Peer Reviewers:

James V. Bruckner, Ph.D.

Deborah A. Cory

- Slechta, Ph.D.

Jamie C. DeWitt, Ph.D. Jeffrey W. Fisher, Ph.D.

William L. Hayton, Ph.D.

Matthew P. Longnecker, Sc.D, M.D Angela L. Slitt, Ph.D.

Contract No. EP

- C

- 13

- 010

Task Order 2013

- 06 

# Peer Review Summary Report

External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS )

N ovember 17 , 2014

Prepared for: U.S. Environmental Protection Agency

Of fice of Science and Technology/Office of Water

1 200 Pennsylvania Aven ue, NW Washington, DC 20460 Prepared by:
Versar, Inc.

6850 Versar Center Springfield, VA 22151External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

i TABLE OF CONTENTS



### Contents

EXECUTIVE SUMMARY

................................ ................................ ................................ ii

I. INTRODUCTION

................................ ................................ ................................ ....... 1

I.1 Background on Draft Health Effects Documents

................................ .................. 1

I.2 Peer Review Process

................................ ................................ .............................. 1

I.3 Peer Review Meeting

................................ ................................ ............................. 3

II. CHARGE TO REVIEWERS

................................ ................................ ...................... 5

III. GENERAL IMPRESSIONS

................................ ................................ ....................... 7

IV. RESPONSE TO CHARGE QUESTIONS

................................ ................................ 17

Charge Questions

................................ ................................ ................................ ...... 17

Question 1.

................................ ................................ ................................ ........ 17

Question 2.

................................ ................................ ................................ ........ 22

Question 3.

................................ ................................ ................................ ........ 27

Question 5.

................................ ................................ ................................ ........ 34

Question 6.

................................ ................................ ................................ ........ 36

Question 7.

................................ ................................ ................................ ........ 39

Question 8.

................................ ................................ ................................ ........ 43

Question 9.

................................ ................................ ................................ ........ 47

Question 10.

................................ ................................ ................................ ...... 50

Question 11.

................................ ................................ ................................ ...... 53

Question 12

................................ ................................ ................................ ....... 55

V. SPECIFIC OBSERVATIONS

................................ ................................ ..................... 58

APPENDIX A: PEER REVIEWER BIOSKETCHES

................................ ................... A

- 1

APPENDIX B: MEETING AGENDA

................................ ................................ ........... B

- 1

APPENDIX C: MEETING ATTENDEE LIST

................................ .............................. C

- 1External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

ii EXECUTIVE SUMMARY

Versar , Inc. (Versar)

, a contractor for the Environmental Protection Agency (EPA)

, coordinated an external peer review of EPA’s draft health effects documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS), an d organized a two

- day public peer review meeting in Arlington, Virginia on August 21 and 22, 2014 . The peer review of EPA’s draft

health effects documents was initiated with a pre

- meeting written peer review manag ed by Versar and conducted by seven independent expert peer reviewers. The role of the peer

reviewers was to evaluate the scientific and technical merit of the d raft documents and provide their responses to 12 charge questions . Peer reviewers were charged only with evaluating the quality of the science included in EPA’s draft health effects documents a nd were not charged with making any regulatory recommendations or reaching consensus in either their deliberations

or writte n comments. The two

- day peer review meeting, which directly followed the written peer review period, was held to discuss the scientific basis supporting EPA’s draft health effects

documents and t o provide members of the public with an opportunity to observe the peer

reviewer deliberation s

.

On the first day of the meeting , Versar began by providing i nformation on the overall peer review process and introducing the p eer reviewers . In addition, EPA provided background information on the draft documents and approach used in the development of the documents. Following opening remarks by Versar and EPA, the peer reviewers began their discussions on

the PFOA draft document, moderated by the Chair, Dr. Deborah Cory

- Slechta. The discussions centered on individual responses to EPA’s 12 charge questions. Additionally, some comments submitted to the public docket prior to the meeting were also discussed. Th e second day of the meeting began with brief remarks from Versar followed by discussions focusing on the PFOS

draft document.

T he reviewers began the discussion on the first day with the recognition of the significant

amount of data that is available for both PFOA and PFOS and thus to be considered by the EPA for incorporat ion into the document and for the ultimate derivation of reference d ose (RfD) values. In general, the reviewers commended EPA in doing a very good job of pulling together this signific ant and extensive body of information and of condensing it into its most critical pieces for the derivation of RfD values. This was especially the case given that there is significant human and animal data available for both chemicals, as well as inconsist encies in the

data.

The reviewers did offer numerous suggestions for improving the documents, many of which are applicable to both PFOA and PFOS. In general, the suggestions relate to the statement of the problem and defining the database that was utili zed, clarity and ease of presentation, and transparency of the reports

, as well as to issues of modeling, use of human data

, and liver weight increases as the most sensitive endpoint. These comments are summarized below

, but p lease

note that this does not reflect a consensus or group perspecti ve.

 All reviewers agreed that it would be extremely useful to include an opening section of the document describing in some detail the literature that was reviewed. More specifically, this would include a description of the dates that were included in theExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

iii lite rature review, whether requirements for peer

- reviewed materials were imposed, and what criteria were used in eliminating studies from consideration. This would assist in identifying studies that should potentially have been included. Many additional studie s of importance were suggested for review at the meeting by the peer reviewers.

 T he reviewers also agreed that there are some significant studies that have been published recently on PFOA and PFOS that may be relevant, and these should be

included in the documents during the current revisions.

 Reviewers also felt that the Tables in the documents could be made much more useful to readers with the addition of columns that included more study details. This would

minimize the amount of back

- and

- forth to the te xt that was required to assess

conclusions.

 Additionally, there was a strong agreement that the Hazard Identification section of the

document s should include a more systematic review of study strengths and limitations. These were noticeably absent in the h uman studies, where all studies were considered equivalent but also applicable to animal studies.

 Reviewers also felt that each Chapter should include introductory paragraphs

, as well as concluding paragraphs, that would provide better integration of the m aterial across the Chapters , as well as summarize the conclusions arrived at from the text in the Chapter.

This would also facilitate the ability of readers to follow the presentations provided in Chapter 5 of the derivation of RfDs.

 As pointed out at the peer review meeting, the authors would be well advised to base these documents on the new documents being produced by EPA’s National Center for Environmental Assessment (NCEA) for the Integrated Risk Information System ( IRIS ) program. While the reviewers r ecognize that the PFOA and PFOA health effect documents will not be used specifically for th e same purpose, they will still likely be

held to the same standards of presentation, clarity and transparency and thus

, the new NCEA formats provide good models to follow.

 With respect to content of the documents, there was initial confusion around some of the modeling outcomes and assumptions based on values that were provided in the

documents. However

, following explanation from EPA personnel in response to

clarif ying questions from the reviewers at the peer review meeting

, it became clear that there were errors in these values and once these were corrected, concerns about the specific values used in some modeling were allayed.

 Even with the corrections to the mod eling, there were residual concerns among reviewers based on toxicokinetic properties of these chemicals, with respect to applying the same candidate RfD values to both short and long

- term exposures

, which in animal studies ranged from 11 to 182 days with reviewers expressing a need for this to be re

considered.

 There were mixed comments by reviewers with respect to the decision not to use human data in the derivation of RfD values. All reviewers generally agreed that the rationales provided for the exclusi on of the human data were not actually appropriate, as it is certainly not the case that such use would be precluded by the fact that there wereExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

iv multiple and not sole exposures to either PFOA or PFOS. There was discussion as to the possibility of whether h uman data could be utilized in these assessments, and that ranged from an opinion that these data would not be useful to the view that it might be possible to use these data, despite the absence of information on route of exposure. Collectively,

th e range of opinion s suggest s that the issue should be revisited

, and that appropriate

rationales should be provided for the decision that is reached in the revised version

.

 For both PFOA and PFOS, the RfDs were ultimately based on increases in liver weights in animal studies. There was significant discussion among reviewers as to the appropriateness of this endpoint for the derivation of the RfDs. That range of opinion

spann ed from an interpretation that these did constitute adverse effects in that they are a direct effect of a chemical exposure, whereas other reviewers saw these as adaptive

effects. Collectively, the range of opinions suggests that the issue should be revisi ted, and that appropriate rationales should be provided for the decision that is reached in the

revised version.

Following the meeting, peer reviewers were given additional time to complete their individual written reviews, which were submitted to Versar upon completion. These final written

comments are contained in Section s III

, IV , V of this report and fall into three categories:

gen eral impressions, responses to charge q uestions, and specific observations. Written peer review comments, as well as comme nts submitted to the docket by members of the public, will be considered by EPA as it revise s the draft documents.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

1 I. INTRODUCTION

I.1 Background on Draft Health Effects Documents

On February 28, 2014, the EPA’s Office of Water (OW) a nnounced in the Federal Register the

release of the draft health effects documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS ) for purposes of public comment (sc ientific views) and peer

review ( http://www.gpo.gov/fdsys/pkg/FR

- 2014

- 02

- 28/pdf/2014

- 04455.pdf )

. The draft

documents and charge questions were prepared by the Health and Ecological Criteria Division (HECD) , within EPA ’s OW

, in order to support future regulatory evaluations and decisions

. EPA will consider the public comments and peer reviewer comments when revising the

documents. Once the PFOA and PFOS health effects documents are finalized, they will be

utilized t o develop lifetime health advisory values for each chemical. P FOA and PFOS are listed

on the t hir d C ontaminant Candidate List (CCL3) 1 and both chemicals are currently being monitored under the third Unregulated Contaminant Monitoring Rule (UCMR3) 2

.

