A EPA
EPA/635/R-20/326a
Interagency Science Consultation Draft
www.epa.gov/iris
Toxicological Review of Perfluorohexanoic Acid (PFHxA)
and Related Compounds Ammonium and Sodium Perfluorohexanoate
(PFHxA-NH4 and PFHxA-Na)
[CASRN 307244
CASRN 21615474
CASRN 2923264]
August 2021
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 Perfluorohexanoic Acid (PFHxA)
DISCLAIMER
This document is an interagency science consultation draft for review purposes only. This
information is distributed solely for the purpose of interagency science consultation. It has not
been formally disseminated by EPA. It does not represent and should not be construed to represent
any Agency determination or policy. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
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 1-3
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 1-9
1.2.3. Data Extraction 1-10
1.2.4. Evidence Synthesis and Integration 1-10
1.2.5. Dose-Response Analysis 1-11
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. TOXICOKINETICS, EVIDENCE SYNTHESIS, AND EVIDENCE INTEGRATION 3-1
3.1.TOXICOKINETICS 3-1
3.1.1. Absorption 3-2
3.1.2. Distribution 3-2
3.1.3. Metabolism 3-6
3.1.4. Elimination 3-7
3.1.5. PBPK Models 3-13
3.1.6. Summary 3-13
3.2. NONCANCER EVIDENCE SYNTHESIS AND INTEGRATION 3-17
3.2.1. Hepatic Effects 3-17
3.2.2. Developmental Effects 3-39
3.2.3. Renal Effects 3-49
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
3.2.4. Hematopoietic Effects
3-59
3.2.5. Endocrine Effects
3-70
3.2.6. Male Reproductive Effects
3-77
3.2.7. Female Reproductive Effects
3-85
3.2.8. Immune Effects
3-92
3.2.9. Nervous System Effects
3-99
3.3. CARCINOGENICITY
3-103
3.3.1. Cancer
3-103
4. SUMMARY OF HAZARD IDENTIFICATION CONCLUSIONS
4-1
4.1. SUMMARY OF CONCLUSIONS FOR NONCANCER HEALTH EFFECTS
4-1
4.2. SUMMARY OF CONCLUSIONS FOR CARCINOGENICITY
4-2
4.3. CONCLUSIONS REGARDING SUSCEPTIBLE POPULATIONS AND LIFESTAGES
4-2
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)
5-32
5.3. CANCER TOXICITY VALUES
5-32
REFERENCES
R-l
SUPPLEMENTAL INFORMATION
(see Volume 2)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
TABLES
Table ES-1. Health effects with evidence available to synthesize and draw summary judgments
and derived toxicity values xiv
Table 1-1. Physicochemical properties of PFHxA 1-2
Table 1-2. PFHxA levels at 10 military installations and National Priority List sites 1-5
Table 3-1. Summary of PK evidence for PFHxA 3-15
Table 3-2. Evaluation results for animal studies assessing effects of PFHxA exposure on the
hepatic system 3-19
Table 3-3. Percent increase in relative liver weight due to PFHxA exposure in short-term and
subchronic oral toxicity studies 3-20
Table 3-4. Incidence of hepatocellular hypertrophy findings in adult rats due to PFHxA exposure
in short-term and subchronic oral toxicity studies 3-21
Table 3-5. Percent change in alanine aminotransferase due to PFHxA exposure in short-term,
subchronic, and chronic oral toxicity studies 3-24
Table 3-6. Percent change in aspartate aminotransferase due to PFHxA exposure in short-term,
subchronic, and chronic oral toxicity studies 3-25
Table 3-7. Percent change in alkaline phosphatase due to PFHxA exposure in short-term,
subchronic, and chronic oral toxicity studies 3-26
Table 3-8. Percent change in total protein (TP) and globulin (G) due to PFHxA exposure in
short-term, subchronic, and chronic oral toxicity studies 3-29
Table 3-9. Evidence profile table for hepatic effects 3-36
Table 3-10. Study design characteristics and outcome-specific study confidence for
developmental endpoints 3-39
Table 3-11. Incidence of perinatal mortality following PFHxA ammonium salt exposure in a
developmental oral toxicity study 3-41
Table 3-12. Percent change relative to control in offspring body weight due to PFHxA sodium or
ammonium salt exposure in developmental oral toxicity studies 3-43
Table 3-13. Percent change relative to control in eye opening due to PFHxA ammonium salt
exposure in a developmental oral toxicity study 3-45
Table 3-14. Evidence profile table for developmental effects 3-47
Table 3-15. Renal endpoints for PFHxA and associated confidence scores from repeated-dose
animal toxicity studies 3-50
Table 3-16. Percent increase in relative and absolute kidney weight due to PFHxA exposure in
short-term, subchronic, and chronic oral toxicity studies 3-51
Table 3-17. Evidence profile table for renal effects 3-56
Table 3-18. Hematopoietic endpoints for PFHxA and associated confidence scores from
repeated-dose animal toxicity studies 3-60
Table 3-19. Percent change in red blood cells due to PFHxA exposure in short-term, subchronic,
and chronic oral toxicity studies 3-62
Table 3-20. Percent change in hematocrit due to PFHxA exposure in short-term, subchronic, and
chronic oral toxicity studies 3-64
Table 3-21. Percent change in hemoglobin due to PFHxA exposure in short-term, subchronic,
and chronic oral toxicity studies 3-64
Table 3-22. Percent change in reticulocytes due to PFHxA exposure in short-term, subchronic,
and chronic oral toxicity studies 3-66
Table 3-23. Evidence profile table for hematopoietic effects 3-68
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Table 3-24. Endocrine endpoints for PFHxA and associated confidence scores from
repeated-dose animal toxicity studies 3-71
Table 3-25. Percent change in thyroid hormone levels following PFHxA exposure in a 28-day oral
toxicity study 3-72
Table 3-26. Incidence of thyroid follicular epithelial cell hypertrophy following PFHxA
ammonium salt exposure in a 90-day oral toxicity study 3-73
Table 3-27. Evidence profile table for endocrine effects 3-75
Table 3-28. Study design, exposure characteristics, and individual outcome ratings 3-79
Table 3-29. Evidence profile table for male reproductive effects 3-83
Table 3-30. Study design characteristics 3-86
Table 3-31. Evidence profile table for female reproductive effects 3-90
Table 3-32. Study design characteristics and individual outcome ratings for immune endpoints 3-93
Table 3-33. Evidence profile table for immune effects 3-97
Table 3-34. Nervous system endpoints for PFHxA and associated confidence scores from
repeated-dose animal toxicity studies 3-99
Table 3-35. Evidence profile table for nervous system effects 3-101
Table 3-36. Summary of PFHxA genotoxicity studies 3-105
Table 5-1. Endpoints considered for dose-response modeling and derivation of points of
departure 5-2
Table 5-2. Benchmark response levels selected for BMD modeling of PFHxA health outcomes 5-6
Table 5-3. Summary of serum half-lives and estimated clearance for PFHxA 5-13
Table 5-4. Two options for rat, mouse, and human clearance values and data-informed
dosimetric adjustment factor (DAF) 5-14
Table 5-5. PODs considered for the derivation of the RfD 5-17
Table 5-6. Uncertainty factors for the development of the RfD for PFHxA 5-20
Table 5-7. Candidate values for PFHxA 5-22
Table 5-8. Confidence in the organ/system-specific RfDs for PFHxA 5-22
Table 5-9. Organ/system-specific RfD (osRfD) values for PFHxA 5-24
Table 5-10. PODs considered for the derivation of the subchronic RfD 5-26
Table 5-11. Candidate values for deriving the subchronic RfD for PFHxA 5-28
Table 5-12. Confidence in the subchronic organ/system-specific RfDs for PFHxA 5-29
Table 5-13. Subchronic osRfD values for PFHxA 5-31
FIGURES
Figure 1-1. Linear chemical structures of (from left to right) perfluorohexanoic acid (PFHxA),
ammonium perfluorohexanoate (PFHxA-NH4), and sodium perfluorohexanoate
(PFHxA-Na) 1-2
Figure 2-1. Literature search and screening flow diagram for perfluorohexanoic acid (PFHxA)
and related compounds ammonium and sodium perfluorohexanoate (PFHxA-
NH4 and PFHxA-Na) 2-2
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 3-18
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Figure 3-2. Liver weights (absolute and relative) after short-term and subchronic PFHxA
exposures (full details available by clicking the HAWC link) 3-20
Figure 3-3. Clinical chemistry findings (serum enzymes) after short-term, subchronic, and
chronic PFHxA exposures (full details available by clicking the HAWC link) 3-24
Figure 3-4. Blood protein findings after short-term, subchronic, and chronic PFHxA exposures
(full details available by clicking the HAWC link) 3-28
Figure 3-5. Hepatobiliary findings in rats exposed by gavage to PFHxA or PFHxA sodium salt (full
details available by clicking the HAWC link) 3-30
Figure 3-6. Peroxisomal beta oxidation activity in rats exposed by gavage to PFHxA or PFHxA
sodium salt (full details available by clicking the HAWC link) 3-31
Figure 3-7. Developmental effects on offspring viability in mice exposed to PFHxA ammonium
salt (HAWC: PFHxA - Animal Toxicity Developmental Effects link) 3-41
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) 3-43
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 Eye Effects link) 3-45
Figure 3-10. Study evaluation for human epidemiological studies reporting findings from PFHxA
exposures (full details available by clicking HAWC link) 3-49
Figure 3-11. Animal toxicological renal histopatholoy 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 3-52
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 urea nitrogen (BUN) and creatinine were
described as null, but findings were not quantitatively reported 3-54
Figure 3-13. Hematological findings (hematocrit [HCT], hemoglobin [HGB], and red blood cells
[RBC]) in rats exposed by gavage to PFHxA or PFHxA sodium salt (full details
available by clicking the HAWC link) 3-61
Figure 3-14. Hematological findings (mean cell hemoglobin [MCH], mean cell hemoglobin
concentration [MCHC], and mean cell volume [MCV]) in rats exposed by gavage
to PFHxA or PFHxA sodium salt (full details available by clicking the HAWC link) 3-63
Figure 3-15. Hematological findings (reticulocytes) in rats exposed by gavage to PFHxA or PFHxA
sodium salt (full details available by clicking the HAWC link) 3-65
Figure 3-16. Hemostasis findings in rats exposed by gavage to PFHxA or PFHxA sodium salt (full
details available by clicking the HAWC link) 3-67
Figure 3-17. Study evaluation for human epidemiologic studies reporting toxicity findings from
PFHxA exposures (HAWC: PFHxA - Human Toxicity Endocrine Effects link) 3-70
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 HAWC link) 3-72
Figure 3-19. Study evaluation for human epidemiological studies reporting male reproductive
findings from PFHxA exposures (HAWC: PFHxA - Human Toxicity Male
Reproductive Effects link) 3-78
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) 3-80
This document is a draft for review purposes only and does not constitute Agency policy.
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Figure 3-21. Male reproductive effects on epididymis and testis weight in rats exposed to PFHxA
or PFHxA sodium salt (HAWC: PFHxA - Animal Toxicity Male Reproductive
Effects link) 3-81
Figure 3-22. Study evaluation for human epidemiological studies reporting female reproductive
findings from PFHxA exposures (HAWC: PFHxA - Human Toxicity Female
Reproductive link) 3-85
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) 3-87
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) 3-88
Figure 3-25. Study evaluation for human epidemiological studies reporting findings from PFHxA
exposures (HAWC: PFHxA - Human Toxicity Immune Effects link) 3-92
Figure 3-26. Immune organ weights in rats exposed by gavage to PFHxA or PFHxA sodium salt
(HAWC: PFHxA - Animal Toxicity Immune Effects link) 3-94
Figure 3-27. Immune cell counts in rats exposed by gavage to PFHxA or PFHxA sodium salt
(HAWC: PFHxA - Animal Toxicity Immune Effects link) 3-95
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
ABBREVIATIONS AND ACRONYMS
ADME
absorption, distribution, metabolism,
ISI
Influential Scientific Information
and excretion
IUR
inhalation unit risk
AFFF
aqueous film-forming foam
i.v.
intravenous
A:G
albumin:globulin ratio
LDH
lactate dehydrogenaseLOQ limitof
AIC
Akaike's information criterion
quantitation
ALP
alkaline phosphatase
LOAEL
lowest-observed-adverse-effect level
ALT
alanine aminotransferase
LOD
limit of detection
APTT
activated partial thromboplastin time
LOEC
lowest observed effect concentration
AST
aspartate aminotransferase
MCH
mean cell hemoglobin
atm
atmosphere
MCHC
mean cell hemoglobin concentration
ATSDR
Agency for Toxic Substances and
MCV
mean cell volume
Disease Registry
MOA
mode of action
AUC
area under the curve
MW
molecular weight
BMD
benchmark dose
NCTR
National Center for Toxicological
BMDL
benchmark dose lower confidence limit
Research
BMDS
Benchmark Dose Software
NOAEL
no-observed-adverse-effect level
BMR
benchmark response
NPL
National Priorities List
BUN
blood urea nitrogen
NTP
National Toxicology Program
BW
body weight
ORD
Office of Research and Development
Cmax
maximum concentration
OECD
Organisation for Economic
CAR
constitutive androstane receptor
Co-operation and Development
CASRN
Chemical Abstracts Service registry
OSF
oral slope factor
number
osRfD
organ/system-specific oral reference
CBC
complete blood count
dose
CHO
Chinese hamster ovary (cell line cells)
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
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RBC red blood cells
RD relative deviation
RfC reference concentration
RfD oral reference dose
RNA ribonucleic acid
ROS reactive oxygen species
RXR retinoid X receptor
SD standard deviation
TP total protein
TRI Toxics Release Inventory
TSCATS Toxic Substances Control Act Test
Submissions
TSH thyroid stimulating hormone
UF uncertainty factor
UFa interspecies uncertainty factor
UFc composite uncertainty factor
UFd evidence base deficiencies uncertainty
factor
UFh human variation uncertainty factor
UFl LOAEL to NOAEL uncertainty factor
UFs subchronic to chronic uncertainty
factor
V2 volume of distribution of peripheral
compartment (two-compartment PK
model)
Vd volume of distribution
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
AUTHORS | CONTRIBUTORS | REVIEWERS
Assessment Team (Lead Authors)
Michelle M. Angrish, Ph.D. U.S. EPA/Office of Research and Development/Center
Laura Dishaw, Ph.D. for Public Health and Environmental Assessment
Authors
J. Allen Davis, M.S.P.H. U.S. EPA/Office of Research and Development/Center
Jeffery Dean, Ph.D. for Public Health and Environmental Assessment
Elizabeth G. Radke, Ph.D.
Paul Schlosser, Ph.D.
Shana White, Ph.D.
Jay Zhao, Ph.D., M.P.H., DABT
Todd Zurlinden, Ph.D.
Yu-Sheng Lin, Ph.D.
Contributors
Xabier Arzuaga, Ph.D.
Belinda Hawkins, Ph.D.
Johanna Congleton, M.S.P.H., Ph.D
Ingrid Druwe, Ph.D.
Kelly Garcia, B.S.
Carolyn Gigot, B.A.
Andrew Greenhalgh, B.S.
Belinda Hawkins, Ph.D.
Shahreen Hussain, B.S.
J. Phillip Kaiser, Ph.D., DABT
Jason C. Lambert, Ph.D., DABT
Elizabeth Oesterling Owens, Ph.D
Brittany Schulz, B.A.
Michele Taylor, Ph.D.
Andre Weaver, Ph.D.
Amina Wilkins, M.P.H.
Michael Wright, Sc.D.
Production Team
Maureen Johnson
Ryan Jones
Dahnish Shams
Vicki Soto
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U.S. EPA/Office of Research and Development/Center for
Public Health and Environmental Assessment
U.S. EPA/Office of Research and Development/Center for
Public Health and Environmental Assessment
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Executive Direction
James Avery
Wayne Casio
Barbara Glenn
Kay Holt
Samantha Jones
Andrew Kraft
Janice Lee
Viktor Morozov
Ravi Subramaniam
Kristina Thayer
Paul White
CPAD Associate Division Director
CPHEA Center Director
CPHEA/CPAD/Science Assessment Methods Branch Chief
CPHEA Deputy Center Director
CPHEA Associate Director
CPAD Senior Science Advisor, Integrated Risk Information System,
IRIS PFAS Team Lead
CPHEA/CPAD/Toxic Effects Assessment [RTP] Branch Chief
CPHEA/CPAD/Quantitative Assessment Branch Chief
CPHEA/CPAD/Toxic Effects Assessment (DC) Branch Chief
CPAD Division Director
CPAD Senior Science Advisor
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
EXECUTIVE SUMMARY
Summary of Occurrence and Health Effects
Perfluorohexanoic acid (PFHxA, CASRN 307-24-4), ammonium perfluorohexanoate
(PFHxA-NH4, CASRN 21615-47-4), and sodium perfluorohexanoate (PFHxA-Na, CASRN 2923-26-4)
are members of the group 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 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) (see December 2018 IRIS Program Outlook) at the request of EPA National
Programs. The systematic review protocol (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 on the basis of those public
comments. Appendix A includes the updated version of the protocol and summarizes the history of
the revisions.
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
quality 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 (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.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Overall, the available evidence indicates that PFHxA exposure is likely to cause hepatic,
developmental, and hematopoietic effects in humans, given relevant exposure circumstances.
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. Selected
quantitative data from these identified hazards were used to derive toxicity values (Table ES-1).
In addition, evidence in rats suggests the potential for PFHxA exposure to affect endocrine
(i.e., thyroid) responses. Due to limitations in the currently available studies, these data were not
considered for use in deriving toxicity values. Although some human and animal evidence was also
identified for cardiometabolic, 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 under relevant exposure circumstances and were not used to derive
toxicity values
Table ES-1. Health effects with evidence available to synthesize and draw
summary judgments and derived toxicity values
Organ/
System
Integration
judgment
Toxicity
value
Value
(mg/kg-
day)
Confid-
ence
UFa
UFh
UFs
ufl
UFd
UFC
Basis
Hepatic
Evidence
indicates
(likely)
osRfD
3 x icr4
Medium
3
10
3
1
3
300
Increased
hepatocellular
hypertrophy in
adult rats
(Loveless et al..
2009)
Subchronic
osRfD
9 x icr4
Medium
3
10
1
1
3
100
Increased
hepatocellular
hypertrophy in
adult rats
(Loveless et al..
2009)
Hematopoietic
Evidence
indicates
(likely)
osRfD
4 x icr3
High
3
10
1
1
3
100
Decreased red
blood cells in
adult rats
(Klaunig et al..
2015)
Subchronic
osRfD
6 x icr4
High
3
10
1
1
3
100
Decreased red
blood cells in
adult rats
(Chengelis et al..
2009b)
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Organ/
System
Integration
judgment
Toxicity
value
Value
(mg/kg-
day)
Confid-
ence
ufa
UFh
UFS
ufl
ufd
UFC
Basis
Develop-
mental
Evidence
indicates
(likely)
osRfD
4 x icr4
Medium
3
10
1
1
3
100
Decreased Fi
body weight at
PND 0 (Loveless
etal.. 2009)
Subchronic
osRfD
4 x icr4
Medium
3
10
1
1
3
100
Decreased Fi
body weight at
PND 0 (Loveless
etal.. 2009)
Overall RfD
4 x icr4
Medium
3
10
1
1
3
100
Decreased Fi
body weight at
PND 0 (Loveless
etal.. 2009)
Overall
Subchronic RfD
4 x icr4
Medium
3
10
1
1
3
100
Decreased Fi
body weight at
PND 0 (Loveless
etal.. 2009)
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- or system-specific reference dose (in mg/kg-d); UFA = animal to human
uncertainty factor; UFC = composite uncertainty factor; UFD = evidence base deficiencies uncertainty factor;
UFh = human variation uncertainty factor; UFL = LOAEL to NOAEL uncertainty factor; UFS = subchronic to chronic
uncertainty factor.
Chronic Oral Reference Dose (RfD) for Noncancer Effects
From the identified hazards of potential concern (i.e., hepatic, hematopoietic, and
developmental toxicity), decreased offspring body weight in neonatal mice (Loveless etal.. 2009)
was selected as the basis for the RfD of 4 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 0.039 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), interindividual differences in human susceptibility
(UFh = 10), and deficiencies in the toxicity evidence base (UFD = 3). The developmental
organ/system-specific (os) RfD is based on the lowest overall PODhed and UFC; therefore, the
selected RfD based on decreased offspring body weight is assumed to be protective of the observed
health effects associated with lifetime PFHxA exposure because this is considered a sensitive
lifestage and, in the current evidence base, effects on body weight were strongest during the early
postnatal window.
This document is a draft for review purposes only and does not constitute Agency policy.
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1 Confidence in the Oral Reference Dose (RfD)
2 The study conducted by Loveless etal. f20091 reported developmental effects following
3 administration of PFHxA sodium salt to pregnant Sprague-Dawley rats dosed by gavage for
4 approximately 70 days prior to cohabitation through gestation and lactation, for a total of 126 days
5 daily gavage with 0, 20,100, or 500 mg/kg-day sodium PFHxA. This study was rated as high
6 confidence based on study evaluation results (click the HAWC link for full study evaluation details)
7 and study design characteristics that make it suitable for deriving toxicity values. The overall
8 confidence in the RfD is medium and is primarily driven by medium confidence in the overall
9 evidence base for hepatic effects, high confidence in the Loveless etal. f20091 study, and medium
10 confidence in quantitation of the POD (Table 5-8).
11 Noncancer Effects Following Inhalation Exposure
12 No studies that examine toxicity in humans or experimental animals following inhalation
13 exposure and no physiologically based pharmacokinetic (PBPK) models are available to support
14 route-to-route extrapolation; therefore, no RfC was derived.
15 Evidence for Carcinogenicity
16 Under EPA's Guidelines for Carcinogen Risk Assessment fU.S. EPA. 20051. EPA concluded
17 there is inadequate information to assess carcinogenic potential for PFHxA by either oral or
18 inhalation routes of exposure. Therefore, the lack of data on the carcinogenicity of PFHxA
19 precludes the derivation of quantitative estimates for either oral (oral slope factor [OSF]) or
20 inhalation (inhalation unit risk [IUR]) exposure.
21 Subchronic Oral Reference Dose (RfD) for Noncancer Effects
22 In addition to providing RfDs for chronic oral exposures in multiple systems, a subchronic
23 RfD was derived for PFHxA. The same study and endpoint (Loveless etal.. 2009) and decreased Fi
24 body weight) was selected as the basis for the subchronic RfD of 4 x 10~4 mg/kg-day (Table ES-1).
25 Details are provided in Section 5.2.1.
This document is a draft for review purposes only and does not constitute Agency policy.
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1.OVERVIEW OF BACKGROUND INFORMATION
AND ASSESSMENT METHODS
1.1. BACKGROUND INFORMATION ON PFHxA AND RELATED AMMONIUM
AND SODIUM SALTS
Section 1.1 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).
1.1.1. Physical and Chemical Properties
PFHxA and its 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. 2013. 2016. 2017).
PFHxA and its related salts are classified as a perfluorinated carboxylic acids (PFCAs) fOECD. 20151.
Because PFHxA and its associated salts contain fewer than seven perfluorinated carbon groups,
they are considered short-chain PFAS fATSDR. 20181. The linear chemical structures of these
chemicals are presented in Figure 1-1, and select physiochemical properties are provided in
Table 1-1.
This document is a draft for review purposes only and does not constitute Agency policy.
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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)
perfluorohexanoic acid (PFHxA), ammonium perfluorohexanoate
(PFHxA-Nm), and sodium perfluorohexanoate (PFHxA-Na).
Source: EPA CompTox Chemicals Dashboard.
Table 1-1. Physicochemical properties of PFHxA
Property (unit)
PFHxA value
PFHxA-NH4 value
PFHxA-Na value
Formula
CF3(CF2)4COOH
C6H4FiiN02
CsFnNa02
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 io"10
2.35 x io"10
2.35 x io"10
Water solubility (mol/L)
9.34 x 10"5(a)
1.10b
8.78 x 10"5(a)
PKa
-0.16°
-
-
LOgP Octanol-Water
2.85a
3.97b
0.70a
Soil adsorption coefficient (L/kg)
l,070b
1070b
1070b
Bioconcentration factor
49.3b
5.47b
49.3b
aU.S. EPA (2018a). CompTox Chemicals Dashboard; access date 2/18/2021. Median or average experimental
values.
bAverage or median predicted values; - indicates data not available.
cReported by NLM (2016); access date 05/06/2019.
This document is a draft for review purposes only and does not constitute Agency policy.
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1.1.2. Sources, Production, and Use
PFAS are not naturally occurring in the environment fATSDR. 2018: U.S. EPA. 2002b. 2007.
2013. 2019c. 20201. They are manmade compounds that 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. This class of chemicals has 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. 2018: U.S. EPA. 2002b. 2007. 2013. 2019c. 20201. 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 fATSDR. 2018: U.S. EPA. 2002b. 2007. 2013. 2019c. 20201. In addition, PFAS have
been found at private and federal facilities associated with various material or processes involving
aqueous film-forming foam (AFFF), chrome plating, and PFAS production and are associated with
other industries using PFAS (e.g., textiles, carpets) fATSDR. 2018: U.S. EPA. 2002b. 2007. 2013.
2019c. 20201. In AFFF, PFHxA has been detected at concentrations ranging from 0.1 to 0.3 g/L
fBaduel etal.. 2015: Houtz etal.. 20131.
No quantitative PFHxA information on production volume is available fU.S. EPA. 2019al.
and EPA's Toxics Release Inventory (TRI) contains no information on releases to the environment
from facilities manufacturing, processing, or otherwise using PFHxA (ATSDR. 2018: U.S. EPA.
2018b).
Wang etal. (20141 estimated 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
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 fATSDR. 20181. and estimates are available of PFAS
accumulation in vegetation when plants are grown in PFAS-contaminated soil. Yoo etal. (2011)
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
(20141 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
fNLM. 20161. PFHxA bioaccumulates in foods grown on PFAS-containing soils. Blaine etal. f20131
This document is a draft for review purposes only and does not constitute Agency policy.
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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 (Appendix A). Infants potentially have higher exposure due to greater ingestion of food
per body weight. Further, although studies of human breast milk in the U.S. population have not
observed PFHxA, it has been detected in human breast milk from France, Korea, and Spain
(summarized in Table 5 of Anderson etal. f201911. Exposure can also occur through hand-to-
mouth transfer of materials containing these compounds (ATSDR. 2018).
The oral route of exposure is considered the dominant exposure pathway for the general
population (Klaunig etal.. 2015). for which contaminated drinking water is likely a significant
source of exposure. 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 fShao et al.. 20161.
Air and Dust
PFHxA has not been evaluated under the National Air Toxics Assessment program and no
additional information on air levels was identified. PFAS, including PFHxA, have 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 fATSDR. 20181. For example, Kato etal. 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 (2008) 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. (2012) 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. f20131 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.
This document is a draft for review purposes only and does not constitute Agency policy.
