2821611
Experiment
Animal Description Observation Time
PFHxA Developmental Effects: Offspring Body Weight
1-generation reproductive (GD 6-18) F1 Mouse, CD-1 (rr'V)
Loveless, 2009, 2850369
developmental (GD 6-20)
reproductive (56 d)
F1 Mouse. CD-1 (:*)
F1 Mouse, CD-1 (J)
F1 Rat, Crl:CD(SD) (: )
F1 Rat, Crl:CD(SD) ( )
F1 Rat, Crl:CD(SD) (_¦')
F1 Rat. Crl:CD(SD) ( )
PND 0
PND 4
PND 7
PND 14
PND 20
PND 21
PND 28
PND 35
PND 41
PND 21
PND 28
PND 35
PND 41
GD 21
PND 0
PND 4 (pre-cull)
PND 7
PND 14
PND 21
PND 28
PND 35
PND 39
PND 60
PND 28
PND 35
PND 39
PND 60
Body Weight Change Iwai. 2014. 2821611 1-generation reproductive (GD 6-18)
Loveless, 2009, 2850369
reproductive (56 d)
F1 Mouse, CD-1 (cf) PND 21-41
F1 Mouse, CD-1 (5) PND 21 -41
F1 Rat, Crl:CD(SD) (<*) PND 21 -60
F1 Rat,Crl:CD(SD)C) PND21-60
i No significant change
A Significant increase ~ Significant decrease |
50 100 150 200 250 300 350 400 450 500 550 600
mg/kg-day
Figure 3-8. Developmental effects on offspring body weight in mice exposed to
PFHxA ammonium salt and rats exposed to PFHxA sodium salt (HAWC: PFHxA
- Animal Toxicity Developmental Effects link).
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Toxicological Review of PFHxA and Related Salts
Table 3-14. Percent change relative to control in offspring body weight due to
PFHxA sodium or ammonium salt exposure in developmental oral toxicity
studies
Postnatal date (GD 6-18) and sex
(Iwai and Hoberman, 2014)
Dose (mg/kg-d)
r-.
o
fM
CO
o
o
*—1
r-.
*—1
o
m
CO
o
o
m
PND 0, male and female (combined) mice
0
0
-6*
-13*
-13*
-13*
PND 4, male and female (combined) mice
0
7
-7
_4*
-27*
-20*
PND 7, male and female (combined) mice
0
5
-7
0
*
00
1
1
-11
PND 14, male and female (combined) mice
-1
3
-8
0
-14
-8
PND 20, male and female (combined) mice
-2
6
-11
2
-20
-12
PND 21, male mice
3
4
*
LO
1
1
-1
*
00
1
1
-14
PND 28, male mice
2
3
-10
0
-10
-8
PND 35, male mice
1
1
-4
-1
-3
-5
PND 41, male mice
1
-1
-2
-3
-3
-4
PND 21, female mice
0
6
-14*
1
-17*
-8
PND 28, female mice
0
4
-1
*
ID
1
1
-7
PND 35, female mice
-1
2
-4
-1
*
O
1
1
-7*
PND 41, female mice
-3
-1
-4
-3
-8*
-4
Fetal bodv weight, developmental exposure (GD 6-20) (Loveless et al., 2009)
GD 21, male and female (combined) rats
-2
0
-9
Postnatal bodv weight, one-generation reproductive exposure (Loveless et al., 2009)
PND 0, male and female (combined) rats
-4
-11
*
00
1
1
PND 7, male and female (combined) rats
0
-6
-17*
PND 14, male and female (combined) rats
3
-6
-17*
PND 21, male and female (combined) rats
3
-5
*
00
1
1
PND 28, male rats
2
-1
-5
PND 35, male rats
1
-1
-3
PND 39, male rats
2
-1
-3
PND 60, male rats
2
-1
-3
PND 28, female rats
1
-5
-4
PND 35, female rats
1
-4
-1
PND 39, female rats
-1
-5
-3
PND 60, female rats
-1
-5
-3
* Indicates instances where statistical significance (p < 0.05) compared to controls was reported by study authors;
shaded cells represent doses not included in the individual studies.
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Toxicological Review of PFHxA and Related Salts
Eye Opening
Potential effects of PFHxA exposure on eye opening were evaluated in a developmental
study in mice flwai and Hoberman. 20141. On PND 14, Iwai and Hoberman f20141 reported a
statistically significant delay in eye opening, with less than 50% of pups in the 350 and 500 mg/kg-
day PFHxA ammonium salt exposure groups having reached this milestone compared to 85%
among vehicle controls (see Figure 3-9). Although pup body weight changes were not statistically
significantly at this timepoint, they were decrements of a magnitude considered to be potentially
biologically significant (8%-14%) and some developmental landmarks are correlated with
postnatal body weight gain fU.S. EPA. 2016cl. Delays in eye opening persisted in the 350 and
500 mg/kg-day dose groups on PND 15 but were not statistically significant. Eye opening in mice
typically occurs between PND 11 and PND 14, with full functionality a few days later fBrust etal..
20151. Delays in eye opening can have long-term impacts on vision by interfering with sensory
input during the critical window of visual cortex development fEspinosa and Strvker. 2012: Wiesel.
19821. The results are summarized in Figure 3-9 and Table 3-15.
Endpoirit Study Name Experiment Animal Description Observation Time
PFHxA Developmental Effects: Developmental Milestone
Eye Opening Iwai, 2014, 2821611 1-generation reproductive (GD 6-18) F1 Mouse, CD-1 PND 10
• • •
•
PND 11
~ • •
•
PND 12
• • •
•
PND 13
• • •
•
PND 14
• V V
PND 15
• • •
•
PND 16
« • •
PND 17
• • •
• No significant change A Significant increase V Significant decrease |
0 50 100 150 200 250 300 350 400 450 500 550 600
mg/kg-day
Figure 3-9. Developmental effects on eye opening (percent change relative to
control) in mice exposed to PFHxA ammonium salt (HAWC: PFHxA - Animal
Toxicity Developmental Eve Effects link!.
Table 3-15. Percent change relative to control in eye opening due to PFHxA
ammonium salt exposure in a developmental oral toxicity study
Study design and reference
Dose (mg/kg-d)
r-.
CO
o
o
*—1
r-.
*—1
o
m
CO
o
o
m
PND 13, male and female (combined) mice (Iwai and Hoberman, 2014)
-6
34
-56
-21
-58
-55
PND 14, male and female (combined) mice (Iwai and Hoberman, 2014)
2
4
-17
-8
-49*
-39*
PND 15, male and female (combined) mice (Iwai and Hoberman, 2014)
0
0
-10
-5
-23
-25
PND 16, male and female (combined) mice (Iwai and Hoberman, 2014)
0
0
-1
0
-9
-1
* Indicates instances where statistical significance (p < 0.05) compared to controls was reported by study authors.
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Toxicological Review of PFHxA and Related Salts
Malformations and Variations
Potential effects of PFHxA exposure on fetal malformations and variations were evaluated
in a single developmental study fLoveless etal.. 20091. No treatment-related effects were found on
fetal malformations or variations in rats following gestational (GD 6-20) exposure to up to
500 mg/kg-day PFHxA sodium salt
Evidence Integration
The only available human study examining potential developmental effects was considered
uninformative; therefore, there is indeterminate human evidence of developmental effects.
In animals, three high confidence studies reported in two publications examined
developmental effects following maternal perinatal exposure to PFHxA salts flwai and Hoberman.
2014: Loveless etal.. 20091. Treatment-related effects, including decreased offspring body weight,
increased mortality, and delayed eye opening, were observed in mice following exposure to PFHxA
ammonium salt at doses as low as 100 mg/kg-day flwai and Hoberman. 20141. Reductions in
offspring body weight were also found in the one-generation reproductive and developmental
studies in rats, although effects were less pronounced than those observed in mice. Animals in the
reproductive cohort exposed throughout gestation and lactation showed body weight reductions
that may be biologically significant (>5%) at exposure to >100 mg/kg-day and statistically
significant at 500 mg/kg-day that persisted to PND 21, whereas the developmental cohort was
reduced (9%) only at the high dose (500 mg/kg-day).
In general, effects on development were greatest in magnitude from PND 0 to PND 7,
suggesting that the early postnatal period might be a sensitive window for developmental effects
associated with PFHxA exposure. Although the evidence base is small, the data are strengthened by
coherent evidence across outcomes, consistency of effects on body weight across two
species/studies, and the severity of some of the outcomes (e.g., increased offspring mortality). In
addition, a similar pattern of effects on development (i.e., offspring body weight reductions and
delays in developmental milestones) has been observed with other PFAS, including PFBS (U.S. EPA.
2021b) and PFBA (U.S. EPA. 2022a). providing additional support for these specific findings.
Importantly, reduced growth during early life is associated with increased risk of developing
adverse health effects in humans, including cardiovascular disease, type 2 diabetes, and early
mortality in later life fThompson and Regnault. 2011: Kaiantie etal.. 2005: Barker. 20041. Similarly,
delays in eye opening can have lasting adverse impacts preventing visual input during a critical
period of development for the visual cortex (Espinosa and Strvker. 2012: Wiesel. 1982).
The potential for maternal effects to act as a driver for the observed developmental effects
was considered. In Iwai and Hoberman (2014). there was no effect on maternal body weight in
either cohort. Some deaths were observed among the cohort 1 dams, but these did not appear to be
dose related as there was aa similar incidence observed across controls and exposed groups.
Reductions in maternal body weight were noted in the developmental study by Loveless et al.
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Toxicological Review of PFHxA and Related Salts
(20091. Dams exposed to 500 mg/kg-day from GD 6-20 showed a slight but statistically significant
5% decrease in total net body weight (i.e., terminal body weight minus the gravid uterine weight)
and body weight gain on GD 21 fLoveless etal.. 20091. In the one-generation reproductive study,
Loveless etal. f20091 reported a statistically significant reduction in maternal weight gain in the
highest dose group (500 mg/kg-day); however, this effect was limited to early gestation (GD 0-7).
Importantly, there was no effect on maternal body weight gain over the entire gestational window
(GD 0-21), nor was there any observed effects on total or net maternal body weights. Thus, the
effects on offspring body weight in this study are not expected to be driven by maternal toxicity.
Given this interpretation of an effect on development and based on the multiple adverse changes in
pups, there is moderate animal evidence of developmental effects.
Overall, the currently available evidence indicates that PFHxA likely causes developmental
effects in humans given sufficient exposure conditions (see Table 3-16)..6 This judgment is based
primarily on several high confidence, gestational exposure experiments in mice (and supportive
findings in rats), with effects occurring after oral PFHxA exposure at >100 mg/kg-day. These
findings are interpreted as relevant to humans in the absence of evidence to the contrary. This
assumption is based on Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA. 19911.
6 The "sufficient exposure conditions" are more fully evaluated and defined for the identified health effects through
dose-response analysis in Section 5.
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Toxicological Review of PFHxA and Related Salts
Table 3-16. Evidence profile table for developmental effects
Evidence stream summary and interpretation
Evidence integration
summary judgment
Evidence from studies of exposed humans
®©o
Evidence indicates
(likely)
Primary basis:
Three high confidence
studies in rats and mice
including gestational (rats
and mice) and continuous
one-generational
reproductive (rats)
exposures, generally
observing effects at
>100 mg/kg-d PFHxA
ammonium or sodium
salt.
Human relevance:
Without evidence to the
contrary, effects in rats
and mice are considered
relevant to humans.
Cross stream coherence:
N/A (human evidence
indeterminate).
• Susceptible
populations and
lifestages:
Studies and confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key
findings
Evidence stream
judgment
• There were no informative human studies available from the PFHxA evidence base.
ooo
Indeterminate
Evidence from animal studies
Studies and confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key
findings
Evidence stream
judgment
Offspring Mortalitv
2 hiqh confidence studies
in rats and mice:
• GD 6-18 (mice)
• 1-generation
reproductive (rats)
• High confidence
studies
• Concerning severity of
effect- increased
mortality
• Unexplained
inconsistency across
species
• Increased perinatal
mortality at
>350 mg/kg-d in mice
®©o
Moderate
Developmental
effects observed in
multiple high
confidence studies
conducted in two
species exposed to
different PFHxA salts
under different
exposure scenarios.
Effects on body
weight were
observed at doses
that were not
associated with
offspring mortality or
maternal toxicity.
Bodv Weight
3 high confidence studies
in rats and mice:
• GD 6-18 (mice)
• GD 6-20 (rats)
• 1-generation
reproductive (rats)
• High confidence
studies
• Consistency across
studies and species
• Dose response
observed in mouse
study
• No factors noted
• Postnatal body
weight decreased at
>100 mg/kg-d in rats
and mice
• Fetal body weight
decreased at
500 mg/kg-d in rats
Eve Opening
1 hiqh confidence study
in mice:
• High confidence study
• No factors noted
• Eye opening was
delayed in mice
prenatally exposed to
3-52
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Toxicological Review of PFHxA and Related Salts
Evidence stream summary and interpretation
Evidence integration
summary judgment
• GD 6-18
PFHxA ammonium
salt at >350 mg/kg-d
• The available evidence
indicates that
development may be a
susceptible lifestage
for exposure to PFHxA.
Malformations and
variations
1 high confidence study
in rats:
• GD 6-20
• High confidence study.
• No factors noted.
• No fetal
malformations or
variations observed at
<500 mg/kg-d
Mechanistic evidence and supplemental information
Biological events or
pathways
Summary of key findings, limitations, and interpretation
Evidence stream
summary
• There were no informative mechanistic studies available from the PFHxA evidence base.
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Toxicological Review of PFHxA and Related Salts
3.2.3. Renal Effects
Human
Three epidemiological studies investigated the relationship between PFHxA exposure and
effects on the renal system. Two cross-sectional studies of adults in Korea and older adults in China
(Zhang etal.. 2019: Seo etal.. 20181 were considered uninformative due to lack of consideration of
confounding, including age and sex. The remaining study was a cross-sectional study of primarily
government employees in China (Wang etal.. 20191 and was classified as low confidence primarily
due to significant concerns for reverse causality that could result if there is decreased elimination of
PFAS with reduced renal function (this is also a concern for the uninformative studies). They
observed a significant decrease in estimated glomerular filtration rate (eGFR) with higher serum
PFHxA levels ((3: -0.3 change in eGFR as mL/min/1.73 m2 per 1 ln-unit PFHxA [95% CI: -0.6,
-0.01]). No association was observed with chronic kidney disease. Due to the potential for reverse
causality and limited sensitivity due to narrow exposure contrast for PFHxA, there is substantial
uncertainty in the results of this study. A summary of the study evaluations is presented in
Figure 3-10, and additional details can be obtained by clicking the HAWC link.
Participant selection -
Exposure measurement -
Outcome ascertainment -
Confounding
Analysis -
Sensitivity -
Selective Reporting -
Overall confidence
J I L
Legend
Good (metric) or High confidence (overall)
Adequate (metric) or Medium confidence (overall)
Deficient (metric) or Low confidence (overall)
Critically deficient (metric) or Uninformative (overall)
Figure 3-10. Study evaluation for human epidemiological studies reporting
findings from PFHxA exposures (full details available by clicking HAWC link).
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Toxicological Review of PFHxA and Related Salts
Animal
Four short-term (28-day), subchronic, or chronic animal studies evaluated potential renal
effects of PFHxA or PFHxA sodium salt in rats. Most of the outcome-specific study ratings were
rated high confidence. For Chengelis etal. f2009bl. limitations were identified that influenced some
outcome-specific ratings. Specifically histopathology was rated low confidence because of issues
related to observational bias, endpoint sensitivity and specificity, and results presentation. Urinary
biomarker outcomes in the same study were rated medium confidence because of results
presentation (only qualitative results were reported). The results of the outcome-specific
confidence judgments are summarized in Table 3-17, and full study evaluation details can be
viewed by clicking the HAWC link.
Table 3-17. Renal endpoints for PFHxA and associated confidence scores from
repeated-dose animal toxicity studies
Author (year)
Species, strain (sex)
Exposure
design
Exposure route
Blood biomarkers
Urinary biomarkers
Histopathology
Organ weight
NTP(2018)
Rat, Harlan
Sprague-Dawley
(male and female)
Short term
(28 d)
Gavage3
Male and female: 0,
62.5, 125, 250, 500,
1,000 mg/kg-d
++
NM
+ +
+ +
Chengelis et al.
(2009b)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and female)
Subchronic
(90 d)
Gavage3
Male and female: 0,
10, 50, 200 mg/kg-d
NR
+
"
+ +
Loveless et al.
(2009)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and female)
Subchronic
(90 d)
Gavageb
Male and female: 0,
20,100, 500 mg/kg-d
++
++
+ +
+ +
Klaunig et al.
(2015)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and female)
2-yr cancer
bioassay
Gavage3
Male: 0, 2.5,15,
100 mg/kg-d
Female: 0, 5, 30,
200 mg/kg-d
++
++
+ +
NM
++ Outcome rating of high confidence; + outcome rating of medium confidence; - outcome rating of low
confidence; NR, outcome not reported; NM, outcome not measured.
a bStudy evaluation for animal toxicological renal endpoints reported from studies with male and female rats
receiving PFHxA3 or PFHxA sodium saltb by gavage. Study evaluation details for all outcomes are available by
clicking the HAWC link.
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Toxicological Review of PFHxA and Related Salts
Organ Weight
Increases in relative kidney weight were observed in both sexes in all three studies that
reported this endpoint fNTP. 2018: Chengelis et al.. 2009b: Loveless etal.. 20091. There were
statistically significant findings in male rat dose groups at PFHxA doses as low as 10 mg/kg-day in
the subchronic study f Chengelis etal.. 2009bl. Except for the results from Chengelis etal. f2009bl.
effects on relative kidney weights generally showed a weak or no dose-response gradient (see
Table 3-18). Craig etal. (20151 analyzed oral chemical exposure data extracted from subchronic
and chronic rat studies and found a statistically significant correlation between absolute, but not
relative, kidney weight, and kidney histopathology (even at doses where terminal body weights
were decreased) for most chemicals (32/35) examined indicating that absolute kidney weight is
more sensitive to the effects of chemical exposure than relative kidney weight. Absolute kidney
weight was increased, but only in one of the three studies reporting on this endpoint fNTP. 20181.
and only in female rats atthe highest dose group (1,000 mg/kg-day). The decrease in relative, but
not absolute, kidney weight could be explained by body weight gain decreases in the affected dose
groups: 1,000 mg/kg-day male dose group (13% decrease) (NTP. 20181. 50 and 200 mg/kg-day
male dose group [8%—11% decrease with similar trends in females fChengelis etal.. 2009bl], and
500 mg/kg-day male dose group (19% decrease, no change in females) fLoveless etal.. 20091.
Findings and full details of PFHxA effects on kidney weights can be viewed by clicking the HAWC
link.
Table 3-18. Percent increase in relative and absolute kidney weight due to
PFHxA exposure in short-term, subchronic, and chronic oral toxicity studies
Endpoint and reference
Dose (mg/kg-d)
o
*—1
o
fM
o
m
in
-------
Toxicological Review of PFHxA and Related Salts
Endpoint and reference
Dose (mg/kg-d)
o
*—1
o
o
lO
LO
rsi
ID
o
o
*—1
lO
fsj
*—1
O
o
fsj
o
LO
fsj
O
o
LO
OOO'I
28-d, male rat (NTP, 2018)
Absolute kidney weight
90-d, female rat (Chengelis et al., 2009b)
0
7
4
Absolute kidney weight
90-d, male rat (Chengelis et al., 2009b)
-1
-6
2
Absolute kidney weight
90-d, female rat (Loveless et al., 2009)
0
1
14
Absolute kidney weight
90-d, male rat (Loveless et al., 2009)
0
8
4
* Indicates instances where statistical significance (p < 0.05) compared to controls was reported by study authors;
shaded cells represent doses not included in the individual studies.
Histopathologv
Renal histopathological subchronic findings were qualitatively reported as null (Chengelis
etal.. 2009b: Loveless etal.. 20091. The short-term study findings included increases in minimal
chronic progressive nephropathy (CPN) that were significant (incidence 8/10) in the
1,000 mg/kg-day female dose group (see Figure 3-11) fNTP. 20181. consistent with increased
absolute kidney weight. Male renal histopathological findings from the chronic study were also null,
whereas findings for female rats included increased papillary necrosis (17/70 vs. 0/60 in controls)
and tubular degeneration (7/70 vs. 1 /60 in controls) in the highest dose group 200 mg/kg-day
(Klaunig etal.. 20151. Full details are available by clicking the HAWC link.
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Toxicological Review of PFHxA and Related Salts
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0 10 20 30 ±0 50 60 70 88 98 188
pUp^fc*** afaciid M 5ia*-i fcarrt Ca" &ar«! ia Cufii^j
Figure 3-11. Animal toxicological renal histopathology after PFHxA exposure
(full details available by clicking the HAWC link). Findings from the subchronic
studies were reported as null and not included in the above visualization.
Blood and Urinary Biomarkers
Blood biomarkers of renal function were inconsistent across study designs and exposure
durations. Both creatinine and blood urea nitrogen (BUN) are removed from the blood by the
kidneys and often used as indicators of kidney function. Creatinine is a waste product of creatine
metabolism (primarily in muscle), and BUN is a waste product of protein metabolism in the liver.
No observations of changes in urea nitrogen or creatinine were reported from Chengelis et al.
f2009bl and Klaunigetal. f20151. In the short-term study fNTP. 20181. BUN was unchanged in
both sexes in all dose groups. Changes in creatinine were inconsistent across sexes with null
findings in females, whereas a 13% decrease (p < 0.05) was found in the male 500 mg/kg-day dose
group fNTP. 20181. In a subchronic study, Loveless etal. f20091 reported a sex-specific increase in
BUN in the male 200 mg/kg-day dose group, whereas creatinine was decreased in both male and
female rats dosed with 200 mg/kg-day PFHxA sodium.
Urinalysis findings included total urine volume and other measures of urine concentrating
ability (e.g., specific gravity, urobiloginen). The urinalysis findings were more consistent than the
blood biomarkers, but still difficult to interpret as adverse or nonadverse. Urinalysis findings were
not measured in the short-term study fNTP. 20181 and were reported as null in a subchronic study
(Chengelis etal.. 2009b). Findings from the other subchronic study (Loveless etal.. 2009) identified
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Toxicological Review of PFHxA and Related Salts
changes in urine concentration reflected as decreased (50%-88%) urine protein combined with
increased (207%-300%) total urine volume in males and females in the 500 mg/kg-day dose
groups. Coherent with increased urine volume, osmolality was decreased (47%, p < 0.05), but only
in the male 500 mg/kg-day dose group fLoveless etal.. 20091. Urobilinogen and pH findings were
null in both male and females in the subchronic study fLoveless etal.. 20091. Findings from the
chronic study lacked consistency between sexes and did not exhibit a clear dose-response
relationship fKlaunig etal.. 20151. Specifically, total urine volume was increased in the female
200 mg/kg-day dose group and null in all male dose groups. Urine specific gravity was increased
(p < 0.05) in the male 15 mg/kg-day dose group and similar to controls in the 100 mg/kg-day dose
group, although specific gravity was increased (p < 0.05) in the female 200 mg/kg-day dose group.
Urine pH was low in males (compared to controls) only in the 100 mg/kg-day dose groups at 26
and 52 weeks fKlaunig etal.. 20151. Total urine volume findings were null in males, whereas an
increase was found in female rats from the 200 mg/kg-day dose group at 26 weeks that returned to
control levels at 52 weeks study duration fKlaunig etal.. 20151. Findings are summarized in
Figure 3-12, and full details are available in the HAWC link.
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Toxicological Review ofPFHxA and Related Salts
tiideant
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Figure 3-12. PFHxA Effects on blood and urine biomarkers of renal function
(full details available by clicking the HAWC link). The dashed blue line divides
blood (top] from urinary biomarkers. Note that blood urea nitrogen and creatinine
were described as null, but findings were not quantitatively reported.
Evidence Integration
The human evidence was limited to a single low confidence study reporting an inverse
association between PFHxA exposure and eGFR, although notable uncertainty in this result exists
due to potential for reverse causality. Based on these data, there is indeterminate human evidence
for renal effects.
The evidence base for renal effects in experimental animals was drawn from generally high
confidence studies including one short-term, two subchronic studies, and one chronic study.
Findings were, in general, null except for histopathology and some urinary biomarkers. Kidney
histopathology was the most significant finding in the short term and chronic studies. In the short-
term study, increased incidence of CPN was observed in female rats at the highest dose
(1,000 mg/kg-day PFHxA) and consistent with increased absolute kidney weight (females only).
Histopathological findings were null in both subchronic studies at doses up to 500 mg/kg-day. In
the chronic study, the incidence of papillary necrosis and tubular degeneration were increased in
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Toxicological Review of PFHxA and Related Salts
females compared to controls at the highest dose (200 mg/kg-day) that is twice the highest dose
received by male rats in the chronic study. Some changes occurred in urinary biomarkers
(decreased urine pH, increased urine volume) and potentially correlated changes were observed in
female histopathology in the chronic study. However, inconsistencies across studies at similar
observation times and doses were notable. Based on these results, there is slight animal evidence of
renal effects.
Overall, the currently available evidence is inadequate to assess whether PFHxA may
causes renal effects in humans (see Table 3-19).
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Toxicological Review of PFHxA and Related Salts
Table 3-19. Evidence profile table for renal effects
Evidence stream summary and interpretation
Evidence integration
summary judgment
Evidence from studies of exposed humans
OOO
Evidence inadequate
Primary basis:
Indeterminate
evidence in humans
and animal evidence is
largely null or lacking
biological coherence,
overall effects not
clearly adverse
Human relevance:
Without evidence to
the contrary, effects in
rats are considered
relevant to humans
Cross-stream
coherence:
N/A (human evidence
indeterminate)
Susceptible lifestages:
• No evidence to
inform
Studies and confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key findings
Evidence stream
judgment
Kidney Function
1 low confidence study
• No factors noted
• Low sensitivity
• Potential for
reverse causality
• Weak association of
PFHxA with decrease in
estimated eGFR
ooo
Indeterminate
Evidence from animal studies
Studies and confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key findings
Evidence stream
judgment
Organ Weight
3 hiqh confidence
studies in adult rats:
• 28-d
• 90-d (2 studies)
• Consistent increases,
all studies
• Weak Weak/no
dose response
• Increased relative kidney
weight at >10 mg/kg-d.
• Increase absolute kidney
weight at 1,000 mg/kg-d;
28-d study, females only
©oo
Slight
Findings of adversity
were considered
uncertain based on lack
of coherence between
effects (organ weight,
histopathology, blood,
and urine biomarkers)
inconsistency between
sexes, and lack of
coherence across
exposure designs
Histopathologv
3 hiqh confidence
studies in adult rats:
• 28-d
• 90-d
• 2-yr
1 low confidence study
in adult rats:
• 90-d
• Large magnitude of
effect, up to 24.3%
for papillary necrosis;
up to 80% for chronic
progressive
nephropathy
• No factors noted
• Increased incidence
papillary necrosis,
tubular degeneration,
chronic progressive
nephropathy at
>200 mg/kg-d; female
rats only, 28-d and
chronic studies
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Toxicological Review of PFHxA and Related Salts
Evidence stream summary and interpretation
Evidence integration
summary judgment
Blood Biomarkers
4 hiqh confidence
studies in adult rats:
• 28-d
• 90-d (2 studies)
• 2-yr
• No factors noted
• Lack of coherence
with other
histopathological
findings; chronic
study
• Lack of consistent
effect across
studies
• Increased BUN at
500 mg/kg-d; males only,
90-d study.
• Decreased creatinine at
>500 mg/kg-d), both
sexes, 1 subchronic study
• Decreased creatine at
1,000 mg/kg-d; males
only, 28-d study
• No treatment related
creatinine kinase
findings; both sexes, 28-d
study
Urinarv Biomarkers
3 hiqh confidence
studies in adult rats:
• 28-d
• 90-d
• 2-yr
1 medium confidence
study in adult rats:
• 90-d
• Coherence of urine
protein, urine
volume, urine
specific gravity, and
decreased osmolality
• Lack of coherence
with
histopathological
findings.
• Decreased osmolality
500 mg/kg-d; males only,
1 subchronic study
• Decreased urine protein
and increased urine
volume in at 500 kg/kg-d;
both sexes, 1 subchronic
study
• Increased total urine
volume at >200 mg/kg-d;
both sexes — 1
subchronic study,
females only, 12-yr study
• Decreased urine pH at
100 mg/kg-d; males only,
1 2-yr study
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Toxicological Review of PFHxA and Related Salts
Evidence stream summary and interpretation
Evidence integration
summary judgment
• No treatment related
findings for urobilinogen;
both sexes, 1 subchronic
study and 12-yr study
Mechanistic evidence and supplemental information
Biological events or
pathways
Primary evidence evaluated
Key findings, interpretation, and limitations
Evidence stream
summary
Molecular
Events—Oatplal
Key findings and interpretation:
Sex hormone-dependent regulation of Oatplal mRNA and protein level (see
Section 3.1.4).
Sex-specific Oatplal
expression appears to
lead to sex-specific PFHxA
elimination, leading to
longer PFHxA half-life in
male rats compared with
females, which may
explain sex-specific renal
findings.
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Toxicological Review of PFHxA and Related Salts
3.2.4. Hematopoietic Effects
Hematology is a subgroup of clinical pathological parameters concerned with morphology,
physiology, and pathology of blood and blood-forming tissues. Hematological parameters are
measured using blood tests such as complete blood counts (CBC) and a clinical chemistry panel. The
CBC is a blood test that measures (red blood cells, white blood cells, hemoglobin, hematocrit, and
platelets), whereas the clinical chemistry panel measures the proteins, enzymes, chemicals, and
waste products in the blood. Hematological measures, when evaluated together and with
information on other biomarkers, are informative diagnostic tests for blood-forming tissues
(i.e., bone marrow, spleen, liver) and organ function. In animals, blood tests are influenced by the
feeding protocol, blood collection site, animal age, and other factors.
Human Studies
One human study (Tiang etal.. 2014) evaluated blood counts in samples drawn from a
population of 141 pregnant women living in Tianjin, China. The study was considered
uninformative, however, due to lack of consideration of confounding, including age, which is
expected to substantially bias the results. Full study evaluation for Tiang etal. f20141 is available by
clicking the HAWC link.
