A EPA
EPA/635/R-21 /312a
Extendi Review Draft
www.epa.gov/iris
Toxicological Review of Perfluorohexanoic Acid [CASRN 307244]
and Related Salts
February 2022
Integrated Risk Information System
Center for Public Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC

-------
Toxicological Review ofPFHxA and Related Salts
DISCLAIMER
This document is an external review draft for review purposes only. This information is
distributed solely for the purpose of public comment 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.
ii	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
CONTENTS
AUTHORS| CONTRIBUTORS| REVIEWERS	xi
EXECUTIVE SUMMARY	xiii
1.	OVERVIEW OF BACKGROUND INFORMATION AND ASSESSMENT METHODS	1-2
1.1.	BACKGROUND INFORMATION ON PFHXA AND RELATED AMMONIUM AND SODIUM
SALTS	1-2
1.1.1.	Physical and Chemical Properties	1-2
1.1.2.	Sources, Production, and Use	1-5
1.1.3.	Environmental Fate and Transport	1-5
1.1.4.	Potential for Human Exposure and Populations with Potentially Greater Exposure	1-6
1.2.	SUMMARY OF ASSESSMENT METHODS	1-9
1.2.1.	Literature Search and Screening	1-9
1.2.2.	Evaluation of Individual Studies	1-11
1.2.3.	Data Extraction	1-12
1.2.4.	Evidence Synthesis and Integration	1-12
1.2.5.	Dose-Response Analysis	1-13
2.	SUMMARY OF LITERATURE IDENTIFICATION AND STUDY EVALUATION RESULTS	2-1
2.1.	LITERATURE SEARCH AND SCREENING RESULTS	2-1
2.2.	STUDY EVALUATION RESULTS	2-3
3.	PHARMACOKINETICS, EVIDENCE SYNTHESIS, AND EVIDENCE INTEGRATION	3-1
3.1.	PHARMACOKINETICS	3-1
3.1.1.	Absorption	3-2
3.1.2.	Distribution	3-2
3.1.3.	Metabolism	3-7
3.1.4.	Elimination	3-8
3.1.5.	PBPK Models	3-14
3.1.6.	Summary	3-14
3.2.	NONCANCER EVIDENCE SYNTHESIS AND INTEGRATION	3-18
3.2.1.	Hepatic Effects	3-18
3.2.2.	Developmental Effects	3-42
3.2.3.	Renal Effects	3-53
This document is a draft for review purposes only and does not constitute Agency policy.
iii	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
3.2.4.	Hematopoietic Effects	3-64
3.2.5.	Endocrine Effects	3-76
3.2.6.	Male Reproductive Effects	3-84
3.2.7.	Female Reproductive Effects	3-92
3.2.8.	Immune Effects	3-100
3.2.9.	Nervous System Effects	3-107
3.3. CARCINOGENICITY	3-111
3.3.1. Cancer	3-111
4.	SUMMARY OF HAZARD IDENTIFICATION CONCLUSIONS	4-1
4.1.	SUMMARY OF CONCLUSIONS FOR NONCANCER HEALTH EFFECTS	4-1
4.2.	CONCLUSIONS REGARDING SUSCEPTIBLE POPULATIONS AND LIFESTAGES	4-2
4.3.	SUMMARY OF CONCLUSIONS FOR CARCINOGENICITY	4-1
5.	DERIVATION OF TOXICITY VALUES	5-2
5.1.	HEALTH EFFECT CATEGORIES CONSIDERED (CANCER AND NONCANCER)	5-2
5.2.	NONCANCER TOXICITY VALUES	5-2
5.2.1.	Oral Reference Dose (RfD) Derivation	5-3
5.2.2.	Inhalation Reference Concentration (RfC)	5-33
5.3.	CANCER TOXICITY VALUES	5-33
REFERENCES	R-l
This document is a draft for review purposes only and does not constitute Agency policy.
iv	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
TABLES
Table ES-1. Health effects with evidence available to synthesize and draw summary judgments
and derived toxicity values	xv
Table 1-1. Physicochemical properties of PFHxA	1-4
Table 1-2. PFHxA levels at 10 military installations and National Priority List sites	1-7
Table 3-1. Summary of PK evidence for PFHxA	3-16
Table 3-2. Evaluation results for animal studies assessing effects of PFHxA exposure on the
hepatic system	3-20
Table 3-3. Percent increase in relative liver weight due to PFHxA exposure in short-term and
subchronic oral toxicity studies	3-21
Table 3-4. Incidence of hepatocellular hypertrophy findings in adult rats due to PFHxA exposure
in short-term and subchronic oral toxicity studies	3-22
Table 3-5. Percent change in alanine aminotransferase (ALT) due to PFHxA exposure in short-
term, subchronic, and chronic oral toxicity studies	3-25
Table 3-6. Percent change in aspartate aminotransferase (AST) due to PFHxA exposure in short-
term, subchronic, and chronic oral toxicity studies	3-26
Table 3-7. Percent change in alkaline phosphatase (ALP) due to PFHxA exposure in short-term,
subchronic, and chronic oral toxicity studies	3-27
Table 3-8. Percent change in total protein (TP) due to PFHxA exposure in short-term,
subchronic, and chronic oral toxicity studies	3-30
Table 3-9. Percent change in globulins (G) due to PFHxA exposure in short term, subchronic, and
chronic oral toxicity studies	3-31
Table 3-10. Genes Targets Identified from EPA Chemicals Dashboard After PFHxA Treatment in
Human Liver Cell Lines	3-34
Table 3-11. Evidence profile table for hepatic effects	3-39
Table 3-12. Study design characteristics and outcome-specific study confidence for
developmental endpoints	3-42
Table 3-13. Incidence of perinatal mortality following PFHxA ammonium salt exposure in a
developmental oral toxicity study	3-44
Table 3-14. Percent change relative to control in offspring body weight due to PFHxA sodium or
ammonium salt exposure in developmental oral toxicity studies	3-47
Table 3-15. Percent change relative to control in eye opening due to PFHxA ammonium salt
exposure in a developmental oral toxicity study	3-49
Table 3-16. Evidence profile table for developmental effects	3-51
Table 3-17. Renal endpoints for PFHxA and associated confidence scores from repeated-dose
animal toxicity studies	3-55
Table 3-18. Percent increase in relative and absolute kidney weight due to PFHxA exposure in
short-term, subchronic, and chronic oral toxicity studies	3-56
Table 3-19. Evidence profile table for renal effects	3-61
Table 3-20. Hematopoietic endpoints for PFHxA and associated confidence scores from
repeated-dose animal toxicity studies	3-65
Table 3-21. Percent change in red blood cells due to PFHxA exposure in short-term, subchronic,
and chronic oral toxicity studies	3-67
Table 3-22. Percent change in hematocrit due to PFHxA exposure in short-term, subchronic, and
chronic oral toxicity studies	3-69
This document is a draft for review purposes only and does not constitute Agency policy.
v	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 3-23. Percent change in hemoglobin due to PFHxA exposure in short-term, subchronic,
and chronic oral toxicity studies	3-69
Table 3-24. Percent change in reticulocytes due to PFHxA exposure in short-term, subchronic,
and chronic oral toxicity studies	3-71
Table 3-25. Evidence profile table for hematopoietic effects	3-74
Table 3-26. Endocrine endpoints for PFHxA and associated confidence scores from
repeated-dose animal toxicity studies	3-77
Table 3-27. Percent change in thyroid hormone levels following PFHxA exposure in a 28-day oral
toxicity study	3-78
Table 3-28. Incidence of thyroid follicular epithelial cell hypertrophy following PFHxA
ammonium salt exposure in a 90-day oral toxicity study	3-79
Table 3-29. Evidence profile table for endocrine effects	3-82
Table 3-30. Study design, exposure characteristics, and individual outcome ratings	3-86
Table 3-31. Evidence profile table for male reproductive effects	3-90
Table 3-32. Study design characteristics	3-93
Table 3-33. Evidence profile table for female reproductive effects	3-98
Table 3-34. Study design characteristics and individual outcome ratings for immune endpoints	3-101
Table 3-35. Evidence profile table for immune effects	3-105
Table 3-36. Nervous system endpoints for PFHxA and associated confidence scores from
repeated-dose animal toxicity studies	3-107
Table 3-37. Evidence profile table for nervous system effects	3-109
Table 3-38. Summary of PFHxA genotoxicity studies	3-113
Table 5-1. Endpoints considered for dose-response modeling and derivation of points of
departure	5-3
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 factors3 for the development of the RfD for PFHxA	5-21
Table 5-7. Candidate values for PFHxA	5-22
Table 5-8. Confidence in the organ/system-specific RfDs for PFHxA	5-23
Table 5-9. Organ/system-specific RfD (osRfD) values for PFHxA	5-25
Table 5-10. PODs considered forthe derivation of the subchronic RfD	5-27
Table 5-11. Candidate values for deriving the subchronic RfD for PFHxA	5-29
Table 5-12. Confidence in the subchronic organ/system-specific RfDs for PFHxA	5-30
Table 5-13. Subchronic osRfD values for PFHxA	5-32
FIGURES
Figure 1-1. Linear chemical structures of (from left to right) perfluorohexanoic acid (PFHxA),
ammonium perfluorohexanoate (PFHxA-NH4), and sodium perfluorohexanoate
(PFHxA-Na)	1-4
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
This document is a draft for review purposes only and does not constitute Agency policy.
vi	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
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	3-19
Figure 3-2. Liver weights (absolute and relative) after short-term and subchronic PFHxA
exposures (full details available by clicking the HAWC link)	3-21
Figure 3-3. Clinical chemistry findings (serum enzymes) after short term, subchronic, and chronic
PFHxA exposures (full details available by clicking the HAWC link)	3-25
Figure 3-4. Blood protein findings after short term, subchronic, and chronic PFHxA exposures
(full details available by clicking the HAWC link)	3-29
Figure 3-5. Hepatobiliary findings in rats exposed by gavage to PFHxA or PFHxA sodium salt (full
details available by clicking the HAWC link)	3-32
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)	3-33
Figure 3-7. Developmental effects on offspring viability in mice exposed to PFHxA ammonium
salt (HAWC: PFHxA - Animal Toxicity Developmental Effects link)	3-44
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)	3-46
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)	3-48
Figure 3-10. Study evaluation for human epidemiological studies reporting findings from PFHxA
exposures (full details available by clicking HAWC link)	3-54
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	3-57
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	3-59
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)	3-66
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)	3-68
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)	3-70
Figure 3-16. Hemostasis findings in rats exposed by gavage to PFHxA or PFHxA sodium salt (full
details available by clicking the HAWC link)	3-72
Figure 3-17. Study evaluation for human epidemiologic studies reporting toxicity findings from
PFHxA exposures (HAWC: PFHxA - Human Toxicity Endocrine Effects link)	3-76
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)	3-78
This document is a draft for review purposes only and does not constitute Agency policy.
vii	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Figure 3-19. Study evaluation for human epidemiological studies reporting male reproductive
findings from PFHxA exposures (HAWC: PFHxA - Human Toxicity Male
Reproductive Effects link)	3-85
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)	3-87
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)	3-88
Figure 3-22. Study evaluation for human epidemiological studies reporting female reproductive
findings from PFHxA exposures (HAWC: PFHxA - Human Toxicity Female
Reproductive link)	3-92
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)	3-95
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)	3-96
Figure 3-25. Study evaluation for human epidemiological studies reporting findings from PFHxA
exposures (HAWC: PFHxA - Human Toxicity Immune Effects link)	3-100
Figure 3-26. Immune organ weights in rats exposed by gavage to PFHxA or PFHxA sodium salt
(HAWC: PFHxA-Animal Toxicity Immune Effects link)	3-102
Figure 3-27. Immune cell counts in rats exposed by gavage to PFHxA or PFHxA sodium salt
(HAWC: PFHxA-Animal Toxicity Immune Effects link)	3-103
This document is a draft for review purposes only and does not constitute Agency policy.
viii	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
ABBREVIATIONS AND ACRONYMS
ADME
absorption, distribution, metabolism,
IUR
inhalation unit risk

and excretion
i.v.
intravenous
AFFF
aqueous film-forming foam
LDH
lactate dehydrogenase
A:G
albumin:globulin ratio
LOQ
limit of quantitation
AIC
Akaike's information criterion
LOAEL
lowest-observed-adverse-effect level
ALP
alkaline phosphatase
LOD
limit of detection
ALT
alanine aminotransferase
LOEC
lowest observed effect concentration
APTT
activated partial thromboplastin time
MCH
mean cell hemoglobin
AST
aspartate aminotransferase
MCHC
mean cell hemoglobin concentration
atm
atmosphere
MCV
mean cell volume
ATSDR
Agency for Toxic Substances and
MOA
mode of action

Disease Registry
MW
molecular weight
AUC
area under the curve
NCTR
National Center for Toxicological
BMD
benchmark dose

Research
BMDL
benchmark dose lower confidence limit
NOAEL
no-observed-adverse-effect level
BMDS
Benchmark Dose Software
NPL
National Priorities List
BMR
benchmark response
NTP
National Toxicology Program
BUN
blood urea nitrogen
ORD
Office of Research and Development
BW
body weight
OECD
Organisation for Economic
Cmax
maximum concentration

Co-operation and Development
CAR
constitutive androstane receptor
OSF
oral slope factor
CASRN
Chemical Abstracts Service registry
osRfD
organ/system-specific oral reference

number

dose
CBC
complete blood count
PBPK
physiologically based pharmacokinetic
CI
confidence interval
PC
partition coefficient
CL
clearance
PECO
populations, exposures, comparators,
CLa
clearance in animals

and outcomes
CLh
clearance in humans
PFAA
perfluoroalkyl acids
CPHEA
Center for Public Health and
PFAS
per- and polyfluoroalkyl substances

Environmental Assessment
PFBA
perfluorobutanoic acid
CPN
chronic progressive nephropathy
PFBS
perfluorobutane sulfonate
DAF
dosimetric adjustment factor
PFCA
perfluorinated carboxylic acid
DNA
deoxyribonucleic acid
PFDA
perfluorodecanoic acid
DTXSID
DSSTox substance identifier
PFHxA
perfluorohexanoic acid
eGFR
estimated glomerular filtration rate
PFHxS
perfluorohexane sulfonate
EPA
Environmental Protection Agency
PFNA
perfluorononanoic acid
ER
extra risk
PFOA
perfluorooctanoic acid
FTOH
fluorotelomer alcohol
PFOS
perfluorooctane sulfonate
GD
gestation day
PK
pharmacokinetic
GGT
y-glutamyl transferase
PND
postnatal day
HAWC
Health Assessment Workplace
POD
point of departure

Collaborative
PODhed
human equivalent dose POD
HCT
hematocrit
PPAR
peroxisome proliferated activated
HED
human equivalent dose

receptor
HERO
Health and Environmental Research
PQAPP
programmatic quality assurance

Online

project plan
HGB
hemoglobin
PT
prothrombin time
HSA
human serum albumin
QA
quality assurance
IQR
interquartile range
QAPP
quality assurance project plan
IRIS
Integrated Risk Information System
QMP
quality management plan
ISI
Influential Scientific Information
RBC
red blood cells
This document is a draft for review purposes only and does not constitute Agency policy.
ix	DRAFT-DO NOT CITE OR QUOTE

-------
RD
RfC
RfD
RNA
ROS
RXR
SD
TP
TRI
TSCATS
TSH
UF
UFa
UFc
UFd
UFh
UFl
UFs
Vi
Vi
Toxicological Review ofPFHxA and Related Salts
relative deviation
reference concentration
oral reference dose
ribonucleic acid
reactive oxygen species
retinoid X receptor
standard deviation
total protein
Toxics Release Inventory
Toxic Substances Control Act Test
Submissions
thyroid stimulating hormone
uncertainty factor
interspecies uncertainty factor
composite uncertainty factor
evidence base deficiencies uncertainty
factor
human variation uncertainty factor
LOAEL to NOAEL uncertainty factor
subchronic to chronic uncertainty
factor
volume of distribution of peripheral
compartment (two-compartment PK
model)
volume of distribution
This document is a draft for review purposes only and does not constitute Agency policy.
10	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
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.	U.S. EPA/Office of Research and Development/Center for
Johanna Congleton, M.S.P.H., Ph.D.	Public Health and Environmental Assessment
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.
*No longer with U.S. EPA; **ORD/Office of Science Advisor, Policy, and Engagement; ***ORD/Center
for Computational Toxicology and Exposure
Production Team
Maureen Johnson
Ryan Jones
Dahnish Shams
Vicki Soto
Jessica Soto-Hernandez
Ashlei Williams
This document is a draft for review purposes only and does not constitute Agency policy.
xi	DRAFT-DO NOT CITE OR QUOTE
U.S. EPA/Office of Research and Development/Center for
Public Health and Environmental Assessment

-------
Toxicological Review ofPFHxA and Related Salts
Executive Direction
J. Allen Davis
Barbara Glenn
Kay Holt
Samantha Jones
Andrew Kraft
Janice Lee
Viktor Morozov
Ravi Subramaniam
Kristina Thayer
Tim Watkins
Paul White
CPAD Senior Science Advisor, Integrated Risk Information System
(Acting)
CPHEA/CPAD/Science Assessment Methods Branch Chief
CPHEA Deputy Center Director
CPHEA Associate Director
CPAD Associate Division Director, IRIS PFAS Team Lead (Acting)
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
CPHEA Center Director (Acting)
CPAD Senior Science Advisor
This document is a draft for review purposes only and does not constitute Agency policy.
xii	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Toxicological Review ofPFHxA and Related Salts
EXECUTIVE SUMMARY
Summary of Occurrence and Health Effects
Perfluorohexanoic acid (PFHxA, CASRN 307-24-4) and its related salts are members of the
group per- and polyfluoroalkyl substances (PFAS). This assessment applies to PFHxA as well as
salts ofPFHxA, including ammonium perfluorohexanoate (PFHxA-NH4, CASRN 21615-47-4), and
sodium perfluorohexanoate (PFHxA-NA, CASRN 2923-26-4), and other nonmetal and alkali metal
salts of PFHxA, that would be expected to fully dissociate in aqueous solutions of pH ranging from
4-9 (e.g., in the human body). Notably, due to the possibility of PFHxA-independent contributions
of toxicity, this assessment would not necessarily apply to nonalkali metal salts ofPFHxA (e.g.,
silver undecafluorohexanoate; CASRN 336-02-7). The synthesis of evidence and toxicity value
derivation presented in this assessment focuses on the free acid ofPFHxA and related ammonium
and sodium salts given the currently available toxicity data.
Concerns about PFHxA and other PFAS stem from the resistance of these compounds to
hydrolysis, photolysis, and biodegradation, which leads to their persistence in the environment.
PFAS are not naturally occurring in the environment; they are manmade compounds that have been
used widely over the past several decades in industrial applications and consumer products
because of their resistance to heat, oil, stains, grease, and water. PFAS in the environment are
linked to industrial sites, military fire training areas, wastewater treatment plants, and commercial
products (Appendix A, Section 2.1.2)
The Integrated Risk Information System (IRIS) Program is developing a series of five PFAS
assessments (i.e., perfluorobutanoic acid [PFBA], perfluorohexanoic acid [PFHxA], perfluorohexane
sulfonate [PFHxS], perfluorononanoic acid [PFNA], perfluorodecanoic acid [PFDA], and their
associated salts) at the request of EPA National Programs and Regions. The systematic review
protocol (see Appendix A) for these five PFAS assessments outlines the related scoping and
problem formulation efforts, including a summary of other federal and state assessments ofPFHxA.
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 links to the
updated version of the protocol and summary of 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
conduct of the studies (studies were generally low confidence); the few studies per health outcome;
This document is a draft for review purposes only and does not constitute Agency policy.
xiii	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Toxicological Review ofPFHxA and Related Salts
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 ofPFHxA exposure exclusively examined the oral exposure route, and
therefore no inhalation assessment was conducted nor was an RfC derived (see Section 5.2.2). The
available animal studies of oral PFHxA exposure examined a variety of noncancer and cancer
endpoints, including those relevant to hepatic, developmental, renal, hematopoietic, endocrine,
reproductive, immune, and nervous system effects.
Overall, the available evidence indicates that PFHxA 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 (see Table ES-1).
In addition, evidence suggests the potential for PFHxA exposure to affect endocrine
(i.e., thyroid) responses, based on studies in rats. However, 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
This document is a draft for review purposes only and does not constitute Agency policy.
xiv	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
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
for
PFHxA
(mg/kg-
d)
Value
for
PFHxA-
NAa
(mg/kg-
d)
Confid-
ence in
osRfD
ufa
UFh
UFS
ufl
ufd
UFC
Basis
Hepatic
Evidence
indicates
(likely)
osRfD
4 x icr4
4 x icr4
Medium
3
10
3
1
3
300
Increased
hepatocellular
hypertrophy in
adult rats
(Loveless et al.,
2009)


Subchronic
osRfD
l x icr3
l x icr3
Medium
3
10
1
1
3
100
Increased
hepatocellular
hypertrophy in
adult rats
(Loveless et al.,
2009)
Hemato-
poietic
Evidence
indicates
(likely)
osRfD
5 x icr3
6 x icr3
High
3
10
1
1
3
100
Decreased red
blood cells in
adult rats
(Klaunig et al.,
2015)


Subchronic
osRfD
8 x icr4
8 x icr4
High
3
10
1
1
3
100
Decreased red
blood cells in
adult rats
(Chengelis et
al., 2009b)
Develop-
mental
Evidence
indicates
(likely)
osRfD
5 x icr4
5 x icr4
Medium
3
10
1
1
3
100
Decreased Fi
body weight at
PND 0 (Loveless
et al., 2009)
This document is a draft for review purposes only and does not constitute Agency policy.
xv	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Organ/
System
Integration
judgment
Toxicity
value
Value
for
PFHxA
(mg/kg-
d)
Value
for
PFHxA-
NAa
(mg/kg-
d)
Confid-
ence in
osRfD
UFA
UFh
UFs
ufl
UFd
UFC
Basis


Subchronic
osRfD
5 x icr4
5 x icr4
Medium
3
10
1
1
3
100
Decreased Fi
body weight at
PND 0 (Loveless
etal.,2009)
Overall RfD


5 x icr4
5 x icr4
Medium
3
10
1
1
3
100
Decreased Fi
body weight at
PND 0 (Loveless
etal.,2009)
Overall
Subchronic
RfD


