DRAFT External Peer Review Charge Questions for the
Draft IRIS Toxicological Review of Perfluorohexanesulfonic Acid (PFHxS)

and Related Salts

July 2023

Introduction

The U.S. Environmental Protection Agency (EPA) is seeking a scientific peer review of the draft IRIS
Toxicological Review of Perfluorohexanesulfonic Acid (PFHxS) and Related Salts developed in support
of the Agency's online database, the Integrated Risk Information System (IRIS). IRIS is prepared
and maintained by EPA's Center for Public Health and Environmental Assessment within the Office
of Research and Development IRIS assessments contain information about chemicals that
encompasses hazard identification and dose-response assessment, two of the four steps in the
human health risk assessment process. When used by risk managers in combination with
information on human exposure and other considerations, IRIS assessments support the Agency's
regulatory activities and decisions to protect public health.

There is no existing IRIS assessment for PFHxS. The draft Toxicological Review of PFHxS is based
on a comprehensive review of the available scientific literature on the potential for noncancer and
cancer health effects in humans exposed to PFHxS or salts of PFHxS. The systematic review
protocol for PFHxS and other appendices for toxicokinetic information, dose-response modeling,
and other supporting materials are provided as Supplemental Information (see Appendices A to G)
to the draft Toxicological Review.

REVIEW MATERIALS PROVIDED

•	Draft PFHxS Toxicological Assessment

•	Supplemental Information (PFHxS Appendices)

CHARGE QUESTIONS

In response to the numbered charge questions below organized by topic area (italicized headers),
the advice provided as part of this peer review would be most useful when prioritized to indicate its
relative importance as follows:

•	Tier 1: Necessary Revisions - Use this category for any revisions you believe are necessary
to adequately support and substantiate the analyses or scientific basis for the assessment
conclusions.

•	Tier 2: Suggested Revisions - Use this category for any revisions you encourage EPA to
implement to strengthen the analyses or scientific basis for the assessment conclusions, or
to improve the clarity of the presentation in the PFHxS Toxicological Review.

•	Tier 3: Future Considerations - Use this category for any advice you have for scientific
exploration that might inform future work. While these recommendations are generally
outside the immediate scope or needs of the PFHxS Toxicological Review, they could inform
future reviews or research efforts.

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Literature Search Methods and Documentation

1.	The Toxicological Review for PFHxS describes and applies a systematic review protocol for
identifying and screening pertinent studies. The protocol is described in brief detail in
Section 1.2.1 (Literature Searching and Screening) and in full detail in Appendix A (Systematic
Review Protocol for the PFAS IRIS Assessments). Please:

a.	Comment on whether the literature search strategy and screening criteria for PFHxS are
appropriate and clearly described.

b.	Identify additional peer-reviewed studies of PFHxS that EPA should consider
incorporating prior to finalizing the assessment.

c.	EPA fully synthesized the literature published through April 2022 in the PFHxS
external review draft. EPA also screened studies published through April 2023, but
only incorporated into the external review draft those studies interpreted to have a
material impact on the draft conclusions (i.e., changing which hazards are identified
or notably affecting the RfDs) or directly informing the identified key science issues.
These decisions are documented in a tabular format in Appendix B.3 (Table B-5).
Specifically, EPA identified five pharmacokinetic studies from the April 2023
literature search update that inform an identified key science issue and thus
incorporated these studies into the PFHxS PK analysis in the PFHxS external review
draft No other supplemental studies from the 2023 search were interpreted to
warrant incorporation. EPA also characterized the epidemiological studies meeting
the PECO criteria from the April 2023 literature search and determined that none of
these epidemiological studies published since April 2022 would have a material
impact on the draft conclusions. No animal studies meeting the PECO criteria were
identified in the April 2023 literature search review. Please review EPA's
characterization and provide tiered recommendations regarding which additional
studies, if any, would have a material impact on the draft's conclusions and should be
incorporated into the assessment before finalizing, as well as your interpretation of
the impact of those studies to be incorporated.

Noncancer Hazard Identification

2.	For each health effect considered in the assessment and outlined below, please comment on
whether the available data have been clearly and appropriately synthesized to describe the
strengths and limitations, including whether the presentation and analysis of study results are
clear, appropriate, and effective to allow for scientifically supported syntheses of the findings
across sets of studies. Please comment on whether the study confidence conclusions for the
PFHxS studies are scientifically justified, giving appropriate consideration to important
methodological features of the assessed outcomes1. Please specify any study confidence
conclusions that are not justified and explain any alternative study evaluation decisions. For
each, please also comment on whether the weight-of-evidence decisions for hazard

1 The Toxicological Review provides an overview of individual study evaluations within each evidence synthesis section, and the
results of those outcome-specific evaluations are made available in the Health Assessment Workplace Collaborative linked here
HAWC. Note that a "HAWC FAQ for assessment readers" document, linked here (scroll to the bottom of the page, and the
document is available for download under "attachments"), is intended to help the reviewer navigate this on-line resource.