Animal Studies
Several animal toxicological studies were available that assessed hematopoietic parameters
including a high confidence short-term study (NTP. 2018). high confidence (Chengelis etal.. 2009b)
and high confidence (Loveless etal.. 2009) subchronic studies, and a high confidence chronic study
fKlaunig etal.. 20151. Cell counts for the blood components associated with immune system
responses are summarized under in Immune Effects, see Section 3.2.8. Study findings are discussed
below and summarized in Table 3-20 (full details are available by clicking the HAWC link! and
summary details are available in PFHxA Tableau visualization.
Table 3-20. Hematopoietic endpoints for PFHxA and associated confidence
scores from repeated-dose animal toxicity studies
Author (year)
Species, strain (sex)
Exposure
design
Exposure route and dose range
Hematology
and
hemostasis
NTP(2018)
Rat, Harlan Sprague-Dawley
(male and female)
Short term
(28 d)
Gavage3
Male and female: 0, 62.5,125, 250,
500,1,000 mg/kg-d
+ +
Chengelis et al.
(2009b)
Rat, Crl:CD(SD) Sprague-
Dawley (male and female)
Subchronic
(90 d)
Gavage3
Male and female: 0,10, 50,
200 mg/kg-d
+ +
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Toxicological Review of PFHxA and Related Salts
Author (year)
Species, strain (sex)
Exposure
design
Exposure route and dose range
Hematology
and
hemostasis
Loveless et al.
(2009)
Rat, Crl:CD(SD) Sprague-
Dawley (male and female)
Subchronic
(90 d)
Gavageb
Male and female: 0, 20,100,
500 mg/kg-d
+ +
Klaunig et al.
(2015)
Rat, Crl:CD(SD) Sprague-
Dawley (male and female)
2-yr cancer
bioassay
Gavage3
Male: 0, 2.5,15,100 mg/kg-d
Female: 0, 5, 30, 200 mg/kg-d
+ +
++ Outcome rating of high confidence.
a bStudy evaluation for animal toxicological hematopoietic endpoints reported from studies with male and female
rats receiving PFHxA3 or PFHxA sodium saltb by gavage. Study evaluation details for all outcomes are available by
clicking the HAWC link.
Hematology
Several findings were consistent (i.e., decreased red blood cells [RBCs], hematocrit, and
hemoglobin) across studies and study designs that, when interpreted together, suggest PFHxA-
related adverse hematological effects such as anemia (see Figure 3-13). Indications were also
present that red blood cells were swollen and made up a larger proportion of the blood volume
(increased mean cell volume [MCV, a measure of the average red blood cell size]). These changes
were correlated with potential secondary erythrogenic responses to PFHxA exposure including
increased reticulocyte (immature RBCs) counts that were consistently increased >10% across
study designs and exposure durations, including the chronic study Klaunig etal. (2015) where the
highest dose levels were 2-5 times lower than those tested in the subchronic studies. Specifically, a
dose-responsive decrease occurred in red blood cells (see Table 3-21), hematocrit (see Table 3-22),
and hemoglobin (see Table 3-23) in the short-term study with decreases at doses ranging from
62.5 mg/kg-day in male rats to 250 mg/kg-day in female rats fNTP. 20181. These findings also were
observed in both subchronic studies in the highest dose groups [200 mg/kg-day in males only
(Chengelis etal.. 2009b) and 500 mg/kg-day in both sexes (Loveless etal.. 2009)]. Of note,
decreases in both hemoglobin and hematocrit were 1.5-2-fold greater in the subchronic study
fLoveless et al.. 20091 than in the short-term study fNTP. 20181 for both males and females at the
same dose (500 mg/kg-day).
Findings from the chronic study fKlaunig et al.. 20151 were generally null or observed at
dose levels >100 mg/kg-day (100 mg/kg-day in males and 200 mg/kg-day in females) at 25 and 51
weeks. Quantitative measures of hematology beyond 52 weeks in the chronic study might be
complicated due to natural diseases occurring in rodents and test variability leading to decreased
sensitivity and increasing variability with the results (AACC. 1992). Klaunig etal. (2015) did,
however, qualitatively evaluate blood and reported no PFHxA treatment effects on blood smear
morphology. Loveless etal. f20091 also evaluated blood smears up to test day 92 with PFHxA
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Toxicological Review ofPFHxA and Related Salts
sodium salt exposure and noted nucleated blood cells in smears indicative of bone marrow damage
or stress, but only for one female and one male.
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Toxicological Review ofPFHxA and Related Salts
Endpoint
Study
Experiment
Animal Description
Observation Time
PFHxA Hematopoietic Effects: MCHC, MCV, MCH
Mean Cell Hemoglobin (MCH)
NTP. 2018. 4309149
28-Day Oral
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~ Significant decrease Significant Trend |
i i i
100 0 100
i i
200 300
1 1 1 1 1 !
400 500 600 700 800 900
i i
1.0001.100
Dose (mgAg-day)
Figure 3-14. Hematological findings (MCH, MCHC, and MCV) in rats exposed by
gavage to PFHxA or PFHxA sodium salt (full details available by clicking the
HAWC linkl.
MCH = mean cell hemoglobin; MCHC = mean cell hemoglobin concentration; MCV = mean cell volume.
Table 3-22. Percent change in hematocrit due to PFHxA exposure in
short-term, subchronic, and chronic oral toxicity studies
Study design and reference
Dose (mg/kg-d)
LO
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Toxicological Review of PFHxA and Related Salts
Study design and reference
Dose (mg/kg-d)
in
-------
Toxicological Review ofPFHxA and Related Salts
Dose (mg/kg-d)
Study design and reference
in
pj
o
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o
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lO
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-4
-9
* indicates instances where statistical significance (p < 0.05) compared to controls was reported by study authors;
shaded cells represent doses not included in the individual studies.
Increased reticulocyte (immature RBCs formed during the erythroid regenerative process]
counts were consistently found across all four animal toxicological studies (see Table 3-24 and
Figure 3-15) and correlated with decreases in RBCs. PFHxA treatment-related increases in
reticulocyte counts were potentially a compensatory biological response to the PFHxA anemia
effect. Reticulocytes were increased (>10%) across all study designs and exposure durations at
200 mg/kg-day (Klaunig et al.. 2015: Chengelis et al.. 2009bl. 250 mg/kg-day (NTP. 2018). or
500 mg-kg/day (Loveless et al.. 2009). Reticulocyte measures were also available from Klaunig et
al. f2015), where increases were identified only in the high dose (200 mg/kg-day) female rat group.
The observation of increased reticulocytes was coherent with histological findings of increased
splenic extramedullary hematopoiesis and bone marrow erythroid hyperplasia incidence in both
the males and females dosed with 500 mg/kg-day fNTP. 2018: Loveless etal.. 20091 (summary
details are available in PFHxA Tableau visualization!. Collectively, the histological findings
considered together with red blood cell parameters suggest PFHxA interacts with the
erythropoietic pathways including outcomes such as anemia that can lead to compensatory
erythrogenic responses in the bone marrow and spleen.
Kndpoint Study
Experiment
Animal Description
Observation Time
PFHxA Hematopoietic EITecLs: Reticulocytes
Reticulocytes NTP. 2018,4309149
28-Day Oral
Rat, Harlan Spraguc-Dawlcy (9)
Day 29
A
—A
Rat. Harlan Sprague-Dawley ((f)
Day 29
0—¦
A
A
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90- Day Oral
Rat, Crl:CD(SD) (9)
Day 90
«
Rat. Crl:CD(SD)
Week 104
SM i I I ' I I I I I I I I I
Significant increase v Significant decrease Q Significant Trend -100 0 100 200 300 400 500 600 700 800 900 1,0001,100
Dose (mg/kg-day)
Figure 3-15. Hematological findings (reticulocytes) in rats exposed by gavage
to PFHxA or PFHxA sodium salt (full details available by clicking the IIAVVC
link).
3-71
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Toxicological Review of PFHxA and Related Salts
Table 3-24. Percent change in reticulocytes due to PFHxA exposure in
short-term, subchronic, and chronic oral toxicity studies
Study design and reference
Dose
mg/kg-d)
in
20 mg/kg-day, whereas APTT was decreased in the 500 mg/kg-day female rat dose group. The
observed increase in platelets and decreased clotting time (along with increased reticulocytes)
were coherent changes indicative of an erythropoietic response in the bone marrow, suggesting
bone marrow was not adversely affected by PFHxA exposure.
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Toxicological Review of PFHxA and Related Salts
Eridpoint Study
Experiment
Animal Description
Observation Time
Platelets (PLT) NTP, 2018, 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley (3)
Day 29
Rat, Harlan Sprague-Dawley (2)
Day 29
Chengelis, 2009, 2850404
90-Day Oral
Rat, Crl:CD(SD) (c?)
Day 90
Rat, Crl:CD(SD)(£)
Day 90
Loveless, 2009, 2850369
90-Day Oral
Rat, Cri:CD(SD) (rj)
Day 92
Rat, Crl:CD(SD)(V)
Day 93
Klaunig, 2015,2850075
2-Year Cancer Bioassay
Rat, Crl:CD(SD)(i)
Week 25
Rat, Crl:CD(SD)(l)
Week 25
Rat, Crl:CD(SD) (o)
Week 51
Rat, Crl:CD(SD) (2)
Week 51
Rat, Crl:CD(SD) )
Day 93
Activated Partial Thromboplastin Loveless, 2009, 2850369
Time (APTT)
90-Day Oral
Rat, Crl:CD(SD)(o)
Day 92
Rat, Crl:CD(SD) (-)
Day 93
| • No significant change^^ Significant increase
V Significant decrease ^ Significant Trend |
PFHxA Hematopoietic Effects: Hemostasis
J7 V
-100 0 100 200 300 400 500 600 700 800 900 1
Dose (mg,'kg-day)
Figure 3-16. Hemostasis findings in rats exposed by gavage to PFHxA or PFHxA
sodium salt (full details available by clicking the HAWC link).
Evidence Integration
The only available human study examining potential hematopoietic effects was considered
uninformative; therefore, there is indeterminate human evidence of hematopoietic effects.
Collectively the animal toxicological information provided coherent evidence indicative of
macrocytic anemia (characterized by low hemoglobin and large red blood cells) that is consistent
across multiple laboratories and experimental designs. Findings informing the overall judgment
included consistent observations of decreased red blood cells, hematocrit, and hemoglobin at doses
as low as 200 mg/kg-day generally in both sexes (summary level details are available in the
Tableau link! This finding was considered an adverse response to PFHxA exposure and correlated
with a compensatory increase in reticulocytes, an indicator of erythroid cell regeneration
supported by histological findings of splenic extramedullary hematopoiesis and bone marrow
erythroid hyperplasia. The responses across hematologic parameters in the chronic study (Klaunig
et al.. 2015] were only observed at the highest dose (200 mg/kg-day) in females. However, the
highest dose (200 mg/kg-day in females, 100 mg/kg-day in males) in Klaunig etal. (2015) was at or
lower than the observed effect level in the other available short term and subchronic studies.
Further, the null responses at lower doses (2.5,15, and 100 mg/kg-day in male rats; 5 and
30 mg/kg-day in female rats) are consistent with null responses in hematologic endpoints at
similar dose levels in the short term and subchronic studies. Overall, these collective erythroid
responses provide evidence for PFHxA treatment-related effects on erythropoiesis in animals.
Based on these data, there is robust animal evidence of hematopoietic effects. Effects on red
blood cell parameters including decreased hemoglobin, decreased red blood cells, and increased
reticulocytes are consistent across both subchronic and chronic studies in the 200 mg/kg-day dose
groups. The animal evidence came from studies using the same outbred Crl:CD(SD) rat model
originating from the same location and supplier that made available clinical hematology parameters
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Toxicological Review of PFHxA and Related Salts
indicating no sex difference in the parameters reported here. Given species conservation between
rodent models it was assumed that rat hematology was similar to that of the mouse where there are
some hematologic differences between humans, including smaller erythrocytes, higher percentage
of circulating reticulocytes (or polychromasia), physiologic splenic hematopoiesis and iron storage,
and more numerous and shorter-lived erythrocytes and platelets fO'Connell etal.. 20151. These
differences could explain the possible regenerative response in the spleen and bone and the
increase in reticulocytes (i.e., erythrogenesis and RBC turnover more rapid in rodent versus
human). Therefore, while some uncertainty around the human relevance of rodent hematopoietic
findings exists, these species-specific differences were controlled for in the experimental design.
Based on indeterminate evidence in humans and robust animal evidence with potentially
uncertain human relevance, the currently available evidence indicates that PFHxA likely causes
hematopoietic effects in humans given sufficient exposure conditions (see Table 3-25).7 This
conclusion is based on four high confidence studies in rats showing consistent (across durations
and study types) and coherent effects (across various outcome measures of hematopoietic
function), generally at >200 mg/kg-day following short-term (28-day), subchronic (90-day), or
chronic (2-year) exposures.
7 The "sufficient exposure conditions" are more fully evaluated and defined for the identified health effects through
dose-response analysis in Section 5.
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Toxicological Review of PFHxA and Related Salts
Table 3-25. Evidence profile table for hematopoietic effects
Evidence integration summary
Evidence stream summary and interpretation
judgment
Evidence from studies of exposed humans
0®Q
Studies and
Factors that increase
Factors that decrease
Evidence stream
Evidence indicates (likely)
confidence
certainty
certainty
Summary and key findings
judgment
Primary basis:
ooo
Indeterminate
• There were no informative human studies available from the PFHxA evidence base.
Four high confidence studies in
rats ranging from short term to
Evidence from animal studies
chronic exposure durations, in
both sexes, generally at
Studies and
Factors that increase
Factors that decrease
Summary and key findings
Evidence stream
>200 mg/kg-d
confidence
certainty
certainty
judgment
Hematology
4 hiqh confidence
studies in adult
rats:
• 28-d
• 90-d (2 studies)
• 2-yr
• Consistent changes
• No factors noted
• Decreased red blood
©©©©
Human relevance:
Some species differences
(decreases in
hematocrit,
hemoglobin, red
blood cells, and
MCHC and increases
in reticulocytes, MCV,
and MCH) across
studies
cells, hematocrit, and
hemoglobin at
>62.5 mg/kg-d; both
sexes
• Increased MCH and MCV
at >250; males more
sensitive
Robust
Findings considered
adverse based on
coherent evidence
that was consistent
across multiple
laboratories and
between human and rodent
hematology exist, but these are
not interpreted to impact
judgments on the collection of
rodent findings specific to
PFHxA.
Cross-stream coherence:
• Coherence of red
blood cells, HCT, and
HGB and reticulocytes
• Large magnitude of
effect as high as 356%
for reticulocytes
• Increased reticulocytes at
>200 mg/kg-d; both
sexes, all studies
• Coherence of red blood
cells and reticulocytes
with splenic
extramedullar
experimental
designs. Consistent
findings of
decreased red blood
cells, hematocrit,
and hemoglobin at
>200 mg/kg-d
coherent with
N/A (human evidence
indeterminate)
Susceptible populations and
lifestages:
No evidence to inform
• High confidence
hematopoiesis and bone
erythroid cell
studies
marrow erythroid
hyperplasia
regeneration
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Toxicological Review of PFHxA and Related Salts
Evidence stream summary and interpretation
Evidence integration summary
judgment
Hemostasis
4 hiqh confidence
studies in adult
rats:
• 28-d
• 90-d (2 studies)
• 2-yr
• Consistent treatment
related effect on
platelet levels
• Consistency across
study designs
• High confidence
studies
• No factors noted
• Increased platelet levels
>10 mg/kg-d; both sexes,
1 28-d, 2 90-d studies
• Decreased activated
partial thromboplastin
(APTT) at >20 mg/kg-d;
males only, 1 90-d study
• Decreased prothrombin
(PT) time at 500 mg/kg-d;
males only, 1 90-d study
Mechanistic evidence and supplemental information
Biological events
or pathways
Species or model
systems
Key findings, limitations, and interpretation
Evidence stream
summary
• No informative studies identified.
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Toxicological Review of PFHxA and Related Salts
3.2.5. Endocrine Effects
Human
Thyroid Hormones
Two studies examined the association between PFHxA exposure and thyroid hormones in
humans (see Figure 3-17). One was considered uninformative due to lack of consideration of
confounding, including age, sex, and other factors which is expected to substantially impact the
results (Seo etal.. 20181. The other study was a cross-sectional study of the general population in
China and was considered low confidence fLi etal.. 20171 due to concerns around participant
selection, outcome measures, consideration of confounding, and decreased sensitivity. Regarding
the latter concern, the exposure levels were low and contrast narrow in Li etal. f20171 (median
[range]: 0.01 [ S«°
&
Legend
Good (metric) or High confidence (overall)
Adequate (metric) or Medium confidence (overall)
Deficient (metric) or Low confidence (overall)
Critically deficient (metric) or Uninformative (overall)
Figure 3-17. Study evaluation for human epidemiologic studies reporting
toxicity findings from PFHxA exposures (HAWC: PFHxA - Human Toxicity
Endocrine Effects link!.
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Toxicological Review of PFHxA and Related Salts
Animal
Four short-term (28-day), subchronic, and chronic animal studies evaluated potential
endocrine effects of PFHxA or PFHxA sodium salt in rats. Most of the outcome-specific study ratings
were rated high confidence. Histopathology for Chengelis etal. f2009bl was rated low confidence
because of issues related to observational bias, concerns about endpoint sensitivity and specificity,
and results presentation. A summary of the studies and the interpretations of confidence in the
results for the different outcomes based on the individual study evaluations is presented in
Table 3-26, and details are available by clicking the HAWC link.
Table 3-26. Endocrine endpoints for PFHxA and associated confidence scores
from repeated-dose animal toxicity studies
Author (year)
Species, strain (sex)
Exposure
design
Exposure route and dose range
Organ weight
Histopathology
Thyroid hormones
NTP (2018)
Rat, Harlan
Sprague-Dawley
(male and female)
Short term
(28 d)
Gavage3
Male and female: 0, 62.5,125,
250, 500,1,000 mg/kg-d
++
+ +
++
Chengelis et al.
(2009b)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and female)
Subchronic
(90 d)
Gavage3
Male and female: 0,10, 50,
200 mg/kg-d
++
"
NM
Loveless et al.
(2009)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and female)
Subchronic
(90 d)
Gavageb
Male and female: 0, 20,100,
500 mg/kg-d
++
+ +
NM
Klaunig et al.
(2015)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and female)
2-yr cancer
bioassay
Gavage3
Male: 0, 2.5,15,100 mg/kg-d
Female: 0, 5, 30, 200 mg/kg-d
NM
+ +
NM
++ Outcome rating of high confidence; - outcome rating of low confidence; NM, outcome not measured.
a bStudy evaluation for animal toxicological endocrine endpoints reported from studies with male and female rats
receiving PFHxA3 or PFHxA sodium saltb by gavage. Study evaluation details for all outcomes are available by
clicking the HAWC link.
++ Outcome rating of high confidence; - outcome rating of low confidence; NM, outcome not measured.
Thyroid Hormones
A single study evaluated potential PFHxA effects on endocrine function specific to thyroid
hormones in rats exposed for 28 days fNTP. 20181. Specifically, males showed statistically
significant, dose-dependent decreases in thyroid hormones. These outcomes showed a clear dose-
dependent pattern of effect with treated animals showing reductions of 25%-73% or 20%-58% for
free or total T4, respectively. Smaller decreases in T3 in males also were observed (18%-29%),
although the dose-dependence of this effect was less clear. No treatment-related changes were
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Toxicological Review of PFHxA and Related Salts
observed for T3 or T4 in females or for TSH in either sex (NTP. 20181. Results are summarized in
Figure 3-18 and Table 3-27.
Endpoint Study
Experiment
Animal Description
Observation Time
PFHxA Endocrine Effects: Hormones
Thyroid stimulating hormone (TSH) NTP, 2018, 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley ( )
Day 29
•—•—•-
•
•
•
Rat, Harlan Sprague-Dawley ( ?)
Day 29
•—• •
•
*
•
Thyroxine (T4) NTP, 2018,4309149
28-Day Oral
Rat, Harlan Sprague-Dawley (2)
Day 29
» • •
*
•
~
Rat, Harlan Sprague-Dawley ( ')
Day 29
vv
~
V
V
Thyroxine (T4), Free (Free T4) NTP, 2018, 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley (2)
Day 29
» • •
—•
•
+
Rat, Harlan Sprague-Dawley ( ')
Day 29
vv
V
V
V
Triiodothyronine (T3) NTP, 2018, 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley (2)
Day 29
•—• •
•
•
+
Rat, Harlan Sprague-Dawley ( ?)
Day 29
w
V
V
| • No significant changeA Significant increase
\/ Significant decrease Q Significant Trendj
100 0 100
200 300
400 500 600 700 800
Dose (mg/kg-day)
900 1,0001,100
Figure 3-18. Thyroid hormone measures from the serum of rats exposed by
gavage to PFHxA or PFHxA sodium salt (full details available by clicking the
HAWCJink).
Table 3-27. Percent change in thyroid hormone levels following PFHxA
exposure in a 2 8-day oral toxicity study
Dose (mg/kg-d)
Study design and reference
Hormone
in
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Toxicological Review of PFHxA and Related Salts
following subchronic (90-day) or chronic (2-year) exposure to PFHxA. Notably Chengelis et al.
(2009b) did not specify what outcomes were evaluated. Therefore, whether thyroid follicular cell
hypertrophy was measured is unclear. No other treatment-related histopathological effects were
noted in the PFHxA evidence base. Results are summarized in Table 3-28.
Table 3-28. Incidence of thyroid follicular epithelial cell hypertrophy
following PFHxA or PFHxA ammonium salt exposures in rats
Sex
(Timepoint)
0
2.5
5
10
15
20
30
50
62.5
100
125
200
250
500
1000
28-d exposure in rats (NTP, 2018)
Female
0/10
0/10
0/10
0/10
0/10
0/10
Male
0/10
0/10
0/10
0/10
0/10
0/10
90-d exposure in rats (Loveless et al., 2009)
Female
(Exposure,
Day 90)
0/10
0/10
0/11
4/10
*
Male
(Exposure,
Day 90)
0/10
0/10
1/10
*
2/10
*
Female
(Recovery
Day 30)
0/10
6/10
*
Male
(Recovery
Day 30)
0/10
3/10
*
Female
(Recovery,
Day 90)
0/10
0/10
0/9
0/10
Male
(Recovery,
Day 90)
0/10
0/10
0/10
2/10
*
2-vr exposure in rats (Klaunig et al., 2015)
Female
0/21
0/25
0/20
0/14
Male
0/18
0/24
0/23
0/24
* Indicates instances where statistical significance (p < 0.05) compared to controls was reported by study authors;
shaded cells represent doses not included in the individual studies.
Organ Weights
Three studies evaluated effects on thyroid and adrenal weights (NTP. 2018: Chengelis etal..
2009b: Loveless etal.. 2009). Although no effects on relative thyroid weight were observed at the
end of the 90-day exposure period in either subchronic study Loveless etal. (2009) qualitatively
reported a statistically significant increase in relative thyroid weight for female rats at the highest
tested dose (500 mg/kg-day) ofPFHxA sodium salt at the 30-dav recovery. NTP f20181 observed a
trend (p < 0.05) for decreased absolute adrenal gland weight in male rats exposed to 500 mg/kg-
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Toxicological Review of PFHxA and Related Salts
day. No other treatment-related effects on endocrine organ weights were observed (NTP. 2018:
Chengelis etal.. 2009b: Loveless etal.. 20091.
Mechanistic Evidence and Supplemental Information
Thyroid Hormone Levels in Zebrafish
(Zhang etal.. 20221 evaluated the effects of a 96-hour exposure to PFHxA (0, 0.48, 2.4, and
12 mg/L) on whole body T3 and T4 levels in early-life stage zebrafish. Whole body total T3 showed
a statistically significant increase in high dose animals. For T4 there was a significant dose-
dependent reduction in at the low and mid dose, but a significant increase at the high dose.
Binding with transport proteins and nuclear receptor
Four studies were identified that investigated the ability of PFHxA to bind to two thyroid
hormone serum transport proteins, transthyretin (Harriers etal.. 2020: Ren etal.. 2016: Weiss etal..
20091 and thyroid binding globulin (Ren etal.. 20161. as well as the thyroid hormone receptor (Ren
etal.. 20151. All studies reported that PFHxA has a low binding affinity for transthyretin and the
thyroid hormone receptors, and no detectable binding was observed for thyroid binding globulin.
Expression of HPT-related genes and proteins
Three studies evaluated the effects of PFHxA exposure on expression of mRNA and proteins
related to thyroid function in early life-stage zebrafish (Zhang etal.. 20221. embryonic avian
neuronal cells (Vongphachan etal.. 20111. and rat hepatoma cells (H4IIE). In zebrafish larvae
exposed to up to 12 mg/L PFHxA for 96 h, whole body expression was increased for genes related
to HPT regulation (crh, trh), thyroid hormone synthesis [tg, nis), transport (ttr), and nuclear
receptors (tra, trp) after PFHxA exposure. The same study also evaluated protein levels of
thyroglobulin and transthyretin, which are important for thyroid hormone synthesis and transport,
respectively, and reported treatment-related increases.
Two studies reported changes in mRNA expression of genes involved in metabolism of
thyroid hormones (Zhang etal.. 2022: Vongphachan etal.. 20111. Deiodinases convert thyroid
hormones to active and inactive forms through outer and/or inner ring deiodination. Differing
patterns of expression were observed for deiodinases, with Vongphachan etal. f20111 finding an
increase in deiodinase 2 and 3 in primary chicken neuronal cell cultures whereas Zhang et al.
f20221 reported decreases in deiodinases 1 and 2 in early lifestage zebrafish. Ugtlab, which
mediates clearance of thyroid hormones from tissues, was decreased in whole body zebrafish
larvae.
Naile etal. (20121 evaluated the effect of PFHxA on mRNA expression of genes related to
thyroid development in rat hepatoma cells (H4IIE). Cells exposed to 0.1-100 [J.M for 72 hours
showed increases (19.1-29.29-fold) in expression of Hex, which is important for cell differentiation
during development of the thyroid. The effect on Pax8, a gene that regulates proliferation and
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Toxicological Review of PFHxA and Related Salts
differentiation of cells that produce thyroxine, was less consistent. In general, there was an increase
in expression (2.46-, 1.8-, and 3.73-fold for 0.1,10, and 100 [J.M, respectively); however, there was a
slight (0.92-fold) decrease in the 1 [im treated cells. The study did not report statistical significance
of the results and the data are not sufficient for running statistical analyses as reported.
Evidence Integration
A single low confidence study provided some evidence of an association between PFHxA
exposure and decreased T3 and TSH in humans, although methodological concerns reduce the
reliability of these findings. Based on these results, there is indeterminate human evidence of
endocrine effects.
Evidence supporting potential endocrine effects of PFHxA exposure is largely based on two
high confidence rat studies showing decreases in serum thyroid hormone levels and increased
thyroid epithelial cell hypertrophy in rats, but interpretation of these results is complex. The only
available animal study that evaluated thyroid hormone levels showed a large magnitude of change
in T4 (up to 73% decrease) and T3 (up to 20% decrease) with a clear dose-response for T4 (free
and total) following exposure to PFHxA as low as 62.5 mg/kg-day, and these effects were observed
only in males fNTP. 20181. A second study found increased incidence of thyroid epithelial cell
hypertrophy following a 90-day exposure to PFHxA sodium salt fLoveless etal.. 20091. Effects on
thyroid hormone levels were also reported in larval zebrafish exposed to PFHxA during early
development. Consistent with the available rodent data, whole body T4 levels were decreased,
however T3 levels were increased in zebrafish which is the opposite direction of effects observed in
the rat studies. The differences in effects on T3 may be explained by differences in the species
(mammalian versus non-mammalian), methods (whole body levels versus serum), and lifestage
(developing versus adult).
For the histopathological findings, treatment-related changes were reported for both males
and females administered 500 mg/kg-day PFHxA sodium salt. The incidence of thyroid
hypertrophy was higher in females than in males, although effects in males persisted longer after
exposures had ceased. Also, no clear dose-response was found, with effects generally observed only
at the highest dose tested. Three other studies evaluated thyroid histopathology but found no
effects in either sex following a wide range of PFHxA exposure durations (28 days to 2 years) and
doses (up to 1,000 mg/kg-day) fNTP. 2018: Klaunigetal.. 2015: Chengelis etal.. 2009bl. No clear
pattern of treatment-related effects was reported for endocrine organ weights.
Although the only available animal study examining thyroid hormones showed strong
effects on T4 and T3 after short-term exposure, no effects were observed on TSH. The observed
pattern of effects on the thyroid (i.e., decreased total and free T4 without a compensatory increase
in TSH) after PFHxA exposure is consistent with thyroid perturbations following exposure to other
PFAS, including PFBA (U.S. EPA. 2022b) and PFBS (U.S. EPA. 2021b). Rodents and humans share
many similarities in the production, regulation, and functioning of thyroid hormones. Although
differences exist, including the timing of in utero thyroid development and hormone turnover rates,
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Toxicological Review of PFHxA and Related Salts
rodents are considered a good model for evaluating the potential for thyroid effects in humans
(Zoeller et al.. 20071. While there is uncertainty in the reliability of the TSH measurements and
patterns of TH changes in animals may not translate perfectly to human clinical definitions, the
observed decreases in total or free T4 in the absence of increases in TSH are considered biologically
relevant to humans fCrofton. 2004: Lau etal.. 20031. TSH is an indicator that the thyroid system has
been perturbed, but it does not always change when serum T4 is decreased fHood etal.. 19991 and
decreases in T4 alongside normal levels of TSH is consistent with the human clinical condition
referred to as hypothyroxinemia [see additional discussion in (U.S. EPA. 2018b)]. Adverse
neurological outcomes have been demonstrated following decreased T4 levels during the early
neonatal period with no changes in T3 or TSH fCrofton. 20041. During pregnancy and early
development, even transient perturbations in thyroid function can have permanent impacts on
normal growth and neurodevelopment in the offspring. Although currently available evidence on
thyroid hormones is limited to effects in males, there is evidence to support effects in females.