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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. 2016. 2019b). PFHxA
was recently added to UCMR5 for public water system monitoring and applies to 2022-2026, with
sample collection proposed 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. 20161. PFHxA also was 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 (Postet
al.. 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. f20151 and Braunig etal. f20171 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 |J.g/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.
Military and National Priorities List (NPL) Sites
PFHxA levels in environmental samples have been measured at military and National
Priorities List (NPL) sites in the United States. Table 1-2 provides the concentrations at these sites
(Anderson etal.. 2016: ATSDR. 2018).
Table 1-2. PFHxA levels at 10 military installations and National Priority
List sites
Media
PFHxA value
Site
Source
Surface soil
Military3
Anderson et al. (2016)
Frequency of detection (%)
70.33
Median (ppb)
1.75
Maximum (ppb)
51.0
Subsurface soil
Frequency of detection (%)
Median (ppb)
Maximum (ppb)
65.38
1.04
140
Military3
Anderson et al. (2016)
This document is a draft for review purposes only and does not constitute Agency policy.
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Media
PFHxA value
Site
Source
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 (2018)
Soil (ppb)
Median
Geometric mean
1,175
1,175
NPLb
ATSDR (2018)
Air (ppbv)
Median
Geometric mean
ND
ND
NPLb
ATSDR (2018)
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. T20121 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. f20181 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 liver. Heo etal.
(2014) 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. f 20141 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. (2014) 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, respectively. The study did not find PFHxA in any of the Serbian samples. PFHxA was
This document is a draft for review purposes only and does not constitute Agency policy.
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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. f20141 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
Section 1.2 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 in Appendix A and is available online. 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 will continue to be updated until shortly before the
release of the document for public comment). The literature search queries the following databases
(no literature was restricted by language):
• PubMed fNational Library of Medicine 1
• Web of Science fThomson Reuters!
• Toxline (moved to PubMed December 2019)
• TSCATS (Toxic Substances Control Act Test Submissions)
In addition, relevant literature not found through evidence base searching was identified
by:
• Review of studies cited in U.S. state, U.S. federal, and international assessments, including
parallel assessment efforts in progress (e.g., the draft Agency for Toxic Substances and
Disease Registry [ATSDR] assessment released publicly in 2018).
• Review of studies submitted to federal regulatory agencies and brought to EPA's attention.
• Identification of studies during screening for other PFAS. For example, searches focused on
one of the other four PFAS currently being assessed by the IRIS Program sometimes
identified epidemiological studies relevant to PFHxA.
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1 • Other gray literature (i.e., primary studies not indexed in typical evidence bases, such as
2 technical reports from government agencies or scientific research groups; unpublished
3 laboratory studies conducted by industry; or working reports/white papers from research
4 groups or committees) brought to EPA's attention.
5 All literature, including literature search updates, is tracked in the EPA Health and
6 Environmental Research Online (HERO) database.-i
7 The PECO criteria identify the evidence that addresses the specific aims of the assessment
8 and focuses the literature screening, including study inclusion/exclusion. In addition to those
9 studies meeting the PECO criteria, studies containing supplemental material potentially relevant to
10 the specific aims of the assessment were inventoried during the literature screening process.
11 Although these studies did not meet PECO criteria, they were not excluded. Rather, they were
12 considered for use in addressing the identified key science issues (Appendix A, Section 2.4) and
13 other major scientific uncertainties identified during assessment development but unanticipated at
14 the time of protocol posting. Studies categorized as "potentially relevant supplemental material"
15 included the following:
16 • In vivo mechanistic or mode-of-action studies, including non-PECO routes of exposure
17 (e.g., intraperitoneal injection) and non-PECO populations (e.g., nonmammalian models)
18 • In vitro and in silico models
19 • Absorption, distribution, metabolism, and excretion (ADME) and pharmacokinetic (PK)
20 studies (excluding models)2
21 • Exposure assessment or characterization (no health outcome) studies
22 • Human case reports or case-series studies
23 • Studies of other PFAS (e.g., perfluorooctanoic acid [PFOA] and perfluorooctane sulfonate
24 [PFOS])
25 The literature was screened by two independent reviewers with a process for conflict
26 resolution, first at the title and abstract level and subsequently the full-text level, using structured
27 forms in DistillerSR (Evidence Partners). Literature inventories for studies meeting PECO criteria
28 and studies tagged as "potentially relevant supplemental material" during screening were created
29 to facilitate subsequent review of individual studies or sets of studies by topic-specific experts.
'EPA'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.
2Given 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.9.2 for details).
This document is a draft for review purposes only and does not constitute Agency policy.
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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
(Appendix A.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; thus, they are described in detail in Appendices A.6.2 and A.6.3, respectively.
• 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). In terms of the process for
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) 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 (listed from best to worst methodological
conduct; see Appendix A, Section 6.1 for definitions) to each evaluation domain
representing the different characteristics of the study methods that were evaluated on the
basis of the criteria outlined in HAWC.
Once all domains were evaluated, the identified strengths and limitations were considered
as a whole 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 (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).
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1.2.3. Data Extraction
The detailed data extraction approach is provided in Appendix A.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 described in HAWC. Not all studies that meet the PECO criteria go through data
extraction: For example, studies evaluated as being uninformative are not considered further and
therefore do not undergo data extraction. The same could be true for low confidence studies if
enough medium and high confidence studies (e.g., on an outcome) are available. All findings 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 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 Appendices A.9 and A.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 to draw an overall
judgment for each health effect The available human and animal evidence pertaining to the
potential health effects are synthesized separately, with each synthesis resulting in a summary
discussion of the available evidence that addresses considerations regarding causation adapted
from Hill fl9651. Mechanistic evidence and other supplemental information is also synthesized to
address key science issues or to help inform key decisions regarding the human and animal
evidence.
The syntheses focus on describing aspects of the evidence that best inform causal
interpretations, including the exposure context examined in the sets of available studies. Syntheses
of the evidence for human and animal health effects are based primarily on studies of high and
medium confidence. Low confidence studies might be used if few or no studies with higher
confidence are available 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 risk of bias and sensitivity
with potential impacts on the conclusions of the evidence synthesis is included in the narrative.
The synthesis of mechanistic evidence and other supplemental information informs the integration
of health effects evidence for hazard identification (i.e., biological plausibility of the available
human or animal evidence, inferences regarding human relevance, or the identification of
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susceptible populations and lifestages across the human and animal evidence) and for
dose-response evaluation.
For each assessed health effect, following the evidence syntheses, integrated judgments are
drawn across all lines of evidence. During evidence integration, a structured and documented
process is 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 (Hill. 19651. 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 strength of the animal and human evidence was considered together in light of
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.
• A summary judgment is drawn as to whether the available evidence base for each potential
human health effect as a whole: "evidence demonstrates," "evidence indicates (likely),"
"evidence suggests," "evidence is inadequate," or "evidence strongly supports no effect" that
PFHxA exposure has the potential to cause the health effect in humans.
The decision points within the structured evidence integration process are summarized in
an evidence profile table for each assessed health effect.
1.2.5. Dose-Response Analysis
The details for the dose-response analysis completed for this assessment are in
Appendix All. Briefly, although procedures for dose-response assessments were developed for
both noncancer and cancer health hazards, and for both oral and inhalation routes 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.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 (U.S. EPA. 2002c).
Specifically, for noncancer outcomes this assessment includes dose-response assessments when the
evidence integration judgments indicate evidence demonstrates and evidence indicates (likely).
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 (U.S. EPA. 2005. 2012a). Within the observed dose range, the preferred approach is to use
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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 fU.S.
EPA. 2012al] as elaborated in Appendix A.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. Extrapolations to exposures lower than the POD involved the application
of five uncertainty factors to estimate candidate noncancer toxicity values, as described in
Appendix All.
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.
Next, evaluation of these osRfDs, including confidence in the evidence base supporting each
potential hazard and other factors (see Appendix All), resulted in the selection of a single RfD to
cover all health outcomes across all organs/systems. Although this overall RfD represents the focus
of the dose-response assessment, the osRfDs can be useful for subsequent cumulative risk
assessments. In addition, a less-than-lifetime, "subchronic" RfD was similarly estimated.
Uncertainties in these toxicity values are transparently characterized and discussed.
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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 339 unique records, with 18 records identified from
posted National Toxicology Program (NTP) study tables and review of reference lists from other
authoritative sources (ATSDR. 2018) (see Figure 2-1). Of the 339 studies identified, 194 were
excluded at the title and abstract level and 77 were reviewed at the full-text level. Of the 77
screened at the full-text level, 26 were considered to meet the PECO criteria (see Appendix A,
Section 4.2.2). The studies meeting PECO at the full-text level included 14 human health effect
studies, 6 in vivo animal studies, 3 in vitro ge no toxicity studies, and 3 ADME/PK studies. In
addition, high-throughput screening data on perfluorohexanoic acid (PFHxA) were available from
EPA's CompTox Chemicals Dashboard fU.S. EPA. 2018a! A literature inventory of the included
animal toxicological studies is available in an literature inventory heatmap accessible via PFHxA
Tableau Link.
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PFHxA
Literature Searches (through Feb 2020)
Pub Med WOS ToxLine TSCATS
(n = 351) (n = 372) (n = 16) (n = 0)
Other
ToxNet (n = 23)
ATSDR assessment (n = 17)
Submitted to EPA (n = 0)
NTP Report (n = 1)
T
TITLE AND ABSTRACT
Title & Abstract Screening
(339 records after duplicate removal)
FULL TEXT SCREENING
Full-Text Screening
(n = 77)
\
Studies Meeting PECO (n =
26)
• Human health effects studies (
n = 14)
• Animal health effect studies (n
= 6)
• Genotoxicity studies (n = 3)
• ADME TK (n = 3)
Excluded (n= 194)
Not relevant to PECO
Excluded {n = 7)
• Not relevant to PECO (n = 3); Review,
commentary, or letter (n = 2); Other (n - 2)
Tagged as Supplemental (n = 118)*
ADME (n - 40); Background/exposure
references (n = 42); Case report or case
study (n - 2); Mechanistic or MOA (n = 9);
Mixture-only (n = 3); Non-PECO route of
exposure (n - 2); Qualitative exposure only
(n = 12); Susceptible population (n = 4);
Other(n = 15)
*Some studies were assigned multiple tags
Figure 2-1. Literature search and screening flow diagram for
perfluorohexanoic acid (PFHxA) and related compounds ammonium and
sodium perfluorohexanoate (PFHxA-NH* and PFHxA-Na).
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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. Thirteen epidemiological studies were identified that report on
the potential association between PFHxA and human health effects. Of these, four were considered
uninformative due to critical deficiencies in one or more domains, including participant selection,
exposure measurement, confounding, or analysis (Tiang etal.. 2014: Kim etal.. 2016a: Seo etal..
2018: Zhang etal.. 20191. The remaining nine studies were rated medium fBao etal.. 2017: Dong et
al.. 2013: Nian etal.. 2019: Zeng etal.. 20151 or low confidence fFu etal.. 2014: Li etal.. 2017: Oin et
al.. 2017: Song etal.. 2018: Wang etal.. 2019: Zhou etal.. 2016).
Of the six unique reports of animal studies meeting PECO criteria, five were considered for
dose-response. The remaining study, Kirkpatrick (20051. was considered uninformative due to
reporting deficiencies (i.e., all summary data [pages 110-1,334] were missing). The available
evidence base of animal toxicity studies on PFHxA and the related ammonium and sodium salts
consists of five reports in rats and mice including short-term fNTP. 20181. subchronic fChengelis et
al.. 2009b: Loveless etal.. 20091. chronic fKlaunig etal.. 20151. and reproductive/developmental
(Iwai and Hoberman. 2014: Loveless etal.. 2009) 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).
Detailed rationales for each domain and overall confidence rating are available in HAWC.
Results for human studies are available here and animal studies are available here. Graphical
representations of the outcome-specific ratings are presented in the organ/system-specific
integration sections (in Section 3.2).
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3.PHARMACOKINETICS, EVIDENCE SYNTHESIS, AND
EVIDENCE INTEGRATION
3.1. PHARMACOKINETICS
Only a few PK studies on PFHxA are available in humans but they provide sufficient data to
estimate its half-life. Several studies such as Ericson etal. (20071 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 f Russell etal.. 20131 using data from an
observational study by Nilssonetal. (20131. Luz etal. (20191 describes a reanalysis of these data
but based only on the three participants with the most rapid clearance.
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.. 20171. particularly to blood, liver, skin, and kidney (Gannon etal.. 20111.
Dzierlenga et al. f20191 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 (Vi = 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 females 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 f Gannon etal.. 20111. Thus, apparent sex-related
quantitative differences in PFHxA PK occur in rats and mice. On the other hand, the AUC in
monkeys 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 fChengelis etal.. 2009a: Gannon etal.. 2011: Iwai. 20111.
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3.1.1. Absorption
Absorption is rapid in rodents and monkeys fChengelis etal.. 2009a: Gannon etal.. 2011:
Iwabuchi et al.. 20171. PFHxA was extensively absorbed with an average time to reach maximum
concentration (Tmax) of 1 h in Sprague-Dawley rats given 26-day repeated gavage doses of 50,150
or 300 mgPFHxA/kg (Chengelis etal.. 2009a). After gavage at2 or 100 mg [l-14C]PFHxA/kgusing
a single dose or 14 daily consecutive doses, Gannon etal. (2011) also observed a short Tmax of 30
and 15 min, respectively, in male and female Sprague-Dawley rats. Similarly, rapid absorption was
also observed in CD-I mice (Gannon etal.. 2011). 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 h, butthe data in male rats indicate either a 25% reduction in absorption or a
corresponding increase in clearance between these two dose levels f Chengelis etal.. 2009a: Gannon
etal.. 20111.
In a recent PK study by Dzierlenga etal. (2019). Sprague-Dawley rats were given PFHxA,
PFOA, and perfluorodecanoic acid (PFDA; C10) 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 at
predetermined schedules, 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. Systemic exposure of PFHxA, as assessed by dose-normalized
area under the plasma AUC and Cmax, was generally lower than systemic exposure to PFOA or PFDA.
Nevertheless, estimated oral bioavailability for all three PFAAs was >100% (Dzierlenga etal..
2019): this result simply could reflect experimental and analytical uncertainty in estimating the
serum concentration AUC from intravenous vs. oral exposure, but also might be due to increased
reabsorption from the intestinal lumen by intestinal transporters of material excreted in the bile.
The researchers also noticed that Tmax slightly increased with increasing oral PFHxA dose levels for
both sexes. For instance, Tmax increased from 0.668 ± 0.154 to 0.890 ± 0.134 h (mean ± standard
error) and from 0.529 ± 0.184 to 0.695 ± 0.14 h with increased gavage doses of PFHxA for male and
female rats, respectively. A similar pattern was observed for PFDA in both male and female rats
and for PFOA exposure in male rats, but not in females (for which Tmax was about constant)
(Dzierlengaetal.. 2019).
3.1.2. Distribution
PFHxA has an aqueous solubility of 15.7 g/L (Zhou etal.. 2010). 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 fGannon etal.. 2011: Russell etal.. 20131. The largest
concentrations were found in liver, skin, heart, lung, and kidney and concentrations peaked within
hours f Gannon et al.. 2 011: Iwabuchi etal.. 20171. 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 h
in rats dosed with 100 mg/kg fGannon 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 etal.. 20171. As described in detail below, the volume of distribution (Vd) was
generally similar (within a factor of 3) among male and female mice, rats, and monkeys fRussell et
al.. 20131.
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," that implies 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. 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 such accumulation over time. These tissue concentrations
could also represent approximate steady-state concentrations achieved in the weeks or months
prior to death, with no subsequent accumulation. More generally, these data cannot inform the
specific exposure scenarios that might have occurred before the time of death.
Perez etal. f20131 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
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.
Fabregaetal. (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. In contrast, Chengelis etal. (2009a) estimated Vd of 0.18 and 0.47 L/kg,
respectively, in male and in female rats. For monkeys, the individual estimates of Vd Chengelis et al.
(2009a) 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, Ke\:
Vd = dose/[mean(AUC0-oo) x mean(ifei)]- (3-1)
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The resulting values of Va 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 5, 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 lipid
content in blood vs. the rest of body. Partitioning or distribution is primarily a function of the
physicochemical properties of a tissue vs. blood (lipid content being a significant component) and
are typically similar across species, not differing by orders of magnitude as suggested by the
difference between the results of Fabrega etal. f20151for 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. T20151 are an artifact of combining data from nonmatched human samples Perez etal. T20131
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 vs. 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
below) 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.
Zhang etal. (2013a) 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.
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) flin etal..
20161. The results showed that although the estimated mass fraction in plasma generally increased
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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 etal. f 20171 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 (Poothong etal.. 2017).
Role of Plasma Protein Binding
Some evidence suggests plasma protein binding (e.g., serum albumin) could also play a role
in PFHxA TK. 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 on the basis of data for selected
HSA ligands including oleic acid, phenylbutazone, and ibuprofen—could affect its
pharmacokinetics.
Organic anion transporters, a family of transmembrane proteins, had been suggested to
play a role in the renal reabsorption of PFAAs fKudo. 2015: Weaver etal.. 20101 (see further
discussion for rats below). Weaver etal. (2010) found that renal transport of PFAAs with different
chain lengths (C2-C18) could occur via specific transporters (Oatl, 0at2, 0at3, 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 fWeaver etal.. 20101.
On the other hand, although Bischel etal. (2011) measured the binding of PFHxA to bovine
serum albumin 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 h in
rats, for example. If 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. 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 faction 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 on the basis of albumin binding.
Distribution in Animal (Rats, Mice, and Monkeys) and In-Vitro Studies
In the study by Chengelis etal. (2009a) described above, both Sprague-Dawley rats and
cynomolgus monkeys (3/sex) were also given PFHxA (10 mg/kg) via a single i.v. injection to
determine PFHxA PK using noncompartmental analysis. In monkeys they observed a distribution
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phase of 8 h and an apparent Va 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 h in both sexes. Serum concentrations of PFHxA were up to
17-fold higher for male than female rats after i.v. dosing, and the AUC after oral dosing was over
4-fold higher in males than females given a 50 mg/kg gavage dose. 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 et al. (20171 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 h
for all tissues, the Tmax for other PFAAs was 12 h 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 min and 1 h 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 min) and 1.5% (at 1 h) 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.
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 on the basis of 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 (Iwabuchi 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.
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 fChengelis etal.. 2009a: Gannon etal.. 20111. Although PFHxA is resistant to
metabolism, fluorotelomer-alcohols and sulfonates can undergo biotransformation to form PFHxA
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or its glucuronide and sulfate conjugates in rodents and humans (Kabadi etal.. 2018: Russell etal..
2015).
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 during 24 h post-dosing regardless of sex
followingi.v. injection (Chengelis etal.. 2009a). Daikin Industries (2009a. 2009b) 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 h after the single or last dose in male and female rats and mice. Likewise Dzierlenga et
al. f20191 reported that liver and kidney concentrations peaked by 30 min in male rats and by 1 h
in female rats after gavage and decreased steadily thereafter (observations at 0.5,1, 3, 6, 9 and
12 h). The tissues concentrations of PFHxA tended to be very low or not quantifiable 24 h after
dosing in both sexes of mice and rats (Gannon etal.. 2011: Iwabuchi etal.. 20171.
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 on the
basis of 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 h, perfluorobutane sulfonate (PFBS) = 4.7 h, pentafluorobenzoic acid
(PFBA) = 9.2 h, ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate (GenX) = 72 h,
PFOA = 136 h, and PFOS = 644 h (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 (2009a) compared PFHxA dosimetry in
naive male and female rats to results after 25 days of dosing (50-300 mg/kg/d) and found no
significant difference in the parameters evaluated, with the serum half-life remaining in the range
of 2-3 h.
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
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carcass. Comparable findings were observed with the multiple oral dose administration (14 daily
doses) scenarios flwai. 20111.
Chengelis etal. f2009al 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 h compared to 1.0 h) with
a single dose of 10 mg/kg i.v. injection. Likewise, Gannon etal. f20111 reported elimination half-
lives for PFHxA of 1.7 and 1.5 h in male rats and 0.5 and 0.7 h 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. (2009a) found the serum terminal half-life ofPFHxA
was generally in the range of 2-3 h regardless of sex. Comparable urinary elimination half-lives
following single 10 mg/kg i.v. were also observed (males: 2.1 h; females 2.5 h) f Chengelis etal..
2009a). 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 ofPFHxA Dosimetry).
As noted above, Daikin Industries f2009a. 2009bl evaluated urinary and fecal excretion in
Sprague-Dawley rats after 50 mg/kg oral doses for 1 or 14 days. The elimination pattern is
consistent with other studies described here, with approximately 90% of the dose recovered in
feces and urine by 24 h. Because excretion was only evaluated at 6 h (urine only), 24 h, and
multiple days thereafter, these specific studies are not considered quantitatively informative for
evaluation of half-life or clearance.
Russell etal. T20151 conducted PK modeling analysis of 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 h in male and female rats, respectively, from single-day exposures, with the estimated
yield ofPFHxA ranging from 0.5 to 1.9 mol%. The model assumes, however, that the yield ofPFHxA
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. f 20181 reanalyzed the 1-day data of Russell etal. f20151 and obtained the
same half-life values (1.3 and 0.5 h in males and females).
A recent study by Dzierlenga etal. (2019) and NTP (2017) 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 h for male rats and 2.3-7.3 h 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 h [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 h
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(i.e., the serum concentrations were similar for all dose levels at 6 h and beyond). Also, the data at
the last time point (24 h) varied considerably, resulting in large uncertainty in the estimated
terminal half-lives fDzierlenga et al.. 20191.
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 h for a single dose of
100 M-g/kg BW or 2.9 h for exposures in drinking water of 1 or 3 months (Iwabuchi etal.. 2017).
Using a single-compartment PK model with an elimination constant defined as 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) on the
basis of 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 fBergwerk et al.. 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.. 2002).
A separate study (Lu etal.. 1996) 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. f20021 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 (see next paragraph), 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. f2009al and Dzierlenga etal. f20191. 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. (2009) examined the role of PFAS (C4-C12) in inhibiting the uptake of
estrone-3-sulfate (ES3) using Oatplal-expressing Chinese hamster ovary (CHO) cells. They
showed the level of inhibition of E3S uptake increased as the chain length increased; for example,
PFHxA inhibited ES3 uptake with an inhibition constant (KQ 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
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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 vs. 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.
Collectively, the evidence provides a biologically plausible explanation for the observed sex-
specific PFHxA elimination in rats (i.e., the two- to three-fold longer half-life in male versus female
rats), although uncertainties remain fChengelis et al.. 2009b: Gannon etal.. 2011: Han etal.. 20121.
Most notably, whether this apparent sex difference in reuptake 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 et
al.. 2007). Kudo etal. (2001) 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 (Chengelis etal.. 2009a: Kudo etal.. 2001)
suggest this is a common underlying mechanism for PFAS elimination.
Mouse Studies
As stated above, Iwai f20111 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. (2011) 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 (2009a. 2009b) evaluated urinary and fecal excretion in
CD-I mice after 50 mg/kg oral doses for 1 or 14 days. The elimination pattern is consistent with
Iwai (2011). with approximately 90% of the dose recovered in the urine and feces (total) after 24 h.
Because excretion was only evaluated at 6 h (urine only), 24 h, 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 f20101 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 h. The estimated half-life was between 0.9 and 1.2 h for the three dose groups
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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. f20111 and Daikin Industries f20101 were
reevaluated by EPA as described with the derivation of the HED in Section 5.2.1 (Approach for
Animal-Human Extrapolation of PFHxA Dosimetry) and Appendix C to obtain overall
pharmacokinetic parameters.
Monkey Studies
In the aforementioned 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 h) than in females (2.4 ± 1.7 h) 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 h
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 Nilsson etal. (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. 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. 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 (Olsen etal.. 2007).
A recent analysis by Luz etal. (2019) found no significant species- or sex-related differences
in the elimination kinetics of PFHxA. The PK analysis, however, is attributed to a meeting abstract
fBuck and Gannon. 20171 and provides no details of the methods the authors used. The text of Luz
etal. f 20191 indicates the analysis of Buck and Gannon f 20171 used data from only 3 of the 11
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subjects of Nilssonetal. (2013). specifically the 3 with the most rapid elimination, reducing the
extent to which the conclusion can be assumed to represent the study population as a whole. Luz et
al. T20191 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 slower than others due to interindividual variability. In the absence of
independent evidence that ongoing exposure occurred in the eight 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 vs. 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. A detailed description of EPA's analysis 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 individual and year specific for when exposure stopped and the elimination began.
Specifically, we used a one-compartment i.v.-infusion model to fit the data:
—-— (l — e_fee t), if t < tinf
tinf'ke (3-2)
r-2— ¦ (l - e~ke'tinf) ¦ e~ke tinf
Where A = dose/Vd, 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 1.48 (0.89-2.44) month-1. Using an average Vd of 0.7315 L/kg for male and female
monkeys from Chengelis etal. (2009a). the resulting mean (CI) for human clearance is
CL = Vd-ke = 1.50 (0.90 - 2.48) mL/kg-h.
Xiao etal. (2011) measured the serum concentrations of 10 PFAA chemicals in 227
nonoccupationally 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
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.
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3.1.5. PBPK Models
No PBPK model is available for PFHxA in rats, mice, or monkeys. Fabregaetal. f20151
described a PBPK model for multiple PFAS in humans, including PFHxA. However, Fabrega etal.
f20151 state two key parameters that determine the rate of resorption from glomerular filtrate in
the kidney were identified using the data from the Ericson et al. (2007) epidemiological survey of
PFAS exposure in residents of Catalonia, Spain. Because PFHxA was not detected in any individuals
sampled by Ericson et al. (20071. 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. f20071 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 in Humans), the tissue: blood partition coefficients Fabregaetal. (2015)
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 Fabregaetal. f20151 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 described above
are important for interpreting and quantifying health outcomes potentially associated with PFHxA
exposure, as discussed in later sections of this assessment. 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 (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 (768 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 fU.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 than rats. Given half-lives in the range of 0.4-14 h among male and female rats (Table 3-1),
one would then predict half-lives of 1.6-57 h 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. Thus,
based on the PFHxA-specific PK data, use of BW0 75 for dosimetric extrapolation could lead to an
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1 underprediction of human elimination by 1-2 orders of magnitude. Therefore, use of BW0 75 as an
2 alternative means of extrapolation is not considered further for PFHxA, and the preferred, data-
3 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 ±2.1
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 inhalation* (0.5 ppm)
1.3
NDf
107
NR
Kabadi et al. (2018)
Single inhalation* (5.0 ppm)
1.3
NDf
277
NR
Female
Single inhalation* (0.5 ppm)
0.5
NDf
107
NR
Single inhalation* (5.0 ppm)
0.5
NDf
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
768 (336-1,176)
337 (205-561)
ND
ND
1.50 (0.90-2.48)
ND
Russell et al. (2013)
Current analysis
1 i.v. = intravenous; ND = not determined; NR = not reported.