5 x icr4
5 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-d) 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.
aTo calculate candidate values for salts of PFHxA, multiply the candidate value of interest by the ratio of molecular weights of the free acid and the salt. For
example, for the sodium salt of PFHxA, the candidate value would be calculated by multiplying the free acid candidate value by 1.070 (MW free acid/MW
sodium salt = 336/314 = 1.070). This same conversion can be applied to other salts of PFHxA, such as the ammonium salt.
This document is a draft for review purposes only and does not constitute Agency policy.
xvi	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Toxicological Review ofPFHxA and Related Salts
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 fLoveless etal.. 20091
was selected as the basis for the RfD of 5 x 10~4 mg/kg-day. A BMDLsrd of 10.62 mg/kg-day was
identified for this endpoint and was used as the point of departure (POD). The human equivalent
dose POD (PODhed) of 0.048 mg/kg-day was derived by applying the ratio of the clearance between
female rats and humans and a normalization from the sodium salt to the free acid using a molecular
weight conversion. The overall RfD for PFHxA was calculated by dividing the PODhed by a
composite uncertainty factor of 100 to account for pharmacodynamic uncertainty in the
extrapolation from rats to humans (UFa = 3), interindividual differences in human susceptibility
(UFh = 10), and deficiencies in the toxicity evidence base (UFd = 3).
Confidence in the Oral Reference Dose (RfD)
The study conducted by Loveless etal. (2009) reported developmental effects following
administration of PFHxA sodium salt to pregnant Sprague-Dawley rats dosed by gavage for
approximately 70 days prior to cohabitation through gestation and lactation, for a total of 126 days
daily gavage with 0, 20,100, or 500 mg/kg-day sodium PFHxA. The overall confidence in the osRfD is
medium and is primarily driven by medium confidence in the overall evidence base for developmental
effects, high confidence in the study (click the HAWC link for full study evaluation details), and
medium confidence in quantitation of the POD (see Table 5-8). High confidence in the study was not
interpreted to warrant changing the overall confidence from medium.
Subchronic Oral Reference Dose (RfD) for Noncancer Effects
In addition to providing RfDs for chronic oral exposures in multiple systems, a less-than-
lifetime subchronic RfD was derived for PFHxA. The same study and endpoint (Loveless etal..
20091 and decreased Fi body weight) and value was selected as the basis for the subchronic RfD of
5 x 10"4 mg/kg-day (see Table ES-1). Details are provided in Section 5.2.1
This document is a draft for review purposes only and does not constitute Agency policy.
xvii	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
1
2	Noncancer Effects Following Inhalation Exposure
3	No studies that examine toxicity in humans or experimental animals following inhalation
4	exposure and no physiologically based pharmacokinetic (PBPK) models are available to support
5	route-to-route extrapolation; therefore, no RfC was derived.
6	Evidence for Carcinogenicity
7	Under EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA. 20051. EPA concluded
8	there is inadequate information to assess carcinogenic potential for PFHxA by either oral or
9	inhalation routes of exposure. The lack of data on the carcinogenicity of PFHxA precludes the
10	derivation of quantitative estimates for either oral (oral slope factor [OSF]) or inhalation
11	(inhalation unit risk [IUR]) exposure.
This document is a draft for review purposes only and does not constitute Agency policy.
1-1	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Toxicological Review ofPFHxA and Related Salts
1.OVERVIEW OF BACKGROUND INFORMATION
AND ASSESSMENT METHODS
A series of five PFAS assessments (perfluorobutanoic acid [PFBA], perfluorohexanoic acid
[PFHxA], perfluorohexane sulfonate [PFHxS], perfluorononanoic acid [PFNA], perfluorodecanoic
acid [PFDA], and their associated salts) are being developed by the Integrated Risk Information
System (IRIS) Program at the request of the U.S. Environmental Protection Agency (EPA) National
Programs and Regions. Appendix A is the systematic review protocol for these five PFAS
assessments. The protocol outlines the scoping and problem formulation efforts relating to these
assessments, including a summary of other federal and state reference values for PFHxA. The
protocol also lays out the systematic review and dose-response methods used to conduct this
review (see also Section 1.2). This systematic review protocol was released for public comment in
November 2019 and was subsequently updated on the basis of those public comments. Appendix A
includes the updated version of the protocol, including a summary of the updates in the protocol
history section (see Appendix A, Section 12).
1.1. BACKGROUND INFORMATION ON PFHxA AND RELATED AMMONIUM
AND SODIUM SALTS
This section provides a brief overview of aspects of the physiochemical properties, human
exposure, and environmental fate characteristics of perfluorohexanoic acid (PFHxA, CASRN
307-24-4), ammonium perfluorohexanoate (PFHxA-NFU, CASRN 21615-47-4), and sodium
perfluorohexanoate (PFHxA-Na, CASRN 2923-26-4).n This overview is not intended to provide a
comprehensive description of the available information on these topics. The reader is encouraged
to refer to source materials cited below, more recent publications on these topics, and the
assessment systematic review protocol (see Appendix A).
1.1.1. Physical and Chemical Properties
PFHxA and related sodium and ammonium PFHxA salts covered in this assessment are
members of the group of per- and polyfluoroalkyl substances (PFAS). Concerns about PFHxA and
other PFAS stem from the resistance of these compounds to hydrolysis, photolysis, and
biodegradation, which leads to their persistence in the environment fNLM. 2017. 2016. 20131.
PFHxA and related salts are classified as a perfluorinated carboxylic acids (PFCAs) fOECD. 20151.
PFHxA and its associated salts are considered short-chain PFAS fATSDR. 20181. The linear
This document is a draft for review purposes only and does not constitute Agency policy.
1-2	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
chemical structures1 of these chemicals are presented in Figure 1-1, and select physiochemical
properties are provided in Table 1-1.
1 The assessment applies to other non-linear isomers of PFHxA and related salts.
This document is a draft for review purposes only and does not constitute Agency policy.
1-3	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
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
l,070b
l,070b
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.
1-4	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Toxicological Review ofPFHxA and Related Salts
1.1.2.	Sources, Production, and Use
PFAS are not naturally occurring in the environment fU.S. EPA. 2020. 2019c: ATSDR. 2018:
U.S. EPA. 2013. 2007. 2002b). 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)
fU.S. EPA. 2020. 2019c: ATSDR. 2018: U.S. EPA. 2013. 2007. 2002b). PFAS also have been detected
from foam used in firefighting and in industrial surfactants, emulsifiers, wetting agents, additives,
and coatings; they are also used in aerospace, automotive, building, and construction industries to
reduce friction fU.S. EPA. 2020. 2019c: ATSDR. 2018: U.S. EPA. 2013. 2007. 2002b). 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) (U.S. EPA. 2020. 2019c: ATSDR.
2018: U.S. EPA. 2013. 2007. 2002b). In AFFF, PFHxA has been detected at concentrations ranging
from 0.1 to 0.3 g/L fBaduel etal.. 2015: Houtz etal.. 20131.
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.
2018c).
Wang etal. (2014) estimates global emissions of 39 to 1,691 tons of PFHxA from direct and
indirect (i.e., degradation of precursors) sources between 1951 and 2030. The lower estimate
assumes manufacturers cease production and use of long-chain PFCAs and that their precursors
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
(2014) 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. PFHxAbioaccumulates 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.
1-5	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Toxicological Review ofPFHxA and Related Salts
conducted a series of greenhouse and field experiments to investigate the potential for PFAS uptake
by lettuce, tomatoes, and corn when grown in industrially impacted and biosolids-amended soils.
Blaine etal. f20131 calculated PFHxA bioaccumulation factors of 9.9-11.7 for lettuce and 2.9-6.8 for
tomatoes (no bioaccumulation factor was reported for corn).
1.1.4. Potential for Human Exposure and Populations with Potentially Greater Exposure
The general population can be exposed to PFAS via inhalation of air or dust, ingestion of
drinking water and food, and dermal contact with PFAS-containing products and during susceptible
lifestages (see Appendix A). The oral route of exposure is considered the dominant exposure
pathway for the general population (Klaunig etal.. 20151. 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 (Shao etal..
2016).
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 French Korean, and Spanish
populations (summarized in Table 5 of Anderson etal. f201911. Exposure can also occur through
hand-to-mouth transfer of materials containing these compounds fATSDR. 20181 or in infants
through ingestion of formula reconstituted with contaminated drinking water.
Air and Dust
PFHxA has not been evaluated under the National Air Toxics Assessment program and no
additional information on atmospheric concentration was identified. PFAS, including PFHxA, have
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 et al.
(2009) 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. (2016) 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. f20121 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. (2013) 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.
1-6	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Toxicological Review ofPFHxA and Related Salts
Water
EPA conducted monitoring for several PFAS in drinking water as part of the third and fifth
Unregulated Contaminant Monitoring Rules (UCMR3 and UCMR5) fU.S. EPA. 2019b. 2016b). 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. 2016b). 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 et al. f 20171 observed mean concentrations ofPFHxA of 0.6 |ig/L in water,
8.4 |ig/kg dry weight in soil, and 3.0 |J.g/kg wet weight in grass at an Australian town where the
groundwater had been impacted by PFAS from a nearby firefighting training facility. Houtz et al.
f20131 analyzed samples of groundwater, soil, and aquifer solids collected at an Air Force
firefighting training facility in South Dakota where AFFF had been used. PFAS concentrations in
groundwater decreased with increased distance from the burn pit, and PFHxA was detected at a
median concentration of 36 |ig/L. PFHxA was detected in surficial soil at a median concentration of
11 Hg/kg and in aquifer solids at a median concentration of 45 |J.g/kg.
Military and National Priorities List (NPL) Sites
PFHxA levels in environmental samples collected in 2014 have been measured at military
and National Priorities List (NPL) sites in the United States. Table 1-2 provides the concentrations
at these sites (ATSDR. 2018: Anderson etal.. 2016).
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.
1-7	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Toxicological Review ofPFHxA and Related Salts
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 et al.
(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 ofPFHxA 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.
1-8	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
1	detected in microwave popcorn packaging materials at a range of 3.4 to 497 ng/g, but was not
2	detected in the corn or popcorn fMoreta and Tena. 20141.
3	Stahl etal. T20141 characterized PFAS in freshwater fish from 164 U.S. urban river sites and
4	157 near-shore Great Lakes sites. PFHxA was not detected in the fish from U.S. urban rivers but
5	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
6	This section summarizes the methods used for developing this assessment. A detailed
7	description of these methods is provided in the PFAS Systematic Review Protocol for the PFDA,
8	PFNA, PFHxA, PFHxS, and PFBA IRIS Assessments (see Appendix A and online! The protocol
9	includes additional problem formulation details, including the specific aims and key science issues
10	identified for this assessment.
1.2.1. Literature Search and Screening
11	The detailed search approach, including the query strings and populations, exposures,
12	comparators, and outcomes (PECO) criteria, are provided in Appendix A, Table 3-1. The results of
13	the current literature search and screening efforts are documented in Section 2.1. Briefly, a
14	literature search was first conducted in 2017 and regular yearly updates have been performed (the
15	literature fully considered in the assessment was until April 2021. The literature search queries the
16	following databases (no literature was restricted by language):
17	• PubMed fNational Library of Medicine 1
18	• Web of Science fThomson Reuters!
19	• Toxline (moved to PubMed December 2019)
20	• TSCATS (Toxic Substances Control Act Test Submissions)
21	In addition, relevant literature not found through evidence base searching was identified
22	by:
23	• Review of studies cited in U.S. state, U.S. federal, and international assessments, including
24	parallel assessment efforts in progress (e.g., the draft Agency for Toxic Substances and
25	Disease Registry [ATSDR] assessment released publicly in 2018).
26	• Review of studies submitted to federal regulatory agencies and brought to EPA's attention.
27	• Identification of studies during screening for other PFAS. For example, searches focused on
28	one of the other four PFAS currently being assessed by the IRIS Program sometimes
29	identified epidemiological studies relevant to PFHxA.
This document is a draft for review purposes only and does not constitute Agency policy.
1-9	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
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.^ The PECO criteria identify the evidence that
7	addresses the specific aims of the assessment and focuses the literature screening, including study
8	inclusion/exclusion. In addition to those studies meeting the PECO criteria, studies containing
9	supplemental material potentially relevant to the specific aims of the assessment were inventoried
10	during the literature screening process. Although these studies did not meet PECO criteria, they
11	were not excluded. Rather, they were considered for use in addressing the identified key science
12	issues (see Appendix A, Section 2.4) and other major scientific uncertainties identified during
13	assessment development but unanticipated at the time of protocol posting. Studies categorized as
14	"potentially relevant supplemental material" included the following:
15	• In vivo mechanistic or mode-of-action studies, including non-PECO routes of exposure
16	(e.g., intraperitoneal injection) and non-PECO populations (e.g., nonmammalian models);
17	• In vitro and in silico models;
18	• Absorption, distribution, metabolism, and excretion (ADME) and pharmacokinetic (PK)
19	studies (excluding models)3;
20	• Exposure assessment or characterization (no health outcome) studies;
21	• Human case reports or case-series studies; and
22	• Studies of other PFAS (e.g., perfluorooctanoic acid [PFOA] and perfluorooctane sulfonate
23	[PFOS]).
24	The literature was screened by two independent reviewers with a process for conflict
25	resolution, first at the title and abstract level and subsequently the full-text level, using structured
26	forms in DistillerSR fEvidence Partners! Literature inventories for studies meeting PECO criteria
27	and studies tagged as "potentially relevant supplemental material" during screening were created
28	to facilitate subsequent review of individual studies or sets of studies by topic-specific experts.
2EPA'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.
3Given the known importance of ADME data, this supplemental tagging was used as the starting point for a
separate screening and review of PK data (see Appendix A, Section9.2 for details).
This document is a draft for review purposes only and does not constitute Agency policy.
1-10	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Toxicological Review ofPFHxA and Related Salts
1.2.2. Evaluation of Individual Studies
The detailed approaches used for the evaluation of epidemiological and animal toxicological
studies used in the PFHxA assessment are provided in the systematic review protocol (See
Appendix A, Section 6). The general approach for evaluating health effect studies meeting PECO
criteria is the same for epidemiological and animal toxicological studies although the specifics of
applying the approach differ; thus, they are described in detail in Appendix A (see Sections 6.2 and
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
This document is a draft for review purposes only and does not constitute Agency policy.
1-11	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Toxicological Review ofPFHxA and Related Salts
carried forward to inform the synthesis (see Section 1.2.4) within each body of evidence for a given
health effect (i.e., study confidence determinations were not used to inform judgments in isolation).
1.2.3.	Data Extraction
The detailed data extraction approach is provided in Appendix A, Section 8, and data
extraction and content management is carried out using HAWC (see Appendix C). Data extraction
elements that may be collected from epidemiological, controlled human exposure, animal
toxicological, and in vitro studies are available in HAWC. As described in the systematic review
protocol (see Appendix A), not all studies that meet the PECO criteria go through data extraction:
For example, studies evaluated as being uninformative are not considered further and therefore do
not undergo data extraction. 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, Sections 9 and 10 for full details). For each
assessed health effect, the evidence syntheses provides a summary discussion of each body of
evidence considered in the review that directly informed the integration across evidence that was
used to draw an overall judgment for each health effect The available human and animal evidence
pertaining to the potential health effects were synthesized separately, with each synthesis resulting
in a summary discussion of the available evidence that addresses considerations regarding
causation adapted from Hill f!965I
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. Mechanistic evidence and other supplemental information was also
synthesized to address key science issues or to help inform key decisions regarding the human and
animal evidence. In certain instances (i.e. few or no studies with higher confidence are available)
low confidence studies might be used to help evaluate consistency, or if the study designs of the low
confidence studies address notable uncertainties in the set of high or medium confidence studies on
a given health effect However, no low confidence studies were used in the evidence syntheses for
PFHxA included in the narrative. Inclusion in the syntheses of mechanistic evidence and other
supplemental information was intended to inform the integration of health effects evidence for
hazard identification (i.e., biological plausibility of the available human or animal evidence,
This document is a draft for review purposes only and does not constitute Agency policy.
1-12	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Toxicological Review ofPFHxA and Related Salts
inferences regarding human relevance, adaptive versus adverse responses, etc.) and for
dose-response evaluation.
For each assessed health effect, following the evidence syntheses, integrated judgments
were drawn across all lines of evidence. During evidence integration, a structured and documented
process was used, as follows:
•	Building from the separate syntheses of the human and animal evidence, the strength of the
evidence from the available human and animal health effect studies was summarized in
parallel, but separately, using a structured evaluation of an adapted set of considerations
first introduced by Bradford Hill (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 A,
Section 11. Briefly, although procedures for dose-response assessments were developed for both
noncancer and cancer health hazards, and for the oral route of exposure following exposure to
PFHxA, the existing data for PFHxA only supported derivation of an oral reference dose (RfD) for
noncancer hazards (see Appendix A, Section 11 for the health hazard conclusions necessary for
deriving other values). An RfD is an estimate, with uncertainty spanning perhaps an order of
magnitude, of an exposure to the human population (including susceptible subgroups) that is likely
without an appreciable risk of deleterious health effects over a lifetime (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. 2012a. 2005). Within the observed dose range, the preferred approach is to use
This document is a draft for review purposes only and does not constitute Agency policy.
1-13	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Toxicological Review ofPFHxA and Related Salts
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. 2012a)] as elaborated in Appendix A, Section 11. Thus, modeling to derive a POD attempted to
include an exposure level near the lower end of the range of observation, without significant
extrapolation to lower exposure levels. Extrapolations to exposures lower than the POD involved
the application of five uncertainty factors to estimate candidate noncancer toxicity values, as
described in Appendix A, Section 11.
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 A, Section 11), 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.
This document is a draft for review purposes only and does not constitute Agency policy.
1-14	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
Toxicological Review ofPFHxA and Related Salts
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 genotoxicity studies, and 3 PK studies. In addition, high-
throughput screening data on PFHxA were available from EPA's CompTox Chemicals Dashboard
fU.S. EPA. 2018al A literature inventory of the included animal toxicological studies is available in
an interactive literature inventory heatmap accessible via PFHxA Tableau Link.
This document is a draft for review purposes only and does not constitute Agency policy.
2-1	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
PFHxA
Literature Searches (through Feb 2020)
PubMed 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
*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-NH4 and PFHxA-Na).
This document is a draft for review purposes only and does not constitute Agency policy.
2-2	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Toxicological Review ofPFHxA and Related Salts
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. Fourteen 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 (Zhang etal.. 2019: Seo et al.. 2018: Kim etal..
2016a: Tiang etal.. 20141. The remaining nine studies were rated medium fNian etal.. 2019: Bao et
al.. 2017: Zeng etal.. 2015: Dong etal.. 20131 or low confidence fWang etal.. 2019: Song etal.. 2018:
Li etal.. 2017: Zhou etal.. 2016: Fu etal.. 2014).
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). All outcomes rated low confidence or higher were used for
evidence synthesis and integration.
This document is a draft for review purposes only and does not constitute Agency policy.
2-3	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Toxicological Review ofPFHxA and Related Salts
3.PHARMACOKINETICS, EVIDENCE SYNTHESIS, AND
EVIDENCE INTEGRATION
3.1. PHARMACOKINETICS
Only a few human PK studies on PFHxA are available, but the studies provide sufficient data
to estimate PFHxA half-life, a dependent variable for the estimation of clearance (along with volume
of distribution). Several studies such as Ericson et al. f20071 reported PFHxA in blood or serum of
human populations (e.g., in relation to age and sex) but, because exposure levels are not known for
the subjects and the concentrations are not measured over time in specific subjects for whom the
exposure level is known to be zero, such observations cannot be used to obtain ADME information.
Several other studies that investigate specific aspects of PFHxA ADME in humans are discussed
briefly below but were not used in the derivation of toxicity values. One analysis provides an
estimate ofPFHxA elimination in humans (Russell etal.. 2013) using data from an observational
study by Nilssonetal. f20131. Luz etal. T20191 describes a reanalysis of these data but based only
on the three participants with the most rapid clearance. While EPA considers the data reported by
Nilsson etal. (2013) to be sufficient for the estimation of a half-life in humans, the approaches used
by (Russell etal.. 2013) and Luz etal. (2019) were not considered adequate. Therefore, the data of
Nilsson etal. (2013) have been re-analyzed as described in Approach for Animal-Human
Extrapolation ofPFHxA Dosimetry (See Section 5.2.1).
Animal experiments in rats, mice, and monkeys have provided valuable information on PK
processes ofPFHxA. In brief, PFHxA and other perfluoroalkyl acids (PFAA) have similar PK aspects:
They are well absorbed following oral exposure and quickly distribute throughout the body
(Iwabuchi etal.. 2017). particularly to blood, liver, skin, and kidney (Gannon etal.. 2011).
Dzierlenga et al. (2019) noted that following intravenous (i.v.) administration of 40 mg/kg PFHxA,
the PK profiles were generally similar between sexes, but a lower dose-normalized area under the
curve (AUC, 3.05 mM-h/mmol/kg), a faster clearance (CL, 327 mL/h-kg), and a lower volume of
distribution of peripheral compartment (V2 = 59.6 mL/kg) was observed in female Sprague-Dawley
rats, as compared to their male counterparts (dose-normalized AUC = 7.38 mM-h/(mmol/kg),
CL = 136 mL/h-kg, and V2 = 271 mL/kg, respectively). Likewise, kinetic parameters (e.g., the
maximum concentration [Cmax]) were comparable between sexes following an oral dose of
40 mg/kg, except that 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
This document is a draft for review purposes only and does not constitute Agency policy.
3-1	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Toxicological Review ofPFHxA and Related Salts
monkeys given a 10 mg/kg i.v. dose ofPFHxA was only slightly lower in females than in males (75
vs. 84 mg-h/L), suggesting no significant sex difference in nonhuman primates.
PFHxA is resistant to metabolic transformation, and urinary excretion is the main
elimination route, followed by feces fGannon et al.. 2011: Iwai. 2011: Chengelis etal.. 2009al.
3.1.1.	Absorption
Absorption is rapid in rodents and monkeys (I wabuchi et al.. 2 017: Gannon etal.. 2011:
Chengelis etal.. 2009a). PFHxA was extensively absorbed with an average time to reach maximum
concentration (Tmax) of 1 hour in Sprague-Dawley rats given 26-day repeated gavage doses of 50,
150 or 300 mg PFHxA/kg (Chengelis etal.. 2009a). After gavage at2 or 100 mg [l-14C]PFHxA/kg
using a single dose or 14 daily consecutive doses, Gannon etal. f20111 also observed a short Tmax of
30 and 15 minutes, respectively, in male and female Sprague-Dawley rats. Similarly, rapid
absorption was also observed in CD-I mice (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 AUC0^i68 hour, but the data in male rats indicate either a 25%
reduction in absorption or a corresponding increase in clearance between these two dose levels
(Gannon etal.. 2011: Chengelis etal.. 2009a).
In a recent PK study by Dzierlenga etal. f20191. Sprague-Dawley rats were given PFHxA, by
i.v. injection (40 mg/kg) or gavage (40, 80, and 160 mg/kg). Besides collection of blood samples to
evaluate the time course of plasma PFHxA for each dose and route, liver, kidney, and brain samples
were collected to determine the distributions ofPFHxA in tissues following 80 mg/kg gavage dose.
A two-compartmental model was used to evaluate the PK profiles. The estimated oral
bioavailability for PFHxA was >100% fDzierlenga etal.. 20191: 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 data indicate thatTmax
increased slightly but not significantly with increasing oral PFHxA dose levels for both sexes. For
instance, Tmax increased from 0.668 ± 0.154 to 0.890 ± 0.134 hour (mean ± standard error) and
from 0.529 ± 0.184 to 0.695 ± 0.14 hour with increased gavage doses of PFHxA for male and female
rats, respectively fDzierlenga et al.. 20191.
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 (Russell etal.. 2013: Gannon et al.. 2 0111. The largest
concentrations were found in liver, skin, heart, lung, and kidney and concentrations peaked within
hours f I wabuchi et al.. 2 017: Gannon etal.. 20111. For example, Gannon etal. f20111 reported
This document is a draft for review purposes only and does not constitute Agency policy.
3-2	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Toxicological Review ofPFHxA and Related Salts
heart, kidneys, liver, and lungs had detectable but not quantifiable concentrations ofPFHxA at 24
hours 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 three) among male and female mice,
rats, and monkeys (Russell etal.. 2013).
Distribution in Animal (Rats, Mice, and Monkeys) and In Vitro Studies
Chengelis etal. f2009al gave both Sprague-Dawley rats andcynomolgus monkeys (3/sex)
PFHxA (10 mg/kg) via a single i.v. injection to determine PFHxA PK using noncompartmental
analysis. In monkeys they observed a distribution phase of 8 hours and an apparent Vd of 0.77 and
0.35 L/kg in males and females, respectively. In male and female rats, Vd was reported as 0.18 and
0.47 L/kg, respectively, and the distribution phase after gavage dosing was about 1-2 hours in both
sexes. Serum concentrations ofPFHxA were up to 17-fold higher for male than female rats after i.v.
dosing. In a separate experiment male and female Sprague-Dawley rats were given oral gavage
doses of 50,150, or 300 mg/kg/d PFHxA for 25 days (6 rats/sex/dose) and the PK evaluated on the
first and last day of dosing. The AUC after oral dosing was approximately 4-fold higher in males
than females given a 50 mg/kg gavage dose on both day 1 and day 25. The half-life in males,
however, was only 2.5 times greater than females after i.v. dosing and was similar to that in females
after oral dosing. Together these lead to the conclusion of higher Vd for females than for males.
Using a one-compartment model, Iwabuchi 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
hour for all tissues, the Tmax for other PFAAs was 12 hours in the tissues except the brain (72 h) and
whole blood (24 h), indicating PFHxA was distributed rapidly throughout the body. Peak
concentrations occurred between 15 minutes and 1 hour after dosing, depending on the tissue. Of
examined tissues, the highest concentrations of PFHxA were found in the serum and kidney,
equivalent to 7.9% and 7.1% of the administered PFHxA, respectively. Note that the peak
concentrations measured in liver and brain were roughly 40% (at 15 minutes) and 1.5% (at 1 hour)
of the corresponding peak serum levels (4.6% and 0.027% of administered PFHxA dose),
respectively. The earlier peak in liver concentration is likely due to initial delivery there from oral
absorption, although the results show low delivery to the brain.
Dzierlenga et al. (20191 measured levels ofPFHxA in rat liver, kidney and brain over 12
hours following an 80 mg/kg oral gavage dose. In general tissue distribution was rapid, with peak
concentration occuring at 0.5 hours (first time-point) in male rat liver and kidney or 1 hour (second
time-point) in male rat brain and in female rat liver, kidney and brain. The concentrations declined
This document is a draft for review purposes only and does not constitute Agency policy.
3-3	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Toxicological Review ofPFHxA and Related Salts
exponentially after the peak, with tissue: plasma ratios mostly remaining in a limited range. For
example, in male and female rat kidney and female rat liver the tissue: plasma ratio only varied
between 0.5 and 0.75, though the liver: plasma ratio varied between 1 and 0.5 in male rats, though
without a clear pattern. However, the kidney: plasma ratio in female rats showed a steady increase
from around 0.8 at 0.5 hours to around 1.7 at 3 hours, after which it slowly declined to around 1.4
at 12 hours (Dzierlenga etal.. 2019). Since tissue: plasma ratios are generally less than 1, this result
in the female rat kidney indicates a mechanism that wasn't active in the liver or male rats, perhaps
involving active transport into the tissue.
For the 1- or 3-month exposures, rats were given a mixture of four PFAA dose levels: 0,1, 5
and 25 ng/L in drinking water with similar intake rate across dose groups
(0.072-0.077 L/kg BW-day) flwabuchi etal.. 20171. In general, the long-term tissue
concentrations ofPFHxA 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 ofPFHxA remained relatively
constant over time flwabuchi etal.. 20171.
An in vitro study using lung epithelial cells (NCI-H292) and adipocytes (3T3-L1K) made
similar observations of no appreciable cellular accumulation and retention ofPFHxA (Sanchez
Garcia etal.. 20181.
Distribution in Humans
The tissue distribution ofPFHxA and other PFAAs were analyzed in 99 human autopsy
samples (brain, liver, lung, bone, and kidney) (Perez etal.. 2013). Perez etal. (2013) used the term
"accumulation," which in PK terminology describes a steady increase in the amount of a substance
in the body tissues over an extended time while exposure continues at a relatively constant level.
So, to demonstrate accumulation, one must have repeated measures of the blood or tissue
concentration in an individual over a significant period of time. If the body quickly reaches a
constant level (with ongoing exposure), that would not be called "accumulation." Because the study
data were collected from cadavers, they show only the tissue levels in the individuals at time of
death, and thus do not actually demonstrate accumulation but simply that exposure, absorption,
and distribution have occurred. These tissue concentrations could represent approximate steady-
state concentrations that were achieved quickly after the start of exposure, without accumulation.
More generally, these data cannot inform the specific exposure scenarios that might have occurred
before the time of death, in particular the duration of exposure that was required to reach the
observed concentrations.
Perez etal. (2013) 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
This document is a draft for review purposes only and does not constitute Agency policy.
3-4	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Toxicological Review ofPFHxA and Related Salts
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.
Because blood plasma concentrations could not be evaluated in the cadavers, the data of
Perez etal. f20131 lack this component of total PFHxA body burden. Plasma is a small fraction of
total body mass (~ 4% in humans), but due to PFHxA's substantial binding to serum proteins it will
carry a disproportionate amount of the PFHxA. For example, if the overall volume of distribution in
humans is 0.5 L/kg, plasma will then contain about 8% of the PFHxA.
Fabregaetal. f20151 attempted to estimate tissue:blood partition coefficients (PCs) for
PFHxA using the data of Perez etal. f20131. Because Perez etal. f20131 did not measure or report
blood concentrations, Fabrega etal. f20151 used the mean blood concentration reported 4 years
earlier for residents of the same county fEricson et al.. 20071. The resulting set of PCs ranged from
6 (unitless ratio) in the kidney to 202 in the brain, indicating a Vd in the human body around 40
L/kg or higher.
Zhang etal. (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 ofPFHxA and 15 perfluoroalkyl substances (C6-C11) between plasma and
blood cells was investigated using blood samples collected from human subjects (n = 60) flin etal..
2016). The results showed that although the estimated mass fraction in plasma generally increased
with the carbon chain length, PFHxA appeared to have lowest mass fraction in plasma (0.24) as
compared with other PFAA chemicals (0.49 to 0.95). In a study population of 61 adults in Norway,
Poothong etal. (2017) 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 fPoothong et al.. 20171.
Synthesis of Distribution Across Species
In contrast to the estimated PCs of Fabrega etal. (2015). 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 etal. f2009al reported varied widely for each sex; for example, the
coefficient of variation among the three females was 74%. Therefore, EPA recalculated male and
This document is a draft for review purposes only and does not constitute Agency policy.
3-5	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Toxicological Review ofPFHxA and Related Salts
female values for this analysis from the mean values of AUCo-oo and the beta-phase elimination
constant, Kei:
Vd = dose/[mean(AUCo-oo) x mean(Kei)].	(3-1)
The resulting values of Vd were 0.77 L/kg and 0.35 L/kg for male and female monkeys, respectively.
Although the reported values for rats and these re-estimated values for monkeys were within
similar ranges, spanning less than a factor of five, the difference between males and females of each
species is larger than expected. The underlying data indicate significant PK differences between
males and females of each species.
The average Vd for rats (0.33 L/kg) is only 40% lower than the average for monkeys
(0.56 L/kg), a modest species difference that could occur due to differences in the relative
concentration of binding proteins and phospholipids in blood (e.g., albumin) vs. the rest of body
(Sanchez Garcia etal.. 2018). Partitioning or distribution is primarily a function of the
physicochemical properties of a tissue vs. blood (binding site content and phospholipid
concentration being significant components for PFAS) and are typically similar across mammalian
species, not differing by orders of magnitude as suggested by the difference between the results of
Fabregaetal. f20151 for humans and the animal PC data. This raises a significant question about
reasons for the apparent disparity. EPA is unaware of a specific mechanism that could explain this
discrepancy, particularly one that differs between monkeys and humans to such a large extent but
not between monkeys and rats.
Therefore, the most likely explanation for the differences in the PCs estimated by Fabrega et
al. (2015) are an artifact of combining data from nonmatched human samples Perez etal. (2013)
whereas Ericson etal. (2007) 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
above, Distribution in Animals) and an assumption of similar partitioning in humans versus other
mammals has been successfully used for many PBPK models, this assumption is considered modest
with minimal associated uncertainty.
A generally accepted assumption in pharmacokinetics is that renal clearance (via
glomerular filtration) is limited to the fraction unbound in plasma (Tanku. 1993). PFAS
accumulation in tissues appears to correlate with phospholipid binding and content and like lipids
the relative distribution of phospholipids, albumin, and other binding sites is not expected to differ
by orders of magnitude between humans and other animals (Sanchez Garcia et al.. 2018). Some
evidence suggests plasma protein binding (e.g., serum albumin) could also play a role in PFHxA
This document is a draft for review purposes only and does not constitute Agency policy.
3-6	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Toxicological Review ofPFHxA and Related Salts
toxicokinetics. 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 (Kudo. 2015: Weaver etal.. 20101 (see further
discussion below for rat studies. Weaver etal. (20101 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 ofPFHxA (Weaver etal.. 2010). The role of Oatplal
and its regulation by sex hormones is discussed at further length below (Rat Studies).
On the other hand, although Bischel etal. f20111 measured the binding of PFHxA to bovine
serum albumin (BSA) in vitro, the measured fraction bound is 99%, which appears quantitatively
inconsistent with the empirical observation that the elimination half-life is on the order of 2-3
hours in rats, for example. In particular, renal elimination is generally predicted to be proportional
to the fraction of a compound unbound in plasma (e.g., (Tanku and Zvara. 1993. pp. author-
yearauthor-vearl). Transporter-mediated renal resorption would only reduce elimination to a
greater extent. If the binding of PFHxA to BSA is indicative of its overall fraction bound in serum
and glomerular filtration could remove only 1% (i.e., the free faction) ofPFHxA carried in the
corresponding serum flow, the elimination half-life should be much longer than is observed. Thus,
although plasma protein binding could play some role in PFHxA distribution and elimination, one
must be careful in quantitatively interpreting such results. Because it is reversible, protein binding
could have a limited impact on distribution and elimination, despite a relatively high fraction of
plasma protein binding at equilibrium. Therefore, the empirically determined distribution and
elimination rates for PFHxA in various species and sexes are used rather than the rate one might
predict on the basis of albumin binding.
3.1.3. Metabolism
Similar to other PFAA compounds, PFHxA is not readily metabolized as evidenced by the
findings that no metabolites were recovered from either the liver or urine following oral dosing of
mice or rats (Gannon etal.. 2011: Chengelis etal.. 2009a). Although PFHxA is resistant to
metabolism, fluorotelomer-alcohols and sulfonates can undergo biotransformation to form PFHxA
or its glucuronide and sulfate conjugates in rodents and humans fKabadi etal.. 2018: Russell etal..
20151.
This document is a draft for review purposes only and does not constitute Agency policy.
3-7	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Toxicological Review ofPFHxA and Related Salts
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 ofPFHxA appeared in the urine of rats during 24 hours post-dosing regardless
of sex following i.v. injection (Chengelis etal.. 2009a). Daikin Industries recovered approximately
90% of an oral dose of 50 mg/kg PFHxA, either as a single dose or on the 14th day of dosing by 24
hours after the single or last dose in male and female rats and mice (Daikin Industries. 2009a. b).
Likewise Dzierlenga etal. (20191 reported that liver and kidney concentrations peaked by 30 min
in male rats and by 1 hour in female rats after gavage and decreased steadily thereafter
(observations at 0.5,1, 3, 6, 9 and 12 hours). The tissues concentrations ofPFHxA tended to be very
low or not quantifiable 24 hours after dosing in both sexes of mice and rats flwabuchi etal.. 2017:
Gannon etal.. 2011).
The comparable weight-normalized blood elimination half-life ofPFHxA 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 hours, perfluorobutane sulfonate (PFBS) = 4.7 hours, pentafluorobenzoic acid
(PFBA) = 9.2 hours, ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate
(GenX) = 72 hours, PFOA = 136 hours, and PFOS = 644 hoursfGomis etal.. 2018). 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 et al.
(2009a) compared PFHxA dosimetry in naive male and female rats to results after 25 days of dosing
(50-300 mg/kg-day) and found no significant difference in the parameters evaluated, with the
serum half-life remaining in the range of 2-3 hours.
Rat Studies
Iwai f20111 evaluated PFHxA excretion in Sprague-Dawley rats and CD-I mice treated with
single and multiple (4 days) oral dose(s) at 50 mg/kg of [14C] ammonium perfluorohexanoate
(APFHx). Urine and feces samples were collected for 0-6 hours (urine only) and 6-24 hours and
then followed 24-hour intervals until 72 hours after dosing. Expired air was collected over 0-24
and 24-48 hours following oral exposure. For the single dose administration in rats, 97-100% of
administered PFHxA dose was recovered within 24 hours with urine as the major route of
elimination (73.0-90.2%), followed by feces (7.0-15.5% of the administered dose). No appreciable
PFHxA was found in expired air. Two percent of the dose remained in the gastrointestinal tract and
This document is a draft for review purposes only and does not constitute Agency policy.
3-8	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Toxicological Review ofPFHxA and Related Salts
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 ofPFHxA in serum was about
2.4-fold shorter for female Sprague-Dawley rats than for male rats (0.42 hours compared to
1.0 hour) with a single dose of 10 mg/kg i.v. injection. Likewise, Gannon etal. f20111 reported
elimination half-lives for PFHxA of 1.7 and 1.5 hours in male rats and 0.5 and 0.7 hours in female
rats for doses of 2 and 100 mg/kg, respectively. On the other hand, after repeated oral
administration (50-300 mg/kg-day) ofPFHxA, Chengelis etal. (2009a) found the serum terminal
half-life ofPFHxA was generally in the range of 2-3 hours regardless of sex. Comparable urinary
elimination half-lives following single 10 mg/kg i.v. were also observed (males: 2.1 hours; females
2.5 hours) f Chengelis etal.. 2009al. It is unclear why Chengelis etal. f2009al obtained different
half-lives for males versus females from some of their results, but not in others. Evaluation of the
half-life from any PK data set depends on the study design, especially the number and spacing of
data points relative to the half-life, the type of PK analysis done, and analytic sensitivity. EPA
analyzed PFHxA half-lives that combined data across studies to obtain sex-specific values,
described in Section 5.2.1 (Approach for Animal-Human Extrapolation ofPFHxA Dosimetry).
As noted above, Daikin Industries evaluated urinary and fecal excretion in Sprague-Dawley
rats after 50 mg/kg oral doses for 1 or 14 days fDaikin Industries. 2009a. b). The elimination
pattern is consistent with other studies described here, with approximately 90% of the dose
recovered in feces and urine by 24 hours. Because excretion was only evaluated at 6 hours (urine
only), 24 hours, and multiple days thereafter, these specific studies are not considered
quantitatively informative for evaluation of half-life or clearance.
Russell etal. f20151 conducted PK modeling analysis of 3,3,4,4,5,5,6,6,7,7,8,8,8-
Tridecafluorooctanol (6:2 FTOH) inhalation (0.5 or 5 ppm) in rats, including its metabolite PFHxA,
as described above. The estimated PFHxA half-lives were 1.3 and 0.5 hours in male and female rats,
respectively, from single-day exposures, with the estimated yield of PFHxA ranging from 0.5 to 1.9
mol%. The model assumes, however, that the yield of PFHxA from 6:2 FTOH is independent of time.
This apparent time-dependence in the half-life could be an artifact of that assumption if induction
of metabolism during the dosing period leads to a higher yield with later times. A more
comprehensive multiday PK analysis would be needed to demonstrate time-dependent PFHxA
clearance unequivocally. Using a noncompartmental PK analysis Kabadi etal. f20181 reanalyzed
the 1-day data of Russell etal. f20151 and obtained the same half-life values (1.3 and 0.5 hours in
males and females).
A recent study by Dzierlenga etal. (2019) and NTP (2017) showed no apparent pattern in
ty2i p among the i.v. (40 mg/kg) and two lower oral doses (40 and 80 mg/kg) for each sex (ranges
5.74-9.3 hours for male rats and 2.3-7.3 hours for female rats), which likely reflects experimental
variability. The ty2, p for the 160 mg/kg oral dose appeared higher than the other three
measurements (13.7 ± 14.2 and 12.2 ± 23.6 hours [mean ± standard error of the mean] for males
This document is a draft for review purposes only and does not constitute Agency policy.
3-9	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Toxicological Review ofPFHxA and Related Salts
and females, respectively), but a loss of dose-concordance occurred among the PK data starting at
6 hours (i.e., the serum concentrations were similar for all dose levels at 6 hours and beyond). Also,
the data at the last time point (24 hours) varied considerably, resulting in large uncertainty in the
estimated terminal half-lives fDzierlenga et al.. 20191.
Similar to the elimination half-life in male Sprague-Dawley rats, the estimated serum
elimination half-life ofPFHxA in male Wistar rats (6 weeks old) was about 2.6 hours for a single
dose of 100 M-g/kg BW or 2.9 hours for exposures in drinking water of 1 or 3 months (Iwabuchi et
al.. 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 ofPFHxA, 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..
2002). and PFAS (Han etal.. 2012: Yang etal.. 2010). In male rats, Oatplal mRNA expression was
2.5-fold greater than in females, undetectable in castrated rats, and inducible in male rats by
treatment with estradiol (Kudo etal.. 20021.
A separate study (Lu etal.. 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, or simply aspects of experimental design and sampling that measure
the PK parameters better in some studies than others. The empirical results of Chengelis et al.
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 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 (Ki) of 1,858 |iM, as compared with 84 |iM for
PFOA. This high Ki for PFHxA (i.e., the concentration required to inhibit one-half the Oatplal
This document is a draft for review purposes only and does not constitute Agency policy.
3-10	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Toxicological Review ofPFHxA and Related Salts
activity, 584 [ig/mL] indicates a low affinity ofPFHxA for the transporter and thus leads to
predictions of a low impact of Oatplal expression on PFHxA elimination kinetics, contrary to the
empirical PK data discussed above. Chengelis etal. f2009al clearly showed more rapid elimination
in female rats 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 fHan etal.. 2012: Gannon etal.. 2011: Chengelis etal.. 2009bl.
Most notably, whether this apparent sex difference in re-uptake exists in humans or in species
other than rats is unclear. Organic-anion transporters are known to be under hormonal regulation
in rat and mouse kidney, with gender-specific differences in their expression likely regulated by
sex-hormone receptors. Some evidence suggests similar sex-related differences in humans (Sabolic
etal.. 20071. Kudo etal. f20011 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 (Elimination, Rat Studies), Iwai (2011) 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 evaluated urinary and fecal excretion in CD-I mice after
50 mg/kg oral doses for 1 or 14 days (Daikin Industries. 2009a. b). The elimination pattern is
consistent with Iwai (2011). with approximately 90% of the dose recovered in the urine and feces
(total) after 24 hours. Because excretion was only evaluated at 6 hours (urine only), 24 hours, and
multiple days after the PFHxA dosing ended, however, the studies cited are not considered
quantitatively informative for evaluation of half-life or clearance.
Daikin Industries f20101 evaluated the time-course ofPFHxA in female Crl:CD(lCR) mouse
plasma after single oral gavage doses of 35,175, and 350 mg/kg, with concentrations measured at
This document is a draft for review purposes only and does not constitute Agency policy.
3-11	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Toxicological Review ofPFHxA and Related Salts
0.5, 2, 4, 6, 8, and 24 hours. The estimated half-life was between 0.9 and 1.2 hours for the three
dose groups butlacked 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 itvaried between 5.1 and 6.5 kg-
h/L, indicating that clearance was not dose-dependent
The plasma time-course data from Gannon etal. (2011) and Daikin Industries (2010) 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 study on cynomolgus monkeys by Chengelis etal. f2009al. three males and three
females received 10 mg/kg PFHxA by i.v. injection. The mean clearance was nearly the same in
both sexes (0.122 L/h-kg in males and 0.136 L/h-kg in females), but the estimated half-life
appeared longer in males (5.3 ± 2.5 hours) than in females (2.4 ± 1.7 hours) with a corresponding
apparent difference in Vd (0.989 L/kg in males and 0.474 L/kg in females). The similarity of the
clearance values and the nearly identical serum values for males and females after the first 4 hours
suggest no striking sex differences in the pharmacokinetics ofPFHxA in monkeys.
Human Studies
No controlled exposure PK studies ofPFHxA 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 ofPFHxA in humans by analyzing biomonitoring data collected from professional ski wax
technicians and then compared the human estimates ofPFHxA elimination to that for mice, rats,
and monkeys. For the human monitoring study, blood samples (n = 94) were collected from male
professional ski wax technicians (n = 11) and analyzed for PFHxA in plasma and serum. (Individual
data for eight of the technicians are shown in Appendix C.2; complete data are available as the
supplemental information for Nilsson etal. (2013)). Personal and area air concentration
monitoring of the ski wax subjects and facilities demonstrated both the metabolic precursor, 6:2
FTOH, and PFHxA were present in all locations, but the arithmetic mean concentration of 6:2 FTOH
ranged from over 100 times higher than PFHxA to almost 100 times lower, across the monitoring
locations. A one-compartment model with first-order kinetics was used for PK analyses. The
estimated geometric mean half-life of PFHxA was 32 days with a range of 14-49 days in the studied
population (Russell etal.. 2013). PFHxA plasma concentrations declined below the plasma
detection limit of 0.05 ng/mL within a period of 2-4 months after exposure ceased, reflecting the
relatively rapid elimination rate ofPFHxA. In contrast, the half-life of PFHxS in humans was
estimated to range from 5 to 9 years fOlsen etal.. 20071.
This document is a draft for review purposes only and does not constitute Agency policy.
3-12	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Toxicological Review ofPFHxA and Related Salts
Analysis by Luz etal. (2019) found no significant species- or sex-related differences in the
elimination kinetics ofPFHxA. The PK analysis, however, is attributed to a meeting abstract (Buck
and Gannon. 20171 and provides no details of the methods the authors used. The text of Luz et al.
f20191 indicates the analysis of Buck and Gannon f20171 used data from only 3 of the 11 subjects of
Nilsson etal. f20131. specifically the 3 with the most rapid elimination, reducing the extent to which
the conclusion can be reliably extrapolated to the population as a whole. Luz etal. (2019) state
slower apparent elimination could occur in some subjects because of ongoing exposure. Although
ongoing exposure could cause this effect, it is also possible that elimination in some individuals is
slower than others due to interindividual variability. In the absence of independent evidence that
ongoing exposure occurred in other human subjects of Nilsson etal. f20131 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. (2013). 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. Finally, only the data collected encompassing the 2007-2008 ski
season, during which only 8 of the 11 technicians were sampled, includes post-exposure samples
needed to quantify elimination. A detailed description of EPA's analysis of the 8 technicians
sampled during the 2007-2008 season is provided in Appendix C.2. Briefly, each ski-wax
technician in the study was presumed to have a constant rate of exposure up to a date that is
different for each individual when exposure stopped and elimination began. Specifically, we used a
one-compartment i.v.-infusion model to fit the data:
Where A = dose/Va, tinf is the time period of exposure (treated as an infusion), and ke is the
elimination rate. The model is analyzed through hierarchical Bayesian analysis, with A and tinf
estimated independently for each individual technician although the technician-level ke is drawn
from a population-level distribution. Note blood concentrations were measured only once a month
and no other data on exposure is available. Thus, although the model clearly simplifies the
exposure estimation, estimating a larger number of parameters reliably would not be possible. As
such, the model allows for estimating variation among individuals without subjectively selecting a
subset of the technicians for analysis. The resulting distribution of ke had a mean (90% confidence
interval, CI) of 0.00252 (0.00136-0.00477) h_1. Using an average Vd of 0.7315 L/kg (731.5 mL/kg)
for male and female monkeys from Chengelis etal. (2009a). the resulting mean for human clearance
is CL = Vd-ke = 1.84 mL/kg-h. Given the expected similarity of Vd across mammalian species, EPA
(3-2)
This document is a draft for review purposes only and does not constitute Agency policy.
3-13
DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Toxicological Review ofPFHxA and Related Salts
considers the average value estimated for rats (0.33 L/kg) to be a reasonable lower bound for
humans and the highest value reported by Chengelis et al. f2009al for an individual (male) monkey
(1.54 L/kg) to be a reasonable upper bound. Combining these with the 90% CI for ke (0.00136-
0.00477 h_1) yields a possible range for human clearance of 0.45-7.35 mL/kg-h, a range of 16-fold
from 4-fold above to 4-fold below the estimated mean.
Xiao etal. (2011) measured the serum concentrations of 10 PFAA chemicals in 227
nonoccupational^ exposed individuals aged 0.3-90 years (133 males and 94 females) in China.
Significant positive correlations were observed between age and serum levels of PFAA chemicals
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 ofPFHxA in human bodies, which is
consistent with the relatively short half-life.
3.1.5.	PBPK Models
No PBPK model is available for PFHxA in rats, mice, or monkeys. Fabrega etal. (2015)
described a PBPK model for multiple PFAS in humans, including PFHxA. However, Fabrega etal.
(2015) 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. f20071 epidemiological survey of
PFAS exposure in residents of Catalonia, Spain. Because PFHxA was not detected in any individuals
sampled by Ericson et al. (2007). 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. (2007) with urinary clearance rates are not
reported in either paper. Thus, uniquely identifying two parameters with a single combination of
average PFHxA exposure and average blood concentration is impossible. Finally, as described
above (Distribution, Distribution in Humans), the tissue: blood partition coefficients Fabrega etal.
(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 Fabrega etal. (2015) is not considered sufficiently suitable for use in this
assessment.
3.1.6.	Summary
The PFHxA elimination half-lives and clearance values reported in studies are important for
interpreting and quantifying health outcomes potentially associated with PFHxA exposure. The
most notable finding was the apparent sex-specific PK differences between male and female mice
and rats where female rodents eliminate PFHxA 2-3 times faster than males (see Table 3-1).
Although monkeys have half-lives and clearance values in the same range as mice and rats, the
clearance in female monkeys is only 11% faster than in males. This indicates that the significant
sex differences observed in rodents does not appear to apply to primates. Humans have a much
This document is a draft for review purposes only and does not constitute Agency policy.
3-14	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Toxicological Review ofPFHxA and Related Salts
longer serum elimination half-life (EPA estimate: 275 hours) than rodents and monkeys (2-7
hours). The difference could be a consequence of species differences in the expression or activity of
the renal transporters that reabsorb PFAS, but this has not been demonstrated. All available PK
evidence is summarized below in Table 3-1.
According to EPA's BW0-75 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 hours among male and female rats (Table
3-1), one would then predict half-lives of 1.6-57 hours in humans, 20-200 times lower than the
range estimated by Russell etal. f20131 and 10-100 times lower than the range estimated by EPA
(Table 3-1). Thus, based on the PFHxA-specific PK data, use of BW0 75 for dosimetric extrapolation
could lead to an underprediction of human elimination by 1-2 orders of magnitude. Therefore, use
of BW°75 as an alternative means of extrapolation is not considered further for PFHxA, and the
preferred, data-driven approach will be used for the dosimetric extrapolation.
This document is a draft for review purposes only and does not constitute Agency policy.
3-15	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 3-1. Summary of PK evidence for PFHxA
Species/Sex
Study design (dose)
Elimination half-
life (beta) (h)
AUC/dose
(kg-h/L)
Clearance
(mL/h-kg)
Volume of
distribution (mL/kg)
Reference
Rats
Male
Single i.v. dose (10 mg/kg)
1.0
8.7
116
175
Chengelis et al. (2009a)

Single oral dose (50 mg/kg)
2.2
10.0
NR
NR


Single oral dose (150 mg/kg)
2.4
6.1
NR
NR


Single oral dose (300 mg/kg)
2.5
8.4
NR
NR

Female
Single i.v. dose (10 mg/kg)
0.42
1.3
775
466


Single oral dose (50 mg/kg)
2.6
2.4
NR
NR


Single oral dose (150 mg/kg)
2.2
2.2
NR
NR


Single oral dose (300 mg/kg)
2.1
3.5
NR
NR

Male
Single i.v. dose (40 mg/kg)
8.0 ±2.2
7.4 ±0.7
136 ± 13
430 ±112
Dzierlenga et al. (2019)

Single oral dose (40 mg/kg)
9.3 ±20.8
9.7 ± 1.3
103 ± 13
601± 470
NTP (2017)

Single oral dose (80 mg/kg)
5.7 ±4.6
6.6 ±0.5
153 ± 11
496 ±81


Single oral dose (160 mg/kg)
14 ± 14
6.8 ±0.6
147 ± 14
615 ± 367

Female
Single i.v. dose (40 mg/kg)
7.3 ±2.0
3.1 ±0.3
327 ±33
223 ±45


Single oral dose (40 mg/kg)
2.3 ±213
6.1 ± 1.1
164 ± 29
327± 149


Single oral dose (80 mg/kg)
5.5 ±2.6
3.2 ±0.4
314 ±39
560 ±113


Single oral dose (160 mg/kg)
12 ±24
3.7 ±0.5
274 ±37
473 ±158

Male
Single oral dose (2 mg/kg)
1.7 ±0.6
8± 1.5
NR
NR
Gannon et al. (2011)

Single oral dose (100 mg/kg)
1.5 ±0.2
6.5 ± 1.4
NR
NR

Female
Single oral dose (2 mg/kg)
0.5 ±0.1
2.5 ±0.5
NR
NR


Single oral dose (100 mg/kg)
0.7 ±0.3
2.5 ±0.7
NR
NR

This document is a draft for review purposes only and does not constitute Agency policy.
3-16	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Species/Sex
Study design (dose)
Elimination half-
life (beta) (h)
AUC/dose
(kg-h/L)
Clearance
(mL/h-kg)
Volume of
distribution (mL/kg)
Reference
Male
Single i.v. dose (0.1 mg/kg)
2.7
9.8
NR
400
Iwabuchi et al. (2017)
Male
Single inhalation3 (0.5 ppm)
1.3
NDb
107
NR
Kabadi et al. (2018)
Single inhalation3 (5.0 ppm)
1.3
NDb
277
NR
Female
Single inhalation3 (0.5 ppm)
0.5
NDb
107
NR
Single inhalation3 (5.0 ppm)
0.5
NDb
277
NR
Mice
Male
Single oral dose (2 mg/kg)
ND
12
NR
NR
Gannon et al. (2011)
Single oral dose (100 mg/kg)
ND
12
NR
NR
Female
Single oral dose (2 mg/kg)
ND
4
NR
NR
Single oral dose (100 mg/kg)
ND
6.4
NR
NR
Monkeys
Male
Single i.v. dose (10 mg/kg)
5.3 ±2.5
8.4 ± 1.8
122 ± 24
989 ± 579
Chengelis et al. (2009a)
Female
Single i.v. dose (10 mg/kg)
2.4 ± 1.7
7.5 ± 1.3
136 ± 22
474 ± 349
Humans
Males and females
Post-exposure observation
768 (336-1,176)
275 (145-509)
ND
ND
1.84 (0.45-7.35)
ND
Russell et al. (2013)
Current analysis
1	i.v. = intravenous; ND = not determined; NR = not reported.
2	a6-hour inhalation exposure to 6:2 FTOH.
3	bDose of PFHxA unknown.
This document is a draft for review purposes only and does not constitute Agency policy.
3-17	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Toxicological Review ofPFHxA and Related Salts
3.2. NONCANCER EVIDENCE SYNTHESIS AND INTEGRATION
For each potential health effect discussed below, the synthesis describes the evidence base
of available human and animal studies. The PFHxA animal literature inventory summarizes the
evidence base on potential health effects (organized by organ or system) from the available high
and medium confidence short-term, developmental, subchronic, and chronic studies in mice and
rats (NTP. 2018: Klaunig etal.. 2015: Iwai and Hoberman. 2014: Chengelis etal.. 2009b: Loveless et
al.. 20091. 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
Two epidemiological studies report on the relationship between PFHxA exposure and liver
enzymes. Of these, one (Tiang etal.. 2014). a cross-sectional study of pregnant women in China, was
critically deficient in the confounding domain and was considered overall uninformative. There was
no consideration of potential confounding in the study design and analysis, including potential
confounding by age, alcohol consumption, medical history, and socioeconomic status. Based on
these deficiencies, the study was excluded from further analysis (see Figure 3-1). The remaining
study (Nian etal.. 2019) was cross-sectional and was classified as medium confidence (see Figure 3-
1). Exposure levels for PFHxA, however, were low and contrast across the study population was
narrow (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
This document is a draft for review purposes only and does not constitute Agency policy.
3-18	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
Toxicological Review ofPFHxA and Related Salts
alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein, alkaline
phosphatase (ALP), y-glutamyl transferase (GGT), total bilirubin, or cholinesterase.
j5S^' vfc®5"
Participant selection -
Legend
Outcome ascertainment - +
Exposure measurement
+
y Good (metric) or High confidence (overall)
+ Adequate (metric) or Medium confidence (overall)
- Deficient (metric) or Low confidence (overall)
NR Not reported
Confounding
a:
Critically deficient (metric) or Uninformative (overall]
Not applicable
Analysis -
Sensitivity - N/A
Selective Reporting - N/A
Overall confidence
Figure 3-1. Study evaluation for human epidemiological studies reporting
hepatic system findings from PFHxA exposures (full details available by
clicking the HAWC link). Note that for N/A, critical deficiencies in confounding
domains were identified and the study was judged as uninformative; thus, the
remaining domains were not evaluated.
Animal
Hepatic outcomes were evaluated in multiple short-term, subchronic, or chronic studies in
rats and mice (NTP. 2018: Klaunig etal.. 2015: Iwai and Hoberman. 2014: Chengelis etal.. 2009b:
Loveless etal.. 2009). Generally, studies were rated as medium or high confidence for the hepatic
outcomes, but some outcome-specific considerations for study evaluation were influential on the
overall confidence ratings for hepatic effects. Histopathology for Chengelis etal. f2009bl was rated
low confidence because of issues related to observational bias, endpoint sensitivity and specificity,
and results presentation. Results of the outcome-specific confidence evaluations are presented in
Table 3-2 below, and details are available by clicking the HAWC link.
This document is a draft for review purposes only and does not constitute Agency policy.
3-19
DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Toxicological Review ofPFHxA and Related Salts
Table 3-2. Evaluation results for animal studies assessing effects of PFHxA
exposure on the hepatic system
Author (year)
Species, strain
(sex)
Exposu re
design
Exposure route and
dose range
Organ weight
Histopathology
Clinical chemistry
Peroxisomal beta
oxidation
NTP (2018)
Rat, Harlan
Sprague-Dawley
(male and female)
Short term
(28 d)
Gavage3
Male and female: 0,
62.5, 125, 250, 500,
1,000 mg/kg-d
+ +
+ +
++
NM
Chengelis et al.
(2009b)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and female)
Subchronic
(90 d)
Gavage3
Male and female: 0,10,
50, 200 mg/kg-d
+ +