Changes in thyroid histopathology were observed in both sexes, with a higher incidence in females
(Loveless etal.. 20091. Given the potential consistency of these findings with those observed for
other PFAS, the availability of a single short-term study of thyroid hormones represents a
significant data gap for PFHxA. The lack of consistency of the histopathology findings also reduces
the strength of the available evidence; however, this variability across studies could be driven by
differences in the dose levels examined (i.e., high dose ranged from 100 to 1,000 mg/kg-day) and
exposure duration (i.e., short-term, subchronic, and chronic exposures).
While males exhibited a clear dose-dependent reduction in T4 and T3 at doses as low as
62.5 mg/kg-day in males but no effect in females at doses as high as 1000 mg/kg-day. These
disparate results may be explained by sex-specific differences in the pharmacokinetics of PFHxA
and its effect on induction of metabolizing enzymes. As discussed in Section 3.1.4, some evidence
suggests sex-specific differences in the pharmacokinetics of PFHxA, with plasma concentrations
measured 2-3 times higher in male rats when compared to females f Chang etal.. 2008: Lau etal..
2006: Lau etal.. 20041. Additionally, thyroid hormones may be reduced through PPARa activation
and induction of microsomal enzyme inducers, such as CAR and PXR (Vansell. 20221. Males are
more sensitive to PFHxA-induced PPARa activation (see Section 3.2.1). While these differences
might explain why treatment-related effects on thyroid hormones were observed only in male rats,
it is unclear why a similar sex-specific pattern was not observed for the reported thyroid
histopathological changes observed at similar or higher doses.
Several mechanisms are implicated in the disruption of thyroid homeostasis. At present
there is insufficient evidence identify the MOA(s) by which PFHxA induces the observed thyroid
effects, but the mechanistic and supplemental information provide provides some support for
PFHxA-related effects on thyroid function. PFHxA may affect thyroid hormone metabolism and bind
to transport proteins and the nuclear receptor. Two mechanistic studies show effects on mRNA
expression of deiodinases, which are important for conversion of thyroid hormones between active
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Toxicological Review of PFHxA and Related Salts
and inactive forms (Zhang etal.. 2022: Vongphachan etal.. 20111 and glucuronidases which are
important for excretion. Additionally, there is evidence that some PFAS may alter thyroid function
via interaction with thyroid hormone receptors and transport proteins; however, the current data
show only weak binding for PFHxA fHamers etal.. 2020: Ren etal.. 2016: Ren etal.. 2015: Weiss et
al.. 20091. Based on available data, there is moderate animal evidence of endocrine effects. In the
absence of evidence to the contrary, effects in rats are considered relevant to humans.
Overall, the currently available evidence indicates that PFHxA likely causes endocrine
effects in humans given sufficient exposure conditions (see Table 3-29).8 This conclusion is based
on four animal studies generally rated as high confidence that reported treatment-related
decreases in thyroid hormone levels and more uncertain evidence of thyroid histopathology after
exposure to PFHxA at >62.5 mg/kg-day.
8 The "sufficient exposure conditions" are more fully evaluated and defined for the identified health effects through
dose-response analysis in Section 5.
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Table 3-29. Evidence profile table for endocrine effects
Evidence stream summary and interpretation
Evidence
integration
judgment
Evidence from studies of exposed humans
®©o
Studies and
confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key findings
Evidence stream
judgment
Evidence
indicates (likely)
Thyroid
Hormones
1 low
confidence
study
• No factors noted
• Lack of coherence
across related
thyroid hormone
measures
• Low confidence
study
• Inverse associations between free T3 and TSH and
PFHxA in a single low confidence study
ooo
Indeterminate
Primary basis:
Two animal
studies generally
rated as high
confidence that
reported
treatment related
changes in thyroid
Evidence from animal studies
hormone levels,
thyroid
histopathology
after exposure to
Studies and
confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key findings
Evidence stream
judgment
Thvroid
Hormones
1 hiqh
confidence
study in
adult rats:
• 28-d
• High confidence
study.
• Dose response
gradient observed
for free and total
T4
• Large effect
magnitude; up to
73%
• No factors noted
• Decreased T4 (free and total) and T3 observed in
males only at >62.5 mg/kg-d
®©o
Moderate
Some evidence
of thyroid effects
based on
hormone and
histopathological
changes in two
rat studies.
Thyroid-related
effects in
PFHxA at
>63.5 mg/kg-d.
Human relevance:
Without evidence
to the contrary,
effects in rats are
considered
relevant to
humans.
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Evidence stream summary and interpretation
Evidence
integration
judgment
Histopathol
ogy
3 high
confidence
studies in
adult rats:
• 28-d
• 90-d
• 2-yr
1 low
confidence
study in
adult rats:
• 90-d
Organ
Weight
High
confidence:
3 high
confidence
studies in
adult rats:
• 28-d
• 90-d (2
studies)
High confidence
studies
• High confidence
studies
Unexplained
inconsistency across
studies
• Unexplained
inconsistency across
studies
• Increased incidence of thyroid epithelial cell
hypertrophy at >100 mg/kg-d for 90 d; persisted
up to 90 d after exposure
• No effects observed in 28 d study at up to
1,000 mg/kg-d
• Relative thyroid weights were increased only in
females 30 d after exposure
• Right adrenal weights decreased but no other
adrenal effects were reported
rodents are
supported by
mechanistic and
supplemental
information
showing changes
in thyroid
hormone levels
in zebrafish
larvae, weak
binding to
thyroid transport
proteins/recepto
r, and changes in
thyroid related
mRNA/protein
expression.
Cross-stregm
coherence:
N/A (human
evidence
indetermingte).
Susceptible
populgtions grid
lifestgges:
No evidence to
inform
Other inferences:
Some mechanistic
data and
supplemental
information were
identified on this
health effect were
identified that
provide additional
support for
potential PFHxA
mediated
endocrine effects
but are
insufficient to
inform a potential
MOA. Notably,
the pattern of the
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Toxicological Review of PFHxA and Related Salts
Evidence stream summary and interpretation
Evidence
integration
judgment
Mechanistic evidence and supplemental information
limited human
and animal
findings for PFHxA
are consistent
with
hypothyroxinemia
seen for other
PFAS
Biological
events or
pathways
Key findings, interpretation, and limitations
Evidence stream
summary
Thyroid
Hormones
Key findings and interpretation:
• Altered whole body thyroid hormone levels CM3, 4/T4) in zebrafish larvae
• Effects T3 shows opposite direction of effect of what was observed in both rodents and humans
Limitations: Small evidence base with some inconsistencies in the pattern of effect observed in human
and rodent data
Some support
for potential
effects of PFHxA
on thyroid
function.
Receptor/
Transport
Protein
Binding
Key findings and interpretation:
• Low binding potency for transthyretin and thyroid hormone receptor
• No observed binding to thyroid binding globulin
Limitations: Small evidence base showing weak effect.
mRNA/
Protein
Expression
Key findings and interpretation:
• Altered expression of several thyroid-related mRNA and proteins observed in vivo (larval zebrafish)
and in vitro (primary chicken neuronal cells; rat hepatoma, H4IIE)
• Potential targets included pathways relevant to all aspects of thyroid regulation including, synthesis,
release, transport, and metabolism of thyroid hormones as well as regulation of thyroid
development, but in general each target evaluated by a single study
• Lack of consistency of effect in two studies that evaluated mRNA expression of deiodinase 2
Limitations: Small evidence base lacking consistency across different studies (when available) for
deiodinase 2
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Toxicological Review of PFHxA and Related Salts
3.2.6. Male Reproductive Effects
Human
Sperm Parameters
One low confidence study (Song etal.. 20181 examined the association between PFHxA
exposure and semen parameters and reported no decrease in concentration or motility with higher
levels of PFHxA in serum (see Figure 3-19). A significant negative correlation between PFHxA levels
in semen and sperm motility was found in this study (correlation coefficient = -0.35, p < 0.01), but
analytical measurement of PFAS in semen is less established than in blood and the relevance to
exposure is unclear. Still, exposure levels in the study based on serum measurements were high
(median: 29 ng/mL, 5th-95th percentile: 11-70 ng/mL), so the study had reasonable sensitivity to
detect an effect
Reproductive Hormones
A single study rated low confidence due to low sensitivity and high potential for
confounding (see Figure 3-19) found some support for associations between PFHxA and
reproductive hormones in a population of adolescent (13-15 years old) males in Taiwan fZhou et
al.. 20161. Overall, authors reported no significant associations between PFHxA and serum
testosterone and estradiol; however, when the data were stratified by sex, a significant negative
association between testosterone and PFHxA exposure level ((3 = -0.31, 95% CI: -0.59, -0.02) was
found in boys. Based on serum measurements, the exposure levels in this study were low and the
range narrow (median: 0.2 ng/mL, IQR 0.1-0.3 ng/mL), which might have reduced study
sensitivity. The presence of an association despite reduced sensitivity could be due to either high
potency of the exposure to cause these effects or potential confounding by other correlated PFAS,
including PFOS, PFDA, and PFNA.
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Toxicological Review of PFHxA and Related Salts
^ ',,1^
gcf®'
Participant selection -
Exposure measurement -
Outcome ascertainment
Confounding
Analysis
Sensitivity
Selective Reporting
Overall confidence
Legend
Good (metric) or High confidence (overall)
Adequate (metric) or Medium confidence (overall)
Deficient (metric) or Low confidence (overall)
Not reported
Critically deficient (metric) or Uninformative (overall)
N/Al Not applicable
Figure 3-19. Study evaluation for human epidemiological studies reporting
male reproductive findings from PFHxA exposures (HAWC: PFHxA - Human
Toxicity Male Reproductive Effects link!.
Animal
Several short-term (28-day), subchronic, and chronic animal studies evaluated sperm
parameters, reproductive organ weights, and other reproductive male outcomes in rats receiving
oral exposures of PFHxA and PFHxA sodium salt. Most outcome-specific study ratings were rated
high confidence; however, some specific concerns were identified that resulted in low confidence
ratings. Although generally a well-conducted study, NTP f20181 was rated low confidence for sperm
parameters due to issues related to exposure duration and concerns for potential insensitivity.
Histopathological results for Chengelis etal. f2009bl were rated low confidence because of issues
related to observational bias, concerns about endpoint sensitivity and specificity, and results
presentation. The results of the outcome-specific study evaluations are presented in Table 3-30,
and details are available by clicking the HAWC link.
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Toxicological Review of PFHxA and Related Salts
Table 3-30. Study design, exposure characteristics, and individual outcome
ratings
Study
Species, strain
(sex)
Exposure design
Exposure route and
dose
Sperm
parameters
Organ weight
Histopathology
Hormone
levels
Reproductive
system
development
NTP
(2018)
Rat, Harlan
Sprague-Dawley
(male and
female)
Short term
(28 d)
Gavage3
Male and female: 0,
62.5, 125, 250, 500,
1,000 mg/kg-d
++
+ +
++
NM
Chengelis
et al.
(2009b)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
Subchronic
(90 d)
Gavage3
Male and female: 0,
10, 50, 200 mg/kg-d
NM
++
NM
NM
Loveless
et al.
(2009)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
Subchronic (90 d)
One-generation
reproductive: Po
females dosed 70 d
prior to
cohabitation,
through gestation
and lactation (126
d); Po males dosed
for 110 d
Developmental:
Gestation Days 6-20
Gavageb
Male and female: 0,
20,100,
500 mg/kg-d
++
++
+ +
NM
++
Klaunig et
al. (2015)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
2-yr cancer bioassay
Gavage3
Male: 0, 2.5,15,
100 mg/kg-d
Female: 0, 5, 30,
200 mg/kg-d
NM
NM
+ +
++
NM
Iwai and
Hoberman
(2014)°
Mouse,
Crl :CD1(ICR);
Charles
River
Laboratories,
Inc.
Gestation Days 6-18
Gavaged
Phase 1: 0,100,
350, 500 mg/kg-d
Phase 2: 0, 7, 35,
175 mg/kg-d
NM
NM
NM
NM
++
++ Outcome rating of high confidence; - outcome rating of low confidence; NM, outcome not measured.
a b dStudy evaluation for animal toxicological endpoints reported from male reproductive studies with rats receiving
PFHxA,3 PFHxA sodium salt,b or PFHxA ammonium saltd by gavage. Study evaluation details for all outcomes are
available by clicking the HAWC link.
cPhase 1 was a range-finding study used to determine the appropriate dose range for identification of a NOAEL in
Phase 2.
Sperm Parameters
Evidence from a 28-day fNTP. 2018] and one-generation reproductive study fLoveless et al..
20091 included sperm parameters useful in evaluating potential male reproductive effects (see
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Toxicological Review of PFHxA and Related Salts
Figure 3-20). In male rats receiving PFHxA daily by gavage for 28 days, a trend (p < 0.05) for
decreased sperm count in the cauda epididymis was identified with a significant (25% change from
control) decrease in the 1,000 mg/kg-day dose group. Animals in this dose group showed a
significant decrease in body weight (13% change from control) at the end of the study but no other
overt toxicity was indicated (e.g., mortalities or significant clinical observations) fNTP. 20181.
Notably, these effects were observed despite concerns about sensitivity due to the short exposure
duration of the study by NTP (2018) which does not encompass a full 6-week spermatogenic cycle
in rats. In the one-generation reproductive study, Loveless etal. (2009) found no treatment-related
effects for sperm parameters following a 10-week premating exposure in male rats to PFHxA
sodium salt at doses up to 500 mg/kg-day. Results are summarized in Figure 3-20.
Endpoint
Study
Experiment
Animal Description
Observation Time
Reproductive Effects: Sperm Parameters
Testicular Spermatids (per Testis)
Loveless. 2009. 2850369
1-Generation Reproductive
PO Rat, Cr1:CD(SD) ( ?)
Day 105
«•—• •
Testicular Spermatid Count (per g
Loveless. 2009. 2850369
1-Generation Reproductive
P0 Rat, Crl:CD(SD) (?)
Day 105
Testis)
Testicular Spermatid Count
NTP, 2018, 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley ( ')
Day 29
• • • •
Testicular Spermatid Count (per mg
NTP. 2018, 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley (c?)
Day 29
Testis)
Cauda Epididymis Sperm Count
NTP, 2018,4309149
28-Day Oral
Rat, Harlan Sprague-Dawley ( )
Day 29
— . ~
Epididymal Sperm Count (per Cauda)
Loveless. 2009. 2850369
1-Generation Reproductive
PO Rat, Crl:CD(SD) ( • 0
Sperm Motility
Loveless, 2009. 2850369
1-Generation Reproductive
PO Rat, Crl:CD(SD) (,?)
Day 105
«• • 0
Sperm Morphology
Loveless. 2009. 2850369
1-Generation Reproductive
PO Rat, Crl:CD(SD) (.?)
Day 105
#• • *
I • No significant change^ Significant increa
e ~ Significant decrease A Significant Trendl
100 0 100 200 300 400 500 600 700 800 900 1.0001,100
Dose (mg/kg-day)
Figure 3-20. Male reproductive effects on sperm parameters in male rats
exposed to PFHxA or sodium salt for 28 or 90 days (HAWC: PFHxA - Animal
Toxicity Male Reproductive Effects link).
Reproductive Organ Weights
Reproductive studies commonly report both absolute and relative organ weights; however,
for the testes, absolute weights are considered most informative for hazard evaluation f Bailey etal..
2004). Three studies (28- or 90-day exposure durations) reported data on the effects of PFHxA or
PFHxA sodium salt exposure on male reproductive organ weights (i.e., testes, epididymis) in rats
(see Figure 3-21) (NTP. 2018: Chengelis etal.. 2009b: Loveless etal.. 2009). Two studies reported a
modest, but statistically significant (p < 0.05; 13%-16% change from control), increase in relative,
but not absolute, testis weight in rats exposed to 1,000 mg/kg-day for 28 days fNTP. 20181 or
500 mg/kg-day for 90 days fLoveless etal.. 20091. No treatment-related effects on male
reproductive organ weights were reported by Chengelis etal. f2009bl.
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Toxicological Review of PFHxA and Related Salts
Endpoint
Study
Experiment
Animal Description
Observation Time
Male Reproductive Effects: Organ Weights
Cauda Epididymis Weight, Absolute NTP, 2018, 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley ( ')
Day 29
• • • +
Epididymides Weight, Absolute
Loveless, 2009, 2850369
90-Day Oral
Rat, Cr1:CD(SD) (
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Toxicological Review of PFHxA and Related Salts
availability of a single study for each outcome and the high risk for bias in these evaluations. Based
on these results, there is indeterminate human evidence of male reproductive effects.
In animals, the evidence supporting potential effects of PFHxA exposure on male
reproduction was primarily limited to decreased sperm count fNTP. 20181 and increased relative
testis weights fNTP. 2018: Loveless etal.. 20091 at the highest tested doses in these studies (1,000
and 500 mg/kg-day, respectively). Decreased sperm count reported by NTP f20181 was considered
low confidence due to the 28-day exposure duration and concerns that such short exposures would
not capture the full spermatogenic cycle. Although finding effects in the presence of an insensitive
exposure duration could indicate a sensitive window for chemical-specific perturbations, similar
results were not observed in a high confidence subchronic study performed in the same rat strain
fLoveless etal.. 20091. albeit the highest tested dose was 500 as compared to 1,000 mg/kg-day in
the short-term study. In addition, evidence of overt toxicity (i.e., 13% reduction in terminal body
weight relative to controls) was found in the male rats dosed 1,000 mg/kg-day in the NTP (20181
study.
Two studies reported increased relative testis weight; however, the preferred metric of
absolute testis weight did not change in either study and no changes in organ weight were observed
in a second subchronic study fChengelis etal.. 2009bl. Reproductive hormone (i.e., testosterone
and luteinizing hormone) levels were reduced in the only chronic study; however, the effect was
small in magnitude, was not dose-dependent, and was observed only at the 26-week time point
(Klaunig etal.. 20151. Similar results on testosterone were not reported in the short-term high
confidence study (NTP. 20181. No other coherent findings (i.e., reproductive histopathology and
male reproductive system development) supporting reproductive toxicity were identified in the
animal evidence base. Based on these results there is indeterminate animal evidence of male
reproductive effects.
Overall, the currently available evidence is inadequate to assess whether PFHxA might
cause male reproductive effects in humans (see Table 3-31).
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Toxicological Review of PFHxA and Related Salts
Table 3-31. Evidence profile table for male reproductive effects
Evidence stream summary and interpretation
Evidence integration
summary judgment
Evidence from studies of exposed humans
OOO
Studies and confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key findings
Evidence stream
judgment
Evidence inadequate
Primary Basis:
Evidence is low confidence
or largely null
Human relevance:
N/A (indeterminate animal
Sperm Parameters
1 low confidence study
• No factors noted
• Low confidence
study.
• Association between
PFHxA levels in semen
and decreased sperm
motility
ooo
Indeterminate
Reproductive
Hormones
1 low confidence study
• No factors noted
• Low confidence
study
• Significant inverse
association between
PFHxA exposure and
testosterone despite
poor sensitivity
evidence)
Cross stream coherence:
N/A (human evidence
indeterminate)
Susceptible population and
lifestages:
Evidence from animal studies
Studies and confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key findings
OOO
Indeterminate
No evidence to inform
Sperm Parameters
1 hiqh confidence study
in adult rats:
• 90-d
1 low confidence in
adult rats
• 28-d
• No factors noted
• Unexplained
inconsistency
across studies
• Decreased sperm count
in the cauda epididymis
at 1,000 mg/kg-d
The data are largely
null. Some evidence of
reproductive effects but
interpretation limited by
unexplained
inconsistency at effects
observed only at the
high dose that elicited
high overt toxicity
(i.e., 13% decrease in
body weight).
Organ Weights
3 hiqh confidence
studies in adult rats:
• High confidence
studies
• No factors noted
• Increased relative testis
weight at >500 mg/kg-d;
no change in absolute
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Toxicological Review of PFHxA and Related Salts
Evidence stream summary and interpretation
Evidence integration
summary judgment
• 28-d
• 90-d (2 studies)
• Dose response
with longer
exposure duration
testis weights (preferred
metric)
Reproductive
Hormones
2 hiqh confidence
studies in adult rats:
• 28-d
• 2-yr
• High confidence
studies
• No factors noted
• Transient decrease of
small magnitude in
luteinizing hormone and
testosterone
Histopathology and
Male Reproductive
System Development
4 hiqh confidence
studies in rats and mice:
• 28-d (rat)
• 90-d (rat)
• GD 6-18 (mouse)
• 2-yr (rat)
1 low confidence study
in adult rats:
• 90-d
• High confidence
studies
• No factors noted
• No treatment-related
effects reported at
<1,000 mg/kg-d
Mechanistic evidence and supplemental information
Biological events of
pathways
Biological events of
pathways
Biological events of pathways
Biological events of
pathways
• No studies identified
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Toxicological Review of PFHxA and Related Salts
3.2.7. Female Reproductive Effects
Human
Reproductive Hormones
A single low confidence study (see Figure 3-22) evaluated associations between PFHxA and
reproductive hormones in a population of Taiwanese adolescents (13-15 years old) (Zhou etal..
2016). Overall, the authors reported nonsignificant inverse associations between PFHxA and serum
testosterone and estradiol in females when the data were stratified by sex. Exposure levels to
PFHxA were low, which might have reduced study sensitivity, as described above in Section 3.2.6.
Male Reproductive Effects.
Participant selection
Exposure measurement -
Outcome ascertainment-
Confounding
Analysis
Sensitivity -
Selective Reporting -
Overall confidence-
Legend
Good (metric) or High confidence (overall)
Adequate (metric) or Medium confidence (overall)
Deficient (metric) or Low confidence (overall)
Critically deficient (metric) or Uninformative (overall)
Figure 3-22. Study evaluation for human epidemiological studies reporting
female reproductive findings from PFHxA exposures (HAWC: PFHxA - Human
Toxicity Female Reproductive link!.
Animal
Five animal studies evaluated outcomes related to female reproduction in rats and mice
receiving PFHxA via gavage, PFHxA sodium salt, or PFHxA ammonium salt. Study designs included
short-term (28-day), subchronic (90-day), and chronic (2-year) one-generation reproductive and
developmental exposures. In general, the outcome-specific study ratings were high confidence. One
study was rated low confidence for histopathology due to concerns about observational bias,
endpoint sensitivity and specificity, and results presentation fChengelis etal.. 2009bl. The results of
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Toxicological Review of PFHxA and Related Salts
study evaluation for female reproductive outcomes are presented in Table 3-32 and details are
available by clicking the HAWC link.
Table 3-32. Study design characteristics
Study
Species, strain
(sex)
Exposure design
Exposure route
and dose
Fertility and
pregnancy
Organ weight
Histopathology
Reproductive
hormones
Reproductive
system
development
NTP(2018)
Rat, Harlan
Sprague-Dawley
(male and
female)
Short term
(28 d)
Gavage3
Male and female:
0, 62.5, 125, 250,
500,1,000 mg/kg-d
++
+ +
++
++
NM
Chengelis et
al. (2009b)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
Subchronic
(90 d)
Gavage3
Male and female:
0,10, 50,
200 mg/kg-d
NM
+ +
NM
NM
Loveless et
al. (2009)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
Subchronic (90 d)
One-generation
reproductive: Po
females dosed 70 d
prior to cohabitation,
through gestation and
lactation (126 d); Po
males dosed for 110 d
Developmental:
Gestation Days 6-20
Gavageb
Male and female:
0, 20,100,
500 mg/kg-d
++
+ +
++
NM
++
Klaunig et
al. (2015)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
2-yr cancer bioassay
Gavage3
Male: 0, 2.5,15,
100 mg/kg-d
Female: 0, 5, 30,
200 mg/kg-d
NM
NM
++
++
NM
Iwai and
Hoberman
(2014)°
Mouse, Crl:
CDl(ICR) ()
(male and
female)
Developmental:
Gestation Days 6-18
Gavaged
Phase 1: 0,100,
350, 500 mg/kg-d
Phase 2: 0, 7, 35,
175 mg/kg-d
++
NM
++
NM
++
++ Outcome rating of high confidence; - outcome rating of low confidence; NM, outcome not measured.
a bStudy evaluation for animal toxicological endpoints reported from female reproductive studies with rats
receiving PFHxA,3 PFHxA sodium salt,b or PFHxA ammonium saltd by gavage. Study evaluation details for all
outcomes are available by clicking the HAWC link.
cPhase 1 was a range-finding study used to determine the appropriate dose range for identification of a NOAEL in
Phase 2.
++ Outcome rating of high confidence; - outcome rating of low confidence; NM, outcome not measured.
Fertility and Pregnancy Outcomes
Three studies published in two reports evaluated outcomes related to fertility and
pregnancy following exposure by gavage with PFHxA or PFHxA salts in rats or mice flwai and
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Toxicological Review of PFHxA and Related Salts
Hoberman. 2014: Loveless etal.. 20091. Some effects on maternal body weight change (i.e., gain or
loss) were noted. In both the developmental and one-generation reproductive rat studies (Loveless
etal.. 20091. statistically significant reductions in maternal body weight change were observed
during gestation in the high dose group (500 mg/kg-day). In the developmental study fLoveless et
al.. 20091. there was a statistically significant decrease in total maternal body weight gain (19%
relative to control) and when correcting for gravid uterine weight (26% relative to control) from
GD 6-21 in the 500 mg/kg-day dose group. In the one-generation reproductive study, similar
effects were observed but were limited to early gestation (Loveless etal.. 20091. From GD 0-7, body
weight gain in dams exposed to 500 mg/kg-day was reduced by 31% relative to controls. There was
no treatment-related effect on maternal weight gain over the entire gestational period (GD 0-21)
and the high dose (500 mg/kg-day) showed a statistically significant increase in body weight
change relative to controls during lactation (PND 0-21) fLoveless etal.. 20091. No changes in
maternal body weight gain were identified in mice (Iwai and Hoberman. 20141.
Only one of the three available studies reported effects on absolute maternal body weight.
In the developmental rat study, dams exposed to 500 mg/kg-day (GD 6-20) showed a statistically
significant decrease in terminal body weight (7% relative to control) fLoveless etal.. 20091. Deficits
remained when correcting for gravid uterine weight (5% relative to control), indicating the effects
on body weight were driven by maternal body weight rather than reductions in fetal body weight
or number of fetuses. However, this level of change may not be biologically significant fU.S. EPA.
19911. There was no effect on absolute maternal body weight in the one-generation reproductive
rat or mouse study (Iwai and Hoberman. 2014: Loveless etal.. 20091. These results are presented in
Figure 3-23.
No treatment-related effects on mating, pregnancy incidence, gestation length, number of
implantations, or litter size were reported in either study that evaluated these outcomes flwai and
Hoberman. 2014: Loveless etal.. 20091. Estrous cyclicity in rats exposed as adults or during
gestation was also unaffected in two studies fNTP. 2018: Loveless etal.. 20091.
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Toxicological Review of PFHxA and Related Salts
Endpoint
Study
Experiment
Animal Description Observation Time
Female Reproductive Effects: Body Weight
Body Weight Change, Gestation
Loveless, 2009, 2850369
14-Day Developmental
P0 Rat, Crl:CD(SD) (?)
GD 6-21
M • ~
1-Generation Reproductive
P0 Rat, Cr1:CD(SD)(?)
GD 0-7
M • ~
P0 Rat, Cr1:CD(SD) (?)
GD 0-21
M • ~
Iwai, 2014, 2821611
1-Generation Reproductive
P0 Mouse, CD-1 (?)
GD 6-18
~ • • ~
P0 Mouse, CD-1 (2)
GD 6-18
Body Weight Change, Gestation
Loveless, 2009, 2850369
14-Day Developmental
P0 Rat. Crl:CD(SD) (?)
GD 6-21
• • • v
(Minus Gravid Uterine Weight)
Body Weight Change, Lactation
Loveless, 2009, 2850369
1-Generation Reproductive
P0 Rat, Cr1:CD(SD) (?)
PND 0-21
M • ~
Iwai, 2014, 2821611
1-Generation Reproductive
P0 Mouse, CD-1 ($)
PND 0-20
• • • #
P0 Mouse, CD-1 ($)
PND 0-20
•
Body Weight, Absolute
Loveless, 2009, 2850369
1-Generation Reproductive
PO Rat, Crl:CD(SD) (?)
GD 0
M • •
P0 Rat, Cr1:CD(SD) (?)
GD 7
M • ~
P0 Rat, Cr1:CD(SD) (?)
GD 14
H • 1
P0 Rat, Cr1:CD(SD) (?)
GD 21
H • ~
Body Weight, Terminal (Minus Gravid
Loveless, 2009, 2850369
14-Day Developmental
P0 Rat, Crl:CD(SD)(?)
GD 21
• • • v
Uterine Weight)
Terminal Body Weight, Absolute
Loveless, 2009, 2850369
14-Day Developmental
P0 Rat, Cr1:CD(SD)(?)
GD 21
M • ~
Iwai, 2014, 2821611
1-Generation Reproductive
P0 Mouse. CD-1 ($)
PND 20
• • • 1
P0 Mouse, CD-1 ($)
PND 20
I • No significant change^ Significant increase V Significant
decrease |
-50 0 50 100 150 200 250 300 350 400 450 500 550
Dose (mg/kg-day)
Figure 3-23. Effects on body weight in female rats and mice exposed to PFHxA
or PFHxA ammonium salt in reproductive studies (HAWC: PFHxA - Animal
Toxicity Female Reproductive Supporting Table!.
Histopathology
Four studies evaluated effects on histopathology of reproductive organs (i.e., uterus and
ovaries) in rodents following exposure to PFHxA fNTP. 2018: Klaunig etal.. 2015: Chengelis etal..
2009b) or PFHxA sodium salt fLoveless etal.. 20091. Only NTP T20181 reported an effect of
exposure, with females showing a statistically significant increase in the incidence of bilateral
uterine horn dilation in all but the vehicle controls and highest dose group (see Figure 3-24).
Whereas the control and high-dose group had 10 animals per group, however, groups showing a
statistically significant increase had only 1-3 animals per group, complicating interpretation of
these findings. The total incidence ranges from 1 to 3 animals/treatment group, regardless of
sample size or PFHxA dose (see Figure 3-24). The biological significance of these results is unclear.