2 *6-hour inhalation exposure to 6:2 fluorotelomer alcohol (FTOH)
3 tDose 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 (Chengelis etal.. 2009b: Iwai and Hoberman. 2014: Klaunig etal.. 2015: Loveless et al.. 2009:
NTP. 20181. Some organs/systems for which data were available (i.e., dermal,
musculoskeletal/connective tissue, sensory, ocular) had no evidence of an effect even at the highest
administered dose, and others (i.e., respiratory, gastrointestinal system, cardiovascular, and
metabolic effects) were limited findings of unclear toxicological relevance (e.g., outcome not
necessarily adverse or considered nonspecific). Thus, these data are not synthesized in detail
below, but are summarized in the animal literature inventory. Similarly, other effects, including
body weights and survival, which had no effect or lowest effect levels at the highest administered
dose were not the drivers for hazard identification but were used to aid interpretation of other
potential health effects. They are summarized in the animal literature inventory under the
appropriate systemic/whole body system. Studies considered suitable for dose-response were
given a more detailed summarization of study methods and findings using HAWC. For hepatic
changes some individual or constellation of liver endpoints might be considered adaptive in nature.
Therefore, to draw inferences regarding the adversity of this type of liver effect, the panel
recommendations outlined by Hall etal. (2012) were used to develop conclusions around adversity
while also considering that Hall etal. f20121 developed adaptive/adversity criteria in the context
liver tumor formation.
3.2.1. Hepatic Effects
Human
Serum Enzymes
Two epidemiological studies report on the relationship between PFHxA exposure and liver
enzymes. Of these, one fliang etal.. 20141 was considered critically deficient in the confounding
domain and was considered overall uninformative. Based on these deficiencies, the study was
excluded from further analysis (Figure 3-1). The remaining study (Nian et al.. 2019) was cross-
sectional and was classified as medium confidence (Figure 3-1). Exposure levels for PFHxA,
however, were low (detected in 70% of the study population, adult residents of Shenyang, China,
median [interquartile range, IQR] = 0.2 [0.01-0.5]), which would reduce the study's ability to detect
an association if present. The study did not observe an association between PFHxA levels and
serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein, alkaline
phosphatase (ALP), y-glutamyl transferase (GGT), total bilirubin, or cholinesterase.
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7.0^
ySF®'
Participant selection
Exposure measurement
Outcome ascertainment -
Confounding
Selective Reporting
Overall confidence
'
+
-
+
+
+
B
+
++
N/A
"
N/A
+
+
NR
H
N/A
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
NfA Not applicable
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.
1 Animal
2 Hepatic outcomes were evaluated in multiple short-term, subchronic, or chronic studies in
3 rats and mice fChengelis etal.. 2009b: Iwai and Hoberman. 2014: Klaunig etal.. 2015: Loveless et
4 al.. 2009: NTP. 2018). Generally, studies were rated as medium or high confidence for the hepatic
5 outcomes, but some outcome-specific considerations for study evaluation were influential on the
6 overall confidence ratings for hepatic effects. Histopathology for Chengelis etal. f2009bl was rated
7 low confidence because of issues related to observational bias, endpoint sensitivity and specificity,
8 and results presentation. Results of the outcome-specific confidence evaluations are presented in
9 Table 3-2 below, and details are available by clicking the HAWC link.
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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 days)
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 days)
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 days)
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-year cancer
bioassay
Gavage3
Male: 0, 2.5,15,
100 mg/kg-d
Female: 0, 5, 30,
200 mg/kg-d
NM
+ +
++
NM
Study 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.
++ Outcome rating of high confidence; + outcome rating of medium confidence; - outcome rating of low
confidence; - outcome rating of uninformative; NR, outcome not reported; NM, outcome not measured.
Organ Weight
Overall, findings of increased liver weights after oral PFHxA or PFHxA sodium salt
exposures in rats were consistent (Figure 3-2; exposure response array link! Relative liver weights
(Table 3-3), are generally considered more reliable than absolute liver weights because they take
into account 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
(NTP. 2018). In the subchronic studies, relative liver weights were increased (22%) at
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1 200 mg/kg-day in males (with no change in females) in one study (Chengelis etal.. 2009b). and the
2 other study observed increases of 63% and 37% at 500 mg/kg-day in males and females,
3 respectively fLoveless etal.. 20091. Note that PFHxA effects on relative liver weights had resolved
4 by 30 days in the recovery group fChengelis etal.. 2009bl. Liver weights were not evaluated in the
5 chronic study fKlaunig etal.. 20151.
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( ')
Day 29
A
A
Rat, Hartan Spraguc Dawley(2)
Day 29
• A
A
Chengelis. 2009,2850404
90-Day Oral
Rat, Crl:CD(SO)<#)
Day 90
••
•
Rat, Crl:CD(SO){ y)
Day 90
••—
•
Loveless, 2009,28503B9
90-Day Oral
Rat,Crl:CD(SO)0)
Day 92
m—•—
A
RaLCr1:CD(SO){$}
Day 93
••—•—
—A
Liver Weight, Absolute. Rccowry Chengelis. 2009,2850404
90 Day Oral
Rat, Crl:CD(SO){(?)
Day118
•—
•
Rat, Cf1:CD(SO){ $}
Day1l8
•—
*
Litei Weight, Relative NTP, 2018,4309149
28-Day Oral
Rat, Harfan Sprague-Dawleyi ')
Day 29
<>r~-
A A
A
Rat, Harfan Sprague-Dawley(,)
Day 29
• A
A
Chengelis. 2009,2850404
90-Day Oral
Rat, Crl:C0(SOHcf)
Day 90
•• A
Rat, Crl:CD(SD){$)
Day 90
•
Lowless.2009,2850369
90-Day Oral
Rat.Crt:CD(SO)<;>)
Day 92
«•—•—
—A
RatCrl:CD(SO){?)
Day 93
A
Liver Weight, Relative, Recovery Chengelis. 2009,2850404
90-Day Oral
Rat,Crl:CD(SO}0>
Day118
•—
•
Kat,Crl:CD(SOHV}
DayllB
•—
•
• No significant changed Significant increase ~ Significant decrease A Significant Trend ® ^ ^00 300 400 500 60© 700
I Dose (mgfcg-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
Reference
Dose (mg/kg-d)
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Histopathologv
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 (Hall etal.. 2012: Thoolen etal.. 2010: U.S. EPA. 2002a). 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 four
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.
2002a). Increased hepatocellular hypertrophy was observed in adult male and female rats in the
high confidence short-term (NTP. 2018) and high confidence subchronic (Loveless etal.. 2009)
studies at doses >100-500 mg/kg-day. In the low confidence subchronic study, centrilobular
hepatocellular hypertrophy was found at 200 mg/kg-day in male rats only (Chengelis etal.. 2009b).
In the chronic study (Klaunig etal.. 2015). no change in hepatocellular hypertrophy was found,
although the highest administered dose was 2-10 times lower (100 mg/kg-day in males or
200 mg/kg-day in females) than the highest dose in other studies where effects on hypertrophy
were observed. Coherent with findings on liver weight, the observations of hepatocellular
hypertrophy were dose-dependent and male rats were more sensitive than females.
Table 3-4. Incidence of hepatocellular hypertrophy findings in adult rats due
to PFHxA exposure in short-term and subchronic oral toxicity studies
Reference
Dose (mg/kg-d)
o
*—1
o
o
lO
LO
rsi
ID
o
o
*—i
lO
fsj
*—1
o
o
o
LO
fsj
o
o
LO
OOO'I
28-dav, female rat (NTP, 2018)
0/10
0/10
0/10
0/10
9/10
28-dav, male rat (NTP, 2018)
0/10
0/10
0/10
9/10
10/10
90-day, female rat
(Chengelis et al., 2009b)
0/10
0/10
0/10
90-day, male rat
(Chengelis et al., 2009b)
0/10
0/10
7/10
90-day, female rat
(Loveless et al., 2009)
0/10
0/10
5/10
90-day, male rat
(Loveless et al., 2009)
0/10
4/10
10/10
90-day, female rat, 30-day recovery
(Loveless et al., 2009)
4/10
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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-day, female rat, 90-day recovery
(Loveless et al., 2009)
0/10
90-day, male rat, 30-day recovery
(Loveless et al., 2009)
9/10
90-day, male rat, 90-day recovery
(Loveless et al., 2009)
6/10
Bold values indicate 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 slight increase in male rats [n = 1/10 vs. 0/10 in other
groups) reported in a subchronic study at 200 mg/kg-day PFHxA, the highest dose tested
(Chengelis etal.. 2009b). In the chronic study Klaunigetal. (2015) reported hepatocellular
necrosis in females that was characterized as hepatocellular necrosis [n = 12/70 vs. 2/60 in
controls, p < 0.05) or hepatocellular, centrilobular necrosis [n = 4/70 vs. 1/60 in controls) in the
200 mg/kg-day dose group (the highest dose tested) in the high confidence chronic study. 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 fKlaunig etal..
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 = 1/46 vs. 0/42 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.
11/60 in controls) (Klaunigetal.. 2015). Incidence of necrosis were not observed in the short-term
study fNTP. 20181. and the subchronic study by Loveless etal. f20091 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 (NTP. 2018). 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
diagnostic tests of organ function and when interpreted together with histopathology are useful for
the assessment of adverse liver effects.
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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 (Amacher
etal.. 1998: Hall etal.. 2012). 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 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 (NTP. 2018). 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 fNTP. 20181.
In the subchronic studies, ALT increases were observed only in male rats at PFHxA sodium
salt exposures as low as 20 mg/kg-day in one subchronic study fLoveless etal.. 20091 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 (Loveless etal.. 2009).
Chengelis etal. (2009b) reported increased AST in males only in the 200 mg/kg-day dose group
that resolved after the 30-day recovery (Table 3-6).
ALP was increased in both subchronic studies with significant increases observed in the
highest exposure groups [200 fLoveless etal.. 20091 and 500 mg/kg-day f Chengelis etal.. 2009bl]
that resolved by the 30-day recovery (Chengelis etal.. 2009b) (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.
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Gnd point
Study
Eiperwnerrt
Animal Description
Observation Time
PFHxA Hepatic Effocts: Serum Biomarfcers
Alafsir>eAroirw5tra»iatera»i9 (ALT)
NTT*. 2018.4309149
28-Day Oral
Rat Harlan Spraigue-Oawley (.")
Day 29
* ~ •
—A
—A
R.nl Hjirlim Spntgiin-Onwley (, )
0ny?9
0 ~ •
A
A
Cliongalis,2006,2850404
00 Day Oral
Roi.cn cd.;so)< ">
DayBO
• •
A
-..'.I, 1< D SO 1
Day BO
—
•
Loveless, 2009- 2950369
00-Day Oral
Rat, Cr1:CO
Day93
•»—#-
—«
Klaww. 2015,2850075
2-Steui Ciwioor Btoaaaay
Ral. Ci1COySC)( '~
Weak 28
• •
Hal, Crl CD(SO). Reoowry
CHenoaiiB. 2006.2850404
00-Day Oral
Rat.Crt CD
Dayi18
•
•
Ral, Crl CD<7 I
Ony11B
•
-~
/ikoairw Photpholaea (M.P)
NIP. 2018,4309148
28 -Day dial
Rat Harlan Sproflf 0 Oarwloy ( ')
Day 29
—A—
—A
Rat Harlan Spra*guo-Oawloy('i)
Day 20
—#-—
—m—
A
Chengeiie. 2009,2850404
00-Day Oral
Rat, Cr1:CD<50)0
DayOO
A
Ral, CrlCD-SCH 1 ~
DaySO
••
~
Lowtoss. 200®. 2860369
00-Day Oral
Rftl, Cr1 CD(SO>< *>
Qay02
m •
- -A
Rat, Crl CD
QayG3
m •
—•
Xlatwi'Q.2015.28500/5
2-ltoar Canoof Bwasaay
War.Crl CD
Week 26
—
-~
Rat Cd:CD4SC) < ">
Week 52
m •
Rot, Crl C0
Day 118
Ral, Cil CD>,SO)4.)
Day 118
•
As&artaloATiiflotransterase (AST)
NTP. 2018.4309149
28-Day Oral
Rat Harlan Sprague-Oawley ( ')
Day 29
—it" ~
—A
Rat Harlan Sprtbgcje-Gowley (c,)
Day 23
A
A
CHenflfliiS . 2006, 2850404
00-Day Oral
Ret, Ci1:CD
(hyW
• A
—A
Rat Crl CDiSCH , )
QaySS
—— •—
—m
Klaunlg. 2016.2850075
2-Vfear Cancer B®asaay
Rat Crl CD'ISOH f>
Week 26
m •
Ral, Crl CDVSOh , >
Wook 2fi
•»—
-~
RatCrt CO( ">
Wcok 52
m—*
Rnl, Ci1 CD
OmytW
—~
Rat, Crl CDiSOH , I
QayfiO
—•
•100 0 >00 200 30© 400 500 600 TOO 800 000 1,0001.100
Dokh (mgiVgday)
Figure 3-3. Clinical chemistry findings (serum enzymes) after short-term,
subchronic, and chronic PFHxA exposures (full details available by clicking
the HAWCJink).
Table 3-5. Percent change in alanine aminotransferase due to PFHxA
exposure in short-term, subchronic, and chronic oral toxicity studies
Reference
Dose (mg/kg-d)
LO
rsj
lO
o
*—1
IT)
rH
O
fsj
o
ro
O
lO
LO
rsj
UD
o
o
*—1
un
(N
o
o
LO
OOO'I
28-day, female rat
(NTP, 2018)
11
15
10
35
44
28-dav, male rat (NTP, 2018)
4
4
8
26
64
90-day, female rat
(Chengelis et al., 2009b)
60
29
3
90-day, male rat
(Chengelis et al., 2009b)
12
22
237
90-day, female rat
(Loveless et al., 2009)
-46
-25
-4
This document is a draft for review purposes only and does not constitute Agency policy,
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Reference
Dose (mg/kg-d)
in
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Reference
Dose (mg/kg-d)
in
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
1 particularly a decrease, can be indicators of protein loss due to kidney disease or impeded
2 production in the liver, such as in liver disease fBoron and Boulpaep. 20171. Blood protein
3 measures (total protein and globulin) were in general decreased across short-term and subchronic
4 studies, with consistent and coherent dose-dependent findings across study designs. No PFHxA-
5 related treatment effects on blood proteins were found in the chronic study at doses up to 100 or
6 200 mg/kg-day PFHxA (the highest doses administered) in male or female rats, respectively. The
7 pattern of findings suggests a primary effect on blood globulins (decreased) in response to PFHxA
8 exposure that was driving decreases in total protein and increases in the albumin:globulin ratio
9 (A:G). These findings are discussed below and detailed information can be viewed in Figure 3-4 or
10 by clicking on the HAWC link.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Entipomt
Study
Experiment
Animal Description
Observation Time
PFHxA Hepatic Effects: Serum Proteins
jWbmmh (A)
NTP. 2016, 4303149
28-Day Oral
Rat, Harlan S-prague-Oawleyt )
Day 29
V
Rat, Harlan SpragueDawluyt-)
Day 29
•
• •
Chengelfs, 2009,2650404
90-OayOral
Rat,Cr1:CD{SO) fc*)
Day 90
*•
•
Ral, Crl:CD(SD) }
Day 90
Loveless, 2009.2850369
90-Day Oral
Rat, CrliCCKSO) (¦)
Day 92
—•
Rat,Crl:CD(SD)( '?)
Day 93
Klauntg, 2015,28500/5
2-Year Cancel Bioassay
Rat, Crl:CD(SDH ")
Week 26
m —•
Rat, Crl:CD(SD) ( }
Week 26
mm—
•
Rat.Crl:CD(SD)0
Week 52
•—•
Ra!,Crt:CD{SD) (.}
Week 52
mm—
-•
/Vbumin (A). R&oowry
Chengelis, 2009.2850404
90-Day Oral
Rat, Crl:CD{SD) (
Day 118
*—
-•
Rat, Crl:CD(SD) (. }
Day 118
-•
Mbumlni'Globiilin (AK3) Ratio
NTP.2018,4309149
28-Day Oral
Rat, Harlan Sprague-Dawlsy < }
Day 29
A
A A
Rat, Harlan Sprague-DawJey( }
Day 29
—•
—• A
Chengelis, 2009.2850404
90-Day Oral
Rat, Crl:CD(SD)( >
Day 90
• •—
«
Rat, Crl:CD(SD) (?)
Day 90
—
-•
Klaumg, 2015,2850075
2-Yfear Cancer BioBsaay
Rat, Cr1:CD(SD) ( '>
Week 26
•—•
Rat, Cr1:CD(SD) ( )
Week 26
mm—
-•
Rat, Crl:CD(SO) (')
Week 52
•—•
Rat, Crl:CD{SO) ( }
Week 52
••—
-•
M>umiinK3lol>utin (Alt3) Ratio. Chengelis, 2009.2850404
90-Day Oral
Rat,Crl:CO(SO) ( >
Day 118
A
Recovary
Rat, Crl:CD(SD)(.)
Day 118
•—
•
Globulin (G)
NTP, 2018,-1309149
28-DayOral
Rat, Harlan Sprague-Dawleyt }
Day 29
$ • V
V
V V
Rat, Harlan Sprague-DawJeyt }
Day 29
(h+-+-
. V
Chengeks, 2009,2850404
90-DayOral
Rat, Crl:CD(SD) ( >
Day 90
mm—
V
Rat, Crl:CD(SD)( '?)
Day 90
mm
V
Loveless, 2009.2850369
90-DayOral
RaLCrl:CD(SD»f
Day 92
•» V
V
Rat,Crl:CD(SD)( )
Day 93
mm m
—*
Klaunig, 2015.2850075
2-\teat Cancer Bioassay
Rat, Crl:CD{SD) (,?)
Week 26
m—•
Rat, Crl:CD(SO)< >
Week 26
mm—
-•
Rat, Crt:CD{SO)(
Week 52
•—m
Rat, Crl:CD{SD)(
Week 52
mm—
-•
Globulin (G)» Recovery
Chengelhs, 2009.2850404
90-DayOral
Rat, Crl:CD(SD)(
Day118
m—
V
Rat, Crl:CD{SD) ( -)
Day 118
• •
Total Protein (TP)
NTP, 2018,4309149
28-Day Oral
Rat, Harlan Sprague-Dawleyf )
Day 29
0-mW-
~
V V
Rat. Harlan Spraguc DawJeyt i}
Day 29
. V
Chengelfcs, 2009,2850404
90-DayOral
Rat,Cr1;CD{SO)< ;<£>
Day 90
mm—
V
Rat. Cr1:CD{SD)( >
Day 90
mm
-•
Loveless, 2009.2850369
90-DayOral
Kat, Cri:CD{SOH-,>
Day 92
*» V
V
~ V
V
Rat, Crl:CO(SD)(r')
Day 93
—«
Klaunig, 2015,285Q075
2-Year Cancer Btoassay
Rat,Crl:CD[SD)( ')
Week 26
m—m
Rat, Crl:CD(SD) (-}
Week 26
mm—
•
Rat,Crl:CD{S0)O
Week 52
•—*
Rat, Crt:CO(SD) (.}
Week 52
mm—
Total Protefn (TP), Recovery
Chengelis. 2009.2850404
90-DayOral
Rat,Crl:CD£SD)(>
Day 118
•—
-•
= 1 1 1 1 1 1 1 1 1 1 1 1
Significant increase ~ Significant decrease < ) Significant Trendl -100 0 100 200 300 400 500 600 700 800 900 t.0001,100
Dose (mgfltg-day)
Figure 3-4. Blood protein findings after short-term, subchronic, and chronic
PFHxA exposures (full details available by clicking the HAWC link).
Effects on total protein (TP; Table 3-8), the total amount of albumin and globulin found in
blood, is associated with chronic liver disease fWhalan. 20141. was decreased up to 20% in male
rats receiving a dose >125 mg/kg-day in the 28-day study (with a significant trend) (NTP. 2018). A
dose-responsive decrease (6-14%, >100 mg/kg-day) in TP also was observed in male rats
fChengelis et ai. 2009b: Loveless et al.. 20091 with decreased levels observed in males (-6%, 200
mg/kg-day) at the 30-day recovery (Chengelis et al.. 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
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
disease, subacute hepatitis, hepatocellular damage, ascites, cirrhosis, and chronic alcoholism
fWhalan. 20141. Slight decreases (p < 0.05) in albumin were reported only in males exposed for
28 days to 125 mg/kg-day (6% decrease) and 1,000 mg/kg-day (7% decrease) PFHxA fNTP. 20181.
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 2-year study (Table 3-9). Globulin decreases were
observed in both male and female rats treated with PFHxA in the short-term study at
>250 mg/kg-day and 1,000 mg/kg-day, respectively fNTP. 20181. Consistent with the short-term
study, decreases were also observed in both males and females in the highest dose groups
[200 f Chengelis etal.. 2009bl and 500 mg/kg-day fLoveless et al.. 20091], Notably, globulin
decreases (10%) persisted at the 30-day recovery in males (200 mg/kg-day) and returned to
normal in females (Chengelis etal.. 2009b).
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 fNTP. 20181. Chengelis et al. f2009bl 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 (TP) and globulin (G) due to PFHxA
exposure in short-term, subchronic, and chronic oral toxicity studies
Reference
Dose (mg/kg-d)
in
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Reference
Dose (mg/kg-d)
in
—
«
~
~
Rat, Harlan Sprague-Dawley(,)
Day 29
• • ~
i r i i i i r i i 1 i
• No significant changA Significant irrcreasev Significant decrease § Significant Trend 0 100 200 300 400 500 600 700 800 900 1.0001,100
' Dose (mgflcg day}
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, decreased total protein (driven by
decreased globulin), and decreased bilirubin effects in rats exposed to PFHxA. Although multiple
pathways might be involved in the observed liver effects (e.g., abnormal storage of water, glycogen,
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
lipids; (Thoolen et al.. 2010: U.S. EPA. 2002a): organelle or cellular proliferation; increased
metabolizing enzyme induction), the available mechanistic evidence was limited to effects on
peroxisomal beta oxidation with some evidence for receptor (e.g., PPARa and CAR) activation.
Peroxisomal beta oxidation
Peroxisomal proliferation can be induced within the peroxisomes to perform beta oxidation
of lipids into acetyl CoA and hydrogen peroxide (H2O2) (Reddv. 2004). 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 et al.. 2009) (Figure 3-6) and both were considered medium or
high confidence for this outcome. Chengelis etal. f2009bl reported an increase (p < 0.05,1.37-fold)
in males treated with 200 mg/kg-day at 13 weeks. Loveless etal. f20091 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. Male rats were more sensitive than females,
with males in the 100 mg/kg-day group also showing increased peroxisomal beta oxidation.
Endpoint Study Experiment Animal Description Observation Time
Perosiwmal Beta Oxidation
Loveless, 2009,2850369
90-Day Oral
Rat, Crt:CD(SD) ( ")
Day 10
A
Rat, Crl:CD
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Separately, in other in vitro studies, C0S1 cells were 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 treatment (24 h) in a
treatment-related manner with PFHxA being a more potent activator of the human (lowest
observed effect concentration, LOEC = 10 |j.M) than the mouse (LOEC = 20 |j.M) receptor (Wolfetal..
20081.
In a short-term study of in vivo PFHxA exposure, NTP (20181 reported significant and dose-
related increases in the liver expression of the PPARa-related genes acyl-CoA oxidase[Acoxl, up to
two-fold increase) and cytochrome P450 4al (Cyp4al, up to 12.5-fold increase), constitutive
androstane receptor (CAR)-related genes cytochrome P450 2bl (Cyp2bl, up to seven-fold increase)
and cytochrome P450 2b2 (Cyp2b2, up to three-fold increase). NTP T20181 provided further
evidence of PPARa activation by PFHxA exposure, with increases in Acyl-CoA oxidase activity (up to
16-fold) in male rats receiving >250 mg/kg-day PFHxA (not measured in females).
Collectively, the in vitro and in vivo results indicate that PFHxA can activate PPARa. The
data also suggest this PPARa activation occurs in both rodents and humans to a similar extent (at
least in vitro). The data suggest pathways such as PPARa and CAR activation can contribute to
some of hepatic changes caused by PFHxA exposure, including hypertrophy. However, PFHxA-
specific data informing possible biological pathways leading to the observed hepatic effects are
sparse, and many uncertainties remain.
Other PFAS
Although no direct in vivo evidence is available for PFHxA effects in rodents, PFAS
exposures in PPARa null and humanized mouse models are available. In Rosen etal. (20171.
transcript profiling in male wild-type and null mice identified PFNA, PFOA, PFOS, and PFHxS
exposure induced hepatic gene expression profiles similar to agonists for CAR, PPARa, PPARy,
estrogen receptor alpha (ERa), while suppressing signal transducer and activator of transcription 5
B (STAT5B). In the same study, Rosen etal. (20171 also compared transcript profiles between
vehicle and PFAS-exposed wild-type and null mice and identified that 11-24% of the genes
differentially regulated by PFAS exposure were PPARa independent In a separate study, Das et al.
(20171 reported findings of increased hepatocyte area and decreased DNA content along with
increased hepatic triglyceride content and increased hepatocellular lipid content (except for PFNA)
indicating hepatocyte hypertrophy and steatosis in adult male SV129 wild-type SV and PPARa null
and mice exposed to 10 mg/kg-day PFOA, PFNA, or PFHxS for 7 days. Further, Foreman etal.
(20091 also observed increased liver weight, hepatic lipid accumulation, ALT increases >two-fold,
and pathologically similar (severity and incidence) hepatocellular hypertrophy in male SV129 wild-
type SV and humanized PPARa mice exposed to PFBA. Collectively, these findings suggest
pathways in addition to PPARa can mediate the hepatic effects (including increased liver weight
and hepatocellular hypertrophy) for those PFAS tested. Based on structural similarity between
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
PFHxA and PFOA, PFNA, and PFBA it is inferred that PFHxA exposure in these genetic mouse model
systems would elicit similar effects.
Consideration for Potentially Adaptive Versus Adverse Responses
Considering the hepatic effects of PFHxA exposure were observed in rodents that have
species-specific responses to chemical-induced liver toxicity, the evidence was considered together
for potentially adaptive versus adverse responses. For PFHxA, and ammonium or sodium salts,
evidence demonstrates increased liver weight, increased hepatocellular hypertrophy, increased
ALT/AST/ALP (increases between 1.5- and 3.5-fold), decreased blood protein (driven primarily by
decreased globulin), increased peroxisomal beta oxidation activity, the induction of CAR and PPRAa
metabolic enzyme gene expression in 28-day and subchronic rodent studies fChengelis etal..
2009b: Loveless et al.. 2009: NTP. 20181. and activation of mouse and human PPRAa fWolf et al..
2014: Wolf etal.. 20081.