++

Loveless et al.
(2009)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and female)
Subchronic
(90 d)
Gavageb
Male and female: 0, 20,
100, 500 mg/kg-d
+ +
+ +
++
++
Klaunig et al.
(2015)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and female)
2-yr cancer
bioassay
Gavage3
Male: 0, 2.5,15,
100 mg/kg-d
Female: 0, 5, 30,
200 mg/kg-d
NM
+ +
++
NM
Study evaluation for animal toxicological hepatic endpoints reported from studies with male and female rats
receiving by gavage PFHxA3 or PFHxA sodium saltb. 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 (see Figure 3-2; exposure response array link! Relative liver
weights (see Table 3-3), are generally considered more reliable than absolute liver weights because
they take into account large variations in body weight that could skew organ weight interpretation
fHall etal.. 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
This document is a draft for review purposes only and does not constitute Agency policy.
3-20	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
1	200 mg/kg-day in males (with no change in females) in one study (Chengelis etal.. 2009bl. 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
Liver Weight, Absolute
Study
NTP, 2018,4309149
Experiment
28-Day Oral
Chengelis,2009,2850404 90-DayOra
Loveless, 2009, 2850369 90-DayOra
Liver Weight,/^solute, Recovery Chengelis, 2009,2850404 90-DayOra
Liver Weight, Relative
NTP, 2018,4309149 28-DayOra
Chengelis,2009,2850404 90-DayOra
Loveless, 2009, 2850369 90-Day Ora
Liver Weight, Relative, Recovery Chengelis, 2009,2850404 90-Day Ora
Animal Description
Rat, Harlan Sprague-Dawley(cv)
Rat, Harlan Sprague-Dawley(v)
Rat, Crl:CD(SD) 0)
Rat, Crl:CD(SD) ($)
Rat, Crl:CD(SD) 0)
Rat, Crl:CD(SD) ($)
Rat, Crl:CD(SD) 0)
Rat, Crl:CD(SD) ($)
Observation Time
Day29
Day29
Day90
Day90
Day92
Day93
Day 118
Day 118
PFHxA Hepatic Effects: Liver Weight
Rat, Harlan Sprague-Dawley(cv)
Rat, Harlan Sprague-Dawley(v)
Rat, Crl:CD(SD) 0)
Rat, Crl:CD(SD) ($)
Rat, Crl:CD(SD) 0)
Rat, Crl:CD(SD) ($)
Rat, Crl:CD(SD) 0)
Rat, Crl:CD(SD) ($)
N o significant changg^ Significant increase^ Significant decrease ^ Significant Trencj
Day29
Day29
Day90
Day90
Day92
Day93
Day 118
Day 118
-A
A
100 0 100 200 300 400 500 600 700 800 900 1,0001,100
Dose (mg/kg-day)
Figure 3-2. Liver weights (absolute and relative) after short-term and
subchronic PFHxA exposures (full details available by clicking the HAWC link).
Table 3-3. Percent increase in relative liver weight due to PFHxA exposure in
short-term and subchronic oral toxicity studies
Study Design and
Reference
Dose (mg/kg-d)
in

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Toxicological Review ofPFHxA and Related Salts
Histopathology
Treatment-related increases in liver weight can result from various changes in hepatic
morphology including hyperplasia of any resident liver cell type, cellular hypertrophy
inflammation, fibrosis, increase in hepatocyte size, neoplasia, congestion, or metabolic enzyme
induction (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 short term
and subchronic studies. Hepatic hypertrophy can refer to an increase in liver weight and size; an
increase in hepatocyte size caused by abnormal storage of water, glycogen, lipids, or organelle
proliferation; and an increase in hepatic enzyme induction fHall etal.. 2012: Thoolen etal.. 2010:
U.S. EPA. 2002al. Coherent with findings on liver weight, the observations of hepatocellular
hypertrophy were dose-dependent and male rats were more sensitive than females. Specifically,
increased hepatocellular hypertrophy was observed in adult male and female rats in the high
confidence short-term (NTP. 20181 and high confidence subchronic (Loveless etal.. 20091 studies
at doses >100-500 mg/kg-day. In the subchronic study, hypertrophy persisted 30 and 90 days
after recovery in males, and 30 days after recovery in females fLoveless et al.. 20091. In the low
confidence (for histopathology outcomes) subchronic study, centrilobular hepatocellular
hypertrophy was observed in male rats only (incidence 7/10, 200 mg/kg-day) and resolved after
28-day recovery (Chengelis etal.. 2009b). In the chronic study (Klaunig etal.. 2015). hepatocellular
hypertrophy findings were null consistent with null observations at similar doses in the short-term
and subchronic studies.
Table 3-4. Incidence of hepatocellular hypertrophy findings in adult rats due
to PFHxA exposure in short-term and subchronic oral toxicity studies
Study Design and Reference
Dose (mg/kg-d)
o
*—1
o
o
lO
LO
rsi
ID
o
o
*—i
lO
fsj
*—1
o
o
o
LO
fsj
o
o
LO
OOO'I
28-d, female rat(NTP, 2018)



0/10

0/10

0/10
0/10
9/10
28-d, male rat (NTP, 2018)



0/10

0/10

0/10
9/10
10/10
90-d, female rat
(Chengelis et al., 2009b)
0/10

0/10



0/10



90-d, male rat
(Chengelis et al., 2009b)
0/10

0/10



7/10



90-d, female rat
(Loveless et al., 2009)

0/10


0/10



5/10

90-d, male rat
(Loveless et al., 2009)

0/10


4/10



10/10

This document is a draft for review purposes only and does not constitute Agency policy.
3-22	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Toxicological Review ofPFHxA and Related Salts
Study Design and Reference
Dose (mg/kg-d)
o
*—1
o
fsj
O
lO
LO
rsi
ID
O
O
*—1
lO
fsj
*—1
O
o
fsj
O
lO
fsj
O
O
lO
OOO'I
90-d, female rat, 30-day recovery
(Loveless et al., 2009)








4/10

90-d, female rat, 90-day recovery
(Loveless et al., 2009)








0/10

90-d, male rat, 30-day recovery
(Loveless et al., 2009)








9/10

90-d, 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 reported in a short term
study at 1,000 mg/kg-day PFHxA fNTP. 20181 and a subchronic study (200 mg/kg-day (Chengehs
et al.. 2009b)). In the high confidence chronic study, Klaunig etal. (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). The authors noted most necrosis findings
were in animals that died or were euthanized prior to scheduled necropsy and the increased
mortality was not treatment related, but was due to mechanical injury, gavage trauma, reflux injury,
or spontaneous disease processes (Klaunig 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) fKlaunig etal.. 20151. 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. 20181], All results reported above can be viewed using the HAWC link.
Clinical Chemistry
A clinical chemistry panel measures the proteins, enzymes, chemicals, and waste products
in the blood. These measures, when evaluated together and with other biomarkers are informative
This document is a draft for review purposes only and does not constitute Agency policy.
3-23	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Toxicological Review ofPFHxA and Related Salts
diagnostic tests of organ function and when interpreted together with histopathology are useful for
the assessment of adverse liver effects.
Serum Enzymes
Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are often useful
indicators of hepatic enzyme induction or hepatocellular damage as increased serum levels are
thought to be due to hepatocyte damage resulting in release into the blood, whereas ALP is
localized to the bile canalicular membrane and more indicative of hepatobiliary damage (Hall etal..
2012: Amacher et al.. 19981. 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 (Klaunig etal.. 2015). but the highest dose was 100 or 200 mg/kg-day PFHxA in male
or female rats, respectively. Full study details are available in Figure 3-3 and by clicking the HAWC
link. Percent changes in treated relative to controls are provided in Table 3-5, Table 3-6, and
Table 3-7.
Specifically, in the short-term study, ALT, AST, and ALP were increased in a dose-response
gradient in adult male rats at doses as low as 500 mg/kg-day fNTP. 20181. In female rats, ALT and
AST measures were increased in a dose-response gradient at doses as low as 500 mg/kg-day,
whereas ALP was increased only in the highest (1,000 mg/kg-day) dose group (NTP. 2018).
ALT increases were observed only in male rats at PFHxA sodium salt exposures as low as 20
mg/kg-day in one subchronic study (Loveless et al.. 2009) and in the highest PFHxA dose group
(200 mg/kg-day) in the other subchronic study fChengelis et al.. 2009bl. AST was increased in only
one subchronic study in males at >20 mg/kg-day (Loveless et al.. 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 (see Table 3-6).
ALP was increased in both subchronic studies with significant increases observed in the
highest exposure groups [200 (Loveless etal.. 2009) and 500 mg/kg-day (Chengelis etal.. 2 009b)]
that resolved by the 30-day recovery (see Table 3-7). The chronic study did not include a 13-week
endpoint that would have been useful for group mean comparisons with the test measures in the
subchronic studies (as clinical pathology test values often change with animal age) (AACC. 1992).
This document is a draft for review purposes only and does not constitute Agency policy.
3-24	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Endpoint
Study
Experiment
Animal Description
Observation Time

PFHxA Hepatic Effects: Serum Biomarkers
Alanine Aminotransferase (ALT)
NTP, 2018,4309149
28-Day Oral
Rat, Harlan Sprague-Dawley(i?)
Day 29
^^



Rat, Harlan Sprague-Dawley(2)
Day 29
V"*—•
• A A

Chengelis, 2009, 2850404
90-Day Oral
Rat
Crl:CD(SD) ((J)
Day 90
••	A
Rat
Crl:CD(SD)(2)
Day 90


Loveless, 2009, 2850369
90-Day Oral
Rat
Crl:CD(SD) ((J)
Day 92
«A-A-
	A
Rat
Crl:CD(SD)(2)
Day 93


Klaunig, 2015,2850075
2-Year Cancer Bioassay
Rat
Crl:CD(SD) ((J)
Week 26
•—•
Rat
Crl:CD(SD)(2)
Week 26

Rat
Crl:CD(SD) ((J)
Week 52
•—•
Rat
Crl:CD(SD)(2)
Week 52

Alanine Aminotransferase (ALT), Recovery
Chengelis, 2009, 2850404
90-Day Oral
Rat
Crl:CD(SD) ((J)
Day 118
•	~



Rat
Crl:CD(SD)(2)
Day 118
~	

Alkaline Phosphatase (ALP)
NTP, 2018,4309149
28-Day Oral
Rat, Harlan Sprague-Dawley(i?)
Day 29

• A A



Rat, Harlan Sprague-Dawley(2)
Day 29

-------
Toxicological Review ofPFHxA and Related Salts
Study Design and Reference
Dose (mg/kg-d)
in

-------
Toxicological Review ofPFHxA and Related Salts
Study Design and Reference
Dose (mg/kg-d)
in

-------
1
2
3
4
5
6
7
8
9
10
11
Toxicological Review ofPFHxA and Related Salts
particularly a decrease, can be indicators of protein loss due to kidney disease or impeded
production in the liver, such as in liver disease fBoron and Boulpaep. 20171. Blood protein
measures (total protein and globulin) were, in general, decreased across short-term fNTP. 20181.
and subchronic fChengelis etal.. 2009b: Loveless etal.. 20091 studies, with consistent and coherent
dose-dependent findings across study designs. No PFHxA-related treatment effects on blood
proteins were found in the chronic study at doses up to 100 or 200 mg/kg-day PFHxA (the highest
doses administered) in male or female rats, respectively. The pattern of findings suggests a
primary effect on blood globulins (decreased) in response to PFHxA exposure that was driving
decreases in total protein and increases in the albumin:globulin ratio (A:G). These findings are
discussed below and detailed information can be viewed in Figure 3-4 or by clicking on the HAWC
link.
This document is a draft for review purposes only and does not constitute Agency policy.
3-28	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
End point
Study Experiment
Animal Description
Observation Time


PFHxA Hepatic Effects: Serum Proteins
Albumin (A)
NTP, 2018,4309149 28-DayOral
Rat, Harlan Sprague-Dawley ((f)
Day 29
¥-
•-Y

—•	~


Rat, Harlan Sprague-Dawley (*)
Day 29


Chengelis, 2009, 2850404 90-Day Oral
Rat, Crl:CD(SD) ((f)
Day 90



Rat, Crl:CD(SD) (2)
Day 90


Loveless, 2009, 2850369 90-DayOral
Rat, Crl:CD(SD) ((f)
Day 92



Rat, Crl:CD(SD) (2)
Day 93


Klaunig, 2015, 2850075 2-Year Cancer Bioassay
Rat, Crl:CD(SD) ((f)
Week 26
m—#


Rat, Crl:CD(SD) (2)
Week 26



Rat, Crl:CD(SD) ((f)
Week 52
•—•


Rat, Crl:CD(SD) (-2)
Week 52

Albumin (f§, Recovery
Chengelis, 2009, 2850404 90-Day Oral
Rat, Crl:CD(SD) ((f)
Day 118
•	•


Rat, Crl:CD(SD) (2)
Day 118
•-



• m l. i- r> l-
NTP, 2018,4309149 28-DayOral
Rat, Harlan Sprague-Dawley ((f)
Day 29


-r -
- - x 		~k~ '

• - " " "


Rat, Harlan Sprague-Dawley (2)
Day 29
A


m A


~




Chengelis, 2009, 2850404 90-Day Oral
Rat, Crl:CD(SD) ((f)
Day 90



Rat, Crl:CD(SD) (2)
Day 90


Klaunig, 2015, 2850075 2-Year Cancer Bioassay
Rat, Crl:CD(SD) ((f)
Week 26
•—•


Rat, Crl:CD(SD) (2)
Week 26



Rat, Crl:CD(SD) ((f)
Week 52
m—•


Rat, Crl:CD(SD) (2)
Week 52

Pi bum in/Globulin (A/G) Ratio,
Chengelis, 2009, 2850404 90-Day Oral
Rat, Crl:CD(SD) ((f)
Day 118


-A

Recovery









Rat, Crl:CD(SD) (2)
Day 118
•-



Globulin (G)
NTP, 2018,4309149 28-DayOral
Rat, Harlan Sprague-Dawley ((f)
Day 29

m V
w
W W
V





Rat, Harlan Sprague-Dawley (2)
Day 29
A


¦ V


yr




Chengelis, 2009, 2850404 90-Day Oral
Rat, Crl:CD(SD) ((f)
Day 90
++—~


Rat, Crl:CD(SD) (2)
Day 90
m—~

Loveless, 2009, 2850369 90-DayOral
Rat, Crl:CD(SD) ((f)
Day 92



—¥


Rat, Crl:CD(SD) (2)
Day 93


Klaunig, 2015, 2850075 2-Year Cancer Bioassay
Rat, Crl:CD(SD) ((f)
Week 26
•—•


Rat, Crl:CD(SD) (2)
Week 26



Rat, Crl:CD(SD) ((f)
Week 52
•—*


Rat, Crl:CD(SD) (2)
Week 52

Globulin (G), Recovery
Chengelis, 2009, 2850404 90-Day Oral
Rat, Crl:CD(SD) ((f)
Day 118
•	~


Rat, Crl:CD(SD) (2)
Day 118




Total Protein (TP)
NTP, 2018,4309149 28-DayOral
Rat, Harlan Sprague-Dawley ((f)
Day 29



v ~
V





Rat, Harlan Sprague-Dawley (2)
Day 29
A





V




Chengelis, 2009, 2850404 90-Day Oral
Rat, Crl:CD(SD) ((f)
Day 90
M	~


Rat, Crl:CD(SD) (2)
Day 90


Loveless, 2009, 2850369 90-DayOral
Rat, Crl:CD(SD) ((f)
Day 92



—~





~

—~


Rat, Crl:CD(SD) (2)
Day 93


Klaunig, 2015, 2850075 2-Year Cancer Bioassay
Rat, Crl:CD(SD) ((f)
Week 26
•—•


Rat, Crl:CD(SD) (2)
Week 26



Rat, Crl:CD(SD) ((f)
Week 52
•—•


Rat, Crl:CD(SD) (2)
Week 52

Total Protein (TP), Recovery
Chengelis, 2009, 2850404 90-Day Oral
Rat, Crl:CD(SD) ((f)
Day 118
•	 ~


Rat, Crl:CD(SD) (2)
Day 118
* •
• No significant chango^ Significant increase^^Signilicant decrease ) Significant Trendj -100 0 100 200 300 400 500 600 700 800 900 1,0001,100
Dose (mg/kg-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; see Table 3-8)—the total amount of albumin and globulin found
in blood, is associated with chronic liver disease fWhalan. 20151—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.
20181. A dose-responsive decrease (6-14%, >100 mg/kg-day) in TP also was observed in male rats
f Chengelis etal.. 2009b: Loveless et al.. 20091 with decreased levels observed in males (-6%, 200
mg/kg-day) at the 30-day recovery (Chengelis etal.. 2009b). Albumin is a major blood protein that
binds fatty acids, cations, bilirubin, thyroxine (T4), and other compounds. Decreased albumin
levels are associated with decreased synthesis in the liver, increased catabolism, severe diffuse liver
This document is a draft for review purposes only and does not constitute Agency policy.
3-29	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Toxicological Review ofPFHxA and Related Salts
disease, subacute hepatitis, hepatocellular damage, ascites, cirrhosis, and chronic alcoholism
fWhalan. 20151. Slight decreases (p < 0.05) in albumin were reported only in males exposed for
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 chronic study (see Table 3-9). Globulin decreases
were observed in both male and female rats treated with PFHxA in the short-term study at
>125 mg/kg-day and 1,000 mg/kg-day, respectively fNTP. 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 100 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) due to PFHxA exposure in
short-term, subchronic, and chronic oral toxicity studies
Study Design and
Reference
Dose (mg/kg-d)
in

-------
Toxicological Review ofPFHxA and Related Salts

Dose (mg/kg-d)
Study Design and
Reference
in

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Toxicological Review ofPFHxA and Related Salts
Hepatobiliary Components
Other indicators of potential liver dysfunction or injury included impacts on bile
components essential for normal lipid metabolism and red blood cell breakdown. ALP (discussed
with serum enzymes and in Table 3-7) is an indicator of bile duct obstruction and was consistently
increased in male and female rats in the short-term study (NTP. 2018) and subchronic studies
(Chengelis etal.. 2009b: Loveless et al.. 2009). In the short-term study (NTP. 2018). bile acids were
increased at the highest dose (1,000 mg/kg-day) with a significant trend (a possible indication of
cholestatic liver injury), and bilirubin was decreased in a dose-response gradient across both the
short-term and subchronic fLoveless etal.. 20091 studies (see Figure 3-5). Lower than normal
bilirubin levels are usually not a concern and can be reduced in response to increased conjugation
rates after hepatic enzyme induction and excretion into bile fHall etal.. 20121.
Bidpoint	Study	Study Type	Animal Description	Observation Time	PFHxA Hepatic Effects: Hepatobiliary Components
Bile Salt/A:ids
NTP, 2018, 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley^)
Day29
.... A



Rat, Harlan Sprague-Dawley($)
Day29
.... A
Direct Bilirubin
NTP, 2018, 4309149
28-Day Oral
Rat, Harlan Sprague-Dawley^)
Day29
•-
• •
—•	
	•	
»



Rat, Harlan Sprague-Dawley($)
Day29
•-
• •
—•	
	•	
	#

Loveless, 2009, 2850369
90-Day Oral
Rat, Crl:CD(SD) (
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Toxicological Review ofPFHxA and Related Salts
(males, 3.1- and 4.36-fold, respectively; females, 1.45- and 2.67-fold, respectively). Notably,
increased activity persisted after the 30-day recovery and male rats were more sensitive than
females, with males in the 100 mg/kg-day group also showing increased peroxisomal beta
oxidation fLoveless etal.. 20091.
Kndpoint
Study
Experiment
Animal Description
Observation Time
Peroxisomal Beta Oxidation
Perosixomal Beta Oxidation
Loveless, 2009,2850369
90-Day Oral
Rat, Crl:CD(SD) (	A



Rat, Crl:CD(SD) (9)
Day 10
» •	A



Rat. Crl:CD(SD) ((f)
Day 92
-A A



Rat, Crl:CD(SD) (9)
Day 93
« •	A
Peroxisomal Beta Oxidation
Chengelis, 2009, 2850404
90-Day Oral
Rat, Crl:CD(SD) ((f)
Day 90
• A



Rat, Crl:CD(SD) (9)
Day 90

Peroxisomal Beta Oxidation (30-Day
Recovery)
Loveless, 2009, 2850369
90-Day Oral
Rat, Crl:CD(SD) ((f)
Day 30 PE
•	~



Rat, Crl:CD(SD) (9)
Day 30 PE
• A
	T	=	J-	1 -100 0 100 200 300 400 500 600 700 800 900 1,0001,100
• No significant change /\ Significant increase Significant decrease W Significant Trend I	„ . „ . .
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).
Considerations related to human relevance
The induction of both PPARa and CAR target gene expression were observed after PFHxA
exposure in both the short-term and subchronic rodent studies (NTP. 2018: Chengelis etal.. 2009b:
Loveless etal.. 2009). Specifically, in the short-term study, NTP (2018). in vivo PFHxA exposure
elicited significant and dose-related increases in the liver expression of the PPARa target genes
acyl-CoA oxidase[Acoxl, up to 2-fold increase) and cytochrome P450 4al (Cyp4al, up to 12.5-fold
increase). In the same short-term study, constitutive androstane receptor (CAR) target genes
cytochrome P450 2bl (Cyp2bl, up to 7-fold increase) and cytochrome P450 2b2 (Cyp2b2, up to 3-
fold increase) were also induced after PFHxA exposure. Functional evidence of PPARa activation by
PFHxA exposure was provided by the NTP (2018) short-term study where increases (up to 16-fold)
in Acyl-CoA oxidase activity in male rats receiving >250 mg/kg-day PFHxA (not measured in
females).
The hepatic effects ofPFHxA exposure observed in rodents (including increased liver
weight, hepatocellular hypertrophy, and peroxisomal beta oxidation) could reflect species-specific
responses to chemical-induced liver toxicity. There was some evidence in vitro as to whether the
PFHxA similarly activated human PPARa. Wolf etal. f20081 examined, in vitro, PPARa activation by
PFHxA in C0S1 cells transfected with reporter gene constructs containing either the mouse or
human PPARa ligand binding domain fused to a Gal4 DNA binding domain under control of an SV-
40 promoter in a luciferase reporter plasmid. These assays indicated that both mouse and human
PPARa are activated by PFHxA in a treatment-related manner with PFHxA being a more potent
activator of the human (lowest observed effect concentration, LOEC = 10 |iM) than the mouse
(LOEC = 20 |j.M) receptor fWolfetal.. 20081. While the transactivation studies of Wolf etal. f20081
indicated PFHxA activation of both the mouse and human PPARa, significant effects were reported
This document is a draft for review purposes only and does not constitute Agency policy.
3-33	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Toxicological Review ofPFHxA and Related Salts
only for treated vs control within a species. The mean and variance were not reported by study
authors and it is not clear if there are significant differences in the degree of activation between the
examined species (mouse and human plasmids) in response to PFHxA treatment.
Further in vitro high throughput screening evidence for PFHxA effects in human cell lines
(including HepG2 and HepaRG cells) are available from EPA's CompTox Chemicals Dashboard fU.S.
EPA. 2018a) by clicking the following
("https://comptox.epa.gov/dashboard/dsstoxdb/results?search=DTXSID3031862" \1 "invitrodb").
After filtering out results flagged to be uncertain (e.g., high degree of variability) or occuring at high
concentrations associated with cytotoxicity fU.S. EPA. 2018b: Filer etal.. 2016: Filer. 20151.19
assay targets remained from human liver cell-based assays (at up to 200 [J.M PFHxA).
Transactivation assays in HepG2 cells indicated PFHxA treatment effects that included activation of
the transcription factors PPARa and hypoxia inducible factor 1 subunit alpha (HIFla, a
transcriptional regulator of genes involved in the hypoxia response). Gene expression assays in
HepaRG cells identified the induction of 16 genes including several cytochrome P450 family
members, transporters, kinases, and oxidase/oxidoreductase related activities that are primarily
involved in detoxification and / or lipid metabolism (see Table 3-10). Bioactivity data were not
available for PFHxA sodium salt or PFHxA ammonium salt Several of these genes were PPARa
targets, suggesting PFHxA activates human PPARa in vitro.
Table 3-10. Genes Targets Identified from EPA Chemicals Dashboard After
PFHxA Treatment in Human Liver Cell Lines
GENE SYMBOL
GENE NAME
AC50a
LOGAC50
BMADb
ABCG2*
ATP-binding cassette, sub-family G (WHITE), member 2 (Junior
blood group)
9.49
0.977
0.201
ACOX1*
acyl-CoA oxidase 1, palmitoyl
9.47
0.976
0.135
ADK
adenosine kinase
2.88
0.459
0.166
CYP2B6*
cytochrome P450, family 2, subfamily B, polypeptide 6
19.1
1.28
0.251
CYP2C19
cytochrome P450, family 2, subfamily C, polypeptide 19
5.21
0.717
0.187
CYP2C8*
cytochrome P450, family 2, subfamily C, polypeptide 8
7.88
0.896
0.216
CYP2C9
cytochrome P450, family 2, subfamily C, polypeptide 9
6.53
0.815
0.314
CYP3A7
cytochrome P450, family 3, subfamily A, polypeptide 7
10.3
1.01
0.259
CYP4A11
cytochrome P450, family 4, subfamily A, polypeptide 11
45.3
1.66
0.213
CYP4A22
cytochrome P450, family 4, subfamily A, polypeptide 22
57.6
1.76
0.269
FABP1*
fatty acid binding protein 1, liver
22.3
1.35
0.161
FM03
flavin containing monooxygenase 3
13.3
1.12
0.145
HMGCS2*
3-hydroxy-3-methylglutaryl-CoA synthase 2 (mitochondrial)
4.13
0.616
0.175
PDK4*
pyruvate dehydrogenase kinase, isozyme 4
20.9
1.32
0.161
SLCOIBI
solute carrier organic anion transporter family, member 1B1
9.82
0.992
0.181
UGT1A1
UDP glucuronosyltransferase 1 family, polypeptide A1
15.1
1.18
0.221
aAC50 - active concentration that elicited half maximal response.
bBMAD - baseline median absolute deviation.
* PPARa target gene (http://www.ppargene.org/index.php).
This document is a draft for review purposes only and does not constitute Agency policy.
3-34	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Toxicological Review ofPFHxA and Related Salts
Collectively, the available in vivo and in vitro evidence for PFHxA include potential for
rodent responses to be relevant to human exposure. The data also suggest similar PPARa
activation occurs in both rodents and humans (at least in vitro). Potential pathways such as PPARa
and CAR activation can contribute to some of the hepatic changes caused by PFHxA exposure,
including hypertrophy. Studies of the prototypical PPARa agonist, WY-14643, indicate an increased
sensitivity of rodents as compared to humans; however, the PFHxA-specific data do not
demonstrate such clear differences with this structurally different compound. PFHxA-specific data
informing possible biological pathways leading to the observed hepatic effects are sparse and many
uncertainties remain.
Evidence from other PFAS
Although no direct in vivo evidence is available for PFHxA effects in PPARa null rodent
models, PFAS exposures in PPARa null and humanized mouse models are available and useful for
considering human relevance. 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 etal. (2017) 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. (2009) also observed increased liver weight, hepatic lipid
accumulation, ALT increases >2-fold, and pathologically similar (severity and incidence)
hepatocellular hypertrophy in male SV129 wild-type SV and humanized PPARa mice exposed to
PFBA. 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 PFHxA and PFOA, PFNA, and PFBA it is inferred that PFHxA
exposure in these genetic mouse model systems would elicit similar effects.
Considerations for Potentially Adaptive Versus Adverse Responses
Considering that the hepatic effects ofPFHxA exposure (increased liver weight and
hepatocyte hypertrophy) observed in rodents could have been adaptive responses to chemical-
induced hepatotoxicity, the potential adversity of these effects was a key consideration and
analyzed. I n the absence of a known mechanism leading to increased liver weight, hepatocellular
hypertrophy, and necrosis, the evidence for PFHxA-mediated hepatotoxicity was evaluated to
inform interpretations regarding adversity utilizing guidance from Hall etal. f20121. Specifically,
Hall etal. f20121 states that, "when assessing a histological change caused by an increase in liver
This document is a draft for review purposes only and does not constitute Agency policy.
3-35	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Toxicological Review ofPFHxA and Related Salts
weight, in order to conclude whether the adverse or not, a number of steps must be carefully
considered:
1.	Is there histological evidence of structural degenerative or necrotic changes:
•	Hepatocyte necrosis, fibrosis, inflammation, and steatotic vacuolar degeneration
•	Biliary/oval cell proligeration, degeneration, fibrosis, and cholestasis
•	Necrosis and degeneration of other resident cells within the liver
2.	In the absence of histological changes, using a weight-of-evidence approach, is there clinical
pathology evidence of hepatocyte damage characterized by a dose dependent and
biologically significant and consistent increase in at least two liver parameters:
•	At least x 2 to x 3 increase in ALT
•	A biologically significant change in other biomarkers of hepatobiliary change (ALP, AST,
yGT, GLDH, etc.)
•	A biologically significant change in another clinical pathology marker indicating liver
dysfunction (albumin, bilirubin, bile acids, coagulation factors, cholesterol, triglycerides,
etc.)"
With regard to Step 1 above, histological evidence of structural change included necrosis in
females rats only (incidence of 12/70) receiving 200 mg/kg-day in the chronic study (note the
highest dose in male rats was half the female doselfKlaunigetal.. 20151. No proliferative indices
were noted and as discussed above, uncertainties remain regarding potential biological pathways
(including PPARa) leading to the PFHxA-mediated observed findings. Incidence of necrosis were
not observed in rats (male or female) from the short-term study (NTP. 2018). and the subchronic
studies by (Chengelis et al.. 2009b: Loveless etal.. 2009). Histological findings did include
increased incidence of hepatocellular hypertrophy from the short term and both subchronic
studies. Notably, hypertrophy findings persisted in both male and female rats 90-day after
recovery fLoveless etal.. 20091. With regard to Step 2 above, other liver parameter effects were
observed after PFHxA exposure and included increased peroxisomal beta oxidation in both
subchronic studies (Chengelis etal.. 2009b: Loveless et al.. 2009) that persisted at 30 days recovery
in both male and female rats (Loveless etal.. 2009). The serum enzymes ALT, AST, and ALP were
increased in a dose-responsive manner at the same or lower doses than the observed increases in
hepatocellular hypertrophy. Other parameters characterized by a dose-dependent PFHxA-
mediated effect included decreased globulin, decreased total protein, and decreased bilirubin.
Considering the Hall etal. f20121 criteria above, the observed increase in relative liver
weight and hepatocellular hypertrophy in rats exposed to PFHxA are interpreted as adverse, human
relevant, and potentially leading to increasingly severe outcomes such as necrosis.
This document is a draft for review purposes only and does not constitute Agency policy.
3-36	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Toxicological Review ofPFHxA and Related Salts
Evidence Integration
The human evidence base is limited to a single medium confidence study reporting null
associations between serum biomarker levels and PFHxA exposure. Based on these data, there is
indeterminate human evidence of hepatic effects.
The hepatic findings in rodents exposed to PFHxA included increased relative liver weight
observed with increased hepatocellular hypertrophy at doses as low as 100 mg/kg-day (Loveless et
al.. 20091 and 200 mg/kg-day (Chengelis etal.. 2009b) in male rats thatpersisted after 30 and 90
day recovery. Corroborative evidence for adverse hepatotoxicity included increased serum
enzymes, (e.g., ALT increased >2-fold) in the subchronic studies, although a dose-responsive
relationship was observed in the short term, but not the subchronic, studies. Serum enzyme
changes were not observed in the chronic study fKlaunig etal.. 20151. Hepatocellular necrosis was
observed in male rats in a high confidence short term study (NTP. 2018) at 1,000 mg/kd-day, low
confidence subchronic study (Chengelis etal.. 2009b) and in the high confidence chronic study
(female rats) (Klaunig et al.. 20151 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.. 20081. peroxisomal beta oxidation activity (NTP. 2018: Chengelis etal.. 2009b:
Loveless etal.. 20091. changes in gene expression for CAR and PPARa cytochrome P450 gene
expression (NTP. 20181. and in vivo PPARa knockout and humanized genetic mouse models
exposed to PFAS structurally similar to PFHxA fDas etal.. 2017: Rosen etal.. 2017: Foreman etal..
20091. Wolf etal. f20081 and Wolf etal. f 20141 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 (Chengelis et al.. 2009b: Loveless et al.. 2009) at a
dose as low as 100 mg/kg-day and this effect persisted after the 30-day recovery (Loveless etal..
2009). 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: Rosen et al..
2017: Foreman et al.. 20091 that found similar levels of increased liver weight and incidence of
This document is a draft for review purposes only and does not constitute Agency policy.
3-37	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
1	hepatocellular hypertrophy when comparing between PPARa knockout, humanized, and wild-type
2	mouse models.
3	Overall, the currently available evidence indicates that PFHxA likely causes hepatic effects
4	in humans under relevant exposure circumstances. This conclusion is based on studies of animals
5	showing increased liver weight, hepatocellular hypertrophy, increased serum enzymes (>2-fold
6	ALT), and decreased serum globulins generally occurring at >100 mg/kg-day within the evidence
7	base of four primarily high confidence studies of short-term, subchronic, and chronic PFHxA
8	exposure in (primarily male) rats. The findings in rats were determined to be adverse and relevant
9	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.
3-38	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 3-11. Evidence profile table for hepatic effects
Evidence stream summary and interpretation
Evidence integration summary
judgment
Evidence from studies of exposed humans

Studies and
confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key
findings
Evidence stream
judgment
Evidence indicates (likely)
Serum
Biomarkers
1 low confidence
study
• No factors noted
• Low confidence
study (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-d
90-d (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 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-d
90-d
2-yr
•	Consistent cellular
hypertrophy across
studies and sexes
•	Coherence with liver
weight
•	Dose-response for
hypertrophy
•	Concerning severity of
effect— necrosis (with
• No factors noted
•	Cellular
hypertrophy at
>100 mg/kg-d;
stronger in
males
•	Necrosis in
males at 200
and 1,000
mg/kg-d and
This document is a draft for review purposes only and does not constitute Agency policy.
3-39	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts





Evidence integration summary

Evidence stream summary and interpretation

judgment
1 low confidence
short term, subchronic,

females at


study in adult
and chronic exposure)

200 mg/kg-d


rats:
• High confidence studies




90-d





Serum
• Consistent increases in
• No factors noted
• Increased ALT,


Biomarkers of
ALT, AST, and ALP, and

AST, ALP, and


Hepatic Iniurv
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-d
• Dose-response for total

respectively;


90-d (2 studies)
protein

stronger in


2-yr
• 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 in association with hepatic effects in a short-term
contributing to hepatic


oral exposure 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.
3-40	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
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.
3-41	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Toxicological Review ofPFHxA and Related Salts
3.2.2. Developmental Effects
Human
No studies were identified that evaluated potential developmental effects of PFHxA
exposure in humans.
Animal
Three studies described in two publications 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 etal.. 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 3-12, and details are available by clicking the HAWC
link. Effects on male and female reproductive system development following developmental
exposure are discussed in the male and female reproductive effects sections, respectively (see
Sections 3.2.6 and 3.2.7).
Table 3-12. Study design characteristics and outcome-specific study
confidence for developmental endpoints
Study
Species, strain (sex)
Exposure design
Exposure route and dose
Offspring
viability
Offspring body
weight
Developmental
milestones
Loveless
Rat, Crl:CD(SD)
Reproductive study: P0
Gavage3
++
++
++
et al.
Sprague-Dawley
females dosed 70 d prior to
Female: 0, 20,100, 500 mg/kg-d



(2009)
(male and female)
cohabitation, through





gestation and lactation






(126 d); P0 males dosed for






110 d






Developmental study:






GD 6-20




Iwai and
Hoberman
(2014)c
Mouse, Crl:
CDl(ICR); Charles
River Laboratories,
Inc.
Developmental study:
GD 6-18
Gavageb
Phase 1: 0,100, 350, 500 mg/kg-d
Phase 2: 0, 7, 35,175 mg/kg-d
++
++
++
Study evaluation for animal toxicological developmental endpoints reported from studies with rats receiving
PFHxA sodium salt3 or PFHxA ammonium saltb by gavage. Study evaluation details for all outcomes are available
by clicking the HAWC link.
cPhase 1 was a range finding study used to determine the appropriate dose range for identification of a NOAEL in
Phase 2.
++ Outcome rating of high confidence
This document is a draft for review purposes only and does not constitute Agency policy.
3-42	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Toxicological Review ofPFHxA and Related Salts
Offspring Mortality
Potential effects of PFHxA exposure on offspring viability were evaluated in a
developmental study flwai and Hoberman. 20141 and a one-generation reproductive study
fLoveless etal.. 20091. Mice exposed to PFHxA ammonium salts during gestation (GD 6-18)
showed a dose-dependent increase in the incidence of offspring mortalities occurring both pre- and
postnatally (Iwai and Hoberman. 2014). Most deaths occurred between postnatal day (PND) 0-7,
with a statistically significant increase observed in the 350 and 500 mg/kg-day dose groups on PND
1-4. Early postnatal losses are reflected in treatment-related effects on several measures of
offspring viability for the 500 mg/kg-day dose group. Specifically, statistically significant changes
were observed in the following related outcomes: decreased viability index for PND 0-4 and PND
0-7, fewer surviving pups per litter on PND 20, and increased incidence of total litter loss between
PND 0-3 (5 of 17 for the 500 mg/kg-day group compared to 1 of 19 dams for concurrent controls).
A dose-dependent increase in the number of stillbirths, a measure of prenatal mortality, was also
reported across the two phases ofthe experiment (incidence of 3/241, 5/245, and 19/177 for the
175, 350, and 500 mg/kg-d dose groups, respectively). Results are summarized in Figure 3-7 and
Table 3-13
This document is a draft for review purposes only and does not constitute Agency policy.
3-43	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Endpoint
Offspring Surwwal
Study
Iwai, 2014,2821611
Experiment	Animal Description
1-generation reproductive (GD 6-18) F1 Mouse, CD-1 (c¥)
Iwai, 2014,2821611 1-generation reproductive (GD 6-18) F1 Mouse, CD-1 (c¥)
Loveless, 2009,2850369
reproductive (56 d)
F1 Rat,Cri:CD(SD)(c¥)
Observation Time
PND0
PND0
PND 4
PND 4
PND 7
PND 7
PND 14
PND 14
PND 20
PND 20
PND 0-4
PND 0-4
PND 0-7
PND 0-7
PND 4-20
PND 4-20
PND 0
PND 0-4
PND 4-21
FHxA Developmental Effects: Offspring Mortality
No. of Pups, Stillborn
Viability, Litters with Stillborn Pups
Iwai, 2014,2821611 1-generation reproductive (GD 6-18) F1 Mouse, CD-1 (c¥)
Iwai, 2014,2821611 1-generation reproductive (GD 6-18) F1 Mouse, CD-1 (c¥)
Pups Found DeadyPresumed Cannibalized Iwai, 2014,2821611 1-generation reproductive (GD 6-18) F1 Mouse, CD-1 (c¥)
Total Litter Loss
Iwai, 2014,2821611 1-generation reproductive (GD 6-18) P0 Mouse, CD-1 (^)
PND 0
PND 0
PND 0
PND 0
PND 0
PND 0
PND 1-4
PND 1-4
PND 5-7
PND 5-7
PND 8-14
PND 8-14
PND 15-20
PND 15-20
PND 0
PND 0
PND 0-3
PND 0-3
PND 4-20
PND 4-20
i No significant changeA Significant increase^ Significant decreas^j ®
50 100 150 200 250 300 350 400 450 500 550 600
Figure 3-7. Developmental effects on offspring viability in mice exposed to
PFHxA ammonium salt (HAWC: PFHxA - Animal Toxicity Developmental
Effects link!.
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
NOAELin Phase 2.
Table 3-13. Incidence of perinatal mortality following PFHxA ammonium salt
exposure in a developmental oral toxicity study
Study Design and Reference
Dose (mg/kg-d)

0
(Phase 1)
0
(Phase 2)

lO
CO
o
o
*—i
lO
*—i
o
LO
CO
o
o
lO
Stillbirths, male and female (combined) mice (Iwai
and Hoberman, 2014)
4
0
0a
0
0
3
5a
19a
This document is a draft for review purposes only and does not constitute Agency policy.
3-44	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Toxicological Review ofPFHxA and Related Salts
Study Design and Reference
Dose (mg/kg-d)

0
(Phase 1)
0
(Phase 2)
r-.
CO
o
o
rH
r-.
rH
o
m
CO
o
o
m
Mortalities, PND 0, male and female (combined)
mice (Iwai and Hoberman, 2014)
0
0
0
0
0
4
3a
21a
Mortalities, PNDs 1-4, male and female
(combined) mice (Iwai and Hoberman, 2014)
2
r
3a
2
2a
0a
13a
15a
Mortalities, PNDs 5-7, male and female
(combined) mice (Iwai and Hoberman, 2014)
0a
i
0a
0
0b
3a
2a
0a
Mortalities, PNDs 8-14, male and female
(combined) mice (Iwai and Hoberman, 2014)
0
0
0
0
0a'b
0a
0a
0a
Mortalities, PNDs 15-20, male and female
(combined) mice (Iwai and Hoberman, 2014)
0
0
0
0
2b
1
0
0
Total pups delivered, male and female (combined)
mice (Iwai and Hoberman, 2014)
221
249
211
232
250
241
245
177
Cumulative perinatal mortality/total pups
delivered, male and female (combined) mice (Iwai
and Hoberman, 2014)
6/
221
2/
249
3/
211
2/
232
4/
250
11/
241
23/
245
55/
177
The Iwai and Hoberman (2014) study was conducted in two phases. Phase 1 was a range-finding study (100, 350,
or 500 mg/kg-d) used to determine the appropriate doses (7, 35,175 mg/kg-d) to identify a NOAEL in Phase 2.
Bold values indicate instances where statistical significance (p < 0.05) compared to controls was reported by study
authors.
aExcludes animals that were missing and presumed cannibalized or where vital status at birth was uncertain due to
maternal cannibalization or autolysis.
bExcludes offspring mortalities that occurred following death of the dam; deaths assumed not treatment related.
Offspring Body Weight
Offspring body weights were available from two developmental studies flwai and
Hoberman. 2014: Loveless etal.. 20091 and a one-generation reproductive study (Loveless etal..
20091. In mice, offspring body weights were statistically significantly decreased at PND 0-7 in
animals exposed gestationally (GD 6-18) to >100 mg/kg-day PFHxA ammonium salt. These effects
were observed across two experimental cohorts with different dose ranges. In addition, although at
some timepoints not statistically significant, consistent body weight deficits >5% relative to control,
a level of change that may be biologically significant during early development (U.S. EPA. 2012a.
19911. generally persisted to the end of lactation (Table 3-14). After weaning, some body weight
deficits persisted, with females with the 350 mg/kg-day dose group showing a statistically
significant reduction through the end of the experiment (PND 41) flwai and Hoberman. 20141.
Similar results were reported in two experiments with rats exposed to PFHxA sodium salt
(Loveless etal.. 2009). In the developmental study, fetal body weights (GD 21) of animals exposed
gestationally (GD 6-20) to 500 mg/kg-day were decreased by 9% relative to controls, although this
change was not statistically significant, but no effects were observed at the lower doses. In the
This document is a draft for review purposes only and does not constitute Agency policy.
3-45	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
Toxicological Review ofPFHxA and Related Salts
one-generation reproductive study, a dose-related decrease (4,11, and 18% decrease relative to
controls for 20,100, and 500 mg/kg-day, respectively, reaching statistical significance atthe
highest dose) was found in pup body weights across all dose groups at PND 0. Similar to results in
the mouse study flwai and Hoberman. 20141. body weight deficits >5% relative to control were
observed through the end of lactation (PND 21) in the 100 and 500 mg/kg-d dose groups, but
resolved after weaning (Loveless etal.. 2009).
Neither study reported treatment-related effects on body weight change (i.e., gains or
losses) between weaning and the end of testing (PND 21-41 for mice; PND 21-60 for rats) (Iwai and
Hoberman. 2014: Loveless etal.. 20091. Results are presented in Figure 3-8 and Table 3-14.
Endpoint
Study
Experiment
Animal Description Observation Time
PFHxA Developmental Effects: Offspring Body Weight
Body Weight, Absolute Iwai, 2014,2821611 1-generation reproductive (GD 6-18) F1 Mouse, CD-1 0?)
Loveless,2009,2850369
developmental (GD 6-20)
reproductive (56 d)
F1 Mouse, CD-1 0)
F1 Mouse, CD-1 (?)
F1 Rat,Crl:CD(SD)0?)
F1 Rat,Crl:CD(SD)0?)
F1 Rat,Cr1:CD(SD)(cv)
F1 Rat,Cr1:CD(SD)(?)
PND 0
PND 4
PND 7
PND 14
PND 20
PND 21
PND 28
PND 35
PND 41
PND 21
PND 28
PND 35
PND 41
GD 21
PND 0
PND 4 (pre-cull)
PND 7
PND 14
PND 21
PND 28
PND 35
PND 39
PND 60
PND 28
PND 35
PND 39
PND 60
Body Weight Change Iwai, 2014,2821611 1-generation reproductive (GD 6-18) F1 Mouse, CD-1 0)	PND 21-41
F1 Mouse, CD-1 (?)	PND 21-41
Loveless, 2009,2850369	reproductive (56 d)	F1 Rat, Cr1:CD(SD) 0)	PND 21-60
F1 Rat, Cr1:CD(SD) (?)	PND 21-60
I • No significant changeA Significant increase^ Significant decrease I
50 100 150 200 250 300 350 400 450 500 550 600
mg/kg-day
Figure 3-8. Developmental effects on offspring body weight in mice exposed
to PFHxA ammonium salt and rats exposed to PFHxA sodium salt (HAWC:
PFHxA - Animal Toxicity Developmental Effects link).
This document is a draft for review purposes only and does not constitute Agency policy.
3-46	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 3-14. Percent change relative to control in offspring body weight due to
PFHxA sodium or ammonium salt exposure in developmental oral toxicity
studies

Dose (mg/kg-d)
Postnatal Date (GD 6-18) and Sex (Iwai and Hoberman,



o

o
o
2014)
r-.
o
fM
CO
o
*—1
r-.
*—1
m
CO
o
m
PND 0, male and female (combined) mice
0

0
-6
-13
-13
-13
PND 4, male and female (combined) mice
0

7
-7
-4
-27
-20
PND 7, male and female (combined) mice
0

5
-7
0
-18
-11
PND 14, male and female (combined) mice
-1

3
-8
0
-14
-8
PND 20, male and female (combined) mice
-2

6
-11
2
-20
-12
PND 21, male mice
3

4
-15
-1
-18
-14
PND 28, male mice
2

3
-10
0
-10
-8
PND 35, male mice
1

1
-4
-1
-3
-5
PND 41, male mice
1

-1
-2
-3
-3
-4
PND 21, female mice
0

6
-14
1
-17
-8
PND 28, female mice
0

4
-9
-1
-16
-7
PND 35, female mice
-1

2
-4
-1
-10
-7
PND 41, female mice
-3

-1
-4
-3
-8
-4
Fetal Bodv Weight, Developmental Exposure (GD 6-20) (Loveless et al., 2009)
GD 21, male and female (combined) rats

-2

0


-9
Postnatal Bodv Wight, One-Generation Reproductive Exposure (Loveless et al., 2009)
PND 0, male and female (combined) rats

-4

-11


-18
PND 7, male and female (combined) rats

0

-6


-17
PND 14, male and female (combined) rats

3

-6


-17
PND 21, male and female (combined) rats

3

-5


-18
PND 28, male rats

2

-1


-5
PND 35, male rats

1

-1


-3
PND 39, male rats

2

-1


-3
PND 60, male rats

2

-1


-3
PND 28, female rats

1

-5


-4
PND 35, female rats

1

-4


-1
PND 39, female rats

-1

-5


-3
PND 60, female rats

-1

-5


-3
This document is a draft for review purposes only and does not constitute Agency policy.
3-47	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Toxicological Review ofPFHxA and Related Salts
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.
Eye Opening
Potential effects ofPFHxA exposure on eye opening were evaluated in a developmental
study in mice (Iwai and Hoberman. 2014). On PND 14, Iwai and Hoberman (2014) reported a
statistically significant delay in eye opening, with less than 50% of pups in the 350 and 500 mg/kg-
day PFHxA ammonium salt exposure groups having reached this milestone compared to 85%
among vehicle controls (see Figure 3-9). Although pup body weight changes were not statistically
significantly at this timepoint, they were decrements of a magnitude considered to be potentially
biologically significant (8-14%) and some developmental landmarks are correlated with postnatal
body weight gain fU.S. EPA. 2016al. Delays in eye opening persisted in the 350 and 500 mg/kg-day
dose groups on PND 15 but were not statistically significant. Eye opening in mice typically occurs
between PND 11 and PND 14, with full functionality a few days later (Brustetal.. 2015). Delays in
eye opening can have long-term impacts on vision by interfering with sensory input during the
critical window of visual cortex development (Espinosa and Strvker. 2012: Wiesel. 1982). The
results are summarized in Figure 3-9 and Table 3-15.
Endpoint	Study Name	Experiment	Animal Description Observation Time	PFHxA Developmental Effects: Developmental Milestone
Eye Opening Iwai, 2014, 2821611 1-generation reproductive (GD 6-18) F1 Mouse, CD-1 (A-j) PND 10
PND 11
PND 12
PND 13
PND 14
PND 15
PND 16
PND 17
0 No significant chango^ Significant increase^^ Significant decrease!
50 100 150 200 250 300 350 400 450 500 550 600
mg/kg-day
Figure 3-9. Developmental effects on eye opening (percent change relative to
control) in mice exposed to PFHxA ammonium salt (HAWC: PFHxA - Animal
Toxicity Developmental Eve Effects link!.
This document is a draft for review purposes only and does not constitute Agency policy.
3-48	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Toxicological Review ofPFHxA and Related Salts
Table 3-15. Percent change relative to control in eye opening due to PFHxA
ammonium salt exposure in a developmental oral toxicity study
Study Design and Reference
Dose (mg/kg-d)
r-.
CO
o
o
*—1
r-.
*—1
o
m
CO
o
o
m
PND 13, male and female (combined) mice (Iwai and Hoberman, 2014)
-6
34
-56
-21
-58
-55
PND 14, male and female (combined) mice (Iwai and Hoberman, 2014)
2
4
-17
-8
-49
-39
PND 15, male and female (combined) mice (Iwai and Hoberman, 2014)
0
0
-10
-5
-23
-25
PND 16, male and female (combined) mice (Iwai and Hoberman, 2014)
0
0
-1
0
-9
-1
Bold values indicate instances where statistical significance (p < 0.05) compared to controls was reported by study
authors.
Malformations and Variations
Potential effects ofPFHxA 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 ofPFHxA;
therefore, there is indeterminate human evidence of developmental effects.
In animals, three high confidence studies reported in two publications examined
developmental effects following maternal perinatal exposure to PFHxA salts flwai and Hoberman.
2014: Loveless etal.. 20091. Treatment-related effects, including decreased offspring body weight,
increased mortality, and delayed eye opening were observed in mice following exposure to PFHxA
ammonium salt at doses as low as 100 mg/kg-day (Iwai and Hoberman. 2014). Reductions in
offspring body weight were also found in the one-generation reproductive and developmental
studies in rats, although effects were less pronounced than those observed in mice. Animals in the
reproductive cohort exposed throughout gestation and lactation showed body weight reductions
that may be biologically significant (>5%) at exposure to >100 mg/kg-day and statistically
significant at 500 mg/kg-day that persisted to PND 21, whereas the developmental cohort was
reduced (9%) only at the high dose (500 mg/kg-day).
In general, effects on development were greatest in magnitude from PND 0 to PND 7,
suggesting that the early postnatal period might be a sensitive window for developmental effects
associated with PFHxA exposure. Although the evidence base is small, the data are strengthened by
coherent evidence across outcomes, consistency of effects on body weight across two
species/studies, and the severity of some of the outcomes (e.g., increased offspring mortality). In
addition, a similar pattern of effects on development (i.e., offspring body weight reductions and
This document is a draft for review purposes only and does not constitute Agency policy.
3-49	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Toxicological Review ofPFHxA and Related Salts
delays in developmental milestones) has been observed with other PFAS, including PFBS and PFBA,
providing additional support for these specific findings.
The potential for systemic and maternal to act as a driver for the observed developmental
effects was considered. In Iwai and Hoberman f20141. delays in eye opening were observed only at
doses that were associated with decreased body weights (8-14%) and overt toxicity (i.e., increased
perinatal mortality) in the pups. Additionally, reductions in maternal body weight were noted in
the developmental Loveless etal. (2009). Dams exposed to 500 mg/kg-day from GD 6-20 showed a
slight but statistically significant 5% decrease in total net body weight (i.e., terminal body weight
minus the gravid uterine weight) and body weight gain on GD 21 fLoveless etal.. 20091. In the one-
generation reproductive study, Loveless etal. f20091 reported a statistically significant reduction in
maternal weight gain in the highest dose group (500 mg/kg-day), however this effect was limited to
early gestation (GD 0-7). Importantly, there was no effect on maternal body weight gain over the
entire gestational window (GD 0-21), nor was there any observed effects on total or net maternal
body weights. Thus, the effects on offspring body weight in this study are not expected to be driven
by maternal toxicity. Given this interpretation of an effect on development and based on the
multiple adverse changes in pups, there is moderate animal evidence of developmental effects.
Overall, the currently available evidence indicates that PFHxA likely causes developmental
effects in humans 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.
3-50	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 3-16. Evidence profile table for developmental effects
Evidence stream summary and interpretation
Evidence integration
summary judgment
Evidence from studies of exposed humans
®©o
Studies and confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key
findings
Evidence stream
judgment
Evidence indicates
(likely)
• There were no human studies available from the PFHxA evidence base.
ooo
Indeterminate
Primary basis:
Three high confidence
Evidence from animal studies
studies in rats and mice
including gestational (rats
and mice) and continuous
one-generational
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 perinatal
mortality at >350
mg/kg-d in mice
®©o
Moderate
Developmental
effects observed in
multiple high
confidence studies
conducted in two
species exposed to
different PFHxA salts
under different
exposure scenarios.
Effects on body
weight were
reproductive (rats)
exposures, generally
observing effects at > 100
mg/kg-d PFHxA
ammonium or sodium
salt.
Bodv Weight
3 high confidence studies
in rats and mice:
•	GD 6-18 (mice)
•	GD 6-20 (rats)
•	1-generation
reproductive (rats)
•	High confidence
studies
•	Consistency across
studies and species
•	Dose-response
observed in mouse
study
• No factors noted
•	Postnatal body weight
decreased at >100
mg/kg-d in rats and
mice
•	Fetal body weight
decreased at 500
mg/kg-d in rats
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:
Eve Opening
1 high confidence study
in mice:
• GD 6-18
• High confidence study
• No factors noted
• Eye opening was
delayed in mice
prenatally exposed to
PFHxA ammonium salt
at >350 mg/kg-d
observed at doses
that were not
associated with frank
effects or maternal
toxicity.
This document is a draft for review purposes only and does not constitute Agency policy.
3-51	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
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-d

The available evidence
indicates that
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 mechanistic studies available from the PFHxA evidence base.
This document is a draft for review purposes only and does not constitute Agency policy.
3-52	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Toxicological Review ofPFHxA and Related Salts
3.2.3. Renal Effects
Human
Three epidemiological studies investigated the relationship between PFHxA exposure and
effects on the renal system. Two cross-sectional studies of adults in Korea and older adults in China
(Zhang etal.. 2019: Seo etal.. 2018) were considered uninformative due to lack of consideration of
confounding, including age, sex, socioeconomic status, and other risk factors for renal disease. The
remaining study was a cross-sectional study of primarily government employees in China fWanget
al.. 20191 and was classified as low confidence primarily due to significant concerns for reverse
causality that could result if there is decreased elimination of PFAS with reduced renal function and
poor sensitivity because the exposure contrast for PFHxA was narrow. They observed a significant
decrease in estimated glomerular filtration rate (eGFR) with higher serum PFHxA levels ((3: -0.3
change in eGFR as mL/min/1.73 m2 per 1 ln-unit PFHxA [95% CI: -0.6, -0.01]). No association was
observed with chronic kidney disease. Due to the potential for reverse causality and 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.
This document is a draft for review purposes only and does not constitute Agency policy.
3-53	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Participant selection
Exposure measurement -
Outcome ascertainment -
Confounding
Analysis -
Sensitivity -
Selective Reporting -
Overall confidence
*****

Legend
Good (metric) or High confidence (overall)
Adequate (metric) or Medium confidence (overall)
Deficient (metric) or Low confidence (overall)
Critically deficient (metric) or Uninformative (overall)
Figure 3-10. Study evaluation for human epidemiological studies reporting
findings from PFHxA exposures (full details available by clicking HAWC link).
1	Animal
2	Four short-term (28-day), subchronic, or chronic animal studies evaluated potential renal
3	effects of PFHxA or PFHxA sodium salt in rats. Most of the outcome-specific study ratings were
4	rated high confidence. For Chengelis etal. f2009bl limitations were identified that influenced
5	some outcome-specific ratings. Specifically histopathology was rated low confidence because of
6	issues related to observational bias, endpoint sensitivity and specificity, and results presentation.
7	Urinary biomarker outcomes in the same study were rated medium confidence because of results
8	presentation (only qualitative results were reported). The results of the outcome-specific
9	confidence judgments are summarized in Table 3-17, and full study evaluation details can be
10 viewed by clicking the HAWC link.
This document is a draft for review purposes only and does not constitute Agency policy.
3-54	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Toxicological Review ofPFHxA and Related Salts
Table 3-17. 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; 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 (NTP. 2018: Chengelis etal.. 2009b: Loveless etal.. 20091. There were
statistically significant findings in male rat dose groups at PFHxA doses as low as 10 mg/kg-day in
the subchronic study (Chengelis etal.. 2009b). With the exception of the results from Chengelis et
al. f2009bl. effects on relative kidney weights generally showed a weak or no dose-response
gradient (see Table 3-18). Craig etal. f20151 analyzed oral chemical exposure data extracted from
subchronic and chronic rat studies and found a statistically significant correlation between
absolute, but not relative, kidney weight, and kidney histopathology (even at doses where terminal
body weights were decreased) for most chemicals (32/35) examined. Absolute kidney weight was
increased, but only in one of the three studies reporting on this endpoint (NTP. 2018). and only in
female rats at the highest dose group (1,000 mg/kg-day). The decrease in relative, but not absolute,
kidney weight could be explained by body weight gain decreases in the affected dose groups:
1,000 mg/kg-day male dose group (13% decrease) fNTP. 20181. 50 and 200 mg/kg-day male dose
This document is a draft for review purposes only and does not constitute Agency policy.
3-55	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
1	group [8-11% decrease with similar trends in females (Chengelis etal.. 2009b)]. and
2	500 mg/kg-day male dose group (19% decrease, no change in females) fLoveless etal.. 20091.
3	Findings and full details of PFHxA effects on kidney weights can be viewed by clicking the HAWC
4	link.
Table 3-18. Percent increase in relative and absolute kidney weight due to
PFHxA exposure in short-term, subchronic, and chronic oral toxicity studies
Endpoint and reference
Dose (mg/kg-d)
o
*—1
o
fM
o
m
in

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Toxicological Review ofPFHxA and Related Salts
absolute kidney weight Male renal histopathological findings from the chronic study were also
null, whereas findings for female rats included increased papillary necrosis (17/70 vs. 0/60 in
controls) and tubular degeneration (7/70 vs. 1/60 in controls) in the highest dose group
200 mg/kg-day fKlaunig etal.. 20151. Full details are available by clicking the HAWC link.
Endpoint name	Study
Kidney, Nephropathy, Chronic Progressixe NTP, 2018,4309149
Animal Description	Dose	Incidence
Rat, Harlan Sprague-Dawley(c5) 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(i2) 0	2/10(20.0%)
62.6	4/10(40.0%)
125	4/10(40.0%)
250	1/10(10.0%)
500	3/10(30.0%)
1,000	8/10(80.0%)
Kidney Histopathology Incidence
Kidney, Necrosis, Papillary Klaunig, 2015,2850075 Rat, Crl:CD(SD) 0)
0
2.5
0/60 (0.0%)
0/60 (0.0%)

15
0/60 (0.0%)

100
0/70 (0.0%)
Rat,Crl:CD(SD)(ii)
0
0/60 (0.0%)

5
0/60 (0.0%)

30
0/60 (0.0%)

200
17/70 (24.3%)
Kidney, Tubular Degeneration Klaunig, 2015,2850075 Rat, Crl:CD(SD) (o)
0
2.5
4/60 (6.7%)
2/60 (3.3%)

15
0/60 (0.0%)

100
3/70 (4.3%)
Rat,Crl:CD(SD)(ii)
0
1/60(1.7%)

5
0/60 (0.0%)

30
2/60 (3.3%)

200
7/70(10.0%)
|| I percent affected I I Significant Com pared to Contro]
40 50 60
% Affected
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.
Blood and Urinary Biomarkers
Blood biomarkers of renal function were inconsistent across study designs and exposure
durations. Both creatinine and blood urea nitrogen (BUN) are removed from the blood by the
kidneys and often used as indicators of kidney function. Creatinine is a waste product of creatine
metabolism (primarily in muscle) and BUN is a waste product of protein metabolism in the liver.
No observations of changes in urea nitrogen or creatinine were reported from Chengelis et al.
(2009b) and Klaunigetal. (2015). In the short-term study (NTP. 2018). BUN was unchanged in
both sexes in all dose groups. Changes in creatinine were inconsistent across sexes with null
findings in females, whereas a 13% decrease (p < 0.05) was found in the male 500 mg/kg-day dose
group (NTP. 2018). In a subchronic study, Loveless etal. (2009) 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.
This document is a draft for review purposes only and does not constitute Agency policy.
3-57	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Toxicological Review ofPFHxA and Related Salts
Urinalysis findings included total urine volume and other measures of urine concentrating
ability (e.g., specific gravity, urobiloginen). The urinalysis findings were more consistent than the
blood biomarkers, but still difficult to interpret as adverse or nonadverse. Urinalysis findings were
not measured in the short-term study fNTP. 20181 and were reported as null in a subchronic study
fChengelis etal.. 2009bl. Findings from the other subchronic study fLoveless et al.. 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 fLoveless etal.. 20091. Urobilinogen and pH findings were null in
both male and females in the subchronic study fLoveless etal.. 20091. Findings from the chronic
study lacked consistency between sexes and did not exhibit a clear dose-response relationship
fKlaunigetal.. 20151. Specifically, total urine volume was increased in the female 200 mg/kg-day
dose group and null in all male dose groups. Urine specific gravity was increased (p < 0.05) in the
male 15 mg/kg-day dose group and similar to controls in the 100 mg/kg-day dose group, although
specific gravity was increased (p < 0.05) in the female 200 mg/kg-day dose group. Urine pH was
low in males (compared to controls) only in the 100 mg/kg-day dose groups at 26 and 52 weeks
fKlaunigetal.. 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 at52 weeks study duration (Klaunig etal.. 2015). Findings are summarized in Figure 3-12,
and full details are available in the HAWC link.
This document is a draft for review purposes only and does not constitute Agency policy.
3-58	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
Toxicological Review ofPFHxA and Related Salts
Endpoint
Study Experiment
Animal Description
Observation Time
Blood Urea Nitrogen (BUN)
NTP, 2018,4309149 28-Day Oral
Rat, Harlan Sprague-Dawley(cJ)
Day29


Rat, Harlan Sprague-Dawley(v)
Day29

Loveless, 2009,2850369 90-DayOral
Rat, Crl:CD(SD)0)
Day92


Rat, Crl:CD(SD)($)
Day93
Creatine Kinase (CK)
NTP, 2018,4309149 28-Day Oral
Rat, Harlan Sprague-Dawley(cJ)
Day29


Rat, Harlan Sprague-Dawley(v)
Day29
Creatinine (CREAT)
NTP, 2018,4309149 28-Day Oral
Rat, Harlan Sprague-Dawley(cJ)
Day29


Rat, Harlan Sprague-Dawley(v)
Day29

Loveless, 2009,2850369 90-DayOral
Rat, Crl:CD(SD)(cv)
Day92


Rat,Crl:CD(SD)(?)
Day93
Osmolality
Loveless, 2009,2850369 90-DayOral
Rat, Crl:CD(SD)(cv)
Day92


Rat,Crl:CD(SD)(?)
Day93
Urine Specific Gravity
Klaunig, 2015,2850075 2-Year Cancer Bioassay
Rat, Crl:CD(SD)(cv)
Week 26
Week 52


Rat,Crl:CD(SD)(?)
Week 26
Week 52
Urine Total Protein (TP)
Loveless, 2009,2850369 90-DayOral
Rat, Crl:CD(SD)(cv)
Day92


Rat,Crl:CD(SD)(?)
Day93
Urine U)lume (UVOL)
Loveless, 2009,2850369 90-DayOral
Rat, Crl:CD(SD)(cv)
Day92


Rat,Crl:CD(SD)(?)
Day93

Klaunig, 2015,2850075 2-Year Cancer Bioass ay
Rat, Crl:CD(SD)(cv)
Week 26
Week 52


Rat,Crl:CD(SD)(?)
Week 26
Week 52
Urine pH
Loveless, 2009,2850369 90-DayOral
Rat, Crl:CD(SD)(cv)
Day92


Rat,Crl:CD(SD)(?)
Day93

Klaunig, 2015,2850075 2-Year Cancer Bioass ay
Rat, Crl:CD(SD)(cv)
Week 26
Week 52


Rat,Crl:CD(SD)(?)
Week 26
Week 52
Urobilinogen
Loveless, 2009,2850369 90-DayOral
Rat, Crl:CD(SD)(cv)
Day92


Rat,Crl:CD(SD)(?)
Day93

Klaunig, 2015,2850075 2-Year Cancer Bioass ay
Rat, Crl:CD(SD)(cv)
Week 26
Week 52


Rat,Crl:CD(SD)(?)
Week 26
Week 52
PFHxA Renal Effects: Blood and Urine Biomarkers
• No significant changg^ Significant increase ^ Significant decrease ^ Significant Trend|
-~
-w'
V
-~
A
100 200 300 400 500 600 700 800 900 1,0001,100
Dose (mg/kg-day)
Figure 3-12. 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 generally high
confidence studies including one short-term, two subchronic studies, and one chronic studies
Findings were, in general, null except for histopathology and some urinary biomarkers. Kidney
histopathology was the most significant finding in the short term and chronic studies. In the short-
term study, increased incidence of CPN was observed in female rats at the highest dose (1,000
mg/kg-day PFHxA) and consistent with increased absolute kidney weight (females only).
Histopathological findings were null in both subchronic studies at doses up to 500 mg/kg-day. In
the chronic study, the incidence of papillary necrosis and tubular degeneration were increased in
This document is a draft for review purposes only and does not constitute Agency policy.
3-59	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
1	females compared to controls at the highest dose (200 mg/kg-day, twice the highest male dose).
2	Some changes occurred in urinary biomarkers (decreased urine pH, increased urine volume) and
3	potentially correlated changes were observed in female histopathology in the chronic study.
4	However, inconsistencies across studies at similar observation times and doses were notable.
5	Based on these results, there is slight animal evidence of renal effects.
6	Overall, the currently available evidence is inadequate to assess whether PFHxA may
7	causes renal effects in humans under relevant exposure circumstances (see Table 3-19).
This document is a draft for review purposes only and does not constitute Agency policy.
3-60	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 3-19. Evidence profile table for renal effects
Evidence stream summary and interpretation
Evidence integration
summary judgment
Evidence from studies of exposed humans
OOO
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
PFHxAwith 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-d
•	90-d (2 studies)
• Consistent increases,
all studies
• No factors noted
•	Increased relative kidney
weight at >10 mg/kg-d.
•	Increase absolute kidney
weight at 1,000 mg/kg-d;
28-d study, females only
©oo
Slight
Findings of adversity
were considered
uncertain based on lack
the contrary, effects in
rats are considered
relevant to humans
Cross-stream
coherence:
N/A (human evidence
indeterminate)
Susceptible lifestages:
No evidence to inform
Histopathologv
3 hiqh confidence
studies in adult rats:
•	28-d
•	90-d
•	2-yr
1 low confidence study
in adult rats:
90-d
• Large magnitude of
effect, up to 24.3% for
papillary necrosis; up
to 80% for chronic
progressive
nephropathy
• No factors noted
• Increased incidence
papillary necrosis, tubular
degeneration, chronic
progressive nephropathy
at >200 mg/kg-d; female
rats only, 28-d and
chronic studies
of coherence between
effects (organ weight,
histopathology, blood
and urine biomarkers)
inconsistency between
sexes, and lack of
coherence across
exposure designs
This document is a draft for review purposes only and does not constitute Agency policy.
3-61	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts





Evidence integration

Evidence stream summary and interpretation

summary judgment
Blood Biomarkers
• No factors noted
• Lack of coherence
• Increased BUN at


4 hiph confidence

with other
500 mg/kg-d; males only,


studies in adult rats:

histopathological
90-d study.


• 28-d

findings; chronic
• Decreased creatinine at


• 90-d (2 studies)

study
>500 mg/kg-d), both


• 2-yr


sexes, 1 subchronic study
•	Decreased creatine at
1,000 mg/kg-d; males
only, 28-d study
•	No treatment related
creatinine kinase findings;
both sexes, 28-d study


Urinarv Biomarkers
• Coherence of urine
• Lack of coherence
• Decreased osmolality 500


3 hiah confidence
protein, urine volume,
with
mg/kg-d; males only, 1


studies in adult rats:
urine specific gravity,
histopathological
subchronic study


• 28-d
and decreased
findings.
• Decreased urine protein


• 90-d
osmolality

and increased urine


• 2-yr


volume in at 500 kg/kg-d;
both sexes, 1 subchronic


1 medium confidence
study in adult rats:
• 90-d


study
• Increased total urine
volume at >200 mg/kg-




day; both sexes - 1
subchronic study, females
only, 1 2-yr study
•	Decreased urine pH at
100 mg/kg-d; males only,
1 2-yr study
•	No treatment related
findings for urobilinogen;
both sexes, 1 subchronic
study and 1 2-yr study


This document is a draft for review purposes only and does not constitute Agency policy.
3-62	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
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
This document is a draft for review purposes only and does not constitute Agency policy.
3-63	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Toxicological Review ofPFHxA and Related Salts
3.2.4. Hematopoietic Effects
Hematology is a subgroup of clinical pathological parameters concerned with morphology,
physiology, and pathology of blood and blood-forming tissues. Hematological parameters are
measured using blood tests such as complete blood counts (CBC) and a clinical chemistry panel.
The CBC 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, including age, socioeconomic
status, and medical history, which is expected to substantially bias the results. Full study
evaluation for Tiang etal. f20141 is available by clicking the HAWC link.
Animal Studies
Several animal toxicological studies were available that assessed hematopoietic parameters
including a high confidence short-term study (NTP. 2018). high confidence (Chengelis etal.. 2009b)
and high confidence 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, see Section 3.2.8. Study findings are discussed
below and summarized in Table 3-20 (full details are available by clicking the HAWC linkl. and
summary details are available in PFHxA Tableau visualization.
This document is a draft for review purposes only and does not constitute Agency policy.
3-64	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Toxicological Review ofPFHxA and Related Salts
Table 3-20. Hematopoietic endpoints for PFHxA and associated confidence
scores from repeated-dose animal toxicity studies
Author (year)
Species, strain (sex)
Exposure
design
Exposure route and dose range
Hematology
and
hemostasis
NTP(2018)
Rat, Harlan Sprague-Dawley
(male and female)
Short term
(28 d)
Gavage3
Male and female: 0, 62.5,125, 250,
500,1,000 mg/kg-d
+ +
Chengelis et al.
(2009b)
Rat, Crl:CD(SD) Sprague-
Dawley (male and female)
Subchronic
(90 d)
Gavage3
Male and female: 0,10, 50,
200 mg/kg-d
+ +
Loveless et al.
(2009)
Rat, Crl:CD(SD) Sprague-
Dawley (male and female)
Subchronic
(90 d)
Gavageb
Male and female: 0, 20,100,
500 mg/kg-d
+ +
Klaunig et al.
(2015)
Rat, Crl:CD(SD) Sprague-
Dawley (male and female)
2-yr cancer
bioassay
Gavage3
Male: 0, 2.5,15,100 mg/kg-d
Female: 0, 5, 30, 200 mg/kg-d
+ +
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.
Hematology
Several findings were consistent (i.e., decreased red blood cells [RBCs], hematocrit, and
hemoglobin) across studies and study designs that, when interpreted together, suggest PFHxA-
related adverse hematological effects such as anemia (see Figure 3-13). Indications were also
present that red blood cells were swollen and made up a larger proportion of the blood volume
(increased mean cell volume [MCV, a measure of the average red blood cell size]). These changes
were correlated with potential secondary erythrogenic responses to PFHxA exposure including
increased reticulocyte (immature RBCs) counts that were consistently increased >10% across
study designs and exposure durations, including the chronic study Klaunigetal. f20151 where the
highest dose levels were 2-5 times lower than those tested in the subchronic studies. Specifically, a
dose-responsive decrease occurred in red blood cells (see Table 3-21), hematocrit (see Table 3-22),
and hemoglobin (see Table 3-23) in the short-term study with decreases at doses ranging from 62.5
mg/kg-day in male rats to 250 mg/kg-day in female rats (NTP. 2018). These findings also were
observed in both subchronic studies in the highest dose groups [200 mg/kg-day in males only
fChengelis etal.. 2009bl and 500 mg/kg-day in both sexes fLoveless etal.. 20091], Of note,
decreases in both hemoglobin and hematocrit were 1.5-2-fold greater in the subchronic study
(Loveless etal.. 2009) than in the short term study (NTP. 2018) for both males and females at the
same dose (500 mg/kg-day).
This document is a draft for review purposes only and does not constitute Agency policy.
3-65	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
1	Findings from the chronic study (Klaunig etal.. 20151 were generally null or observed at
2	dose levels >100 mg/kg-day (100 mg/kg-day in males and 200 mg/kg-day in females) at 25 and 51
3	weeks. Measures of hematology beyond 52 weeks in the chronic study might be complicated due to
4	natural diseases occurring in rodents and test variability leading to decreased sensitivity and
5	increasing variability with the results fAACC. 19921. Klaunig etal. T20151 did, however,
6	qualitatively evaluate blood and reported no PFHxA treatment effects on blood smear morphology.
7	Loveless etal. (20091 also evaluated blood smears up to test day 92 with PFHxA sodium salt
8	exposure and noted nucleated blood cells in smears indicative of bone marrow damage or stress,
9	but only for 1 female and 1 male.
Endpoint	Study	Experiment	Animal Description	Observation Time	PFHxA Hematopoietic Effects: Red Blood Cells
Hematocrit (HCT) NTP, 2018,4309149 28 Day Oral Rat, Harlan Sprague-Dawley(v) Day29
<>
<1
~

V
Rat, Harlan Sprague-Dawley0) Day29
~~ ~	
~

V
Loveless, 2009,2850369 90-DayOral Rat, Crl
CD(SD) 0) Day 92
~ • V
Rat, Crl
CD(SD) ($) Day93
«•—•—
T


Chengelis, 2009,2850404 90 Day Oral Rat, Crl
CD(SD) (<$) Day 90
—~
Rat, Crl
CD(SD) ($) Day90

Klaunig, 2015,2850075 2-Year Cancer Bioassay Rat, Crl
CD(SD) (<$) Week 25
m—•
Week 51
#—*
Week 104
•—•
Rat, Crl:CD(SD)($) Week 25

Week 51
#•	«
Week 104
Hemoglobin (HGB) NTP, 2018,4309149 28 Day Oral Rat, Harlan Sprague-Dawley(v) Day29
••	•



• • V



Rat, Harlan Sprague-Dawley0) Day29




V V v
V

V
Loveless, 2009,2850369 90-DayOral Rat, Crl
CD(SD) 0) Day 92
» • ~
Rat, Crl
CD(SD) ($) Day93
» • V
Chengelis, 2009,2850404 90 Day Oral Rat, Crl
CD(SD) (<$) Day 90
-• T
Rat, Crl
CD(SD) ($) Day90
-• T
Klaunig, 2015,2850075 2-Year Cancer Bioassay Rat, Crl
CD(SD) (<$) Week 25
m m
Week 51
m •
Week 104
m •
Rat, Crl:CD(SD)($) Week 25

Week 51
T
Week 104
Red Blood Cell (RBC) NTP, 2018,4309149 28 Day Oral Rat, Harlan Sprague-Dawley(v) Day29




• • V



Rat, Harlan Sprague-Dawley0) Day29
~~ T
~

V
Loveless, 2009,2850369 90-DayOral Rat, Crl
CD(SD) 0) Day 92
~ • V
Rat, Crl
CD(SD) ($) Day93
» • ~
Chengelis, 2009,2850404 90 Day Oral Rat, Crl
CD(SD) (<$) Day 90
-• ~
Rat, Crl
CD(SD) ($) Day90
-• T
Klaunig, 2015,2850075 2-Year Cancer Bioassay Rat, Crl
CD(SD) (<$) Week 25
m •
Week 51
m •
Week 104
m •
Rat, Crl:CD(SD)($) Week 25

Week 51
T
Week 104

• No significant chang^^ Significant increase^^ Significant decrease SignificantTrendl -100 0 100 200 300 400 500 600 700 800 900 1,0001,100
Dose (mg/kg-day)
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).
This document is a draft for review purposes only and does not constitute Agency policy.
3-66	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 3-21. Percent change in red blood cells due to PFHxA exposure in
short-term, subchronic, and chronic oral toxicity studies
Study Design and Reference
Dose (mg/kg-d)
in

-------
Toxicological Review ofPFHxA and Related Salts
Mean Corpuscular Hemoglobin NTP, 2018,4309149
(MCH)
Experiment
28 Day Oral
Animal Description Observation Time
Rat Harlan Sprague-Dawley(ij) Day29
Rat Harlan Sprague-Dawley(r') Day29
PFHxA Hematopoietic Effects: MCHC, MCV, MCH
Loveless, 2009,2850369 90-Day Oral
Rat CrkCD(SD) (n)
Day 92
M •


Rat Crl:CD(SD)(^)
Day 93
M •

Chengelis, 2009, 2850404 90 Day Oral
Rat CrkCD(SD) (n)
Day 90
«•
•

Rat Crl:CD(SD)(^)
Day 90
• •
•
Klaunig, 2015, 2850075 2-Year Cancer Bioassay
Rat Crl:CD(SD) (r-')
Week 25
«• •



Week 51
«• •



Week 104
m •


Rat Crl:CD(SD)(^)
Week 25
••
•


Week 51
••
•


Week 104
••
•
Mean Corpuscular Hemoglobin NTP, 2018,4309149
Concentration (MCHC)
Rat Harlan Sprague-Dawley(^) Day29
Rat Harlan Sprague-Dawley(r') Day29
Loveless,2009, 2850369
90-Day Oral
Rat Crl:CD(SD) (/-')
Day 92


Rat Crl:CD(SD)(^)
Day 93
Chengelis, 2009, 2850404
90 Day Oral
Rat Crl:CD(SD)(n)
Day 90


Rat Crl:CD(SD)(^)
Day 90
Klaunig, 2015,2850075 2-Ye
ar Cancer Bioassay
Rat CrkCD(SD) (r')
Week 25



Week 51



Week 104


Rat Crl:CD(SD)(^)
Week 25



Week 51



Week 104
NTP, 2018,4309149
28 Day Oral
Rat Harlan Sprague-Dawley(^)
Day 29


Rat Harlan Sprague-Dawley(r')
Day 29
Loveless, 2009, 2850369
90-Day Oral
Rat Crl:CD(SD) (/-')
Day 92


Rat Crl:CD(SD)(^)
Day 93
Chengelis, 2009, 2850404
90 Day Oral
Rat Crl:CD(SD) (r ')
Day 90


Rat Crl:CD(SD)(^)
Day 90
Klaunig, 2015,2850075 2-Ye
ar Cancer Bioassay
Rat Crl:CD(SD) (k)
Week 25



Week 51



Week 104
Rat Crl:CD(SD)(i!)
> No significant chang^^ Significant increase Significant decrease «
Week 25
Week 51
Week 104
Significant Trend!