Uterine horn dilation can indicate an estrogenic effect, but no coherent changes in serum estradiol
or estrous cyclicity were observed in this study. Similarly, no other treatment-related effects on
female reproductive histopathology were reported fNTP. 2018: Klaunig etal.. 2015: Chengelis etal..
2009b: Loveless etal.. 2009).
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Toxicological Review of PFHxA and Related Salts
Endpoint Study Animal Description Dose Incidence Female Reproductive Histopathology
Uterus, Bilateral Dilation NTP, 2018, 4309149 Rat, Harlan Sprague-Dawley ( J) 0 1/10 (10.0%)
]
62.6 2/2(100.0%)
125 1/1 (100.0%)
250 2/2(100.0%)
500 3/3(100.0%)
1,000 1/10(10.0%)
1 1 1 I I 1 I 1 1
|l I percent affected I I Significant Compared to Control 1 ® ^0 ^ 40 80
1 1 % Affected
Figure 3-24. Female reproductive effects on uterine horn dilation in rats
exposed to PFHxA for 28 days (HAWC: PFHxA - Animal Toxicity Female
Reproductive link!.
Organ Weights
Three studies evaluated effects of PFHxA exposure on uterine and ovarian weights (NTP.
2018: Chengelis etal.. 2009b: Loveless etal.. 20091. Authors reported no treatment-related effects
for these outcomes.
Reproductive Hormones
Two studies measured effects of PFHxA or PFHxA ammonium salt on testosterone fNTP.
2018: Klaunig etal.. 20151. estradiol, and luteinizing hormone (Klaunigetal.. 20151. No
treatment-related effects were reported in either study.
Female Reproductive System Development
Two studies evaluated the potential for reproductive development effects following
developmental exposure to PFHxA ammonium or sodium salts. Iwai and Hoberman (20141 and
Loveless etal. (20091 found no effects on age at vaginal opening, a measure of puberty onset
Evidence Integration
A single low confidence human study reported a weak inverse association between PFHxA
exposure measures and serum levels of reproductive hormone levels in adolescents fZhou etal..
20161. Based on these results, there is indeterminate human evidence of female reproductive
effects.
In animals, evidence supporting effects of PFHxA exposure female reproduction was largely
limited to effects on maternal weight gain during gestation in rats exposed to 500 mg/kg-day
(Loveless etal.. 20091. These effects corresponded with a small but statistically significant absolute
body weight in the high confidence developmental rat study only fLoveless etal.. 20091. however
the level of the decrease (5%-7%) may not be biologically significant. There were no effects on
maternal weight or weight gain in the in the mouse study. The reported effects on uterine horn
dilation appears to be influenced by differences in sample sizes, as the total incidence of the finding
is similar across controls and all dosing groups. Furthermore, this finding is generally associated
with estrogenic effects, but no coherent changes were observed that would be indicative of
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Toxicological Review of PFHxA and Related Salts
estrogenic changes in females. No treatment-related changes were reported for other female
reproductive outcomes fNTP. 2018: Klaunig etal.. 2015: Iwai and Hoberman. 2014: Chengelis etal..
2009b: Loveless etal.. 20091. Based on these results, there is indeterminate animal evidence of
female reproductive effects.
Overall, the currently available evidence is inadequate to assess whether PFHxA might
cause female reproductive effects in humans (see Table 3-33).
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Toxicological Review of PFHxA and Related Salts
Table 3-33. Evidence profile table for female reproductive effects
Evidence stream summary and interpretation
Evidence integration
summary judgment
Evidence from studies of exposed humans
OOO
Evidence inadequate
Studies and
confidence
Factors that
increase
strength
Factors that decrease
certainty
Summary and key
findings
Evidence stream judgment
Reproductive
Hormones
1 low confidence
study
• No factors
noted
• Low confidence
study
• Nonsignificant inverse
association between
PFHxA exposure and
testosterone and
estradiol
ooo
Indeterminate
Evidence from animal studies
Studies and
confidence
Factors that
increase
strength
Factors that decrease
certainty
Summary and key
findings
Evidence stream judgment
Primary Basis:
Evidence is low
confidence or largely
null.
Human relevance:
• N/A (human and
animal evidence
both
indeterminate)
Cross stream
coherence:
• N/A (human and
animal evidence
both
indeterminate)
Fertilitvand
Pregnancv Outcomes
3 hiqh confidence
studies in rats and
mice:
• 28-d (rat)
• 90-d (rat)
• GD 6-18 (mouse)
• High
confidence
studies
• Unexplained
inconsistency
across studies
• Decreases in maternal
weight gain during
gestation in rats
exposed to
500 mg/kg-d
OOO
Indeterminate
The animal evidence is largely null. Some
evidence of female reproductive effects
but body weight effects lacked
consistency across studies.
Histopathology effects were not dose-
dependent and lacked coherent evidence
to support the biological significance of
the findings
HistoDathologv
4 high confidence
studies in rats and
mice:
• 28-d (rat)
• High
confidence
studies
• Unexplained
inconsistency
across studies
• Lack of expected
coherence with
• Increase in bilateral
uterus dilation
reported for all
groups except the
highest dose
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Toxicological Review of PFHxA and Related Salts
Evidence stream summary and interpretation
Evidence integration
summary judgment
• 90-d (rat)
• 2-yr (rat)
• GD 6-18 (mouse)
1 low confidence
study in adult rats:
• 90-d
other estrogen
related outcomes
Susceptible
populations:
• None identified
Organ Weights,
Reproductive
Hormones,
Reproductive System
Development
6 hiqh confidence
studies in rats and
mice:
• 28-d (rat)
• 90-d (rat, 2
studies)
• 2-yr (rat)
• GD 6-18 (mouse)
• GD 6-20 (rat)
• High
confidence
studies
• No factors noted
• No treatment-related
effects were reported
at <1,000 mg/kg-d
Mechanistic evidence and supplemental information
Biological events of
pathways
Biological
events of
pathways
Biological events of pathways
Biological events of pathways
• No studies Identified
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Toxicological Review of PFHxA and Related Salts
3.2.8. Immune Effects
Human
Asthma. Immune Markers, and Potentially Related Respiratory Outcomes
One medium confidence case-control study in Taiwan of asthma was reported in three
publications (Oin etal.. 2017: Zhou etal.. 2017: Dong etal.. 20131. Dong etal. (20131 includes
results from all three studies that examined the potential association between PFHxA exposure and
asthma incidence or severity, control of asthma symptoms and related immune markers (see
Figure 3-25). The study also measured pulmonary function. The only finding of note was a
nonmonotonic positive association between incident asthma (i.e., diagnosis in the previous year)
and PFHxA exposure (odds ratio [95% CI] for Q2: 1.2 [0.7, 2.1], Q3: 0.9 [0.5, 1.6], Q4: 1.6 [0.9, 2.9])
that was not statistically significant. No clear association was found with asthma severity or control
of asthma symptoms (Dong etal.. 20131. pulmonary function measured with spirometry (Oin etal..
20171. or immune markers (Dong etal.. 20131 among children with asthma. The exposure levels in
this study were low and contrast narrow (median [IQR]: 0.2 ng/mL [0.1-0.3 ng/mL]), which may
have reduced study sensitivity.
O0
Participant selection- +
Exposure measurement- +
Outcome ascertainment- +
Confounding- +
Analysis
Sensitivity
Selective Reporting
Overall confidence
Figure 3-25. Study evaluation for human epidemiological studies reporting
findings from PFHxA exposures (HAWC: PFHxA - Human Toxicity Immune
Effects link!.
The evaluation of Dong et al. (2013) encompasses all publications related to this study.
&¦
TP
Legend
Good (metric) or High confidence (overall)
Adequate (metric) or Medium confidence (overall)
Deficient (metric) or Low confidence (overall)
Critically deficient (metric) or Uninformative (overall)
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Toxicological Review of PFHxA and Related Salts
Animal
Several animal studies, including short-term (28-day), subchronic, reproductive, and
designs, evaluated toxicological findings of immune effects in rats receiving oral exposures of
PFHxA and PFHxA sodium salt. Most of the outcome-specific study ratings were considered high or
medium confidence; however, some specific concerns were identified that resulted in a low
confidence rating. Histopathology for Chengelis etal. f2009bl was rated low confidence because of
issues related to observational bias, concerns about endpoint sensitivity and specificity, and results
presentation. The results of the outcome-specific study evaluations are presented in Table 3-34 and
details are available by clicking the HAWC link.
Table 3-34. Study design characteristics and individual outcome ratings for
immune endpoints
Study
Species, strain (sex)
Exposure design
Exposure route and
dose
Organ weight
Histopathology
Immune cell
counts
NTP(2018)
Rat, Harlan
Sprague-Dawley
(male and female)
Short term
(28 d)
Gavage3
Male and female: 0,
62.5, 125, 250, 500,
1,000 mg/kg-d
+ +
++
++
Kirkpatrick
(2005a)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and female)
Reproductive: Po animals
dosed 14 d prior to mating
through end of mating
period (male) or PND 4
(female)
Gavage3
Male and female: 50,
150 and
300/450° mg/kg/d
+
+
+
Chengelis et
al. (2009b)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and female)
Subchronic
(90 d)
Gavage3
Male and female: 0,
10, 50, 200 mg/kg-d
+ +
"
++
Loveless et al.
(2009)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and female)
Subchronic (90 d)
Gavageb
Male and female: 0,
20,100, 500 mg/kg-d
+ +
++
++
Klaunig et al.
(2015)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and female)
2-yr cancer bioassay
Gavage3
Male: 0, 2.5,15,
100 mg/kg-d
Female: 0, 5, 30,
200 mg/kg-d
NM
++
++
++ Outcome rating of high confidence; + outcome rating of medium confidence; - outcome rating of low
confidence; NM, outcome not measured.
a bStudy evaluation for animal toxicological immune endpoints reported from studies with male and female rats
receiving PFHxA3 or PFHxA sodium saltb by gavage. Study evaluation details for all outcomes are available by
clicking the HAWC link.
cDue to high toxicity at the 450 mg/kg-d exposed animals, dosage was reduced to 300 mg/kg-d on exposure day 4.
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Toxicological Review of PFHxA and Related Salts
Organ Weights
Four studies evaluated effects on spleen and thymus weights in response to PFHxA fNTP.
2018: Chengelis etal.. 2009b: Kirkpatrick. 2005al or PFHxA sodium salt fLoveless etal.. 20091
exposure.
The available evidence identified, in general, decreased absolute or relative thymus weights.
Two studies reported statistically significant decreases in absolute thymus weights in males
exposed to 500 mg/kg-day PFHxA sodium salt for 90 days (Loveless etal.. 20091 or
450/300 mg/kg-day in reproductive study (Kirkpatrick. 2005al. Similarly, downward trends in
both relative and absolute thymus weights thymus were reported in males and females receiving
PFHxA in the short term fNTP. 20181. A single study qualitatively reported no treatment-related
effects on thymus weights fChengelis etal.. 2009bl.
Spleen weights did not show a clear pattern of effect across studies. In the short-term study,
a trend of increased weights in males and females receiving PFHxA (NTP. 20181 was observed,
whereas spleen weights were decreased in males receiving PFHxA sodium salt in the 90-day study
by Loveless etal. (20091. Both Chengelis etal. (2009bl and Kirkpatrick (2005al reported no
treatment-related effects on spleen after exposure to <450 mg/kg-day PFHxA for up to 90 days.
Results are summarized in Figure 3-26.
Endpoint
Study
Experiment
Animal Description
Observation Time
Immune Effects: Organ Weights
Spleen Weight, Absolute
Kirkpatrick, 2005, 4940398
Reproductive Oral PFHxA
PO Rat, Cr1:CD(SD) (?)
PND 4
•
PO Rat, Cr1:CD(SD) (c?)
Primary necropsy
•
Loveless, 2009, 2850369
90-Day Oral
Rat, Crl:CD(SD) ( ?)
Day 92
Rat. Crl:CD(SD) ( ?)
Day 92
V
NTP. 2018. 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley (- ]
Day 29
Rat, Harlan Sprague-Dawley (
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Toxicological Review of PFHxA and Related Salts
Histopathologv
Five studies examined spleen, thymus, lymph nodes, or bone marrow for histopathological
changes fNTP. 2018: Klaunig etal.. 2015: Chengelis etal.. 2009b: Loveless et al.. 2009: Kirkpatrick.
2005a). Some evidence of effects on immune related histopathology was reported by NTP f20181
reported an increased incidence of extramedullary hematopoiesis in the spleens of males and
females at 1,000 mg/kg-day after a 28-day exposure. Minimal to mild extramedullary
hematopoiesis also was found in the spleens of male rats receiving 500 mg/kg-day PFHxA sodium
salt (Loveless etal.. 20091. This effect was coincident with erythroid hyperplasia of the bone
marrow of males and females and might be related to the effects on red blood cells (discussed in
"Hemostasis" of Section 3.2.4) rather than an immune-specific effect. These changes did not persist
after the 30-day recovery and specific incidence data were not reported fLoveless etal.. 20091. A
reproductive study in rats reported increased incidence of histopathologic changes in the spleen,
thymus, and lymph nodes in the high dose group (450/300 mg/kg-day). Notably, these findings
were limited to unscheduled deaths (i.e., found dead or euthanized in extremis) in animals showing
signs of overt toxicity, therefore, these results may not reflect an immune-specific effect. No other
effects were reported for immune-related tissues fNTP. 2018: Klaunig etal.. 2015: Chengelis etal..
2009b: Loveless etal.. 2009: Kirkpatrick. 2005al.
Immune Cell Counts
Five animal studies evaluated hematological indicators of immunotoxicity (NTP. 2018:
Klaunig etal.. 2015: Chengelis etal.. 2009b: Loveless etal.. 2009: Kirkpatrick. 2005 a). Of these
studies, NTP (2018) and Loveless etal. (2009) reported increased neutrophils at doses as low as
20 mg/kg-day and decreased basophils in males receiving >250 and 500 mg/kg-day PFHxA or
PFHxA sodium salt, respectively. No effects were observed on basophils or neutrophils in the other
three rat studies (reproductive, -d90-, and -y2-) at exposures to PFHxA as high as 450 mg/kg-day
(Klaunig et al.. 2 015: Chengelis etal.. 2009b: Kirkpatrick. 2005a). Eosinophils were decreased only
in males exposed to PFHxA sodium salt for 90 days (Loveless etal.. 2009). No other
treatment-related effects were reported for specific white blood cell populations or total white
blood cell counts following PFHxA or PFHxA sodium salt exposures in rats (NTP. 2018: Klaunig et
al.. 2015: Chengelis etal.. 2009b: Loveless etal.. 2009: Kirkpatrick. 2005al. Results are summarized
in Figure 3-27.
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Toxicological Review ofPFHxA and Related Salts
Endpoint
Study
Experiment
Animal Description
Observation Time
Immune Effects: Immune Cell Counts
Basophils
NTP, 2018. 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley ( ')
Day 29
• • V
\7
T
Rat, Harlan Sprague-Dawley {:)
Day 29
Loveless, 2009, 2850369
90-Day Oral
Rat, Ci1:CD(SD) \ i
Day 93
Kirkpatrick, 2005, 4940398
Reproductive Oral PFHxA
ru rsal, wn.UUpU) ^ )
Day 28
• • • •
P0 Rat, Cri:CD(SD) (?)
pwn a
^ m .
KINU 4
Lymphocyte
NTP, 2018. 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley ( ')
Day 29
Rat, Harlan Sprague-Dawley (.)
Day 29
Loveless. 2009, 2850369
90-Day Oral
Rat, Cr»:CD(SD) (¦¦?)
Day 92
RaL Crt:CD(SD) (J)
Day 93
Kirkpatrick. 2005. 4940398
Reproductive Oral PFHxA
P0 Rat. Cri:CD(SD) ( ')
Day 28
• •—• ~
P0 Rat. Cri:CD(SD) (y)
PND 4
Lymphocyte, Total
NTP, 2018. 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley (_')
Day 29
Rat, Harlan Sprague-Dawley ( )
Day 29
Monocytes
NTP. 2018. 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley ( )
Day 29
-
Rat, Harlan Sprague-Dawley (- )
Day 29
Loveless. 2009. 2850369
90-Day Oral
Rat, Crt:CD(SD) (•")
Day 92
Rat, Cr1:CD(SD) (?)
Day 93
Kirkpatrick. 2005, 4940398
Reproductive Oral PFHxA
P0 Rat. Crl:CD(SD) ( ')
P0 Rat, Cri:CD(SD) ( ?)
Day 28
PND 4
• • • ¦*
Neutrophils
NTP 2018 4309149
¦jr n n i
Rat, Harlan Sprague-Dawley (-')
n 70
ay urai
Day ^3
W •
Rat, Harlan Sprague-Dawley (1)
Day 29
A • • ¦
A
V • • •
a
Loveless. 2009, 2850369
90-Day Oral
Rat, Crt:CD(SD) ( ?)
Day 92
A
A
ci
Rat, Crl:CD(SD)(r)
Day 93
Kirkpatrick. 2005, 4940398
Reproductive Oral PFHxA
P0 Rat. Crt:CD(SD)( ^)
Day 28
*
P0 Rat. Crt:CD(SD) (y)
PND 4
• •—• «
White Blood Cell (WBC)
Klaunig. 2015. 2850075
2-Year Cancer Bioassay
Rat, Cr1:CD(SD) (*)
o^i fH-rrvQni t \
Week 25
Week 51
Week 104
•—•
*—•
•—•
r\ai, ^n.bL/(oui ( i
vveeK
Week 51
Week 104
-—•
NTP. 2018. 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley ( ')
Day 29
• • • •
Rat, Harlan Sprague-Dawley ('z.)
Day 29
Chengelis, 2009.2850404
90-Day Oral
Rat, CH:CD(SD) (c?)
Day 90
*-• ~
Rat, Cri:CD(SD) (?)
Day 90
~
Loveless, 2009, 2850369
90-Day Oral
Rat, Crt:CD(SD) ( ?)
Day 92
Rat, Crt:CD(SD) (2)
Day 93
Kirkpatrick. 2005, 4940398
Reproductive Oral PFHxA
P0 Rat, Crt:CD(SD) (-')
Day 28
P0 Rat, Crf:CD(SD) ( t»)
Rat, Crt:CD(SD) (,.")
PND 4
• • • •
White Blood Cell (WBC). Recovery
Chengelis, 2009. 2850404
90-Day Oral
Day 118
• •
Rat, Cr1:CD(SD) (
Day 118
• •
i i i i
1 1 1 1 1
400 500 600 700 800
i i
[ • No significant change^ Significant increa
se ~ Significant decrease Q Significant Trend | 100 0 100 200 30C
900 1,000 1.100
Dose (mg/kg-bw/day)
Figure 3-27. Immune cell counts in rats exposed by gavage to PFHxA or PFHxA
sodium salt (HAWC: PFHxA - Animal Toxicity Immune Effects link!.
Evidence Integration
The human evidence was limited to one medium confidence study that showed no clear
association between PFHxA exposure and immune-related health outcomes, although the authors
did observe a nonsignificant trend toward an association with asthma diagnosis in the previous
year. Based on these results, there is indeterminate human evidence of immune effects.
Except for changes in thymus weight, the available animal toxicological evidence did not
show a clear pattern of effect across studies. Specifically, three studies reported treatment-related
changes in thymus and spleen weights in rats, but the direction of effect on spleen weights was not
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Toxicological Review of PFHxA and Related Salts
consistent across studies. Extramedullary hematopoiesis was the only histopathological finding of
note, but this is interpreted as possibly secondary to the effects on red blood cells rather than an
immune-specific effect and is discussed in that context in Section 3.2.4. Increases in neutrophils and
decreases in basophils showed a consistent direction of effect across two studies (of the five
available). Eosinophils also were decreased, but only in males in a single study. No other treatment-
related changes were observed for immune cell counts (i.e., specific cell populations or total white
blood cells), and discerning the biological significance of this pattern is difficult in isolation.
The evidence supporting the potential immunotoxicity to humans is limited by several
factors, including the lack of consistency across studies for several of the affected outcomes.
Furthermore, the evaluated outcomes are limited to changes in the structural components of the
immune system, which are less predictive indicators of immunotoxicity flPCS. 20121. Notably, there
is evidence indicating that other PFAS, including PFOS and PFOA, may affect immune system
function through suppression of antibody response and induction of hypersensitivity (Dewittetal..
2019). Additional studies, particularly those that evaluate changes in immune function would be
beneficial for understanding the potential for adverse effects of PFHxA exposure on the immune
system. Based on these results, there is indeterminate animal evidence of immune effects.
Overall, the currently available evidence is inadequate to determine whether PFHxA
exposure might cause immune system effects in humans (see Table 3-35).
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Toxicological Review of PFHxA and Related Salts
Table 3-35. Evidence profile table for immune effects
Evidence stream summary and interpretation
Evidence integration
summary judgment
Evidence from studies of exposed humans
OOO
Evidence inadequate
Primary basis:
Studies and
confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key findings
Evidence stream
judgment
Asthma
1 medium
confidence study
• No factors noted
• Imprecision
• Lack of coherence - no
associations with other
measures of pulmonary
function
• Nonsignificant association
with asthma diagnosis, but
other asthma-related
outcomes were not
affected.
ooo
Indeterminate
Evidence is low
confidence or limited
Human relevance:
• N/A (indeterminate
animal evidence)
Cross-stream
coherence:
N/A (human evidence
Evidence from animal studies
Studies and
confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key findings
Evidence stream
judgment
Histopathology
4 hiqh and medium
confidence studies
in adult rats:
• 28-d
• 90-d
• 2-yr
• Reproductive
1 low confidence
study in rats;
• 90-d
• High and medium
confidence studies
• Consistency across
studies for
extramedullar
hematopoiesis
• No factors noted
• Increased splenic
extramedullar
hematopoiesis was
observed male and female
rats at 500 mg/kg-d;
coincident with minimal
erythroid hyperplasia of the
bone marrow
• Several immune-related
histopathology findings
reported in male and
female rats at
300/450 mg/kg-d, but only
in animals with
unscheduled deaths
OOO
Indeterminate
Some evidence of
immune system but
limited by unexplained
inconsistency, lack of
coherence, and
potential for non-
immune related causes
[see Section 3.2.4 for
additional discussion].
Available evidence was
consisted of
observational outcomes
that are less predictive
of immune system
indeterminate)
Susceptible populations
and life stages:
• No evidence to
inform
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Toxicological Review of PFHxA and Related Salts
Evidence stream summary and interpretation
Evidence integration
summary judgment
Immune Cell Counts
5 high and medium
confidence studies
in rats:
• 28-d
• 90-d (2 studies)
• 2-yr
• Reproductive
• High and medium
confidence studies
• Cons/stency-studies for
neutrophils and basophils
• Lack of coherence with
other immune markers
• Decreased basophil counts
and increased neutrophil
cell counts at >20 mg/kg-d
toxicity and in one study
were only observed in
that died or were
removed from the study
due to overt toxicity.
Organ Weight
4 hiqh and medium
confidence studies
in rats:
• 28-d
• 90-d (2 studies)
• Reproductive
• High and medium
confidence studies
• Unexplained
inconsistency across
studies for spleen
weights
• Thymus weights decreased
at 500 mg/kg-d in
short-term, subchronic, and
reproductive studies
• Changes in spleen weight
were inconsistent in the
direction of effect across
studies
Mechanistic evidence and supplemental information
Biological events of
pathways
Biological events of
pathways
Biological events of pathways
Biological events of
pathways
• No studies Identified
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Toxicological Review of PFHxA and Related Salts
3.2.9. Nervous System Effects
Human
No studies were identified that evaluated the effects of PFHxA on the nervous system in
humans.
Animal
Four short-term (28-day), subchronic, and chronic animal studies evaluated the effects of
PFHxA or PFHxA sodium salt in rats. Most outcome-specific study ratings were high or medium
confidence. One study was rated low confidence for histopathology due to concerns about
observational bias, endpoint sensitivity and specificity, and data presentation fChengelis et al..
2009b). A summary of the studies and the interpretations of confidence in the results for the
different outcomes based on the individual study evaluations is presented in Table 3-36, and details
are available by clicking the HAWC link.
Table 3-36. Nervous system endpoints for PFHxA and associated confidence
scores from repeated-dose animal toxicity studies
Author (year)
Species, strain
(sex)
Exposure
design
Exposure route and
dose range
Brain weight
Histopathology
Behavior
NTP(2018)
Rat, Harlan
Sprague-Dawley
(male and female)
Short term
(28 d)
Gavage3
Male and female: 0, 62.5,125,
250, 500,1,000 mg/kg-d
++
++
NM
Chengelis et al.
(2009b)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
Subchronic
(90 d)
Gavage3
Male and female: 0,10, 50,
200 mg/kg-d
++
+
Loveless et al. (2009)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
Subchronic
(90 d)
Gavageb
Male and female: 0, 20,100,
500 mg/kg-d
++
++
++
Klaunig et al. (2015)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
2-yr cancer
bioassay
Gavage3
Male: 0, 2.5,15,100 mg/kg-d
Female: 0, 5, 30, 200 mg/kg-d
NM
++
++
++ Outcome rating of high confidence; + outcome rating of medium confidence; - outcome rating of low
confidence; NM, outcome not measured.
a bStudy evaluation for animal toxicological nervous system endpoints reported from studies with male and female
rats receiving PFHxA3 or PFHxA sodium saltb by gavage. Study evaluation details for all outcomes are available by
clicking the HAWC link.
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Toxicological Review of PFHxA and Related Salts
Brain Weight
Three studies evaluated effects of PFHxA or PFHxA sodium salt on the nervous system in
animals fNTP. 2018: Chengelis etal.. 2009b: Loveless etal.. 20091. Two studies reported increases
in relative but not absolute brain weights after exposure to PFHxA or PFHxA sodium salt for 28 or
90 days, respectively fChengelis etal.. 2009b: Loveless etal.. 20091. These effects were observed at
the highest dose tested (200 or 500 mg/kg-day) and affected only males in one study (Loveless et
al.. 20091 and only females in the other (Chengelis etal.. 2009b). Notably, relative weights are not
considered appropriate for brain weight measurements because this measure is not typically
affected by fluctuations in body weight fU.S. EPA. 19981: therefore, absolute brain weights are
preferred.
Other Nervous System Effects
No treatment-related effects were observed on other nervous system outcomes, including
behavior (i.e., open field locomotor activity, functional observational battery) and histopathology
(NTP. 2018: Klaunigetal.. 2015: Chengelis etal.. 2009b: Loveless etal.. 20091.
Mechanistic Evidence and Supplemental Information
Two studies evaluated effects of PFHxA exposure on neurodevelopment in zebrafish using
wildtype fGuo etal.. 2021: Gaballah etal.. 20201 and transgenic [Tg (HuC-GFP)] fGuo etal.. 20211
strains exposed during embryonic and early larval development Both reported effects on larval
swimming behavior, with larvae showing an increase in swimming activity at low to moderate
doses but no effect at the higher doses. Guo etal. (20211 evaluated several other nervous system
related outcomes in wildtype larvae, including acetylcholinesterase activity, and neurotransmitter
levels. Acetylcholinesterase activity was statistically significantly reduced at all dose levels.
Treatment-related effects on neurotransmitter levels was largely limited to the high exposure
group (12 mg/L), with dopamine, DOPAC, and GABA levels showing 92% to 174% increases
relative to controls. Acetylcholine was slightly reduced in the 2.4 mg/L exposure group and there
was no effect on serotonin. A transgenic strain, Tg (HuC-GFP), that expresses GFP in neuronal cells
was used to evaluate effects on neurodevelopment and proliferation in the central nervous system
in vivo. Larvae in the high exposure group showed a 48% decreased in GFP expression, but there
were no effects in the lower exposure groups.
Two studies evaluated effects on expression of genes and proteins related to
neurodevelopment in primary avian neuronal cells fVongphachan etal.. 20111 and larval zebrafish
(Guo etal.. 20211. Only one gene was evaluated in both studies, mbp, which is essential for
myelination of nerves, showed increased expression in both models, although this was only
observed at the lowest tested concentration in the zebrafish study. Guo etal. (20211 examined 17
additional genes that are involved in various aspects of nervous system function and development
in zebrafish larvae exposed up to 120 hours post fertilization. The results are summarized in in
Table 3-37. Statistically significant changes in gene expression were reported for every gene.
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Although some genes showed increased expression (mbp, dat, bdnf, nr4a2b), most were decreased.
Protein expression data from the same study provide some support for these results, al-tubulin,
elavl3, and gap43 protein levels were reduced in animals exposed to med or high concentrations
showing a similar pattern and direction of effect as the gene expression results. Vongphachan et al.
f20in also measured effects on rc3, which is important for Ca2+ signal transduction and may play
a role in long term potentiation but found no treatment-related effects.
Table 3-37. Changes in expression of genes related to neurodevelopment in
early life stage zebrafish
Gene
Function/Role
0.48 mg/L
PFHxA
2.4 mg/L
PFHxA
12 mg/L
PFHxA
chrna7
Acetylcholine receptor
-
-
ache
Acetylcholinesterase
-
mbp
Myelination
-
-
al-tubulin
Axon/dendrite growth
-
-
shha
Axon growth
-
elavl3
Differentiation of ganglion, amacrine cells
-
-
gap43
Synaptogenesis; axon development/regeneration
-
syn2a
Synaptogenesis
gfap
Astroglia development
-
-
th2
Serotoninergic neuron marker
-
htrlab
Serotonin receptor
4,
htrlb
Serotonin receptor
4,
htr2a
Serotonin receptor
4,
htrlaa
Serotonin receptor
4,
htr5a
Serotonin receptor
-
-
4,
dat
Dopamine reuptake
-
-
bdnf
Neuronal differentiation
-
nr4a2b
Development of dopaminergic neurons
- no statistically significant change relative to control; 4^, statistically significant decrease relative to control;
statistically significant increase relative to control.
Evidence Integration
No human studies were identified to inform the potential nervous system effects of PFHxA
or PFHxA salts; therefore, there is indeterminate human evidence of nervous system effects.
In animals, the only available evidence to support an effect of PFHxA or PFHxA salts the
nervous system stems from increase in relative brain weights, which is not considered a reliable
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measure of neurotoxicity fU.S. EPA. 19981. No treatment-related effects were reported for other
nervous system outcomes.