Several biological pathways lead to chemical-induced increases in liver weight and
hepatocellular hypertrophy, including hepatocyte swelling due to abnormal storage of water,
glycogen, lipids; organelle (i.e., mitochondria, endoplasmic reticulum, peroxisome) proliferation;
and increased immune cell infiltration (Thoolenetal.. 2010: U.S. EPA. 2002a). Although the
available clinical and histopathological data limited evaluation of all these pathways, evidence
indicated the hepatocellular changes induced by PFHxA exposure in rodents could become adverse
with long-term exposure at doses up to 200 mg/kg-day in female rats (the highest dose tested in
males was 100 mg/kg-day) where necrosis was observed with an incidence of 17.1% (12/70).
Evidence also showed increased PPARa activation and peroxisomal beta oxidation activity after
PFHxA exposure (in the 28-day and subchronic studies described above) that are possibly
biological pathways toward hepatocellular hypertrophy and increased liver weight PPARa
activation has been proposed as a potential MOA for the liver effects induced after exposure to
some PFAS (Klaunig etal.. 2012). but primarily in the context of PPARa-mediated pathways for
nongenotoxic carcinogens (Klaunig etal.. 2003). Notably, evidence showed that PFHxA exposure
did not lead to hepatic carcinogenesis in the high confidence chronic study (Klaunig etal.. 2015).
Further, evidence from other PFAS exposures in genetic mouse models indicated possible pathways
leading to increased liver weight and hypertrophy other than PPARa (described above under "other
PFAS").
In the absence of a known mechanism leading to increased liver weight, hepatocellular
hypertrophy, and necrosis, the evidence for PFHxA-mediated hepatotoxicity was evaluated. There
was evidence of increased serum enzymes ALT, AST, and ALP that were dose-responsive in the
28-day study at doses >500 mg/kg-day (NTP. 2018). Of these changes in serum enzymes, ALT was
increased 3.3-fold and ALP was increased 1.3-fold in male rats receiving a subchronic dose to 200
mg/kg-day PFHxA fChengelis etal.. 2009bl. In the other subchronic study fLoveless etal.. 20091.
ALT was increased (1.56-2.33-fold) at >20 mg/kg-day, AST was increased (1.25-1.39-fold) at
>100 mg/kg-day, and ALP was increased 2.6-fold at 500 mg/kg-day PFHxA sodium salt. Of these
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
clinical pathological measures, hepatocellular hypertrophy, hepatic congestion, and hepatocellular
necrosis were found in rats exposed to PFHxA. Although these changes in serum enzymes were not
found in PFHxA-exposed females, the recommendation from Hall etal. f20121 that serum changes
in ALT in the range of "2-4 times or higher in individual or group mean data when compared with
concurrent controls should raise concern as an indicator of potential hepatic injury unless a clear
alternative explanation is found." Hepatic effects were considered adverse based on changes in
clinical chemistry accompanied by increased liver weight, increased hepatocellular hypertrophy,
decreased total protein, observations of macrocytic anemia (see Section 3.2.4) in the subchronic
studies, and increased incidence of necrosis in the chronic study.
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 collective 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 etal.. 20091 and 200 mg/kg-day (Chengelis etal.. 2009b) in male rats. 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 etal.. 2015). Hepatocellular necrosis was observed in male rats in a low
confidence subchronic study (Chengelis etal.. 2009b) and in the high confidence chronic study
(female rats) fKlaunig et al.. 2 0151 at 200 mg/kg-day (note that the highest dose tested in males
was 100 mg/kg-day, 2-fold less than in females). Other clinical findings altered by PFHxA exposure
included decreased bilirubin and decreased total protein mainly driven by decreases in
immunoglobulins (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 100 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.. 2008). peroxisomal beta oxidation activity (Chengelis etal.. 2009b: Loveless et al..
2009: NTP. 20181. 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: Foreman et al.. 2009: Rosen etal..
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2017). Wolf etal. (2008) and Wolfetal. (2014) found evidence for PFHxA activation of
human>rodent PPARa receptor activity. Dose-responsive increases in peroxisomal beta oxidation
activity were observed in two subchronic studies fChengelis et al.. 2009b: Loveless et al.. 20091 at a
dose as low as 100 mg/kg-day and this effect persisted after the 30-day recovery f 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 (Das etal.. 2017: Foreman etal..
2009: Rosen etal.. 2017) that found similar levels of increased liver weight and incidence of
hepatocellular hypertrophy when comparing between PPARa knockout, humanized, and wild-type
mouse models.
Overall, the currently available evidence indicates that PFHxA likely causes hepatic effects
in humans under relevant exposure circumstances. This conclusion is based on studies of animals
showing increased liver weight, hepatocellular hypertrophy, increased serum enzymes (>2-fold
ALT), and decreased serum globulins generally occurring at > 200 mg/kg-day (with some effects
noted at lower doses) within the evidence base of four primarily high confidence studies of short-
term, subchronic, and chronic PFHxA exposure in (primarily male) Sprague-Dawley 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.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Table 3-9. 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
Evidence indicates (likely)
Serum
Biomarkers
1 low confidence
study
• No factors noted
• 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,
generally in males at >100 mg/kg-
Evidence from animal studies
d PFHxA
Studies and
confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key
findings
Evidence stream
judgment
Human relevance:
Given the induction of human
Organ Weight
3 high
confidence:
28-day
90-day (2 studies)
• Consistent increases, all
studies and sexes
• Dose-response in all
studies
• Coherence with
histopathology
• 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
potential for progression
to more severe
phenotypes, including
necrosis with longer-term
exposure, and strong
support for liver injury
from serum biomarkers
PPARa by PFHxA, as well as
support for involvement of both
PPARa-dependent and
independent pathways, effects in
rats are considered potentially
relevant to humans
Cross-stream coherence:
N/A (human evidence
indeterminate)
Susceptible populations and
lifestages:
No evidence to inform
HistoDathologv
3 hiqh confidence
studies in adult
rats:
28-day
90-day
2-year
• Consistent cellular
hypertrophy across
studies and sexes
• Coherence with liver
weight
• Dose-response for
hypertrophy
• Lack of coherence
across sexes (see
narrative summary)
• Cellular
hypertrophy at
>100 mg/kg-d;
stronger in
males
• Necrosis in
females at
200 mg/kg-d
(no change in
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Evidence integration summary
Evidence stream summary and interpretation
judgment
1 low confidence
• Concerning severity of
males at
study in adult
effect— necrosis (with
<100 mg/kg-day)
rats:
chronic exposure)
90-day
• High confidence studies
Serum
• Consistent increases in
• No factors noted
• Increased ALT,
Biomarkers of
ALT, AST, and ALP, and
AST, ALP, and
Hepatic Injury
decreases in bilirubin,
bile salts/acids
4 hiqh confidence
across studies
at >20, >100,
studies in adult
• Magnitude of effect,
>200, and
rats:
>2-fold ALT
500 mg/kg-d,
28-day
• Dose-response for total
respectively;
90-day (2 studies)
protein
stronger in
2-year
• Coherence of ALP and
males
bilirubin
• Decreased total
• High confidence studies
protein and
bilirubin at
>100 mg/kg-day;
stronger in
males
Mechanistic evidence and supplemental information
Biological events
Primary evidence evaluated
Evidence stream
or pathways
Key findings, interpretation, and limitations
judgment
Molecular
Key findings and interpretation:
• Biologically plausible
Initiating
• In vitro induction of PPARa activity in transfection studies. Reporter
support for PPARa-
Events—PPARa
gene activation at lower effective concentrations in human versus
dependent and
mouse constructs.
independent pathways
• Induction of PPARa with hepatic effects in a short-term oral exposure
contributing to hepatic
study.
effects of PFHxA
Limitations: Small evidence base investigating PPARa activation by PFHxA
exposure.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Evidence stream summary and interpretation
Evidence integration summary
judgment
Molecular
Initiating
Events—Other
Pathways
Key findings and interpretation:
• Indirect evidence supporting activation of PPARa-independent
pathways contributing to hepatic effects similar to those observed for
PFHxA in PPARa knockout and humanized mice after short-term oral
exposure to PFAS other than PFHxA.
• In a short-term oral exposure study, PFHxA activated CAR, PPARa,
PPARy, and ERa and suppressed STAT5B. CAR-responsive genes were
increased in association with hepatic effects.
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 days
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.
This document is a draft for review purposes only and does not constitute Agency policy.
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3.2.2. Developmental Effects
Human
No studies were identified that evaluated potential developmental effects of PFHxA
exposure in humans.
Animal
Three studies examined developmental outcomes, including offspring viability, body
weight, and developmental milestones. 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) fLoveless et al.. 20091. 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 3-10, 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 (Sections 3.2.6 and 3.2.7).
Table 3-10. Study design characteristics and outcome-specific study
confidence for developmental endpoints
¦to
>
"O
o
-Q
bjO
(O
+¦>
c
£ w
1 s
Study
Species, strain (sex)
Exposure design
Exposure route and dose
Offsprir
viability
Offsprir
weight
1 s
Q)
> *>
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
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 (Iwai and Hoberman. 2014). Most deaths occurred between postnatal day (PND) 0-7,
with a statistically significant increase for the 350 and 500 mg/kg-day dose groups. These early
postnatal losses are reflected in treatment-related changes in several measures of offspring
viability for the 500 mg/kg-day dose group. Specifically, statistically significant decreases occurred
in the viability index for PND 0-4 and PND 0-7, surviving pups per litter were lower on PND 20,
and the incidence of total litter loss increased 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 3-11.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Endpoint
Offspring Surwatl
Study
Iwal. 2014,2821611
Experiment Animal Description
1 -generation reproductive (GO S- S 8> F1 Mouse, CD*1 {" £}
Watoibty index
Iwai, 2014,2821611 1-generation reproductive (GO 6-18> F1 Mouse, CD-1 ( ' J
Loveless, 2009 28503G9
reproductive (56 d)
F1 Ral, CrlCD(SD) (,)
Observation Time
PNOO
PNDO
PNO 4
PNO 4
PN0 7
PN0 7
PND 14
PNDU
PNO 20
PND 20
PND 0 4
PNO 0-4
PNO 0-7
PND 0-7
PND 4-20
PND 4-20
PNOO
PND 0-4
PND 4-21
PRHxA Developmental Effects:Offspring Mortality
No. of Pups. Stillborn
Viability. LiHers with Stillborn Pups
Iwal. 2014.2821611 1-generatton reproductive (GO 6*18j F1 Mouse, CD-1 ( , J
Iwai. 2014,2821611 1-generation reproductive (GO 6-18) F1 Mouse, CD-1
Pups Found DeadiPresuroed Cannibalized Iwal. 2014,2821611 1 -generation reproductive (GD 6-18} Ft Mouse, CD-1 ( " )
Total Lit'er Loss
Iwal. 2014,2621611 1-generation reproductive (GO 6-18) P0 Mouse. CD-I (i)
PNOO
PNOO
PNDO
PNOO
PNOO
PNOO
PND 1-4
PND 1-4
PND 5-7
PND 5-7
PND 8-14
PND 8-14
PND 15-20
PND 15-20
PNOO
PNOO
PND 0-3
PND 0 3
PND 4-20
PND 4-20
t No significant clang^A Significant
' Significant decrease
50 100 150 200 250 300 350 400 450 500 550 600
mg.*g-
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2
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9
10
11
12
13
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Reference
Dose (mg/kg-d)
0
(Phase 1)
0
(Phase 2)
r-.
CO
o
o
rH
r-.
o
m
CO
o
o
m
Mortalities, PND 0, male and female (combined)
mice (Iwai and Hoberman, 2014)
0
0
0
0
0
4
3
21
Mortalities, PNDs 1-4, male and female
(combined) mice (Iwai and Hoberman, 2014)
2
la
3a
2
r
0
13a
15
Mortalities, PNDs 5-7, male and female
(combined) mice (Iwai and Hoberman, 2014)
0a
1
0a
0
i
3
2a
0
Mortalities, PNDs 8-14, male and female
(combined) mice (Iwai and Hoberman, 2014)
0
0
0
0
0a'b
0a
0a
0
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
220b
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/
220a
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.
Bold values indicate instances where statistical significance (p < 0.05) compared to controls was reported by study
authors.
aExcludes animals that were missing, presumed cannibalized.
bExcludes data from litters where the dam died during late lactation; deaths assumed not treatment related.
Offspring Body Weight
Offspring body weights were available from two developmental studies (Iwai 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 Reductions in
body weight observed at the higher doses across two experimental cohorts with different dose
ranges generally persisted throughout lactation. After weaning, body weight deficits persisted in
females but not males, however body weight gain during this period was unaffected (Iwai and
Hoberman. 20141. Similar results were reported in two separate cohorts of 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, but no effects were observed at the lower doses. In the one-generation reproductive
study, a dose-related decrease (4% to 18% less than controls) was found in pup body weights
across all dose groups at PND 0. Offspring body weight decrements persisted through PND 21 in
the 100 and 500 mg/kg-d dose groups but no treatment-related effects on body weight gains
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
1 occurred between PND 21-41 (Loveless etal.. 20091. Results are presented in Figure 3-8 and
2 Table 3-12.
Endpolnt Study Experiment Animal Description Observation Time PFHxADevelopmentsI Effects: Offspring Body Weight
BOO/Weight Absolute iwat.2014.2821611 i-generalionrepfo PNDO
V V V
PND 4
• * ~
PND 7
• V •
PND 14
• • •
PND 20
• « •
F1 Mouae. C0-1 {^) PND 21
~ * •
PND 28
• ~ «
PND 35
• • •
PND 41
1 • •
Preputial Separation
• ¦ ~
F1 Mouae. CD-1 {.) PND 21
~ ~ •
* • «
~ ~ •
PND 28
PND 35
• V V
PND 41
~ ~ •
V&ginal Patency
• • #
Loveless. 2009,2850369 developmental (GO 6-20) F1 Rat Crl:CD(SD) (r*. > GD 21
• • 1
reproductive(56d) F1 Rat Crt:CD(SO)t-'^ PNDO
•—• ~
PND 4 (prc-cull)
PND 7
• *— V
•—• ~
PND 14
•—< ~
PND 21
• • V
Terminal Body Weight, ^solute hvai, 2014.2821611 1-generalion rep*ocuclive (GO 6-18) F1 ktause. CD-I {') PND 41
»-• •
F1 Ntouse, CD-1 { ) PND 41
• V •
Body Weight Change Loveless. 2009,2850369 leproductms (58d) F1 Ral. Crl;CO(SD)( ) PND21-450
• 1 •
F1 Ral. CriiCCKSD) (*>) PND 21 -«0
• « •
1 1 1 1 1—1 1 1 1 1 1
. N05,gni.cJni,JlJrig,A Sign.rtcanl IrajeaseT Significant decrease | 0 50 ,0° 150 200 !5° 3M 350 '°° JM 500 550 650
mgftg-day
Figure 3-4. 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).
Table 3-12. Percent change relative to control in offspring body weight due to
PFHxA sodium or ammonium salt exposure in developmental oral toxicity
studies
Dose (mg/kg-d)
Reference
O
Psl
U*l
m
o
o
rH
ln
rH
O
1ft
m
o
o
lO
GD 21 (developmental study), male and female (combined)
rats (Loveless et al., 2009)
-2
0
9
PND 0 (one-generation reproductive study), male and
female (combined) rats (Loveless et al., 2009)
-4
-11
18
PND 7 (one-generation reproductive study), male and
female (combined) rats (Loveless et al., 2009)
0
-6
-17
PND 14 (one-generation reproductive study), male and
female (combined) rats (Loveless et al., 2009)
3
-6
-17
This document is a draft for review purposes only and does not constitute Agency policy,
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Reference
Dose (mg/kg-d)
r-.
o
fM
CO
o
o
rH
r-.
rH
o
m
CO
o
o
m
PND 21 (one-generation reproductive study), male and
female (combined) rats (Loveless et al., 2009)
3
-5
-18
PND 0 , male and female (combined) mice (Iwai and
Hoberman, 2014)
0
0
-6
-13
-13
-13
PND 4, male and female (combined) mice (Iwai and
Hoberman, 2014)
0
7
-7
-4
-27
-20
PND 7, male and female (combined) mice (Iwai and
Hoberman, 2014)
0
5
-7
0
-18
-11
PND 14, male and female (combined) mice (Iwai and
Hoberman, 2014)
-1
3
-8
0
-14
-8
PND 20, male and female (combined) mice (Iwai and
Hoberman, 2014)
-2
6
-11
2
-20
-12
Bold values indicate 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.
1 Eye Opening
2 Potential effects of PFHxA exposure on developmental milestones were evaluated in a
3 developmental study flwai and Hoberman. 20141. On PND 14, Iwai and Hoberman f20141 reported
4 a statistically significant delay in eye opening, with less than 50% of pups in the 350 and
5 500 mg/kg-day PFHxA ammonium salt exposure groups having reached this milestone compared
6 to 85% among vehicle controls (Figure 3-9). Delays in eye opening persisted in the 350 and
7 500 mg/kg-day dose groups at PND 15 but were not statistically significant. Delays in eye opening
8 can have long-term impacts on vision by interfering with sensory input during the critical window
9 of visual cortex development fEspinosa and Strvker. 2012: Wiesel. 19821. The results are
10 summarized in Figure 3-9 and Table 3-13.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Endpoint Study Name Experiment Animal Description Observation Time
Eye Opening Iwai, 2014, 2H21611 1-generation reproductive (GD 6-1B) F1 Mouse, CO-1 (J ) PND 10
PND11
PND 12
PND 13
PND 14
PND 15
PND 16
PND 17
• No significant changed Significant increase^ Significant decrease
PFHxA Developmental Effects; Developmental Milestone
» •
» •
» •
50 100 150 200 250 300 350 400 450 500 550 600
mg/kg-day
Figure 3-5. Developmental effects on eye opening (percent change relative to
control) in mice exposed to PFHxA ammonium salt (HAWC: PFHxA - Animal
Toxicity Developmental Eye Effects link).
Table 3-13. Percent change relative to control in eye opening due to PFHxA
ammonium salt exposure in a developmental oral toxicity study
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
Bold values indicate instances where statistical significance (p < 0.05) compared to controls was reported by study
authors.
Malformations and Variations
Potential effects of PFHxA exposure on fetal malformations and variations were evaluated
in a single developmental study (Loveless 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
No human studies were identified to inform the potential developmental effects of PFHxA;
therefore, there is indeterminate human evidence of developmental effects.
In animals, three high confidence studies examined developmental effects following
maternal perinatal exposure to PFHxA salts flwai and Hoberman. 2014: Loveless etal.. 2009).
Treatment-related effects, including decreased offspring body weight, increased mortality, and
delayed eye opening were observed in mice following exposure to PFHxA ammonium salt as low as
100 mg/kg-day. Notably, no effects on maternal weight gain were observed up to the highest tested
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
dose of 500 mg/kg-day in this study (Iwai and Hoberman. 20141. Reductions in offspring body
weight were also found in the one-generation reproductive and developmental studies in rats.
Animals in the reproductive cohort exposed throughout gestation and lactation showed body
weight reductions (>5%) at exposure to >100 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 critical window for developmental changes
associated with PFHxA. 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) were observed with other PFAS, including PFBS and PFBA, providing
additional support for these specific findings.
Reductions in maternal body weights were also noted and might indicate maternal toxicity
(U.S. EPA. 1991). For the developmental cohort, total net body weight (i.e., terminal body weight
minus the gravid uterine weight) of dams in the high dose group was statistically significantly
decreased (5% relative to controls) fLoveless etal.. 20091. Effects on total body weight in the
gestationally exposed dams were associated with a decrease in maternal weight gain at GD 21 in
the 500 mg/kg-day group. No effects on total or net maternal body weights were found in the one-
generation reproductive cohort (Loveless etal.. 2009) but weight gain of dams exposed to
500 mg/kg-day was statistically significantly reduced. The effect on maternal weight gain was
limited to early gestation (GD 0-7). PFHxA sodium salt exposure had no effect on maternal weight
gain over the entire gestational window (GD 0-21), and dams in this exposure group showed an
increase in body weight during lactation. Also, delays in eye opening in the developmental mouse
study were observed only at doses that elicited overt toxicity (i.e., increased offspring mortality) in
the pups (Iwai and Hoberman. 2014). Because treatment-related changes in offspring body weight
and mortality were observed at doses that did not affect maternal weight gain, maternal toxicity is
not expected to be the primary driver of developmental effects. Based on these findings, there is
moderate animal evidence of developmental effects.
Overall, the currently available evidence indicates that PFHxA likely causes developmental
effects in humans under relevant exposure circumstances. This judgment is based primarily on
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 based on similarities in the anatomy and physiology of the developmental system across
rodents and humans.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Table 3-14. 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 at > 100
mg/kg-day 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 life stages:
The available evidence
suggests that
Studies and confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key
findings
Evidence stream
judgment
• There were no 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 Mortality
2 high 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 pre and
postnatal mortality at
>350 mg/kg-day 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 were
observed at doses
that were not
associated with frank
effects 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
• Fetal effects observed
at doses that are
associated with
maternal toxicity
(i.e., substantial
decreases in dam body
weight)
• Postnatal body weight
decreased at >100
mg/kg-day in rats and
mice
• Fetal body weight
decreased at 500
mg/kg-day in rats
Eve Opening
1 high confidence study
in mice:
• GD 6-18
• High confidence study
• Effects observed at
doses are associated
with frank effects in
offspring (i.e., offspring
mortality)
• Eye opening was
delayed in mice
prenatally exposed to
PFHxA ammonium salt
at >350 mg/kg-day
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Evidence stream summary and interpretation
Evidence integration
summary judgment
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-day
development may be a
susceptible lifestage for
exposure to PFHxA.
Mechanistic evidence and supplemental information
Biological events or
pathways
Summary of key findings, limitations, and interpretation
Evidence stream
judgment
• There were no informative studies available from the PFHxA evidence base.
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3.2.3. Renal Effects
Human
Three epidemiological studies investigated the relationship between PFHxA exposure and
effects on the renal system. Two were considered uninformative due to critical deficiencies in
multiple study evaluation domains (Seo etal.. 2018: Zhang etal.. 2019). 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 with this
population and poor sensitivity because the exposure levels for PFHxA were low. They observed a
significant decrease in estimated glomerular filtration rate (eGFR) with higher PFHxA exposure
((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 the poor sensitivity, 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.
0-yC" '
c,e°' ^
Exposure measurement
Outcome ascertainment
Confounding
Analysis ¦
Sensitivity
Selective Reporting
Overall confidence
N/A
+
N/A
-
B
N/A
+ +
i
+
B
D
~
N/A
N/A
N/A
N/A
+
N/A
"
-
"
Legend
X
Good (metric) or High confidence (overall)
+
Adequate (metric) or Medium confidence (overall)
-
Deficient (metric) or Low confidence (overall)
NR
Not reported for metric
B
Critically deficient (metric) or Uninformative (overall)
N/A
Not applicable
Figure 3-6. Study evaluation for human epidemiological studies reporting
findings from PFHxA exposures (full details available by clicking HAWC link!.
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. (2009b). limitations were identified that influenced
some outcome-specific ratings. Specifically, histopathology was rated low confidence because of
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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 3-15, and full study evaluation details can be
viewed by clicking the HAWC link.
Table 3-15. Renal endpoints for PFHxA and associated confidence scores from
repeated-dose animal toxicity studies
Author (year)
Species, strain (sex)
Exposu re
design
Exposure route
Blood biomarkers
Urinary biomarkers
Histopathology
Organ weight
NTP(2018)
Rat, Harlan
Sprague-Dawley
(male and female)
Short term
(28 days)
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 days)
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 days)
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-year cancer
bioassay
Gavage3
Male: 0, 2.5,15,
100 mg/kg-d
Female: 0, 5, 30,
200 mg/kg-d
++
++
+ +
NM
Study 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.
++ Outcome rating of high confidence; + outcome rating of medium confidence; - outcome rating of low
confidence; - outcome rating of uninformative; NR, outcome not reported; NM, outcome not measured.
Organ Weight
Increases in relative kidney weight were observed in both sexes in all three studies that
reported this endpoint (Chengelis etal.. 2009b: Loveless etal.. 2009: NTP. 2018). There were
statistically significant findings in male rat dose groups at PFHxA doses as low as 10 mg/kg-day in
the subchronic study (Chengelis etal.. 2009b). With the exception of the results from Chengelis et
al. (2009b). effects on relative kidney weights generally showed a weak or no dose-response
gradient (Table 3-16). Craig etal. f20151 analyzed oral chemical exposure data extracted from
subchronic and chronic rat studies and found a statistically significant correlation between
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1 absolute, but not relative, kidney weight, and kidney histopathology (even at doses where terminal
2 body weights were decreased) for most chemicals (32/35) examined. Absolute kidney weight was
3 increased, but only in one of the three studies reporting on this endpoint fNTP. 20181. and only in
4 male rats at the highest dose group (1,000 mg/kg-day). The decrease in relative, but not absolute,
5 kidney weight could be explained by body weight gain decreases in the affected dose groups:
6 1,000 mg/kg-day male dose group (13% decrease) (NTP. 2018). 50 and 200 mg/kg-day male dose
7 group (8-11% decrease with similar trends in females (Chengelis etal.. 2009b)). and
8 500 mg/kg-day male dose group (19% decrease, no change in females). Findings and full details of
9 PFHxA effects on kidney weights can be viewed by clicking the HAWC link.
Table 3-16. 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
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
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 (Figure 3-11) (NTP. 2018). 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.. 2015). Full details are available
by clicking the HAWC link.
Endpolntname Study Animal Description Dose Incidence
Kidney, Nephropathy, Chronic Progressive NTP, 2018,4309149 Rat, Harlan Sprague-Dawlcy(^) 0 3/10 (30.0%)
62.5 8/10(80.0%)
125 3/10(30.0%)
250 8/10(80.0%)
500 2/10(20.0%)
1,000 6/10(60.0%)
Rat, Harlan Sprague-DaWley
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19
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
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) were more consistent than the blood biomarkers, but
still difficult to interpret. Urinalysis findings were not measured in the short-term study fNTP.