I.2 Peer Review Process

Consistent with guidelines for the peer review of highly influential scientific assessments,

Versar , an EPA contractor, was tasked with assembling six to seven scientific experts to

evaluate the draft PFOA and PFOS documents. The purpose of the peer review was to provide a documented, independent, and critical review of the draft health effects documents, and identify any necessary improvements to the documents pr ior to being published . In assembling these peer reviewers and coordi nating the peer review, Versar was charged with evaluating the qualifications of peer review candidates, conducting a thorough conflict of interest (COI) screening proce ss, independently selecting the peer reviewers, distributing review materials,

maintain ing contact with the peer reviewers

, organizing and hosting the public peer review meeting

, and developing a final peer review report.

The peer review selection process was initiated with a three

- week public nomination period that was held from February 2 8 , 2014 to March 21, 2014 , as documented in the Federal Register on February 28, 2014 ( http://www.gpo.gov/fdsys/pkg/FR

- 2014

- 02

- 28/pdf/2014

- 04455.pdf ) . D uring this period, members of the public were able to nominate scientific experts with knowledge and experience in one or more of the following areas: (1) epidemiology, (2) toxicology (liver effects, immunotoxicity, neurotoxicity, developmental and reproductive toxicology, etc.), (3) membrane transport, (4) human health risk assessment, (5) pharmacokinetic models, and (6) mode

- of

action for cancer and noncancer effects. Concurrently, Versar conducted an independent search

for qualified scientific experts to augment the list of publically

- nominated candidates. In total,

Versar evaluated 29 interested and available candidates who were either nominated by the public (n=18) or identified by Versar (n=11).

1 CCL3 is a list of contaminants that are currently not subject to any proposed or promulgated national primary drinking water regulations, that are known or anticipated to occur in public water systems, and which may require regulation under the Safe Drinking Water Act (SDWA). Additional information about the CCL3 can be found at the following website: http://water.epa.gov/scitech/drinkingwater/dws/ccl/ccl3.cfm

. 2 EPA uses the Unregulated Contaminant Monitoring (UCM) program to collect data for unregulated contaminants suspected to be present in drinking water. Results from UCMR3 can be examined as they become available at the following website: http://wat er.epa.gov/lawsregs/rulesregs/sdwa/ucmr/ucmr3/

.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

2 Versar considered and screened all 29 candidates against the selection cr iteria described in the Federal Register dated February 2 8 , 2014, which included : (1) having demonstrated expertise in

the areas described above, based on information in their submitted resume, biographical sketch and/or current publications

, (2) being fre e of any COI and the appearance of the lack of impartiality , and (3) being available to participate in

- person in a two

- day peer r eview meeting in the Washington DC area in the July or August 2014 timeframe . Following the screening process,

Versar narrowed the list of poten tial reviewers to 15 candidates and provided to EPA the names

of the candidates selected by Versar to be on the interim list. Additionally, information on the 15 candidates, including their professional affiliations, expertise, education, and professional experience were provided for the interim lis t and published in the Federal Register on April 30, 2014 ( http://www.gpo.gov/fdsys/pkg/FR

- 2014

- 04

- 30/pdf/2014

- 09888. pdf ). The Federal Register also requested the public to submit relevant information or documentation on the

interim list of candidates that Versar should c onsider during the evaluat ion process of selecting

the final six to seven reviewers

.

Following the close of the public comment period on the interim list of potential reviewers,

Versar re

- evaluated each interim candidate’s credentials to select the experts who , collectively, provide d expertise spanning the multiple subject matter areas covered in the draft documents

and provide d a balance of perspectives. In addition , Versar evaluated the availability of each

candidate to ensure all final peer reviewers were available on the same days for the meeting in

the selected timeframe. Once the evaluation process was completed, Versar narrowed the

interim list of 15 candidates and selected the seven final peer reviewers. In addition, Versar

selected Dr. Deborah Cory

- Slechta as Chair of the peer review meeting due to her expertise in toxicology as well as he r strong record of chairing and participating in peer review panels, scientific meetings

, and workshops. A list of the final seven peer reviewers who participated in this review is provided below . In addition, each reviewer’s biographical sketch is include d in Appendix A

.

Following the selection process, Versar distributed EPA’s draft PFOA and PFOS documents and 12 charge questions (see Section II) to the peer reviewers

. The peer reviewers were asked to evaluate the scientific and technical merit of the draft documents and provide their responses to the 12 charge questions

. This included evaluating the appropriateness of the quality, accuracy,

and relevance of the data in the documents. Peer reviewers were not charged with making any regulatory recommendations or reaching consensus in either their written comments or public

deliberations. In addition to being provided the draft documents and charge questions, c omments
submitted to EPA’s public docket (D ocket ID number EPA

– HQ

– OW

– 2014

– 0138) during each

document’s 60

- day public comment period were provided to the peer reviewers ahead of the meeting for their consideration. Also

, a brief s ummary of the public comments was developed by Versar and provided to the reviewers. However, peer reviewers were not asked to evaluate or respond to comments submitted to the docket.

Versar managed the pre

- meeting peer review period, which provided the peer reviewers approximately two months to evaluate the draft health e ffects document and complete their written reviews. Following receipt of the peer reviewers’ draft comments, Versar compiled the comments into a pre

- meeting peer review report and distributed them to the peer reviewers andExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

3 EPA to prepare for the public peer review meeting. These preliminary responses to the charge

q uestions form ed the basis of reviewer discussions on Day s 1 and 2 of the public meeting

.

Peer Reviewers:

James V. Bruckner, Ph.D. University of Georgia

Deborah A. Cory

- S lechta, Ph.D. University of Rochester School of Medicine and Dentistry

Jamie C. DeWitt, Ph.D. East Carolina University

Jeffrey W. Fisher, Ph.D.

U.S. Food & Drug Administration

William L. Hayton, Ph.D. The Ohio State University

Matthew P. Longnecker, Sc.D, M.D

National Institute of Environmental Health Sciences

Angela L. Slitt, Ph.D. University of Rhode Island

I.3 Peer Review Meeting

On August 21 and 22, 2014 , Versar convened a public peer review meeting in Arlington, Virginia

. This meeting was he ld to discuss the scientific basis supporting EPA’s draft health

effects documents and to provide members of the public with an opportunity to observe the peer

reviewer deliberation s

. The meeting follow ed both the documents’ public comment period, during w hich members of the public were able to submit written comments , and the pre

- meeting written peer review period, during which the seven selected peer reviewers evaluated EPA’s

draft health effects documents and provided preli minary comments in response to the c harg e

q uestions

.

Versar managed the pre

- meeting registration period, which allowed members of the public to register to attend the meeting in person or remotely via teleconference and/or webinar. Members of the public were able to register online, v ia Versar’s registration website ( http://peerreview.versar.com/epa/pfoa/ ) as well as by telephone, email, or U.S. mail. In advance

of the meeting, Versar provided all registered attendees with pre

- meet ing handouts , which included the agenda and logistics information

.

On the first day of the meeting, Versar began by providing information on the overall peer review process and introducing the peer reviewers. In addition, EPA provided backgroundExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

4 informat ion on the draft documents and approach used in the development of the documents. Following opening remarks by Versar and EPA, the peer reviewers began their discussions on the PFOA draft document, which centered on individual responses to EPA’s 12 charge questions. The remainder of the day was dedicated to discussions on the PFOA document, which were moderated by the Chair, Dr. Deborah Cory

- Slechta. The second day of the meeting began with brief remarks from Versar followed by discussions focusing on the P FOS draft document. Approximately 2 3 public observers attended the peer review meeting in person and 21 observers attended the meeting via teleconference and/or webinar . Please see Appendix B for the meeting agenda and Appendix C for a list of public attendees.

Following the public peer review meeting, peer reviewers were given additional time to complete their individual written reviews. These final written comments are contained in

Section s III , I V , and V of this report. Written peer revi ew comments, as well as comments submitted to the EPA docket by members of the public, will be considered by EPA as it revises

the draft health effects documents.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

5 II. CHARGE TO REVIEWERS

PFO A and PFOS are environmentally persistent organic f luorocarbons (PFC) that have been identified in ambient waters, ground water, drinking water, and biosolids. They are metabolically inert but have the ability to bind to and interact with a variety of biomolecules leading to responses in living organisms. Both compounds have a substantial database of epidemiological, pharmacokinetic, toxicological and mechanistic studies. The two documents submitted for peer review include health assessment chapters that will be used 1) to provide information to drinking wa ter treatment plant operators regarding the significance of monitoring results with respect to potential health outcomes and 2) to determine whether the perfluorinated compounds currently being monitored at Public Drinking Water Systems require regulation. The health information at that time will be accompanied with chapters on environmental fate, occurrence at public drinking water systems and occurrence in other media. The quantitative aspects of the Health Assessment documents will also be used to develo p lifetime Health Advisories for both compounds.

Charge Questions

1. Please comment on the strengths, weaknesses, and characterization of the studies selected as key for quantification.

2. Please provide citations (and, where possible, pdfs or hard copies) for any references you suggest EPA consider adding to the document. Describe where you suggest th ese references be incorporated.