Although the available animal toxicity data are largely null and derived from low risk of bias
studies, some uncertainties and data gaps remain. The results are limited to a small number of
studies in adult animals, and the evidence base is lacking studies that could inform potential for
nervous system effects when exposure occurs during development. This lifestage is a known critical
window of sensitivity for nervous system effects fU.S. EPA. 19981 and has been identified as a
research area of potential concern for other PFAS known to affect thyroid function. Two studies
provide limited mechanistic evidence and supporting information suggesting the potential for
neurodevelopmental effects of PFHxA. Based on these results, there is indeterminate animal
evidence of nervous system effects and additional studies would be necessary to draw a more
definitive judgment
Overall, the currently available evidence is inadequate to assess whether PFHxA might
cause nervous system effects in humans (see Table 3-38).
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Table 3-38. Evidence profile table for nervous system effects
Evidence stream summary and interpretation
Evidence integration
summary judgment
Evidence from studies of exposed humans
OOO
Studies and
confidence
Factors that
increase
certainty
Factors that decrease certainty
Summary and key
findings
Strength of
evidence
Evidence inadequate
Primary Basis:
No evidence in humans and
animal evidence is largely null
or lacking biological relevance.
• No studies identified
ooo
Indeterminate
Evidence from animal studies
Human relevance:
Studies and
confidence
Factors that
increase
certainty
Factors that decrease certainty
Summary and key
findings
Strength of
evidence summary
N/A (indeterminate animal
evidence)
Brain Weight
3 hiqh confidence
studies in adult rats:
• 28-d
• 90-d (2 studies)
• High
confidence
studies
• No factors noted
• Increased relative
brain weights
(preferred
metric) in
animals at
>200 mg/kg-d;
absolute brain
weight
unaffected
OOO
Indeterminate
Evidence is largely
null. The only
evidence of nervous
system effects was
relative brain
weight increases,
which is not
• Cross stream coherence:
N/A (human evidence
indeterminate).
Susceptible populations and
lifestages:
No evidence to inform.
Other inferences:
Some mechanistic data and
supplemental information
were identified that provide
limited support for potential
PFHxA mediated nervous
system effects. These were
limited to
neurodevelopmental models
for which there are no
available animal data and
Histopathology
3 hiqh confidence
studies in adult rats:
• 28-d
• 90-d
• 2-yr
1 low confidence
study in adult rats:
• High
confidence
studies
• No factors noted
• No
treatment-relate
d effects
reported
considered to be
appropriate for
evaluating nervous
system toxicity.
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Evidence stream summary and interpretation
Evidence integration
summary judgment
• 90-d
there are insufficient to inform
a potential MOA.
Behavior
2 hiqh confidence
studies in adult rats:
• 90-d
• 2-yr
1 medium confidence
study in adult rats:
• 90-d
• High and
medium
confidence
studies
• No factors noted
• No
treatment-relate
d effects
reported
Mechanistic evidence and supplemental information
Biological events or
pathways
Key findings, interpretation, and limitations
Evidence stream
summary
Behavior
Key findings and interpretation:
• Alterations in larval swimming activity observed following early life exposure
in zebrafish
• Increased swimming activity at low to moderate exposure levels in two
studies
• Statistically significant effects seen mostly at lowest exposure level
Limitations:
• Small evidence base with effects not showing a clear dose response
Some support for
potential effects of
PFHxA on
neurodevelopment,
but these are
largely limited to a
single study in early
life stage zebrafish.
Neurodevelopment
Key findings and interpretation:
• Neuron-specific GPF expression statistically significantly reduced in vivo in
larval zebrafish, suggesting treatment-related effects on neuro proliferation
• Effects only observed in high exposure group
Limitations:
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Evidence stream summary and interpretation
Evidence integration
summary judgment
• Small evidence base
Acetylcholinesterase
Activity
Key findings and interpretation:
• Decreased acetylcholinesterase activity observed in larval zebrafish at in all
dose groups
Limitations:
• Small evidence base
Neurotransmitter
Levels
Key findings and interpretation:
• Altered neurotransmitter levels in whole body zebrafish larvae observed at
all dose levels
Limitations:
• Small evidence base
Gene and Protein
Expression
Key findings and interpretation:
• Changes observed in 18 genes and 3 proteins related to nervous system
function and development in larval zebrafish
• Decreases in both gene and protein expression for al-tubulin, elavl3, and
gap43 in zebrafish
• Two studies in zebrafish and avian neuronal cells found decreases in gene
expression of mbp, but no clear dose response in zebrafish (low exposure
group only)
Limitations:
• Small evidence base
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3.3. CARCINOGENICITY
3.3.1. Cancer
Human Studies
One low confidence case-control study of breast cancer and PFHxA exposure is available in
humans (Velarde etal.. 20221. This study reported a positive association in the fourth quartile
(OR = 2.66, 95% CI: 0.95-7.66 vs. Ql), but the association was non-monotonic across quartiles
(inverse associations were reported in the second and third quartiles) and there were serious
concerns for potential selection bias and exposure misclassification (due to lack of temporality in
exposure measurement), so there is considerable uncertainty in this finding.
Animal Studies
A high confidence cancer bioassay conducted in rats evaluated neoplastic and non-
neoplastic lesions in the lungs, kidney, stomach, and liver of male rats dosed with 0, 2.5,15, or
100 mg/kg-day and in female rats dosed with 0, 5, 30, or 200 mg/kg-day fKlaunig et al.. 20151.
Findings for nonneoplastic and neoplastic lesions were reported as null and are summarized in
HAWC and in PFHxA Tableau.
Genotoxicity
Genotoxic, mutagenic, and clastogenic effects of PFHxA have been tested in several
mammalian and prokaryotic cell systems in vitro (see Table 3-39) (Eriksen etal.. 2010: Nobels et
al.. 2010: Loveless et al.. 20091. Sodium perfluorohexanoate (NaPFHx) was negative for
mutagenicity in Escherichia coli strain WP2uvrA and Salmonella typhimurium strains TA98, TA100,
TA1535, and TA1537 in both the presence and absence of exogenous S9 metabolic activation
(Loveless etal.. 20091. Nobels etal. (20101 examined the ability of PFHxA to induce the expression
of 14 prokaryotic stress response genes after exposure of the E. coli K-12 derivative SFi to 0.0156-1
mM PFHxA. The results of this study demonstrated that PFHxA did not significantly induce the
expression of regulatory elements critical for the prokaryotic gene expression response to oxidative
stress (katG, zwf soi28, and nfo), membrane damage [micF and osmY), general cell lesions [uspA and
cIpB), heavy metal stress (merR), and DNA damage [nfo, recA, umuDC, ada, sfiA, and dinD). In
mammalian cells in vitro, PFHxA did not generate reactive oxygen species (ROS) or oxidative
deoxyribonucleic acid damage in the human hepatoma cell line, HepG2 (Eriksen et al.. 20101. Lastly,
NaPFHx failed to induce chromosomal aberrations in human peripheral blood lymphocytes in the
presence and absence of exogenous metabolic activation, suggesting a lack of clastogenic activity
(Loveless etal.. 20091.
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Evidence Integration
A single low confidence human study reported a positive association between PFHxA
exposure measures and breast cancer; however, this association was limited to the fourth quartile
with inverse associations reported for the second and third quartiles fVelarde etal.. 20221. In
animals, one study fKlaunig etal.. 20151 evaluated the potential carcinogenicity of oral PFHxA
exposure via histological evaluation of the lung, kidney, stomach, and liver of male rats, and did not
observe significant treatment-related effects, and the few studies examining markers of potential
genotoxicity were largely null. No studies of potential carcinogenicity in via other exposure routes
were identified. Given the sparse and notably uncertain evidence base, and in accordance with the
Guidelines for Carcinogen Risk Assessment fU.S. EPA. 20051 EPA concluded there is inadequate
information to assess carcinogenic potential for PFHxA for any route of exposure.
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Table 3-39. Summary of PFHxA genotoxicity studies
PFHxA genotoxicity
Endpoint
Test system
Doses/
concentrations
tested
Results3
Comments
References
Without
exogenous
activation
With
exogenous
activation
ROS production
HepG2
(human
hepatoma
cell line)
0.4, 4, 40, 200, 400,
1,000, 2,000 nM
NA
Intracellular reactive oxygen species (ROS) production was
measured using 2',7'-dichlorofluorescein diacetate. ROS
production was measured every 15 min for 3 hr. No clear
concentration-response relationship was observed for PFHxA,
whereas exposure to H2O2 (positive control) generated ROS in
a concentration dependent manner.
Eriksen et al.
(2010)
DNA damage
HepG2
(human
hepatoma
cell line)
100, 400 nM
NA
Comet assay to detect the formation of DNA strand breaks
(including alkali-labile sites) and formamidopyrimidine-DNA-
glycosylase sensitive sites after 24-hr exposure. Cytotoxicity
was monitored by measuring lactate dehydrogenase (LDH)
activity to ensure observed DNA damage was not secondary to
cytotoxicity.
Eriksen et al.
(2010)
Cell stress-
dependent
gene expression
Escherichia
coli
K-12
derivative SFi
0.0156, 0.03125,
0.0625, 0.125, 0.25,
0.5, 1 mM
NA
Promoters of 14 prokaryotic DNA-damage responsive genes
were fused to lacZ cassettes and expressed in E. coli.
Activation of gene expression was measured after 90 min of
exposure by p-galactosidase reduction capacity and
spectrophotometrically at 420 nm. Genes involved in
prokaryotic DNA damage and repair (umuDC and ada) were
upregulated at approximately >1.4-fold but did not reach
statistical significance at any dose. Study authors did not
provide complete data for analysis.
Nobels et al.
(2010)
Mutation
(Ames assay)
Salmonella
typhimurium
strains TA98,
TA100,
TA1535, and
TA1537
333, 667,1,000,
3,333, 5,000 ng/plate
sodium
perfluorohexanoate
(NaPFHx)
Assay performed according to OECD Guideline 471. No
positive mutagenic responses were observed at any dose level
or with any tester strain in the presence or absence of S9
metabolic activation.
Loveless et al.
(2009)
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PFHxA genotoxicity
Endpoint
Test system
Doses/
concentrations
tested
Results3
Comments
References
Without
exogenous
activation
With
exogenous
activation
Mutation
E. coli
WP2uvrA
333, 667,1,000,
3,333, 5,000 ng/plate
sodium
perfluorohexanoate
(NaPFHx)
Assay performed according to OECD Guideline 471. No
positive mutagenic responses were observed at any dose level
or with any tester strain in the presence or absence of S9
metabolic activation.
Loveless et al.
(2009)
Chromosomal
aberration
Human
peripheral
blood
lymphocytes
4h (nonactivated):
2,000, 3,000,
3,860 ng/mL sodium
perfluorohexanoate
(NaPFHx)
4 hr (activated) and
22 hr (nonactivated):
250, 500,
1,000 ng/mL sodium
perfluorohexanoate
(NaPFHx)
Assay performed according to OECD Guideline 473.
Percentage of cells with structural or numerical aberrations
was not significantly increased above that of the vehicle
control at any concentration. Aroclor-induced rat liver S9 was
used for exogenous metabolic activation. Mitomycin C and
cyclophosphamide were used as positive controls. Substantial
toxicity (defined as a reduction in the mitotic index of >50% in
the NaPFHx treated cell culture as compared to vehicle
control) was observed in all test conditions.
Loveless et al.
(2009)
Cell
Proliferation
and neoplastic
transformation
Human
breast
epithelial
cells
(MCF-10A)
500 pM, 1 nM,
10 nM, 100 nM,
500 nM, 1 |jM,
10 |jM, 100 |jM,
500 |jM
Cell proliferation was inferred from cell viability (measured by
the MTT assay) and cell counting after 72 hrs PFAS treatment.
No alterations were identified for PFHxA at any of the
concentrations tested.
Neoplastic transformation was measured by transwell
migration and invasion assays. No positives were identified for
PFHxA compared to controls at any of the concentrations
tested.
(Pierozan et
al., 2022)
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PFHxA genotoxicity
Endpoint
Test system
Doses/
concentrations
tested
Results3
Comments
References
Without
exogenous
activation
With
exogenous
activation
DNA damage in
sperm
Human
sperm cells
obtained
from
nonsmoker
healthy men
(n = 3, 27-34
yr old)
100 piM, 300 piM,
1000 nM
DNA damage was analyzed using the alkaline comet assay. No
significant DNA damage was identified in human sperm cells
treated with PFHxA.
(Emerce and
Cetin, 2018)
a- = negative; NA = not applicable.
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4.SUMMARY OF HAZARD IDENTIFICATION
CONCLUSIONS
4.1. SUMMARY OF CONCLUSIONS FOR NONCANCER HEALTH EFFECTS
For all noncancer health effects, limited or no human epidemiological evidence was
available. Therefore, conclusions were based primarily on animal toxicological studies. The animal
evidence base consists of a short-term daily exposure to 0, 62.5,125, 250, 500,1000 mg/kg PFHxA
for 28 days fNTP. 20181. subchronic daily exposure to 0,10, 50, 200 mg/kg PFHxA fChengelis etal..
2009b), subchronic daily exposure to 0, 20,100, 500 mg/kg PFHxA sodium salt (Loveless etal..
20091. and chronic daily exposure for two years to 0, 5, 30, 200 mg/kg or 0, 2.5,15,100 mg/kg
PFHxA female or male respectively (Klaunig etal.. 20151. Two developmental, gestational exposure,
studies (Iwai and Hoberman. 2014: Loveless etal.. 20091 and a one-generation reproductive study
fLoveless et al.. 20091 with maternal oral doses between 7-500 mg/kg-day also were also available.
The outcome-specific ratings for these studies were generally high confidence.
As described in detail in Section 3, the available evidence indicates that PFHxA exposure is
likely to cause hepatic (Section 3.2.1), developmental (Section 3.2.2), hematopoietic (Section 3.2.4),
and endocrine effects (Section 3.2.5) in humans, given sufficient exposure conditions. As previously
noted, the "sufficient exposure conditions" are more fully evaluated and defined for the identified
health effects through dose-response analysis in Section 5.
The evidence for PFHxA-mediated adverse hepatic effects was based primarily on a set of
consistent and coherent findings in animal studies, including hepatocellular hypertrophy and
increased relative liver weight, generally at >100 mg/kg-day PFHxA. Most of the hepatic effects
observed in rodents changed in a consistent direction between sexes and across studies, however
the magnitude of change was often greater in males compared with females possibly due to faster
PFHxA clearance in females compared with males (see Section 3.1.4). Hepatic hypertrophy
persisted after recovery in the subchronic studies and was coherent with findings of increased
peroxisomal beta oxidation in animals. Hepatic hypertrophy was not observed after chronic
exposure, possibly because of the tested dose levels (e.g., male rats in the chronic study were
maximally exposed to PFHxA doses 2- to 5-fold below the maximal doses in the two subchronic
studies) or other study design differences. Interestingly, necrosis was observed in the female high
dose group (200 mg/kg-day) in the chronic study, suggesting that with longer term exposure,
hepatocellular hypertrophy could progress to more severe outcomes such as necrosis. These effects
in rodents were evaluated together along with supplemental evidence, including mechanistic and
pharmacokinetics information, to consider whether the rodent effects represent adverse or
adaptive changes to PFHxA exposure and their human relevance. Regarding human relevance, the
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hepatocellular hypertrophy findings were coherent with mechanistic findings on the activation of
target genes and transcription factors, including PPARa, in human and rat cell systems treated with
PFHxA. While the role of PPARa was not specifically challenged in rodent models exposed to
PFHxA, evidence that PFHxA activates both human and rodent PPARa at comparable
concentrations, alongside evidence from structurally similar PFAS, indicates that PPARa dependent
and independent hepatic effects are both likely contributing to the hepatic effects of PFHxA and that
these effects are relevant to humans. Regarding adversity, the histologic changes (increased
hypertrophy and necrosis) were coherent with clinical pathology findings (including increased ALT
and decreased serum globulins) and collectively considered in the context of the Hall criteria (see
Section 3.2.1) as adverse.
The data from the animal toxicological studies that support identifying developmental
effects as a potential human hazard included effects from three studies that reported consistent,
dose-responsive, and concerning effects of PFHxA exposure on offspring body weights and
mortality. Delayed eye opening was also reported. The observed developmental effects following
PFHxA exposure are similar to other PFAS, including PFBS (U.S. EPA. 2021b) and PFBA (U.S. EPA.
2022b). providing additional support for these specific findings. Effects on offspring body weight
were observed in two species (rats and mice) exposed to different PFHxA salts (sodium and
ammonium) using different exposure scenarios, although effects on mortality were observed only
in the mouse study. Low birth weight is associated with lasting adverse effects on health, with
increased risk for disease and reductions in lifespan in humans (Thompson and Regnault. 2011:
Kaiantie etal.. 2005: Barker. 2004) and delays in eye opening can impact normal development of
the visual system (Espinosa and Strvker. 2012: Wiesel. 1982).
The primary support for hematopoietic effects included consistent decreases in red blood
cells, hematocrit, and hemoglobin across study designs and exposure durations in male and female
adult rats fNTP. 2018: Chengelis etal.. 2009b: Loveless etal.. 20091. These hematological findings
correlate with increases in reticulocytes, an indicator of erythroid cell regeneration supported by
pathological findings in the spleen and bone marrow (Loveless etal.. 2009). The decreases in
hemoglobin were consistent with the decreased mean corpuscular hemoglobin concentration
observed in both sexes (NTP. 2018: Loveless et al.. 2009). When combined, increased mean
corpuscular hemoglobin concentration (MCHC), and mean corpuscular volume (MCV) are
indicators of anemia. Several of the hematological findings were significant at the highest dose
tested in the subchronic studies and returned to control levels after 30- or 90-day recovery periods
(or both) fChengelis etal.. 2009b: Loveless etal.. 20091. Findings from females in the chronic study
(e.g., HGB, RBC, and reticulocytes) were significant at the highest administered dose
(200 mg/kg-day), whereas no effects were observed in males that received half (100 mg/kg-day)
the female dose. Together, the subchronic and chronic evidence from males and female rats suggest
PFHxA-mediated hematopoietic effects are dependent on both dose and duration.
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For endocrine effects, the evidence to support PFHxA mediated effects is largely based on a
short-term study in rats showing a strong dose-dependent effect on thyroid hormones (decreased
serum T4 and T3, with no significant changes in TSH) in males only fNTP. 20181. This pattern of
thyroid hormone changes in rats is consistent with findings for other short-chain PFAS with more
substantial evidence bases, namely PFBA and PFBS, increasing the certainty in this evidence. These
rodent PFHxA data are also supported by similar findings in early lifestage zebrafish fZhang etal..
20221. Effects on thyroid histopathology and thyroid weight were also investigated in several rat
studies ranging from short-term to chronic exposure durations fNTP. 2018: Klaunig etal.. 2015:
Chengelis etal.. 2009b: Loveless etal.. 20091. Of these, only Loveless etal. (20091 found treatment
related increases in thyroid follicular cell hypertrophy and organ weight. Additionally, some
mechanistic evidence is available that suggests PFHxA may affect thyroid function by altering
expression of thyroid-associated mRNA and proteins or binding with serum transport proteins or
the nuclear receptor.
There evidence is inadequate to determine whether PFHxA has the potential to cause
renal, male, and female reproductive, immune, and nervous system effects in humans, renal, male,
and female reproductive, immune, and nervous system. There was insufficient evidence to
determine whether PFHxA exposure has the potential to cause other health effects (e.g., ocular
effects, cardiovascular, respiratory, gastrointestinal). Other potential health outcomes have not
been evaluated in the context of PFHxA exposure. Thus, important data gaps for PFHxA exists given
the associations observed for other PFAS, such as PFBS, PFOA, PFOS, and GenX (U.S. EPA. 2021a:
MDH. 2020. 2019. 2018: U.S. EPA. 2018b. 2016a. b). See Table 4-1 for a comparison of the
noncancer hazard judgments drawn for PFHxA with the judgments in the final EPA assessments for
PFBS, PFOA, PFOS, and GenX.
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Table 4-1. Hazard conclusions across published EPA PFAS human health assessments
Health outcome
PFAS assessmentsabc
PFHxA
(this
assessment)
PFBA
U.S. EPA (2022b)
PFBSd
U.S. EPA (2018b)
Gen X chemicalsd
U.S. EPA (2021a)
PFOAd
U.S. EPA (2023b)
PFOSd
U.S. EPA (2023a)
Endocrine/Thyroid
+
+
+
ND
Human: +
Animal: +/-
Human: +/-
Animal: +/-
Hepatic/Liver
+
+
+
Human: +
Animal: +
Human: -
Animal: +
Developmental
+
+
¦
+/-
Human: +
Animal: +
Human: +
Animal: +
Reproductive
—
—
+/-
Human: -
Animal: +/-
ND
Immunotoxicity
—
+/-
Human: +
Animal: +
Human: +/-
Animal: +
Renal
-
¦
+/-
Human: +/-
Animal: +/-
ND
Hematopoietic/
Hematological
¦
-
ND
+/-
ND
ND
Ocular
ND
-
ND
ND
ND
ND
Serum Lipids
ND
ND
-
ND
Human: +
Animal: +
Human: +
Hyperglycemia
ND
ND
ND
ND
Human: -
Animal: -
Animal: +/-
Nervous System
-
ND
ND
ND
Human: -
Animal: -
Animal: +/-
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Health outcome
PFAS assessmentsabc
PFHxA
(this
assessment)
PFBA
U.S. EPA (2022b)
PFBSd
U.S. EPA (2018b)
Gen X chemicalsd
U.S. EPA (2021a)
PFOAd
U.S. EPA (2023b)
PFOSd
U.S. EPA (2023a)
Cardiovascular
ND
ND
-
ND
ND
ND
Cancer
-
-
-
+/-
+/-
+/-
Assessments used multiple approaches for summarizing their noncancer hazard conclusion scales; for comparison purposes, the conclusions are presented as
follows: V = evidence demonstrates or evidence indicates (e.g., PFHxA), or evidence supports (e.g., PFBS);= suggestive evidence,= inadequate
evidence (e.g., PFHxA) or equivocal evidence (e.g., PFBS);= sufficient evidence to conclude no hazard (no assessment drew this conclusion); ND = no data
available for this outcome for this PFAS.
bThe assessments all followed the EPA carcinogenicity guidelines (U.S. EPA, 2005) a similar presentation to that used to summarize the noncancer judgments is
applied for the cancer hazard conclusions, as follows: V = carcinogenic to humans or likely to carcinogenic to humans;'+/-' = suggestive evidence of
carcinogenic potential;= inadequate information to assess carcinogenic potential;= not likely to be carcinogenic to humans(no assessment drew this
conclusion); ND = no carcinogenicity data available for this PFAS.
cThe hazard conclusions for the various EPA PFAS assessments presented in this table were not considered during evidence integration and thus did not inform
the evidence integration conclusions presented in the PFHxA assessment.
dThe U.S. EPA PFOA (U.S. EPA, 2016b) and PFOS (U.S. EPA, 2016a) assessments did not use structured language to summarize the noncancer hazard
conclusions. The presentation in this table was inferred from the hazard summaries found in the respective assessments; however, this is for comparison
purposes only and should not be taken as representative of the conclusions from these assessments. Those interested in the specific noncancer hazard
conclusions for PFOA and PFOS must consult the source assessments. Note that new assessments for PFOA and PFOS are currently being finalized to support a
National Primary Drinking Water Regulation; note that hazard conclusions in these updated assessments will differ from those presented in this table as the
new assessments use structured language to summarize the noncancer hazard conclusions. For access to the more recent draft assessment materials please
follow this link.
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4.2. CONCLUSIONS REGARDING SUSCEPTIBLE POPULATIONS AND
LIFESTAGES
No human studies were available to inform the potential for PFHxA exposure to affect
sensitive subpopulations or lifestages.
In adult rats exposed to PFHxA for 28 days to 2 years, toxicological findings were either
consistently observed at lower dose levels in males than females or the findings were observed only
in males (except for necrosis in the chronic study). The reason for this sex dependence is possibly
due to sex-dependent PFHxA elimination caused by sex-specific differences in the expression
(mRNA and protein) of the renal organic anion transporting polypeptide (Oatp) lal (Kudo etal..
2001) as discussed in Section 3.1.4. Currently, whether this sex-specific difference might also exist
in humans is unclear.
Additionally, given the effects seen in the developing organism (i.e., perinatal mortality,
reduced body weights, and delays in time to eye opening), the prenatal and early postnatal window
represents a potentially sensitive lifestage for PFHxA exposure.
4.3. SUMMARY OF CONCLUSIONS FOR CARCINOGENICITY
The evidence is inadequate to make a judgment on whether PFHxA exposure might affect
the development of any specific cancers (see Section 3.3). Given this general lack of evidence and
consistent with EPA guidelines fU.S. EPA. 20051 instruction to apply a standard descriptor as part
of the hazard narrative and to express a conclusion regarding the weight of evidence for the
carcinogenic hazard potential, a descriptor of inadequate information to assess carcinogenic
potential is applied for PFHxA; this descriptor is applicable across all exposure routes.
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5.DERIVATION OF TOXICITY VALUES
5.1. HEALTH EFFECT CATEGORIES CONSIDERED (CANCER AND
NONCANCER)
Multiple noncancer health effects were examined following oral PFHxA exposures. The
evidence integration judgments are based primarily on five animal toxicological studies (NTP.
2018: Klaunig etal.. 2015: Iwai and Hoberman. 2014: Chengelis etal.. 2009b: Loveless etal.. 20091.
These studies were generally rated high confidence in outcome-specific study evaluations. Based on
these studies, it was determined that the evidence indicates PFHxA likely causes hepatic,
developmental, hematopoietic, and endocrine effects in humans given sufficient exposure
conditions. These health effects were considered for derivation of toxicity values. The dose levels
associated with these hazards are further characterized in Section 5.2.1.
For all other health effects (i.e., renal, male, and female reproductive, immune, and nervous
system), the evidence is inadequate to assess potential health effects, thus these were not
considered for derivation of toxicity values.
No studies of inhalation exposure were identified, thus an RfC was not estimated (see
Section 5.2.2). Similarly, the evidence base related to potential carcinogenicity was determined to
contain "inadequate information to assess carcinogenic potential"; therefore, no cancer toxicity
values were estimated for any exposure route (see Section 5.3).
5.2. NONCANCER TOXICITY VALUES
A reference dose (RfD) is the daily oral exposure to the human population (including
sensitive subpopulations) that is likely without appreciable risk of deleterious effects during a
lifetime. In addition to developing an RfD designed to protect against lifetime exposure, a less-than-
lifetime toxicity value (referred to as a "subchronic RfD") is estimated. These subchronic toxicity
values are presented as they might be useful for certain decision purposes (e.g., site-specific risk
assessments with less-than-lifetime exposures). Both RfD and subchronic RfD derivations include
organ/system-specific RfDs (osRfDs) associated with each health effect considered for point of
departure (POD) derivation. Subsequent decisions related to dosimetric extrapolation, application
of uncertainty factors, and confidence in toxicity values are discussed below.
As noted above, a reference concentration (RfC) or subchronic RfC could not be developed.
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5.2.1. Oral Reference Dose (RfD) Derivation
Study Selection
The identified hazards relating to developmental, hepatic, hematopoietic, and endocrine
effects were based on five high confidence animal studies that were considered for use in deriving
an oral reference dose (RfD). The developmental studies Loveless etal. (20091 or Iwai and
Hoberman (20141 were selected to support RfD derivation for developmental effects, given that
endpoints observed from exposures during early lifestages are known to be sensitive for
developmental effects (compared with exposures at later lifestages) and thus more appropriate for
estimating potential effects of lifetime exposure. Both studies used rats or mice as the laboratory
animal species and used vehicle-exposed controls. Animals were exposed to reagent-grade
Na+PFHxA (reported as 100% pure) or NH4+PFHxA (reported as 93.4% pure; impurities not
reported) via a human-relevant route (oral administration via gavage) and for a duration of
exposure (GD 6-18) encompassing critical windows relevant to the developmental effects of
interest
In addition to the developmental studies, subchronic fChengelis etal.. 2009b: Loveless etal..
20091 and chronic fKlaunig et al.. 20151 studies in rats were selected to support RfD derivation for
hepatic and hematopoietic outcomes of PFHxA exposure. Animals were exposed to Na+PFHxA
(reported as 100% pure) or PFHxA (reported as 98.1% pure) via oral administration and for a
duration relevant for estimating potential effects of lifetime exposure (90 days or 2 years). For
endocrine outcomes, endpoints were available from the same subchronic and chronic studies,
however observations were null except for increased thyroid follicular cell hypertrophy from the
90-day study fLoveless etal.. 20091 in males and females. However, the 90-day findings were only
observed at the highest dose, were inconsistent with findings from the other 90-day and chronic
study and lacked support for coherence from other findings. Also available in the PFHxA database is
a short-term (i.e., 28-day) study of PFHxA exposure in rats (NTP. 20181 that was interpreted to
provide critical information supporting information supporting thyroid hazard identification and
included quantitative data useful for dose response analysis. Note that, as discussed below, when
developing a lifetime reference value, chronic or subchronic studies (and studies of developmental
exposure) are generally preferred over short-term or acute studies, so the fNTP. 20181 study was
ultimately not considered for use in deriving candidate lifetime toxicity values.
For hepatic outcomes, a collection of adverse effects was found in rats, with PFHxA
exposure resulting in increased liver weights (absolute and relative) in adult exposed animals
(Chengelis etal.. 2009b: Loveless etal.. 20091. histopathological lesions (i.e., hepatocellular
necrosis), and hypertrophy. Other hepatic findings (increased liver enzymes [i.e., ALT, AST, ALP];
increased peroxisomal beta oxidation; decreased total protein, albumin, and bilirubin; and
mechanistic evidence indicating PPARa activation in humans and rodents at similar concentrations
of PFHxA) supported the final evidence integration judgments for these endpoints and were critical
for identifying adverse, human relevant endpoints for dose-response analysis (as discussed in
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Section 3.2.1). For the purposes of dose-response modeling, hepatic hypertrophy was considered a
more sensitive effect (compared with other hepatic findings such as relative liver weight).