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identification have been clearly described and scientifically justified. Note that the data from
studies considered informative to the assessment are synthesized in the relevant health effect-
specific sections and available in the Health Assessment Workspace Collaborative (HAWC).

a.	For immune effects, the Toxicological Review concludes the available evidence
indicates PFHxS exposure is likely to cause immunosuppression in humans given
sufficient exposure conditions, primarily on the basis of consistent evidence of reduced
antibody responses from epidemiological studies in children and adults. For nearly all
epidemiology studies of PFHxS, there is potential that exposure to other highly
correlated PFAS could contribute to the observed effects. Thus, the synthesis of
epidemiology studies on immune effects included an evaluation of the potential for
confounding across PFAS as well as other sources of confounding. After considering
these factors on the basis of the available data, it was determined that there was
minimal concern for substantial confounding, and it was unlikely to fully explain the
associations seen in the literature. Although immunotoxicity-specific animal studies
were not identified, general toxicity or developmental toxicity studies that included
immune-related endpoints (i.e., basophil cell counts) were identified. However, these
endpoints in animals were nonspecific and not informative to the immune effects
hazard judgment

b.	For thyroid effects, the Toxicological Review concludes the available evidence indicates
PFHxS exposure is likely to cause thyroid effects in humans given sufficient exposure
conditions, on the basis of a series of short-term studies in rats demonstrating
consistent and coherent effects with a clear biological gradient The thyroid findings for
PFHxS were similar to those observed for other structurally related long-chain PFAS
and determined to be adverse and relevant to humans.

c.	For developmental effects, the Toxicological Review concludes the available evidence
suggests but is not sufficient to infer whether PFHxS exposure has the potential to
cause developmental effects in humans given sufficient exposure conditions. This
judgement is based primarily on the consistent but notably uncertain evidence of
decreased birth weight and length from studies of exposed humans in which PFHxS was
measured during or shortly after pregnancy. However, the strength of the epidemiology
evidence is reduced due to concern for confounding due to sample timing (pregnancy
hemodynamics). Evidence in experimental animals from three high confidence studies
in rats and mice was indeterminate.

d.	For hepatic effects, the Toxicological Review concludes the available evidence suggests
but is not sufficient to infer whether PFHxS exposure has the potential to cause liver
effects in humans given sufficient exposure conditions. This conclusion is based on
slight evidence of an association between PFHxS exposure and small changes in serum
markers of liver disease in humans that was inconsistent across studies, and evidence
from experimental animal studies in rats and mice that was also considered slight.

e.	For neurodevelopmental effects, the Toxicological Review concludes the available
evidence suggests but is not sufficient to infer whether PFHxS exposure has the
potential to cause neurodevelopmental effects in humans given sufficient exposure
conditions. This conclusion is based on slight epidemiological evidence of some positive

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associations between PFHxS exposure and ADHD or behaviors potentially related to
ADHD. Evidence from experimental animal studies was indeterminate.

f.	For cardiometabolic effects, the Toxicological Review concludes the available evidence
suggests but is not sufficient to infer whether PFHxS exposure has the potential to
cause cardiometabolic effects in humans given sufficient exposure conditions. This
conclusion is based primarily on consistent increases in cholesterol in humans.
However, limitations in the available epidemiological studies introduced significant
uncertainty. Evidence from experimental animal studies was indeterminate.

g.	For hematopoietic effects, male and female reproductive effects, and renal effects the
Toxicological Review concludes there is inadequate evidence to determine whether
PFHxS exposure has the potential to cause these effects in humans on the basis of the
sparsity of the available evidence.

Noncancer Toxicity Value Data Selection and Modeling

3. For PFHxS, no RfC was derived for inhalation exposure. Organ/system-specific RfDs were

derived for immune and thyroid effects and considered for use in deriving the oral RfD. The RfD
was based on immune effects observed in humans. The study chosen for use in deriving the
immune osRfD was Budtz-Jorgensen and Grandjean (2018) and Grandjean et al. (2012), which
reported decreased serum anti-tetanus antibody concentrations in children (male and female)
at age seven years and PFHxS measured at age five years. Are the selection of the studies for the
immune effects (Butz-J0rgensen and Grandjean, 2018; and Grandjean, 2012) for use in deriving
the RfD for PFHxS scientifically justified? Are the modeling approaches appropriate?

a.	If so, please provide an explanation.

b.	If not, please provide an alternative study(ies) or effect(s) that should be used to
support the derivation of the lifetime RfD and detail the rationale for use of such an
alternative.

c.	As part of the recommendations in "a" or "b" above, please comment on whether the
effects selected are appropriate for use in deriving the lifetime RfD, including
considerations regarding adversity (or appropriateness in representing an adverse
change) and the scientific support for their selection2. Please also see charge questions
2b and 2c.

d.	EPA used benchmark dose modeling (BMD) (U.S. EPA, 2012) to identify points-of-
departure (PODs) for PFHxS. Are the BMD modeling approaches, selection, and
justification of benchmark response levels, and selection of the BMD models used to
identify each POD for toxicity value derivation scientifically justified and clearly
described?

2 For the decreased antibody responses, Selgrade (Tox Sci 2007;100:328-332] suggests that these specific
immunotoxic effects may be broadly indicative of developmental immunosuppression impacting these children's
ability to protect against a range of immune hazards.