3. The OW concluded that the human epidemiology data for PFOS/PFOA do not provide adequate quantifiable dose

- response information for use as the basis of a candidate RfD because of uncertainty regarding the routes, levels and timing of exposures plus the confounding influences of other PFCs present in serum. Please comment of the OW

characterization of the data.

4. Please comment on the transparency and characterization of the epidemiological data.

5. The OW has concluded that the cancer classifications for PFOA and PFOS are most consistent with respective classifications of suggestive evidence for carcinogenicity as

described the EPA Guidelines for Carcinogen Risk Assessment (pp. 2

- 56, 2

- 57). Please comment on the strengths and weaknesses of this classification.

6. Significant interspecies differences in pharmacokinetics exist for both PFOA and PFOS. Adjusting for interspecies differenc es was an important step in developing candidate RfDs given the totality of the human and animal data. Please comment on the strengths and weaknesses of the pharmacokinetic model adjustments to accommodate the impact of albumin binding and renal tubule tra nsporters in determining average serum values.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

6 7. Table 5

- 5 in the PFOA document and Table 5

- 7 in PFOS document list the parameters used for the ORD pharmacokinetic models that provide the final serum and AUC values for calculating the internal dose point of departure for the RfD calculation. Please comment on the strengths and weaknesses of the selected parameters.

8. The volume of distribution (Vd) and half

- life values are critical in the derivation of the interspecies uncertainty factor applied in derivation of candidate RfDs from a NOAEL, LOAEL or a BMDL. The available data for both values are provided in Section 3.5.2 and 3.5.3 of both documents. Please comment the strengths and weaknesses of the values

selected.

9. A variety of endpoints and studies were use d to compare points of departure and the resultant RfDs for both PFOA and PFOS. In addition, comparisons were provided across RfD outcomes based on the model outputs compared to those for the NOAEL, LOAEL and BMDL points of departure. The range of candidat e RfDs derived from the different points of departure is fairly narrow. Please comment on the strengths, weaknesses and transparency of this analysis.

10. The RfDs for PFOS and PFOA are derived from the modeled steady state serum

concentrations and their association with effects that include short term and longer term exposures with associated diverse effects. The studies considered included effects due to exposure durations that ranged from 11 to 182 days, and occur at comparable human equivalent dose (HE D) levels. The current, draft RfDs do not include an uncertainty factor for study duration because of the apparent concordance HEDs despite duration differences. Given this pattern of response, is it appropriate to conclude that the candidate RfDs are appl icable to both short

- term and lifetime exposures?

11. In addition to using the average serum values from animal studies to calculate internal doses for humans, the animal to human extrapolation can be accomplished by dividing animal average serum values by th e human to animal clearance ratios to project a human average serum point of departure in units of mg/L serum. Please provide recommendations for applying uncertainty factors to the extrapolated average human serum values to determine

serum

- based threshold s that are protective for humans. A NOAEL expressed in average human serum units would be useful in interpreting NHANES population monitoring data.

12. Please describe any suggestions you have for improving the clarity, organization, and/or transparency of th e draft documents.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

7 III. GENERAL IMPRESSIONS

James V. Bruckner

PFOA

- specific comments This is one of the most comprehensive Health Effects Documents I have reviewed. The clarity and accuracy of accounts of pertinent research reports/publications are ex cellent. It is obvious considerable time and efforts were devoted to its composition. If anything, the amount of detail is so great that it is difficult to distill the mass of information on each topic and capture its “essence”. This is likely the result o f directions the authors were given for writing the document. Some topics in the Hazard Identification section do have summarizing sentences, in which the key/critical studies and their finding(s) are integrated and conclusions reached. It would be very

he lpful to devote much more attention to this for more topics, perhap s as an addition to S ection

4.4 Hazard Characterization.

I do have a real problem with the scientific basis and soundness of certain conclusions in the document. The primary effect of PFOA in different species is increased absolute and/or relative liver weight. These are quite modest, reversible, non

- specific effects that usually are not considered toxicologically significant. Livers of mice and rats dosed with PFOA typically exhibited hype rtrophy characterized by increased peroxisomes, numerous mitochondria, reduced rough endoplasmic reticulum (RER), proliferation of smooth endoplasmic reticulum (SER), and increased autophagosomes or lipid

- like droplets. Such morphological changes, particul arly those in RER and SER, are manifestations of microsomal enzyme induction. This is considered adaptive, rather than adverse. Hall et al. (2012) point s out that activation of a battery of genes involved in xenobiotic metabolism and transport serve to maintain homeostasis by enhancing the systemic elimination of the foreign chemical. Although PFOA is very poorly metabolized, it does persistently induce mi crosomal enzymes and the accompanying hepatocellular morphological changes. Upregulation of genes responsible for biliary excretion may be beneficial, since excretion of bilirubin, bile acids and conjugates of toxic chemicals/metabolites

would be enhanced.

There are substantial qualitative and quantitative differences in responses of rodents and humans to PPARα activation. Therefore, many of the PFOA

- induced alterations in lipid metabolism/homeostasis and associated biological processes in mice will be abs ent or an order of magnitude less pronounced at comparable doses in humans. Many of PFOA’s effects on the liver of rodents are dependent on PPARα activation, though some effects appear to be PPARα

independent. Studies in PPARα

- knockout mice show activation of other nuclear receptors by PFOA, including PXR, CAR, LXRA and FXR. Bjork et al. (2011) observed markedly lower transcriptional responses of PPARα, PXR, CAR and FXR to PFOA in cultured human than in cultured rat hepatocytes. These more subtle effects l ead the investigators to conclude the changes in human cells reflected an adaptive metabolic remodeling rather than overt metabolic dysregulation, or disorder occurring in rat cells. The PFOA document’s authors should go into detail discussing and summariz ing the relative toxicological significance of non

- PPARα effects

in rodents versus humans.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

8 It is important to recognize that clearly adverse effects of PFOA are seen. Lovelass et al. (2008), Cui et al. (2009) and others have seen focal necrosis and degen erative changes in the liver of mice and rats given relatively high doses of PFOA, as well as modest elevations in serum (hepatic) enzyme activities. Wolf et al. (2008a) observed a variety of degenerative structural changes in the liver of PPARα

- null mice dosed with PFOA. Sakr et al. (2007a,b) and Olsen and Zobel (2007) reported associations between serum PFOA levels and slightly elevated serum enzyme activities in some occupationally

- exposed populations. The increases in enzymes may have been attributable to factors other than PFOA. In light of the foregoing, it would be preferable to utilize hepatic morphological changes in rodents and/or elevated serum enzymes as the critical effect(s), rather than increased liver weight. These are clearly adverse effect s seen in

both rodents and humans.

An internationa l panel of experts (Hall et al. , 2012) opined that an increase in liver weight of ≤ 150%, at doses of chemical that do not produce structural or biochemical evidence of hepatocellular damage, would not be considered adverse . Absolute and relative liver weights were not increased as much as 50% by most PFOA doses in the majority rodent and monkey studies. Perkins et al. (2004), for example, reported dose

- dependent increases in liver/body weight in rats f

ed 1, 10, 30 and 100 ppm PFOA for 13 weeks of 0, 10, 30, and 41%

, respectively . Butenhoff et al. (2002) measured increases of 17, 21 and 37.5% and relative liver weight in monkeys given 3, 10 or 30/20 mg PFOA/kg/day for 26 weeks, respectively. Liver hyper trophy of this magnitude does not warrant such a low RfD. By adhering to EPA policies of calculating a BMDL 10 and using multiple UFs, regardless of the (lack of) severity of the critical effect and relatively low level of concern about other potential heal th effects, the end result is a vanishingly low RfD (i.e. 0.00002 mg/kg/day). A great deal of time and effort were spent on the PFOA hazard assessment, toxicokinetic modeling and extrapolations, dose metric and POD considerations, etc. Despite all of thes e scientifically

- credible exercises and deliberations, the end result (RfD) seems to this reviewer to have been preordained

-- to be extremely low.

Logic expressed on page 5

- 6, in support of use of liver weight gain as a critical effect and biomarker of loss of hepatocellular homeostasis seems flawed. As pointed out in the second paragraph, liver weight changes were not observed in PFOA

- treated mice w ith a humanized PPARα receptor. It is noted that changes in gene products that modulate lipid metabolism do occur in these mice. EPA argues that this supports adoption of increased liver weight as a

biomarker/critical effect. It has not been established th at these changes in gene expression are

adverse , or whether they are sufficient in magnitude to significantly alter lipid metabolism. It would be expected that repeated dosing wi th enough of a molecule (i.e., PFOA) that resembles a fatty acid would affect expression of such genes. Reversible changes in total cholesterol, bile acids, bilirubin, etc. have been observed. It has not been established, however, whether mild fluctuations in these indices are detrimental. No increases in mortality from cerebrevascu lar

disease or ischemic heart disease have been found in PFOA

- exposed humans. How then does the concurrence of alteration of expression of such genes and of liver weight gain support the latter as toxicologically

- significant effect that should be prevented by setting the RfD low enough?