Hepatocellular hypertrophy was observed in both sexes by Loveless etal. f2009I whereas
Chengelis etal. f2009bl observed hypertrophy in the livers of male mice, not females.
For hematopoietic outcomes, a collection of adverse effects including decreased HGB and
decreased RBCs, which were coherent with other correlative changes in other red blood cell
indicators as discussed in Section 3.2.4, were observed in PFHxA exposed rats. The observed HGB
and RBC findings were considered the most sensitive and specific compared to other hematological
findings (e.g., blood proteins, hematocrit) and both advanced for POD derivation as there was no
reason to choose one endpoint over the other. Hemoglobin decreases were considered similar in
sensitivity to decreases in red blood cell counts and, while hemoglobin is used as an indicator of
disease of the blood (i.e., anemia) fWHO. 20221. there was no reason to advance one endpoint over
the other. Hematopoietic effects were observed in both sexes in the subchronic studies, whereas the
only significant observation from the chronic study was in females, likely due to females receiving
the highest dose administered in the study (Klaunig etal.. 2015). As described in Section 3.2.4,
quantitative hematology measures in rats after 52 weeks are likely less reliable due to increasing
naturally occurring diseases that decrease sensitivity; thus, no hematopoietic data after 51 weeks
were considered for POD derivation.
For developmental outcomes adverse effects were observed that included decreased Fi pup
body weight and increased perinatal mortality after gestational exposure (Iwai and Hoberman.
2014: Loveless etal.. 2009). Effects on Fi pup body weight were strongest during the early
postnatal period so these timepoints were prioritized. Perinatal mortality (still birth and postnatal
deaths from PND 0-21) showed a clear dose-response across two experimental cohorts with
overlapping dose ranges. Data were pooled for dose-response analysis. Eye opening delays were
also observed but were not advanced for POD derivation because the PFHxA doses causing these
effects were higher than those that lead to pup body weight deficits and perinatal mortality.
For endocrine outcomes, adverse effects related to the thyroid consisted of decreased free
and total T4 and T3 levels observed in exposed rats in the short-term study (NTP. 2018). and
increased thyroid follicular hypertrophy in a subchronic study (Loveless etal.. 2009). Decreased
thyroid hormone levels are judged relevant to human health, given the many similarities in the
production, regulation, and functioning of thyroid hormones between rodents and humans. In
addition, rodents are more sensitive to increases in thyroid follicular hypertrophy that was
observed in one subchronic study compared with null findings from all other studies at similar and
higher PFHxA dose levels (including in a chronic study). Thus, changes in thyroid hormone levels
are considered more relevant for deriving human health toxicity values and increased thyroid
hypertrophy was not considered further for RfD derivation. Total T4 assay measurements are more
reliable than those provided by the assays available to measure free T4 due to the very small
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quantity of unbound (i.e., 'free') T4 in circulation fFaix. 2013: Thienpontetal.. 20131. For this
reason, total, but not free, T4 was moved forward for POD and candidate value derivation.
A summary of endpoints considered for toxicity value derivation is presented in Table 5-1.
Table 5-1. Endpoints considered for dose-response modeling and derivation
of points of departure
Endpoint
Study,
endpoint confidence,
exposure duration
Strain,
species, sex
POD
derivation3
Rationale for deriving POD
Hepatic
Increased relative
liver weight
Chengelis et al. (2009b)
High confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, both
No
Liver weights were considered a less
sensitive and specific measure of
toxicity than other available hepatic
findings such as increased
hepatocellular hypertrophy.
Loveless et al. (2009)
High confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, both
No
Increased
hepatocellular
hypertrophy
Chengelis et al. (2009b)
Low confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, both
No
Endpoint considered adverse based
on collection of hepatic findings (see
Section 3.2.1) and a biologically
plausible precursor event of more
severe outcomes such as necrosis.
Although male-specific effects were
observed in Chengelis et al. (2009b),
the evaluation of this outcome in this
study was considered low confidence
and therefore not advanced. Both
sexes were affected in Loveless et al.
(2009) and advanced.
Loveless et al. (2009)
High confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, both
Yes
Increased
hepatocellular
necrosis
Klaunig et al. (2015)
High confidence,
Chronic (104 w)
Crl:CD(SD)
rat, female
Yes
Although significant effects were only
observed at the highest dose in
females, and largely in animals that
died an unscheduled death, necrosis
was advanced for further
consideration as it represents a severe
and specific indication of hepatic
toxicity.
Hematopoietic
Decreased blood
proteins (total
protein and
globulin)
Chengelis et al. (2009b)
High confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, both
No
Several hematologic endpoints are
available and some (e.g., red blood
cells and hemoglobin) are considered
a more direct measure of
hematopoietic effects than these
markers.
Loveless et al. (2009)
High confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, both
No
Klaunig et al. (2015)
High confidence,
Chronic (51 w)
Crl:CD(SD)
rat, both
No
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Endpoint
Study,
endpoint confidence,
exposure duration
Strain,
species, sex
POD
derivation3
Rationale for deriving POD
Decreased
hematocrit
Chengelis et al. (2009b)
High confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, both
No
Hematocrit is a measure of the
percentage by volume of red blood
cells in blood. The endpoint was
considered a less direct measure of
hematopoietic outcomes compared
with other endpoints (e.g., red blood
cells and hemoglobin) that were also
available from the same studies.
Loveless et al. (2009)
High confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, both
No
Klaunig et al. (2015)
High confidence,
Chronic (51 wk)
Crl:CD(SD)
rat, female
No
Decreased
hemoglobin
Chengelis et al. (2009b)
High confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, both
Yes
The endpoint was considered a
sensitive and specific adverse
hematopoietic outcome (see Section
3.2.4). Male endpoints from the
chronic study were mostly null and
not advanced for POD derivation.
Loveless et al. (2009)
High confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, both
Yes
Klaunig et al. (2015)
High confidence,
Chronic (51 wk)
Crl:CD(SD)
rat, female
Yes
Klaunig et al. (2015)
High confidence,
Chronic (51 wk)
Crl:CD(SD)
rat, male
No
Decreased red
blood cells
Chengelis et al. (2009b)
High confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, both
Yes
The endpoint was considered a
biologically significant endpoints that
is sensitive and specific for
hematopoietic outcomes. There was
no clear reason to advance one study
over others.
Loveless et al. (2009)
High confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, both
Yes
Klaunig et al. (2015)
High confidence,
Chronic, (51 wk)
Crl:CD(SD)
rat, female
Yes
Increased
reticulocytes
Chengelis et al. (2009b)
High confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, female
No
Increases were considered to reflect a
compensatory (secondary) response
to decreased red blood cell
parameters.
Loveless et al. (2009)
High confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, both
No
Klaunig et al. (2015)
High confidence,
Chronic, (51 wk)
Crl:CD(SD)
rat, both
No
Developmental
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Endpoint
Study,
endpoint confidence,
exposure duration
Strain,
species, sex
POD
derivation3
Rationale for deriving POD
Decreased,
postnatal (Fi)
pup body weight
Loveless et al. (2009)
High confidence,
One-generation repro-
ductive; measured on PND
0, 4, 7, 14, 21
Crl:CD(SD)
rat, Fi
Yes
Effects on body weight were strongest
during the early postnatal period so
these timepoints were prioritized.
Iwai and Hoberman (2014)
High confidence,
Develop-mental (GD 6-18);
measured on PND 0, 4, 7,
14, 21
CD-I
mouse, Fi
Yes
Decreased, Fi
fetal body weight
Loveless et al. (2009)
High confidence,
Develop-mental (GD 6-20);
measured on GD 21
Crl:CD(SD)
rat, Fi
No
Statistically nonsignificant 9%
decrease only at the highest dose.
Increased,
perinatal
mortality
Iwai and Hoberman (2014)
High confidence, Develop-
mental (GD 6-18);
measured on PND 0-21,
including stillbirths
CD-I
mouse, Fi
Yes
Perinatal mortality (still birth and
postnatal deaths from PND 0-21)
showed a clear dose-response across
two experimental cohorts with
overlapping dose ranges. These data
were pooled for dose-response
analysis.
Delayed eye
opening
Iwai and Hoberman (2014)
High confidence,
Develop-mental (GD
6-18); measured on PND
10-17
CD-I
mouse, Fi
No
Delays were observed at a dose that
elicited body weight deficits and
perinatal mortality.
Endocrine
Decreased
thyroxine (T4),
total
NTP(2018)
High confidence,
Short term (28 d)
Harlan
Sprague-
Dawley rat,
male
Yes
Males showed statistically significant
decreases in free and total T4 and T3
at all doses. No effects were observed
in females. Total T4 was prioritized
NTP(2018)
High confidence,
Short term (28 d)
Harlan
Sprague-
Dawley rat,
female
No
due to a clear dose response pattern
across the tested exposure range and
increased assay reliability (vs. free T4).
Decreased
thyroxine, free
(fT4)
NTP(2018)
High confidence,
Short term (28 d)
Harlan
Sprague-
Dawley rat,
male
No
NTP(2018)
High confidence,
Short term (28 d)
Harlan
Sprague-
Dawley rat,
female
No
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Endpoint
Study,
endpoint confidence,
exposure duration
Strain,
species, sex
POD
derivation3
Rationale for deriving POD
Decreased
triiodothyronine
(T3)
NTP(2018)
High confidence,
Short term (28 d)
Harlan
Sprague-
Dawley rat,
male
No
NTP(2018)
High confidence,
Short term (28 d)
Harlan
Sprague-
Dawley rat,
female
No
Increased
Thyroid epithelial
cell hypertrophy
Loveless et al. (2009)
High confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, both
No
Increased incidence in thyroid
epithelial hypertrophy only observed
in one subchronic study at the high
Chengelis et al. (2009b)
High confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, both
No
dose (Loveless et al., 2009).
Klaunig et al. (2015)
High confidence,
Chronic (104 w)
Crl:CD(SD)
rat, both
No
NTP(2018)
High confidence,
Short term (28 d)
Harlan
Sprague-
Dawley,
both
No
Increased thyroid
weight
NTP(2018)
High confidence,
Short term (28 d)
Harlan
Sprague-
Dawley,
both
No
Treatment-related effects were
limited to a single study that only
qualitatively reported increased
thyroid weight in high dose group
Loveless et al. (2009)
High confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, both
No
females.
Chengelis et al. (2009b)
High confidence,
Subchronic (90 d)
Crl:CD(SD)
rat, both
No
aSee text for rationale for inclusion/exclusion from point-of-departure derivation.
Estimation or Selection of Points of Departure (PODs)
The outcomes determined most appropriate for quantifying the identified noncancer
hazards and advanced for dose-response analysis (see Table 5-1) were modeled using approaches
consistent with EPA's Benchmark Dose (BMD) Technical Guidance document fU.S. EPA. 2012a).
Specifically, the BMD and 95% lower confidence limit on the BMD (BMDL) were estimated using a
benchmark response (BMR) to represent a minimal, biologically significant level of change. BMD
modeling of continuous data was conducted using EPA's Benchmark Dose Software (BMDS, Version
3.2).
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Ideally, the selected BMR is based on data that support the biological relevance of the
outcome being evaluated; however, in some cases there is no clear scientific understanding to
support a biologically based BMR. In these instances, the BMD guidance provides some BMRs that
can be applied to the data. For data drawn from toxicological studies, a suggested BMR of 1
standard deviation (SD) from the control mean for continuous data or a BMR of 10% extra risk (ER)
for dichotomous data can be used to estimate the BMD and BMDL. The selection of these BMRs, as
indicated in Table 5-2, is based on BMD guidance stating that in the absence of information
regarding the level of change considered biologically significant, these BMRs can be used fU.S. EPA.
2012a). For effects on offspring body weights, a BMR of 5% relative deviation (RD) from the control
mean is used for continuous data to account for effects occurring in a sensitive lifestage fU.S. EPA.
2012a).
Table 5-2. Benchmark response levels selected for BMD modeling of PFHxA
health outcomes
Endpoint
BMR
Rationale
Hepatic effects
Hepatocellular
hypertrophy
10% ER
For dichotomous hepatic data, a 10% ER is generally considered a
minimallv biologically significant response level (U.S. EPA, 2012a).
Hepatocellular
necrosis
10% ER
For dichotomous hepatic data, a 10% ER is generally considered a
minimallv biologically significant response level (U.S. EPA, 2012a).
Developmental effects
Postnatal (Fi)
body weight
5% RD
A 5% RD in markers of growth/development in gestational studies
(e.g., fetal weight) has generally been considered a minimally
biologically significant response level and has been used as the BMR
for benchmark dose modeling in prior IRIS assessments (U.S. EPA,
2012b, 2004, 2003).
Offspring
mortality
1% ER
Although 5% ER is generally supported for developmental and
reproductive outcomes (U.S. EPA, 2012a), a lower BMR of 1% ER was
considered appropriate for modeling offspring mortality considering
the severity of the frank effect.
Hematopoietic effects
Red blood cells
1 SD
No biological information is readily available that allows for
determining a minimally biological significant response for these
outcomes. The BMD Technical Guidance (U.S. EPA, 2012a)
recommends a BMR based on 1 SD in such a situation.
Hemoglobin
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Endpoint
BMR
Rationale
Endocrine effects
Total T4
1SD
Evidence to support identification of a minimally biologically
significant response for thyroid hormones is lacking in adult animals.
In developing animals, there is a wide range in the level of response
in thyroid hormones associated with neurodevelopmental effects
(10%-25% for serum T4) in human and rodent studies (Gilbert et al.,
2016; Gilbert, 2011; Haddow et al., 1999).
Given that the biological information is not sufficient to identify the
BMR and the decreases in serum T4 (up to 73%) in male rats exceed
these values, a 1SD for continuous data was selected for this
endpoint, consistent with EPAs Benchmark Dose Technical Guidance
(U.S. EPA, 2012a).
An adequate fit is judged based on yl goodness-of-fit p-value (p >0.1), magnitude of the
scaled residuals in the vicinity of the BMR, and visual inspection of the model fit. In addition to
these three criteria for judging adequacy of model fit, a determination is made as to whether the
variance across dose groups is homogeneous. If a homogeneous variance model is deemed
appropriate based on the statistical test provided by BMDS (i.e., Test 2), the final BMD results are
estimated from a homogeneous variance model. If the test for homogeneity of variance is rejected
(i.e., Test 2; p < 0.05), the model is run again while modeling the variance as a power function of the
mean to account for this nonhomogeneous variance. If this nonhomogeneous variance model does
not adequately fit the data (i.e., Test 3; p < 0.05), the data set is considered unsuitable for BMD
modeling. Among all models providing adequate fit for a given endpoint, the benchmark dose lower
confidence limit (BMDL) from the model with the lowest Akaike's information criterion (AIC) was
selected as a potential POD when BMDL values were sufficiently close (within 3-fold). Otherwise,
the lowest BMDL was selected as a potential POD for each endpoint
Where modeling was feasible, the estimated BMDLs were used as PODs. Further details,
including the modeling output and graphical results for the model selected for each endpoint, can
be found in Supplemental Information, Appendix B. The benchmark dose approach involving
modeling to obtain the BMDL is preferred, but it involves modeling dose levels corresponding to
BMR levels near the low end of the observable range of the data and is not always feasible. When
data sets were not amenable to BMD modeling, no-observed-adverse-effect level (NOAEL) or
lowest-observed-adverse-effect level (LOAEL) values were selected and used as the POD based on
expert judgment, considering the study design features (e.g., severity and rarity of the outcome;
biological significance, considering the magnitude of change at the NOAEL or LOAEL; statistical
significance and power; exposure and outcome ascertainment methods).
For the study by Iwai and Hoberman f20141. the experiment was conducted in two phases.
Except for differences in the dose levels, the design and conduct were the same across the two
phases. Specifically, in addition to concurrent control groups for each phase, animals were exposed
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to 100, 350, or 500 mg/kg-day in Phase 1 and 7, 35 or 175 mg/kg-day in Phase 2. When possible,
the two phases were combined for modeling to provide a more robust dose range. If the combined
data set did not result in adequate model fit, the phases were modeled separately and the results
for the individual phases were presented.
Approach for Animal-Human Extrapolation of PFHxA Dosimetry
The PFAS protocol (Appendix A) recommends the use of physiologically based
pharmacokinetic (PBPK) models as the preferred approach for dosimetry extrapolation from
animals to humans, while allowing for the consideration of data-informed extrapolations (such as
the ratio of serum clearance values) for PFAS that lack a scientifically sound and sufficiently
validated PBPK model. If chemical-specific information is not available, the protocol then
recommends that doses be scaled allometrically using body weight (BW)3/4 methods. This
hierarchy of recommended approaches for cross-species dosimetry extrapolation is consistent with
EPA's guidelines on using allometric scaling for deriving oral reference doses (U.S. EPA. 20111. This
hierarchy preferentially prioritizes adjustments that result in reduced uncertainty in the dosimetric
adjustments (i.e., preferring chemical-specific values to underpin adjustments versus use of default
approaches).
As discussed in Section 3.1.5, no PBPK model is available for PFHxA in rats, mice, or
monkeys. Although a PBPK model for humans was described by Fabrega etal. f20151. it was not
considered sufficiently reliable for use in an IRIS Toxicological Review.
There are, however, pharmacokinetic information available for PFHxA in relevant animal
species (rats, mice, and monkeys) or humans that were useful for data-informed extrapolation
approach for estimating the dosimetric adjustment factor (DAF) for PFHxA. Various PK analyses
can be performed to extract meaningful information from PK data. Because PK data for various
PFAS are available, including for PFBA fChang etal.. 20081. PFBS fOlsen etal.. 20091. PFHxA
fDzierlenga etal.. 20191. PFHxS fSundstrom etal.. 20121. PFNA fTatum-Gibbs etal.. 20111. and
PFOA and PFOS (Kim etal.. 2016b). that show a clear biphasic elimination pattern indicative of
distinct distribution and elimination phases, EPA chose to use a two-compartment PK model,
similar to the analysis of (Fuiii etal.. 2015). The EPA model is characterized by equation 5-1:
C(t) = A-exp(-a-t) + B-exp(-(3-t) - flagorai- (A+B) exp(-ka-t), (5-1)
where a and (3 are first-order rate constants (units of time"1) representing the rate of
distribution and elimination, respectively, ka is a rate constant (units of time"1) for oral absorption,
and flagorai is set to zero when analyzing intravenous dose data or one for oral data. Details of the
model fitting are provided in Appendix B. The model assumes that oral bioavailability is 100%,
consistent with PK data from Dzierlenga et al. (2019) and other studies and that internal dosimetry
and elimination are linear with dose. This is implicitly a two-compartment PK model represented
by the model, for which the rate of elimination corresponds to p. It is presumed that the total
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Toxicological Review of PFHxA and Related Salts
concentration from several consecutive doses would be obtained by simply adding the individual
concentration curves, given the distinct dose times.
This PK model assumes the parameters are independent of time and dose. As discussed in
the "Elimination" section, PK studies that measured tissue concentrations after multiple days of
exposure are consistent with simple PK models parameterized from one-day exposure and support
the assumption that the model parameters are independent of time. Although PK data at lower
doses do not show any trend consistent with dose-dependence, data for the highest dose indicate
that elimination can be reduced (Dzierlenga etal.. 20191: the opposite of what is predicted based on
the hypothesis of saturable resorption. While saturation of reabsorption transporters would lead to
a decreased half-life at higher doses, there are also transporters responsible for elimination of PFAS
to urine, such as Oatl and Oat3, and saturation of these transporters could lead to an increase in
observed half-life. A systematic deviation from the assumption of equal or more rapid clearance at
higher doses has not been observed in the other relevant data flwabuchi etal.. 2017: Gannon etal..
2011: Chengelis etal.. 2009al. Further, because PFHxA is not metabolized, nonlinearity in its
internal dose is not expected due to that mechanism. Parameter estimation, however, was
performed both including and excluding the highest dose data. Had the resulting estimate of (3 been
significantly different when the high-dose data were included, this would have indicated a dose
dependence. The results of the alternative analyses did not indicate such a difference, however,
leading to the conclusion that PFHxA PK is not dose dependent and that the assumption of
nonvarying parameters in the PK model equation is appropriate. Further details are provided in
Appendix C.
Given the fit of this model to a specific data set, the AUC from the time of exposure to infinity
is:
AUCinf = A/a + B/p - flagorai-(A+B)/ka (5-2)
AUC is the integral of the chemical concentration in blood or serum over time, with units of
mass x time / volume (e.g., mg-hr/L), and is considered an appropriate measure of internal dose
when the chemical has an accumulative effect over time.
By definition, the clearance (CL) of a compound is the effective volume of blood cleared of
the compound per unit time (units ofvolume/time). Mathematically, given the PK model described
above, CL = dose/AUCmf. If one assumes that risk increases in proportion to AUC, the ratio of
clearance in animal to that in the human, CLa:CLh, can then be used to convert an oral dose-rate in
animals (mg/kg-day) to a human equivalent dose (HED) rate. A similar approach using the ratio of
the beta-phase half-lives can be used and is outlined in Appendix C, but that approach ignores
differences in the absorption rate and alpha-phase distribution rate that impact AUC and is,
therefore, considered to produce a more uncertain outcome. The relationship between the volume
of distribution (Yd), CL and half-life (ti/2) is given by:
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Toxicological Review of PFHxA and Related Salts
CL = ln(2) x Vd/Ti/2.
Hence:
CLa/CLh - (VdA/VdH)/(tl/2,A/tl/2,H)-
So, if one assumes VdH = VdA, as the EPA has done, then:
CLa/CLh - l/( ti/2,A/ ti/2,H);
i.e., the clearance ratio is equal to one over the half-life ratio, so there is no quantitative
difference between the two options for HED calculation in this case. However, if there were
independent data to identify VdH, the value might not be identical to Vdrat, in which case the
clearance ratio would be different from the half-life ratio. Further, using the estimated clearance
ratio makes the calculation more transparent because it requires an explicit statement of the
assumption that VdH = Vdrat, while use of the ratio of half-lives makes this assumption implicitly,
hiding it from consideration as a factor introducing uncertainty.
Therefore, the HED was explicitly calculated using the ratio of clearance values:
Given the PK model and definition of clearance above, the resulting HED is the dose that
results in the same AUC in humans as is predicted in animals exposed at the POD, if one can obtain a
value of CLh.
In the term CLa[S], the [s] in the subscript refers to the sex-specific value available for
animals but not humans in the case of PFHxA. Because there are sex-specific values (significant
differences between males and females) in clearance among mice and rats, the CL values for female
rodents would be used to extrapolate health effects in female rodents and the CL values for male
rodents would be used to extrapolate male rodent health effects. This choice simply ensures that an
observed effect in male rats, for example, is extrapolated using the expected internal dose for male
rats. When endpoints from both male and female animals are analyzed (i.e., separate dose-response
analyses are conducted for results in males vs. females) resulting in sex-specific PODs, the
corresponding male and female human HEDs would be calculated, using (CLh/CLa[S]).
The volume of distribution in the beta phase (i.e., after the chemical has distributed into the
body as a whole) given the two-compartment model above is:
Except for the i.v. dose data from Dzierlenga etal. (2019). the Vd for rats for all other
experiments and studies for male and female rats were between 0.9 and 1.7 L/kg and the averages
for males and females were virtually indistinguishable: 1.37 and 1.35 L/kg, respectively. For the i.v.
dose data from Dzierlenga et al. f20191. Vd, p was 5.2 L/kg in male rats and 18.7 L/kg in female rats.
HED = (CLh/CLA[S]) x POD
(5-3)
Vd, p = CL/p = dose/[p x (A/a + B/(3 - flagorai x (A+B)/ka)]
(5-4)
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Toxicological Review of PFHxA and Related Salts
In contrast, Vd, p for the i.v. dose data from Chengelis etal. (2009a) was 0.93 L/kg for both male and
female rats. Thus, excluding those specific i.v. experiments, Vd, p in rats does not appear to be sex
specific and an overall average of 1.36 L/kg appears appropriate for that species.
For male and female mice, the corresponding Vd was 0.75 and 0.78 L/kg, respectively, based
on data from Gannon etal. f2011I again not indicating a significant sex difference, although the
value is somewhat lower than in rats.
For male and female monkeys, Chengelis etal. (2009a) reported Vd = 0.99 ± 0.58 L/kg and
0.47 ± 0.35 L/kg, respectively. Although these indicate a possible sex difference, only three animals
of each sex were used and the estimated ranges (0.39-1.5 vs. 0.23-0.87 L/kg) significantly overlap.
Hence, some caution in interpreting these data is required. The overall average Vd for monkeys,
0.73 L/kg, is similar to the value for mice, although also lower than the value in rats.
Because the volume of distribution (Vd) has not been determined in humans, but an
estimate for the human half-life (ti/2) is available, three options for estimating a clearance in
humans can be considered, although this might be viewed as extreme for the purpose of predicting
HED values. The observed ti/2 in humans is presumed to represent the beta or clearance phase,
given the PFHxA study participant evaluation occurred over months after primary exposure to
PFHxA had ended fNilsson etal.. 20101. Hence it is presumed that ti/2 = ln(2)/(3. Rearranging the
two equations, CL = Vd,p x p = Vd,p x ln(2)/ ti/2. Three options were considered, as follows:
1) The Vd for humans is equal to that determined in the next closest species biologically,
monkeys. This assumes the biological and biochemical factors that determine the tissue:
serum concentration ratio and the relative proportion (fraction of BW) for various tissues is
similar in humans and monkeys. This assumption presumes the relative binding of PFHxA
in human serum relative to various other tissues in the body is like that in monkeys but
leads to a conclusion that renal clearance in humans is significantly slower than in other
species.
2) Use the clearance values estimated for mice, rats, and monkeys to estimate the clearance in
humans via allometric scaling. The results for mice, rats, and monkeys in Table 5-3 show
almost no trend with increasing species BW but can be fitted with a power function to
obtain CL = 0.152-BW"0 023 (L/kg), assuming standard BW values of 0.03 and 0.25 kg for
mice and rats, respectively, and the reported BW of monkeys used by Chengelis et al.
f2009al. For a standard human BW of 80 kg, the resulting predicted clearance in humans is
0.137 L/hr-kg. If this is the actual clearance in humans, but ti/2 = 275 hr, human
Vd,p = CL x ti/2/ln(2) = 54 L/kg. Note that human participants were exposed to PFHxA for
months, which could have allowed them to accumulate a deep tissue dose, while the
monkey PK study involved only a single i.v. administration. Thus, a much higher Vd might
have been estimated in monkeys had they been subject to repeated doses.
3) The apparent human half-life estimated by EPA from the data of Nilsson etal. (2013) might
be an artifact of significant ongoing exposure to PFHxA during the period of observation.
Perez etal. f20131 detected PFHxA levels in human tissues higher than other PFAS and
other observational studies regularly detect PFHxA in human serum demonstrating
widespread human exposure to the general population. Thus, there is no reason to believe
the subjects of Nilsson etal. f20131 did not also have some level of ongoing exposure; the
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Toxicological Review of PFHxA and Related Salts
question is whether such exposure was significant relative to the body burden accumulated
from exposure as ski-wax technicians. If the value of CL estimated in (2) (0.137 L/hr-kg) is
an accurate prediction for humans and the Vd is equal to the average estimated for monkeys
(0.73 L/kg), the half-life in humans should be
ti/2 = ln(2) x VA /CL =ln(2) x 0.73 (L/kg)/(0.137 L/hr-kg) = 3.7 hours. If this were the case,
human serum levels would fall 99% in a single day, while the data of Nilsson etal. (20131
show that such a decline takes at least 2 months and, even after a day or two off work, a
technician's serum concentration would be near zero. Further, the serum concentrations
reported Nilsson etal. f20131 do decline to near or below the limit of detection by late
spring or early summer, indicating that other ongoing sources of exposure were not
significant for that population. Thus, this third option seems extremely unlikely and was not
evaluated further.
The two options for human CL estimated above are provided in Table 5-3.
Table 5-3. Summary of serum half-lives and estimated clearance for PFHxA
Species/sex
Study
design
Elimination
half-life (ti/2)
(hr)
Clearance (CL) (L/hr-kg)
Volume of
distribution (Vd)
(L/kg)
References/data
sources
Rat, female
Oral and
i.v.
2.7(0.5-11.2)
0.383
(0.259-0.574)a
1.48
(0.27-4.42)3
Dzierlenga et al. (2019):
Chengelis et al. (2009a):
Gannon et al. (2011)
Rat, male
Oral and
i.v.
5.4(1.6-19.5)
0.163
(0.112-0.228)a
1.31
(0.37-4.4)3
Dzierlenga et al. (2019):
Chengelis et al. (2009a):
Iwabuchi et al. (2017):
Gannon et al. (2011)
Mouse,
female
Oral
7.9(2.8-23)
0.206 (0.137-0.308)a
2.46 (0.82-6.82)3
Gannon et al. (2011):
Daikin Industries (2010)
Mouse, male
Oral
10.6 (2.3-29)
0.0894 (0.053-0.153)a
1.38
(0.31-3.73)3
Gannon et al. (2011)
Monkey,
female
i.v.
2.4
0.136
0.474 ± 0.349b
Chengelis et al. (2009a)
Monkey, male
i.v.
5.3
0.122
0.989 ± 0.579b
Chengelis et al. (2009a)
Human, male
and female
(data derived)
Ecological
275 (145-510)
1.84 x 10_3c
((0.45 -7.35) x 10"3)d
0.73(0.33-1.45)e
Nilsson et al. (2013)
Human, male
and female
(allometric)
NA
275
0.137f
54f
Nilsson et al. (2013)
aFor each experiment (study/route/dose), a separate distribution of CL = dose/AUCinf and VdP = CL/P was generated. Median,
5th, and 95th percentiles of each distribution were calculated and are available on request. Results across experiments/dose
levels were pooled, and the values presented here are statistics for the pooled results, 50th (5th—95th) percentiles for each
species/sex.
bReported mean ± SD from three male or female monkeys.
CCL = Vd x ln(2)/ti/2 with Vd assumed as the average of the estimated values for male and female monkeys and ti/2 estimated as
described in Appendix C.2.
dAs described in Section 3.1.4, the 90% CI for the elimination constant, ke = ln(2)/ti/2, was combined with the estimated range of
Vd to estimate an overall range of human CL.
eUpper and lower bounds of Vd for humans set to average value in rats (lower bound) and highest estimated individual value for
monkeys (see Human Studies in Section 3.1.4), respectively.