20181 and were reported as null in a subchronic study fChengelis etal.. 2009bl. Findings from the
other subchronic study fLoveless etal.. 20091 identified 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
(Loveless etal.. 20091. Urobilinogen and pH findings were null in both male and females in the
subchronic study (Loveless 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 only in
the 100 mg/kg-day dose groups at 26 and 52 weeks and attributed by the author to the slightly
acidic nature of PFHxA (Klaunig 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 f Klaunig etal.. 20151. Findings are
summarized in Figure 3-12, and full details are available in the HAWC link.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
EmJpomt Stud* Experiment Animal Description OWervafion Time PFHkA Re nal Effects: Btootf and Urine BtofnarKcrs
Blood Urea Nitrogen (BUN)
NTP, 2010.4309149
28-Day Oral
Rat, Harlan Sprague-Dawley ( ')
Day 29
•H
Rat, Harlan S prague-Da wleyf,)
Day 29
»
—•
Loveless, 2009,2850369
90 Day Oi a I
Rat, Crl:CD(SD)(-?J
Day 92
•»"
A
Rat, Crl:CD(SD) (9)
Day 93
Creatine Kinase (CK)
NIP, 2018,4309149
28-Day Oral
Rat, Harlan Sprague-Dawley ( ')
Day 29
—•
•
—•
Rat, Harlan Sprague-Dawley (,)
Day 29
•
•
Creatinine (CREAT)
NTP, 2018,4309149
28-Day Oral
Rat, Harlan Sprague-Dawley ( )
Day 29
0^—
—~—
—•—
V
Rat, Harlan Sprague-Oawley (,)
Day 29
Loveless, 2009, 2850369
90-Day Oral
Rat,Crt:CD(SD)< ')
Day 92
••
~
Rat, Crl:CD(SD) ($)
Day 93
V
Osmolality
Loveless, 2009, 2850369
90-Day Oral
Rat, Crt:CD(SD) < }
Day 92
m
f
Rat, CritCD(SD)(V)
Day 93
m
—•
IJrirve Specific Gravity
Klaunig, 2015,2850075
?-Yfear Cancer Rioassay
Rat, Cr1:CD(SO) ( 5)
Week 26
m
Week 52
Ra?, Crt:CD(SD)(V)
Week26
••
~
Week 52
mm
•
Urine Total Protein (TP)
Loveless, 2009, 2850369
90-Day Oral
Rat, Crl:CD(SD) < ')
Day 92
V
Rat, Crt:CD(SQ)(y)
Day 93
m-
V
Urine Vblurne(UVOL)
Loveless, 2009,2850369
90-Day OraJ
Rat, Crl;CD(SD)< I)
Day 92
m
A
Rat,Crl:CD0 ™ ™ 800 900 1'M0,'1O°
I I Dose (mg/kg-day)
Figure 3-8. PFHxA Effects on blood and urine biomarkers of renal function
(full details available by clicking the HAWCJink). The dashed blue line divides
blood (top] from urinary biomarkers. Note that urea nitrogen (BUN) 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 one short-term
study, 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). 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 females compared
to controls at the highest dose (200 mg/kg-day, twice the highest male dose). Some changes
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1 occurred in urinary biomarkers (decreased urine pH, increased urine volume) and potentially
2 correlated changes were observed in female histopathology in the chronic study. However,
3 inconsistencies between sexes and across studies at similar observation times were notable. Based
4 on these results, there is slight animal evidence of renal effects.
5 Overall, the currently available evidence is inadequate to assess whether PFHxA may
6 causes renal effects in humans under relevant exposure circumstances (Table 3-17).
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Table 3-17. Evidence profile table for renal 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:
Indeterminate
evidence in humans
and animal evidence is
largely null or lacking
Low Confidence
1 low confidence study
• No factors noted
• Low sensitivity
• Weak association of
PFHxA with decrease in
estimated eGFR
ooo
Indeterminate
Evidence from animal studies
biological coherence.
Studies and confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key findings
Evidence stream
judgement
Human relevance:
Without evidence to
Organ Weight
3 high confidence
studies in adult rats:
• 28-day
• 90-day (2 studies)
• Consistent increases,
all studies
• Lack of coherence
across sexes (see
Section xx)
• Increased relative kidney
weight at >10 mg/kg-d.
• Increase absolute kidney
weight at 1000 mg/kg-d;
28-day study, males only
©oo
Slight
Findings of adversity
were considered
uncertain based on lack
the contrary, effects in
rats are considered
relevant to humans
Cross-stream
coherence:
N/A (human evidence
indeterminate)
Susceptible lifestages:
No evidence to inform
Histopathology
3 hiqh confidence
studies in adult rats:
• 28-day
• 90-day
• 2-year
1 low confidence study
in adult rats:
90-day
• Large magnitude of
effect, up to 24.3% for
papillary necrosis; up
to 80% for chronic
progressive
nephropathy
• Unexplained
inconsistency
between exposure
durations.
• Lack of dose-
response
• Increased incidence
papillary necrosis, tubular
degeneration, chronic
progressive nephropathy
at >200 mg/kg-d; female
rats only, 28-day and
chronic studies
of coherence between
effects (organ weight,
histopathology, blood
and urine biomarkers)
inconsistency between
sexes, and lack of
coherence across
exposure designs
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Evidence integration
Evidence stream summary and interpretation
summary judgment
Blood Biomarkers
• No factors noted
• Unexplained
• Increased BUN at
4 hiph confidence
inconsistency
500 mg/kg-d; males only,
studies in adult rats:
across exposure
90-day study.
• 28-day
durations, sexes
• Decreased creatinine at
• 90-day (2 studies)
• Lack of coherence
>500 mg/kg-d), both
• 2-year
with other
histopathological
findings; chronic
study
sexes, 1 subchronic study
• Decreased creatine at
1,000 mg/kg-day; males
only, 28-day study
• No treatment related
creatinine kinase findings;
both sexes, 28-day study
Urinarv Biomarkers
• Coherence of urine
• Unexplained
• Decreased osmolality 500
3 hiah confidence
protein, urine volume,
inconsistency
mg/kg-day; males only, 1
studies in adult rats:
urine specific gravity,
between exposure
subchronic study
• 28-day
and decreased
durations and sexes
• Decreased urine protein
• 90-day
osmolality
• Lack of
and increased urine
• 2-year
dose-response
gradient.
volume in at 500 kg/kg-d;
both sexes, 1 subchronic
1 medium confidence
study in adult rats:
• 90-day
• Lack of coherence
with
histopathological
study
• Increased total urine
volume at >200 mg/kg-
findings.
day; both sexes - 1
subchronic study, females
only, 1 2-year study
• Decreased urine pH at
100 mg/kg-day; males
only, 1 2-year study
• No treatment related
findings for urobilinogen;
both sexes, 1 subchronic
study and 12-year study
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Evidence stream summary and interpretation
Evidence integration
summary judgment
Mechanistic evidence and supplemental information
Biological events or
pathways
Primary evidence evaluated
Key findings, interpretation, and limitations
Evidence stream
judgement
Molecular Initiating
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 leading to sex-
specific PFHxA
elimination
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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 measures three primary types of blood cells (red blood cells, white blood cells, and
erythrocytes), 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 in the analysis and inadequate
reporting of population selection criteria. 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 fLoveless et al.. 20091 subchronic studies, and a high confidence chronic study
fKlaunigetal.. 20151. Cell counts for the blood components associated with immune system
responses are summarized under in Immune Effects, Section 3.2.8. Study findings are discussed
below and summarized in Table 3-18 (full details are available by clicking the HAWC linkl. and
summary details are available in PFHxA Tableau visualization.
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Table 3-18. 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 days)
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 days)
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 days)
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-year cancer
bioassay
Gavage3
Male: 0, 2.5,15,100 mg/kg-d
Female: 0, 5, 30, 200 mg/kg-d
+ +
Study 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.
++ Outcome rating of high confidence; + outcome rating of medium confidence; - outcome rating of low
confidence; - outcome rating of uninformative; NR, outcome not reported; NM, outcome not measured.
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 (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, even in the females (that received a dose twice the male dose) in the
chronic studies (Klaunig etal.. 2015). Specifically, a dose-responsive decrease occurred in red
blood cells (Table 3-19), hematocrit (Table 3-20), and hemoglobin (Table 3-21) 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 fLoveless etal.. 200911. Of note, decreases in both hemoglobin and hematocrit were 1.5-
2-fold greater in the subchronic study (Loveless et al.. 2009) than in the 28-day study (NTP. 2018)
for both males and females at the same dose (500 mg/kg-day).
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1 Findings from the 2-year chronic study (Klaunig etal.. 20151 were generally null or
2 observed at dose levels >100 mg/kg-day (100 mg/kg-day in males and 200 mg/kg-day in females)
3 at 25 and 51 weeks. Measures of hematology beyond 52 weeks in the chronic study might be
4 complicated due to natural diseases occurring in rodents and test variability leading to decreased
5 sensitivity and increasing variability with the results fAACC. 19921. Klaunig etal. f20151 did,
6 however, qualitatively evaluate blood and reported no PFHxA treatment effects on blood smear
7 morphology. Loveless etal. f20091 also evaluated blood smears up to test day 92 with PFHxA
8 sodium salt exposure and noted nucleated blood cells in smears indicative of bone marrow damage
9 or stress, but only for 1 female and 1 male.
Endpoint Study Exporlment Animal Description Observation Time PFHxA Hematopoietic Effects: Red Blood Cells
Hematocrit (HCT)
NTP, 2018.4309149
28 Day Oral
Rat, Harlan Sprague-Dawley (
) Day 29
~
V
~
Rat, HarlBn Sprague-Dawley ( '} Day 29
~~
~
~
—~
Loveless, 2009,2850369
90-Day Oral
Rat, CrLCD(SD) < .¦$)
Day 92
H •—
Rat, CrLCD(SO) (?)
Day 93
a# •—
—V
Chengelis. 2009, 2850404
90 Day Oral
Rat. Crl:CD(SD)(^)
Day 90
»-•
V
Rat, Crl:CD{SD)(y)
Day 90
a
Klaunig, 2015,2850075
2-Year Cancer Bjoassay
Rat, CrlrCD(SO) (J)
Week 25
• ~
Week 51
• ~
Week 104
• ~
Rat. Crl.-CD(SD)<2)
Week 25
Week 51
Week 104
••
-a
Hemoglobin (HGB)
NTP. 2018,4309149
28 Day Oral
Rat, Hartan Sprague-Dawley (
) Day 29
~
—~
T
Rat, Hartan Sprague-Dawley (
) Day 29
~~
~
~
~
Loveless. 2009,2850369
90-Day Oral
Rat, Crl:CD{SD) (-f)
Day 92
- ~
Rat, CrlrCDfSDX?)
Day 93
~
Chengelis. 2009. 2850404
90 Day Oral
Rat. Crl:CD{SD) (c?)
Day 90
t
•
i
Rat. Crl:CD(SD)($?)
Day 90
m m
V
Klaunig. 2015.2850075
2-Year CancerBtoassay
Rat. Crl:CD{SD) (5)
Week 25
Week 51
m—a
Week 104
•—a
Rat. Crl£D{SD)(V)
Week 25
••—-
•
Week 51
••
V
Week 104
•*
«
Red Blood Coll (RBC)
NTP. 2018,4309149
28 Day Oral
Rat. Harlan Sprague-Dawley (
} Day29
T
—~
T
Rat, Hartan Sprague-Dawley (') Day29
~
~
Lowless, 2009,2850369
90-Day Oral
Rat. CrLCD(SO) (.?)
Day 92
~
Rat, Crl:CD(SD)(V)
Day 93
~
Chengolls. 2009. 2850404
90 Day Oral
Rat. Crl&DfSD) (tf)
Day 90
m a
V
Rat, Cr1rC0{SD) (9)
Day90
mm
V
Klaunig. 2015.2850075
2-Year Cancer B:oassay
Rat, Crl:CD{SO) (5)
Week 25
•—~
Week 51
a»—a
Week 104
e»—a
Rat. Crl:CD{SD)<5)
Week 25
•a
«
Week 51
*a
V
Week 104
•a
-~
1 1 <
1 i 1 t i » i i s 1 I
• No significant cftanqaA Significant increase X7 Significant decrease Q Significant Trend -100 0 100 200 300 400 500 600 700 800 900 1,0001.100
Dose (mg/kg-day)
Figure 3-9. Hematological findings (hematocrit [HCT], hemoglobin [HGB], and
red blood cells | RBC]) in rats exposed by gavage to PFHxA or PFHxA sodium
salt (full details available by clicking the HAWC link).
This document is a draft for review purposes only and does not constitute Agency policy,
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Table 3-19. Percent change in red blood cells due to PFHxA exposure in
short-term, subchronic, and chronic oral toxicity studies
Reference
Dose (mg/kg-d)
in
500 mg/kg-day in
7 the other studies. MCV, a measure of average blood volume of RBCs was increased in both a
8 short-term and a subchronic study (Loveless etal.. 2009: NTP. 2018).
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Endpoiril Study Experiment Animal Description Observation Time PfflxA Hematopoietic efecls:MCHC, MCV, MCH
Msen Corpuscular Hemoglobin NIP. 2018,4309149 28 Day Oral Rat Harlan Sprague-Oawley ( ) Day 29
(MCH)
Rat, Harlan Sprague-Oawley ( ') Day 29
—A A A
Lowtles 6,2009.2650369 90-DayOrat Rat, Cri:CD) (i) Day 93
«•—• 4
Chengelis. 2009,2850404 SODayOral Rat. Cr1:CD(SO) (") Day90
Rjt, Crl:CD(SO) <.') Day 90
~
•
Klaunig, 2015,2650075 2-\fearCancer Bioassay Rat, CrliCD(SO) ( ") Week 25
Week 51
Week 104
•-—•
•—•
m—•
Rat, Crl:CD(SO) <,) Week 25
Week 51
•• 1
— •
Week 104
Mean Corpuscular Hemoglobin NIP 2018, 4309149 28 0ay0ral Rat, Harlan Sprague-Oawley ( ) Day29
Concentration (MCHC)
•• •
— ~
Rat, Harlan Sprague-Oawley ( > Day 29
Loveless, 2009.2650369 90-DayOral Rat, Crl:CD(SD) ( ') Day92
•—• • ~
«•—-•
Rat Crt:CD Day 90
Waunig, 2015,2650075 2-^ar Cancer Bioassay Rat Crl:CD(SO) (') Week 25
•-« •
•—~
Week 51
m—~
Week 104
e»—~
Rat Crt.CD(SD) (*) Week 25
— •
Week 51
Week 104
— 1
M ~
i r r i i i 1 i i i 1
• Mo significant changq^ SigniScanl increase V Significant decrease ^ Significant TrendI -too o 100 200 300 400 500 600 700 800 900 1,0001.100
Dose (mgftg-day)
Figure 3-10. Hematological findings (mean cell hemoglobin [MCH], mean cell
hemoglobin concentration [MCHC], and mean cell volume [MCV]) in rats
exposed by gavage to PFHxA or PFHxA sodium salt (full details available by
clicking the HAWC link).
This document is a draft for review purposes only and does not constitute Agency policy,
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Table 3-20. Percent change in hematocrit due to PFHxA exposure in
short-term, subchronic, and chronic oral toxicity studies
Reference
Dose (mg/kg-d)
in
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Reference
Dose (mg/kg-d)
in
10%) across all study designs and exposure durations at
200 mg/kg-day (Chengelis etal.. 2009b: Klaunig etal.. 2015). 250 mg/kg-day (NTP. 2018). or
500 mg-kg/day (Loveless etal.. 2009). Reticulocyte levels also were measured by Klaunig et al.
(2015). but only decreased in female rats that received double the dose of males. The observation
of increased reticulocytes was correlated 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 fLoveless etal.. 2009: NTP. 20181 (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.
Endpoint Study Experiment
Animal Description
Observation Time
Reticulocytes (RET) NTP,2018. A309149 28DayOral
Rat, Harlan Sprague-Dawley( )
Day 29
Rat, Harlan Sprague-Dawley(
Day 29
Loveless, 2009.2850369 90-Day Oral
Rat,Ci1:CD(SD>(5)
Day 92
Rat, Crl.CD(SD) ($)
Day 93
Chengelis. 2009,2650404 90 Day Oral
Ral, Cr1:CD(SD) ((JJ
Day 90
Rat. Crl:CO(SO)($)
Day 90
Klaunig, 2015, 2850075 2-Yfear Cancer Bioassay
Rat, Cil CD(SD)( J)
Week 25
Week 51
Week 104
Rat. Ci1CD(SD)(¥)
Week 25
PFHxAHemaotopoietic Effects: Reticulocytes
Week 51
Week 104
No significant chanaaA Significant increase W Significant decrease A Significant Trend I
-100 0 100 200 MO 400 500 600 700 600 900 1,0001.100
Dose (rng/kg-day)
Figure 3-11. Hematological findings (reticulocytes) in rats exposed by gavage
to PFHxA or PFHxA sodium salt (full details available by clicking the HAWC
link).
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Table 3-22. Percent change in reticulocytes due to PFHxA exposure in
short-term, subchronic, and chronic oral toxicity studies
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 Perfluorohexanoic Acid (PFHxA)
Endpoint Study Experiment Animal Description Obsemrtk*, Tme Hematopoietic Effects: Hemostasls
Platefebi (PLT) NTP, 2018,4309149 28-Day 0»al Ral, Harlan Spragi;e-Daw1ey(,:1) DayZ9
Rat. Harlan Sorague-Dawley( ) Day29
Chengelia, 2009,2850404 90-D.iyOml Rat, CrlCD(SD) (J) Day90
Rat, Crl :CD(SD) (9> Day 90
Loveless, 2009,2&50369 90-Day Owl RaLCrl:CD(SD)(*) Day92
Rat,Crl:CD(SD)(?) Day93
Klauntg.2015,2850075 2-Yfear Cancer Bioass ay RatCrl:CD(SD)(j) Week 26
Rat, Crl :CD(SD) (?) Week 25
Rat. Crl :CD(SD) (-7) Week 51
Rat, Crl:CO(SD) (?) Week 51
Rat, Crl :CO(SD) (
Figure 3-12. 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 of Sprague-Dawley rat 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. These collective
erythroid responses provide evidence for PFHxA treatment-related effects on erythropoiesis. Blood
loss could have been secondary to gastral erosion or ulceration (Klaunig et al.. 2 0151 (summary
level details are available in the Tableau link! but Klaunig et al. f20151 reported gastral erosion and
ulceration were likely due to mechanical dosing errors ruling out treatment-related effects on blood
loss.
Based on these data, there is moderate animal evidence of hematopoietic effects. Effects on
red blood cell parameters including decreased hemoglobin and red blood cells, and decreased
reticulocytes are consistent across both subchronic and chronic studies in the 200 mg/kg-day dose
groups. Overall, the currently available evidence indicates that PFHxA likely causes hematopoietic
effects in humans under relevant exposure circumstances. This conclusion is based on four high
confidence studies in rats showing consistent (across durations and study types), dose-related, and
coherent effects (across various outcome measures of hematopoietic function) at >500 mg/kg-day
following short-term (28-day), subchronic (90-day), or chronic (2-year) exposures.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Table 3-23. Evidence profile table for hematopoietic effects
Evidence stream summary and interpretation
Evidence integration summary
judgment
Evidence from studies of exposed humans
0®Q
Evidence indicates (likely)
Primary basis:
Four high confidence studies in
rats ranging from short term to
chronic exposure durations, in
both sexes, generally at >200
mg/kg-day
Human relevance:
Without evidence to the
contrary, effects in rats are
considered relevant to humans
Cross-stream coherence:
N/A (human evidence
indeterminate)
Susceptible populations and
lifestages:
No evidence to inform
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
Hematology
4 hiqh confidence
studies in adult
rats:
• 28-day
• 90-day
(2 studies)
• 2-year
• Consistent changes
(decreases in
hematocrit,
hemoglobin, red blood
cells, and MCHC and
increases in
reticulocytes, MCV,
and MCH) across
studies
• Coherence of red
blood cells, HCT, and
HGB and reticulocytes
• Large magnitude of
effect as high as 356%
for reticulocytes
• High confidence
studies
• Lack of dose-
response gradient
across studies
• Lack of coherence
across sexes
• Decreased red blood cells,
hematocrit, and
hemoglobin at >62.5
mg/kg-d; both sexes
• Increased MCH and MCV
at >250; males more
sensitive
• Increased reticulocytes at
>200 mg/kg-d; both sexes,
all studies
• Coherence of red blood
cells and reticulocytes
with splenic
extramedullar
hematopoiesis and bone
marrow erythroid
hyperplasia
®©o
Moderate
Findings considered
adverse based on
coherent evidence
that was consistent
across multiple
laboratories and
experimental
designs. Consistent
findings of
decreased red blood
cells, hematocrit,
and hemoglobin at
>200 mg/kg-day
correlated with a
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Evidence stream summary and interpretation
Evidence integration summary
judgment
Hemostasis
4 hiqh confidence
studies in adult
rats:
• 28-day
• 90-day
(2 studies)
• 2-year
• Consistent treatment
related effect on
platelet levels
• Consistency across
study designs
• High confidence
studies
• Lack of coherence
across sexes
• Lack of dose-
response gradient
• Increased platelet levels
>10 mg/kg-d; both sexes,
1 28-day, 2 90-day studies
• Decreased activated
partial thromboplastin
(APTT) at >20 mg/kg-d;
males only, 1 90-day
study
• Decreased prothrombin
(PT) time at 500 mg/kg-
day; males only, 190-day
study
compensatory
findings of erythroid
cell regeneration
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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3.2.5. Endocrine Effects
Human
Thyroid Hormones
Two studies examined the association between PFHxA exposure and thyroid hormones in
humans (Figure 3-17). One was considered uninformative due to critical deficiencies in
confounding and statistical analysis (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 and range in Li etal. f20171
were low (median [range]: 0.01 [
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
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 3-24, and details are available by clicking the HAWC link.
Table 3-24. Endocrine endpoints for PFHxA and associated confidence scores
from repeated-dose animal toxicity studies
+-»
-C
M
'
Author (year)
Species, strain (sex)
design
Exposure route and dose range
O
if
-C
1—
NTP (2018)
Rat, Harlan
Sprague-Dawley
(male and female)
Short term
(28 days)
Gavage3
Male and female: 0, 62.5,125,
250, 500,1,000 mg/kg-d
++
++
++
Chengelis et al.
Rat, Crl:CD(SD)
Subchronic
Gavage3
++
-
NM
(2009b)
Sprague-Dawley
(male and female)
(90 days)
Male and female: 0,10, 50,
200 mg/kg-d
Loveless et al.
Rat, Crl:CD(SD)
Subchronic
Gavageb
++
++
NM
(2009)
Sprague-Dawley
(male and female)
(90 days)
Male and female: 0, 20,100,
500 mg/kg-d
Klaunig et al.
Rat, Crl:CD(SD)
2-year cancer
Gavage3
NM
++
NM
(2015)
Sprague-Dawley
(male and female)
bioassay
Male: 0, 2.5,15,100 mg/kg-d
Female: 0, 5, 30, 200 mg/kg-d
Study 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 medium confidence; - outcome rating of low
confidence; - outcome rating of uninformative; NR, outcome not reported; 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 also 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 Perfluorohexanoic Acid (PFHxA)
1 observed for T3 or T4 in females or for TSH in either sex (NTP. 20181. Results are summarized in
2 Figure 3-18 and Table 3-25.
Endpoint Study Experiment Animal Description Observation Time
Thyroid Stimulating Hormone (TSH) NTP, 2018,4309149 28 Day Oral Rat, Harlan Sprague-Dawley (V) Day 29
Rat, Harlan Sprague-Dawley (~~ ~ ~ ~
» » »—¦ 1 •
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
reported no treatment-related effects on thyroid histopathology at doses as high as 200 mg/kg-d
following subchronic (90-day) or chronic (2-year) exposure to PFHxA. Notably Chengelis et al.
f2009bl 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 3-26.
Table 3-26. Incidence of thyroid follicular epithelial cell hypertrophy
following PFHxA ammonium salt exposure in a 90-day oral toxicity study
Reference
Time point
Dose (mg/kg-d)
0
20
100
500
90-dav, female rat (Loveless et al., 2009)
Exposure, Day 90
0/10
0/10
0/11
4/10
90-dav, male rat (Loveless et al., 2009)
0/10
0/10
1/10
2/10
90-dav, female rat (Loveless et al., 2009)
Recovery Day 30
0/10
6/10
90-dav, male rat (Loveless et al., 2009)
0/10
3/10
90-dav, female rat (Loveless et al., 2009)
Recovery, Day 90
0/10
0/10
0/9
0/10
90-dav, male rat (Loveless et al., 2009)
0/10
0/10
0/10
2/10
Bold values indicate 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 (Chengelis etal.. 2009b:
Loveless etal.. 2009: NTP. 20181. Although no effects on relative thyroid weight were observed at
the end of the 90-day exposure period in either subchronic study Loveless etal. f20091
qualitatively reported a statistically significant increase in relative thyroid weight for female rats at
the highest tested dose (500 mg/kg-day) of PFHxA sodium salt at the 30-day recovery. NTP (2018)
observed a trend (p < 0.05) for decreased absolute adrenal gland weight in male rats exposed to
500 mg/kg-day. No other treatment-related effects on endocrine organ weights were observed
(Chengelis etal.. 2009b: Loveless et al.. 2 0 09: NTP. 2018).
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
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
and total), but these effects were observed only in males (NTP. 20181. A second study found
increased incidence of thyroid epithelial cell hypertrophy following a 90-day exposure to PFHxA
sodium salt fLoveless etal.. 20091. 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 d to 2 years) and doses (up to 1,000 mg/kg-day) fChengelis etal.. 2009b: Klaunig et
al.. 2015: NTP. 20181. No clear pattern of treatment-related effects were reported for endocrine
organ weights.
Although the only available study examining thyroid hormones showed strong effects on T4
and T3 after short-term exposure, no effects were observed on TSH; however, a pattern of
decreased T4 without changes in TSH is consistent with hypothyroxinemia and has been observed
for other PFAS with more detailed studies of endocrine function, including PFBA and PFBS. During
pregnancy and early development, perturbations in thyroid function can have impacts on normal
growth and neurodevelopment in the offspring. Given the potential consistency of these findings
with those observed for other PFAS, the availability of only one short-term study of thyroid
hormones represents a significant data gap for PFHxA.
The small number of studies and inconsistent findings for endpoints reported across study
designs reduces the strength of the available evidence; however, some of these inconsistencies
could be explained by differences in the test article (i.e., PFHxA vs. PFHxA salts), 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). 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 (Chang etal.. 2008: Lau etal.. 2004: Lau etal.. 2006). Differences in
pharmacokinetics might explain why effects on thyroid hormones were observed only in male rats,
but why a similar sex-specific pattern was not observed for the reported thyroid histopathological
effects is unclear. Furthermore, that the observed changes in thyroid histopathology are secondary
to hepatic effects is possible. In rats, increases in thyroid epithelial cell hypertrophy are associated
with induction of microsomal liver enzymes and hepatocellular hypertrophy fCesta etal.. 20141.
Based on the results, there is slight animal evidence of endocrine effects.
Overall, the currently available evidence suggests, but is not sufficient to infer, that PFHxA
could cause endocrine effects in humans under relevant exposure circumstances. This conclusion is
based on four animal studies generally rated high confidence that reported treatment-related
changes in thyroid hormone levels and thyroid histopathology after exposure to PFHxA at
>62.5 mg/kg-day (Table 3-27).
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Table 3-27. Evidence profile table for endocrine effects
Evidence stream summary and interpretation
Evidence integration
summary judgement
Evidence from studies of exposed humans
©OO
Studies and
confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key
findings
Evidence stream
judgments
Evidence suggests but is not
sufficient to infer
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:
Four animal studies
generally rated high
confidence that reported
treatment related changes
in thyroid hormone levels,
thyroid histopathology after
exposure to PFHxA at > 63.5
Evidence from animal studies
Studies and
confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key
findings
Evidence stream
judgments
mg/kg-day.
Human relevance:
Without evidence to the
contrary, effects in rats are
considered relevant to
humans.