A
V
V
iA
-tAt*
-A™
-A
100 200 300 400 500 600 700 800 900
Dose (mg/kg-day)
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).
This document is a draft for review purposes only and does not constitute Agency policy.
3-68	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 3-22. Percent change in hematocrit due to PFHxA exposure in
short-term, subchronic, and chronic oral toxicity studies
Study Design and Reference
Dose (mg/kg-d)
in

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Toxicological Review ofPFHxA and Related Salts
Study Design and Reference
Dose (mg/kg-d)
in
10%) across all study designs and exposure durations at
200 mg/kg-day (Klaunig et al.. 2015: Chengelis etal.. 2009bl. 250 mg/kg-day fNTP. 20181. 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 fNTP. 2018: Loveless etal.. 20091 (summary details are
available in PFHxA Tableau visualization). Collectively the histological findings considered
together with red blood cell parameters suggest PFHxA interacts with the erythropoietic pathways
including outcomes such as anemia that can lead to compensatory erythrogenic responses in the
bone marrow and spleen.
Endpoint
Study
Experiment
Animal Description
Observation Time
Reticulocytes
NTP, 2018.4309149
28-Day Oral
Rat, Harlan Sprague-Dawley (9)
Day 29



Rat, Harlan Sprague-Dawley (cf)
Day 29

Chengelis, 2009, 2850404
90-Day Oral
Rat, Crl:CD(SD) (9)
Day 90



Ral, Crl:CD(SD) (cf)
Day 90

Loveless, 2009,2850369
90-Day Oral
Rat, Crl:CD(SD) (cf)
Day 92



Rat, Crl:CD(SD) (9)
Day 93

Klaunig, 2015, 2850075
2-Year Cancer Rioassay
Rat, Crl:CT)(SD) (9)
Week 25



Rat, Crl:CD(SD) (cf)
Week. 25



Rat, Crl:CT)(SD) (9)
Week 51



Rat, Crl:CD(SD) (cf)
Week 51



Rat, Crl:CD(SD) (9)
Week 104



Rat, Crl:CD(SD) (cf)
Week 104
| • No significant change A Significant
increase V Significant decrease ^ Significant Trend |

PFHxA Hemaotopoielic Effects: Reticulocytes
100 200 300 400 500 600 700 800 900 1,0001,1001
Dose (mg/kg-day)
Figure 3-15. Hematological findings (reticulocytes) in rats exposed by gavage
to PFHxA or PFHxA sodium salt (full details available by clicking the HAWC
link).
This document is a draft for review purposes only and does not constitute Agency policy.
3-70	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Toxicological Review ofPFHxA and Related Salts
Table 3-24. Percent change in reticulocytes due to PFHxA exposure in
short-term, subchronic, and chronic oral toxicity studies
Study Design and Reference
Dose (mg/kg-d)
in
20 mg/kg-day, whereas APTT was decreased in the 500 mg/kg-day female rat dose
group. The observed increase in platelets and decreased clotting time (along with increased
reticulocytes) were coherent changes indicative of an erythropoietic response in the bone marrow,
suggesting bone marrow was not adversely affected by PFHxA exposure.
This document is a draft for review purposes only and does not constitute Agency policy.
3-71	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Toxicological Review ofPFHxA and Related Salts
Endpoint
Study
Experiment
Animal Description
Observation Time
Platelets (PLT)
NTP, 2018,4309149
28-Day Oral
Rat, Harlan Sprague-Dawley(cf) Day 29



Rat, Harlan Sprague-Dawley(i
:) Day 29

Chengelis, 2009, 2850404
90-Day Oral
Rat, Crl:CD(SD) (
-------
Toxicological Review ofPFHxA and Related Salts
effects (across various outcome measures of hematopoietic function), generally at >200 mg/kg-day
following short-term (28-day), subchronic (90-day), or chronic (2-year) exposures.
This document is a draft for review purposes only and does not constitute Agency policy.
3-73	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 3-25. 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-d
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-d
•	90-d (2 studies)
•	2-yr
•	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
• No factors noted
•	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
This document is a draft for review purposes only and does not constitute Agency policy.
3-74	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Evidence stream summary and interpretation
Evidence integration summary
judgment
Hemostasis
4 hiqh confidence
studies in adult
rats:
•	28-d
•	90-d (2 studies)
•	2-yr
•	Consistent treatment
related effect on
platelet levels
•	Consistency across
study designs
•	High confidence
studies
• No factors noted
•	Increased platelet levels
>10 mg/kg-d; both sexes,
1 28-d, 2 90-d studies
•	Decreased activated
partial thromboplastin
(APTT) at >20 mg/kg-d;
males only, 1 90-d study
•	Decreased prothrombin
(PT) time at 500 mg/kg-
day; males only, 190-d
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.
This document is a draft for review purposes only and does not constitute Agency policy.
3-75	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Toxicological Review ofPFHxA and Related Salts
3.2.5. Endocrine Effects
Human
Thyroid Hormones
Two studies examined the association between PFHxA exposure and thyroid hormones in
humans (see Figure 3-17). One was considered uninformative due to lack of consideration of
confounding, including age, sex, medical history, and socio-economic status which is expected to
substantially impact the results fSeo etal.. 20181. The other study was a cross-sectional study of
the general population in China and was considered low confidence fLi etal.. 20171 due to concerns
around participant selection, outcome measures, consideration of confounding and decreased
sensitivity. Regarding the latter concern, the exposure levels were low and contrast narrow in Li et
al. (2017) (median [range]: 0.01 [
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Toxicological Review ofPFHxA and Related Salts
Animal
Four short-term (28-day), subchronic, and chronic animal studies evaluated potential
endocrine effects of PFHxA or PFHxA sodium salt in rats. Most of the outcome-specific study
ratings were rated high confidence. Histopathology for Chengelis etal. f2009bl was rated low
confidence because of issues related to observational bias, concerns about endpoint sensitivity and
specificity, and results presentation. A summary of the studies and the interpretations of
confidence in the results for the different outcomes based on the individual study evaluations is
presented in Table 3-26, and details are available by clicking the HAWC link.
Table 3-26. Endocrine endpoints for PFHxA and associated confidence scores
from repeated-dose animal toxicity studies




+-»
-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 d)
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 d)
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 d)
Male and female: 0, 20,100,
500 mg/kg-d



Klaunig et al.
Rat, Crl:CD(SD)
2-yr 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 low confidence; NM, outcome not measured.
Thyroid Hormones
A single study evaluated potential PFHxA effects on endocrine function specific to thyroid
hormones in rats exposed for 28 days fNTP. 2018], Specifically, males showed statistically
significant, dose-dependent decreases in thyroid hormones. These outcomes showed a clear dose-
dependent pattern of effect with treated animals showing reductions of 25-73% or 20-58% for
free or total T4, respectively. Smaller decreases in T3 in males also were observed (18-29%),
although the dose-dependence of this effect was less clear. No treatment-related changes were
This document is a draft for review purposes oniy and does not constitute Agency poiicy.
3-77	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
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 3-27.
Endpoint	Study	Experiment Animal Description	Observation Time
Thyroid Stimulating Hormone (TSH) NTP, 201
3,4309149 28 Day Oral
Rat, Harlan Sprague-Dawley(v)
Day 29
+—•—•—
•
m
~



Rat, Harlan Sprague-Dawley(cv)
Day 29
•—•—•—
•
m
~
Thyroxine (T4), Free
NTP, 201
3,4309149 28 Day Oral
Rat, Harlan Sprague-Dawley(v)
Day 29
*—•—•—
—•	
	•	
	+



Rat, Harlan Sprague-Dawley(cv)
Day 29
^v-
~
V
V
Total Thyroxine (T4)
NTP, 201
3,4309149 28 Day Oral
Rat, Harlan Sprague-Dawley(v)
Day 29
~—•—•—
—•	
	•	
	+



Rat, Harlan Sprague-Dawley(cv)
Day 29
^v-
~
V
V
Triiodothyronine (T3)
NTP, 201
3,4309149 28 Day Oral
Rat, Harlan Sprague-Dawley(v)
Day 29
~—•—•—
—•	
	•	
	+



Rat, Harlan Sprague-Dawley(cv)
Day 29
^v-
~
V
V
• No significant changa^ Significant increase^ Significant decrease Q Significant Trendl	-100 0 100 200 300 400 500 600 700 800 900 1,0001,100
Dose (mg/kg-day)
Figure 3-18. Thyroid hormone measures from the serum of rats exposed by
gavage to PFHxA or PFHxA sodium salt (full details available by clicking the
HAWCJink).
Table 3-27. Percent change in thyroid hormone levels following PFHxA
exposure in a 28-day oral toxicity study


Dose (mg/kg-d)
Study Design and Reference
Hormone
in

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Toxicological Review ofPFHxA and Related Salts
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 3-28.
Table 3-28. Incidence of thyroid follicular epithelial cell hypertrophy
following PFHxA ammonium salt exposure in a 90-day oral toxicity study
Sex and Reference
Time point
Dose (mg/kg-d)
0
20
100
500
90-d, female rat (Loveless et al., 2009)
Exposure, Day 90
0/10
0/10
0/11
4/10
90-d, male rat (Loveless et al., 2009)
0/10
0/10
1/10
2/10
90-d, female rat (Loveless et al., 2009)
Recovery Day 30
0/10


6/10
90-d, male rat (Loveless et al., 2009)
0/10


3/10
90-d, female rat (Loveless et al., 2009)
Recovery, Day 90
0/10
0/10
0/9
0/10
90-d, 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 (NTP. 2018: Chengelis etal..
2009b: Loveless et al.. 20091. 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) ofPFHxA 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 (NTP. 2018:
Chengelis etal.. 2009b: Loveless etal.. 2009).
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 ofPFHxA 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
This document is a draft for review purposes only and does not constitute Agency policy.
3-79	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Toxicological Review ofPFHxA and Related Salts
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 ofPFHxA exposure
durations (28 days to 2 years) and doses (up to 1,000 mg/kg-day) fNTP. 2018: Klaunig etal.. 2015:
Chengelis etal.. 2009bl. 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 ofPFHxA, with plasma
concentrations measured 2-3 times higher in male rats when compared to females f Chang etal..
2008: Lau etal.. 2006: Lau etal.. 2004). 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. There are many
mechanisms by which chemicals have been shown to disrupt thyroid homeostasis. Although there
is evidence that some PFAS may alter thyroid function via interaction with thyroid hormone
receptors and transport proteins, the current data show only weak binding for PFHxA fBorghoff et
al.. 2018: Ren etal.. 2016: Ren etal.. 20151. It is possible that the observed changes in thyroid
histopathology are secondary to hepatic effects. In rats, increases in thyroid epithelial cell
hypertrophy are associated with induction of microsomal liver enzymes and hepatocellular
hypertrophy (Cesta 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
This document is a draft for review purposes only and does not constitute Agency policy.
3-80	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
1	based on four animal studies generally rated as high confidence that reported treatment-related
2	changes in thyroid hormone levels and thyroid histopathology after exposure to PFHxA at
3	>62.5 mg/kg-day (Table 3-27).
This document is a draft for review purposes only and does not constitute Agency policy.
3-81	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 3-29. 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 as 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-d.
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-d
•	High confidence study.
•	Dose-response gradient
observed for free and
total T4
•	Large effect
magnitude; up to 73%
• No factors noted
• Decreased T4 (free and
total) and T3 observed
in males only at > 62.5
mg/kg-d
©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 the four available
studies, and
histopathological changes
may be explained by non-
thyroid related effects
Histopathology
3 hiqh
confidence
studies in adult
rats:
•	28-d
•	90-d
•	2-yr
• High confidence
studies
• Unexplained
inconsistency across
studies
•	Increased incidence of
thyroid epithelial cell
hypertrophy at >100
mg/kg-d for 90 d;
persisted up to 90 d
after exposure
•	No effects observed in
28 d study at up to
1,000 mg/kg-d
Decreases in T3 were
observed in both animal and
human studies, although
results in humans were of
low confidence.
Susceptible populations and
lifestages:
No evidence to inform
This document is a draft for review purposes only and does not constitute Agency policy.
3-82	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Evidence stream summary and interpretation
Evidence integration
summary judgement
1 low confidence
study in adult
rats:
• 90-d




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-d
•	90-d (2
studies)
• High confidence
studies
• Unexplained
inconsistency across
studies
•	Relative thyroid
weights were increased
only in females 30 d
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
This document is a draft for review purposes only and does not constitute Agency policy.
3-83	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Toxicological Review ofPFHxA and Related Salts
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 ofPFHxA in serum (see Figure 3-19). A significant negative correlation between PFHxA
levels in semen and sperm motility was found in this study (correlation coefficient = -0.35,
p < 0.01), but analytical measurement of PFAS in semen is less established than in blood and the
relevance to exposure is unclear. Still, exposure levels in the study based on serum measurements
were fairly high (median: 29 ng/mL, 5th-95th percentile: 11-70 ng/mL), so the study had
reasonable sensitivity to detect an effect.
Reproductive Hormones
A single study rated low confidence due to low sensitivity and high potential for
confounding (see Figure 3-19) found some support for associations between PFHxA and
reproductive hormones in a population of adolescent (13-15 years old) males in Taiwan fZhou et
al.. 2016). Overall, authors reported no significant associations between PFHxA and serum
testosterone and estradiol; however, when the data were stratified by sex, a significant negative
association between testosterone and PFHxA exposure level ((3 = -0.31, 95% CI: -0.59, -0.02) was
found in boys. Based on serum measurements, the exposure levels in this study were low and the
range narrow (median: 0.2 ng/mL, IQR 0.1-0.3 ng/mL), which might have reduced study
sensitivity. The presence of an association despite reduced sensitivity could be due to either high
potency of the exposure to cause these effects or potential confounding by other correlated PFAS,
including PFOS, PFDA, and PFNA.
This document is a draft for review purposes only and does not constitute Agency policy.
3-84	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
Toxicological Review ofPFHxA and Related Salts
^ ',,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 3-19. Study evaluation for human epidemiological studies reporting
male reproductive findings from PFHxA exposures (HAWC: PFHxA - Human
Toxicity Male Reproductive Effects link).
Animal
Several short-term (28-day), subchronic, and chronic animal studies evaluated sperm
parameters, reproductive organ weights, and other reproductive male outcomes in rats receiving
oral exposures of PFHxA and PFHxA sodium salt Most outcome-specific study ratings were rated
high confidence; however, some specific concerns were identified that resulted in low confidence
ratings. Although generally a well-conducted study, NTP (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 3-30, and details are available by clicking the HAWC link.
This document is a draft for review purposes only and does not constitute Agency policy.
3-85	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 3-30. Study design, exposure characteristics, and individual outcome
ratings
Study
Species, strain
(sex)
Exposure design
Exposure route and
dose
Sperm
parameters
Organ weight
Histopathology
Hormone
levels
Reproductive
system
development
NTP
(2018)
Rat, Harlan
Sprague-Dawley
(male and
female)
Short term
(28 d)
Gavage3
Male and female: 0,
62.5,125, 250, 500,
1,000 mg/kg-d

++
+ +
++
NM
Chengelis
et al.
(2009b)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
Subchronic
(90 d)
Gavage3
Male and female: 0,
10, 50, 200 mg/kg-d
NM
++

NM
NM
Loveless
et al.
(2009)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
Subchronic (90 d)
One-generation
reproductive: P0
females dosed 70 d
prior to
cohabitation,
through gestation
and lactation (126
d); P0 males dosed
for 110 d
Developmental:
Gestation Days 6-20
Gavageb
Male and female: 0,
20,100,
500 mg/kg-d
++
++
+ +
NM
++
Klaunig et
al. (2015)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
2-yr cancer bioassay
Gavage3
Male: 0, 2.5,15,
100 mg/kg-d
Female: 0, 5, 30,
200 mg/kg-d
NM
NM
+ +
++
NM
Iwai and
Hoberman
(2014)°
Mouse, Crl:
CDl(ICR);
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,a PFHxA sodium salt,b or PFHxA ammonium saltd by gavage. Study evaluation details for all outcomes are
available by clicking the HAWC link.
cPhase 1 was a range-finding study used to determine the appropriate dose range for identification of a NOAEL in
Phase 2.
++ Outcome rating of high confidence; - outcome rating of low confidence; NM, outcome not measured.
This document is a draft for review purposes only and does not constitute Agency policy.
3-86	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Toxicological Review ofPFHxA and Related Salts
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 (see
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 which does not encompass a full 6-week spermatogenic cycle
in rats. In the one-generation reproductive study, Loveless etal. 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.
Endpoint	Study	Experiment	Animal Description Observation Time	Reproductive Effects: Sperm Parameters
Testicular Spermatids (per Testis)
Loveless, 2009, 2850369 1-Generation Reproductive
P0 Rat, Crl:CD(SD) ( No significant changg^ Significant increase^ Significant decrease > Significant Trencj ""100 0 "100 200 300 400 500 600 700 800 900 1,0001,100
Figure 3-20. Male reproductive effects on sperm parameters in male rats
exposed to PFHxA or sodium salt for 28 or 90 days (HAWC: PFHxA - Animal
Toxicity Male Reproductive Effects link).
Reproductive Organ Weights
Reproductive studies commonly report both absolute and relative organ weights; however,
for the testes, absolute weights are considered most informative for hazard evaluation f Bailey etal..
20041. Three studies (28- or 90-day exposure durations) reported data on the effects ofPFHxA or
PFHxA sodium salt exposure on male reproductive organ weights (i.e., testes, epididymis) in rats
(see Figure 3-21) (NTP. 2018: Chengelis etal.. 2009b: Loveless etal.. 2009). Two studies reported a
modest, but statistically significant (p < 0.05; 13-16% change from control), increase in relative, but
not absolute, testis weight in rats exposed to 1,000 mg/kg-day for 28 days fNTP. 20181 or
500 mg/kg-day for 90 days (Loveless etal.. 2009). No treatment-related effects on male
reproductive organ weights were reported by Chengelis etal. f2009bl.
This document is a draft for review purposes only and does not constitute Agency policy.
3-87	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Toxicological Review ofPFHxA and Related Salts
Endpoint	Study Experiment Animal Description	Observation Time	Male Reproductive Effects: Organ Weights
Cauda Epididymis Weight, Absolute NTP, 2018,4309149	28-DayOral	Rat, Harlan Sprague-Dawley(<-)	Day29
Epididymides Weight, /tosolute	Loveless, 2009,2850369 90-DayOral	Rat, Crl:CD(SD) (r-)	Day92
Epididymides Weight, Relative	Loveless, 2009,2850369 90-DayOral	Rat, Crl:CD(SD) (r-)	Day92
Epididymis Weight, /tosolute	NTP, 2018,4309149 28-DayOral	Rat, Harlan Sprague-Dawley(r-)	Day29
RightTestis Weight, Absolute	NTP, 2018,4309149 28-DayOral	Rat, Harlan Sprague-Dawley(r-)	Day29
Testes Weight, /tosolute	Loveless, 2009, 2850369 90-DayOral	Rat, Crl:CD(SD) (>-)	Day92
Testes Weight, Relative	Loveless, 2009, 2850369 90-Day Oral	Rat, Crl:CD(SD) (r )	Day92
Testis Weight, Absolute	NTP, 2018,4309149 28-DayOral	Rat, Harlan Sprague-Dawley(r-)	Day29
Testis Weight, Right, Relative	NTP, 2018,4309149 28-DayOral	Rat, Harlan Sprague-Dawley(<-.)	Day29
| 9	No significant effected Significant in crease^ Significant decrease cs Significant Tren^
• • • # ~
-100 0 100 200 300 400 500 600 700 800 900 1,0001,100
Dose (mg/kg-day)
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).
Reproductive Hormones
Two studies measured hormone levels (i.e., testosterone, estradiol, and luteinizing
hormone) following exposure to PFHxA (NTP. 2018: Klaunig etal.. 20151. Klaunig etal. (20151
reported a small, transient decrease in testosterone and luteinizing hormone in males at the
26-week time point. Effects were not dose dependent and were not significantly different from
controls at doses up to 100 mg/kg-day PFHxA. This pattern was not observed at the 52-week time
point A short-term study found no effects on testosterone following exposure of up to
1,000 mg/kg-day for 28 days (NTP. 2018). Klaunig etal. (2015) also measured estradiol but found
no treatment-related changes.
Histopathologv
Four studies evaluated effects ofPFHxA or PFHxA sodium salt on histopathology of the
testes and epididymites and reported no treatment-related changes fNTP. 2018: Klaunig etal..
2015: Chengelis et al.. 2009b: Loveless etal.. 20091. One study was rated low confidence for this
outcome (Chengelis etal.. 2009b).
Male Reproductive System Development
Two studies examined outcomes related to male reproductive system development
following developmental exposure to PFHxA ammonium or sodium salts flwai and Hoberman.
2014: Loveless etal.. 20091. No treatment-related effects were reported on the age at preputial
separation, a marker of puberty onset.
Evidence Integration
The available evidence informing the potential for an effect ofPFHxA exposure on male
reproduction in humans was limited to two low confidence studies that provided some indication of
an association between PFHxA exposure and sperm motility (Song etal.. 2018) and reproductive
hormone levels fZhou etal.. 20161. These results are difficult to interpret, however, based on the
This document is a draft for review purposes only and does not constitute Agency policy.
3-88	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Toxicological Review ofPFHxA and Related Salts
availability of a single study for each outcome and the high risk for bias in these evaluations. Based
on these results, there is indeterminate human evidence of male reproductive effects.
In animals, the evidence supporting potential effects ofPFHxA exposure on male
reproduction was primarily limited to decreased sperm count fNTP. 20181 and increased relative
testis weights fNTP. 2018: Loveless etal.. 20091 at the highest tested doses in these studies (1,000
and 500 mg/kg-day, respectively). Decreased sperm count reported by NTP (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 short term study. In addition, evidence of overt toxicity (i.e., 13% reduction in terminal body
weight relative to controls) was found in the male rats dosed 1,000 mg/kg-day in the NTP (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 (see Table 3-
31).
This document is a draft for review purposes only and does not constitute Agency policy.
3-89	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 3-31. Evidence profile table for male reproductive effects
Evidence stream summary and interpretation
Evidence integration
summary judgment
Evidence from studies of exposed humans
OOO
Studies and confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key findings
Evidence stream
judgment
Evidence inadequate
Primary Basis:
Evidence is low confidence
or largely null
Human relevance:
Without evidence to the
contrary, effects in rats are
considered relevant to
humans
Cross stream coherence:
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
N/A (human evidence
indeterminate)
Susceptible population and
Studies and confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key findings
OOO
Indeterminate
Sperm Parameters
1 high confidence study
in adult rats:
•	90-d
1 low confidence in
adult rats
•	28-d
• No factors noted
• Unexplained
inconsistency across
studies
• Decreased sperm count
in the cauda epididymis
at 1,000 mg/kg-d
The data are largely
null. Some evidence of
reproductive effects
but interpretation
limited by unexplained
inconsistency at effects
observed only at the
lifestages:
No evidence to inform
Organ Weights
3 high confidence
studies in adult rats:
•	28-d
•	90-d (2 studies)
•	High confidence
studies
•	Dose-response
with longer
exposure duration
• No factors noted
• Increased relative testis
weight at >500 mg/kg-d;
no change in absolute
testis weights (preferred
metric)
high dose that elicited
high overt toxicity
(i.e., 13% decrease in
body weight).

This document is a draft for review purposes only and does not constitute Agency policy.
3-90	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Evidence stream summary and interpretation
Evidence integration
summary judgment
Reproductive
Hormones
2 high confidence
studies in adult rats:
• 28-d
• High confidence
studies
• No factors noted
• Transient decrease of
small magnitude in
luteinizing hormone and
testosterone


• 2-yr





Histopathology and
Male Reproductive
System Development
4 high confidence
studies in rats and
mice:
•	28-d (rat)
•	90-d (rat)
•	GD 6-18 (mouse)
•	2-yr (rat)
1 low confidence study
in adult rats:
•	90-d
• High confidence
studies
• No factors noted
• No treatment related
effects reported at
<1,000 mg/kg-d


Mechanistic evidence and supplemental information

Biological events of
pathways
Biological events of
pathways
Biological events of pathways
Biological events of
pathways

• No studies identified

This document is a draft for review purposes only and does not constitute Agency policy.
3-91	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Toxicological Review ofPFHxA and Related Salts
3.2.7. Female Reproductive Effects
Human
Reproductive Hormones
A single low confidence study (see Figure 3-22) evaluated associations between PFHxA and
reproductive hormones in a population of Taiwanese adolescents (13-15 years old) (Zhou etal..
2016). Overall, the authors reported nonsignificant inverse associations between PFHxA and
serum testosterone and estradiol in females when the data were stratified by sex. Exposure levels
to PFHxA were low, which might have reduced study sensitivity, as described above in Section
3.2.6. Male Reproductive Effects.
Participant selection
Exposure measurement
Outcome ascertainment
Confounding
Analysis
Sensitivity
Selective Reporting
Overall confidence


A6



Legend
Good (metric) or High confidence (overall)
Adequate (metric) or Medium confidence (overall)
Deficient (metric) or Low confidence (overall)
Critically deficient (metric) or Uninformative (overall)
Figure 3-22. Study evaluation for human epidemiological studies reporting
female reproductive findings from PFHxA exposures (HAWC: PFHxA - Human
Toxicity Female Reproductive link).
Animal
Five animal studies evaluated outcomes related to female reproduction in rats and mice
receiving PFHxA via gavage, PFHxA sodium salt, or PFHxA ammonium salt Study designs included
short-term (28-day), subchronic (90-day), and chronic (2-year) one-generation reproductive and
developmental exposures. In general, the outcome-specific study ratings were high confidence.
One study was rated low confidence for histopathology due to concerns about observational bias,
endpoint sensitivity and specificity, and results presentation (Chengelis etal.. 2009b). The results
This document is a draft for review purposes only and does not constitute Agency policy.
3-92	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
1	of study evaluation for female reproductive outcomes are presented in Table 3-32 and details are
2	available by clicking the HAWC link.
Table 3-32. Study design characteristics
Study
Species, strain
(sex)
Exposure design
Exposure route
and dose
Fertility and
pregnancy
Organ weight
Histopathology
Reproductive
hormones
Reproductive
system
development
NTP(2018)
Rat, Harlan
Sprague-Dawley
(male and
female)
Short term
(28 d)
Gavage3
Male and female:
0, 62.5,125, 250,
500,1,000 mg/kg-d
++
+ +
+ +
++
NM
Chengelis et
al. (2009b)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
Subchronic
(90 d)
Gavage3
Male and female:
0,10, 50,
200 mg/kg-d
NM
+ +

NM
NM
Loveless et
al. (2009)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
Subchronic (90 days)
One-generation
reproductive: P0
females dosed 70 d
prior to cohabitation,
through gestation and
lactation (126 d); P0
males dosed for 110 d
Developmental:
Gestation Days 6-20
Gavageb
Male and female:
0, 20,100,
500 mg/kg-d
++
+ +
+ +
NM
++
Klaunig et
al. (2015)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
2-yr cancer bioassay
Gavage3
Male: 0, 2.5,15,
100 mg/kg-d
Female: 0, 5, 30,
200 mg/kg-d
NM
NM
+ +
++
NM
Iwai and
Hoberman
(2014)°
Mouse, Crl:
CD1(ICR; Charles
River
Laboratories,
Inc.
Gestation Days 6-18
Gavaged
Phase 1: 0,100,
350, 500 mg/kg-d
Phase 2: 0, 7, 35,
175 mg/kg-d
++
NM
+ +
NM
++
Study evaluation for animal toxicological endpoints reported from female reproductive studies with rats receiving
PFHxA,a PFHxA sodium salt,b or PFHxA ammonium saltd by gavage. Study evaluation details for all outcomes are
available by clicking the HAWC link.
cPhase 1 was a range-finding study used to determine the appropriate dose range for identification of a NOAEL in
Phase 2.
++ Outcome rating of high confidence; - outcome rating of low confidence; NM, outcome not measured.
3	Fertility and Pregnancy Outcomes
4	Three studies published in two reports evaluated outcomes related to fertility and
5	pregnancy following exposure by gavage with PFHxA or PFHxA salts in rats or mice flwai and
This document is a draft for review purposes only and does not constitute Agency policy.
3-93	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Toxicological Review ofPFHxA and Related Salts
Hoberman. 2014: Loveless etal.. 20091. Some effects on maternal body weight change (i.e., gain or
loss) were noted. In both the developmental and one-generation reproductive rat studies (Loveless
etal.. 20091. statistically significant reductions in maternal body weight change were observed
during gestation in the high dose group (500 mg/kg-day). In the developmental study fLoveless et
al.. 20091. there was a statistically significant decrease in total maternal body weight gain (19%
relative to control) and when correcting for gravid uterine weight (26% relative to control) from
GD 6-21 in the 500 mg/kg-day dose group. In the one-generation reproductive study, similar
effects were observed but were limited to early gestation (Loveless etal.. 20091. From GD 0-7,
body weight gain in dams exposed to 500 mg/kg-day was reduced by 31% relative to controls.
There was no treatment-related effect on maternal weight gain over the entire gestational period
(GD 0-21) and the high dose (500 mg/kg-day) showed a statistically significant increase in body
weight change relative to controls during lactation (PND 0-21) fLoveless et al.. 20091. No changes
in maternal body weight gain were identified in mice (Iwai and Hoberman. 2014).
Only one of the three available studies reported effects on absolute maternal body weight
In the developmental rat study, dams exposed to 500 mg/kg-day (GD 6-20) showed a statistically
significant decrease in terminal body weight (7% relative to control) fLoveless etal.. 20091.
Deficits remained when correcting for gravid uterine weight (5% relative to control), indicating the
effects on body weight were driven by maternal body weight rather than reductions in fetal body
weight or number of fetuses. However, this level of change may not be biologically significant (U.S.
EPA. 1991). There was no effect on absolute maternal body weight in the one-generation
reproductive rat or mouse study (Iwai and Hoberman. 2014: Loveless etal.. 20091. These results
are presented in Figure 3-23.
No treatment-related effects on mating, pregnancy incidence, gestation length, number of
implantations, or litter size were reported in either study that evaluated these outcomes flwai and
Hoberman. 2014: Loveless etal.. 20091. Estrous cyclicity in rats exposed as adults or during
gestation was also unaffected in two studies (NTP. 2018: Loveless etal.. 2009).
This document is a draft for review purposes only and does not constitute Agency policy.
3-94	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Toxicological Review ofPFHxA and Related Salts
Endpoint	Study	Experiment	Animal Description Observation Time	Female Reproductive Effects: Body Weight
Body Weight Change, Gestation Loveless, 2009, 2850369 14-Day Developmental P0 Rat, Crl:CD(SD) (2) GD 6-21
... v
1-Generation Reproductive P0 Rat, Crl:CD(SD) (2) GD 0-7
P0 Rat, Crl:CD(SD) (*) GD 0-21
... ~
... .
Iwai, 2014, 2821611 1-Generation Reproductive P0 Mouse, CD-1 (Q) GD6-18
P0 Mouse, CD-1 (2) GD 6-18
. .