'Human CL estimated by allometric scaling from values estimated for mice, rats, and monkeys; human ti/2 from EPA estimate
(Section 3.1.4); human Vd = CL x ti/2/ln(2).
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Toxicological Review of PFHxA and Related Salts
Thus, two alternative values of the DAF, CLh:CLa[S]—which is the ratio of clearance values—
can be obtained (see Table 5-4). Even though there is no apparent difference in CL between men
and women, the sex difference in rats and mice means that the internal dose in male rats at a given
applied dose, for example, will be different from the internal dose in female rats given the same
dose. Use of DAFs specific to the sex of the experimental animals in which an endpoint is observed
is needed to account for this difference in dosimetiy among laboratory animals. The HED for 10
mg/kg-day in male rats is expected to be different from the HED for 10 mg/kg-day in female rats
because the internal dose in male rats will be higher than the internal dose in female rats at that
dose.
Table 5-4. Two options for rat, mouse, and human clearance values and data-
informed dosimetric adjustment factor
Sex
Species
Animal clearance
(L/hr- kg)a
Human clearance (L/hr-kg)
DAF (CLh:CLa[si)
Male
Rat
0.163
1.84 x 10"3 b
((0.45 -7.35) x 10"3)c
(mean, estimated rage, using
preferred [data-driven]
approach)
1.1 x 10"2
(2.8 x 10~3- 4.5 x 10"2)d
Mouse
0.0894
2.1 x 10"2
(5.0 x 10"3- 8.2 x 10"2)d
Female
Rat
0.383
4.8 x 10"3
(1.2 x 10"3- 1.9 x 10"2)d
Mouse
0.206
8.9 x 10"3
(2.2 x 10"3- 3.6 x 10"2)d
Male
Rat
0.163
0.1376
(alternative approach)
0.84
Mouse
0.0894
1.5
Female
Rat
0.383
0.36
Mouse
0.206
0.67
Shaded values were applied to derive the PODhed.
aSpecies/sex-specific CL values (see Appendix C).
Calculated from mean human elimination constant, ke, obtained by Bayesian PK analysis and average volume of
distribution for male and female monkeys (see Table 5-3).
Calculated from 90% credible interval for the human elimination constant, ke, obtained by Bayesian PK analysis
and the estimated range of volume of distribution for humans (see Table 5-3).
dDAF uncertainty calculated from estimated range of human clearance value.
Calculated from allometric scaling of CL using results in Table 5-3.
To evaluate whether it is more reasonable to expect CL or Vd to be similar in humans as in
experimental animals, values of CL were examined directly in humans for PFHxS, PFNA, and PFOA
by Zhang etal. f2013bl and can be compared to those for experimental animals. By comparing
human and rat clearance for a set of compounds from the same chemical family, for which data are
available in both species, a "read across" can be done to evaluate the most likely case for PFHxA.
Note that PFHxS has the same carbon chain length as PFHxA (C6) and while PFOA and PFNA have
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Toxicological Review of PFHxA and Related Salts
longer chains (Cs and Cg respectively) they are still much more chemically similar to PFHxA than
any other compounds for which corresponding human data are available. Briefly, Zhang et al.
f2013bl measured PFAS concentrations in serum and matched 24-hour urine samples to directly
measure urinary clearance. To avoid the complicating issue of losses from menstrual blood, results
for men and women over the age of 50 years are evaluated. Median urinary CL values reported by
Zhang etal. f2013blwere 0.015, 0.094, and 0.19 mL/kg-day for PFHxS, PFNA, and total PFOA (all
isomers), respectively.
Kim etal. (2016b) reported renal PFHxS clearance of 0.76 mL/kg-day in rats while Kim et
al. (2016b) and Sundstrom etal. (2012) reported total clearance of 7-9 mL/kg-day. Sundstrom et
al. f20121 also reported total clearance of PFHxS of 3-5 mL/kg-day in male mice and 1.3-
1.9 mL/kg-day in monkeys. Thus, these results for PFHxS show significantly slower clearance in
humans than in mice, rats, and monkeys.
Dzierlenga etal. (2019) evaluated the PK of PFOA (as well as PFHxA) in male rats and
obtained clearance values of 9-16 mL/kg/d, depending on the dose and route. Thus, PFOA is also
cleared much more rapidly in rats than humans.
The reported dose/AUC can be used to derive clearance values for PFNA from the results of
Tatum-Gibbs etal. f20111. The estimated CL in rats is highly variable across the studies evaluated
but ranged from 2 to 66 mL/kg-day in males and from 4 to 106 mL/kg-day in females fTatum-Gibbs
etal.. 2011: Benskin et al.. 2009: De Silva etal.. 2009: Ohmori et al.. 20031. CL in male and female
mice reported by Tatum-Gibbs etal. (2011) ranged from 3 to 10 mL/kg-day. Although the wide
range for rats indicates a degree of uncertainty, these results indicate that clearance in mice and
rats is similar and much larger than the corresponding human value (0.094 mL/kg-day) (Zhang et
al..2013bl.
Thus, three other PFAS, including one with the same carbon-chain length as PFHxA, have
been shown to have much lower clearance in humans than rats. Data for PFDA were not discussed
here since it is a Cio compound, but it also shows a similar rat-human difference in clearance. Hence,
a read-across analysis suggests that option (1) above is more likely to be true.
The alternative, option (2) above, requires one to accept that the Vd in humans is roughly
two orders of magnitude higher than in rats and monkeys, although the biochemical factors that
determine serum-tissue partitioning are expected to be conserved across mammalian species, as
described in the section above on distribution. Hence, option (2) seems highly unlikely.
Therefore, the top set ofDAFs in Table 5-4—based on CLhuman = 1.84 x 10~3 L/kg-hr—are
the preferred set because they are consistent with data for other PFAS, and the reasonable
expectation, based on data from multiple chemicals, is the volume of distribution in humans
does not substantially differ from that in experimental animals.
Representative calculations of the HED for considered health effects follow, using the POD
of 20 mg/kg-day for postnatal (Fi) body weight at PND 0 fLoveless etal.. 20091 as an example and
the female rat DAF of 4.8 x 10"3, based on clearance:
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Toxicological Review of PFHxA and Related Salts
HED = POD {m9/kg-day) x DAF
HED = 20 (m9lkg_day) x 4.8 x 10"' = 0.096 ('n°Skg.day) (5-5)
In general, clearance captures the overall relationship between exposure and internal dose,
specifically the average concentration of a substance in serum, while the half-life does not Use of
half-life makes an intrinsic assumption that Vd is the same in the test species as in humans. There is
a significant difference between rats and monkeys, which leads to the expectation of a difference
between rats and humans (see Table 5-3).
HED based on clearance incorporates the observed differences in Vd among mice, rats, and
primates, and is therefore, the preferred approach for dosimetry extrapolation from animals to humans.
Uncertainty of Animal-human extrapolation of PFHxA dosimetry
Although the variability between, and even within, some data sets for rats (~4-fold for males
and ~6-fold for females between the lowest and highest mean clearance values) is large, the number
of studies provides confidence in the estimated average clearance values for both male and female
rats, which is reflected by the modest 90% CI for rat CL in Table 5-3.
Only one PK study is available for mice, although with two dose levels (Gannon etal.. 2011).
Further, the data for the 100 mg/kg dose approach a plateau, as if clearance stopped when the
concentration was around 0.5 ng/g, although such a plateau was not observed for the 2 mg/kg data.
EPA concluded that the data, which used 14C labeling, were not correctly adjusted for the
background signal or LOD. EPA was able to analyze the two dose levels for male and female mice
successfully, however, by focusing on the data above the concentration at which the plateau
occurred. Because the data from Gannon etal. (2011) for rats is near the middle of the range for
other rat studies and the methods described otherwise are appropriate, it is presumed that this
study has good quality results, except for the LOD correction of this dose in mice, is presumed.
Therefore, some uncertainty remains with the clearance value obtained for mice from this study.
The PK study of Chengelis etal. f2009al is considered high quality, but the results for
monkeys used only three males and three females.
Uncertainty in the application of the DAF based on clearance remains, given that neither Vd
nor CL were measured or determined in humans. To estimate CL in humans, the human Vd was
assumed equal to the average value estimated in male and female monkeys, which seems less
uncertain given the data and analyses described above. The Vd of male and female mice was
assumed the same as in male and female rats, respectively. Because the difference in Vd between
male and female rats was small, using these sex-specific values for mice will give similar results to
using an average.
One alternative approach to using clearance in mice or rats to estimate the average blood
concentrations in those species for each bioassay might be to use the measured serum
concentrations from toxicological studies as BMD modeling inputs and then the estimated human
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Toxicological Review of PFHxA and Related Salts
clearance value to calculate the HED. Three of the four studies being evaluated, however, did not
measure PFHxA serum concentrations (Klaunig etal.. 2015: Iwai and Hoberman. 2014: Chengelis et
al.. 2009b: Loveless etal.. 20091. Although Iwai and Hoberman f20141 attempted to measure serum
concentrations in mice, all serum measurements were below the LOQ. Therefore, this alternative
approach cannot be applied in evaluating these dose-response data.
There is uncertainty in the estimated human clearance because the Vd had to be
extrapolated from animals (nonhuman primates) and the limited human PK data from only eight
individuals with noncontrolled exposures. As discussed in Section 3.1.2, the distribution of PFHxA
between serum and various tissues is determined by biochemical parameters such as the
concentrations of various binding proteins and the affinity of PFHxA for those proteins, that are
largely conserved across mammalian species. However, Vd values estimated for animals ranged
between 0.33 L/kg in rats (Section 3.1.2) to 1.54 L/kg in one of six monkeys studied fChengelis et
al.. 2009a). so uncertainty in the human Vd is presumed to be about 4.7-fold. Together with the
estimated uncertainty in the human first-order elimination constant for which the 90% credible
interval ranges 3.5-fold, an overall range of uncertainty in the human clearance of 16-fold (± 4-fold)
was estimated (see Section 3.1.4 Pharmacokinetics-Elimination-Human Studies).
Application of Animal-Human Extrapolation for PFHxA Dosimetry
Table 5-5 presents the estimated PODhed (mg/kg-day) values for the hepatic,
developmental, and hematopoietic toxicity endpoints considered for RfD derivation based on the
endpoint selection justification described above and in Table 5-1 and preferred DAF values
presented in Table 5-4.
The last column in Table 5-5 includes normalization from the ammonium salt to the free
acid using a molecular weight conversion [MW free acid/MW ammonium salt = 314/331 = 0.949
flwai and Hoberman. 20141] and sodium salt to free acid [MW free acid/MW sodium
salt = 314/336 = 0.935 fLoveless etal.. 20091], The PODhed for postnatal (Fi) body weights used the
female HED, as exposures were to the dams and assumed equal clearance in a developing offspring
as an adult
The free acid of PFHxA is calculated using the ratio of molecular weights, as follows:
PFHxA (free acid) = (-
PFHxA (free acid) =
MW ammonium salt.
.MW sodium salt.
MW free acid
MW free acid
-) = ) = 0.949
tj V331/
.) = (ill] = 0.935
J \336/
(5-6)
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Toxicological Review of PFHxA and Related Salts
Table 5-5. PODs considered for the derivation of the RfD
Endpoint
Study/confidence
Species, strain
(sex)
PODtype/model
POD
(mg/kg-d)
PODhed
PFHxA3
(mg/kg-d)
Hepatic effects
^Hepatocellular
hypertrophy
Loveless et al. (2009)
High confidence
Subchronic (90 d)
Rat, Crl:CD(SD)
(male)
Rat, Crl:CD(SD)
(female)
BMDL10ER
Multistage 1 NCV
NOAELb
(0% response)
10.66
100
0.11°
0.45°
^Hepatocellular
necrosis
Klaunig et al. (2015)
High confidence
Chronic (104 w)
Rat, Crl:CD(SD)
(female)
NOAELb
(5% response vs.
3.3% in controls)
30
0.14
Hematopoietic effects
4/Hemoglobin
Klaunig et al. (2015)
High confidence
Chronic (51 w)
Rat, Crl:CD(SD)
(female)
BMDLisd
Linear CV
122.77
0.59
Chengelis et al.
(2009b)
High confidence
Subchronic (90 d)
Rat, Crl:CD(SD)
(male)
NOAELd
(7% decrease)
50
0.55
Rat, Crl:CD(SD)
(female)
NOAELd
(3% decrease)
50
0.24
Loveless et al. (2009)
High confidence
Subchronic (90 d)
Rat, Crl:CD(SD)
(male)
NOAELd
(6% decrease)
20
0.21°
Rat, Crl:CD(SD)
(female)
BMDLisd
Polynomial 3 CV
127.61
0.57°
4/Red blood cells
Klaunig et al. (2015)
High confidence
Chronic (51 w)
Rat, Crl:CD(SD)
(male)
NOAELb
(4% decrease)
100
1.21
Rat, Crl:CD(SD)
(female)
BMDLisd
Linear CV
109.15
0.52
Chengelis et al.
(2009b)
High confidence
Subchronic (90 d)
Rat, Crl:CD(SD)
(male)
NOAELd
(no change)
50
0.55
Rat, Crl:CD(SD)
(female)
BMDLisd
Exponential 5
CV
16.32
0.078
Loveless et al. (2009)
High confidence
Subchronic (90 d)
Rat, Crl:CD(SD)
(male)
BMDLisd
Linear NCV
44.57
0.46°
Rat, Crl:CD(SD)
(female)
BMDLisd
Linear CV
112.36
0.50°
Developmental effects
4/Postnatal (Fi)
body weight, PND 0
Loveless et al. (2009)
High confidence
Rat, Crl:CD(SD),
Fi (combined)
BMDLsrd
Hill
10.62
0.048°
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Toxicological Review of PFHxA and Related Salts
Endpoint
Study/confidence
Species, strain
(sex)
PODtype/model
POD
(mg/kg-d)
PODhed
PFHxA3
(mg/kg-d)
4/Postnatal (Fi)
body weight, PND 0
Iwai and Hoberman
(2014)
High confidence
Mouse, CD-I, Fi
(combined)
BMDLsrd
Polynomial 3 CV
Phase 2
80.06
0.68e
4/Postnatal (Fi)
body weight, PND 4
BMDLsrd
Exponential-M5
Phase 1 and 2
Polynomial 3 CV
Phase 2
102.94
89.79
0.87e
0.76e
^Perinatal (Fi)
mortality (PND 0-
21, including
stillbirths)
Iwai and Hoberman
(2014)
High confidence
Mouse, CD-I, Fi
(combined)
BMDLieiro
NLogistic
Phase 2
24.77
0.21e
Endocrine effects
4/Total T4
NTP(2018)
High confidence
Short term (28 d)
Rat, Harlan
Sprague-Dawley
(male)
BMDLisd
Hill CV
25.97
0.29
CV = constant variance; NCV = nonconstant variance; SD = standard deviation.
aHED calculations based on the DAF, the ratio of human and animal clearance values (see Table 5-4). DAF values for
female rats and female mice were used for the respective developmental effects on combined male and female
pups of each species. PODhed based on PFHxA free acid.
bResponse only at high dose with responses far above BMR level, data not modeled.
cPODhed multiplied by normalization factor to convert from sodium salt to free acid (MW free acid/MW sodium
salt = 314/336 = 0.935).
dNo models provided adequate fit; therefore, a NOAEL approach was selected.
6P0Dhed multiplied by normalization factor to convert from ammonium salt to the free acid (MW free acid/MW
ammonium salt = 314/331 = 0.949).
Derivation of Candidate Toxicity Values for the RfD
As discussed below, the subchronic, chronic, developmental, and short-term studies for
hepatic, hematopoietic, developmental, and endocrine effects after PFHxA exposure were
considered for use in deriving candidate toxicity values for the RfD. The PODs presented in
Table 5-5 were considered and specific PODs were advanced for candidate toxicity value derivation
over others within each health outcome category based on the confidence in the study, endpoint
sensitivity and specificity, the PODhed, and other considerations including uncertainty (see
Table 5-6), as discussed below. The candidate lifetime toxicity values are presented in Table 5-7.
For hepatic outcomes, candidate toxicity values for both increased hepatocellular
hypertrophy and necrosis were derived. From the PODs for hepatocellular hypertrophy (Loveless
etal.. 20091. the POD for male rats was selected was selected over the POD for female rats as the
PODhed from males was several-fold lower. Although. Although increased hepatocellular
hypertrophy was considered as likely to be precursor event of more severe outcomes such as
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Toxicological Review of PFHxA and Related Salts
necrosis (which might warrant deprioritizing necrosis), a candidate toxicity value for necrosis was
also derived as both studies were of high confidence and the PODheds did not notably differ.
For hematopoietic outcomes, data on RBCs and HGB from three high confidence studies
were advanced for estimation of PODheds. Of those, the lowest PODhed, by a large margin, was for
decreased RBCs in female rats from the high confidence subchronic study fChengelis etal.. 2009bl
(PODhed = 0.078 mg/kg-d). A candidate value for this POD was derived. Given the sensitivity of this
POD over other PODs from the two subchronic studies and no clear reason to advance the other
PODs (e.g., both studies were of high confidence and RBCs and HGB were interpreted to have
similar sensitivity and specificity), none of the other PODs for RBCs or HGB from the subchronic
studies were used to calculate candidate values. However, some caution in this POD is carried
forward to candidate value derivation as the unadjusted POD was far below the observed NOAEL
(50 mg/kg-d), suggesting that variability in the data may be a drive the candidate value lower. The
subchronic PODhed of 0.078 mg/kg-d (Chengelis etal.. 2009b) was also 7-fold lower than the RBC
finding available from the chronic study observed in the same sex, species, dose, and the magnitude
of change in the response at 200 mg/kg-d was also similar (~8% decrease RBC). Although both
subchronic and chronic exposure designs and study durations include the life cycle of a red blood
cell (~60 days in rats), the subchronic study duration design may miss longer term effects on RBC
regeneration. Therefore, although the PODhed was less sensitive, the chronic POD for RBCs was also
advanced for candidate toxicity value derivation.
For developmental outcomes, the high confidence finding of decreased postnatal (Fi) body
weight in rats at PND 0 had the lowest PODhed of 0.048 mg/kg-d (Loveless etal.. 2009). This was 4-
fold lower than the next lowest PODhed for increased perinatal mortality in mice (Iwai and
Hoberman. 2014). and considerably lower than PODheds for offspring body weight decreases in
mice. Ultimately, given the large difference in PODs for body weight changes, the PODs in mice were
not advanced and only a candidate toxicity value for decreased postnatal (Fi) body weight in rats
was derived. Because derived. Because changes in fetal body weight are less severe than fetal
mortality and the PODhed for the fetal body weight change in rats was only 4-fold more sensitive to
PFHxA exposure than the PODhed for offspring mortality in mice and therefore a candidate toxicity
value was also derived for the increases in perinatal mortality in mice (Iwai and Hoberman. 2014).
For endocrine effects, the available POD was limited to thyroid hormone data from a short-
term study in rats fNTP. 20181. Consistent with EPA's A Review of the Reference Dose and Reference
Concentration Processes fU.S. EPA. 2002cl and for the purposes of this assessment, a lifetime
toxicity value is not supported given the high degree of uncertainty when using PODs from a short-
term study to protect against effects of exposure for a lifetime. The general lack of long-term or
developmental studies of endocrine effects introduces uncertainty, as discussed below.
Under EPA's A Review of the Reference Dose and Reference Concentration Processes (U.S.
EPA. 2002c] and Methods for Derivation of Inhalation Reference Concentrations and Application of
Inhalation Dosimetry fU.S. EPA. 19941. five possible areas of uncertainty and variability were
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Toxicological Review of PFHxA and Related Salts
considered in deriving the candidate toxicity values for PFHxA. An explanation of these five possible
areas of uncertainty and variability and the UF values assigned to each of the of the PODhed values
selected for use in deriving candidate toxicity values, as well as the rationales for these decisions
are listed in Table 5-6.
Table 5-6. Uncertainty factors for the development of the RfD for PFHxA
UF
Value
Justification
UFa
3
As described in EPA's A Review of the Reference Dose and Reference Concentration
Processes (U.S. EPA, 2002c), the interspecies uncertainty factor (UFa) is applied to account
for extrapolation of animal data to humans. A UFa of 3 is applied to account for
uncertainty in characterizing the PK and pharmacodynamic differences between species
(i.e., from rats or mice to humans) following oral PFHxA exposure. Some aspects of the
cross-species extrapolation of PK processes have been accounted for by calculating an
HED through application of a DAF based on animal and human clearance; however,
residual uncertainty related to potential pharmacodynamic differences remains. Typically,
a threefold UF is applied for this uncertainty in the absence of chemical-specific
information. This is the case for the hepatic, hematopoietic, and developmental effects of
PFHxA.
UFh
10
A UFh of 10 is applied for interindividual variability in humans in the absence of
quantitative information on potential differences in PKand pharmacodynamics relating
to PFHxA exposure in humans.
UFs
1
(developmental;
hepatic and
hematopoietic
[chronic study])
A UFs of 1 is applied to the developmental endpoint from the one-generation
reproductive studv bv Loveless et al. (2009) and developmental studv bv Iwai and
Hoberman (2014). The developmental period is recognized as a susceptible lifestage and
exposure designs capturing sensitive developmental windows (e.g., gestation) are more
relevant for the induction of developmental effects than lifetime exposures (U.S. EPA,
1991). Although effects on bodv weights are not uniaue to development, the current
evidence for PFHxA suggests this is a sensitive lifestage for body weight effects of PFHxA
exposure based on effects being measured at lower doses than adults.
A UFs of 1 is also applied to hematopoietic (i.e., decreased RBC) and hepatic (i.e.,
necrosis) endpoints in the chronic studv (Klaunig et al., 2015) as the 51 wks of dailv
exposure is considered sufficiently representative of exposure for the rodents' lifetime
and the incidence or severity of these outcomes is not anticipated to increase with longer
exposure duration.
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Toxicological Review of PFHxA and Related Salts
UF
Value
Justification
3 (hepatic and
hematopoietic
[subchronic
study])
A UFs of 3 is applied to hepatocellular hypertrophy for the purpose of deriving a lifetime
RfD. Although the endpoint was derived from a 90-d subchronic studv (Loveless et al.,
2009), which would tvoicallv warrant application of a UFs= 10, there are some other
sparse data that mitigate this uncertainty, to an extent. Specifically, significant
hepatocellular hypertrophy was not observed in the chronic study in male or female rats
(Klaunig et al., 2015). However, a UFs= 1 was not applied as the evidence supports a
pathway where hepatocellular hypertrophy is an adverse event leading to more severe
outcomes with longer exposure durations, such as the necrosis that was observed in
female rats in the chronic study. Additionally, the highest dose levels used in the chronic
study were at or below the LOAEL for this effect in the available subchronic studies (see
Section 3.2.1). Thus, some uncertainty remains and a UFs of 3 is applied.
A UFs of 3 is applied to the hematopoietic endpoint (i.e., decreased RBCs) from the 90-d
subchronic studv (Chengelis et al., 2009b). Specifically, a UFs lower than 10 was
warranted as more significant effects on RBCs were not observed after chronic exposure
at the same PFHxA doses (RBCs decreases of the same magnitude were observed at
matched doses and sexes across exposure durations see Section 3.2.4); however,
uncertainty remains when considering the doses tested in the chronic as compared to the
subchronic study. Further, the subchronic study may poorly predict a chronic exposure
setting across multiple RBC life cycles (one cycle is ~60 d), which could reflect cumulative
effects as greater proportions of RBCs across stages are affected, or possibly even
reduced effects (compensatory responses) warranting a UFs higher than 1. Thus, a UFs of
3 was applied.
UFl
1
A UFl of 1 is applied for LOAEL-to-NOAEL extrapolation when the POD is a BMDL or a
NOAEL.
UFd
3
The database uncertainty factor (UFd) is applied to account for the potential of deriving
an under-protective reference value as a result of incomplete characterization of a
chemical's toxicity (U.S. EPA, 2002c). For PFHxA, a UFD of 3 was selected to account for
deficiencies and uncertainties in the database. Although not large, the available evidence
base spans a number medium and high confidence studies in laboratory animals,
including several short-term studies, two subchronic studies, and one chronic study in
Sprague-Dawley rats, as well as developmental/reproductive studies in Sprague-Dawley
rats and Crl:CDl mice. Limitations in the database, as described in U.S. EPA (2002c) were
used as the basis for a UFd = 3. These limitations included a lack of informative human
studies for most outcomes; subchronic or chronic toxicity studies in more than one
species; studies of potential multigenerational effects; effects; developmental
neurotoxicity studies; and thyroid toxicity studies after PFHxA exposure during
development or after long-term exposure.
UFc
See Table 5-7
and Table 5-11
Composite uncertainty factor = UFa x UFh x UFs x UFl x UFd.
UFa = interspecies uncertainty factor, UFh = interindividual variability in humans uncertainty factor,
UFs = extrapolating from subchronic to chronic uncertainty factor, UFl = LOAEL-to-NOAEL extrapolation
uncertainty factor, UFd = database uncertainty factor.
The uncertainty factors described in Table 5-6 were applied and the resulting candidate
values for use in estimating an RfD for lifetime exposure are shown in Table 5-7.
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Toxicological Review of PFHxA and Related Salts
Table 5-7. Candidate values for PFHxA
Endpoint/study/
confidence
Species,
strain (sex)
PODhed
PFHxA3
(mg/kg-
d)
UFa
UFh
UFs
UFl
UFd
UFc
Candidate
value
PFHxA
(mg/kg-d)
Candidate
value
PFHxA-Nab
(mg/kg-d)
Candidate
value
PFHxA-
NH4b
(mg/kg-d)
Hepatic effects
^Hepatocellular
hypertrophy, 90 d
Loveless et al. (2009)
High confidence
Rat,
Crl:CD(SD)
(male)
0.11
3
10
3
1
3
300
4x 104
4 x 10"4
4 x 10"4
^Hepatocellular
necrosis
2y
Klaunig et al. (2015)
High confidence
Rat,
Crl:CD(SD)
(female)
0.144
3
10
1
1
3
300
5 x 10"4
5 x 10"4
5 x 10"4
Hematopoietic effects
4/Red blood cells, 51
wks
Klaunig et al. (2015)
High confidence
Rat,
Crl:CD(SD)
(female)
0.52
3
10
1
1
3
100
5 x 10"3
6 x 10"3
5 x 10"3
4/Red blood cells
90 d
Chengelis et al.
(2009b)
High confidence
Rat,
Crl:CD(SD)
(male)
0.078
3
10
3
1
3
300
3 x 10"4
3 x 10"4
3 x 10"4
Developmental effects
vJ/Fi body weight,
PND0
Loveless et al. (2009)
High confidence
Rat, Sprague-
Dawley, Fi
(combined)
0.048
3
10
1
1
3
100
5 x 10"4
5 x 10"4
5 x 10"4
^Perinatal (Fi)
mortality (PND 0-21,
including stillbirths)
Iwai and Hoberman
(2014)
High confidence
Mouse, CD-I,
Fi
(combined)
0.21
3
10
1
1
3
100
2 x 10"3
2 x 10"3
2 x 10"3
aHED calculations based on DAF, the ratio of human and animal clearance values (see Table 5-4). DAF values for
female rats and female mice were used for the respective developmental effects on combined male and female
pups of each species.
bTo calculate candidate values for salts of PFHxA, multiply the candidate value of interest by the ratio of molecular
weights of the free acid and the salt. For example, for the sodium salt of PFHxA, the candidate value would be
calculated by multiplying the free acid candidate value by 1.070 (MW free acid/MW sodium
salt = 336/314 = 1.070). This same conversion can be applied to other salts of PFHxA, such as the ammonium salt.
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Toxicological Review of PFHxA and Related Salts
Selection of Lifetime Toxicity Value(s)
Selection of Organ- or System-Specific RfDs
Organ/system-specific (os)RfDs associated with each health effect are presented in
Table 5-8 as they could be useful for certain decision purposes (i.e., site-specific risk assessments).
The rationale for and application of osRfD are described in the PFAS Protocol, Appendix A.
Confidence in each osRfD is described in Table 5-8 and is based on several factors, including
confidence in the study, the evidence base supporting the hazard, and quantitative estimate for
each osRfD.
The candidate toxicity value of 4 x 10"4 mg/kg-d PFHxA for hepatocellular hypertrophy was
selected as the hepatic osRfD. Considering that hepatocellular hypertrophy likely precedes necrosis
and is a slightly more sensitive endpointthan necrosis, hepatocellular hypertrophy from male rats
in the subchronic study (Loveless etal.. 20091 was selected over the candidate value for necrosis.
The candidate toxicity value of 5 x 10"3 mg/kg-d PFHxA for decreased RBCs in female rats
from the chronic study was selected as the hematopoietic osRfD. Although the candidate value for
the subchronic study (3 x 10"4 mg/kg-d PFHxA) was lower than that for the chronic study,
confidence in the POD from the subchronic study was reduced as the unadjusted POD was well
below the observed NOAEL in the study and may have been driven largely by variability in the data.
Note that the unadjusted PODs for decreased RBCs in the other subchronic study datasets were
more similar to the POD from the chronic study as shown in Table 5-7. Further, the subchronic
study may poorly predict a chronic exposure setting across multiple RBC life cycles (one cycle is
~60 days), which could reflect cumulative effects as greater proportions of RBCs across stages are
affected, or possibly even reduced effects (compensatory responses). Therefore, the value from the
chronic study was interpreted as the most appropriate value for use in addressing the potential
hematopoietic effects of lifetime exposure.
The candidate toxicity value of 5 x 10"4 mg/kg-d PFHxA for decreased Fi offspring body
weight at PND 0 rats (Loveless et al.. 2009) was selected as the developmental osRfD. This
candidate value was selected over the candidate value for increased perinatal mortality in mice
(Iwai and Hoberman. 2014) because changes in fetal body weight are less severe and candidate
value was substantially lower than fetal mortality. Therefore, this value is interpreted as protective
of all developmental effects, including the increases in perinatal mortality in mice. In addition, of
the two study designs, Loveless etal. f20091 included a longer exposure that spanned the entirety
of gestation (exposure continued through the end of lactation, but the effect in question was
measured on PND 0) versus Iwai and Hoberman (2014) where mouse offspring were exposed from
GD 6-18; thus, the study in rats may encompass a greater proportion of the relevant critical
windows for the developmental effects of PFHxA exposure.