Cross-stream coherence:
Thyroid
Hormones
1 hiqh
confidence study
in adult rats:
• 28-day
• 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-day
©oo
Slight
Some evidence of thyroid
effects based on hormone
and histopathological
changes in two rat studies;
however, the data is
limited, lacking consistency
across studies, and
histopathological changes
may be explained by non-
thyroid related effects
Histopathology
3 hiqh
confidence
studies in adult
rats:
• 28-day
• 90-day
• 2-year
• High confidence
studies
• Unexplained
inconsistency across
studies
• Increased incidence of
thyroid epithelial cell
hypertrophy at >100
mg/kg-day for 90 days;
persisted up to 90 days
after exposure
Decreases in T3 were
observed in both animal and
human studies, although
results in humans were of
low confidence.
Susceptible populations and
lifestages:
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Evidence stream summary and interpretation
Evidence integration
summary judgement
1 low confidence
study in adult
rats:
• 90-day
No evidence to inform
Other inferences:
No mechanistic data or
supplemental information
on this health effect were
identified to inform a
potential MOA for the
observed effects, although
the pattern of the limited
findings for PFHxA are
consistent with
hypothyroxinemia seen for
other PFAS
Organ Weight
High confidence:
3 hiqh
confidence
studies in adult
rats:
• 28-day
• 90-day (2
studies)
• High confidence
studies
• Unexplained
inconsistency across
studies
• Relative thyroid
weights were increased
only in females 30 days
after exposure
• Right adrenal weights
decreased but no other
adrenal effects were
reported
Mechanistic evidence and supplemental information
Species or
model systems
Key findings, limitations,
and interpretation
Evidence stream summary
Species or model systems
• No informative studies identified
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3.2.6. Male Reproductive Effects
Human
Sperm Parameters
One low confidence study (Songetal.. 2018) examined the association between PFHxA
exposure and semen parameters and reported no decrease in concentration or motility with higher
levels of PFHxA in serum (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 were fairly high (median: 29 ng/mL, 5 th-
95th percentile: 11-70), 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 (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 etal.. 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. The
exposure levels in this study were low and the range narrow (median: 0.2, IQR 0.1-0.3), 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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^ ',,1^
90^'
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 3-15. 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 (2018) was rated low confidence for
sperm parameters due to issues related to exposure duration and concerns for potential
insensitivity. Histopathological results for Chengelis etal. (2009b) 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 3-28, and details are available by clicking the HAWC link.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Table 3-28. 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 days)
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 days)
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 days)
One-generation
reproductive: P0
females dosed 70 d
prior to
cohabitation,
through gestation
and lactation (126
days); P0 males
dosed for 110 days
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-year 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);
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
++
Study 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.
++ Outcome rating of high confidence; + outcome rating of medium confidence; - outcome rating of low
confidence; -- outcome rating of uninformative; NR, outcome not reported; NM, outcome not measured.
This document is a draft for review purposes only and does not constitute Agency policy.
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Sperm Parameters
Evidence from a 28-day fNTP. 2018] and one-generation reproductive study fLoveless etal..
20091 included sperm parameters useful in evaluating potential male reproductive effects
(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) (NTP. 2018).
Notably, these effects were observed despite concerns about sensitivity due to the short exposure
duration of the study by NTP f20181. In the one-generation reproductive study, Loveless et al.
f20091 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.
Endpalnt Slimy EIpfpIlmBrt Animal Description Obs.rvstlon Time Reprofluclta Blectt: sporm parameters
Testicular Spermatids (per Testis)
Loveless, 2009.2850369 1 Generation Reproductive
PO Rat. Crt.CD(SD}(
Day10&
Testicular Spermatic] Count (per g
Lovele33,2009. 2850169 1 -Generation ReproducUw
PO Rat. CrtCD(SD) (J)
Day 105
Testis)
Testicular Spermatid Count
NTP, 2018.4309149 28-DayOral
Rat, Harlan Sprague-Dawtey(')
Day 29
Testicular Spermatid Count (per mg
NTP. 2018.4309149 28-Day Oral
Rat, Harlan Sprague-Daw1ey< n)
Day 29
A
Testis)
W
Cauda Epididymis Sperm Count
NTP. 2018.4309149 28-DayOral
Rat. Harlan Sprague-Dawley< J)
Day 29
A •
V
Eprdidymal Sperm Count (per Cauda) Lovctess, 2009 .2850369 1-Generation Reproductive
PO Rat, Crt.CD(SD) (^)
Day 105
CpidMymal Sperm Count (per g
Loveless. 2009.2050369 i-Generation Reproductive
PO Rat, CrtCD(SD) (5)
Day 105
Cauda)
Percent fcbtite Sperm
NTP, 2018.4309149 28-DayOfal
Rat, Harlan Sprague-Dawtey(rT)
Day 29
*
Sperm Motility
Luwless. 2009.2850369 1-Generation Reptodudiw
PO Rat, Crl:CD(SD) (j)
Day 105
Sperm Morphology
Loveless. 2009.2850369 1-Generahon Reproductive
PORat, CrtCO(SO)(*)
Day 105
• No significant chnngg^ Significant incraase^F"signlllcant decrease Slgnllcant Trend 100 0 TOO 200 300 400 500 600 700 800 900 1.0001.100
Figure 3-16. 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..
20041. 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
(Figure 3-21) (Chengelis etal.. 2009b: Loveless et al.. 2009: NTP. 2018). 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 (Loveless etal.. 2009). No treatment-related effects on male
reproductive organ weights were reported by Chengelis etal. f2009bl.
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Endpoint
Study Experiment
Animal Description Observation Time
Cauda Epididymis Weight, Absolute NTP. 2010,4309149 28-DayOral
Rat, Harlan Sprague-Dawley{") Day 29
Epididymides Weigh!, Absolute
Loveless, 2009,2850369 90-Day Oral
Rat, Crl:C D(SD) (;) Day 92
Epididymides Weight, Relative
Loveless, 2009,2850369 90 Day Oral
Rat Crt:CD(SD) (
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
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 fLoveless etal.. 2009: NTP. 20181 at the highest tested doses in these studies (1,000
and 500 mg/kg-day, respectively). Decreased sperm count reported by NTP (2018) 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 NTP study. In addition, evidence of overt toxicity (i.e., reductions in body weight) was found in
the 1,000 mg/kg-day males in the NTP (2018) 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
(Klaunigetal.. 2015). Similar results on testosterone were not reported in the short-term high
confidence study (NTP. 2018). 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 under relevant exposure circumstances (Table 3-29).
This document is a draft for review purposes only and does not constitute Agency policy.
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Table 3-29. Evidence profile table for male reproductive effects
Evidence stream summary and interpretation
Evidence integration
summary judgment
Evidence from studies of exposed humans
OOO
Evidence inadequate
Primary Basis:
Evidence is low confidence
or largely null
Human relevance:
Without evidence to the
contrary, effects in rats are
considered relevant to
humans
Cross stream coherence:
N/A (human evidence
indeterminate)
Susceptible population and
lifestages:
No evidence to inform
Studies and confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key findings
Evidence stream
judgment
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 from animal studies
Studies and confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key findings
OOO
Indeterminate
The data are largely
null. Some evidence of
reproductive effects
but limited by
unexplained
inconsistency and low
sensitivity.
Sperm Parameters
1 high confidence study
in adult rats:
• 90-day
1 low confidence in
adult rats
• 28-day
• No factors noted
• High dose elicited
overt toxicity
(i.e., decreased
body weight)
• Unexplained
inconsistency across
studies
• Decreased sperm count
in the cauda epididymis
at 1,000 mg/kg-day
Organ Weights
3 high confidence
studies in adult rats:
• 28-day
• 90-day (2 studies)
• High confidence
studies
• Dose-response
with longer
exposure duration
• No factors noted
• Increased relative testis
weight at >500 mg/kg-
day; no change in
absolute testis weights
(preferred metric)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Evidence stream summary and interpretation
Evidence integration
summary judgment
Reproductive
Hormones
2 high confidence
studies in adult rats:
• 28-day
• 2-year
• High confidence
studies
• No factors noted
• Transient decrease of
small magnitude in
luteinizing hormone and
testosterone
Histopathology and
Male Reproductive
System Development
4 high confidence
studies in rats and
mice:
• 28-day (rat)
• 90-day (rat)
• GD 6-18 (mouse)
• 2-year (rat)
1 low confidence study
in adult rats:
• 90-day
• High confidence
studies
• Low sensitivity.
• No treatment related
effects reported at
<1,000 mg/kg-day
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 Perfluorohexanoic Acid (PFHxA)
3.2.7. Female Reproductive Effects
Human
Reproductive Hormones
A single low confidence study (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 the male
reproductive section.
oOV*
¦ja
0^
Participant selection -
Exposure measurement
Outcome ascertainment -
Confounding
Analysis -
Sensitivity
Selective Reporting
Overall confidence -
Legend
p
Good (metric) or High confidence (overall)
+
Adequate (metric) or Medium confidence (overall)
-
Deficient (metric) or Low confidence (overall)
NR
Not reported
D
Critically deficient (metric) or Uninformative (overall)
N/A
Not applicable
Figure 3-18. 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
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
1 of study evaluation for female reproductive outcomes are presented in Table 3-30 and details are
2 available by clicking the HAWC link.
Table 3-30. 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 days)
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 days)
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 days)
One-generation
reproductive: P0
females dosed 70 d
prior to cohabitation,
through gestation and
lactation (126 days);
P0 males dosed for
110 days
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-year 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
++
Study 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 medium confidence; - outcome rating of low
confidence; -- outcome rating of uninformative; NR, outcome not reported; NM, outcome not measured.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Fertility and Pregnancy Outcomes
Three studies evaluated outcomes related to fertility and pregnancy following exposure by
gavage with PFHxA or PFHxA salts in rats or mice flwai and Hoberman. 2014: Loveless etal.. 2009:
NTP. 20181. Loveless etal. f20091 provided evidence for decreased maternal body weight gains
(31% change from control) in rats exposed to 500 mg/kg-d from both developmental and one-
generation reproductive experiments. Dams from the developmental exposure (GD 6-20) showed
a statistically significant decrease in weight gain and in terminal body weight on GD 21. Deficits
remained when correcting for gravid uterine weight, indicating that reductions were being driven
by effects in the dams rather than by the number or size of fetuses. In the one-generation
reproductive, dams continuously exposed from premating through lactation showed a decrease in
weight gain during early gestation (GD 0-7), which was not significant over the entire gestational
period (GD 0-21). These animals showed a statistically significant increase in body weight gains
during lactation. No change in maternal body weights were identified in mice (Iwai and Hoberman.
2014). Results are presented in Figure 3-23. No effects on mating, pregnancy incidence, gestation
length, number of implantations, or litter size occurred 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 fLoveless et al.. 2009: NTP. 20181.
Endpoint
Study
Experiment
Animal Description
Observation Time
Body Weight Cha nge, Gestalion
Iwai, 2014,2821611
1-Generation Reproductive
P0 Mouse,CD-I (£)
P0 Mouse,CD-I (£)
GD 6-18
GD 6 18
Loveless. 2009.2050369
14-Day Developmental
PORal, CdrCD(SD) {?)
GD 6-21
1-Generation Reproductive
PORat, Crt£D(SDH )
P0 Rat, Cri:CD(SD){?)
GD 0-7
GD 0-21
Body Weight Change, Gestalion
(Minus Gra vid Uterine Weight)
Loveless. 2009,2650369
14-Day Developm ental
PORat, Cri;CD{?)
GD 6-21
Body Weight Change, Lactation
Iwai, 2014,2821011
1-Generation Reproductive
P0 Mouse, CD-I (2)
P0 Mouse, CD-I (9)
PND 0-20
PND 0-20
Loveless. 2009,2850369
1-Generation Reproductive
PORat, CrtrCD(SD) {?)
PND 0-21
Body Weight Change, Terminal
Loveless. 2009,2650369 1-GeneratJon Reproduce
PORat, CrtCD(SDKj)
Day 105
Body Weight, Terminal (Mnus Gravid
Uterine Weight)
Loveless. 2009,2850369
14-Day Developm ental
PORaL Crl:CD(SD) (r')
GD 21
Terminal Body Weight,/ibsofute
Iwai, 2014,2821611
1-Generation Reproductive
P0 Mouse, CD-I (5)
P0 Mouse, CD-I (9)
PND 20
PND 20
Loveless. 2009,2650369
14-Day Developmental
PORat, Cri£D(SD){2)
GO 21
Female Reproductive Effects: Body Weight
• No significant charcg
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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 (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 (Chengelis etal.. 2009b: Klaunigetal.. 2015: Loveless et
al„ 2009: NTP. 20181.
Endpoint Study Animal Description Dose Incidence
Uterus, Bilateral Dilation NTP, 2018.4309149 Rat. Harlan Sprague-Dawtey(+) 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%)
|l I percent affected BBSignificant Compared to Controj
Figure 3-20. 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
fChengelis etal.. 2009b: Loveless etal.. 2009: NTP. 20181. Authors reported no treatment-related
effects for these outcomes.
Reproductive Hormones
Two studies measured effects of PFHxA or PFHxA ammonium salt on testosterone (Klaunig
etal.. 2015: NTP. 20181. estradiol, and luteinizing hormone f Klaunig etal.. 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 (Zhou etal..
2016). Based on these results, there is indeterminate human evidence of female reproductive
effects.
This document is a draft for review purposes only and does not constitute Agency policy.
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Female Reproductive Histopathology
40 50 60
% Affected
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In animals, evidence supporting effects of PFHxA exposure female reproduction was largely
limited to effects on maternal weight gain during gestation in rats fLoveless etal.. 20091. Effects on
maternal weight gain, however, were not consistent across studies. The observed uterine horn
dilation appears influenced by differences in sample sizes, as the total incidence is similar across
controls and all dosing groups. Furthermore, this latter finding is generally associated with
estrogenic effects, but no coherent changes were observed that would be indicative of estrogenic
changes in females. No treatment-related changes were reported for other female reproductive
outcomes fChengelis etal.. 2009b: Iwai and Hoberman. 2014: Klaunig etal.. 2015: Loveless etal..
2009: NTP. 20181. 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 under relevant exposure circumstances (Table 3-31).
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Table 3-31. 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:
• In the absence of evidence to
the contrary, the evidence in
rodents is presumed to be
relevant to humans based on
similarities in the anatomy and
physiology of the reproductive
systems across these two
species.
Cross stream coherence:
• The strength of the evidence is
neither increased nor
decreased due to a lack of
Fertilitv and
Pregnancv Outcomes
3 hiqh confidence
studies in rats and
mice:
• 28-day (rat)
• 90-day (rat)
• GD 6-18 (mouse)
• High
confidence
studies
• Unexplained
inconsistency
across studies
• Decreases in
maternal weight gain
during gestation at
500 mg/kg-day
OOO
Indeterminate
The animal evidence is largely
null. Some evidence of female
reproductive effects but results
were not dose-dependent, and
there was no coherent evidence
supporting the biological
significance of the findings
Histopatholoev
4 high confidence
studies in rats and
mice:
• 28-day (rat)
• 90-day (rat)
• High
confidence
studies
• Lack of dose-
response gradient
for uterine horn
dilation
• Increase in bilateral
uterus dilation
reported for all
groups except the
highest dose
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Evidence stream summary and interpretation
Evidence integration summary
judgment
• 2-year (rat)
• GD 6-18 (mouse)
1 low confidence
study in adult rats:
• 90-day
• Unexplained
inconsistency
across studies
• Lack of expected
coherence with
other estrogen
related outcomes
coherence across evidence
streams.
Susceptible populations:
• None identified
Organ Weights,
Reproductive
Hormones,
Reproductive
System
Development
6 hiqh confidence
studies in rats and
mice:
• 28-day (rat)
• 90-day (rat, 2
studies)
• 2-year (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-day
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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3.2.8. Immune Effects
Human
Asthma
One medium confidence case-control study in Taiwan reported in three publications (Dong
etal.. 2013: Oin etal.. 2017: Zhou etal.. 20171 examined the potential association between PFHxA
exposure and asthma, asthma symptoms, pulmonary function, and related immune markers
(Figure 3-25). 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.. 2013).
pulmonary function measured with spirometry (Oin etal.. 2017). or immune markers (Dong etal..
2013) among children with asthma. The exposure levels in this study were low (median [IQR]: 0.2
[0.1-0.3]), which likely reduced study sensitivity.
0°'
&
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)
B
Critically deficient (metric) or Uninformative (overall)
NR
Not reported
N/A
Not applicable
Figure 3-21. 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.
This document is a draft for review purposes only and does not constitute Agency policy.
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Animal
Several short-term (28-day), subchronic, and chronic animal studies 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 confidence; however, some specific
concerns were identified that resulted in a low confidence rating. Histopathology for Chengelis et
al. (2009b) 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 3-32 and details are available by clicking the
HAWC link.
Table 3-32. 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 days)
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 days)
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 days)
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-year
cancer
bioassay
Gavage3
Male: 0, 2.5,15,100 mg/kg-d
Female: 0, 5, 30, 200 mg/kg-d
NM
+ +
+ +
Study 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.
++ Outcome rating of high confidence; + outcome rating of medium confidence; - outcome rating of low
confidence; - outcome rating of uninformative; NR, outcome not reported; NM, outcome not measured.
Organ Weights
Three studies evaluated effects on spleen and thymus weights in response to PFHxA
fChengelis etal.. 2009b: NTP. 20181 or PFHxA sodium salt fLoveless etal.. 20091 exposure.
The available evidence identified, in general, decreased absolute or relative thymus weights.
Statistically significant decreases in absolute weights were found in males exposed to
500 mg/kg-day PFHxA sodium salt for 90 days (Loveless etal.. 2009). and downward trends in both
This document is a draft for review purposes oniy and does not constitute Agency poiicy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
relative and absolute organ weights were reported in males and females receiving PFHxA in the
28-day study fNTP. 20181.
Spleen weights did not show a clear pattern of effect across studies. In the 28-day study a
trend of increased weights in males and females receiving PFHxA fNTP. 20181 was observed,
whereas spleen weights were decreased in males receiving PFHxA sodium salt in the 90-day study
by Loveless etal. (2009). Chengelis etal. (2009b) qualitatively reported no treatment-related
effects on spleen or thymus weights after exposure to <200 mg/kg-day PFHxA for 90 days. Results
are summarized in Figure 3-26.
Endpoint Study Experiment
Animal Description
Observation Time
Immune Effects: Organ Weights
Spleen Weight. Absolute Loveless, 2009,2850369 90 Day Oral
Rat Cr1:CD(SD)(9)
Day 92
Rat Cri:CD(SD)(<$)
Day 92
V
NTP, 2018,4309149 28-DayOral
Rat, Harlan Sprague-Dawley(^)
Day 29
0 ~—
• 9 a
Rat Harlan Sprague-Dawley(J)
Day 29
• » •
1 » •
Spleen Weight, Relative Loveless, 2009,2850369 90-DayQral
Rat Crl:CD(SD)($)
Day 92
a
Rat Crl:CD(SD)(-J)
Day 92
a
NTP, 2018.4309149 28 Day Oral
Rat, Harlan Sprague Davtfey(^)
Day 29
0
A
Rat, Harlan Sprague-Dawley(rv)
Day 29
• ~—•—
A
Thymus Weight, Absolute Loveless, 2009, 2850369 90-Day Oral
Rat Cf1:CD(SD)(?>
Day 92
aa—•—
~
Rat Crl;CD(SD)(o)
Day 92
•a—•—
V
NTP, 2018.4309149 28 Day Oral
Rat, Harlan Sprague Daw1ey(9)
Day 29
0
a a a
Rat, Harlan Sprague Daw1ey(:?>
Day 29
o
—a a a
Thymus Weight Retatiw Loveless, 2009, 2850369 90-DayOral
RatCi1:CD(SD)(9)
Day 92
a
Rat Ci1:CD(SD)(£)
Day 92
a
NTP, 2018.4309149 28-Day Oral
Rat, Harlan Sprague-Dawley(9)
Day 29
• » ~
—a a a
Rat, Harlan Sprague Dawtey(s)
Day 29
0 —
—a a a
1 1 1 1 1 1 1 1 1 1 1
x = 1 -100 0 100 200 300 400 500 600 700 800 900 1,0001,100
• No significant chanoftA Significant increase v Significant decrease O Significant Trend
' Dose (rngftg-bw/day)
Figure 3-22. Immune organ weights in rats exposed by gavage to PFHxA or
PFHxA sodium salt (HAWC: PFHxA - Animal Toxicity Immune Effects link).
Histopathologv
Four studies examined spleen, thymus, lymph nodes, or bone marrow for histopathological
changes (Chengelis etal.. 2009b: Klaunigetal.. 2015: Loveless etal.. 2009: NTP. 2018). Some
evidence of effects in the spleen from two of these studies was found. 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..
2009). 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 notpersist after the 30-day
recovery and specific incidence data were not reported fLoveless etal.. 20091. Spleen
histopathological findings were null in the 90-day PFHxA subchronic study that tested doses up to
200 mg/kg-day (Chengelis etal.. 2009b). All studies reported null results for histopathological
examinations of the thymus, lymph node, and bone marrow (Chengelis etal.. 2009b: Klaunig etal..
2015: NTP. 20181.
This document is a draft for review purposes only and does not constitute Agency policy.
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Immune Cell Counts
Four animal studies had evidence of hematological indicators of immunotoxicity fChengelis
et al.. 2009b: Klaunig et al.. 2015: Loveless etal.. 2009: NTP. 20181. Of these studies, NTP f 20161
and Loveless etal. (20091 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 two subchronic and
rat studies (90 days and 2 years) at exposures to PFHxA as high as 200 mg/kg-day fChengelis et al..
2009b: Klaunig et al.. 20151. Eosinophils were decreased only in males exposed to PFHxA sodium
salt for 90 days fLoveless etal.. 20091. 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 fChengelis et al.. 2009b: Klaunig et al.. 2015: Loveless et al.. 2009:
NTP. 2018). Results are summarized in Figure 3-27.
EncfpoinI Study Experiment Animal Description Observation Time
Basophils
NTP 2018. •1309149 28-DayOral
Ral Hartan Sprague-Dawley( ¦) Day 29
~
—~—
V
Ral Harlan Sprague-Dawley( ) Day 29
• • •
•
•
—•
Loveless. 2009,2860369 90-DayOral
Rat. Cri CO(SD) (-') Day 92
Rat, Cri CD (—
—•
Lymphocyte
NIP. 2018. 4309149 28 Day Oral
Rat, Harlan Sprague Dawley( •") Day 29
• *—•-
•
Rat, Harlan Sprague-Dawley ( ) Day?9
• ~ ~
•
Lawless. 2009,2850369 90-Day Oral
RalCriCD(SD)(0 Day 92
M—»—
~
Rat, Cri CO(SD) (,) Day 93
~
Lymphocyte, Total
NTP. 2018 4309149 28-DayOral
Rat, Harlan Sprague-Oawley( ') Day 29
• • ••
•
—•
Rat. Harlan Sprague-Dawley (.) Day 29
• •—•
•
Monocytes
NTP 2018.4309149 28-DayOral
Rat, Harlan Sprague-Dawley ( ') Day29
• ~- •-
•
Rat, Hartan Sprague-Dawley ( ) Day 29
¦ ~ ¦»
•
Lo wless. 2009.2850369 90-Day Oral
Rat. Cri-CD(SD) (') Day 92
•* •—
~
Rat, Cri CO(SD) (-) Day 93
~
Neutrophils
NTP 2018.4309149 28-OayOral
Rat, Harlan Sprague-Dawley ( ') Day 29
• ~ •
•
Ral Harlan Sprague-Dawley( - ) Day29
0
•
~
Loveless, 2009,2850369 90 Day Oral
Ral Cri CD(SD)
•
—•—
—•
Cftengelis. 2009.2850404 90-Day Oral
Rat. CM CD(SD> (cf) Day 90
* •—
-•
RaiCrtCD(SD)(^) Day 90
m-*—
Loveless, 20O9,2850369 90-Day Oral
Rai Cri CD. Recovery
Cftengelis. 2009.2850404 90-DayQral
Rai CflCDjSD) (¦;') Day 118
¦
RaiCitCD
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
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.
With the exception of changes in thymus weight, the available animal toxicological evidence
did not show a clear pattern of effect across studies. Specifically, two studies reported
treatment-related changes in thymus and spleen weights in rats, but the direction of effect on
spleen weights was not 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 four 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 (WHO. 2012). Additional
studies, particularly those that evaluate changes in immune function (e.g., in response to foreign
challenge) 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 under relevant exposure conditions
(Table 3-33).
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Table 3-33. Evidence profile table for immune effects
Evidence stream summary and interpretation
Evidence integration
summary judgment
Evidence from studies of exposed humans
OOO
Inadequate
Primary basis:
Evidence is low
confidence or limited
Human relevance:
Without evidence to
the contrary, effects in
rats are considered
relevant to humans
Cross-stream
coherence:
N/A (human evidence
indeterminate)
Susceptible populations
and life stages:
• No evidence to
inform
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
• Potential for residual
confounding (e.g., with
other PFAS)
• Imprecision
• Lack of internally
coherent findings (no
associations with other
measures of pulmonary
function)
• Nonsignificant association
with asthma diagnosis, but
other asthma-related
outcomes were not affected
ooo
Indeterminate
Evidence from animal studies
Studies and
confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key findings
Evidence stream
judgment
Histopathology
3 hiqh confidence
studies in adult rats
• 28-day
• 90-day
• 2-year
1 low confidence
study in rats;
• 90-day
• High confidence studies
• Consistency across studies
for extra medullary
hematopoiesis
Lack of biological
gradient/
dose-response.
• Increased splenic
extramedullar
hematopoiesis was
observed male and female
rats at 500 mg/kg-day;
coincident with minimal
erythroid hyperplasia of the
bone marrow
OOO
Indeterminate
Some evidence of
immune system but
limited by low
sensitivity
(observational
outcomes less
predictive of immune
system toxicity), lack of
coherence, and
potential for non-
immune related causes
Immune Cell Counts
4 high confidence
studies in rats:
• 28-day
• High confidence studies
• Consistency-studies for
neutrophils and basophils
• Lack of biological
gradient/
dose-response gradient
• Decreased basophil counts
and increased neutrophil
cell counts at >20 mg/kg-
day
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
Evidence stream summary and interpretation
Evidence integration
summary judgment
• 90-day (2 studies)
• 2-year
• Lack of coherence with
other immune markers
[see Section 3.2.4 for
additional discussion]
Organ Weight
3 hiqh confidence
studies in rats:
• 28-day
• 90-day (2 studies)
• High confidence studies
• Unexplained
inconsistency across
studies for spleen
weights
• Thymus weights decreased
at 500 mg/kg-day in
short-term and subchronic
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
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of Perfluorohexanoic Acid (PFHxA)
3.2.9. Nervous System Effects
1 Human
2 No studies were identified that evaluated the effects of PFHxA on the nervous system in
3 humans.