PFOS

- specific comments This Health Effects Document, like that for PFOA, is quite comprehensive. Its descriptions of the many studies of PFOS are clear, quite complete, and apparently quite accurate. As with theExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

9 PFOA docume nt, so much detail is given about many studies in the Hazard Identification section, that is difficult to compare study designs/dosage regimens/species/indices/findings/etc. and to draw conclusions. The summary tables for single and multiple studies, howev er, are quite helpful in this regard. It would also be very useful to have more summary statements or paragraphs at the end of each topic. These should address the scientific importance of findings, their relevance to humans; and their impact on the weight of evidence on an issue.

The hazard characterization section (4.4) is, for the most part, inclusive and balanced in its presentation and integration of findings of the more important studies in each subject area. This

is true for both non

- cancer and canc er effects in humans and animals. It concerns me, however, that the document’s authors do not focus in the remainder of the document on science (i.e., the candidate critical effects and their relevance to human health), but merely choose the most

sensitive end

- points and stress how similar the RfDs are after dosimetry modeling estimates and adjustments. I am not sure how this similarity of derived points of departure and other values, calculated from dissimilar endpoints, supports or validates the final RfD

.

I recommend that an additional section be written, in which the primary adverse effects of PFOS

are discussed

-- in terms of their relative toxicological significance, their apparent mechanism(s), their relevance to humans, their likelihood in realistic exposure scenarios, and implications of altered experimental indices to actual organ dysfunction.

I am quite concerned about the increased rat pup mortality in several studies at relatively low maternal doses, but not about reversible liver weight change s or centrilobular hypertrophy. Is the decreased pup survival in several studies at relatively low maternal doses of PFOS relevant to

humans?

-- Is the dose

- response curve steep, as suggested by Luebker et al. (2005a), such that

there would be less concern about sub

- threshold doses?

-- What is the most likely mode of action (pulmonary surfactant or maturation, dietary, hormonal)?

-- Is decreased survival PPARα

related?

-- Is the mechanism in rats relevant to other species?

-- Does pup mortality occur in

othe r species at comparable doses?

-- Might there be a dose

- dependent alteration of maternal

fetal partitioning of PFOS?

Deborah A. Cory

- Slechta

Both document s, although the PFOA document to some degree more than PFOS, overall are

more of a tabulation of studies than a critical review of studies from which a rationale is presented for a choice of studies to model and from which to derive associated RfDs. The

Executive summar ies are too abbreviated and do not include suff i cient rationale, description and

detail to provide the reader with an understanding of how decisions described in Chapter 5 were made. Since in some cases, this will be the only sections read, they could provide a more informative summary.

It would be very helpful to provide a section up front that describes all of the parameters of the literature search, including the years that are included in the document review, as well as descriptions of criteria for studies that were included vs. those that were e xcluded. In addition, it should be indicated whether there was a criterion that studies be peer

- reviewed. This is particularly important given the voluminous size of the data base that has accumulated for theseExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

10 two chemicals. Given that revisions will be d one and that such documents do not get updated with any frequency, it would be good to attempt to include as much of the new pertinent literature as possible.

The section on Toxicokinetics in the documents present studies in detail, but no real

conclusion s; this is true of most of the sections in these documents. Chapters 3 and 4 in particular read like tabulations of studies rather than critical reviews and because of that, the documents seem disjointed and Section 5, i.e., derivation of values, tend to b e difficult to read through and require constant searching back to the original chapters in which they are described. It is critical to identify the strengths and weaknesses of the various studies, and which were given weight to use in the final determin a tions. It would be helpful if S ections 3, 4 and 5 included an introductory paragraph describing the goal of the chapter, and that each ends with an overall summary with conclusions. The tables in these chapters also would benefit from the

inclusion of add itional information that ultimately permits comparisons within the Table and does not require continually returning to the text to recall the species, sample sizes, etc.

In the sections on Hazard Identification, it is useful that studies are summarized by target organ,
but there are almost no conclusions and no discussions of strengths or weaknesses of studies and

therefore their use or not in future decisions

. In fact, one is left with the impression that all studies are equal, especially in the section describing human studies. Within Chapter 4, the sub

sections entitled “evaluative and integrative” are actually neither. Data are presented simply as positive or negative with no real discussion of the strengths and limitations and what was

concluded over all. For this reason, Chapter 5 is also lacking. It provides very little in the way of

rationale and conclusions. Thus

, the transparency of the process is really insufficient

.

Jamie C. DeWitt

The information presented throughout the documents appears to be accurate (with one minor exception noted in Table 1 of these comments) and is presented clearly. For PFOA, a reference dose (RfD) of 0.00002 mg/kg/day was determined and evidence of carcinogenicity is considered suggestive with a human equivalent dose (HED) of 0.58 mg/kg/day. The RfD was based on changes in liver weight reported as a common denominator in four rodent (three rat and one mouse) studies and carcinogenicity was based on a limited number of epidemiology studies linking kidney and testicular tumors with exposure and evidence of tumor induction in the liver, testes, and pancreas (the “tumor triad”) in rats. For PFOS, a RfD of 0.00003 mg/kg/day was determined and evidence of carcinogenicity is considered suggestive but with insufficient

evidence to determine human carcinogenic potential. The RfD was based on developmental neurotoxicity and changes in liver weight.

While the carcinogenicity assessment seems appropriate for the two compounds given the limitations of the data sets, changes in liver weight as a basis of both of the RfDs is questionable in terms of its significance to exposed humans. Exposure to these ag ents increases liver weight and hepatocellular hypertrophy in rodents (and the definition of these endpoints as “adverse” or “toxic” also is contentious); this has been demonstrated across various rodent strains and under myriad exposure paradigms. However , there is no consensus in the scientific community regarding the mechanism by which exposure to these compounds increases liver weight andExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

11 induces hepatocellular hypertrophy in rodents and whether any of the putative mechanisms are

sufficient to induce he patotoxicity in exposed humans. Proposed mechanisms include peroxisome proliferator activated receptor alpha (PPARα ) activation, activation of other nuclear receptors, peroxisome proliferation (which may or may not be dependent on PPARα activation),

and ox idative stress. Humans can certainly respond to PPA Rα agonists (i.e., fibrate drugs are used as hypolipidemic agents) and a handful of epidemiological studies of highly exposed human populations have reported associations between PFOA/PFOS and alterations in liver enzymes, but the clinical relevance of the changes to the liver enzymes reported for these studies

is uncertain. These liver

- related changes in humans generally occur at higher doses than required to induce changes in the livers of rodents, which occurs at relatively lower doses than other observed effects. Therefore, a critical endpoint that occurs at very low doses in rodents, has no agreed upon mechanism that may or may not be relevant in humans at relatively high doses, may

not be the best choi ce for the basis of a RfD. Liver weight change has been reported to occur in several species, including non

- human primates, and at low doses, it may be an adaptive response and not a toxicological response. While this response may be protective of human he alth because it is common following low dose exposure to PFOA or PFOS, other endpoints may be more relevant to humans, especially endocrine system effects, including changes to thyroid hormones and mammary gland development, and immune system effects. Endo crine and immune system effects have been reported in exposed humans, suggesting that such endpoints may operate via a mechanism that is more relevant to humans than mechanisms related to changes in liver weights.

In addition, the one developmental neurot oxicity study used, in part, for the PFOS RfD is only weakly supported by additional studies in rodents or other species and is based on behavioral responses that could be influenced by factors other than direct effects on the nervous system.

Additional co nfirmatory studies are necessary for this observation to be considered a critical effect of PFOS exposure.

Finally, while well

- written overall, the documents lacked an overall critical analysis or depth required of a risk assessment. Why specific studies were included or not should be better explicated in the text.

Jeffrey W. Fisher

The document was well written in terms of balance and presenting information. S ummary statements are needed for chapters; a synthesis/analyses of the data are needed in some cases . A

more critical evaluation of the human and non

- human responses to PFOA/PFOS is required to justify not using human or non

- human primate data. A rationale for the modeling approaches is needed given the more recent PBPK models that are available.

William L. Hayton

The literature that pertains to the health effects of PFOA and PFOS is large and presents a major challenge to accurately summarize and analyze it and develop an RfD for PFOA and PFOS. Reported health effects in animals and humans, some times contradictory, include exposure

associated changes in serum cholesterol, lipids, uric acid, and thyroid hormones, obesity

- relatedExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

12 metabolism, immune system function, and effects on reproduction, development of the mammary gland, the nervous system, a nd behavior. Target organ effects (e.g., liver, kidney) have been reported, as well as associations of PFOA and PFOS exposure with testicular, prostate and kidney cancer. Studies in several laboratory animal species have added the complications of

inters pecies comparisons and extrapolation of findings to humans. In humans, there have been a Phase I clinical trial of PFOA, and epidemiological studies of populations exposed to PFOA and PFOS occupationally and in communities with and without water supplies contaminated with PFOA and PFOS. The draft documents have accurately presented in summary form the results of many animal and human studies and used pharmacokinetic methods to link PFOA and PFOS exposure rates to internal dose metrics such as serum concen tration. While the overall effort is commendable, there are two issues that the draft documents raise: 1) t he literature cited does not include many apparently relevant published works. The cut

- off date for cited literature was early 2013 (this should b e indicated in the documents), but commenter’s noted a number of pertinent publications in 2011 and 2012 that were not cited, and there have appeared several highly pertinent papers since the cut

- off date, and 2) w hile the descriptions of individual studies are generally clear and accurate, there is a lack of independent, critical analysis of the studies and a lack of synthesis of results from multiple studies common to a particular health effect.