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Toxicological Review of PFHxA and Related Salts
Table 5-8. Confidence in the organ/system specific RfDs for PFHxA
Confidence
categories
Designation
Discussion
Hepatic osRfD = 4 x 10~4 mg/kg-d PFHxA; 4 x 10~4 mg/kg-d PFHxA-Na and PFHxA-NH4
Confidence in the
study used to derive
osRfD
High
Confidence in the studv (Loveless et al., 2009) is hiah based on the studv
evaluation results (i.e., rated high confidence overall) (HAWC link). The
overall study size, design, and test species were considered relevant for
deriving toxicity values.
Confidence in the
evidence base for
hepatic effects
Medium
Confidence in the oral toxicity evidence base for hepatic effects is medium
based on consistent, dose-dependent, and biologically coherent effects on
organ weight and histopathology observed in multiple high confidence
subchronic and chronic studies. The available mechanistic evidence also
supports biological plausibility of the observed effects. Limitations of the
evidence base for hepatic effects is the lack of human studies, and
measures that would have been useful to inform the pathways for hepatic
effects leading to hepatocellular hypertrophy (e.g., specific histological
stains for hepatic vacuole contents).
Confidence in
quantification of the
PODhed
Medium
Confidence in the quantification of the POD and osRfD is medium, given
the POD was based on BMD modeling within the range of the observed
data and dosimetric adjustment based on PFHxA-specific PK information.
Some residual uncertainty in the application of the dosimetric approach
described above is that Vd and CL were not measured in humans or mice
and were considered equivalent to those for monkeys and rats,
respectively.
Overall confidence in
the hepatic osRfD
Medium
The overall confidence in the osRfD is medium and is primarily driven by
medium confidence in the overall evidence base for hepatic effects, high
confidence in the study, and medium confidence in quantitation of the
POD. High confidence in the study was not interpreted to warrant
changing the overall confidence from medium.
Hematopoietic osRfD = 5 x 10~3 mg/kg-d PFHxA and PFHXA-NH4; 6 x 10~3 mg/kg-d PFHxA-Na
Confidence in study
High
Confidence in the studv (Klaunig et al., 2015) is hiah based on the studv
evaluation results (i.e., rated hiah confidence overall) (HAWC link) and
characteristics that make it suitable for deriving toxicity values, including
relevance of the exposure paradigm (route, duration, and exposure levels),
use of a relevant species, and the study size and design.
Confidence in
evidence base for
hematopoietic
effects
Medium
Confidence in the evidence base for hematopoietic effects was medium
based on consistent and biologically coherent effects on red blood cells,
hemoglobin, and other hematological parameters measured across
multiple high confidence chronic and subchronic studies. The RBC and
hemoglobin findings were correlative with an erythrogenic response
indicated by increased reticulocytes and pathological findings of splenic
extramedullar hematopoiesis and bone marrow erythroid hyperplasia.
Limitations of the hematopoietic evidence base are lack of human studies,
and some hematological measures were observed only at the highest
dose, limiting interpretation of dose-response.
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Toxicological Review of PFHxA and Related Salts
Confidence
categories
Designation
Discussion
Confidence in the
quantification of the
PODhed
Medium
Confidence in the quantification of the POD and osRfD is medium given
the POD was based on BMD modeling within the range of the observed
data and dosimetric adjustment based on PFHxA-specific PK information.
Some residual uncertainty in the application of the dosimetric approach
described above is that Vd and CL were not measured in humans or mice
and were considered equivalent to those for monkeys and rats,
respectively. Some additional residual uncertainty in POD quantitation was
due to the availability of a much lower POD, and the dose spacing was
biased toward low end of the dose range.
Confidence in
hematopoietic osRfD
Medium
The overall confidence in the osRfD is medium and is primarily driven by
medium confidence in the overall evidence base for hematopoietic effects,
and medium confidence in quantitation of the POD. High confidence in the
study was not interpreted to warrant changing the overall confidence
from medium.
Developmental osRfD = 5 x 10~4 mg/kg-d PFHxA; 5 x 10~4 mg/kg-d PFHxA-Na and PFHxA-Nm
Confidence in study
High
Confidence in the studv (Loveless et al., 2009) is hiah based on studv
evaluation results (i.e., rated hiah confidence overall) (HAWC link) and
characteristics that make it suitable for deriving toxicity values, including
relevance of the exposure paradigm (route, duration, and exposure levels),
use of a relevant species, and the study size and design.
Confidence in
evidence base for
developmental
effects
Medium
Confidence in evidence base for developmental effects is medium based
on the availability of data from two studies in different species (i.e., rats
and mice) that consistently observed decreases in offspring body weight
and coherent increases in perinatal mortality. Areas of uncertainty
included lack of human data and multigenerational animal toxicity studies.
Also, data to inform other organ/system-specific hazards (e.g., thyroid,
immune, nervous system) following a developmental exposure are lacking.
Together these present significant data gaps in the potential effects during
this sensitive life stage.
Confidence in the
quantification of the
PODhed
Medium
Confidence in the quantification of the POD and osRfD is medium given
the POD was based on BMD modeling within the range of the observed
data and dosimetric adjustment based on PFHxA-specific PK information.
Some residual uncertainty in the application of the dosimetric approach
described above is that Vd and CL were not measured in humans or mice
and were considered equivalent to those in monkeys and rats,
respectively.
Confidence in
developmental osRfD
Medium
The overall confidence in the osRfD is medium and is primarily driven by
medium confidence in the overall evidence base for developmental
effects, high confidence in the study, and medium confidence in
quantitation of the POD. High confidence in the study was not interpreted
to warrant changing the overall confidence from medium.
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Toxicological Review of PFHxA and Related Salts
Selection of the overall oral reference dose and confidence statements
Table 5-9. Organ/system specific RfD values for PFHxA
System
Basis
PODhed
UFc
osRfD for
PFHxA
(mg/kg-d)
osRfD for
PFHxA-Naa
(mg/kg-d)
osRfD for
PFHxA-
NH4a
(mg/kg-d)
Confidence
Hepatic
Increased
hepatocellular
hypertrophy in
adult male
Crl:CD
Sprague-Dawle
y rats
0.11 mg/kg-d
based on
BMDLioer and
free salt
normalization
(Loveless et al.,
2009)
300
4 x 10"4
4 x 10"4
4 x 10"4
Medium
Hematopoietic
Decreased red
blood cells in
adult female
Crl:CD
Sprague-Dawle
y rats
0.52 mg/kg-d
based on
BMDLisd
(Klaunig et al.,
2015)
100
5 x 10"3
6 x 10"3
5 x 10"3
Medium
Developmenta
1 (selected as
RfD)
Decreased
postnatal (PND
0) body weight
in Fi
Sprague-Dawle
y male and
female rats,
exposed
throughout
gestation and
lactation
0.048 mg/kg-d
based on
BMDLsrd and
free salt
normalization
(Loveless et al.,
2009)
100
5 x 10"4
5 x 10"4
5 x 10"4
Medium
aTo calculate candidate values for salts of PFHxA, multiply the candidate value of interest by the ratio of molecular
weights of the free acid and the salt. For example, for the sodium salt of PFHxA, the candidate value would be
calculated by multiplying the free acid candidate value by 1.070 (MW free acid/MW sodium
salt = 336/314 = 1.070). This same conversion can be applied to other salts of PFHxA, such as the ammonium salt.
From the identified human health effects of PFHxA and derived osRfDs for hepatic,
hematopoietic, and developmental effects (see Table 5-9), an RfD of 5 x 10~4 mg/kg-day PFHxA
based on decreased postnatal (Ft) body weight in rats was selected. As described in Table 5-8,
confidence in the RfD is medium, based on medium confidence in the developmental RfD. The
decision to select the developmental RfD was based on all available osRfDs in addition to overall
confidence and composite uncertainty for those osRfDs. The confidence in the selected RfD is
equivalent to that of the hepatic and hematopoietic RfDs. The developmental
endpoint decreased Fi body weight at PND 0 having the lowest overall PODhed of 0.048 mg/kg-d
PFHxA based on BMDLsrd and free salt normalization (Loveless etal.. 20091 and UFc of 100 was
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Toxicological Review of PFHxA and Related Salts
considered protective across all lifestages. The hepatic RfD was slightly lower but was based on a
higher PODhed (0.11 mg/kg-day PFHxA) and UFc (300). The developmental RfD, therefore, is based
on the lowest PODhed and lowest UFc using a study considered high confidence.
Estimation or Selection of Points of Departure (PODs) for Subchronic RfD Derivation
In addition to providing an RfD for lifetime exposure in health systems, this document also
provides an RfD for less-than-lifetime ("subchronic") exposures. These subchronic RfDs were based
on the endpoints advanced for POD derivation provided in Table 5-1. Data to inform potential
hepatic and hematopoietic effects from the high confidence subchronic studies by (Chengelis etal..
2009b: Loveless etal.. 20091 were considered the most informative for developing candidate
values. The high confidence developmental/reproductive studies flwai and Hoberman. 2014:
Loveless etal.. 20091 were also advanced for candidate value derivation. While it was not advanced
for a lifetime RfD, the high confidence short-term study fNTP. 20181 was considered for subchronic
candidate value derivation for endocrine effects. In general, the rationales for advancing these
endpoints for subchronic value derivation are the same as described and summarized above in
Table 5-1; however, for hematopoietic effects, subchronic data from Chengelis etal. (2009b) and
Loveless etal. f20091 were prioritized over the data from the chronic study by Klaunigetal. f20151
for use in deriving a subchronic RfD.
The endpoints selected for dose-response were modeled using approaches consistent with
EPA's Benchmark Dose Technical Guidance document (U.S. EPA. 2012a). The approach was the same
as described above for derivation of lifetime toxicity values, the BMRs selected for dose-response
modeling and the rationales for their selection (see Table 5-2), and the dosimetric adjustments
using the ratio of the clearance in animal to that in the human and salt to free acid normalization.
Table 5-10 presents the estimated PODhed (mg/kg-day) values for the hepatic, developmental, and
hematopoietic toxicity endpoints considered for subchronic RfD derivation.
Table 5-10. PODs considered for the derivation of the subchronic RfD
Endpoint
Study/confidence
Species,
strain (sex)
PODtype/model
POD (mg/kg-d)
PODhed PFHxA3
(mg/kg-d)
Hepatic effects
^Hepatocellular
hypertrophy
Loveless et al. (2009)
High confidence
Rat,
Crl:CD(SD)
(male)
BMDLioer
Multistage 1 NCV
10.66
0.11°
Rat,
Crl:CD(SD)
(female)
NOAELb
(0% response)
100
0.45°
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Toxicological Review of PFHxA and Related Salts
Endpoint
Study/confidence
Species,
strain (sex)
PODtype/model
POD (mg/kg-d)
PODhed PFHxA3
(mg/kg-d)
Hematopoietic effects
4/Hemoglobin
Chengelis et al. (2009b)
High confidence
Rat,
Crl:CD(SD)
(male)
BMDLisd
Polynomial 3 CV
81.35
0.89
Rat,
Crl:CD(SD)
(female)
NOAELd
(3% decrease)
50
0.24
Loveless et al. (2009)
High confidence
Rat,
Crl:CD(SD)
(male)
NOAELd
(6% decrease)
202
0.21°
Rat,
Crl:CD(SD)
(female)
BMDLisd
Polynomial 3 CV
127.61
0.57°
4/Red blood cell
Chengelis et al. (2009b)
High confidence
Rat,
Crl:CD(SD)
(male)
NOAELd
(no change)
50
0.55
Rat,
Crl:CD(SD)
(female)
BMDLisd
Exponential 5 CV
16.32
0.078
Loveless et al. (2009)
High confidence
Rat,
Crl:CD(SD)
(male)
BMDLisd
Linear NCV
44.57
0.46°
Rat,
Crl:CD(SD)
(female)
BMDLisd
Linear CV
112.36
0.50°
Developmental Effects
4/Postnatal (Fi)
body weight,
PNDO
Loveless et al. (2009)
High confidence
Rat,
Crl:CD(SD), Fi
(combined)
BMDLsrd
Hill
10.62
0.048°
4/Postnatal (Fi)
body weight,
PNDO
Iwai and Hoberman
(2014)
High confidence
Mouse, CD-I,
Fi (combined)
BMDLsrd
Polynomial 3 CV
Phase 2
80.06
0.68e
4/Postnatal (Fi)
body weight,
PND 4
BMDLsrd
Exponential-M5
Phase 1 and 2
Polynomial 3 CV
Phase 2
102.94
89.79
0.87e
0.76e
^Perinatal
Mortality
Iwai and Hoberman
(2014)
High confidence
Mouse, CD-I,
Fi (combined)
BMDLier
Nested Logistic
Phase 2
24.77
0.21e
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Toxicological Review of PFHxA and Related Salts
Endpoint
Study/confidence
Species,
strain (sex)
PODtype/model
POD (mg/kg-d)
PODhed PFHxA3
(mg/kg-d)
Endocrine effects
4, Total T4
NTP(2018)
High confidence
Rat, Harlan
Sprague-
Dawley (male)
BMDLisd
Hill CV
25.97
0.29
1SD = 1 standard deviation, CV = constant variance, NCV = nonconstant variance.
aHED calculations based on the DAF, the ratio of human and animal clearance values (see Table 5-3). DAF values for
female rats and female mice were used for the respective developmental effects on combined male and female
pups of each species. PODhed based on PFHxA free acid.
bResponse only at high dose with responses far above BMR level, data not modeled.
cPODhed multiplied by normalization factor to convert from sodium salt to free acid (MW free acid/MW sodium
salt = 314/336 = 0.935).
dNo models provided adequate fit; therefore, a NOAEL approach was selected.
6P0Dhed multiplied by normalization factor to convert from sodium salt to free acid (MW free acid/MW ammonium
salt = 314/331 = 0.949).
Derivation of Candidate Toxicity Values for the Subchronic RfD
The PODhed values listed in Table 5-10 were further narrowed for selecting candidate
toxicity values for subchronic osRfD derivation and subchronic RfD selection. As described for the
RfD, RBCs were a more sensitive PODhed for hematopoietic effects. Therefore, the red blood cell
endpoint from female rats from Chengelis etal. (2009b) was advanced as the candidate toxicity
value for subchronic RfD derivation over male endpoints for hematocrit and red blood cells based
on RBC being more sensitive and therefore expected to be protective of effects in both sexes.
Applying the rationales described for the selection of the lifetime osRfDs, the same endpoints were
advanced as the candidate toxicity values for derivation of the hepatic and developmental
subchronic osRfDs: male hepatocellular hypertrophy and decreased Fi body weight at PND 0
(Loveless etal.. 20091. For endocrine effects, a candidate toxicity value for the subchronic RfD was
derived based on a short-term study showing decreased total T4 in adult male rats exposed for 28
days. Due to the high uncertainty associated with deriving a lifetime value based on a short-term
study this endpoint was not considered for the RfD but was advanced for the subchronic RfD.
As described above under "Derivation of Candidate Values for the RfD," and in U.S. EPA
f2002cl five possible areas of uncertainty and variability were considered in deriving the
candidate subchronic toxicity values for PFHxA. In general, the explanations for these five possible
areas of uncertainty and variability and the values assigned to each as a designated UF to be applied
to the candidate PODhed values are listed above and in Table 5-6, including the UFd which remained
at 3 due to data gaps (i.e., for most outcomes, a lack of: informative human studies, animal studies
from multiple species or spanning multiple generations, studies of other organ/system-specific
effects associated with other PFAS, including PFOA and PFOS, particularly following developmental
exposure). The exception that a UFs = 1 was applied for all endpoints since no subchronic to chronic
extrapolation was required for the candidate toxicity values for the subchronic RfD. For the
endocrine endpoint, a UFs = 3 was applied. Although the data are derived from a short-term (28-
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Toxicological Review of PFHxA and Related Salts
day) study, the life cycle of T4 occurs on the order days or less [ty2 is approximately 12 hours
(Dohler etal.. 19791]. therefore, the short-term study duration is expected to capture effects for all
components of the T4 life cycle and uncertainty about the ability of PFHxA exposure to affect
following a 28-day exposure is reduced. Considering this along with the short half-life (hours) of
PFHxA in rats (see Section 3.1) and the approximate 3-fold difference in duration between this
study and a guideline subchronic study (i.e., 90 days), a UFs = 3 was deemed most appropriate. The
resulting candidate toxicity values are shown in Table 5-11.
Table 5-11. Candidate subchronic toxicity values for PFHxA
Endpoint/study/
confidence
Species, strain
(sex)
PODhed
PFHxA3
(mg/kg-d)
UFa
UFh
UFs
UFl
UFd
UFc
Candidate
value
PFHxA
(mg/kg-d)
Candidate
value
PFHxA-Nac
(mg/kg-d)
Candidate
value
PFHxA-Nac
(mg/kg-d)
Hepatic effects
^Hepatocellular
hypertrophy, 90 d
Loveless et al. (2009)
High confidence
Rat, Crl:CD(SD)
(male)
0.11b
3
10
1
1
3
100
1 X 10"3
1 X 10"3
1 X 10"3
Hematopoietic effects
4/Red blood cell, 90 d
Chengelis et al.
(2009b)
High confidence
Rat, Crl:CD(SD)
(female)
0.078
3
10
1
1
3
100
8 x 10"4
8 x 10"4
8 x 10"4
Developmental effects
4/Postnatal (Fi) body
weight, PND 0
Loveless et al. (2009)
High confidence
Rat,
Sprague-Dawley,
Fl
(combined)
0.048b
3
10
1
1
3
100
5 x 10"4
5 x 10"4
5 x 10"4
^Perinatal Mortality,
PND 0-21
Iwai and Hoberman
(2014)
High confidence
Mouse, CD-I, Fl
(combined)
0.21
3
10
1
1
3
100
2 x 10"3
2 x 10"3
2 x 10"3
Endocrine effects
4, Total T4
NTP (2018)
High confidence
Rat, Harlan
Sprague-Dawley
(male)
0.29
3
10
3
1
3
300
1 x 10"3
1 x 10"3
1 x 10"3
aThe RfD for the free acid of PFHxA is calculated using the ratio of molecular weights as described above.
bPODHED multiplied by normalization from the sodium salt to free acid (MW free acid/MW sodium
salt = 314/336 = 0.935).
cTo calculate subchronic candidate values, osRfDs or the subchronic RfD for salts of PFHxA, multiply the value of
interest by the ratio of molecular weights of the salt and free acid. For example, for the sodium salt of PFHxA, the
candidate value is calculated by multiplying the free acid candidate value by 1.070: (MW free acid/MW sodium
salt = 336/317 = 1.070).
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Toxicological Review of PFHxA and Related Salts
Selection of Subchronic Organ- or System-Specific RfDs
As described above, subchronic osRfDs associated with each health effect are presented as
they may be useful for certain decision purposes (i.e., site-specific risk assessments with less-than-
lifetime exposures). Confidence in each subchronic osRfD are described in Table 5-12 and consider
confidence in the study used to derive the quantitative estimate, the overall health effect, specific
evidence base, and quantitative estimate for each subchronic osRfD.
Table 5-12. Confidence in the subchronic organ/system specific RfDs for
PFHxA
Confidence categories
Designation
Discussion
Hepatic subchronic osRfD = 1 x 10~3 mg/kg-d PFHxA; 1 x 10~3 mg/kg-d PFHxA-Na or PFHxA-NH4
Confidence in the study
used to derive the
subchronic osRfD
High
Confidence in the studv (Loveless et al., 2009) is hiah based on the
studv evaluation results (i.e., rated high confidence overall) HAWC
link) and characteristics that make it suitable for deriving toxicity
values, including relevance of the exposure paradigm (route, duration,
and exposure levels), use of a relevant species, and the study size and
design.
Confidence in the
evidence base for
hepatic effects
Medium
Confidence in the oral toxicity evidence base for hepatic effects is
medium based on consistent, dose-dependent, and biologically
coherent effects on organ weight and histopathology observed in
multiple high confidence subchronic and chronic studies. The
available mechanistic evidence also supports biological plausibility of
the observed effects. Limitations of the evidence base for hepatic
effects is the lack of human studies, and measures that would have
been useful to inform the pathways for hepatic effects leading to
hepatocellular hypertrophy (e.g., specific stains for hepatic vacuole
contents, specific histological for pathology).
Confidence in
quantification of the
PODhed
Medium
Confidence in the quantification of the POD and subchronic osRfD is
medium given the POD was based on BMD modeling within the range
of the observed data and dosimetric adjustment based on PFHxA-
specific PK information. Some residual uncertainty in the application
of the dosimetric approach described above is that Vd and CL were not
measured in humans or mice and were considered equivalent to
those in monkeys and rats, respectively.
Overall confidence in
the hepatic subchronic
osRfD
Medium
The overall confidence in the subchronic osRfD is medium and is
primarily driven by medium confidence in the overall evidence base
for hepatic effects, high confidence in the study, and medium
confidence in quantitation of the POD. High confidence in the study
was not interpreted to warrant changing the overall confidence from
medium.
Hematopoietic subchronic osRfD = 8 x 10~4 mg/kg-d PFHxA; 8 x 10~4 mg/kg-d PFHxA-Na or PFHxA-NH4
Confidence in study
used to derive the
subchronic osRfD
High
Confidence in the studv (Chengelis et al., 2009b) is hiah based on the
studv evaluation results (i.e., rated high confidence overall) (HAWC
link) and characteristics that make it suitable for deriving toxicity
values, including relevance of the exposure paradigm (route, duration,
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Toxicological Review of PFHxA and Related Salts
Confidence categories
Designation
Discussion
and exposure levels), use of a relevant species, and the study size and
design.
Confidence in evidence
base for hematopoietic
effects
High
Confidence in the evidence base for hematopoietic effects was high
based on consistent and biologically coherent effects on red blood
cells, hemoglobin, and other hematological parameters measured
across multiple high confidence chronic and subchronic studies. The
RBC and hemoglobin findings were also coherent with an erythrogenic
response indicated by increased reticulocytes and pathological
findings of splenic extramedullar hematopoiesis and bone marrow
erythroid hyperplasia. Limitations of the hematopoietic evidence base
are lack of human studies, and some hematological measures were
observed only at the highest dose, limiting interpretation of dose-
response.
Confidence in
quantification of the
PODhed
Low
Confidence in the quantification of the POD and subchronic osRfD is
low given the POD was far below the NOAEL (50 mg/kg-d) and the
osRfD is far below toxicity values derived for the same finding from
other subchronic studies suggesting some underlying variability
driving the POD lower.
Confidence in
hematopoietic
subchronic osRfD
Medium-Low
The overall confidence in the subchronic osRfD is medium-low and is
primarily driven by low quantitation of the POD. High confidence in
the study was not interpreted to warrant changing the overall
confidence from medium-low.
Developmental subchronic osRfD = 5 x 10~4 mg/kg-d PFHxA; 5 x 10~4 mg/kg-d PFHxA-Na or PFHxA-NFU
Confidence in study
used to derive the
subchronic osRfD
High
Confidence in the studv (Loveless et al., 2009) is hiah based on the
studv evaluation results (i.e., rated high confidence overall) (HAWC
link) and characteristics that make it suitable for deriving toxicity
values, including relevance of the exposure paradigm (route, duration,
and exposure levels), use of a relevant species, and the study size and
design.
Confidence in evidence
base for developmental
effects
Medium
Confidence in evidence base for developmental effects is medium
based on the availability of data from two studies in different species
(i.e., rats and mice) that consistently observed decreases in offspring
body weight and coherent increases in mortality. One area of
uncertainty is that there were no multigenerational studies available.
Also, data to inform other organ/system-specific hazards
(e.g., thyroid, immune, nervous system) following a developmental
exposure is lacking. Together these present significant data gaps in
the potential effects during this sensitive life stage.
Confidence in the
quantification of the
PODhed
Medium
Confidence in the quantification of the POD and subchronic osRfD is
medium given the POD was based on BMD modeling, within the range
of the observed data and dosimetric adjustment based on PFHxA-
specific PK information. Some residual uncertainty in the application
of the dosimetric approach described above is that Vd and CL were not
measured in humans or mice and were considered equivalent to
those in monkeys and rats, respectively.
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Toxicological Review of PFHxA and Related Salts
Confidence categories
Designation
Discussion
Confidence in
developmental
subchronic osRfD
Medium
The overall confidence in the subchronic osRfD is medium and is
primarily driven by medium confidence in the overall evidence base
for developmental effects, high confidence in the study, and medium
confidence in quantitation of the POD. High confidence in the study
was not interpreted to warrant changing the overall confidence from
medium.
Endocrine subchronic osRfD = 1 x 10~3 mg/kg-d PFHxA; 1 x 10~3 mg/kg-d PFHxA-Na or PFHxA-NFU
Confidence in study
used to derive the
subchronic osRfD
High
Confidence in the studv (NTP, 2018) is hiah based on the studv
evaluation results (i.e., rated high confidence overall) (HAWC link) and
characteristics that make it suitable for deriving toxicity values,
including relevance of the exposure paradigm (route and exposure
levels), use of a relevant species, and the study size and design.
Confidence in evidence
base for endocrine
effects
Medium-Low
Confidence in the oral toxicity evidence base for endocrine effects is
medium-low based primarily on strong dose-dependent effects on
serum T4 in males reported in a high confidence short-term study.
This finding is consistent with effects observed in a low confidence
human study and a zebrafish study. The available mechanistic
evidence supports biological plausibility of the observed effects in
rodents. Limitations of the evidence base for endocrine effects is the
lack of informative human studies and longer duration studies in
animals. Additionally, studies to inform the pathways for thyroid
effects leading changes in thyroid hormone levels and/or sex specific
differences.
Confidence in the
quantification of the
PODhed
Medium
Confidence in the quantification of the POD and subchronic osRfD is
medium given the POD was based on BMD modeling within the range
of the observed data and dosimetric adjustment based on PFHxA-
specific PK information. Some residual uncertainty in the application
of the dosimetric approach described above is that Vd and CL were not
measured in humans or mice and were considered equivalent to
those in monkeys and rats, respectively.
Confidence in endocrine
subchronic osRfD
Medium
The overall confidence in the subchronic osRfD is medium and is
primarily driven by medium-low confidence in the overall evidence
base for developmental effects, high confidence in the study, and
medium confidence in quantitation of the POD. High confidence in the
study was not interpreted to warrant changing the overall confidence
from medium.
Selection of Subchronic RfD and Confidence Statement
Organ/system-specific subchronic RfD values for PFHxA are summarized in Table 5-13.
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Table 5-13. Subchronic osRfD values for PFHxA
System
Basis
PODhed
UFc
osRfD for
PFHxA
(mg/kg-d)
osRfD for
PFHxA-Naa
(mg/kg-d)
osRfD for
PFHxA-
NH4a
(mg/kg-d)
Confidence
Hepatic
Increased
hepatocellular
hypertrophy in
adult male
Crl:CD
Sprague-Dawley
rats
0.11 mg/kg-d based
on BMDLioer and free
salt normalization
(Loveless et al., 2009)
100
1 X 10"3
1 X 10"3
1 X 10"3
Medium
Hematopoietic
Decreased red
blood cells in
adult female
Crl:CD
Sprague-Dawley
rats
0.078 mg/kg-d based
on BMDLisd
(Chengelis et al.,
2009b)
100
8 x 10"4
8 x 10"4
8 x 10"4
Medium-
Low
Developmental
Decreased
postnatal (PND
0) body weight in
Fi
Sprague-Dawley
male and female
rats, exposed
throughout
lactation and
gestation
0.048 mg/kg-d based
on BMDL5RD and free
salt normalization
(Loveless et al., 2009)
100
5 x 10"4
5 x 10"4
5 x 10"4
Medium
Endocrine
Decreased total
T4 in adult male
Harlan Sprague-
Dawley rats
0.29 mg/kg-d based
on BMDLisd (NTP,
2018)
300
1 x 10"3
1 x 10"3
1 x 10"3
Medium
aTo calculate candidate values for salts of PFHxA, multiply the candidate value of interest by the ratio of molecular
weights of the free acid and the salt. For example, for the sodium salt of PFHxA, the candidate value would be
calculated by multiplying the free acid candidate value by 1.070 (MW free acid/MW sodium
salt = 336/314 = 1.070). This same conversion can be applied to other salts of PFHxA, such as the ammonium salt.
From the identified targets of PFHxA toxicity and derived subchronic osRfDs (see
Table 5-13), a subchronic RfD of 5 x 10~4 mg/kg-day based on decreased postnatal body weight
is selected for less-than-lifetime exposure. Confidence in the subchronic RfD is medium, based on
medium confidence in the developmental subchronic RfD, as described in Table 5-12. The
confidence in the selected subchronic RfD is equivalent to that of the hepatic subchronic RfDs and
higher than the hematopoietic subchronic RfD. The developmental subchronic RfD is expected to be
protective of all life stages. The UFc (see Table 5-13) is lower than or equivalent to the other
subchronic osRfDs and the endpointhas the lowestPODHED (0.048 mg/kg-day, see Table 5-11). The
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Toxicological Review of PFHxA and Related Salts
decision to select the developmental subchronic RfD was based on all the available subchronic
osRfDs in addition to overall confidence and composite uncertainty for those subchronic osRfDs.
5.2.2. Inhalation Reference Concentration (RfC)
No published studies investigating the inhalation effects of subchronic, chronic, or
gestational exposure to PFHxA in humans or animals have been identified. Therefore, an RfC is not
derived.
5.3. CANCER TOXICITY VALUES
As discussed in Sections 3.3 and 4.2, given the sparse evidence base and in accordance with
the Guidelines for Carcinogen Risk Assessment fU.S. EPA. 20051. EPA concluded that there is
inadequate information to assess carcinogenic potential for PFHxA for any route of exposure.
Therefore, consistent with the Guidelines and the lack of adequate data on the potential
carcinogenicity of PFHxA, quantitative estimates for either oral (oral slope factor, OSF) or
inhalation (inhalation unit risk; IUR) exposure were not derived.
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