4 Animal
5 Four short-term (28-day), subchronic, and chronic animal studies evaluated the effects of
6 PFHxA or PFHxA sodium salt in rats. Most outcome-specific study ratings were high or medium
7 confidence. One study was rated low confidence for histopathology due to concerns about
8 observational bias, endpoint sensitivity and specificity, and data presentation fChengelis etal..
9 2009b). A summary of the studies and the interpretations of confidence in the results for the
10 different outcomes based on the individual study evaluations is presented in Table 3-34, and details
11 are available by clicking the HAWC link.
Table 3-34. Nervous system endpoints for PFHxA and associated confidence
scores from repeated-dose animal toxicity studies
Author (year)
Species, strain
(sex)
Exposu re
design
Exposure route and dose
range
Brain weight
Histopathology
Behavior
NTP(2018)
Rat, Harlan
Sprague-Dawley
(male and female)
Short term
(28 days)
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 days)
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 days)
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-year
cancer
bioassay
Gavage3
Male: 0, 2.5,15,100 mg/kg-d
Female: 0, 5, 30, 200 mg/kg-d
NM
+ +
++
Study 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.
++ Outcome rating of high confidence; + outcome rating of medium confidence; - outcome rating of low
confidence; - outcome rating of uninformative; NR, outcome not reported; NM, outcome not measured.
This document is a draft for review purposes oniy and does not constitute Agency poiicy.
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Brain Weight
Three studies evaluated effects of PFHxA or PFHxA sodium salt on the nervous system in
animals f Chengelis etal.. 2009b: Loveless etal.. 2009: NTP. 20181. 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 (Chengelis etal.. 2009b: Loveless etal.. 2009). 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.. 2009] 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 weightfU.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
fChengelis etal.. 2009b: Klaunigetal.. 2015: Loveless etal.. 2009: NTP. 20181.
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
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 (U.S. EPA. 1998) and has been identified as
a research area of potential concern for other PFAS known to affect thyroid function. No
mechanistic data were identified to inform this potential health effect. Based on these results, there
is indeterminate animal evidence of nervous system effects.
Overall, the currently available evidence is inadequate to assess whether PFHxA might
cause nervous system effects in humans under relevant exposure circumstances.
This document is a draft for review purposes only and does not constitute Agency policy.
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Table 3-35. Evidence profile table for nervous system effects
Evidence stream summary and interpretation
Evidence integration summary
judgment
Evidence from studies of exposed humans
OOO
Inadequate
Primary Basis:
No evidence in humans and
animal evidence is largely null
or lacking biological relevance.
Human relevance:
Without evidence to the
contrary, effects in rats are
considered relevant to humans
Cross stream coherence:
N/A (human evidence
indeterminate).
Susceptible populations and
life stages:
No evidence to inform.
Studies and confidence
Factors that
increase certainty
Factors that decrease
certainty
Summary and key
findings
Strength of evidence
• No studies identified
ooo
Indeterminate
Evidence from animal studies
Studies and confidence
Factors that
increase certainty
Factors that decrease
certainty
Summary and key
findings
Strength of evidence
summary
Brain Weight
3 high confidence studies
in adult rats:
• 28-day
• 90-day (2 studies)
• High confidence
studies
• No factors noted
• Increased relative
brain weights in
animals at >200
mg/kg-day; 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
considered to be
appropriate for
evaluating nervous
system toxicity.
Histopathology
3 hiqh confidence studies
in adult rats:
• 28-day
• 90-day
• 2-year
1 low confidence study in
adult rats:
• 90-day
• High confidence
studies
• No factors noted
• No treatment-related
effects reported
Behavior
2 hiqh confidence studies
in adult rats:
• 90-day
• 2-year
• High and medium
confidence
studies
• No factors noted
• No treatment-related
effects reported
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Evidence stream summary and interpretation
Evidence integration summary
judgment
1 medium confidence
study in adult rats:
• 90-day
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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3.3. CARCINOGENICITY
3.3.1. Cancer
Human Studies
No human studies or studies of human cells were available.
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 etal.. 20151. Findings
for non-neoplastic 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 (Table 3-36) fEriksen etal.. 2010: Lau. 2015:
Loveless etal.. 2009: Nobels etal.. 20101. Sodiumperfluorohexanoate (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 ClpB), 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 (Eriksenetal.. 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 fLoveless etal.. 20091.
Evidence Integration
One study (Klaunig et al.. 2 0151 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 (see Section 3.3). No studies of potential carcinogenicity in exposed
humans or via other exposure routes were identified. As discussed above, given the sparse
evidence base, and in accordance with the Guidelines for Carcinogen Risk Assessment (U.S. EPA.
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1 20051 EPA concluded there is inadequate information to assess carcinogenic potential for
2 PFHxA for any route of exposure.
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Table 3-36. Summary of PFHxA genotoxicity studies
PFHxA genotoxicity
Endpoint
Test system
Doses/
Concentrations
tested
Results3
Comments
References
Without
exogenous
activation
With
exogenous
activation
Genotoxicity
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 h. No clear
concentration-response relationship was observed for PFHxA,
whereas exposure to H202 (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-h 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, ImM
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)
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PFHxA genotoxicity
Endpoint
Test system
Doses/
Concentrations
tested
Results3
Comments
References
Without
exogenous
activation
With
exogenous
activation
Mutation
(Ames assay)
Salmonella
typhimurium
strains TA98,
TA100,
TA1535, and
TA1537
333, 667, 1,000,
3,333, 5,000
Hg/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)
Mutation
E. coli
WP2uvrA
333, 667, 1,000,
3,333, 5,000
Hg/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
Hg/mL sodium
perfluorohexanoate
(NaPFHx)
4 h (activated) and
22 h (nonactivated):
250, 500,1,000
Hg/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)
a+ = positive; - = 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 short-term fNTP. 20181. subchronic fChengelis et al.. 2009b: Loveless et
al.. 2009). and chronic (Klaunig et al.. 2 015) studies in adult male and female Sprague-Dawley rats
with exposure durations spanning 28 days to 2 years and with oral doses of 2.5-1,000 mg/kg-day
PFHxA, PFHxA sodium salt, or PFHxA ammonium salt Two developmental studies flwai and
Hoberman. 2014: Loveless etal.. 20091 with gestational exposure durations that represent a critical
lifestage with maternal oral doses between 7-500 mg/kg-day also were 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), and hematopoietic effects
(Section 3.2.4) in humans, given relevant exposure circumstances.
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 Both effects could be adaptive changes to PFHxA exposure;
however, these findings were considered adverse on the basis of their consistent effect between
sexes and across studies. The effects also persisted during the recovery period and correlate with
other endpoints (increased ALT and decreased serum globulins) collectively considered adverse.
Available mechanistic evidence suggests increased peroxisomal beta oxidation and the involvement
of both PPARa-dependent and -independent pathways in response to PFHxA exposure.
The data from the animal toxicological studies that supported identifying developmental
effects as a potential human hazard included effects from two studies that reported consistent,
dose-responsive, and substantial effects of PFHxA exposure on offspring body weights and
mortality. Delayed eye opening was also reported, but only at doses associated with frank effects in
the offspring (i.e., mortality). 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.
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 fChengelis etal.. 2009b: Loveless etal.. 2009: NTP. 20181. These hematological findings
correlate with increases in reticulocytes, an indicator of erythroid cell regeneration supported by
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pathological findings in the spleen and bone marrow (Loveless etal.. 20091. The decreases in
hemoglobin were consistent with the decreased mean corpuscular hemoglobin concentration
observed in both sexes fLoveless etal.. 2009: NTP. 20181. 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)
(Chengelis etal.. 2009b: Loveless et al.. 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.
The current evidence suggests, but is not sufficient to infer, that PFHxA exposure might
cause endocrine effects in humans. This judgment is based on evidence in animals showing
decreases in thyroid hormone levels in male (but not female) rats exposed for 28 days and
increased incidence of thyroid epithelial cell hypertrophy in male and female rats in one subchronic
study (Section 3.2.5).
For all other health effects described in Section 3 (i.e., renal, male and female reproductive,
immune, and nervous system) the evidence is inadequate to assess whether PFHxA might cause
effects in human. The summary level findings from the animal toxicological studies that examined
exposure to PFHxA can be viewed by clicking the PFHxA Tableau link, selecting the "Study Findings"
tab, and filtering for the relevant health system.
The relevant exposure conditions that might lead to these health effects are further
characterized in Section 5.
4.2. SUMMARY OF CONCLUSIONS FOR CARCINOGENICITY
Evidence is insufficient to make a judgment on whether PFHxA exposure might affect the
development of any specific cancers. Consistent with EPA guidance (U.S. EPA. 2005) 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.
4.3. 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. The reason for this sex dependence is possibly due to sex-dependent PFHxA elimination
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1 caused by sex-specific differences in the expression (mRNA and protein) of the renal organic anion
2 transporting polypeptide (Oatp) lal fKudo etal.. 20011 as discussed in Section 3.1.4. Currently,
3 whether this sex-specific difference might also exist in humans is unclear.
4 Additionally, given the effects seen in the developing organism (i.e., perinatal mortality,
5 reduced body weights, and delays in time to eye opening), the prenatal and early postnatal window
6 represents a potentially sensitive lifestage for PFHxA exposure.
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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 in five
animal toxicological studies (Chengelis etal.. 2009b: Iwai and Hoberman. 2014: Klaunig etal.. 2015:
Loveless etal.. 2009: NTP. 20181. 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, and hematopoietic effects in humans under relevant
exposure circumstances. These health effects were considered for derivation of toxicity values.
The dose levels associated with these hazards are further characterized below (Section 5.2.1).
For endocrine effects, the currently available evidence suggests, but is not sufficient to
infer that PFHxA may cause effects in humans. Although there was some evidence of effects on
thyroid system function in rats (i.e., thyroid hormone levels and thyroid epithelial cell hypertrophy)
the results lacked consistency and some of the observed changes could be explained by
nonendocrine-related effects. Based on the limitations of the current evidence base, endocrine
effects were not considered for derivation of toxicity values. 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 (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 (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
(Section 5.2.1.1), a less-than-lifetime toxicity value (referred to as a "subchronic RfD"; see Section
5.2.1.2) 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.
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As noted above, reference concentration (RfC) or subchronic RfC could not be developed.
5.2.1. Oral Reference Dose (RfD) Derivation
Study and Endpoint Selection
The following general considerations were used to identify studies for estimating points of
departure (PODs) for potential use in toxicity value derivation. As described in Sections 2 and 3,
the available epidemiological studies of PFHxA exposure are primarily low confidence and
therefore were not further considered for dose-response analyses of PFHxA exposure. Within the
available animal toxicological studies, preference was given to medium or high confidence
subchronic, chronic, or developmental studies testing multiple dose levels, including doses near the
lower end of the doses tested across the evidence base. These types of studies increase the
confidence in the resultant RfD because they represent data with lower risk of bias and minimize
the need for low-dose and exposure duration extrapolation (Appendix A, Section 11.1).
A summary of endpoints and rationales 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
Reference/
Confidence
Exposure
duration
Strain/
Species
Sexes
studied
POD derivation
Rationale
Hepatic Effects
Relative liver
weight
Chengelis et al.
(2009b)
High confidence
Subchroni
c
Crl:CD(S
D) rat
Both
No
Increases in relative
liver weight were
considered an
adaptive change in
response to PFHxA
exposure
Loveless et al.
(2009)
High confidence
Subchroni
c
Crl:CD(S
D) rat
Both
No
Hepatocellula
r hypertrophy
Chengelis et al.
(2009b)
Low confidence
Subchroni
c
Crl:CD(S
D) rat
Female
No
Increases in
hepatocellular
hypertrophy in
combination with
increased liver
weight, increased
serum enzymes, and
decreased blood
proteins were judged
"likely" and
considered adverse
toxic effects to PFHxA
exposure. The
evidence was
strengthened by
consistency of the
Chengelis et al.
(2009b)
Low confidence
Subchroni
c
Crl:CD(S
D) rat
Male
Yes
Loveless et al.
(2009)
High confidence
Subchroni
c
Crl:CD(S
D) rat
Both
Yes
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Endpoint
Reference/
Confidence
Exposure
duration
Strain/
Species
Sexes
studied
POD derivation
Rationale
effect across species
and study designs,
with the effect
persisting into
recovery periods.
Although many
pathways can lead to
hypertrophy there
was evidence for
increased
peroxisomal beta
oxidation activity.
Increased
hepatocellular
hypertrophy was
considered the toxic
effect altering
homeostasis.
Male-specific effects
in Chengelis et al.
(2009b) at 200
mg/kg-day, both
sexes affected in
Loveless et al. (2009).
Hepatocellula
r necrosis
Klaunig et al.
(2015)
High confidence
Chronic
Crl:CD(S
D) rat
Female
No
Necrosis was
considered an
adverse effect
downstream of
hepatocellular
hypertrophy, a
response to toxic
effects on
homeostasis.
Necrosis observed in
females (at the
highest dose of 200
mg/kg-day) was not
replicated in other
studies or sexes, and
the less overt/more
predictive indicator of
hepatic toxicity
(hepatocellular
hypertrophy) was
available.
Blood
proteins (total
Chengelis et al.
(2009b)
High confidence
Subchroni
c
Crl:CD(S
D) rat
Both
No
Increases in blood
protein findings were
considered an
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Endpoint
Reference/
Confidence
Exposure
duration
Strain/
Species
Sexes
studied
POD derivation
Rationale
protein and
globulin)
Loveless et al.
(2009)
High confidence
Subchroni
c
Crl:CD(S
D) rat
Both
No
adaptive change to
PFHxA exposure.
Klaunig et al.
(2015)
High confidence
Chronic
Crl:CD(S
D) rat
Both
No
Hematopoietic Effects
Hematocrit
Chengelis et al.
(2009b)
High confidence
Subchroni
c
Crl:CD(S
D) rat
Both
No
More direct
measurements of red
blood cells and
hemoglobin are
available.
Loveless et al.
(2009)
High confidence
Subchroni
c
Crl:CD(S
D) rat
Both
No
Klaunig et al.
(2015)
High confidence
Chronic
Crl:CD(S
D) rat
Both
No
Hemoglobin
Chengelis et al.
(2009b)
High confidence
Subchroni
c
Crl:CD(S
D) rat
Both
Yes
Decreases considered
similar in sensitivity
to decreases in red
blood cell counts.
Hemoglobin reflects
the oxygen carrying
capacity of those
cells. In Klaunig et al.
(2015), the effects
were specific to
females.
Loveless et al.
(2009)
High confidence
Subchroni
c
Crl:CD(S
D) rat
Both
Yes
Klaunig et al.
(2015)
High confidence
Chronic
Crl:CD(S
D) rat
Female
Yes
Klaunig et al.
(2015)
High confidence
Chronic
Crl:CD(S
D) rat
Male
No
No treatment-related
effects were
observed in males up
to the high dose of
100 mg/kg-day.
Red blood
cells
Chengelis et al.
(2009b)
High confidence
Subchroni
c
Crl:CD(S
D) rat
Both
Yes
Finding was more
sensitive than other
hematological
findings (other than
hematocrit) and
consistent across
study designs and
exposure durations.
Loveless et al.
(2009)
High confidence
Subchroni
c
Crl:CD(S
D) rat
Both
Yes
Klaunig et al.
(2015)
High confidence
Chronic
Crl:CD(S
D) rat
Both
Yes
Reticulocytes
Chengelis et al.
(2009b)
Subchroni
c
Crl:CD(S
D) rat
Both
No
Increases were
considered to reflect
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Endpoint
Reference/
Confidence
Exposure
duration
Strain/
Species
Sexes
studied
POD derivation
Rationale
High confidence
a compensatory
response to
decreased red blood
cells and therefore
not prioritized for
derivation of toxicity
values.
Loveless et al.
(2009)
High confidence
Subchroni
c
Crl:CD(S
D) rat
Both
No
Klaunig et al.
(2015)
High confidence
Chronic
Crl:CD(S
D) rat
Both
No
Developmental Effects
Postnatal (Fi)
pup body
weight
Loveless et al.
(2009)
High confidence
One-
generatio
n repro-
ductive;
measured
on PND 0,
4,7,14,
21
Crl:CD(S
D) rat
Combine
d
Yes, PND
0
Yes, PND
0
Decrements in body
weights were
observed at doses
that were not
associated with frank
effects and showed
strong
dose-response.
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,
7,14, 21
CD-I
mouse,
Fi
Combine
d
Yes, PNDs
0 and 4
Fi fetal body
weight
Loveless et al.
(2009)
High confidence
Develop-
mental
(GD 6-
20);
measured
on GD 21
Crl:CD(S
D) rat
Combine
d
No
Statistically
nonsignificant 9%
decrease at high dose
was associated with
maternal toxicity
(i.e., reduced weight
gain).
Perinatal
mortality
Iwai and
Hoberman
(2014)
High confidence
Develop-
mental
(GD 6-
18);
measured
on PND
0-21,
including
stillbirths
CD-I
mouse,
Fi
Combine
d
Yes
In mice, perinatal
mortality (still birth
and postnatal deaths
from PND 0-21)
showed a clear dose-
response across two
experimental cohorts
of animals with
overlapping dose
ranges. Effects were
observed at doses not
associated with
maternal toxicity.
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Reference/
Exposure
Strain/
Sexes
Endpoint
Confidence
duration
Species
studied
POD derivation
Rationale
Eye opening
Iwai and
Develop-
CD-I
Combine
No
Delays observed at a
Hoberman
mental
mouse,
d
dose that elicited
(2014)
(GD
Fi
frank effects
High confidence
6-18);
(i.e., increased
measured
offspring mortality.).
on PND
10-17
Estimation or Selection of Points of Departure (PODs) for RfD Derivation
The outcomes determined most relevant to the identified noncancer hazards from the
animal studies advanced for dose-response (Table 5-1) were modeled using approaches consistent
with EPA's Benchmark Dose (BMD) Technical Guidance document fU.S. EPA. 2012al 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 on the Health Assessment Workspace Collaborative (HAWC)
website using EPA's Benchmark Dose Software (BMDS, Version 3.2).
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 (U.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. 2012al.
Table 5-2. Benchmark response levels selected for BMD modeling of PFHxA
health outcomes
Endpoint
BMR
Rationale
Hepatic effects
Hepatocellular
hypertrophy
10% ER
For hepatic toxicity, 10% ER is considered a minimally biologically
significant response level for this endpoint (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
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Endpoint
BMR
Rationale
for benchmark dose modeling in prior IRIS assessments (U.S. EPA,
2003, 2004, 2012b).
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 in light of
the severity of the frank effect.
Hematopoietic effects
Red blood cells
1SD
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 standard deviation (SD) in such a
situation.
Hemoglobin
An adequate fit is judged on the basis ofx2 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 on the basis of 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 three-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 on the
basis of 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).
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For the study by Iwai and Hoberman (2014). the experiment was conducted in two phases.
With the exception of 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 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 IRIS PFAS protocol (Supplemental Information document, 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 PBPK
model. If chemical-specific information is not available, the protocol then recommends that doses
be scaled alio metrically using body weight BW3/4 methods. This hierarchy of recommended
approaches for cross-species dosimetry extrapolation is consistent with EPA's guidance on using
allometric scaling for the derivation of oral reference doses (U.S. EPA. 2011). It also prioritizes the
order of relative uncertainty associated with each approach as follows:
• A PBPK model that is well grounded in multiple data sets (including physiological data, in
vitro distribution data, and in vivo PK data) has the least uncertainty.
• A data-informed extrapolation, based on empirical PK data in the species of interest, has
intermediate uncertainty because it is based on direct observation of the internal dose
(i.e., serum concentration) in experimental animals and humans, typically.
• BW3/4 scaling has the greatest uncertainty, relative to the two above approaches, because it
is based on a general assumption about the relative rate of clearance in humans vs. animals
and makes use of no chemical-specific data. Further, as described in Section 3.1, a
comparison of BW3/4 scaling to the available PK data in rats and humans indicates that use
of BW3/4 would overpredict human clearance, and hence underpredict risk, by 1-2 orders of
magnitude. Thus, BW3/4 scaling was not considered appropriate for this assessment.
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. (2015). it was not
considered sufficiently reliable for use in an IRIS Toxicological Review.
On the other hand, when PK data for PFHxA exist in relevant animal species (rats, mice, and
monkeys) or humans, a data-informed extrapolation approach for estimating the dosimetric
adjustment factor (DAF) can be used. Various PK analyses can be performed to extract meaningful
information from PK data. Because PK data for various PFAS are available, including for PFBA
(Changetal.. 2008). PFBS (Olsenetal.. 2009). PFHxA (Dzierlenga etal.. 2019). PFHxS (Sundstrom et
al.. 2012). PFNA (Tatum-Gibbs etal.. 2011). and PFOA and PFOS (Kim etal.. 2016b). that show a
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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 fFuiii etal.. 20151. The EPA
model is characterized by equation 5-1:
C(t) = A-exp(-a-t) + B-exp(-(3-t) - flagorar (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 etal. f20191 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 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 [Dzierlengaetal. (2019): the opposite of what is predicted based
on the hypothesis of saturable resorption], A systematic deviation from this assumption has not
been observed in the other relevant data (Chengelis etal.. 2009a: Gannon etal.. 2011: Iwabuchi et
al.. 20171. 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:
AUCmf = 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-h/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 of volume/time). Mathematically, given the PK model described
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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 the Supplementary Information, 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. Effectively, using
the half-life ratio assumes that another pharmacokinetic parameter, the volume of distribution, is
the same between species (this is contrary to available data, see below).
To avoid assuming the volume of distribution is equal between rats and humans, the HED
can be calculated using clearance:
HED = (CLh/CLA[S]) x POD 5-3
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, provided that one can
obtain a value of CLH (see below).
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:
Vd,p = CL/p = dose/[p x (A/a + B/(3 - flagorai x (A+B)/ka)] 5-4
With the exception of the i.v. dose data from Dzierlenga et al. (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.
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.
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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 et al.. 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.
(2009a). For a standard human BW of 70 kg, the resulting predicted clearance in humans is
0.138 L/h-kg3. If this is the actual clearance in humans, but ti/2 = 768 h, human
Vd,p = CL x ti/2/ln(2) = 153 L/kg. Note human participants were exposed to PFHxA for a
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 reported by Russell etal. f20131 might be an artifact of
significant ongoing exposure to PFHxA during the period of observation. Perez etal. (2013)
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. (2010) did not also have some level of ongoing exposure; the 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) (0.138 L/h-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 Vd /CL = ln(2) x 0.73
3If a BW of 80 kg is used for humans (U.S. EPA. 2019d). the result is 0.137 L/h-kg. The calculation was
performed using 70 kg for comparability with previous assessments.
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1 (L/kg)/(0.138 L/h-kg) = 5.3 h. If this were the case, human serum levels would fall to 96%
2 in a single day, while the data of Nilssonetal. (20101 show that for such a decline to occur
3 takes at least two months. If this were the case, even after a day or two off work, a
4 technician's serum concentration would be near zero. Further, the serum concentrations
5 reported (Nilsson et al.. 20101 do decline to near or below the limit of detection by late
6 spring or early summer of each year, indicating that other ongoing sources of exposure
7 were not significant for that population. Thus, this third option seems extremely unlikely
8 and will not be evaluated further.
9 The two options for human CL estimated in points (1) and (2) above are provided in
10 Table 5-3.
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Table 5-3. Summary of serum half-lives and estimated clearance for PFHxA
Species/Sex
Study
design
Elimination
half-life (ti/2) (h)
Clearance (CL)
(L/h-kg)
Volume of
distribution
(l/d) (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)a
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
Ecological
337
1.5 x 10"3
0.152d
0.73°
74d
Russell et al. (2013)
aFor each experiment (study/route/dose), a separate distribution of CL = dose/AUCmf 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 3 male or female monkeys.
CCL = Vd x ln(2)/ti/2 with Va assumed as the average of the estimated values for male and female monkeys.
dHuman CL estimated by allometric scaling from values estimated for mice, rats, and monkeys; human
l/d = CL x ti/2/ln(2).
1 Thus, two alternative values of the DAF, CLH:CLA[s]—which is the ratio of clearance values—
2 can be obtained (Table 5-4).
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Table 5-4. Two options for rat, mouse, and human clearance values and data-
informed dosimetric adjustment factor (DAF)
Sex
Species
Animal clearance
(L7h-kg)a
Human clearance (L/h-kg)
DAF (CLH:CU[si)
Male
Rat
0.163
1.50 (0.90-2.48) x 10"3(b)
(mean, 90% CI, using
preferred [data-driven]
approach)
9.2 x 10"3
Mouse
0.0894
1.7 x 10"2
Female
Rat
0.383
3.9 x 10"3
Mouse
0.206
7.3 x 10"3
Male
Rat
0.163
0.152°
(alternative approach)
0.93
Mouse
0.0894
1.7
Female
Rat
0.383
0.40
Mouse
0.206
0.74
Shaded values were applied to derive the PODhed-
aSpecies/sex-specific CL values (Supplementary Information, Appendix C).
Calculated from human ti/2 value, obtained by Bayesian PK analysis and average volume of distribution for male
and female monkeys (Table 5-3).
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 PFDA
by Zhang etal. f2013bl and can be compared to those for experimental animals. Briefly, Zhang et
al. (2013b) 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 here. Median urinary CL values
reported by Zhang et al. (2013b) were 0.015, 0.094, and 0.035 mL/kg-day for PFHxS, PFNA, and
PFDA, respectively.
Kim etal. f2016bl reported renal PFHxS clearance of 0.76 mL/kg-day while Kim et al.
f2016bl and Sundstrom etal. f20121 reported total clearance of 7-9 mL/kg-day. Sundstrom et al.
(2012) 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.
The reported dose/AUC can be used to derive clearance values for PFNA from the results of
Tatum-Gibbs etal. f2011I 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 (Benskinet
al.. 2009: De Silva etal.. 2009: Ohmori etal.. 2003: Tatum-Gibbs etal.. 20111. 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.
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Therefore, the top set ofDAFs in Table 5-4—based on CLhuman = 6.6 x 10~4 L/kg-h—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 human equivalent dose (HED) for considered health
effects follow, using the POD of 20 mg/kg-day for postnatal (Fi) body weight at PND 0 (Loveless et
al.. 20091 as an example and the female rat DAF of 3.9 x 10"3, based on clearance:
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 In
particular, use of half-life makes an intrinsic assumption that Vd is the same in the test species as in
humans. From Table 5-3 one can see a significant difference between rats and monkeys, which
leads one also to expect a difference between rats and humans.
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 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. 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 is small, using
these sex-specific Vd values for mice will give similar results to using an average.
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 variation for females between 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, with the exception of the LOD correction of this dose in mice, is
HED = 20 (mll/kg_iay) X 3.9 X 10-= = 0.078 (m«/kg_iay)
5-5
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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
clearance value to calculate the HED. Three of the four studies being evaluated, however, did not
measure PFHxA serum concentrations fChengelis etal.. 2009b: Iwai and Hoberman. 2014: Klaunig
etal.. 2015: Loveless etal.. 20091. Although Iwai and Hoberman f 20141 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.