Matthew P. Longnecker

The PFOA and PFOS documents ac hieve the goal of identifying RfDs that are well founded. My main criticism is that the rationale for not using the human data to provide a POD needs to be strengthened.

For example, in the PFOA document, on page 5

- 19, first paragraph below the table, it says “human data ... lack the exposure information for dose

- response modeling.” This statement is logically inconsistent with techniques that were used to estimate HED on the basis of serum concentration, as given on page 5

- 17, near the bottom. Or, in som e cases, such as in the C8 study, the exposure estimates that were calculated based on water district were sufficiently good

that a dose

- response analysis would be possible. In other words, because many human studies

have serum concentration of PFOA or re asonable estimated exposure values, the corresponding HED could be estimated, and hence the dose

- response could be modeled. Granted, some assumptions would be needed, but the methods could be serviceable (see response to item 3 below). (Some of the above also applies to pages 5

- 1 and 5

- 2). More compelling arguments for not basing the POD on human data are, e.g., that: 1) the low probability that humans are 1,000 times more sensitive to PFOA than other species (the number is based on the last column in

t able 5

- 9 compared with PFOA values in the C8 study and background exposed populations), especially given the relatively tight agreement between LOAEL (average serum concentration basis) among other species, 2) the possibility that the observed associations in humans were due to unmeasured confounding factors or reverse causality, and 3) other weaknesses in the epidemiologic data such as inconsistent results acro ss studies (selected outcomes), unreplicated findings, or associations with clinical chemistry re sults for which corresponding adverse clinical correlates (i.e., morbidity) are not clearly established.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

13 Angela L Slitt

The documents provide a very thorough evaluation of PFOA and PFOS studies. It is logically organized, presenting findings in a way that the reader can understand the findings related to human, monkey, and rodents. The organization of the document allows the reader to easily find information about each species within the subchapters and summarizes key points in table form.

PFOA is a well

- studied compound, with a substantial amount of toxicokinetic and endpoint studies in rodents. Mechanistic data describing the role of membrane transporters to understand gender difference s in PFOA elimination in rats is fairly well written. Little data exist regarding contribution of membrane transporters to PFOS disposition and elimination. The documents thoroughly describe species differences in PPAR

- alpha signaling that might contribute to observed endpoints in rats, but not humans or monkeys. Overall, bo th documents are very thorough and provide a reliable basis for PFOS and PFOA evaluation.

For PFOA toxicokinetics, mechanisms of PFOA transport are important for understanding species differences in response to PFOA exposure, with focus placed on kidney. Figure 3

- 2 in the PFOA document does not adequately present the localization of renal transporters with relationship to their contribution to the urine compartment or renal reabsorption. A very nice diagram showing the subcellular localizatio n of renal transporters presented by Klaassen and Aleksunes (Pharmacol Rev. 2010 Mar;62(1):1

- 96) clearly depicts the contribution of various transporters to filtrate or blood. This is an easier diagram to put PFOA elimination into context than the one pre sented. Contribution of membrane transporters to species differences in PFOA excretion Section 3 (specifically 3.4.1) would be put in better context if a table could be generated to compare Km and Vmax values for PFOA for various transporters, with specif ic focus on species information for OATPs and OATs. Data regarding information on contribution

for OATps in liver accumulation of PFOS and PFOA is lacking, with specific re gard to species

differences . As PFOS is a likely candidate for hepatic uptake transport, understanding a mechanism to explain species differences in hepatic effects possibly due to difference in hepatic exposure is critical. Understanding impact species specific regulation of OATp expression in liver (e.g. whether species difference in PPAR

- alpha signaling contributes) is also important in putting rodent distribution data into context.

Increased liver weight is considered to be a critical effect, but how increased liver weight rel ates to the observed human and monkey health effects needs to be further explained. First, use of liver weight alone might not be substantiate of an effect for point of departure compared to other liver effects observed at higher concentrations, such as i ncreased serum ALT or AST. The studies that have evaluated these endpoints are well conducted . In layman terms, if someone is walking around with an increased liver weight, is he or she at risk for disease? Will his/her life span be shortened? To inc rease transparency of the document, a more comprehensive explanation is needed to justify why increased liver weight should be considered as a critical endpoint for human health.

However, it should be noted for future consideration that there are a few publications in mice that do also ascribe liver weight changes in increased lipid accumulation along with increased expression of genes that contribute to fatty liver disease. This is considered to be a gap in knowledge for the field. Will relatively lo w dose PFOS exposures associated with hepaticExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

14 steatosis have other endocrine related effects known to be associated with NAFLD (insulin resistance, impaired glucose tolerance)? Emerging studies are evaluating whether PFOS induces hepatic steaosis and whet her it is a PPAR

- α mediated effect. For example a study performed by Wan et al. (2012) administered 0, 1, 5 or 10 mg/kg/day PFOS to adult male CD

- 1 mice for 3, 7, 14 or 21 days. Histological analysis of liver sections, and biochemical/molecular analysis of biomarkers for hepatic lipid metabolism were assessed. Overall, the study reported that PFOS

administration induced hepatic steatosis in a t ime

- and dose

- dependent manner. The study also shows a high correlation between liver weight and lipid content. Increased expression of a lipogenic target (CD36/FAT) was observed at 5 and 10 mg/kg PFOS. A second study by Bijland et al. (2011) illustrated that PFOS administration increased liver weight (+ 107%, p <

0.0001 ), which was accompanied by an increased he patic TG content ( + 192%, p < 0.0001, respectively) in E3L.CETP mice on a C57Bl/6 background. At the age of 8

– 10 weeks, mice were fed a semisynthetic Western

- type diet, containing 0.25% (wt/wt) cholesterol, 1% (wt/wt) corn oil, and 14% (wt/wt) bovine fat for 4 weeks in three independent experiments. Upon randomization according to body weight, plasma TC, and TG levels, mice were fed a Western

type diet without or with PFOS (0.003%,

∼ 3 mg/kg/day) for 4

– 6 weeks

. In summary, there is

evidence that administrat ion of relatively low PFOS doses to mice can result in hepatic lipid accumulation in the absence of overt “wasting .”

Data is lacking as to whether higher species, such as monkeys or humans will also develop

PFOS

- induced steatosis, which is confounding. Studies have profiled gene expression in wild

type and PPAR α

- null mice administered PFOS, finding that there is pathology and gene expression consistent with lipid

- promoting effects in liv er that are independent of PPAR α , as they are observed in PPAR α

- null mice (Rosen et al., 2010). Limitations to the studies are that they did not specifically quantify hepatic lipid content, but inferred that the PFOS

- induced vacuolization in liver pathology observed was potentially related to triglycerides. Studies by Bj ork et al. (2011) comparing rat and human primary hepatocytes treated with PFOS (25 μM) demonstrated that human hepatocytes were slightly less responsive to the induction of lipid oxidation and synthesis genes, as well as induction of carbohydrate metaboli sm. It should be noted that the hepatocytes from the study are from a single human donor, the hepatocyte lipid content was not determine, and hepatocyte culture conditions were standard and not optimized to induce steatosis. In summary, the current liter ature is lacking robust information regarding whether PFOS, which highly concentrates in liver, has a steatotic

- inducing effect in human or monkey liver. The evidence in PPAR α

- null mice indicates that it might have som e PPAR α independent effects related t o hepatic fat accumulation

. Because evidence fo r hepatic lipid

content in PPARα

- null mice after PFOA or PFOS has been described only by pathology, more

robust studies are needed to conclude whether the effect s can occur independent of PPAR α and

the observe d increased liver weight is due not only to hypertrophy due to nuclear receptor activation, but lipid accumulation. However, this reviewer is noting this as a concern for the future and area where a gap in knowledge exists.

Moreover, because the reviewer is noting a concern for hepatic fat accumulation tha t exists in

the absence of PPAR α, it should be appreciated that traditional markers, such as AST and ALT have poor prognosis for NAFLD or toxicant associated steatohepatitis. Most patients with

NAFLD a re asymptomatic and the disease is often diagnosed following findings of elevated aminotransferases, especially when combined with other features of metabolic syndrome. TheseExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

15 abnormal liver function tests usually require the physician to distinguish betwee n NAFLD and alcoholic fatty liver disease (AFLD). Some have suggested that a serum AST/ALT ratio >1 are indicative of AFLD. Other possible signs of AFLD include elevated HDL

- cholesterol with hypertriglyceridemia. While most diagnoses of NAFLD may result f rom altered liver function tests, normal serum aminotransferase tests can be seen in patients with both steatosis and NASH (Ipekci, et al., 2003; Mofrad, et al., 2003). Indeed, it is reported that two

- thirds of NASH patients may have normal aminotransferas e levels at any given time (Oh, et al., 2008; Delgado, 2008; Wieckowska and Feldstein, 2008). Kunde et al. investigated the accuracy of NASH diagnosis by serum ALT in women undergoing gastric bypass surgery (Kunde, et al., 2005). They compared two differen t reference laboratory cutoffs for "normal" ALT levels, the previous guideline of 30U/L, and new lower level of 19U/L that was suggested to aid in the diagnosis of NAFLD. Importantly, the authors reported that the diagnostic utility of serum ALT remained

p oor even at the new lower cutoff. Sensitivity and specificity of serum ALT levels were found to be 42% and 80% (ALT > 30U/L) versus 74% and 42% (ALT > 19U/L). These and other studies (Lizardi

- Cervera, et al., 2006; Amarapurkar and Patel, 2004; Amarapurka, et al., 2006; Chen, et al., 2006; Fracanzani, et al., 2008; Sorrentino, et al., 2004; Mofrad, et al., 2003; Uslusoy, et al., 2009) illustrate the need for a more effective diagnostic measure for NAFLD, especially the NASH stage. In sum, use of ALT and AST elevation to base the po int of departure must be

taken into context because they are poor prognostic markers for increased liver accumulation, NAFLD, or even NASH. Use of ALT and AST might not be an appropriate biomarker for

measurement of PFOA or PFOS

- i nduced adverse effects on liver and should be considered as a gap in our knowledge for future work.