Body Weight Change, Gestation Loveless, 2009, 2850369 14-Day Developmental P0 Rat, Crl:CD(SD) {'2) GD 6-21
(Minus Gravid Uterine Weight)
... ~
Body Weight Change, Lactation Loveless, 2009, 2850369 1-Generation Reproductive P0 Rat, Crl:CD(SD) (2) PND 0-21
... A
Iwai, 2014, 2821611 1-Generation Reproductive P0 Mouse, CD-1 (2) PND 0-20
P0 Mouse, CD-1 (2) PND 0-20
Body Wsight, Absolute Loveless, 2009, 2850369 1-Generation Reproductive P0 Rat, Crl:CD(SD) (2) GD 0
P0 Rat, Crl:CD(SD) (2) GD 7
P0 Rat, Crl:CD(SD) (2) GD 14
P0 Rat, Crl:CD(SD) (2) GD 21
















Body Wsight, Terminal (Minus Gravid Loveless, 2009, 2850369 14-Day Developmental P0 Rat, Crl:CD(SD) (2) GD 21
Uterine Weight)
... ~
Terminal Body Weight, Absolute Loveless, 2009, 2850369 14-Day Developmental P0 Rat, Crl:CD(SD) (2) GD 21
... ~
Iwai, 2014, 2821611 1-Generation Reproductive P0 Mouse, CD-1 (2) PND 20
P0 Mouse, CD-1 (2) PND 20
. •


• No significant changg^ Significant increase^^Significant decrease |
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).
Histopathologv
Four studies evaluated effects on histopathology of reproductive organs (i.e., uterus and
ovaries) in rodents following exposure to PFHxA (NTP. 2018: Klaunig etal.. 2015: Chengelis etal..
2009b) or PFHxA sodium salt fLoveless et al.. 20091. Only NTP T20181 reported an effect of
exposure, with females showing a statistically significant increase in the incidence of bilateral
uterine horn dilation in all but the vehicle controls and highest dose group (see Figure 3-24).
Whereas the control and high-dose group had 10 animals per group, however, groups showing a
statistically significant increase had only 1-3 animals per group, complicating interpretation of
these findings. The total incidence ranges from 1 to 3 animals/treatment group, regardless of
sample size or PFHxA dose ( see Figure 3-24). The biological significance of these results is unclear.
Uterine horn dilation can indicate an estrogenic effect, but no coherent changes in serum estradiol
or estrous cyclicity were observed in this study. Similarly, no other treatment-related effects on
female reproductive histopathology were reported (NTP. 2018: Klaunig etal.. 2015: Chengelis etal..
2009b: Loveless et al.. 2009).
This document is a draft for review purposes only and does not constitute Agency policy.
3-95	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Toxicological Review ofPFHxA and Related Salts
Endpoint	Study	Animal Description	Dose Incidence	Female Reproductive Histopathology
Uterus, Bilateral Dilation NTP, 2018,4309149 Rat, Harlan Sprague-Dawley($) 0
1/10(10.0%)
i
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%)
i
r=j	. „ . . —		—	_ . ,1 0 10 20 30 40 50 60 70
I l percent affected ^¦Significant Compared to Conlroll
I	'	% Affected
Figure 3-24. Female reproductive effects on uterine horn dilation in rats
exposed to PFHxA for 28 days (HAWC: PFHxA - Animal Toxicity Female
Reproductive link).
Organ Weights
Three studies evaluated effects ofPFHxA exposure on uterine and ovarian weights fNTP.
2018: Chengelis etal.. 2009b: Loveless etal.. 20091. Authors reported no treatment-related effects
for these outcomes.
Reproductive Hormones
Two studies measured effects of PFHxA or PFHxA ammonium salt on testosterone fNTP.
2018: Klaunig et al.. 2 0151. estradiol, and luteinizing hormone (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 (2014) and
Loveless etal. (20091 found no effects on age at vaginal opening, a measure of puberty onset.
Evidence Integration
A single low confidence human study reported a weak inverse association between PFHxA
exposure measures and serum levels of reproductive hormone levels in adolescents fZhou etal..
2016). Based on these results, there is indeterminate human evidence of female reproductive
effects.
In animals, evidence supporting effects ofPFHxA exposure female reproduction was largely
limited to effects on maternal weight gain during gestation in rats exposed to 500 mg/kg-day
fLoveless etal.. 20091. These effects corresponded with a small but statistically significant absolute
body weight in the high confidence developmental rat study only fLoveless etal.. 2009). however
the level of the decrease (5-7%) may not be biologically significant. There were no effects on
maternal weight or weight gain in the in the mouse study. The reported effects on uterine horn
dilation appears to be influenced by differences in sample sizes, as the total incidence of the finding
is similar across controls and all dosing groups. Furthermore, this finding is generally associated
with estrogenic effects, but no coherent changes were observed that would be indicative of
This document is a draft for review purposes only and does not constitute Agency policy.
3-96	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
1	estrogenic changes in females. No treatment-related changes were reported for other female
2	reproductive outcomes fNTP. 2018: Klaunig etal.. 2015: Iwai and Hoberman. 2014: Chengelis etal..
3	2009b: Loveless et al.. 20091. Based on these results, there is indeterminate animal evidence of
4	female reproductive effects.
5	Overall, the currently available evidence is inadequate to assess whether PFHxA might
6	cause female reproductive effects in humans under relevant exposure circumstances (see
7	Table 3-33).
This document is a draft for review purposes only and does not constitute Agency policy.
3-97	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 3-33. Evidence profile table for female reproductive effects
Evidence stream summary and interpretation
Evidence integration summary
judgment
Evidence from studies of exposed humans
OOO
Evidence inadequate
Studies and
confidence
Factors that
increase
strength
Factors that
decrease certainty
Summary and key
findings
Evidence stream judgment
Reproductive
Hormones
1 low confidence
study
• No factors
noted
• Low confidence
study
• Nonsignificant
inverse association
between PFHxA
exposure and
testosterone and
estradiol
ooo
Indeterminate
Evidence from animal studies

Studies and
confidence
Factors that
increase
strength
Factors that
decrease certainty
Summary and key
findings
Evidence stream judgment
Primary Basis:
Evidence is low confidence or
largely null.
Human relevance:
•	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-d (rat)
•	90-d (rat)
•	GD 6-18 (mouse)
• High
confidence
studies
• Unexplained
inconsistency
across studies
• Decreases in
maternal weight
gain during gestation
in rats exposed to
500 mg/kg-d
OOO
Indeterminate
The animal evidence is largely null.
Some evidence of female
reproductive effects but body weight
effects lacked consistency across
studies. Histopathology effects were
not dose-dependent and lacked
coherent evidence to support the
biological significance of the findings
Histopathology
4 high confidence
studies in rats and
mice:
• 28-d (rat)
• High
confidence
studies
•	Unexplained
inconsistency
across studies
•	Lack of expected
coherence with
• Increase in bilateral
uterus dilation
reported for all
groups except the
highest dose
This document is a draft for review purposes only and does not constitute Agency policy.
3-98	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Evidence stream summary and interpretation
Evidence integration summary
judgment
•	90-d (rat)
•	2-yr (rat)
•	GD 6-18 (mouse)
1 low confidence
study in adult rats:
•	90-d

other estrogen
related outcomes


coherence across evidence
streams.
Susceptible populations:
• None identified
Organ Weights,
Reproductive
Hormones,
Reproductive
System
Development
6 hiqh confidence
studies in rats and
mice:
•	28-d (rat)
•	90-d (rat, 2
studies)
•	2-yr (rat)
•	GD 6-18 (mouse)
•	GD 6-20 (rat)
• High
confidence
studies
• No factors noted
• No treatment-
related effects were
reported at
<1,000 mg/kg-d


Mechanistic evidence and supplemental information

Biological events of
pathways
Biological
events of
pathways
Biological events of pathways
Biological events of pathways

• No studies Identified

This document is a draft for review purposes only and does not constitute Agency policy.
3-99	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
Toxicological Review ofPFHxA and Related Salts
3.2.8. Immune Effects
Human
Asthma
One medium confidence case-control study in Taiwan was reported in three publications
(Oin etal.. 2017: Zhou etal.. 2017: Dongetal.. 20131. Dongetal. (20131 includes results from all
three studies that examined the potential association between PFHxA exposure and asthma, asthma
symptoms, pulmonary function, and related immune markers (see 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 and contrast narrow (median [IQR]: 0.2 ng/mL [0.1-0.3
ng/mL]), which may have reduced study sensitivity.
Participant selection
Exposure measurement -
Outcome ascertainment-
Confounding-
Analysis -\
O0

&
&


Legend
Good (metric) or High confidence (overall)
Adequate (metric) or Medium confidence (overall)
Deficient (metric) or Low confidence (overall)
Critically deficient (metric) or Uninformative (overall)
Sensitivity ¦
Selective Reporting ¦
Overall confidence -
Figure 3-25. Study evaluation for human epidemiological studies reporting
findings from PFHxA exposures (HAWC: PFHxA - Human Toxicity Immune
Effects link).
The evaluation of Dong et al. (2013) encompasses all publications related to this study.
This document is a draft for review purposes only and does not constitute Agency policy.
3-100	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Toxicological Review ofPFHxA and Related Salts
Animal
Several short-term (28-day), subchronic, and chronic animal studies evaluated toxicological
findings of immune effects in rats receiving oral exposures ofPFHxA 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 3-34 and details are available by clicking the
HAWC link.
Table 3-34. Study design characteristics and individual outcome ratings for
immune endpoints
Study
Species, strain (sex)
Exposu re
design
Exposure route and dose
Organ weight
Histopathology
Immune cell
counts
NTP(2018)
Rat, Harlan
Sprague-Dawley
(male and female)
Short term
(28 d)
Gavage3
Male and female: 0, 62.5,125,
250, 500,1,000 mg/kg-d
+ +
+ +
+ +
Chengelis et
al. (2009b)
Rat, Crl:CD(SD)
Sprague-Dawley (male
and female)
Subchronic
(90 d)
Gavage3
Male and female: 0,10, 50,
200 mg/kg-d
+ +

+ +
Loveless et al.
(2009)
Rat, Crl:CD(SD)
Sprague-Dawley (male
and female)
Subchronic
(90 d)
Gavageb
Male and female: 0, 20,100,
500 mg/kg-d
+ +
+ +
+ +
Klaunig et al.
(2015)
Rat, Crl:CD(SD)
Sprague-Dawley (male
and female)
2-yr cancer
bioassay
Gavage3
Male: 0, 2.5,15,100 mg/kg-d
Female: 0, 5, 30, 200 mg/kg-d
NM
+ +
+ +
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 low confidence; NM, outcome not measured.
Organ Weights
Three studies evaluated effects on spleen and thymus weights in response to PFHxA (NTP.
2018: Chengelis et al.. 2009bl or PFHxA sodium salt fLoveless et al.. 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
relative and absolute organ weights were reported in males and females receiving PFHxA in the
short term (NTP. 2018).
This document is a draft for review purposes oniy and does not constitute Agency poiicy.
3-101	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Toxicological Review ofPFHxA and Related Salts
Spleen weights did not show a clear pattern of effect across studies. In the short term study,
a trend of increased weights in males and females receiving PFHxA fNTP. 2018] was observed,
whereas spleen weights were decreased in males receiving PFHxA sodium salt in the 90-day study
by Loveless etal. f20091. Chengelis etal. f2009bl 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
Spleen Weight, Absolute Loxreless, 2009,2850369 90-DayOral
NTP, 2018, 4309149 28-Day Ora
Spleen Weight, Relati\« Lo\rel ess, 2009,2850369 90-Day Ora
NTP, 2018, 4309149 28-Day Ora
Animal Description
Rat, Crl:CD(SD) (i!2)
Rat, Crl:CD(SD) (o)
Rat, Harlan Sprague-Dawley(iii)
Rat, Harlan Sprague-Dawley(c>)
Rat, Crl:CD(SD) (i!2)
Rat, Crl:CD(SD) (o)
Rat, Harlan Sprague-Dawley(iii)
Rat, Harlan Sprague-Dawley(c>)
Observation Time
Day 92
Day 92
Day 29
Day 29
Day 92
Day 92
Day 29
Day 29
Immune Effects: Organ Weights
Thymus Weight, Absolute Loxreless, 2009,2850369 90-DayOra
NTP, 2018, 4309149 28-Day Ora
Thynus Weight, Relati\« Loxreless,2009,2850369 90-DayOra
NTP, 2018, 4309149 28-Day Ora
Rat, Crl:CD(SD) (i2)	Day 92
Rat, Crl:CD(SD) 0)	Day 92
Rat, Harlan Sprague-Dawley(ii2)	Day29
Rat, Harlan Sprague-Dawley(c5)	Day29
Rat, Crl:CD(SD) (i2)	Day 92
Rat, Crl:CD(SD) 0)	Day 92
Rat, Harlan Sprague-Dawley(ii2)	Day29
Rat, Harlan Sprague-Dawley(c5)	Day29
No significant changg^, Significant increase^^ Significant decrease ^ SignificantTrendl
,
300 400 500 600 700 800 900 1,0001,100
Dose (mg/kg-bw/day)
Figure 3-26. 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 (NTP. 2018: Klaunigetal.. 2015: Chengelis etal.. 2009b: Loveless etal.. 2009). 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 f 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 fChengelis etal.. 2009bl All studies reported null results for histopathological
examinations of the thymus, lymph node, and bone marrow fNTP. 2018: Klaunig etal.. 2015:
Chengelis etal.. 2009b).
This document is a draft for review purposes only and does not constitute Agency policy.
3-102	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
Toxicological Review ofPFHxA and Related Salts
Immune Cell Counts
Four animal studies had evidence of hematological indicators of immunotoxicity fNTP.
2018: Klaunig etal.. 2015: Chengelis etal.. 2009b: Loveless etal.. 20091. Of these studies, NTP
f20181 and Loveless etal. f20091 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 (Klaunig etal..
2015: Chengelis etal.. 2009b). 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 fNTP. 2018: Klaunig etal.. 2015: Chengelis et al.. 2009b: Loveless et
al.. 2009). Results are summarized in Figure 3-27.
Endpoint
Study Experiment
Animal Description
Observation Time

immune Effects: Immune Cell Counts

Basophils
NTP, 2018,4309149 28-Day Oral
Rat Harlan Sprague-Dawley (r-')
Day 29
• •
V

V


Rat Harlan Sprague-Dawley
Day 29
• • •
•
•
•

Loveless, 2009, 2850369 90-DayOral
Rat Crl:CD(SD) (k)
Rat Crl:CD(SD) (^)
Day 92
Day 93
M •
— •

		^
•

Eosinophils
NTP, 2018,4309149 28-DayOral
Rat Harlan Sprague-Dawley (r')
Day 29

•
•
•


Rat Harlan Sprague-Dawley
Day 29
• • •
•
•
•

Loveless, 2009, 2850369 90-DayQral
Rat Crl:CD(SD) (/-;)
Rat Crl:CD(SD) (^)
Day 92
Day 93
M •
M •

V
	~

Lymphocyte
NTP, 2018,4309149 28-DayOral
Rat Harlan Sprague-Dawley(r')
Day 29
• • •
•
•
•


Rat Harlan Sprague-Dawley
Day 29
• • •
•
•
•

Loveless, 2009, 2850369 90-DayOral
Rat Crl:CD(SD) (r')
Rat Crl:CD(SD) (^)
Day 92
Day 93
M •
M •

			•
	•

Lymphocyte, Total
NTP, 2018,4309149 28-DayOral
Rat Harlan Sprague-Dawley (n)
Day 29
• •	•
•
•
•


Rat Harlan Sprague-Dawley
Day 29
• *	•
•
•
•
Monocytes
NTP, 2018,4309149 28-Day Oral
Rat Harlan Sprague-Dawley (r-')
Day 29
• • •
•
•
•


Rat Harlan Sprague-Dawley
Day 29
• • •
•
•
•

Loveless, 2009, 2850369 90-DayQral
Rat Crl:CD(SD)(rv)
Rat Crl:CD(SD) (^)
Day 92
Day 93
M •
M •

		•
—	¦¦¦'•

Neutrophils
NTP, 2018,4309149 28-DayOral
Rat Harlan Sprague-Dawley (rv) Day 29
• •
•
•
•


Rat Harlan Sprague-Dawley
Day 29
• •
•
•
	A

Loveless, 2009,2850369 90-DayQral
Rat CrkCD(SD) (r')
Rat Crl:CD(SD) (^)
Day 92
Day 93
A A
M •

A
	•

White Blood Cell (WBC)
Klaunig, 2015, 2850075 2-Year Cancer Bioassay Rat Crl:CD(SD) (<¦')
Week 25
m •






Week 51
m •






Wfeek104
• •





Rat Crl:CD(SD) (^)
Week 25
Week 51
Week 104
••
•«
•«
•
•
•



NTP, 2018,4309149 28-DayOral
Rat Harlan Sprague-Dawley (rv)
Day 29
• • »
•
•
•


Rat Harlan Sprague-Dawley (i)
Day 29
• • •
•
•
•

Chengelis, 2009,2850404 90-Day Oral
Rat Crl:CD(SD) (n)
Rat Crl:CD(SD) ( i)
Day 90
Day 90
»• - -
m •
•



Loveless, 2009, 2850369 90-DayOral
Rat Crl:CD(SD) {<¦:)
Rat Crl:CD(SD) ( i)
Day 92
Day 93
99 •
M •
~
	¦¦¦•
	•

White Blood Cell (WBC), Recovery
Chengelis, 2009, 2850404 90-Day Oral
Rat Crl:CD(SD) {<¦:)
Rat Crl:CD(SD) ( i)
Day 118
Day 118
•
•
•
•






200 300
400 500 600 700 800
900 1,000

| • No significant changed Significant
ncrease yf Significant decrease @ SignificantTrendl
Dose (mg/kg-bw/day)
Figure 3-27. Immune cell counts in rats exposed by gavage to PFHxA or PFHxA
sodium salt (HAWC: PFHxA - Animal Toxicity Immune Effects link).
This document is a draft for review purposes only and does not constitute Agency policy.
3-103	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Toxicological Review ofPFHxA and Related Salts
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 (IPCS. 2012). Notably,
there is evidence indicating that other PFAS, including PFOS and PFOA, may affect immune system
function through suppression of antibody response and induction of hypersensitivity (Pewit! et al..
20191. Additional studies, particularly those that evaluate changes in immune function would be
beneficial for understanding the potential for adverse effects ofPFHxA 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 (see
Table 3-35).
This document is a draft for review purposes only and does not constitute Agency policy.
3-104	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 3-35. Evidence profile table for immune effects
Evidence stream summary and interpretation
Evidence integration
summary judgment
Evidence from studies of exposed humans
OOO
Inadequate
Primary basis:
Evidence is low
Studies and
confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key findings
Evidence stream
judgment
Asthma
1 medium
confidence study
• No factors noted
•	Imprecision
•	Lack of coherence - no
associations with other
measures of pulmonary
function
• Nonsignificant association
with asthma diagnosis, but
other asthma-related
outcomes were not
affected.
ooo
Indeterminate
confidence or limited
Human relevance:
Without evidence to
the contrary, effects in
rats are considered
Evidence from animal studies
Studies and
confidence
Factors that increase
certainty
Factors that decrease
certainty
Summary and key findings
Evidence stream
judgment
relevant to humans
Cross-stream
coherence:
N/A (human evidence
indeterminate)
Susceptible populations
and life stages:
• No evidence to
inform
Histopathology
3 hiqh confidence
studies in adult rats
•	28-d
•	90-d
•	2-yr
1 low confidence
study in rats;
•	90-d
•	High confidence studies
•	Consistency across studies
for extra medullary
hematopoiesis
• No factors noted
• Increased splenic
extramedullar
hematopoiesis was
observed male and female
rats at 500 mg/kg-d;
coincident with minimal
erythroid hyperplasia of the
bone marrow
OOO
Indeterminate
Some evidence of
immune system but
limited by unexplained
inconsistency, lack of
coherence, and
potential for non-
immune related causes
[see Section 3.2.4 for
additional discussion].
Available evidence was
consisted of
observational
outcomes that are less
Immune Cell Counts
4 high confidence
studies in rats:
•	28-d
•	90-d (2 studies)
•	2-yr
•	High confidence studies
•	Consistency-studies for
neutrophils and basophils
• Lack of coherence with
other immune markers
• Decreased basophil counts
and increased neutrophil
cell counts at >20 mg/kg-d

This document is a draft for review purposes only and does not constitute Agency policy.
3-105	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Evidence stream summary and interpretation
Evidence integration
summary judgment
Organ Weight
3 hiqh confidence
studies in rats:
•	28-d
•	90-d (2 studies)
• High confidence studies
• Unexplained
inconsistency across
studies for spleen
weights
•	Thymus weights decreased
at 500 mg/kg-d in
short-term and subchronic
studies
•	Changes in spleen weight
were inconsistent in the
direction of effect across
studies
predictive of immune
system toxicity.

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.
3-106	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
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 3-36, and details
11	are available by clicking the HAWC link.
Table 3-36. Nervous system endpoints for PFHxA and associated confidence
scores from repeated-dose animal toxicity studies
Author (year)
Species, strain
(sex)
Exposu re
design
Exposure route and dose
range
Brain weight
Histopathology
Behavior
NTP(2018)
Rat, Harlan
Sprague-Dawley
(male and female)
Short term
(28 d)
Gavage3
Male and female: 0, 62.5,125,
250, 500,1,000 mg/kg-d
++
+ +
NM
Chengelis et al.
(2009b)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
Subchronic
(90 d)
Gavage3
Male and female: 0,10, 50,
200 mg/kg-d
++

+
Loveless et al. (2009)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
Subchronic
(90 d)
Gavageb
Male and female: 0, 20,100,
500 mg/kg-d
++
+ +
++
Klaunig et al. (2015)
Rat, Crl:CD(SD)
Sprague-Dawley
(male and
female)
2-yr cancer
bioassay
Gavage3
Male: 0, 2.5,15,100 mg/kg-d
Female: 0, 5, 30, 200 mg/kg-d
NM
+ +
++
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; NM, outcome not measured.
This document is a draft for review purposes oniy and does not constitute Agency poiicy.
3-107	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Toxicological Review ofPFHxA and Related Salts
Brain Weight
Three studies evaluated effects ofPFHxA or PFHxA sodium salt on the nervous system in
animals fNTP. 2018: Chengelis etal.. 2009b: Loveless et al.. 20091. Two studies reported increases
in relative but not absolute brain weights after exposure to PFHxA or PFHxA sodium salt for 28 or
90 days, respectively (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.. 20091 and only females in the other (Chengelis etal.. 2009b). Notably, relative weights are not
considered appropriate for brain weight measurements because this measure is not typically
affected by fluctuations in body weight fU.S. EPA. 19981: therefore, absolute brain weights are
preferred.
Other Nervous System Effects
No treatment-related effects were observed on other nervous system outcomes, including
behavior (i.e., open field locomotor activity, functional observational battery) and histopathology
fNTP. 2018: Klaunig etal.. 2015: Chengelis etal.. 2009b: Loveless etal.. 20091.
Evidence Integration
No human studies were identified to inform the potential nervous system effects ofPFHxA
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.
3-108	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 3-37. 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
lifestages:
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-d
•	90-d (2 studies)
• High confidence
studies
• No factors noted
• Increased relative
brain weights in
animals at >200
mg/kg-d; absolute
brain weight
unaffected
OOO
Indeterminate
Evidence is largely null.
The only evidence of
nervous system effects
was relative brain weight
increases, which is not
considered to be
appropriate for
evaluating nervous
system toxicity.
Histopathology
3 hiqh confidence studies
in adult rats:
•	28-d
•	90-d
•	2-yr
1 low confidence study in
adult rats:
•	90-d
• High confidence
studies
• No factors noted
• No treatment-related
effects reported
Behavior
2 hiqh confidence studies
in adult rats:
•	90-d
•	2-yr
• High and medium
confidence
studies
• No factors noted
• No treatment-related
effects reported
This document is a draft for review purposes only and does not constitute Agency policy.
3-109	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Evidence stream summary and interpretation
Evidence integration summary
judgment
1 medium confidence
study in adult rats:
• 90-d





Mechanistic evidence and supplemental information
Biological events of
pathways
Biological events of
pathways
Biological events of pathways
Biological events of
pathways
• No studies Identified
This document is a draft for review purposes only and does not constitute Agency policy.
3-110	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Toxicological Review ofPFHxA and Related Salts
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 nonneoplastic and neoplastic lesions were reported as null and are summarized in HAWC and in
PFHxA Tableau.
Genotoxicity
Genotoxic, mutagenic, and clastogenic effects ofPFHxA have been tested in several
mammalian and prokaryotic cell systems in vitro (see Table 3-38) fLau. 2015: Eriksen etal.. 2010:
Nobels etal.. 2010: Loveless etal.. 20091. 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 fEriksenetal.. 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. 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. 2005) EPA concluded
This document is a draft for review purposes only and does not constitute Agency policy.
3-111	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
there is inadequate information to assess carcinogenic potential for PFHxA for any route of
exposure.
This document is a draft for review purposes only and does not constitute Agency policy.
3-112	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 3-38. 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 hr. 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-hr exposure. Cytotoxicity
was monitored by measuring lactate dehydrogenase (LDH)
activity to ensure observed DNA damage was not secondary
to cytotoxicity.
Eriksen et al.
(2010)
Cell stress-
dependent
gene
expression
Escherichia
coli
K-12
derivative
SFi
0.0156, 0.03125,
0.0625, 0.125, 0.25,
0.5, 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)
This document is a draft for review purposes only and does not constitute Agency policy.
3-113	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
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 hr (activated) and
22 hr
(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- = negative; NA = not applicable.
This document is a draft for review purposes only and does not constitute Agency policy.
3-114	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Toxicological Review ofPFHxA and Related Salts
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, gestational exposure,
studies flwai and Hoberman. 2014: Loveless etal.. 20091 and a one-generation reproductive study
fLoveless etal.. 20091 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 three studies that reported consistent,
dose-responsive, and substantial effects ofPFHxA 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 fNTP. 2018: Chengelis etal.. 2009b: Loveless etal.. 20091. These hematological findings
correlate with increases in reticulocytes, an indicator of erythroid cell regeneration supported by
This document is a draft for review purposes only and does not constitute Agency policy.
4-1	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Toxicological Review ofPFHxA and Related Salts
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 fNTP. 2018: Loveless etal.. 20091. 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 (see 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. 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
caused by sex-specific differences in the expression (mRNA and protein) of the renal organic anion
transporting polypeptide (Oatp) lal (Kudo etal.. 20011 as discussed in Section 3.1.4. Currently,
whether this sex-specific difference might also exist in humans is unclear.
Additionally, given the effects seen in the developing organism (i.e., perinatal mortality,
reduced body weights, and delays in time to eye opening), the prenatal and early postnatal window
represents a potentially sensitive lifestage for PFHxA exposure.
This document is a draft for review purposes only and does not constitute Agency policy.
4-2	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
4.3. SUMMARY OF CONCLUSIONS FOR CARCINOGENICITY
1	The evidence is insufficient to make a judgment on whether PFHxA exposure might affect
2	the development of any specific cancers. Consistent with EPA guidance fU.S. EPA. 20051 to apply a
3	standard descriptor as part of the hazard narrative and to express a conclusion regarding the
4	weight of evidence for the carcinogenic hazard potential, a descriptor of inadequate information
5	to assess carcinogenic potential is applied for PFHxA.
This document is a draft for review purposes only and does not constitute Agency policy.
4-1	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Toxicological Review ofPFHxA and Related Salts
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 (NTP. 2018: Klaunig etal.. 2015: Iwai and Hoberman. 2014: Chengelis
etal.. 2009b: Loveless etal.. 20091. These studies were generally rated high confidence in outcome-
specific study evaluations. Based on these studies, it was determined that the evidence indicates
PFHxA likely causes hepatic, developmental, 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 in 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 (see
Section 5.2.2). Similarly, the evidence base related to potential carcinogenicity was determined to
contain "inadequate information to assess carcinogenic potential"; therefore, no cancer toxicity
values were estimated for any exposure route (see Section 5.3).
5.2.	NONCANCER TOXICITY VALUES
A reference dose (RfD) is the daily oral exposure to the human population (including
sensitive subpopulations) that is likely without appreciable risk of deleterious effects during a
lifetime. In addition to developing an RfD designed to protect against lifetime exposure, a less-than-
lifetime toxicity value (referred to as a "subchronic RfD") is estimated. These subchronic toxicity
values are presented as they might be useful for certain decision purposes (e.g., site-specific risk
assessments with less-than-lifetime exposures). Both RfD and subchronic RfD derivations include
organ/system-specific RfDs (osRfDs) associated with each health effect considered for point of
departure (POD) derivation. Subsequent decisions related to dosimetric extrapolation, application
of uncertainty factors, and confidence in toxicity values are discussed below.
As noted above, reference concentration (RfC) or subchronic RfC could not be developed.
This document is a draft for review purposes only and does not constitute Agency policy.
5-2	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
Toxicological Review ofPFHxA and Related Salts
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 ofPFHxA exposure are primarily low confidence and
therefore were not further considered for dose-response analyses ofPFHxA 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 (see 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
Exposu re
duration
Strain/
Species
Sexes
studied
POD derivation
Rationale
Hepatic Effects
Relative liver
weight
Chengelis et al.
(2009b)
High confidence
Subchronic
Crl:CD(S
D) rat
Both
No
Hepatic hypertrophy
was considered a
more specific and
reliable measure than
increases in relative
liver weight
Loveless et al.
(2009)
High confidence
Subchronic
Crl:CD(S
D) rat
Both
No
Hepatocellula
r hypertrophy
Chengelis et al.
(2009b)
Low confidence
Subchronic
Crl:CD(S
D) rat
Female
No
Only male-specific
effects were
observed in Chengelis
et al. (2009b),
whereas both sexes
were affected in
Loveless et al. (2009).
Chengelis et al.
(2009b)
Low confidence
Subchronic
Crl:CD(S
D) rat
Male
Yes
Loveless et al.
(2009)
High confidence
Subchronic
Crl:CD(S
D) rat
Both
Yes
Hepatocellula
r necrosis
Klaunig et al.
(2015)
High confidence
Chronic
Crl:CD(S
D) rat
Female
No
Significant effect only
at highest dose in
females, and largely
in animals that died
an unscheduled
death.
This document is a draft for review purposes only and does not constitute Agency policy.
5-3	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Endpoint
Reference/
Confidence
Exposu re
duration
Strain/
Species
Sexes
studied
POD derivation
Rationale
Blood
proteins (total
protein and
globulin)
Chengelis et al.
(2009b)
High confidence
Subchronic
Crl:CD(S
D) rat
Both
No
Increases in blood
proteins are
considered a non-
specific indicator of
hepatic toxicity and
more specific
measures are
available.
Loveless et al.
(2009)
High confidence
Subchronic
Crl:CD(S
D) rat
Both
No
Klaunig et al.
(2015)
High confidence
Chronic
Crl:CD(S
D) rat
Both
No
Hematopoietic Effects
Hematocrit
Chengelis et al.
(2009b)
High confidence
Subchronic
Crl:CD(S
D) rat
Both
No
More direct
measurements of red
blood cells and
hemoglobin are
available.
Loveless et al.
(2009)
High confidence
Subchronic
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
Subchronic
Crl:CD(S
D) rat
Both
Yes
Decreases were
considered similar in
sensitivity to
decreases in red
blood cell counts and
there was no reason
to advance one
endpoint over the
other. Hemoglobin
reflects the oxygen
carrying capacity of
red blood cells. In
Klaunig et al. (2015),
the effects were
specific to females.
Loveless et al.
(2009)
High confidence
Subchronic
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
Red blood
cells
Chengelis et al.
(2009b)
High confidence
Subchronic
Crl:CD(S
D) rat
Both
Yes
Finding was more
sensitive and specific
than other red blood
cell parameters and
there was no reason
to advance one
endpoint over the
other.
Loveless et al.
(2009)
High confidence
Subchronic
Crl:CD(S
D) rat
Both
Yes
Klaunig et al.
(2015)
High confidence
Chronic
Crl:CD(S
D) rat
Both
Yes
This document is a draft for review purposes only and does not constitute Agency policy.
5-4	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Endpoint
Reference/
Confidence
Exposu re
duration
Strain/
Species
Sexes
studied
POD derivation
Rationale
Reticulocytes
Chengelis et al.
(2009b)
High confidence
Subchronic
Crl:CD(S
D) rat
Both
No
Increases were
considered to reflect
a compensatory
(secondary) response
to decreased red
blood cell
parameters.
Loveless et al.
(2009)
High confidence
Subchronic
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-
generation
repro-
ductive;
measured
on PND 0,
4,7,14, 21
Crl:CD(S
D) rat
Combine
d
Yes, PND 0
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 only at the
highest dose.
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 (combined data
across two cohorts)
Perinatal mortality
(still birth and
postnatal deaths from
PND 0-21) showed a
clear dose-response
across two
experimental cohorts
with overlapping dose
ranges. Data were
pooled for dose-
response analysis.
Eye opening
Iwai and
Hoberman
(2014)
High confidence
Develop-
mental
(GD
6-18);
measured
CD-I
mouse,
Fi
Combine
d
No
Delays were observed
at a dose that elicited
body weight deficits
and perinatal
mortality.
This document is a draft for review purposes only and does not constitute Agency policy.
5-5	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Toxicological Review ofPFHxA and Related Salts
Endpoint
Reference/
Confidence
Exposu re
duration
Strain/
Species
Sexes
studied
POD derivation
Rationale


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 (see Table 5-1) were modeled using approaches
consistent with EPA's Benchmark Dose (BMD) Technical Guidance document fU.S. EPA. 2012a!
Specifically, the BMD and 95% lower confidence limit on the BMD (BMDL) were estimated using a
benchmark response (BMR) to represent a minimal, biologically significant level of change. BMD
modeling of continuous data was conducted using EPA's Benchmark Dose Software (BMDS, Version
3.2).
Ideally, the selected BMR is based on data that support the biological relevance of the
outcome being evaluated; however, in some cases there is no clear scientific understanding to
support a biologically based BMR. In these instances, the BMD guidance provides some BMRs that
can be applied to the data. For data drawn from toxicological studies, a suggested BMR of 1
standard deviation (SD) from the control mean for continuous data or a BMR of 10% extra risk (ER)
for dichotomous data can be used to estimate the BMD and BMDL. The selection of these BMRs, as
indicated in Table 5-2, is based on BMD guidance stating that in the absence of information
regarding the level of change considered biologically significant, these BMRs can be used fU.S. EPA.
2012a). For effects on offspring body weights, a BMR of 5% relative deviation (RD) from the
control mean is used for continuous data to account for effects occurring in a sensitive lifestage
fU.S. EPA. 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
for benchmark dose modeling in prior IRIS assessments (U.S. EPA,
2012b, 2004, 2003).
Offspring
mortality
1% ER
Although 5% ER is generally supported for developmental and
reproductive outcomes (U.S. EPA, 2012a), a lower BMR of 1% ER was
This document is a draft for review purposes only and does not constitute Agency policy.
5-6	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Toxicological Review ofPFHxA and Related Salts
Endpoint
BMR
Rationale


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 1SD 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 3-fold). Otherwise, the lowest BMDL was selected as a potential POD for each
endpoint.
Where modeling was feasible, the estimated BMDLs were used as PODs. Further details,
including the modeling output and graphical results for the model selected for each endpoint, can
be found in Supplemental Information, Appendix B. The benchmark dose approach involving
modeling to obtain the BMDL is preferred, but it involves modeling dose levels corresponding to
BMR levels near the low end of the observable range of the data and is not always feasible. When
data sets were not amenable to BMD modeling, no-observed-adverse-effect level (NOAEL) or
lowest-observed-adverse-effect level (LOAEL) values were selected and used as the POD 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).
For the study by Iwai and Hoberman f20141. 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
This document is a draft for review purposes only and does not constitute Agency policy.
5-7	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Toxicological Review ofPFHxA and Related Salts
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 ofPFHxA 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 describes that doses be
scaled allometrically using body weight BW3/4 methods. This hierarchy of approaches for cross-
species dosimetry extrapolation is consistent with EPA's guidance on using allometric scaling for
the derivation of oral reference doses fU.S. EPA. 20111. 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. f20151. 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
fChangetal.. 20081. PFBS fOlsenetal.. 20091. PFHxA fDzierlenga etal.. 20191. PFHxS fSundstrom et
al.. 20121. PFNA fTatum-Gibbs etal.. 20111. and PFOA and PFOS fKim etal.. 2016bl. that show a
clear biphasic elimination pattern indicative of distinct distribution and elimination phases, EPA
chose to use a two-compartment PK model, similar to the analysis of (Fujii etal.. 2015). 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
This document is a draft for review purposes only and does not constitute Agency policy.
5-8	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Toxicological Review ofPFHxA and Related Salts
where a and (3 are first-order rate constants (units of time-1) representing the rate of distribution
and elimination, respectively, ka is a rate constant (units of time-1) for oral absorption, and flagorai is
set to zero when analyzing intravenous dose data or one for oral data. Details of the model fitting
are provided in Appendix B. The model assumes that oral bioavailability is 100%, consistent with
PK data from Dzierlenga et al. 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. (20191: 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 flwabuchi et al.. 2017: Gannon etal.. 2011: Chengelis et al..
2009a). 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-hr/L), and is considered an appropriate measure of internal dose
when the chemical has an accumulative effect over time.
By definition, the clearance (CL) of a compound is the effective volume of blood cleared of
the compound per unit time (units of volume/time). Mathematically, given the PK model described
above, CL = dose/AUCmf. If one assumes that risk increases in proportion to AUC, the ratio of
clearance in animal to that in the human, CLA:CLH, can then be used to convert an oral dose-rate in
animals (mg/kg-day) to a human equivalent dose (HED) rate. A similar approach using the ratio of
the beta-phase half-lives can be used and is outlined in Appendix C, but that approach ignores
differences in the absorption rate and alpha-phase distribution rate that impact AUC and is,
This document is a draft for review purposes only and does not constitute Agency policy.
5-9	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Toxicological Review ofPFHxA and Related Salts
therefore, considered to produce a more uncertain outcome. Effectively, using the half-life ratio
assumes that another PK parameter, the volume of distribution, is the same between species (this is
contrary to available data).
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.
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 * (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. (2019). 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.
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.
This document is a draft for review purposes only and does not constitute Agency policy.
5-10	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
Toxicological Review ofPFHxA and Related Salts
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 (Nilsson etal.. 2010). Hence it is presumed that ti/2 = ln(2)/(3. Rearranging the
two equations, CL = Vd,p x P = Vd,p x ln(2)/ ti/2- Three options were considered, as follows:
1)	The Vd for humans is equal to that determined in the next closest species biologically,
monkeys. This assumes the biological and biochemical factors that determine the
tissue:serum concentration ratio and the relative proportion (fraction of BW) for various
tissues is similar in humans and monkeys. This assumption presumes the relative binding
of PFHxA in human serum relative to various other tissues in the body is like that in
monkeys but leads to a conclusion that renal clearance in humans is significantly slower
than in other species.
2)	Use the clearance values estimated for mice, rats, and monkeys to estimate the clearance
in humans via allometric scaling. The results for mice, rats, and monkeys in Table 5-3 show
almost no trend with increasing species BW, but can be fitted with a power function to
obtain CL = 0.152-BW"0 023 (L/kg), assuming standard BW values of 0.03 and 0.25 kg for
mice and rats, respectively, and the reported BW of monkeys used by Chengelis et al.
f2009al. For a standard human BW of 80 kg, the resulting predicted clearance in humans is
0.137 L/hr-kg. If this is the actual clearance in humans, but ti/2 = 275 hr, human
Vd,p = CL x ti/2/ln(2) = 54 L/kg. Note that human participants were exposed to PFHxA for
months, which could have allowed them to accumulate a deep tissue dose, while the
monkey PK study involved only a single i.v. administration. Thus, a much higher Vd might
have been estimated in monkeys had they been subject to repeated doses.
3)	The apparent human half-life estimated by EPA from the data of Nilsson etal. (2013)
might be an artifact of significant ongoing exposure to PFHxA during the period of
observation. Perez etal. f20131 detected PFHxA levels in human tissues higher than other
PFAS and other observational studies regularly detect PFHxA in human serum
demonstrating widespread human exposure to the general population. Thus, there is no
reason to believe the subjects of Nilsson etal. (2013) 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) (0.137 L/hr-kg) is an accurate prediction for humans and the Vd is equal to
the average estimated for monkeys (0.73 L/kg), the half-life in humans should be
ti/2 = ln(2) x Vd /CL = ln(2) x 0.73 (L/kg)/(0.137 L/hr-kg) = 3.7 hr. If this were the case,
human serum levels would fall 99% in a single day, while the data of Nilsson etal. (2013)
show that such a decline takes at least 2 months and, even after a day or two off work, a
technician's serum concentration would be near zero. Further, the serum concentrations
reported Nilsson etal. f20131 do decline to near or below the limit of detection by late
spring or early summer, indicating that other ongoing sources of exposure were not
significant for that population. Thus, this third option seems extremely unlikely and was
not be evaluated further.
This document is a draft for review purposes only and does not constitute Agency policy.
5-11	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
1	The two options for human CL estimated in points (1) and (2) above are provided in
2	Table 5-3.
This document is a draft for review purposes only and does not constitute Agency policy.
5-12	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 5-3. Summary of serum half-lives and estimated clearance for PFHxA
Species/Sex
Study
design
Elimination
half-life (ti/2) (hr)
Clearance (CL)
(L/hr-kg)
Volume of
distribution
(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
1.84 x 10"3 (c)
0.137d
0.73°
54d
Nilsson 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 and ti/2
estimated as described in Appendix C.2.
dHuman CL estimated by allometric scaling from values estimated for mice, rats, and monkeys; human
Vd = CLx 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 (see Table 5-4).
This document is a draft for review purposes only and does not constitute Agency policy.
5-13	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Toxicological Review ofPFHxA and Related Salts
Table 5-4. Two options for rat, mouse, and human clearance values and data-
informed dosimetric adjustment factor (DAF)
Sex
Species
Animal clearance
(L/hr- kg)a
Human clearance (L/hr-kg)
DAF (CLH:CU[si)
Male
Rat
0.163
1.84(1.00-3.49) x 10"3(b)
(mean, 90% CI, using
preferred [data-driven]
approach)
1.1 x 10"2
Mouse
0.0894
2.1 x 10"2
Female
Rat
0.383
4.8 x 10"3
Mouse
0.206
8.9 x 10"3
Male
Rat
0.163
0.137°
(alternative approach)
0.84
Mouse
0.0894
1.5
Female
Rat
0.383
0.36
Mouse
0.206
0.67
Shaded values were applied to derive the PODhed-
aSpecies/sex-specific CL values (Appendix C).
Calculated from human ti/2 value, obtained by Bayesian PK analysis and average volume of distribution for male
and female monkeys (see 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 PFOA
by Zhang etal. f2013bl and can be compared to those for experimental animals. By comparing
human and rat clearance for a set of compounds from the same chemical family, for which data are
available in both species, a "read across" can be done to evaluate the most likely case for PFHxA.
Note that PFHxS has the same carbon chain length as PFHxA (C6) and while PFOA and PFNA have
longer chains (C8 and C9 respectively) they are still much more chemically similar to PFHxA than
any other compounds for which corresponding human data are available. Briefly, Zhang et al.
f2013bl measured PFAS concentrations in serum and matched 24-hour urine samples to directly
measure urinary clearance. To avoid the complicating issue of losses from menstrual blood, results
for men and women over the age of 50 years are evaluated. Median urinary CL values reported by
Zhang etal. (2013b) were 0.015, 0.094, and 0.19 mL/kg-day for PFHxS, PFNA, and total PFOA (all
isomers), respectively.
Kim etal. f2016bl reported renal PFHxS clearance of 0.76 mL/kg-day while Kim et al.
f2016bl and Sundstrom et al. 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.
Dzierlenga et al. (2019) evaluated the PK of PFOA (as well as PFHxA) in male rats and
obtained clearance values of 9-16 mL/kg/d, depending on the dose and route. Thus, PFOA is also
cleared much more rapidly in rats than humans.
This document is a draft for review purposes only and does not constitute Agency policy.
5-14	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Toxicological Review ofPFHxA and Related Salts
The reported dose/AUC can be used to derive clearance values for PFNA from the results of
Tatum-Gibbs etal. f20111. The estimated CL in rats is highly variable across the studies evaluated
but ranged from 2 to 66 mL/kg-day in males and from 4 to 106 mL/kg-day in females fTatum-Gibbs
etal.. 2011: Benskin et al.. 2009: De Silva etal.. 2009: Ohmori et al.. 20031. CL in male and female
mice reported by Tatum-Gibbs etal. f20111 ranged from 3 to 10 mL/kg-day. Although the wide
range for rats indicates a degree of uncertainty, these results indicate that clearance in mice and
rats is similar and much larger than the corresponding human value (0.094 mL/kg-day) (Zhang et
al.. 2013bl.
Thus, three other PFAS, including one with the same carbon-chain length as PFHxS, have
been shown to have much lower clearance in humans than rats. Data for PFDA were not discussed
here since it is a Cio compound, but it also shows a similar rat-human difference in clearance.
Hence, a read-across analysis suggests that option (1) above is more likely to be true.
The alternative, option (2) above, requires one to accept that the Vd in humans is roughly
two orders of magnitude higher than in rats and monkeys, although the biochemical factors that
determine serum-tissue partitioning are expected to be conserved across mammalian species, as
described in the section above on distribution. Hence, option (2) seems highly unlikely.
Therefore, the top set ofDAFs in Table 5-4—based on CLhuman = 1.84 x 10~3 L/kg-hr—are
the preferred set because they are consistent with data for other PFAS, and the reasonable
expectation, based on data from multiple chemicals, is the volume of distribution in humans
does not substantially differ from that in experimental animals.
Representative calculations of the HED for considered health effects follow, using the POD
of 20 mg/kg-day for postnatal (Fi) body weight at PND 0 (Loveless et al.. 20091 as an example and
the female rat DAF of 4.8 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. There is a significant difference between rats and monkeys, which leads to the expectation
of a difference between rats and humans, (see Table 5-3)
HED based on clearance incorporates the observed differences in Vd among mice, rats, and
primates, and is therefore, the preferred approach for dosimetry extrapolation from animals to humans.
Uncertainty of animal-human extrapolation ofPFHxA dosimetry
Although the variability between, and even within, some data sets for rats (~4-fold for males
and ~6-fold for females between the lowest and highest mean clearance values) is large, the number
HED = 20 (mll/kg_iay) X 4.8 X 10"' = 0.096 (m»/kg_iay)
5-5
This document is a draft for review purposes only and does not constitute Agency policy.
5-15	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Toxicological Review ofPFHxA and Related Salts
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 fGannonetal.. 20111.
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. f20111 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
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 PFHxAserum concentrations (Klaunig etal.. 2015: Iwai and Hoberman. 2014: Chengelis et
al.. 2009b: Loveless etal.. 20091. Although Iwai and Hoberman (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.
There is uncertainty in the estimated human clearance because the Vd had to be
extrapolated from animals (nonhuman primates) and the limited human PK data from only eight
individuals with noncontrolled exposures. As discussed in Section 3.1.2, the distribution ofPFHxA
between serum and various tissues is determined by biochemical parameters such as the
concentrations of various binding proteins and the affinity of PFHxA for those proteins, that are
largely conserved across mammalian species. However, Vd values estimated for animals range
between 0.33 L/kg in rats to 1.54 L/kg in one of six monkeys studied. Together with the estimated
uncertainty in the human half-life for which the 90% confidence interval ranges 3.5-fold, an overall
This document is a draft for review purposes only and does not constitute Agency policy.
5-16	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Toxicological Review ofPFHxA and Related Salts
range of uncertainty in the human clearance of 16-fold (± 4-fold) was estimated (see Section 3.1.4
Pharmacokinetics-Elimination-Human Studies).
PODhfti for RfD derivation
Table 5-5 presents the estimated PODhed (mg/kg-day) values for the hepatic,
developmental, and hematopoietic toxicity endpoints considered for RfD derivation based on the
endpoint selection justification in Table 5-1 and preferred DAF values presented in Table 5-4.
The last column in Table 5-5 includes normalization from the ammonium salt to the free
acid using a molecular weight conversion [MW free acid/MW ammonium salt = 314/331 = 0.949
flwai and Hoberman. 2014)] and sodium salt to free acid [MW free acid/MW sodium
salt = 314/336 = 0.935 fLoveless etal.. 20091], The PODhed for postnatal (Fi) body weights used
the female HED, as exposures were to the dams and assumed equal clearance in a developing
offspring as an adult
The free acid of PFHxA is calculated using the ratio of molecular weights, as follows:
/ MW free acid \ /314\
PFHxA (free acid) = (—-r	:	—) = (rrr) = 0.949
\MW ammonium saltJ \331/
PFHxA (free acid) = ( MWfreeacid ) = (HI) = 0.935	5-6
\MW sodium salt J V336/
Table 5-5. PODs considered for the derivation of the RfD
Endpoint
Study/confidence
Species, strain
(sex)
PODtype/model
POD
(mg/kg-d)
PODhed PFHxA3
(mg/kg-d)
Hepatic effects
^Hepatocellular
hypertrophy
Chengelis et al.
(2009b)
Low confidence
Rat, Crl:CD(SD)
(male)
NOAELb
(0% response)
50
0.55
Loveless et al. (2009)
High confidence
Rat, Crl:CD(SD)
(male)
BMDLioer
Multistage 1 NCV
10.66
0.11°
Rat, Crl:CD(SD)
(female)
BMDLioer
Multistage 3 NCV
96.32
0.43°
Hematopoietic effects
4/Hemoglobin
Klaunig et al. (2015)
High confidence
Rat, Crl:CD(SD)
(female)
BMDLisd
Linear CV
122.77
0.59
Chengelis et al.
(2009b)
High confidence
Rat, Crl:CD(SD)
(male)
BMDLisd
Polynomial 3 CV
81.35
0.89
Rat, Crl:CD(SD)
(female)
NOAELd
(3% decrease)
50
0.24
Loveless et al. (2009)
High confidence
Rat, Crl:CD(SD)
(male)
NOAELd
(6% decrease)
50
0.51°
This document is a draft for review purposes only and does not constitute Agency policy.
5-17	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Endpoint
Study/confidence
Species, strain
(sex)
PODtype/model
POD
(mg/kg-d)
PODhed PFHxA3
(mg/kg-d)