PODhfti for RfD derivation
Table 5-5 presents the estimated PODhed (mg/kg-day) values or the hepatic, developmental,
and hematopoietic toxicity endpoints considered for RfD derivation based on the endpoint selection
justification in Table 5-1 and preferred DAF values 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 = 314/336 = 0.935
(Iwai and Hoberman. 2014)] and sodium salt to free acid [MW free acid/MW sodium
salt = 314/331 = 0.949 (Loveless 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) = I —
\M I
PFHxA (free acid) = (¦
MW free acid
i \ /314\
—- = — = 0.935
salt) \336/
.) = (HI] = 0.949
7 \331/
.MW ammonium salt.
MW free acid
5-6
.MW sodium salt.
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Table 5-5. PODs considered for the derivation of the RfD
Endpoint
Study/Confidence
Species, strain
(sex)
PODtype/model
POD
(mg/kg-d)
PODhed3
(mg/kg-d)
Hepatic effects
^Hepatocellular
hypertrophy
Chengelis et al.
(2009b)
Low confidence
Rat, Crl:CD(SD)
(male)
NOAELb
(0% response)
50
0.46
Loveless et al. (2009)
High confidence
Rat, Crl:CD(SD)
(male)
BMDLioer
Multistage 1 NCV
10.66
0.093°
Rat, Crl:CD(SD)
(female)
BMDLioer
Multistage 3 NCV
96.32
0.36°
Hematopoietic effects
4/Hemoglobin
Klaunig et al. (2015)
High confidence
Rat, Crl:CD(SD)
(female)
BMDLisd
Linear CV
122.77
0.48
Chengelis et al.
(2009b)
Rat, Crl:CD(SD)
(male)
BMDLisd
Polynomial 3 CV
81.35
0.75
High confidence
Rat, Crl:CD(SD)
(female)
NOAELd
(3% decrease)
50
0.19
Loveless et al. (2009)
High confidence
Rat, Crl:CD(SD)
(male)
NOAELd
(6% decrease)
50
0.44°
Rat, Crl:CD(SD)
(female)
BMDLisd
Polynomial 3 CV
127.61
0.47°
4/Red blood cell
Klaunig et al. (2015)
High confidence
Rat, Crl:CD(SD)
(male)
NOAELb
(4% decrease)
100
0.93
Rat, Crl:CD(SD)
(female)
BMDLisd
Linear CV
109.15
0.43
Chengelis et al.
(2009b)
Rat, Crl:CD(SD)
(male)
NOAELd
(no change)
50
0.46
High confidence
Rat, Crl:CD(SD)
(female)
BMDLisd
Exponential 5
CV
16.32
0.06
Loveless et al. (2009)
High confidence
Rat, Crl:CD(SD)
(male)
BMDLisd
Linear NCV
44.57
0.39°
Rat, Crl:CD(SD)
(female)
BMDLisd
Linear CV
112.36
0.42°
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
0.039°
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
0.55e
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Endpoint
Study/Confidence
Species, strain
(sex)
PODtype/model
POD
(mg/kg-d)
PODhed3
(mg/kg-d)
4/Postnatal (Fi)
body weight, PND 4
BMDL5RD
Exponential-M5
Phase 1 and 2
Polynomial 3 CV
Phase 2
103.12
89.79
0.70e
0.61e
^Perinatal (Fi)
mortality (PND 0-
21, including
stillbirths)
Iwai and Hoberman
(2014)
High confidence
Mouse, CD-I, Fi
(combined)8
BMDLier
Nested Logistic
Phase 1
Model Averageh
Phase 2
98.61
102.65
0.67ef
0.70ef
CV = constant variance; NCV = nonconstant variance; SD = standard deviation.
aHED calculations based on the DAF, the ratio of human and animal clearance values (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.
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 = 314/331 = 0.949).
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 = 314/336 = 0.935).
the combined data set from phases 1 and 2 did not provide adequate fit for modeling, so the phases were
modeled separately and both PODs are presented.
gData sets were modeled using BMDS 2.7
hAn average of BMDLs from NCTR (BMDL of 78.90 mg/kg-day) and Rai Van Ryzin (126.4 mg/kg-day) models with an
identical AIC value is selected as the final BMDL (102.65 mg/kg/day)
Derivation of Candidate Toxicity Values for the RfD
The PODs calculated in Table 5-5 were narrowed, within a health effect, for derivation of
candidate lifetime toxicity values based on the POD, certainty in the POD, and biological
understanding (if any) of the mechanisms of potential PFHxA-mediated toxicity. The selection of
the endpoints for which an RfD was determined (Table 5-6) was based on several factors, including
whether the endpoint is protective of a lifetime exposure, whether an endpoint with less
uncertainty or greater sensitivity exists, and whether the endpoint is protective of both sexes and
all life stages. Based on these considerations, the endpoints in Table 5-5 were narrowed to the
following: for hepatic endpoints to hepatocellular hypertrophy from a subchronic study (Loveless
etal.. 20091. for hematopoietic endpoints to RBCs and HGB from the chronic study fKlaunigetal..
2015). and for developmental endpoints to offspring body weight from (Loveless etal.. 2009).
For the hepatic endpoint, hepatocellular hypertrophy was moved forward for POD
determination. This was based on consistent evidence across studies and sexes for increased
hepatocellular hypertrophy accompanied by increased relative liver weight, increased serum
enzymes, and decreased proteins that when interpreted together indicate hepatic toxicity and
altered homeostasis. This alteration in homeostasis is anticipated to lead to adverse toxic
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responses including necrosis. The lowest effect level for hepatocellular hypertrophy was observed
in the subchronic studies in the 100 mg/kg-day male dose group fLoveless etal.. 20091. Males were
more sensitive for this endpointthan females (the lowest effect level was 100 mg/kg-day in males
vs. 500 mg/kg-day in females) although the effect persisted in both sexes 90 days after recovery
(500 mg/kg-day). In the chronic study, the 200 mg/kg-day female dose group was sensitive for
necrosis (note the highest administered dose in males was 100 mg/kg-day). Considering that
hepatocellular hypertrophy likely precedes necrosis and the dose causing necrosis in the chronic
study (Klaunig et al.. 2015) was two times higher than the 100 mg/kg-day PFHxA dose causing
hypertrophy in the subchronic study fLoveless et al.. 20091. hypertrophy from male rats in the
subchronic study fLoveless et al.. 20091 was selected as the appropriate endpoint and advanced for
RfD determination.
For developmental effects, decreased postnatal (Fl) body weight was prioritized over
offspring mortality (Table 5-6). This was based on the severity of the outcome and the lower
PODhed for fetal body weight, versus mortality, and is expected to be protective of all developmental
effects. Of the two body weight data sets, the data from fLoveless et al.. 20091 were advanced
because the study design included a longer exposure that spanned fetal development through
continuous maternal exposure, through gestation, and until the end of lactation) versus Iwai and
Hoberman f20141 where offspring were exposed only through the study GD 6-18.
For hematopoietic effects, endpoints were available from both subchronic studies and the
chronic study. Because these endpoints were available from the chronic study, their suitability for
RfD determination was based on evaluating evidence for the magnitude of change, the deviation
around the mean within a large cohort (7000 rats) of laboratory animals (Matsuzawa etal.. 19931.
and the sensitivity of the endpoint to respond to PFHxA exposure. The magnitude of change for
RBCs (~8% decreased) or HGB (~5% decrease) was similar when comparisons were made
between chronic and subchronic studies. RBCs and HGB were decreased in both males and females
dosed with 200 mg/kg-day in the subchronic study (Chengelis etal.. 2009b) and in females dosed
with 200 mg/kg-day in the chronic study. The biological significance of the magnitude of change
for both RBC and HGB in rats is uncertain, but the effect on red blood cell parameters had a slightly
lower POD than HGB and was concurrent with increased reticulocyte levels, a compensatory
response to anemia. Females were more sensitive in the chronic study when the magnitude of
effect between males and females, at similar dose levels, were compared. Note, however, females
received twice the maximum dose that male rats received, which might explain sex-specific
differences in the chronic study. Therefore, the female RBC hematological endpoint from the
chronic study was prioritized for RfD determination (Klaunig etal.. 2015).
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Table 5-6. Uncertainty factors for the development of the RfD for PFHxA
UF
Value
Justification
ufa
3
A UFa of 3 is applied to account for uncertainty in characterizing the PK and
pharmacodynamic differences between species following oral NaPFHx/NH4PFHxA/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. Therefore, a UFA of 3 was selected for PFHxA; see text above for
further discussion.
UFh
10
A UFh of 10 is applied for interindividual variability in humans in the absence of
quantitative information on potential differences in PK and pharmacodynamics relating to
NaPFHx/NH4+PFHxA/PFHxA exposure in humans.
UFS
1
(developmental
and
hematopoietic
endpoints)
A UFs of 1 is applied to developmental endpoints from the one-generation reproductive
study by Loveless et al. (2009) and Iwai and Hoberman (2014). The developmental period
is recognized as a susceptible lifestage and studies using exposure designs capturing
sensitive developmental windows (i.e., gestation or lactation) are more relevant for
induction of developmental effects than lifetime exposures (U.S. EPA, 1991). Although
effects on body weights are not unique to development and studies evaluating the body
weight effects of postnatal exposure are lacking, 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 endpoints in the studv (Klaunig et al., 2015) as
the 51 weeks of daily exposure represented more than 10% of a rodent life span and the
incidence or severity of these outcomes is not anticipated to increase with increasing
exposure duration.
3 (hepatic)
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-dav subchronic studv (Loveless et al.,
2009), the evidence supports a pathwav where hepatocellular hvpertrophv is the toxic
effect altering homeostasis. The evidence suggests that hepatocellular hypertrophy is an
adverse hepatic response to PFHxA exposure that worsens with longer exposure toxic
effects such as necrosis.
ufl
1
A UFL of 1 is applied for LOAEL-to-NOAEL extrapolation when the POD is a BMDL or a
NOAEL.
UFd
3
A UFd of 3 is applied because the evidence base for hepatic, hematopoietic, and
developmental endpoints included two subchronic studies and one chronic study in
Sprague-Dawley rats and developmental/reproductive studies in Sprague-Dawley rats and
Crl:CDl mice. Limitations, 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, or a
multigenerational study. For developmental outcomes, pups were indirectly exposed via
the dam (i.e., via placental or lactational transfer); thus, the dose received by the pups is
unclear and might be significantly less than that administered to the dams.
UFC
See Table 5-7
and Table 5-11
Composite uncertainty factor = UFA x UFH x UFS x UFL x UFD.
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Under EPA's A Review of the Reference Dose and Reference Concentration Processes (U.S. EPA.
2002c), five possible areas of uncertainty and variability were considered in deriving the candidate
values for PFHxA. An explanation of these five possible areas of uncertainty and variability and the
values assigned to each as a designated uncertainty factor (UF) to be applied to the candidate
PODhed values are listed in Table 5-6.
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, which inherently accounts for uncertainty regarding the PK and
pharmacodynamic differences between species. The PK uncertainty is accounted for through the
application of dosimetric approaches for estimation of human equivalent doses as described above.
However, this leaves some residual uncertainty understanding dose to target sites of toxicity (PK)
and how adverse effects occur when molecules reach the target sites (pharmacodynamics). For
developmental and hematopoietic outcomes, the evidence base lacked chemical- and species-
specific information that would have been useful for informing the UFa; therefore, a UFA of 3 was
applied. For hepatic effects, mechanistic and supplemental information useful for further
evaluating the interspecies uncertainty factor was available. This evidence was PPARa pathway
rich, likely due to the known species specificity for PPARa -linked oncogenic pathways fKlaunig et
al.. 20031. PFHxA, however, was noncarcinogenic in Sprague-Dawley rats fKlaunig etal.. 20151.
As described in Section 3.2.1, PFHxA activates several receptors, and multiple pathways
lead to hepatocellular hypertrophy and increased liver weight The pharmacodynamic relationship
between these PFHxA receptor-mediated interactions is not clear from the available evidence, but
there are pathways with which these receptors are involved. Although some prototypical PPARa
activators exhibit an exaggerated activation (and downstream response) in rodent as compared to
human receptors, some evidence from in vitro studies suggests that PFHxA might induce human
PPARa at similar (or lower) concentrations to mouse PPARa. Interpretation of these results is
limited, however, as the data are derived from two experiments from same group (Wolfetal.. 2014:
Wolfetal.. 2008). Given the suggestion of similar sensitivities in PPARa activation by PFHxA across
species and possible PPARa-independent contributions to the observed hepatic effects, the
possibility that humans might exhibit pharmacodynamic sensitivity for hepatic effects different
from rats cannot be ruled out. Thus, based on the residual uncertainty surrounding the
interspecies differences in pharmacodynamics described above, a factor of 3 is applied to account
for the pharmacodynamic uncertainty of the UFa for all potential health effect consequences of
PFHxA exposure.
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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Table 5-7. Candidate values for PFHxA
Endpoint
Study/
Confidence
Species,
strain (sex)
POD
(mg/kg-d)
PODhed3
(mg/kg-d)
UFa
UFh
UFS
ufl
UFd
UFC
Candidate
value
(mg/kg-d)
^Hepatocellular
hypertrophy,
90 day
Loveless et
al. (2009)
High
confidence
Rat,
Crl:CD(SD)
(male)
10.66
0.093b
3
10
3
1
3
300
3 x 10"4
4/Red blood cells,
51 weeks
Klaunig et al.
(2015)
High
confidence
Rat,
Crl:CD(SD)
(female)
109.15
0.43b
3
10
1
1
3
100
4 x 10"3
4/Fi body weight,
PNDO
Loveless et
al. (2009)
High
confidence
Rat,
Sprague-
Dawley, Fi
(combined)
10.62
0.039b
3
10
1
1
3
100
4 x 10"4
aHED calculations based on DAF, the ratio of human and animal clearance values (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.
bPODHED multiplied by normalization factor to convert from sodium salt to free acid (MW free acid/MW sodium
salt = 314/331 = 0.949).
1 Selection of Lifetime Toxicity Value(s)
2 Selection of Organ- or System-Specific RfDs
3 Organ/system-specific (os)RfDs associated with each health effect are presented in
4 Table 5-8 as they could be useful for certain decision purposes (i.e., site-specific risk assessments).
5 The rationale for and application of osRfD are described in the Protocol, Appendix A. Confidence in
6 each osRfD is described in Table 5-8 and is based on several factors, including confidence in the
7 study, the evidence base supporting the hazard, and quantitative estimate for each osRfD.
Table 5-8. Confidence in the organ/system-specific RfDs for PFHxA
Confidence
categories
Designation
Discussion
Hepatic osRfD = 3 x 10 4 mg/kg-d
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
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Confidence
categories
Designation
Discussion
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 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 Va 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 = 4 x 10 3 mg/kg-d
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
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 correlative with an erythrogenic response
indicated by increased reticulocytes and pathological findings of splenic
extra medullary 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 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 Va and CL were not measured in humans or mice
and were considered equivalent to those in monkeys and rats,
respectively.
Confidence in
hematopoietic osRfD
High
The overall confidence in the osRfD is high and is primarily driven by high
confidence in the overall evidence base for hematopoietic effects, high
confidence in the study, and medium confidence in quantitation of the
POD.
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Confidence
categories
Designation
Discussion
Developmental osRfD = 4 x 10"4 mg/kg-d
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.
Additionally, the actual dose received by the offspring is unclear because
the pups were indirectly exposed via the dams. 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 Va 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.
1 Selection of RfD and Confidence Statement
2 Organ/system-specific RfD values for PFHxA selected in the previous section are
3 summarized in Table 5 9.
Table 5-9. Organ/system-specific RfD (osRfD) values for PFHxA
System
Basis
PODhed
UFC
osRfD
(mg/kg-d)
Confidence
Hepatic
Increased
hepatocellular
hypertrophy in
adult male Crl:CD
Sprague-Dawley
rats
0.093 mg/kg-d
based on
BMDLioer and
free salt
normalization
(Loveless et al.,
2009)
300
3 x 10"4
Medium
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System
Basis
PODhed
UFC
osRfD
(mg/kg-d)
Confidence
Hematopoietic
Decreased red
blood cells in
adult female
Crl:CD
Sprague-Dawley
rats
0.43 mg/kg-d
based on
BMDLisn (Klaunig
etal., 2015)
100
4 x 10"3
High
Developmental
Decreased
postnatal (PND 0)
body weight in Fi
Sprague-Dawley
male and female
rats, exposed
throughout
lactation and
gestation
0.039 mg/kg-d
based on
BMDLsrd and free
salt
normalization
(Loveless et al.,
2009)
100
4 x 10"4
Medium
From the identified human health effects of PFHxA and derived osRfDs for hepatic,
hematopoietic, and developmental effects (Table 5-9), an RfD of 4 x 10~4 mg/kg-day 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 osRfD. The decision to
select the developmental osRfD 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 osRfDs but lower than the hematopoietic osRfD. The developmental
endpoint decreased F1 body weight at PND 0 having the lowest overall PODHED of 0.039 mg/kg-d
based on BMDLsrd and free salt normalization (Loveless etal., 2009) and UFc of 100. The
developmental osRfD was considered protective across all lifestages, including developmental. The
hepatic osRfD was slightly lower but was based on a higher PODHED (0.093 mg/kg-day) and UFC
(300). The developmental osRfD, therefore, is based on the lowest PODHED and lowest UFC using a
study considered high confidence. The developmental osRfD is expected to be protective across all
life stages, including developmental.
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 in Table 5-1. Data to inform potential hepatic
and hematopoietic effects from the high confidence subchronic studies by fChengelis 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..
2009) were also advanced for candidate value derivation. The high confidence short-term study
(NTP. 2018) was not advanced based on the same rationale as described above for the lifetime RfD.
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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. f2009bl and Loveless etal. f20091 were prioritized over the
data from the chronic study by Klaunig etal. 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)
POD type/model
POD (mg/kg-d)
PODhed3
(mg/kg-d)
Hepatic effects
^Hepatocellular
hypertrophy
Chengelis et al. (2009b)
Low confidence
Rat,
Crl:CD(SD)
(male)
NOAELb
(0% response)
50
0.46
Loveless et al. (2009)
High confidence
Rat,
Crl:CD(SD)
(male)
BMDLioer
Multistage 1 NCV
10.66
0.093°
Rat,
Crl:CD(SD)
(female)
BMDLioer
Multistage 3 NCV
96.32
0.36°
Hematopoietic effects
4/Hemoglobin
Chengelis et al. (2009b)
High confidence
Rat,
Crl:CD(SD)
(male)
BMDLisd
Polynomial 3 CV
81.35
0.75
Rat,
Crl:CD(SD)
(female)
NOAELd
(3% decrease)
50
0.19
Loveless et al. (2009)
High confidence
Rat,
Crl:CD(SD)
(male)
NOAELd
(6% decrease)
50
0.44°
Rat,
Crl:CD(SD)
(female)
BMDLisd
Polynomial 3 CV
127.61
0.47°
4/Red blood cell
Chengelis et al. (2009b)
High confidence
Rat,
Crl:CD(SD)
(male)
NOAELd
(no change)
50
0.46
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Endpoint
Study/Confidence
Species,
strain (sex)
POD type/model
POD (mg/kg-d)
PODhed3
(mg/kg-d)
Rat,
Crl:CD(SD)
(female)
BMDLisd
Exponential 5 CV
16.32
0.064
Loveless et al. (2009)
High confidence
Rat,
Crl:CD(SD)
(male)
BMDLisd
Linear NCV
44.57
0.39°
Rat,
Crl:CD(SD)
(female)
BMDLisd
Linear CV
112.36
0.42°
Developmental Effects
4/Postnatal (Fi)
body weight,
PNDO
Loveless et al. (2009)
High confidence
Rat,
Crl:CD(SD), Fi
(combined)
BMDLsrd
Hill
10.62
0.039°
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
0.55e
4/Postnatal (Fi)
body weight,
PND 4
BMDLsrd
Exponential-M5
Phase 1 and 2
Polynomial 3 CV
Phase 2
103.12
89.79
0.70e
0.61e
^Perinatal
Mortality
Iwai and Hoberman
(2014)
High confidence
Mouse, CD-I,
Fi
(combined)8
BMDLier
Nested Logistic
Phase 1
Model Averageh
Phase 2
98.61
102.65
0.67ef
0.70ef
1SD = 1 standard deviation, CV = constant variance, NCV = nonconstant variance.
aHED calculations based on the DAF, the ratio of human and animal clearance values (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.
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 = 314/331 = 0.949).
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 sodium
salt = 314/331 = 0.934).
the combined data set from phases 1 and 2 did not provide adequate fit for modeling, so the phases were
modeled separately and both PODs are presented.
gData sets were modeled using BMDS 2.7
hAn average of BMDLs from NCTR (BMDL of 78.90 mg/kg-day) and Rai Van Ryzin (126.4 mg/kg-day) models with an
identical AIC value is selected as the final BMDL (102.65 mg/kg/day)
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Derivation of Candidate Toxicity Values for the Subchronic RfD
The PODhed values listed in Table 5-10 were further narrowed for subchronic osRfD
derivation and subchronic RfD selection. RBCs were a more sensitive PODhed for hematopoietic
effects. Therefore, the red blood cell endpoint from female rats from Chengelis etal. f2009blwas
advanced for subchronic RfD derivation over male endpoints for hematocrit and red blood cells
based on female 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 for derivation of the hepatic and developmental subchronic osRfDs: male
hepatocellular hypertrophy and decreased Fi body weight at PND 0 fLoveless etal.. 20091.
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 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 subchronic RfD. The resulting candidate values are
shown in Table 5-11.
Table 5-11. Candidate values for deriving the subchronic RfD for PFHxA
Endpoint
Study/Confidence
Species, strain
(sex)
POD
(mg/kg-d)
PODhed3
(mg/kg-d)
UFa
UFh
UFS
ufl
UFd
UFC
Candidate
value
(mg/kg-d)
^Hepatocellular
hypertrophy, 90 day
Loveless et al.
(2009)
High confidence
Rat, Crl:CD(SD)
(male)
10.66
0.093b
3
10
1
1
3
100
9 x 10"4
4/Red blood cell, 90
day
Chengelis et al.
(2009b)
High confidence
Rat, Crl:CD(SD)
(female)
16.32
0.064
3
10
1
1
3
100
6 x 10"4
4/Postnatal (Fi) body
weight, PND 0
Loveless et al.
(2009)
High confidence
Rat,
Sprague-Dawley,
Fi
(combined)
10.62
0.039b
3
10
1
1
3
100
4 x 10"4
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 = 314/331 = 0.949).
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1 Selection of Subchronic Organ- or System-Specific RfDs
2 As described above, subchronic osRfDs associated with each health effect are presented as
3 they may be useful for certain decision purposes (i.e., site-specific risk assessments with less-than-
4 lifetime exposures). Confidence in each osRfD are described in Table 5-12 and consider confidence
5 in the study used to derive the quantitative estimate, the overall health effect, specific evidence
6 base, and quantitative estimate for each osRfD.
Table 5-12. Confidence in the subchronic organ/system-specific RfDs for
PFHxA
Confidence categories
Designation3
Discussion
Hepatic subchronic osRfD = 9 x 10 4 mg/kg-d
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 Va 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 = 6 x 10"4 mg/kg-d
Confidence in study
used to derive the
subchronic osRfD
High
Confidence in the studv (Chengelis et al., 2009b) is high based on the
studv evaluation results (i.e., rated high confidence overall) (HAWC
link) and characteristics that make it suitable for deriving toxicity
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Confidence categories
Designation3
Discussion
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
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 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.
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 Va and CL were not
measured in humans or mice and were considered equivalent to
those in monkeys and rats, respectively.
Confidence in
hematopoietic
subchronic osRfD
High
The overall confidence in the subchronic osRfD is high and is primarily
driven by high confidence in the overall evidence base for
hematopoietic effects, high confidence in the study, and medium
confidence in quantitation of the POD.
Developmental subchronic osRfD = 4 x 10 4 mg/kg-d
Confidence in study
used to derive the
subchronic osRfD
High
Confidence in the study (Loveless et al., 2009) is high 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. Additionally, the actual dose received by the
offspring is unclear since the pups were indirectly exposed via the
dams. 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
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Confidence categories
Designation3
Discussion
of the dosimetric approach described above is that Va and CL were not
measured in humans or mice and were considered equivalent to
those in monkeys and rats, respectively.
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.
1 Selection of Subchronic RfD and Confidence Statement
2 Organ/system-specific subchronic RfD values for PFHxA selected in the previous section are
3 summarized in Table 5-13.
Table 5-13. Subchronic osRfD values for PFHxA
System
Basis
PODhed
UFC
osRfD
(mg/kg-d)
Confidence
Hepatic
Increased
hepatocellular
hypertrophy in adult
male Crl:CD
Sprague-Dawley rats
0.093 mg/kg-d based on BMDLioer
and free salt normalization
(Loveless et al., 2009)
100
9 x 10"4
Medium
Hematopoietic
Decreased red blood
cells in adult female
Crl:CD
Sprague-Dawley rats
0.064 mg/kg-d based on BMDLisd
(Chengelis et al., 2009b)
100
6 x 10"4
High
Developmental
Decreased postnatal
(PND 0) body weight
in Fi Sprague-Dawley
male and female rats,
exposed throughout
lactation and
gestation
0.039 mg/kg-d based on BMDL5RD
and free salt normalization
(Loveless et al., 2009)
100
4 x 10"4
Medium
4 From the identified targets of PFHxA toxicity and derived subchronic osRfDs (Table 5-13),
5 an RfD of 4 x 10~4 mg/kg-day based on decreased postnatal body weight is selected for less-
6 than-lifetime exposure. Confidence in the RfD is medium, based on medium confidence in the
7 developmental osRfD, as described in Table 5-12. The confidence in the selected RfD is equivalent
8 to that of the hepatic osRfDs but lower than the hematopoietic osRfD. The developmental osRfD is
9 expected to be protective of all life stages, including developmental. The UFc (Table 5-13) is
10 equivalent to the other osRfDs and the endpoint has the lowest PODhed (0.039 mg/kg-day, Table 5-
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1 11). The decision to select the developmental osRfD was based on all of the available osRfDs in
2 addition to overall confidence and composite uncertainty for those osRfDs.
5.2.2. Inhalation Reference Concentration (RfC)
3 No published studies investigating the inhalation effects of subchronic, chronic, or
4 gestational exposure to PFHxA in humans or animals have been identified. Therefore, an RfC is not
5 derived.
5.3. CANCER TOXICITY VALUES
6 As discussed in Sections 3.3 and 4.2, given the sparse evidence base and in accordance with
7 the Guidelines for Carcinogen Risk Assessment fU.S. EPA. 20051. EPA concluded that there is
8 inadequate information to assess carcinogenic potential for PFHxA for any route of exposure.
9 Therefore, consistent with the Guidelines and the lack of adequate data on the potential
10 carcinogenicity of PFHxA, quantitative estimates for either oral (oral slope factor, OSF) or
11 inhalation (inhalation unit risk; IUR) exposure were not derived.
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AACC (American Association for Clinical Chemistry). (1992). Clinical pathology testing
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