In the review panel discussion, there was discussion regarding a publication by Hall et al. (2012), which summarized the outcome of a workshop regarding li ver hypertro phy and hepatocarcinogenesis. Overall, the workshop concluded that “hepatomegaly as a consequence of hepatocellular hypertrophy without histologic or clinical pathology alterations indicative of liver toxicity was considered an adaptive and a n on

- adverse reaction”. This conclusion is taken in the context of hepatic hypertrophy caused by nuclear receptor activation. This differs from others that have concluded in mouse NTP studies where correlations between liver weight increases and histologic al parameters and carcinogenesis were assessed, the authors concluded that ‘‘the best single predictor of liver cancer in mice was hepatocellular hypertrophy’’ (Allen et al. 2004). Based on the conclusions presented in Hall et al. (2012), increased liver weight might not be considered an appropriate POD because of lack of overt toxicity and hepatomegaly being considered an adverse effect. It should be noted that this reviewer still has concerns regarding this conclusion for PFOS and PFOA because the patho logy described in PPAR α

- null mice reflect increased hepatic lipids and not hepatomegaly due to nuclear receptor activation that is described in this opinion publication. If one considers the pathology examples of hypertrophy presented in Hall et al. (2012 ), it is quite different from the pathology described for PFOS and PFOA. For clarity, the document should try to delineate the cellular components that are contributing to increased liver weight caused by PFOA and PFOS administration, if such a publicatio n exists (e.g. how much of the liver is associated with protein/peroxisome proliferation increase versus lipid increase). Because the literature is not clear regarding what exactly in the liver is causing increased liver weight, studies documenting hepati c lipid accumulation should not be currently considered for POD. Given the recent opinion cited by Hall et al. (2012), it isExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

16 recommended that studies documenting liver damage, such as ALT and AST elevation be currently used as the POD.

The documents of

ten have redundancy in information, especially in regard to hormone effects (there are very similar write ups in sections about effects on thyroid hormone) and metabolic/cardiovascular disease risk factors (e.g. lipid endpoints).External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

17 IV

. RESPONSE TO CHARGE QU ESTIONS

Charge Questions

Question 1. Please comment on the strengths, weaknesses, and characterization of the studies selected as key for quantification.

James V. Bruckner

PFOA

- specific comments The document’s authors have done a good job describing and integrating the findings of the numerous studies in which liver weight gain was observed. Although there is a consensus about the effect and the dosage required to elicit it in different species, this reviewer does not believe it should be utilized, as described above. There are several clearly adverse effects such as elevated serum (hepatic) enzyme activities, focal hepatocellular necrosis, bile duct degeneration and fibrosis, etc. These effects are generally seen in response to relatively high PFOA do ses, so the PODs will be higher than with liver weight increase. Alternatively, a human endpoint such as elevated serum cholesterol could be considered. See responses to Charge Question 3.

PFOS

- specific comments

There have been a substantial number of well

- conducted toxicological studies of PFOS. My major concern, as expressed above, is its potential to cause adverse effects in children. Other than that, PFOS doesn’t appear to produce effects other than those anticipated from a repetitive,

cumulative do se of an 8

- carbon fatty acid.

Deborah A. Cory

- Slechta

In general, it appears that, at least with resp ect to the animal studies, the choices are appropriate

both in the case of PFOA and PFOS. The derivation of the RfDs/RfCs are based on studies of

suffici ent strength, duration and represent the most sensitive endpoints.

Having said that, in both documents, the reader is forced to that conclusion with no real assistance from the text itself. There is virtually no discussion of the strengths and weaknesses of the studies overall. Human study outcomes for the most part are simply enumerated, although an occasional statement will be made about a limitation (usually) of one o f those studies. There is no discussion in the human studies of the power to detect ef fects, the sample sizes, etc. Much weight seems to be given to occupational studies in some cases, being used to essentially dismiss effects in a community cohort as the same effect was not seen in occupationally exposed workers, when in fact finding effec ts in a population with seemingly longer, albeit lower exposure levels actually makes the outcome more robust. Also, population studies with smaller sample sizes that nevertheless find significant effects are in fact more compelling and suggest

robust effe cts which can be detected even with a small sample size. This deficiency is manifest in statements such as those in the PFOS document (p. 5

- 1) that ‘in most cases the findings are suggestive and not conclusive of an effect’.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

18 There is a bit more discussio n of the animal studies in both documents, at least with respect to methods, but as with the human studies, there is little text addressing which studies represent stronger studies or what the weaknesses are. From these increase liver weight has been chose n as the endpoint from which to derive RfDs. This reviewer does not have an issue with that choice, as while it has been described as adaptive by some, it represents a response to an involuntary exposure with a direction of effect that is potentially assoc iated with adverse consequences. The fact that it is reversible when exposure ends seems irrelevant as reversal of exposure is not happening in the human environment.

Jamie C. DeWitt

Strengths: The studies selected as key for quantification were generally well

- conducted studies, employing a range of doses and sample sizes large enough for detecting statistical differences. Additionally, the doses associated with LOAELs for the identified critical endpoints were not associated with signs of overt o r systemic toxicity in the animal models and nearly all of the studies measured serum and/or tissue concentrations of the parent compounds.

Weaknesses: No obvious experimental design weaknesses were noted in any of the studies selected as key for quantification.

Characterization: The studies selected as key for quantification for PFOA are all rodent studies while at least one study selected for PFOS quantification includes a non

- human primate study. It is therefore surprising that the PFOA databas e does not include, as a study key for quantification, the Butenhoff et al. 2002 study of non

- human primates. Additionally variability in putative mechanisms among species was not adequately addressed in the characterization of the selective studies, altho ugh all of the selective studies were descriptive and not mechanistic.

Jeffrey W. Fisher

PFOA and PFOS: D ata bases are massive and both need to be updated. Several human studies

and a few non

- human primate toxicity studies are available . The authors ne ed to explain why these studies are not adequate for causality (dose

- response).

William L. Hayton

An advantage to assessment of health effects for both PFOA and PFOS is the large amount of published work that informs the topic. While the draft health

- effects documents have summarized the results of many pertinent studies, the literature reviewed was n ot comprehensive, which projects an appearance of weakness. The documents do not state whether

the intention was to include all relevant health

- effects literature, or to be selective and summarize those studies judged to be most relevant. Such a statemen t at the beginning of the documents would be helpful; a cut

- off date for the literature review would also frame expectations of readers. If the intention was to be selective, a description of selection criteria would help allay concerns of readers about p apers that were not included. If the intention was to comprehensively review all the PFOA and PFOS health

- effects literature, then it appears thatExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

19 more work should be done to include omitted works. Public comments list a number of works

to consider for i nclusion.

A general, albeit minor weakness of the literature is that PFOA and PFOS serum concentrations in control animals were not measured for many studies

– they were likely non

- zero and, since there is no information on how high they were, it is possi ble that baseline health

- effects metrics

were affected and that dose

- response relationships were affected, especially in the low dose range. It is perhaps worthwhile to mention this shortcoming somewhere in the health effects

documents.

Matthew P. Longne cker

EPA may want to consider the article by AP Hall et al. 2012, about liver hypertrophy. The defense of increased liver weight as the POD (or a POD) could be strengthened by evaluating

the evi dence in the context of Hall’s F igure 9, where evidence rega rding hepatoxicity and toxic

mechanisms are also considered. In this case

, the possibility of an unknown mechanism exists that could be relevant to humans, and long

- term exposure could have effects tha t have not yet

been detected. See Hall p age 986, wher e it defines adverse as: “...affects [response] to an additional environmental challenge”. Thus, an adverse effect, via an unknown mechanism, by this definition is possible and has not been studied in animals or humans.

While AP Hall’s article is not all t hat supportive of using increased liver weight as a point of departure (unless certain criteria are met), they are focused on animal studies, especially those done in rodents. If increased relative liver weight were to occur in a human population, I

suspe ct that it would be considered an adverse outcome, whether or not there was evidence of hepatotoxicity or a specific mechanism. Note also that for PFOA, in monkeys, there was an increase in relative liver weight with chronic exposure (PFOA document, page 4

- 66), so increase in liver weight in the animal experimen ts may be relevant to humans.

An additional comment of relevance here pertains to whether the human data support hepatoxicity. While there are studies that report elevated liver function tests in subjects with higher serum concentrations of perfluorakyl substances, these elevations do not clearly support the presence of toxicity. Again, AP Hall’s discussion of what constitutes evidence of hepatoxicity is relevant here, and takes into account the number of LFTs elevated, the specific LFTs involved, and the magnitude of their elevation.