Rat, Crl:CD(SD)
(female)
BMDLisd
Polynomial 3 CV
127.61
0.57°
4/Red blood cell
Klaunig et al. (2015)
High confidence
Rat, Crl:CD(SD)
(male)
NOAELb
(4% decrease)
100
1.21


Rat, Crl:CD(SD)
(female)
BMDLisd
Linear CV
109.15
0.52

Chengelis et al.
(2009b)
Rat, Crl:CD(SD)
(male)
NOAELd
(no change)
50
0.55

High confidence
Rat, Crl:CD(SD)
(female)
BMDLisd
Exponential 5
CV
16.32
0.078

Loveless et al. (2009)
High confidence
Rat, Crl:CD(SD)
(male)
BMDLisd
Linear NCV
44.57
0.46°


Rat, Crl:CD(SD)
(female)
BMDLisd
Linear CV
112.36
0.50°
Developmental effects
4/Postnatal (Fi)
body weight, PND 0
Loveless et al. (2009)
High confidence
Rat, Crl:CD(SD),
Fi (combined)
BMDLsrd
Hill
10.62
0.048°
4/Postnatal (Fi)
body weight, PND 0
Iwai and Hoberman
(2014)
High confidence
Mouse, CD-I, Fi
(combined)
BMDLsrd
Polynomial 3 CV
Phase 2
80.06
0.68e
4/Postnatal (Fi)
body weight, PND 4


BMDLsrd
Exponential-M5
Phase 1 and 2
Polynomial 3 CV
Phase 2
103.12
89.79
0.87e
0.76e
^Perinatal (Fi)
mortality (PND 0-
21, including
stillbirths)
Iwai and Hoberman
(2014)
High confidence
Mouse, CD-I, Fi
(combined)
BMDLieiro
N Logistic
Phase 2
24.77
0.21e
CV = constant variance; NCV = nonconstant variance; SD = standard deviation.
aHED calculations based on the DAF, the ratio of human and animal clearance values (see Table 5-4). DAF values
for female rats and female mice were used for the respective developmental effects on combined male and
female pups of each species. PODhed based on PFHxAfree acid.
bResponse only at high dose with responses far above BMR level, data not modeled.
cPODHed multiplied by normalization factor to convert from sodium salt to free acid (MW free acid/MW sodium
salt = 314/336 = 0.935).
dNo models provided adequate fit; therefore a NOAEL approach was selected.
6P0DHed multiplied by normalization factor to convert from ammonium salt to the free acid (MW free acid/MW
ammonium salt = 314/331 = 0.949).
This document is a draft for review purposes only and does not constitute Agency policy.
5-18	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Toxicological Review ofPFHxA and Related Salts
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 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 fLoveless etal.. 20091. for
hematopoietic endpoints to RBCs and HGB from the chronic study fKlaunig etal.. 20151. and for
developmental endpoints to offspring body weight from fLoveless etal.. 20091.
For the hepatic endpoint, hepatocellular hypertrophy was moved forward for POD
determination. This decision 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
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 endpoint than 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 fKlaunig et al.. 20151 was two times higher than the 100 mg/kg-day PFHxA dose causing
hypertrophy in the subchronic study (Loveless et al.. 2009). hypertrophy from male rats in the
subchronic study (Loveless et al.. 2009) was selected as the appropriate endpoint and advanced for
RfD determination.
For developmental effects, decreased postnatal (Fi) body weight was prioritized over
offspring mortality. 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 Loveless etal. f20091 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 (2014)
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 (7,000 rats) of laboratory animals fMatsuzawa et al.. 19931.
This document is a draft for review purposes only and does not constitute Agency policy.
5-19	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Toxicological Review ofPFHxA and Related Salts
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 fChengelis etal.. 2009bl and in females dosed
with 200 mg/kg-day in the chronic study fKlaunig etal.. 20151. Note that the maximum dose in the
chronic study (200 mg/kg-day for females) was the lowest effective dose at which most responses
were observed across all studies. The maximum dose in the chronic study (females, 200 mg/kg-
day) was 2.5-fold lower than the maximum dose in the available subchronic studies. However,
PFHxA-dependent decreases in RBC and HGB levels were correlated with other red blood cell
indices including decreased MCH along with increased MCHC and MCV that, when interpreted
together, presents coherent evidence for PFHxA induced hematological effects such as anemia.
Further, increased reticulocyte counts are a possible indicator of compensatory erythroid cell
regeneration, which is supported by histological findings of splenic extramedullary hematopoiesis
and bone marrow erythroid hyperplasia, adding further support for this interpretation. The effect
on red blood cell parameters had a slightly lower POD than HGB, thus the female RBC hematological
endpoint from the chronic study was prioritized for RfD determination fKlaunig etal.. 20151.
Under EPA's A Review of the Reference Dose and Reference Concentration Processes fU.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.
This document is a draft for review purposes only and does not constitute Agency policy.
5-20	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Table 5-6. Uncertainty factors3 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 PKand 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 wks 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-d 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.
This document is a draft for review purposes only and does not constitute Agency policy.
5-21	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Toxicological Review ofPFHxA and Related Salts
aUFA = interspecies uncertainty factor, UFH = interhumans uncertainty factor, UFS = extrapolating from subchronic
to chronic uncertainty factor, UFL = LOAEL-to-NOAEL extrapolation uncertainty factor, UFD = database uncertainty
factor.
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 fWolfetal.. 2014:
Wolfetal.. 20081. 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.
Table 5-7. Candidate values for PFHxA
Endpoint/study/
confidence
Species,
strain (sex)
PODhed
PFHxA3
(mg/kg-d)
UFA
UFh
UFS
ufl
UFd
UFC
Candidate
value
PFHxA
(mg/kg-d)
Candidate
value
PFHxA-Nab
(mg/kg-d)
^Hepatocellular
hypertrophy,
90 d
Loveless et al.
(2009)
High confidence
Rat,
Crl:CD(SD)
(male)
0.11
3
10
3
1
3
300
4 x 10"4
4 x 10"4
4/Red blood
cells, 51 wks
Klaunig et al.
(2015)
High confidence
Rat,
Crl:CD(SD)
(female)
0.52
3
10
1
1
3
100
5 x 10"3
6 x 10"3
4/Fi body weight,
PND0
Loveless et al.
(2009)
High confidence
Rat, Sprague-
Dawley, Fi
(combined)
0.048
3
10
1
1
3
100
5 x 10"4
5 x 10"4
This document is a draft for review purposes only and does not constitute Agency policy.
5-22	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
aHED calculations based on DAF, the ratio of human and animal clearance values (see Table 5-4). DAF values for
female rats and female mice were used for the respective developmental effects on combined male and female
pups of each species.
bTo calculate candidate values for salts of PFHxA, multiply the candidate value of interest by the ratio of molecular
weights of the free acid and the salt. For example, for the sodium salt of PFHxA, the candidate value would be
calculated by multiplying the free acid candidate value by 1.070 (MW free acid/MW sodium
salt = 336/314 = 1.070). This same conversion can be applied to other salts of PFHxA, such as the ammonium salt.
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 = 4 x 10 4 mg/kg-d PFHxA; 4 x 10"4 mg/kg-d PFHxA-Na
Confidence in the
study used to derive
osRfD
High
Confidence in the studv (Loveless et al., 2009) is hiah based on the studv
evaluation results (i.e., rated high confidence overall) (HAWC link). The
overall study size, design, and test species were considered relevant for
deriving toxicity values.
Confidence in the
evidence base for
hepatic effects
Medium
Confidence in the oral toxicity evidence base for hepatic effects is medium
based on consistent, dose-dependent, and biologically coherent effects on
organ weight and histopathology observed in multiple high confidence
subchronic and chronic studies. The available mechanistic evidence also
supports biological plausibility of the observed effects. Limitations of the
evidence base for hepatic effects is the lack of human studies, and
measures that would have been useful to inform the pathways for hepatic
effects leading to hepatocellular hypertrophy (e.g., specific 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 \/d 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.
This document is a draft for review purposes only and does not constitute Agency policy.
5-23	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Confidence
categories
Designation
Discussion
Hematopoietic osRfD = 5 x 10 3 mg/kg-d PFHxA; 6 x 10 3 mg/kg-d PFHxA-Na
Confidence in study
High
Confidence in the studv (Klaunig et al., 2015) is hiah based on the studv
evaluation results (i.e., rated 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
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.
Developmental osRfD = 5 x 10"4 mg/kg-d PFHxA; 5 x 10 4 mg/kg-d PFHxA-Na
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.
This document is a draft for review purposes only and does not constitute Agency policy.
5-24	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Confidence
categories
Designation
Discussion
Confidence in the
quantification of the
PODhed
Medium
Confidence in the quantification of the POD and osRfD is medium given
the POD was based on BMD modeling within the range of the observed
data and dosimetric adjustment based on PFHxA-specific PK information.
Some residual uncertainty in the application of the dosimetric approach
described above is that 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 for
PFHxA
(mg/kg-d)
osRfD for
PFHxA-Naa
(mg/kg-d)
Confidence
Hepatic
Increased
hepatocellular
hypertrophy in
adult male Crl:CD
Sprague-Dawley
rats
0.11 mg/kg-d
based on
BMDLioer and
free salt
normalization
(Loveless et al.,
2009)
300
4 x 10"4
4 x 10"4
Medium
Hematopoietic
Decreased red
blood cells in
adult female
Crl:CD
Sprague-Dawley
rats
0.52 mg/kg-d
based on
BMDLisn (Klaunig
et al., 2015)
100
5 x 10"3
6 x 10"3
High
Developmental
Decreased
postnatal (PND 0)
body weight in Fi
Sprague-Dawley
male and female
rats, exposed
throughout
lactation and
gestation
0.048 mg/kg-d
based on
BMDL.5rd and free
salt
normalization
(Loveless et al.,
2009)
100
5 x 10"4
5 x 10"4
Medium
This document is a draft for review purposes only and does not constitute Agency policy.
5-25	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Toxicological Review ofPFHxA and Related Salts
aTo calculate candidate values for salts of PFHxA, multiply the candidate value of interest by the ratio of molecular
weights of the free acid and the salt. For example, for the sodium salt of PFHxA, the candidate value would be
calculated by multiplying the free acid candidate value by 1.070 (MW free acid/MW sodium
salt = 336/314 = 1.070). This same conversion can be applied to other salts of PFHxA, such as the ammonium salt.
From the identified human health effects ofPFHxA and derived osRfDs for hepatic,
hematopoietic, and developmental effects (see Table 5-9), an RfD of 5 x 10~4 mg/kg-day PFHxA
based on decreased postnatal (Ft) body weight in rats was selected. As described in Table 5-8,
confidence in the RfD is medium, based on medium confidence in the developmental RfD. The
decision to select the developmental RfD was based on all available osRfDs in addition to overall
confidence and composite uncertainty for those osRfDs. The confidence in the selected RfD is
equivalent to that of the hepatic RfDs but lower than the hematopoietic RfD. The developmental
endpoint decreased Fi body weight at PND 0 having the lowest overall PODhed of 0.048 mg/kg-d
PFHxA based on BMDLsrd and free salt normalization (Loveless etal.. 2009) and UFc of 100 was
considered protective across all lifestages. The hepatic RfD was slightly lower but was based on a
higher PODhed (0.11 mg/kg-day PFHxA) and UFC (300). The developmental RfD, therefore, is based
on the lowest PODhed and lowest UFC using a study considered high confidence.
Estimation or Selection of Points of Departure (PODs) for Subchronic RfD Derivation
In addition to providing an RfD for lifetime exposure in health systems, this document also
provides an RfD for less-than-lifetime ("subchronic") exposures. These subchronic RfDs were
based on the endpoints advanced for POD derivation provided in Table 5-1. Data to inform
potential hepatic and hematopoietic effects from the high confidence subchronic studies by
(Chengelis etal.. 2009b: Loveless etal.. 2009) were considered the most informative for developing
candidate values. The high confidence developmental/reproductive studies (Iwai and Hoberman.
2014: Loveless et al.. 20091 were also advanced for candidate value derivation. The high confidence
short-term study fNTP. 20181 was not advanced based on the same rationale as described above
for the lifetime RfD. In general, the rationales for advancing these endpoints for subchronic value
derivation are the same as described and summarized above in Table 5-1; however, for
hematopoietic effects, subchronic data from Chengelis etal. (2009b) and Loveless etal. (2009)
were prioritized over the data from the chronic study by Klaunigetal. f20151 for use in deriving a
subchronic RfD.
The endpoints selected for dose-response were modeled using approaches consistent with
EPA's Benchmark Dose Technical Guidance document fU.S. EPA. 2012al. 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.
This document is a draft for review purposes only and does not constitute Agency policy.
5-26	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
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)
PODhed PFHxA3
(mg/kg-d)
Hepatic effects
^Hepatocellular
hypertrophy
Chengelis et al. (2009b)
Low confidence
Rat,
Crl:CD(SD)
(male)
NOAELb
(0% response)
50
0.55

Loveless et al. (2009)
High confidence
Rat,
Crl:CD(SD)
(male)
BMDLioer
Multistage 1 NCV
10.66
0.11°


Rat,
Crl:CD(SD)
(female)
BMDLioer
Multistage 3 NCV
96.32
0.43°
Hematopoietic effects
4/Hemoglobin
Chengelis et al. (2009b)
High confidence
Rat,
Crl:CD(SD)
(male)
BMDLisd
Polynomial 3 CV
81.35
0.89


Rat,
Crl:CD(SD)
(female)
NOAELd
(3% decrease)
50
0.24

Loveless et al. (2009)
High confidence
Rat,
Crl:CD(SD)
(male)
NOAELd
(6% decrease)
50
0.51°


Rat,
Crl:CD(SD)
(female)
BMDLisd
Polynomial 3 CV
127.61
0.57°
4/Red blood cell
Chengelis et al. (2009b)
High confidence
Rat,
Crl:CD(SD)
(male)
NOAELd
(no change)
50
0.55


Rat,
Crl:CD(SD)
(female)
BMDLisd
Exponential 5 CV
16.32
0.078

Loveless et al. (2009)
High confidence
Rat,
Crl:CD(SD)
(male)
BMDLisd
Linear NCV
44.57
0.46°


Rat,
Crl:CD(SD)
(female)
BMDLisd
Linear CV
112.36
0.50°
Developmental Effects
4/Postnatal (Fi)
body weight,
PNDO
Loveless et al. (2009)
High confidence
Rat,
Crl:CD(SD), Fi
(combined)
BMDLsrd
Hill
10.62
0.048°
This document is a draft for review purposes only and does not constitute Agency policy.
5-27	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Toxicological Review ofPFHxA and Related Salts
Endpoint
Study/confidence
Species,
strain (sex)
POD type/model
POD (mg/kg-d)
PODhed PFHxA3
(mg/kg-d)
4/Postnatal (Fi)
body weight,
PND 0
Iwai and Hoberman
(2014)
High confidence
Mouse, CD-I,
Fi (combined)
BMDLsrd
Polynomial 3 CV
Phase 2
80.06
0.68e
4/Postnatal (Fi)
body weight,
PND 4
BMDLsrd
Exponential-M5
Phase 1 and 2
Polynomial 3 CV
Phase 2
103.12
89.79
0.87e
0.76e
^Perinatal
Mortality
Iwai and Hoberman
(2014)
High confidence
Mouse, CD-I,
Fi (combined)
BMDLier
Nested Logistic
Phase 2
24.77
0.21e
1SD = 1 standard deviation, CV = constant variance, NCV = nonconstant variance.
aHED calculations based on the DAF, the ratio of human and animal clearance values (see Table 5-3). DAF values
for female rats and female mice were used for the respective developmental effects on combined male and
female pups of each species. PODhed based on PFHxAfree acid.
bResponse only at high dose with responses far above BMR level, data not modeled.
cPODHed multiplied by normalization factor to convert from sodium salt to free acid (MW free acid/MW sodium
salt = 314/336 = 0.935).
dNo models provided adequate fit therefore a NOAEL approach was selected.
6P0DHed multiplied by normalization factor to convert from sodium salt to free acid (MW free acid/MW ammonium
salt = 314/331 = 0.949).
Derivation of Candidate Toxicity Values for the Subchronic RfD
The PODhed values listed in Table 5-10 were further narrowed for 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 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 (Loveless 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
This document is a draft for review purposes only and does not constitute Agency policy.
5-28	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
1	chronic extrapolation was required for the subchronic RfD. The resulting candidate values are
2	shown in Table 5-11.
Table 5-11. Candidate values for deriving the subchronic RfD for PFHxA
Endpoint/Study/Confidence
Species, strain
(sex)
PODhed
PFHxA3
(mg/kg-d)
UFa
UFh
UFS
ufl
UFD
UFC
Candidate
value PFHxA
(mg/kg-d)
Candidate
value
PFHxA-Nac
(mg/kg-d)
^Hepatocellular
hypertrophy, 90 d
Loveless et al. (2009)
Rat, Crl:CD(SD)
(male)
0.11b
3
10
1
1
3
100
1 X 10"3
1 X 10"3
High confidence
4/Red blood cell, 90 d
Chengelis et al. (2009b)
Rat, Crl:CD(SD)
(female)
0.078
3
10
1
1
3
100
8 x 10"4
8 x 10"4
High confidence
4/Postnatal (Fi) body
weight, PND 0
Loveless et al. (2009)
Rat,
Sprague-Dawley,
Fi
(combined)
0.048b
3
10
1
1
3
100
5 x 10"4
5 x 10"4
High confidence
aThe RfD for the free acid of PFHxA is calculated using the ratio of molecular weights as described above.
bPODHED multiplied by normalization from the sodium salt to free acid (MW free acid/MW sodium
salt = 314/336 = 0.935).
cTo calculate subchronic candidate values, osRfDs or the subchronic RfD for salts of PFHxA, multiply the value of
interest by the ratio of molecular weights of the salt and free acid. For example, for the sodium salt of PFHxA, the
candidate value is calculated by multiplying the free acid candidate value by 1.070: (MW free acid/MW sodium
salt = 336/317 = 1.070)
3
This document is a draft for review purposes only and does not constitute Agency policy.
5-29	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
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 = 1 x 10 3 mg/kg-d PFHxA; 1 x 10 3 mg/kg-d PFHxA-Na
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 = 8 x 10"4 mg/kg-d PFHxA; 8 x 10"4 mg/kg-d PFHxA-Na
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
This document is a draft for review purposes only and does not constitute Agency policy.
5-30	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
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 = 5 x 10 4 mg/kg-d PFHxA; 5 x 10"4 mg/kg-d PFHxA-Na
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
This document is a draft for review purposes only and does not constitute Agency policy.
5-31	DRAFT-DO NOT CITE OR QUOTE

-------
Toxicological Review ofPFHxA and Related Salts
Confidence categories
Designation3
Discussion