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e. Given the lack of studies on inhalation exposure to PFHxS, no reference concentration
(RfC) is derived. Please comment on this decision.

4.	In addition, for PFHxS, an RfD for less-than-lifetime ("subchronic") exposures is derived. No
"subchronic" RfC is derived. The same studies, outcomes, and comparisons were chosen for use
in deriving the lifetime and subchronic RfD. Are the selection of these studies and these effects
for the derivation of the subchronic RfD for PFHxS scientifically justified?

a.	If so, please provide an explanation.

b.	If not, please provide an alternative study(ies) or effect(s) that should be used to
support the derivation of the subchronic RfD and detail the rationale for use of such an
alternative.

c.	As part of the recommendations in "a" or "b" above, please comment on whether the
effects selected are appropriate for use in deriving the subchronic RfD, including
considerations regarding adversity (or appropriateness in representing an adverse
change) and the scientific support for their selection.

d.	Given the lack of studies on inhalation exposure to PFHxS, no "subchronic" RfC is
derived. Please comment on this decision.

Nonccmcer Toxicity Value Pharmacokinetic Extrapolation and Uncertainty Factors

5.	Appendix E describes a Bayesian pharmacokinetic (PK) analysis of the available data for mice,
rats, and monkeys, in order to obtain key PK parameters with rigorous confidence ranges.
Appendix E also describes the application of the resulting parameters in a one-compartment PK
model for rats to evaluate its potential for use in extrapolating PFHxS toxicity data to humans.
Section 3.1 evaluates and synthesizes the PK data in relevant species and sexes, and among
human lifestages, up to the derivation of key PK parameters used in the subsequent analysis.
However, the evaluation of existing PBPK models and the one-compartment PK model found
that these options were not sufficiently reliable for use. Given the information available on
potential interspecies differences in PFHxS PK, EPA applied a data-derived extrapolation factor
(DDEF) to POD values from toxicity studies in laboratory animals to estimate corresponding
human equivalent doses (HEDs) in the derivation of the respective RfDs. Similarly, estimated
human clearance (CL) values were used to convert internal dose POD (PODint) values from
epidemiological analyses to corresponding HEDs (i.e., human dose levels that are equivalent to
the identified PODint values). In selecting the CL values, EPA evaluated published data for
PFHxS relevant to PFHxS dosimetry in women of childbearing age (see question 5c below),
including during pregnancy and lactation.

a.	Is the decision and method for applying DDEFs for rat-human extrapolation
scientifically justified, given the available science? If not, please provide an explanation
and detail on a more appropriate approach.

b.	Is use of the human sex- and lifestage-specific clearance values to estimate HEDs from
internal dose PODs in humans scientifically justified? If not, please provide an
explanation and detail on a more appropriate approach.

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c. Have the uncertainties in the DDEFs and human CL been adequately evaluated and
described? In answering this question, please explicitly consider EPA's approach to
adjusting for menstrual fluid loss in the draft assessment

6.	EPA has evaluated and applied where appropriate uncertainty factors to account for
intraspecies variability (UFh), interspecies differences (UFa), database limitations (UFd),
duration (UFs), and LOAEL-to-NOAEL extrapolation (UFl) for PFHxS.

a.	Is uncertainty in the derivation of the toxicity values scientifically justified and clearly
described given that the assessment evaluates and considers the available evidence on
potential susceptibility to PFHxS within different populations or lifestages, including
any potential impacts from early life exposure to PFHxS on children's health or health
effects later in life? If not, please explain. Please describe and provide comments, if
needed.

b.	Please specifically comment on whether use of the intra-human uncertainty factor, UFh
= 10, in combination with the selected sex- and lifestage-specific human CL values,
appropriately accounts for both the pharmacokinetic and pharmacodynamic
uncertainties and differences (variability) among humans?

c.	For immune effects, a UFs of 1 was selected. A UFs of 10 was not considered as the
developmental period is recognized as a susceptible lifestage for these types of effects
and therefore exposure during this time window can be considered more relevant than
exposure in adulthood (U.S. EPA, 1991). Also important is the fact that, given PFHXS
long half-life and the expectation that the children and their mothers have been exposed
to elevated levels of PFHxS for many years, the observed effects on immune response
are considered to be the result of a cumulative, prolonged exposure. Uncertainties with
regards to additional susceptible life stages (e.g., old age) are addressed as part of the
UFd. Does the provided scientific rationale support this decision? If not, please explain.

Carcinogenicity Hazard Identification and Toxicity Value Derivation

7.	The Toxicological Review concludes there is inadequate information to assess carcinogenic
potential for PFHxS and that this descriptor applies to all routes of human exposure. Please
comment on whether the available human, animal and mechanistic studies, and the analysis
presented in the Toxicological Review are scientifically justified and clearly described.

8.	Given the conclusion there is inadequate information to assess carcinogenic potential for PFHxS,
the Toxicological Review does not derive quantitative estimates for cancer effects for oral or
inhalation exposures. Is this decision scientifically justified and clearly described?

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