Finally, as discussed at the meeting, for the PFOA document on p age 5

- 23 (“RfD Selection ”), and the PFOS document on p age 5

- 26 (“RfD selection”), I suggest minor ed itorial changes to deemphasize the “consistency of response” point and instead focus a little more on how the RfD

is robust to choice of POD endpoints. If the selection of RfD does not hinge on inc reased liver weight as a POD, it will be more defendable.

Transparency might be increased by saying why (more clearly, or more clearly by implicit reasoning) the Macon et al. 2011 study, in which the LOAEL was 0.01 mg PFOA/kg from GD10 to GD17, based on delayed mammary gland development, was not considered as a POD, and why the Hines et al. 2009 study, in which the LOAEL was 0.01 mg PFOA/kg from GD1 toExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

20 GD17, based on various outcomes, was not considered as a POD. The PFOS studies with low

LOAELs were considered in the dose

- response assessment (no suggestions for improvement there).

Angela L Slitt

My response is basically the same as my General Impressions above

.

The documents provide a very thorough evaluation of PFOA and PFOS studies. It is logically organized, presenting findings in a way that the reader can understand the findings related to human, monkey, and rodents. The organization of the document makes allows the reader to easily find information about each species within the subchapters and summariz es key points in

table form. PFOA is a well

- stud ied compound, with a substantial amount of toxicokinetic and endpoint studies in rodents. Mechanistic data describing the role of membrane transporters to understand gender difference s in PFOA elimination in rats is fairly well written. Little data exist regarding contribution of membrane transporters to PFOS disposition and elimination. The documents thoroughly describe species differences in PPAR

- alpha signaling that might contribute to observed endpoints in rats, but not humans or monkeys. Overall, b oth documents are very thorough are provide a reliable basis for PFOS and PFOA evaluation.

For PFOA toxicokinetics, mechanisms of PFOA transport are important for understanding species differences in response to PFOA exposure, with focus placed on kidney. Figure 3

- 2 in the PFOA document does not adequately present the localization of renal transporters with relationship to their contribution to the urine compartment or renal reabsorption. A very nice diagram showing the subcellular localization of renal transporters presented by Klaassen and Aleksunes (Pharmacol Rev. 2010 Mar;62(1):1

- 96) clearly depicts the contribution of various transporters to filtrate or blood. This is an easier diagram to put PFOA elimination into context than the one presented. Co ntribution of membrane transporters to species differences in PFOA excretion Section 3 (specifically 3.4.1) would be put in better context if a table could be generated to compare Km and Vmax values for PFOA for various transporters, with specific

focus on species information for OAT p s and OATs. Data regarding information on contribution

for O AT ps in liver accumulation of PFOS and PFOA is lacking, with specific re gard to species

differences . As PFOS is a likely candidate for hepatic uptake transport, unde rstanding a mechanism to explain species differences in hepatic effects possibly due to difference in hepatic exposure is critical. Understanding impact species specific regulation of O AT p expression in liver (e.g. wh ether species difference in PPAR

- alpha signaling contributes) is also important in putting rodent distribution data into context.

Increased liver weight is considered to be a critical effect, but how increased liver weight relates to the observed human and monkey health effects needs to be fu rther explained. In layman terms, if someone is walking around with an increased liver weight, is he or she at risk for disease? Will his/her life span be shortened? To increase transparency of the document, a more comprehensive explanation is needed to justify why increased liver weight should be considered as a critical endpoint for human health.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

21 Use of humanized PPARα mice are a sexy tool to delineate species differences in effects associated with peroxisome proliferation. For transparency, the docu ment should acknowledge the limitations of that model. Specifically, lack of response may not necessarily correlate to a lack of response for human PPARα because of species differences in binding to cogate DNA elements (e.g. a human receptor may have lowe r binding capacity to mouse DNA due to structural differences and species differences in co

- activator/co

- repressor interactions ) . Wording in the documents using these mice should acknowledge this limitation.

The documents often have redundancy in informa tion, especially in regard to hormone effects (there are very similar write ups in sections about effects on thyroid hormone) and metabolic/cardiovascular disease risk factors (e.g. lipid endpoints).External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

22 Question 2. Please provide citations (and, where possible, pdfs or hard copies) for any references you suggest EPA consider adding to the document. Describe where you suggest these references be incorporated.

James V. Bruckner

PFOA

- specific comments Fabrega, F. et al. (2014). PBPK modeling for PFOS and PFOA: Validation with human experimental data. Toxicol. Lett. On line. (Hard copy available)

Stahl, T., Mattern D and Brunn, H. (2011). Toxicology of perfluorinated compounds. Environ. Sci. Europe 23: 38

- 60.

Hall, A. P. , et al. 2012. Liver hypertrophy: A review of adaptive (adverse and non

- adverse) changes

- Conclusions from the 3 rd International ESTP Expert Workshop. Toxicol. Pathol. 40:

971

- 994.

Bjork, J. A., Butenhoff, J. L., and Wallace, K. B. 2011. Multiplicity of n uclear receptor activation by PFOA and PFOS in primary human and rat hepatocytes. Toxicology 228: 8

- 17.

PFOS

- specific comments

No additional references were located.

Deborah A. Cory

- Slechta

For both PFOA and PFOS, the document should include a descripti on of the process through which studies were identified and how they were processed for inclusion or not. It is not clear what the exact dates of the studies examined included, i.e., what the cut

- off date was for these

studies . This makes it difficult to evaluate whether there are missing studies. That said, this reviewer is not aware of any specific omissions in the peer

- reviewed literature other than those

that were discussed at the face

- to

- face meeting.

Jamie C. DeWitt

Granum, B., Haug, L.S., Namork, E., et al. 2013. Pre

- natal exposure to perfluoroalkyl substances may be associated with altered vaccine antibody levels and immune

- related health outcomes in early childhood. J Immunotoxicol. 10:373

– 379; Looker, C., Luster, M.I., Calafat, A.M., et al. 2014. Influenza vaccine response in adults exposed to perfluorooctanoate and perfluorooctanesulfonate. Toxicol. Sci. 138:76

– 88.

Any time the Grandjean et al. (2012) findings related to PFAS and vaccine responses are discussed, these references could/should be discussed as well as they report related findings in human populations. Although t hey also are confounded by multiple PFAS (as was the Grandjean et al. study), they lend additional support to immunotoxicity as an endpoint w orthy of consideration. However, it is noted that these references were published after the cutoff date for

consideration for inclusion in the document.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

23 Lopez

- Espinosa, M.J., et al. 2012. Thyroid function and perfluoroalkyl acids in children living

near a chemical plant. Environ.H ealth Perspect. 120:1036

– 1041. This study is missing from the discussion of thyroid hormone disruption. It reports a positive correlation between hypothyroidism and PFOA in children from the C8 population aged 1

- 17.

Corsini E., et al. 2011. In vitro evaluation o f the immunotoxic potential of perfluorinated compounds (PFCs). Toxicol. Appl. Pharmacol. 250:108

- 116. Corsini E. et al. 2012. In vitro characterization of the immunotoxic potential of several perfluorinated compounds (PFCs)

. Toxicol. Appl. Pharmacol. 258:248

- 255. These studies are in vitro/ex vivo studies of human

derived cells that provide evidence that in vitro measures of immunocompetence in mice may be relevant to the human experience.

Jeffrey W. Fisher

For completeness sake, at least, please update lab animal studies conducted since 2012.

William L. Hayton

A review of PFOA health

- effects literature (GB Post et al. (2012) Environ. Res. 116: 93

- 117) provides an excellent, in

- depth discussion of many is sues covered in the PFOA health effects document. Consider citing this review in the document.

The literature on PFOA and PFOS toxicokinetics (Section 3) has been comprehensively covered in the health effects documents, with the notable omission of Wam baugh et al., Dosimetric Anchoring of In Vivo and In Vitro Studies for Perfluorooctanoate and Perfluorooctanesulfonate.

Toxicol. Sci. 136:308

- 327, 2013. This paper informed a significant part of the health effects

documents.

Commenter’s have suggested a number of references to consider with regard to Section 4 Hazard Identification. Many recent publications report on toxicity associated with PFOA/PFOS exposure. For the Dose

- Response Assessment (Section 5) it is desirable to focus on those

toxicities tha t have occurred at the lowest PFOA/PFOS exposures. For PFOA, the literature that is used in Section 5 to determine an RfD was published prior to 2009 (Tables 5

- 8

– 5

- 11). The benchmark response chosen based on the Section 4 literature was a 10% increase in liver weight, which was the biological response that occurred at the lowest PFOA exposure; it was acknowledged that this response “... is a biomarker for systemic exposure in rodents, rather than a biomarker of adversity ...” (p. 5

- 6). More recent studies of hazard have identified potential adverse effects that result from, or are associated with, PFOA exposures that are lower than the LOAEL for a 10% increase in liver weight. For example

, adverse effects on fetal, neonatal and early childhood stages of de velopment may occur at lower exposures than does liver weight gain, which suffers in addition from not being a biomarker of adversity, and which therefore raises a question about the validity of any RfD based upon it. Macon et al. 2011 reported an

LOAEL f or delayed mammary gland development of 0.01 mg/kg administered to pregnant CD

- 1 mice during GD10

– GD17. As this relatively brief exposure was well below that required for steady state, it is possible that had the dams been at steady state at the time of conception (about 9 weeks of exposure) a much lower LOAEL may have been observed; i.e., a much lower doseExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

24 rate at steady state would have produced the same exposure to the fetal pups as did the 0.01 mg/kg administered to the dams during GD10

– GD17. The steady state situation is more relevant to adverse effects in humans than is a brief exposure.