of the dosimetric approach described above is that Vd and CL were not
measured in humans or mice and were considered equivalent to
those in monkeys and rats, respectively.
Confidence in
developmental
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 i are summarized in Table
3	5-13.
Table 5-13. Subchronic osRfD values for PFHxA
System
Basis
PODhed
UFC
osRfD for
PFHxA
(mg/kg-d)
osRfD for
PFHxA-Naa
(mg/kg-d)
Confidence
Hepatic
Increased
hepatocellular
hypertrophy in adult
male Crl:CD
Sprague-Dawley rats
0.11 mg/kg-d based on
BMDLioer and free salt
normalization (Loveless et
al„ 2009)
100
1 X 10"3
1 X 10"3
Medium
Hematopoietic
Decreased red blood
cells in adult female
Crl:CD
Sprague-Dawley rats
0.078 mg/kg-d based on
BMDLisn (Chengelis et al.,
2009b)
100
8 x 10"4
8 x 10"4
High
Developmental
Decreased postnatal
(PND 0) body weight
in Fi Sprague-Dawley
male and female rats,
exposed throughout
lactation and
gestation
0.048 mg/kg-d based on
BMDL5rd and free salt
normalization (Loveless et
al., 2009)
100
5 x 10"4
5 x 10"4
Medium
aTo calculate candidate values for salts of PFHxA, multiply the candidate value of interest by the ratio of molecular
weights of the free acid and the salt. For example, for the sodium salt of PFHxA, the candidate value would be
calculated by multiplying the free acid candidate value by 1.070 (MW free acid/MW sodium
salt = 336/314 = 1.070). This same conversion can be applied to other salts of PFHxA, such as the ammonium salt.
4	From the identified targets of PFHxA toxicity and derived subchronic osRfDs (see Table 5-
5	13), an RfD of 5 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 RfD, as described in Table 5-12. The confidence in the selected RfD is equivalent to
8	that of the hepatic RfDs but lower than the hematopoietic RfD. The developmental RfD is expected
This document is a draft for review purposes only and does not constitute Agency policy.
5-32	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
Toxicological Review ofPFHxA and Related Salts
to be protective of all life stages. The UFc (see Table 5-13) is equivalent to the other osRfDs and the
endpointhas the lowest PODhed (0.048 mg/kg-day, see Table 5-11). The decision to select the
developmental RfD was based on all of the available osRfDs in addition to overall confidence and
composite uncertainty for those osRfDs.
5.2.2. Inhalation Reference Concentration (RfC)
No published studies investigating the inhalation effects of subchronic, chronic, or
gestational exposure to PFHxA in humans or animals have been identified. Therefore, an RfC is not
derived.
5.3. CANCER TOXICITY VALUES
As discussed in Sections 3.3 and 4.2, given the sparse evidence base and in accordance with
the Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005). EPA concluded that there is
inadequate information to assess carcinogenic potential for PFHxA for any route of exposure.
Therefore, consistent with the Guidelines and the lack of adequate data on the potential
carcinogenicity ofPFHxA, quantitative estimates for either oral (oral slope factor, OSF) or
inhalation (inhalation unit risk; IUR) exposure were not derived.
This document is a draft for review purposes only and does not constitute Agency policy.
5-33	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
Toxicological Review ofPFHxA and Related Salts
REFERENCES
AACC (American Association for Clinical Chemistry). (1992). Clinical pathology testing
recommendations for nonclinical toxicity and safety studies. Toxicol Pathol 20: 539-543.
Amacher. DE: Schomaker. ST: Burkhardt. IE. (1998). The relationship among microsomal enzyme
induction, liver weight and histological change in rat toxicology studies. Food Chem Toxicol
36: 831-839. http://dx.doi.org/10.1016/S0278-6915r98100066-0
Anderson. IK: Luz. AL: Goodrum. P: Durda. 1. (2019). Perfluorohexanoic acid toxicity, part II:
Application of human health toxicity value for risk characterization. Regul Toxicol
Pharmacol 103: 10-20. http://dx.doi.org/10.1016/i.vrtph.2019.01.020
Anderson. RH: Long. GC: Porter. RC: Anderson. IK. (2016). Occurrence of select perfluoroalkyl
substances at U.S. Air Force aqueous film-forming foam release sites other than fire-training
areas: Field-validation of critical fate and transport properties. Chemosphere 150: 678-685.
http://dx.doi.Org/10.1016/j.chemosphere.2016.01.014
ATSDR (Agency for Toxic Substances and Disease Registry). (2018). Toxicological profile for
perfluoroalkyls. Draft for public comment [ATSDR Tox Profile], Atlanta, GA: U.S. Department
of Health and Human Services, Centers for Disease Control and Prevention.
https://www.atsdr.cdc.gov/toxprofiles/tp200.pdf
Baduel. C: Paxman. CI: Mueller. IF. (2015). Perfluoroalkyl substances in a firefighting training
ground (FTG), distribution and potential future release. J Hazard Mater 296: 46-53.
http ://dx. doi. or g/10.1016 /i .ihazmat 2015.03.007
Bailey. SA: Zidell. RH: Perry. RW. (2004). Relationships between organ weight and body/brain
weight in the rat: What is the best analytical endpoint? Toxicol Pathol 32: 448-466.
http: //dx.doi.org/10.1080/01926230490465874
Bao. WW: Oian. ZM: Geiger. SD: Liu. E: Liu. Y: Wang. SO: Lawrence. WR: Yang. BY: Hu. LW: Zeng. XW:
Dong. GH. (2017). Gender-specific associations between serum isomers of perfluoroalkyl
substances and blood pressure among Chinese: Isomers of C8 Health Project in China. Sci
Total Environ 607-608: 1304-1312. http://dx.d0i.0rg/l0.1016/i.scitotenv.2017.07.124
Benskin. TP: De Silva. AO: Martin. LI: Arsenault. G: Mccrindle. R: Riddell. N: Maburv. SA: Martin. TW.
(2009). Disposition of perfluorinated acid isomers in Sprague-Dawley rats; part 1: single
dose. Environ Toxicol Chem 28: 542-554. http://dx.doi.Org/10.1897/08-239.l
Bergwerk. AT: Shi. X: Ford. AC: Kanai. N: Tacquemin. E: Burk. RD: Bai. S: Novikoff. PM: Stieger. B:
Meier. PI: Schuster. VL: Wolkoff. AW. (1996). Immunologic distribution of an organic anion
transport protein in rat liver and kidney. Am J Physiol 271: G231-G238.
http://dx.doi.Org/10.1152/aipgi.1996.271.2.G231
Bischel. HN: Macmanus-Spencer. LA: Zhang. CI: Luthv. RG. (2011). Strong associations of short-
chain perfluoroalkyl acids with serum albumin and investigation of binding mechanisms.
Environ Toxicol Chem 30: 2423-2430. http: //dx.doi.org/10.1002 /etc.647
Blaine. AC: Rich. CD: Hundal. LS: Lau. C: Mills. MA: Harris. KM: Higgins. CP. (2013). Uptake of
perfluoroalkyl acids into edible crops via land applied biosolids: field and greenhouse
studies. Environ SciTechnol 47: 14062-14069. http://dx.doi.org/10.1021/es403094q
This document is a draft for review purposes only and does not constitute Agency policy.
R-l	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Toxicological Review ofPFHxA and Related Salts
Borghoff. ST: Fitch. S: Rager. IE: Huggett. D. (2018). A hypothesis-driven weight-of-evidence analysis
to evaluate potential endocrine activity of perfluorohexanoic acid [Review], Regul Toxicol
Pharmacol 99: 168-181. http://dx.doi.Org/10.1016/i.yrtph.2018.09.001
Boron. W: Boulpaep. E. (2017). Medical physiology. In Medical Physiology (3rd ed.). Philadelphia,
PA: Elsevier, https://www.elsevier.com/books/medical-physiology/boron/978-l-4557-
4377-3
Braunig. 1: Baduel. C: Heffernan. A: Rotander. A: Donaldson. E: Mueller. IF. (2017). Fate and
redistribution of perfluoroalkyl acids through AFFF-impacted groundwater. Sci Total
Environ 596-597: 360-368. http://dx.doi.Org/10.1016/i.scitotenv.2017.04.095
Brust. V: Schindler. PM: Leweiohann. L. (2015). Lifetime development of behavioural phenotype in
the house mouse (Mus musculus). Frontiers in Zoology 12: S17.
http://dx.doi.org/10.1186/1742-9994-12-Sl-S17
Buck. R: Gannon. S. (2017). Perfluorohexanoic acid pharmacokinetics in mouse, rat, microminipig,
pig, monkey and human. In Abstracts of Papers of the 253rd National meeting of the
American Chemical Society. Washington, DC: American Chemical Society.
Burkemper. TL: Aweda. TA: Rosenberg. AT: Lunderberg. DM: Peaslee. GF: Lapi. SE. (2017).
Radiosynthesis and biological distribution of F-18-labeled perfluorinated alkyl substances.
Environ Sci Technol Lett 4: 211-215. http://dx.doi.org/10.1021/acs.estlett7b00042
Cesta. MF: Malarkev. DE: Herbert. RA: Brix. A: Hamlin. MH: Singletarv. E: Sills. RC: Bucher. TR:
Birnbaum. LS. (2014). The National Toxicology Program Web-based nonneoplastic lesion
atlas: a global toxicology and pathology resource. Toxicol Pathol 42: 458-460.
http://dx.doi. org/10.1177/0192623313517304
Chang. S: Das. K: Ehresman. DT: Ellefson. ME: Gorman. GS: Hart. TA: Noker. PE: Tan. Y: Lieder. PH:
Lau. C: Olsen. GW: Butenhoff. TL. (2008). Comparative pharmacokinetics of
perfluorobutyrate in rats, mice, monkeys, and humans and relevance to human exposure via
drinking water. Toxicol Sci 104: 40-53. http://dx.doi.org/10.1093/toxsci/kfn057
Chen. WL: Bai. FY: Chang. YC: Chen. PC: Chen. CY. (2018). Concentrations of perfluoroalkyl
substances in foods and the dietary exposure among Taiwan general population and
pregnant women. J Food Drug Anal 26: 994-1004.
http://dx.doi.Org/10.1016/j.jfda.2017.12.011
Chengelis. CP: Kirkpatrick. IB: Myers. NR: Shinohara. M: Stetson. PL: Sved. DW. (2009a).
Comparison of the toxicokinetic behavior of perfluorohexanoic acid (PFHxA) and
nonafluorobutane-1-sulfonic acid (PFBS) in cynomolgus monkeys and rats. Reprod Toxicol
27: 400-406. http://dx.doi.Org/10.1016/j.reprotox.2009.01.013
Chengelis. CP: Kirkpatrick. IB: Radovskv. A: Shinohara. M. (2009b). A 90-day repeated dose oral
(gavage) toxicity study of perfluorohexanoic acid (PFHxA) in rats (with functional
observational battery and motor activity determinations). Reprod Toxicol 27: 342-351.
http://dx.doi.Org/10.1016/j.reprotox.2009.01.006
Craig. EA: Yan. Z: Zhao. 01. (2015). The relationship between chemical-induced kidney weight
increases and kidney histopathology in rats. J Appl Toxicol 35: 729-736.
http ://dx. doi. or g/10.10 0 2 /j at. 3 0 3 6
This document is a draft for review purposes only and does not constitute Agency policy.
R-2	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
Toxicological Review ofPFHxA and Related Salts
D'eon. TC: Simpson. AT: Kumar. R: Baer. AT: Maburv. SA. (2010). Determining the molecular
interactions of perfluorinated carboxylic acids with human sera and isolated human serum
albumin using nuclear magnetic resonance spectroscopy. Environ Toxicol Chem 29: 1678-
1688. http ://dx.doi.org/10.1002/etc.204
Daikin Industries (Daikin Industries Limited). (2009a). The excretion and tissue distribution of
[14C]-ammonium perflourohexanoate in the mouse and the rat following a multiple oral
administration of 50 mg/kg. Osaka, Japan.
Daikin Industries (Daikin Industries Limited). (2009b). The excretion of [14C]-ammonium
perfluorohexanoate in the mouse and the rat following a single oral administration at 50
mg/kg. Osaka, Japan.
Daikin Industries (Daikin Industries Limited). (2010). Oral (gavage) acute pharmacokinetic study of
PFH ammonium salt (ammonium salt of perflourinated hexanoic acid) in mice. Osaka, Japan.
Das. KP: Wood. CR: Lin. MT: Starkov. AA: Lau. C: Wallace. KB: Corton. TC: Abbott. BP. (2017).
Perfluoroalkyl acids-induced liver steatosis: Effects on genes controlling lipid homeostasis.
Toxicology 378: 37-52. http://dx.doi.Org/10.1016/j.tox.2016.12.007
De Silva. AO: Benskin. TP: Martin. LI: Arsenault. G: Mccrindle. R: Riddell. N: Martin. TW: Maburv. SA.
(2009). Disposition of perfluorinated acid isomers in Sprague-Dawley rats; part 2:
subchronic dose. Environ Toxicol Chem 28: 555-567. http://dx.doi.Org/10.1897/08-254.l
Dewitt. TC: Blossom. ST: Schaider. LA. (2019). Exposure to per-fluoroalkyl and polyfluoroalkyl
substances leads to immunotoxicity: epidemiological and toxicological evidence [Review], J
Expo Sci Environ Epidemiol 29: 148-156. http://dx.doi.org/10.1038/s41370-018-0097-y
Dong. GH: Tung. KY: Tsai. CH: Liu. MM: Wang. D: Liu. W: Tin. YH: Hsieh. WS: Lee. YL: Chen. PC.
(2013). Serum polyfluoroalkyl concentrations, asthma outcomes, and immunological
markers in a case-control study of Taiwanese children. Environ Health Perspect 121: 507-
513, 513e501-508. http://dx.doi.org/10.1289/ehp.1205351
Dzierlenga. AL: Robinson. VG: Waidvanatha. S: Devito. MI: Eifrid. MA: Gibbs. ST: Granville. CA:
Blvstone. CR. (2019). Toxicokinetics of perfluorohexanoic acid (PFHxA), perfluorooctanoic
acid (PFOA) and perfluorodecanoic acid (PFDA) in male and female Hsd:Sprague dawley SD
rats following intravenous or gavage administration. Xenobiotica 50: 1-11.
http://dx.d0i.0rg/l 0.1080/00498254.2019.1683776
Ericson. I: Gomez. M: Nadal. M: van Bavel. B: Lindstrom. G: Domingo. TL. (2007). Perfluorinated
chemicals in blood of residents in Catalonia (Spain) in relation to age and gender: a pilot
study. Environ Int 33: 616-623. http://dx.doi.Org/10.1016/i.envint.2007.01.003
Eriksen. KT: Raaschou-Nielsen. 0: Sarensen. M: Roursgaard. M: Loft. S: Mailer. P. (2010). Genotoxic
potential of the perfluorinated chemicals PFOA, PFOS, PFBS, PFNA and PFHxA in human
HepG2 cells. MutatRes 700: 39-43. http://dx.doi.Org/10.1016/i.mrgentox.2010.04.024
Espinosa. IS: Strvker. MP. (2012). Development and plasticity of the primary visual cortex [Review],
Neuron 75: 230-249. http://dx.doi.Org/10.1016/j.neuron.2012.06.009
Fabrega. F: Kumar. V: Benfenati. E: Schuhmacher. M: Domingo. TL: Nadal. M. (2015). Physiologically
based pharmacokinetic modeling of perfluoroalkyl substances in the human body. Toxicol
Environ Chem 97: 814-827. http://dx.d0i.0rg/l 0.1080/02772248.2015.1060976
Filer. D. (2015). The Toxcast™ analysis pipeline: An R package for processing and modeling
chemical screening data. Washington, DC: Environmental Protection Agency.
https://www.epa.gOv/sites/production/files/2015-08/documents/pipeline overview.pdf
This document is a draft for review purposes only and does not constitute Agency policy.
R-3	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
Toxicological Review ofPFHxA and Related Salts
Filer. PL: Kothiva. P: Setzer. RW: Tudson. RS: Martin. MT. (2016). tcpl: The ToxCast Pipeline for
High-Throughput Screening Data. Bioinformatics 33: 618-620.
http://dx.doi.org/10.1093/bioinformatics/btw68Q
Foreman. IE: Chang. SC: Ehresman. DT: Butenhoff. TL: Anderson. CR: Palkar. PS: Kang. BH: Gonzalez.
FT: Peters. 1M. (2009). Differential Hepatic Effects of Perfluorobutyrate Mediated by Mouse
and Human PPAR-alpha. Toxicol Sci 110: 204-211.
http: / /dx.doi. or g/10.10 9 3 /toxsci /kfp 077
Fraser. AT: Webster. TF: Watkins. DT: Strvnar. MT: Kato. K: Calafat. AM: Vieira. VM: Mcclean. MP.
(2013). Polyfluorinated compounds in dust from homes, offices, and vehicles as predictors
of concentrations in office workers' serum. Environ Int 60: 128-136.
http://dx.doi.Org/10.1016/j.envint2013.08.012
Fu. Y: Wang. T: Fu. 0: Wang. P: Lu. Y. (2014). Associations between serum concentrations of
perfluoroalkyl acids and serum lipid levels in a Chinese population. Ecotoxicol Environ Saf
106: 246-252. http://dx.doi.org/10.1016/i.ecoenv.2014.04.039
Fujii. Y: Niisoe. T: Harada. KH: Uemoto. S: Ogura. Y: Takenaka. K: Koizumi. A. (2015). Toxicokinetics
of perfluoroalkyl carboxylic acids with different carbon chain lengths in mice and humans. J
Occup Health 57: 1-12. http://dx.doi.org/10.1539/ioh.14-0136-OA
Gannon. SA: lohnson. T: Nabb. PL: Serex. TL: Buck. RC: Loveless. SE. (2011). Absorption,
distribution, metabolism, and excretion of [l-14C]-perfluorohexanoate ([14C]-PFHx) in rats
and mice. Toxicology 283: 55-62. http://dx.doi.Org/10.1016/i.tox.2011.02.004
Gomis. MI: Vestergren. R: Borg. P: Cousins. IT. (2018). Comparing the toxic potency in vivo of long-
chain perfluoroalkyl acids and fluorinated alternatives. Environ Int 113: 1-9.
http://dx.doi.Org/10.1016/j.envint2018.01.011
Goodrow. SM: Ruppel. B: Lippincott. RL: Post. GB: Procopio. NA. (2020). Investigation of levels of
perfluoroalkyl substances in surface water, sediment and fish tissue in New Jersey, USA. Sci
Total Environ 729: 138839. http://dx.doi.Org/10.1016/j.scitotenv.2020.138839
Gotoh. Y: Kato. Y: Stieger. B: Meier. PI: Sugivama. Y. (2002). Gender difference in the Oatpl-
mediated tubular reabsorption of estradiol 17beta-P-glucuronide in rats. Am J Physiol
Endocrinol Metab 282: E1245-E1254. http://dx.doi.org/10.1152/aipendo.00363.2001
Hall. AP: Elcombe. CR: Foster. TR: Harada. T: Kaufmann. W: Knippel. A: Kiittler. K: Malarkev. PE:
Maronpot. RR: Nishikawa. A: Nolte. T: Schulte. A: Strauss. V: York. MT. (2012). Liver
hypertrophy: a review of adaptive (adverse and non-adverse) changes-conclusions from
the 3rd International ESTP Expert Workshop [Review], Toxicol Pathol 40: 971-994.
http://dx.d0i.0rg/l 0.1177/0192623312448935
Han. X: Nabb. PL: Russell. MH: Kennedy. GL: Rickard. RW. (2012). Renal elimination of
perfluorocarboxylates (PFCAs) [Review], Chem Res Toxicol 25: 35-46.
http: / /dx. doi. or g/10.10 21 /tx2 0 0 3 6 3w
Hata. S: Wang. P: Eftychiou. N: Ananthanaravanan. M: Batta. A: Salen. G: Pang. KS: Wolkoff. AW.
(2003). Substrate specificities of rat oatpl and ntcp: implications for hepatic organic anion
uptake. Am J Physiol Gastrointest Liver Physiol 285: G829-G839.
http://dx.doi.org/10.1152/ajpgi.00352.2002
Heo. IT: Lee. TW: Kim. SK: Oh. IE. (2014). Foodstuff analyses show that seafood and water are major
perfluoroalkyl acids (PFAAs) sources to humans in Korea. J Hazard Mater 279: 402-409.
http: / /dx. doi. or g/10.1016 /i .ihazmat 2014.07.004
This document is a draft for review purposes only and does not constitute Agency policy.
R-4	PRAFT-PO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
Toxicological Review ofPFHxA and Related Salts
Hill. AB. (1965). The environment and disease: Association or causation? Proc R Soc Med 58: 295-
300.
Houtz. EF: Higgins. CP: Field. TA: Sedlak. PL. (2013). Persistence of perfluoroalkyl acid precursors in
AFFF-impacted groundwater and soil. Environ Sci Technol 47: 8187-8195.
http://dx.doi.org/10.1021/es4018877
IPCS (International Programme on Chemical Safety). (2012). Harmonization project document no.
10: Guidance for immunotoxicity risk assessment for chemicals. (Harmonization Project
Document No. 10). Geneva, Switzerland: World Health Organization.
http://www.inchem.org/documents/harmproi/harmproi/harmproilO.pdf
Iwabuchi. K: Senzaki. N: Mazawa. D: Sato. I: Hara. M: Ueda. F: Liu. W: Tsuda. S. (2017). Tissue
toxicokinetics of perfluoro compounds with single and chronic low doses in male rats. J
Toxicol Sci 42: 301-317. http: / /dx.doi.org/10.2131 /its.42.301
Iwai. H. (2011). Toxicokinetics of ammonium perfluorohexanoate. Drug Chem Toxicol 34: 341-346.
http: //dx.doi.org/10.3109 /01480545.2011.585162
Iwai. H: Hoberman. AM. (2014). Oral (Gavage) Combined Developmental and Perinatal/Postnatal
Reproduction Toxicity Study of Ammonium Salt of Perfluorinated Hexanoic Acid in Mice. Int
J Toxicol 33: 219-237. http://dx.doi.org/10.1177/1091581814529449
Tanku. I.. T1993I Physiological modelling of renal drug clearance. Eur J Clin Pharmacol 44: 513-519.
http://dx.d0i.0rg/l 0.1007%2FBF02440850
Tanku. I: Zvara. K. (1993). Quantitative analysis of drug handling by the kidney using a physiological
model of renal drug clearance. Eur J Clin Pharmacol 44: 521-524.
Tiang. W: Zhang. Y: Zhu. L: Deng. 1. (2014). Serum levels of perfluoroalkyl acids (PFAAs) with isomer
analysis and their associations with medical parameters in Chinese pregnant women.
Environ Int 64: 40-47. http://dx.doi.Org/10.1016/i.envint.2013.12.001
Tin. H: Zhang. Y: Tiang. W: Zhu. L: Martin. TW. (2016). Isomer-Specific Distribution of Perfluoroalkyl
Substances in Blood. Environ Sci Technol 50: 7808-7815.
http://dx.doi.org/10.1021/acs.est6b01698
Kabadi. SV: Fisher. 1: Aungst. I: Rice. P. (2018). Internal exposure-based pharmacokinetic evaluation
of potential for biopersistence of 6:2 fluorotelomer alcohol (FTOH) and its metabolites.
Food Chem Toxicol 112: 375-382. http://dx.doi.Org/10.1016/j.fct.2018.01.012
Karaskova. P: Venier. M: Melvmuk. L: Becanova. 1: Voita. S: Prokes. R: Diamond. ML: Klanova. I.
(2016). Perfluorinated alkyl substances (PFASs) in household dust in Central Europe and
North America. Environ Int 94: 315-324. http://dx.doi.Org/10.1016/i.envint.2016.05.031
Kato. K: Calafat. AM: Needham. LL. (2009). Polyfluoroalkyl chemicals in house dust. Environ Res
109: 518-523. http://dx.doi.Org/10.1016/i.envres.2009.01.005
Kim. DH: Kim. UT: Kim. HY: Choi. SD: Oh. IE. (2016a). Perfluoroalkyl substances in serum from South
Korean infants with congenital hypothyroidism and healthy infants - Its relationship with
thyroid hormones. Environ Res 147: 399-404.
http://dx.doi.0rg/lO.lOl6/i.envres.2Ol6.O2.O37
Kim. ST: Heo. SH: Lee. PS: Hwang. IG: Lee. YB: Cho. HY. (2016b). Gender differences in
pharmacokinetics and tissue distribution of 3 perfluoroalkyl and polyfluoroalkyl substances
in rats. Food Chem Toxicol 97: 243-255. http://dx.doi.Org/10.1016/i.fct.2016.09.017
This document is a draft for review purposes only and does not constitute Agency policy.
R-5	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
Toxicological Review ofPFHxA and Related Salts
Kirkpatrick. IB. (2005). Final report: A combined 28-day repeated dose oral toxicity study with
reproductive/developmental screening study test of perfluorohexanoic acid and
lH,lH,2H,2H-tridecafluoro-l-octanol in rats, with recovery. Ashland, OH: WIL Research
Laboratories, https:/ /www, agc-
chemicals.com/ip /en/fluorine /products /detail /feature /index.html?pCode=TP-EN-F001
Klaunig. IE: Shinohara. M: Iwai. H: Chengelis. CP: Kirkpatrick. TB: Wang. Z: Bruner. RH. (2015).
Evaluation of the chronic toxicity and carcinogenicity of perfluorohexanoic acid (PFHxA) in
Sprague-Dawley rats. Toxicol Pathol 43: 209-220.
http: //dx.doi.org/10.1177/0192623314530532
Knobeloch. L: Imm. P: Anderson. H. (2012). Perfluoroalkyl chemicals in vacuum cleaner dust from
39 Wisconsin homes. Chemosphere 88: 779-783.
http://dx.doi.Org/10.1016/i.chemosphere.2012.03.082
Kudo. N. (2015). Metabolism and pharmacokinetics. In JC DeWitt (Ed.), Toxicological Effects of
Perfluoroalkyl and Polyfluoroalkyl Substances (pp. 151-175). New York, NY: Springer
International Publishing, http://dx.doi.org/10.1007/978-3-319-15518-0 6
Kudo. N: Katakura. M: Sato. Y: Kawashima. Y. (2002). Sex hormone-regulated renal transport of
perfluorooctanoic acid. Chem Biol Interact 139: 301-316.
http: //dx.doi.org/10.1016/S0009-2797r02100006-6
Kudo. N: Suzuki. E: Katakura. M: Ohmori. K: Noshiro. R: Kawashima. Y. (2001). Comparison of the
elimination between perfluorinated fatty acids with different carbon chain length in rats.
Chem Biol Interact 134: 203-216. http://dx.doi.Org/10.1016/S0009-2797f01100155-7
Lau. C. (2015). Perfluorinated compounds: An overview. In JC DeWitt (Ed.), Toxicological effects of
perfluoroalkyl and polyfluoroalkyl substances (pp. 1-21). New York: Springer.
http://dx.doi.org/10.1007/978-3-319-15518-0 1
Lau. C: Butenhoff. TL: Rogers. TM. (2004). The developmental toxicity of perfluoroalkyl acids and
their derivatives [Review], Toxicol Appl Pharmacol 198: 231-241.
http://dx.doi.Org/10.1016/i.taap.2003.ll.031
Lau. C: Thibodeaux. TR: Hanson. RG: Narotskv. MG: Rogers. TM: Lindstrom. AB: Strvnar. Ml. (2006).
Effects of perfluorooctanoic acid exposure during pregnancy in the mouse. Toxicol Sci 90:
510-518. http: //dx.doi.org/10.1093 /toxsci/kfj 105
Li. Y: Cheng. Y: Xie. Z: Zeng. F. (2017). Perfluorinated alkyl substances in serum of the southern
Chinese general population and potential impact on thyroid hormones. Sci Rep 7: 43380.
http://dx.doi.org/10.1038/srep43380
Loveless. SE: Slezak. B: Serex. T: Lewis. I: Mukerji. P: O'Connor. TC: Donner. EM: Frame. S. R.:
Korzeniowski. SH: Buck. RC. (2009). Toxicological evaluation of sodium perfluorohexanoate.
Toxicology 264: 32-44. http://dx.doi.Org/10.1016/i.tox.2009.07.011
Lu. R: Kanai. N: Bao. Y: Wolkoff. AW: Schuster. VL. (1996). Regulation of renal oatp mRNA
expression by testosterone. Am J Physiol 270: F332-F337.
http://dx.doi.Org/10.1152/aiprenal.1996.270.2.F332
Luz. AL: Anderson. IK: Goodrum. P: Durda. I. (2019). Perfluorohexanoic acid toxicity, part I:
Development of a chronic human health toxicity value for use in risk assessment Regul
Toxicol Pharmacol 103: 41-55. http://dx.doi.org/10.1016/i.vrtph.2019.01.019
Matsuzawa. T: Nomura. M: Unno. T. (1993). Clinical pathology reference ranges of laboratory
animals. Nihon Juigaku Zasshi 55: 351-362. http://dx.doi.org/10.1292 /ivms.55.351
This document is a draft for review purposes only and does not constitute Agency policy.
R-6	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Toxicological Review ofPFHxA and Related Salts
Moreta. C: Tena. MT. (2014). Determination of perfluorinated alkyl acids in corn, popcorn and
popcorn bags before and after cooking by focused ultrasound solid-liquid extraction, liquid
chromatography and quadrupole-time of flight mass spectrometry. J Chromatogr A 1355:
211-218. http://dx.doi.Org/10.1016/j.chroma.2014.06.018
Nian. M: Li. 00: Bloom. M: Oian. ZM: Svberg. KM: Vaughn. MG: Wang. SO: Wei. 0: Zeeshan. M:
Gurram. N: Chu. C: Wang. 1: Tian. YP: Hu. LW: Liu. KK: Yang. BY: Liu. R0: Feng. D: Zeng. XW:
Dong. GH. (2019). Liver function biomarkers disorder is associated with exposure to
perfluoroalkyl acids in adults: Isomers of C8 Health Project in China. Environ Res 172: 81-
88. http://dx.doi.Org/10.1016/i.envres.2019.02.013
Nilsson. H: Karrman. A: Rotander. A: van Bavel. B: Lindstrom. G: Westberg. H. (2013).
Biotransformation of fluorotelomer compound to perfluorocarboxylates in humans. Environ
Int 51: 8-12. http://dx.doi.org/10.1016/i.envint2012.09.001
Nilsson. H: Karrman. A: Westberg. H: Rotander. A: van Bavel. B: Lindstrom. G. (2010). A time trend
study of significantly elevated perfluorocarboxylate levels in humans after using fluorinated
ski wax. Environ Sci Technol 44: 2150-2155. http://dx.doi.org/10.1021/es9034733
NLM (National Library of Medicine). (2013). HSDB: Perfluoro-n-nonanoic acid. Available online at
https://pubchem.ncbi.nlm.nih.gov/source/hsdb/804Q
NLM (National Library of Medicine). (2016). HSDB: Perfluorohexanoic acid. Available online at
https://pubchem.ncbi.nlm.nih.gov/source/hsdb/8299
NLM (National Library of Medicine). (2017). HSDB: Perfluorohexanesulfonic acid. Available online
at https://pubchem.ncbi.nlm.nih.gov/source/hsdb/8274
Nobels. I: Dardenne. F: De Coen. W. im: Blust. R. (2010). Application of a multiple endpoint bacterial
reporter assay to evaluate toxicological relevant endpoints of perfluorinated compounds
with different functional groups and varying chain length. Toxicol In Vitro 24: 1768-1774.
http://dx.doi.Org/10.1016/i.tiv.2010.07.002
NTP (National Toxicology Program). (2017). Toxicokinetic evaluation (C20613) of
perfluorohexanoic acid (307-24-4) in Harlan Sprague-Dawley rats exposed via gavage or
intravenous injection. Durham, NC. Retrieved from
https://tools.niehs.nih.gov/cebs3/views/?action=main.dataReview&bin id=2566
NTP (National Toxicology Program). (2018). 28-day evaluation of the toxicity (C20613) of
perfluorohexanoic acid (PFHxA) (307-24-4) on Harlan Sprague-Dawley rats exposed via
gavage. Available online at
https://tools.niehs.nih.gov/cebs3/views/?action=main.dataReview&bin id=3879
OECD (Organisation for Economic Co-operation and Development). (2015). Working towards a
global emission inventory of PFASS: focus on PFCAS - status quo and the way forward. Paris,
France.
http://www.oecd.org/chemicalsafetv/Working%20Towards%20a%20Global%20Emission
%20Tnventory%20of%2 OPFASS.pdf
Ohmori. K: Kudo. N: Katavama. K: Kawashima. Y. (2003). Comparison of the toxicokinetics between
perfluorocarboxylic acids with different carbon chain length. Toxicology 184: 135-140.
http://dx.doi.org/10.1016/S0300-483Xr02100573-5
This document is a draft for review purposes only and does not constitute Agency policy.
R-7	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
Toxicological Review ofPFHxA and Related Salts
Olsen. GW: Burris. TM: Ehresman. DT: Froehlich. TW: Seacat. AM: Butenhoff. TL: Zobel. LR. (2007).
Half-life of serum elimination of perfluorooctanesulfonate,perfluorohexanesulfonate, and
perfluorooctanoate in retired fluorochemical production workers. Environ Health Perspect
115: 1298-1305. http://dx.doi.Org/l 0.1289/ehp.l 0009
Olsen. GW: Chang. SC: Noker. PE: Gorman. GS: Ehresman. DT: Lieder. PH: Butenhoff. TL. (2009). A
comparison of the pharmacokinetics of perfluorobutanesulfonate (PFBS) in rats, monkeys,
and humans. Toxicology 256: 65-74. http: //dx.doi.Org/10.1016/i.tox.2008.ll.008
Perez. F: Llorca. M: Kock-Schulmever. M: Skrbic. B: Oliveira. LS: da Boit Martinello. K: Al-Dhabi. NA:
Antic. I: Farre. M: Barcelo. D. (2014). Assessment of perfluoroalkyl substances in food items
at global scale. Environ Res 135: 181-189. http://dx.doi.Org/10.1016/i.envres.2014.08.004
Perez. F: Nadal. M: Navarro-Ortega. A: Fabrega. F: Domingo. TL: Barcelo. D: Farre. M. (2013).
Accumulation of perfluoroalkyl substances in human tissues. Environ Int 59: 354-362.
http: / /dx. doi. or g/10.1016/i. envint 2013.06.004
Poothong. S: Thomsen. C: Padilla-Sanchez. TA: Papadopoulou. E: Haug. LS. (2017). Distribution of
novel and well-known poly- and perfluoroalkyl substances (PFASs) in human serum,
plasma, and whole blood. Environ Sci Technol 51: 13388-13396.
http://dx.doi.org/10.1021/acs.est7b03299
Post. GB: Cohn. PD: Cooper. KR. (2012). Perfluorooctanoic acid (PFOA), an emerging drinking water
contaminant: a critical review of recent literature [Review], Environ Res 116: 93-117.
http://dx.doi.Org/10.1016/i.envres.2012.03.007
Oin. XD: Oian. ZM: Dharmage. SC: Perret. 1: Geiger. SD: Rigdon. SE: Howard. S: Zeng. XW: Hu. LW:
Yang. BY: Zhou. Y: Li. M: Xu. SL: Bao. WW: Zhang. YZ: Yuan. P: Wang. 1: Zhang. C: Tian. YP:
Nian. M: Xiao. X: Chen. W: Lee. YL: Dong. GH. (2017). Association of perfluoroalkyl
substances exposure with impaired lung function in children. Environ Res 155: 15-21.
http://dx.doi.Org/10.1016/i.envres.2017.01.025
Ravne. S: Forest. K. (2010). Theoretical studies on the pKa values of perfluoroalkyl carboxylic acids.
J Mol Struct Theochem 949: 60-69. http://dx.doi.Org/10.1016/i.theochem.2010.03.003
Reddv. IK. (2004). Peroxisome proliferators and peroxisome proliferator-activated receptor alpha:
biotic and xenobiotic sensing. Am J Pathol 164: 2305-2321.
http://dx.d0i.0rg/l 0.1016/s0002-9440ri 0163787-x
Ren. XM: Oin. WP: Cao. LY: Zhang. 1: Yang. Y: Wan. B: Guo. LH. (2016). Binding interactions of
perfluoroalkyl substances with thyroid hormone transport proteins and potential
toxicological implications. Toxicology 366-367: 32-42.
http://dx.doi.0rg/lO.lOl6/j.tox.2Ol6.O8.Oll
Ren. XM: Zhang. YF: Guo. LH: Oin. ZF: Lv. 0Y: Zhang. LY. (2015). Structure-activity relations in
binding of perfluoroalkyl compounds to human thyroid hormone T3 receptor. Arch Toxicol
89: 233-242. http://dx.d0i.0rg/l0.1007/s00204-014-1258-v
Rosen. MB: Das. KP: Roonev. 1: Abbott. B: Lau. C: Corton. TC. (2017). PPARa-independent
transcriptional targets of perfluoroalkyl acids revealed by transcript profiling. Toxicology
387: 95-107. http://dx.d0i.0rg/l 0.1016/i.tox.2017.05.013
Russell. MH: Himmelstein. MW: Buck. RC. (2015). Inhalation and oral toxicokinetics of 6:2 FTOH
and its metabolites in mammals. Chemosphere 120: 328-335.
http://dx.doi.Org/10.1016/i.chemosphere.2014.07.092
This document is a draft for review purposes only and does not constitute Agency policy.
R-8	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Toxicological Review ofPFHxA and Related Salts
Russell. MH: Nilsson. H: Buck. RC. (2013). Elimination kinetics of perfluorohexanoic acid in humans
and comparison with mouse, rat and monkey. Chemosphere 93: 2419-2425.
http://dx.doi.Org/10.1016/i.chemosphere.2013.08.060
Sabolic. I: Asif. AR: Budach. WE: Wanke. C: Bahn. A: Burckhardt. G. (2007). Gender differences in
kidney function [Review], Pflugers Arch 455: 397-492. http://dx.doi.org/DOI
10.1007/s00424-007-0308-l
Sanchez Garcia. D: Siodin. M: Hellstrandh. M: Norinder. U: Nikiforova. V: Lindberg. 1: Wincent. E:
Bergman. A: Cotgreave. I: Munic Kos. V. (2018). Cellular accumulation and lipid binding of
perfluorinated alkylated substances (PFASs) - A comparison with lysosomotropic drugs.
Chem Biol Interact 281: 1-10. http://dx.doi.Org/10.1016/i.cbi.2017.12.021
Schecter. A: Malik-Bass. N: Calafat. AM: Kato. K: Colacino. TA: Gent. TL: Hvnan. LS: Harris. TR: Malla.
S: Birnbaum. L. (2012). Polyfluoroalkyl compounds in Texas children from birth through 12
years of age. Environ Health Perspect 120: 590-594.
http: / /dx. doi. or g/10.12 89 /ehp. 1104325
Seo. SH: Son. MH: Choi. SD: Lee. DH: Chang. YS. (2018). Influence of exposure to perfluoroalkyl
substances (PFASs) on the Korean general population: 10-year trend and health effects.
Environ Int 113: 149-161. http://dx.doi.Org/10.1016/i.envint2018.01.025
Shao. M: Ding. G: Zhang. 1: Wei. L: Xue. H: Zhang. N: Li. Y: Chen. G: Sun. Y. (2016). Occurrence and
distribution of perfluoroalkyl substances (PFASs) in surface water and bottom water of the
Shuangtaizi Estuary, China. Environ Pollut216: 675-681.
http://dx.doi.Org/10.1016/i.envpol.2016.06.031
Shitara. Y: Sugivama. D: Kusuhara. H: Kato. Y: Abe. T: Meier. PI: Itoh. T: Sugivama. Y. (2002).
Comparative inhibitory effects of different compounds on rat oatpl (slc21al)- and 0atp2
(Slc21a5)-mediated transport. Pharm Res 19: 147-153.
http: //dx.doi.org/10.1023 /a:l 014264614637
Song. X: Tang. S: Zhu. H: Chen. Z: Zang. Z: Zhang. Y: Niu. X: Wang. X: Yin. H: Zeng. F: He. C. (2018).
Biomonitoring PFAAs in blood and semen samples: Investigation of a potential link between
PFAAs exposure and semen mobility in China. Environ Int 113: 50-54.
http: / /dx. doi. or g/10.1016/i. envint 2018.01.010
Stahl. LL: Snyder. BP: Olsen. AR: Kincaid. TM: Wathen. IB: Mccarty. HB. (2014). Perfluorinated
compounds in fish from U.S. urban rivers and the Great Lakes. Sci Total Environ 499: 185-
195. http://dx.doi.org/10.1016/i.scitotenv.2014.07.126
Strvnar. MI: Lindstrom. AB. (2008). Perfluorinated compounds in house dust from Ohio and North
Carolina, USA. Environ Sci Technol 42: 3751-3756. http://dx.doi.org/10.1021/es7032058
Sundstrom. M: Chang. SC: Noker. PE: Gorman. GS: Hart. TA: Ehresman. PI: Bergman. A: Butenhoff. TL.
(2012). Comparative pharmacokinetics of perfluorohexanesulfonate (PFHxS) in rats, mice,
and monkeys. Reprod Toxicol 33: 441-451.
http://dx.doi.Org/10.1016/j.reprotox.2011.07.004
Tatum-Gibbs. K: Wambaugh. IF: Pas. KP: Zehr. RP: Strvnar. MI: Lindstrom. AB: Pelinskv. A: Lau. C.
(2011). Comparative pharmacokinetics of perfluorononanoic acid in rat and mouse.
Toxicology 281: 48-55. http://dx.doi.Org/10.1016/j.tox.2011.01.003
This document is a draft for review purposes only and does not constitute Agency policy.
R-9	PRAFT-PO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
Toxicological Review ofPFHxA and Related Salts
Thoolen. B: Maronpot. RR: Harada. T: Nvska. A: Rousseaux. C: Nolte. T: Malarkev. DE: Kaufmann. W:
Kiittler. K: Deschl. U: Nakae. D: Gregson. R: Vinlove. MP: Brix. AE: Singh. B: Belpoggi. F:
Ward. TM. (2010). Proliferative and nonproliferative lesions of the rat and mouse
hepatobiliary system [Review], Toxicol Pathol 38: 5S-81S.
http://dx.doi.org/10.1177/0192623310386499
U.S. EPA (U.S. Environmental Protection Agency). (1991). Guidelines for developmental toxicity risk
assessment. Fed Reg 56: 63798-63826.
U.S. EPA (U.S. Environmental Protection Agency). (1998). Guidelines for neurotoxicity risk
assessment [EPA Report] (pp. 1-89). (EPA/630/R-95/001F). Washington, DC: U.S.
Environmental Protection Agency, Risk Assessment Forum.
http://www.epa.gov/risk/guidelines-neurotoxicity-risk-assessment
U.S. EPA (U.S. Environmental Protection Agency). (2002a). Hepatocellular hypertrophy. HED
guidance document #G2002.01 [EPA Report], Washington, DC.
U.S. EPA (U.S. Environmental Protection Agency). (2002b). Perfluoroalkyl sulfonates; significant
new use rule [SNUR], (EPA-HQ-OPPT-2002-0043-0001).
https://www.regulations.gOv/document/EPA-H0-OPPT-2002-0043-0001
U.S. EPA (U.S. Environmental Protection Agency). (2002c). A review of the reference dose and
reference concentration processes. (EPA630P02002F). Washington, DC.
https://www.epa.gov/sites/production/files/2014-12/documents/rfd-final.pdf
U.S. EPA (U.S. Environmental Protection Agency). (2003). Toxicological review of methyl ethyl
ketone (CAS No. 78-93-3): In support of summary information on the Integrated Risk
Information System (IRIS). (EPA 635/R-03/009). Washington, DC.
http: //cfpub.epa.gov/ncea/iris /iris documents/documents/toxreviews /0071tr.pdf
U.S. EPA (U.S. Environmental Protection Agency). (2004). Toxicological review of boron and
compounds. In support of summary information on the Integrated Risk Information System
(IRIS) [EPA Report], (EPA/635/04/052). Washington, DC: U.S. Environmental Protection
Agency, IRIS. http://nepis.epa.gov/exe/ZyPURL.cgi?Dockey=P 1006CK9.txt
U.S. EPA (U.S. Environmental Protection Agency). (2005). Guidelines for carcinogen risk assessment
[EPA Report], (EPA630P03001F). Washington, DC.
https://www.epa.gov/sites/production/files/2013-
09/documents/cancer guidelines final 3-25-05.pdf
U.S. EPA (U.S. Environmental Protection Agency). (2007). Perfluoroalkyl sulfonates; significant new
use rule. (EPA-HQ-OPPT-2005-0015-0040). https://www.regulations.gov/document/EPA-
HO-QPPT-2005-0015-0040
U.S. EPA (U.S. Environmental Protection Agency). (2011). Recommended use of body weight 3/4 as
the default method in derivation of the oral reference dose. (EPA100R110001). Washington,
DC. https://www.epa.gov/sites/production/files/2013-09/documents/recommended-use-
of-bw34.pdf
U.S. EPA (U.S. Environmental Protection Agency). (2012a). Benchmark dose technical guidance
[EPA Report], (EPA/100/R-12/001). Washington, DC: U.S. Environmental Protection
Agency, Risk Assessment Forum, https: //www.epa.gov/risk/benchmark-dose-technical-
guidance
This document is a draft for review purposes only and does not constitute Agency policy.
R-10	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
Toxicological Review ofPFHxA and Related Salts
U.S. EPA (U.S. Environmental Protection Agency). (2012b). Toxicological review of tetrahydrofuran.
In support of summary information on the integrated risk information system (IRIS) (pp. 1-
207). (EPA/635/R-11/006F). Washington, DC.
U.S. EPA (U.S. Environmental Protection Agency). (2013). Significant new uses: Perfluoroalkyl
sulfonates and long-chain perfluoroalkyl carboxylate chemical substances. (EPA-HQ-OPPT-
2012-0268-0034). https://www.regulations.gov/document/EPA-H0-OPPT-2012-0268-
0034
U.S. EPA (U.S. Environmental Protection Agency). (2016a). North American Free Trade Agreement
(NAFTA) Technical Working Group on Pesticides (TWG) Developmental Neurotoxicity
Study GuidanceDocument (712B16001).
https://nepis.epa.gov/exe/ZyPURL.cgi?Dockey=P100YH73.txt
U.S. EPA (U.S. Environmental Protection Agency). (2016b). The Third Unregulated Contaminant
Monitoring Rule. Available online at https: //www.epa.gov/dwucmr/third-unregulated-
contaminant-monitoring-rule
U.S. EPA (U.S. Environmental Protection Agency). (2018a). Chemistry Dashboard. Washington, DC.
Retrieved from https: //comptox.epa.gov/dashboard
U.S. EPA (U.S. Environmental Protection Agency). (2018b). ToxCast data pipeline R package.
Retrieved from
https://figshare.com/articles/GitHub ToxCast Data Pipeline R Package/6062788
U.S. EPA (U.S. Environmental Protection Agency). (2018c). Toxic Release Inventory (TRI). Retrieved
from https://www.epa.gov/enviro/tri-search
U.S. EPA (U.S. Environmental Protection Agency). (2019a). ChemView [Database], Retrieved from
https://chemview.epa.gov/chemview
U.S. EPA (U.S. Environmental Protection Agency). (2019b). Development of the Proposed
Unregulated Contaminant Monitoring Rule for the Fifth Monitoring Cycle (UCMR 5): Public
Meeting and Webinar Held July 16, 2019. (815A19001).
https://nepis.epa.gov/exe /ZyPURL.cgi?Dockev=P 10 0XWUH.txt
U.S. EPA (U.S. Environmental Protection Agency). (2019c). EPA's per- and polyfluoroalkyl
substances (PFAS) action plan [EPA Report], (EPA 823R18004). Washington, DC.
https://www.epa.gov/sites/production/files/2019-
02/documents/pfas action plan 021319 508compliant l.pdf
U.S. EPA (U.S. Environmental Protection Agency). (2020). Significant new use rule: Long-chain
perfluoroalkyl carboxylate and perfluoroalkyl sulfonate chemical substances. (EPA-HQ-
OPPT-2013-0225-0232). https://www.regulations.gov/document/EPA-HQ-OPPT-2013-
0225-0232
Venkatesan. AK: Halden. RU. (2014). Loss and in situ production of perfluoroalkyl chemicals in
outdoor biosolids-soil mesocosms. Environ Res 132: 321-327.
http://dx.doi.Org/10.1016/j.envres.2014.04.024
Wang. 1: Zeng. XW: Bloom. MS: Oian. Z: Hinvard. LI: Belue. R: Lin. S: Wang. SO: Tian. YP: Yang. M:
Chu. C: Gurram. N: Hu. LW: Liu. KK: Yang. BY: Feng. D: Liu. RO: Dong. GH. (2019). Renal
function and isomers of perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS):
Isomers of C8 Health Project in China. Chemosphere 218: 1042-1049.
http://dx.doi.Org/10.1016/i.chemosphere.2018.ll.191
This document is a draft for review purposes only and does not constitute Agency policy.
R-ll	DRAFT-DO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Toxicological Review ofPFHxA and Related Salts
Wang. X: Halsall. C: Codling. G: Xie. Z: Xu. B: Zhao. Z: Xue. Y: Ebinghaus. R: Tones. KC. (2014).
Accumulation of perfluoroalkyl compounds in tibetan mountain snow: temporal patterns
from 1980 to 2010. Environ Sci Technol 48: 173-181.
http://dx.doi.org/10.1021/es4044775
Weaver. YM: Ehresman. Dl: Butenhoff. 1L: Hagenbuch. B. (2010). Roles of rat renal organic anion
transporters in transporting perfluorinated carboxylates with different chain lengths.
Toxicol Sci 113: 305-314. http: / /dx. doi. o r g/10.10 9 3/toxsci/kfp275
Whalan. IE. (2015). A toxicologist's guide to clinical pathology in animals: Hematology, clinical
chemistry, urinalysis. Switzerland: Springer International Publishing.
http: //dx.doi.org/10.1007/978-3-319-15853-2
Wiesel. TN. (1982). The postnatal development of the visual cortex and the influence of
environment [Review], Biosci Rep 2: 351-377. http://dx.doi.org/10.1007/bf01119299
Wolf. CI: Rider. CV: Lau. C: Abbott. BP. (2014). Evaluating the additivity of perfluoroalkyl acids in
binary combinations on peroxisome proliferator-activated receptor-a activation. Toxicology
316: 43-54. http://dx.doi.org/10.1016/i.tox.2013.12.002
Wolf. CI: Takacs. ML: Schmid. IE: Lau. C: Abbott. BP. (2008). Activation of mouse and human
peroxisome proliferator-activated receptor alpha by perfluoroalkyl acids of different
functional groups and chain lengths. Toxicol Sci 106: 162-171.
http ://dx.doi. or g/10.10 9 3/toxsci/kfnl 6 6
Xiao. L: TianOing. Z: Wei. L: XiaoNa. L: Xin. Z: YouSheng. 1: Tian. Z: YiHe. 1. (2011). Serum levels of
perfluorinated compounds in the general population in Shenzhen, China. Chin Sci Bull 56:
3092-3099. http:/ /dx.doi.org/10.1007/sl 1434-011 -4616-7
Yang. CH: Glover. KP: Han. X. (2009). Organic anion transporting polypeptide (Oatp) lal-mediated
perfluorooctanoate transport and evidence for a renal reabsorption mechanism of Oatplal
in renal elimination of perfluorocarboxylates in rats. Toxicol Lett 190: 163-171.
http://dx.doi.Org/10.1016/j.toxlet.2009.07.011
Yang. CH: Glover. KP: Han. X. (2010). Characterization of cellular uptake of perfluorooctanoate via
organic anion-transporting polypeptide 1A2, organic anion transporter 4, and urate
transporter 1 for their potential roles in mediating human renal reabsorption of
perfluorocarboxylates. Toxicol Sci 117: 294-302. http://dx.doi.org/10.1093/toxsci/kfq219
Yoo. H: Washington. TW: Tenkins. TM: Ellington. 11. (2011). Quantitative determination of
perfluorochemicals and fluorotelomer alcohols in plants from biosolid-amended fields using
LC/MS/MS and GC/MS. Environ Sci Technol 45: 7985-7990.
http://dx.doi.org/10.1021/esl02972m
Zeng. XW: Oian. Z: Emo. B: Vaughn. M: Bao. 1: Oin. XP: Zhu. Y: Li. 1: Lee. YL: Pong. GH. (2015).
Association of polyfluoroalkyl chemical exposure with serum lipids in children. Sci Total
Environ 512-513: 364-370. http://dx.doi.Org/10.1016/i.scitotenv.2015.01.042
Zhang. T: Sun. H: Lin. Y: Oin. X: Zhang. Y: Geng. X: Kannan. K. (2013a). Pistribution of poly- and
perfluoroalkyl substances in matched samples from pregnant women and carbon chain
length related maternal transfer. Environ Sci Technol 47: 7974-7981.
http://dx.doi.org/10.1021/es400937y
This document is a draft for review purposes only and does not constitute Agency policy.
R-12	PRAFT-PO NOT CITE OR QUOTE

-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Toxicological Review ofPFHxA and Related Salts
Zhang. T: Zhang. B: Bai. X: Yao. Y: Wang. L: Shu. Y: Kannan. K: Huang. X: Sun. H. (2019). Health Status
of Elderly People Living Near E-Waste Recycling Sites: Association of E-Waste Dismantling
Activities with Legacy Perfluoroalkyl Substances (PFASs). Environ Sci Technol Lett 6: 133-
140. http://dx.doi.org/10.1021/acs.estlett.9b00085
Zhang. Y: Beesoon. S: Zhu. L: Martin. TW. (2013b). Biomonitoring of perfluoroalkyl acids in human
urine and estimates of biological half-life. Environ Sci Technol 47: 10619-10627.
http://dx.doi.org/10.1021/es401905e
Zhou. 0: Deng. S: Yu. 0: Zhang. 0: Yu. G: Huang. 1: He. H. (2010). Sorption of perfluorooctane
sulfonate on organo-montmorillonites. Chemosphere 78: 688-694.
http://dx.doi.Org/10.1016/i.chemosphere.2009.12.005
Zhou. Y: Hu. LW: Oian. ZM: Chang. 11: King. C: Paul. G: Lin. S: Chen. PC: Lee. YL: Dong. GH. (2016).
Association of perfluoroalkyl substances exposure with reproductive hormone levels in
adolescents: By sex status. Environ Int 94: 189-195.
http ://dx. doi. or g/10.1016/i. envint 2016.05.018
Zhou. Y: Hu. LW: Oian. ZM: Geiger. SD: Parrish. KL: Dharmage. SC: Campbell. B: Roponen. M: Talava.
P: Hirvonen. MR: Heinrich. 1: Zeng. XW: Yang. BY: Oin. XD: Lee. YL: Dong. GH. (2017).
Interaction effects of polyfluoroalkyl substances and sex steroid hormones on asthma
among children. Sci Rep 7: 899. http: //dx.doi.org/10.1038/s41598-017-01140-5
This document is a draft for review purposes only and does not constitute Agency policy.
R-13	DRAFT-DO NOT CITE OR QUOTE

-------