Matthew P. Longnecker

I suggest you include the following citation and include a discussion of the evidence presented: Paula I. Johnson, Patrice Sutton, Dylan S. Atchley, Erica Koustas, Juleen Lam, Saunak Sen, Karen A. Robinson, Daniel A. Axelrad, and Tracey J. Woodruff. The Navigation Guide

— Evidence

- Based Medicine Meets Environmental Health: Systematic Review of Human Evidence

for PFOA Effects on Fetal Growth. Environ Health Perspect; DOI:10.1289/ehp.1307893 (in press and available through the journal’s website).

Based on the meta

- analysis in this paper, the evidence that PFOA is associated with lower birthweight is consistent. Thus, the rationale for not ba sing the POD on the human data needs to be strengthened, as noted above. The Johnson et al. report could be discussed in the section on anthropometric endpoints that begins on p 4

- 22.

The relationship between birthweight and PFOA or PFOS may be confoun ded because glomerular filtration (and hence excretion of the compounds) is proportional to birthweight, as

discussed in:

Morken NH, Travlos GS, Wilson RE, Eggesbø M, Longnecker MP. Maternal glomerular filtration rate in pregnancy and fetal size. PLoS One . 2014 Jul 8;9(7):e101897

In the PFOA document, on page 4

- 1 8, you might want to also cite:

Taylor KW, Hoffman K, Thayer KA, Daniels JL. Polyfluoroalkyl chemicals and menopause among women 20

- 65 years of age (NHANES). Environ Health Perspect. 2014 Feb;12 2(2): 145

- 50.

The Taylor et al., like the Knox et al. report (already cited in the PFOA document) is from a large

- cross sectional study. Both studies, in their discussion sections, note that the association of PFOA or PFOS concentration in serum with age at menopause could be expected because postmenopausal women have lost a route of excretion for the compound and will have higher serum concentrations on that basis. It would be worth noting this possible explanation in the PFOA document on page 4

- 18, and in the PFOS document on page 4

- 8.

Additional data are available on the potential carcinogenicity of PFOA:

 Steenland K, Woskie S. Cohort mortality study of workers exposed to perfluorooctanoic

acid. Am J Epidemiol. 2012;176(10):909

- 17.

 Barry V, Winquist A, Steenland K. Perfluorooctanoic acid (PFOA) exposures and incident cancers among adults living near a chemical plant. Environ Health Perspect. 2013;121(11

- 12):1313

- 8

 Hall AP et al. Toxicol Pathol 2012:40:971

- 94. (About liver hypertrophy.)External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

25 The S teenland and Barry reports could be discussed in Section 4.1.2, on pages 4

- 28 and 4

- 29, respectively.

Angela L. Slitt

1) Evidence is presented for PFOA and PFOS as substrates for the related O AT p1d1 in zebra fish. Establishing whether PFOS is an O AT p transporter substrate is needed to better understand PFOS accumulation in liver. This study suggests that it might be. The following finding should be included in the PFOS do cument in S ection 3.2.3 and the PFOA document

in S ection 3.0:

a. Popovic M, Zaja R, Fent K, Smital T

. Toxicol Appl Pharmacol. 2014 Interaction of environmental contaminants with zebrafish organic anion transporting polypeptide,

O AT p1d1 (Slco1d1).

2) This publication presents the finding that PFOS inhibits Pgp, Mrp1, and Mrp4 activity. T he following finding should be included in the PFOS document in S ection 3.2.3 and the PFOA

document in S ection 3.0 :

a. Dankers AC1, Roelofs MJ, Piersma AH, Sweep FC, Russel FG, van den Berg M, van
Duursen MB, Masereeuw R. Toxicol Sci. 2013 Dec;136(2):382

- 91 . Endocrine disruptors differentially target ATP

- binding cassette transporters in the blood

- testis barrier and affect Leydig cell testosterone secretion in vitro.

3) PFOS induced ABC transporters in grey mullets.

a. de Cerio OD1, Bilbao E, Cajaraville MP, Can cio I. Gene. 2012 Apr 25;498(1):50

- 8. Regulation of xenobiotic transporter genes in liver and brain of juvenile thicklip grey mullets (Chelon labrosus) after exposure to Prestige

- like fuel oil and to perfluorooctane sulfonate.

4) These are new publications r egarding epidemiology findings for PFOS and PFOA exposure and serum lipids:

a. Fitz

- Simon N, Fletcher T, Luster MI, Steenland K, Calafat AM, Kato K, Armstrong

B. Epidemiology. 2013 Jul;24(4):569

- 76. doi: 10.1097/EDE.0b013e31829443ee. Erratum in: Epidemiology . 2013 Nov;24(6):941.

b. Starling AP, Engel SM, Whitworth KW, Richardson DB, Stuebe AM, Daniels JL, Haug LS, Eggesbø M, Becher G, Sabaredzovic A, Thomsen C, Wilson RE, Travlos GS, Hoppin JA, Baird DD, Longnecker MP. Perfluoroalkyl substances and lipid

concen trations in plasma during pregnancy among women in the Norwegian Mother and Child Cohort Study. Environ Int. 2014 Jan;62:104

- 12.

c. Fu Y, Wang T, Fu Q, Wang P, Lu Y. Associations between serum concentrations of perfluoroalkyl acids and serum lipid levels in a Chinese population. Ecotoxicol Environ Saf. 2014 Aug;106:246

- 52. 5) These are publications regarding PFOS exposure and hepatic steatosis :

a. Lv Z, Li G, Li Y, Ying C, Chen J, Chen T, Wei J, Lin Y, Jiang Y, Wang Y, Shu B, Xu B, Xu S. Glucose and lipid homeos tasis in adult rat is impaired by early

- life exposure to perfluorooctane sulfonate. Environ Toxicol. 2013 Sep;28(9):532

- 42. doi: 10.1002/tox.20747. Epub 2011 Aug 24. PMID: 23983163 Select item 22484034External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

26 b. Wan HT, Zhao YG, Wei X, Hui KY, Giesy JP, Wong CK. P FOS

- induced hepatic steatosis, the mechanistic actions on β

- oxidation and lipid transport. Biochim Biophys Acta. 2012 Jul;1820(7):1092

- 101. doi: 10.1016/j.bbagen.2012.03.010. Epub 2012 Mar 28. PMID: 22484034 [PubMed

- indexed for MEDLINE] Free Article

c. Bijland S, Rensen PC, Pieterman EJ, Maas AC, van der Hoorn JW, van Erk MJ, Havekes LM, Willems van Dijk K, Chang SC, Ehresman DJ, Butenhoff JL, Princen HM. Perfluoroalkyl sulfonates cause alkyl chain length

- dependent hepatic steatosis and hypolipidemia ma inly by impairing lipoprotein production in APOE*3

- Leiden CETP mice. Toxicol Sci. 2011 Sep;123(1):290

- 303. doi: 10.1093/toxsci/kfr142. Epub 2011 Jun 24.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

27 Question 3

. The OW concluded that the human epidemiology data for PFOS/PFOA do not provide adequate quantifiable dose

- response information for use as the basis of a candidate RfD because of uncertainty regarding the routes, levels and timing of exposures plus the confounding influences of other PFCs present in serum. Please comment of the OW
characteriza tion of the data.

James V. Bruckner

PFOA

- specific comments The document’s authors have done a good job summarizing and accurately characterizing the epidemiology liter ature for various endpoints in S ection 4.4

- Hazard Characterization. It is true there are a number of confounding factors that make estimation of PFOA exposures difficult. The EPA might consider, however, utilization of reverse dosimetry modeling. There is a reasonable body of data on serum PFOA levels, which could be used to estimate a range of PFOA exposures that would result in such internal doses.

PFOS

- specific comments I agree that human epidemiology data do not provide an adequate basis for calculation of a RfD or RfC. A reverse dosimetry modeling approach, however, could be use d to estimate a range of PFOS exposures that could have resulted in measured body burdens. The human data might then

be utilized in the risk assessment.

Deborah A. Cory

- Slechta

It is not clear that such an assertion should be used in the construction of this document. It is not clear why the route of exposure should be raised to a concern in the calculations, in fact in the human environment, there are exposures from multiple routes, no doubt and thus this is consistent with human environmental exposures . Further, if there is data on serum levels, it should reflect that cumulative exposure across exposure routes. Indeed, at the end, the goal is to arrive at an RfD based on serum levels. There is, moreover, no guarantee that there is no

contamination in st udies in animals from food, glassware etc.

Furthermore, in many epidemiological studies in which mixed exposures are the norm, controlling for other exposures is utilized to address this concern and to therefore make conclusions about individual exposures . In point of fact, in every single human study, there will invariably be other exposures and not a single exposure, and thus this strategy essentially says that no human studies can ever be used for any risk assessments. The stated rationales for not

usin g human data based on these statements is not adequate. This is why it is important as well to evaluate the strengths and weaknesses of each of the studies in terms of whether appropriate controlling for other known exposures was carried out and sample siz es sufficient etc. to arrive at some conclusions with respect to their ultimate usability in constructing RfDs.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

28 Jamie C. DeWitt, Ph.D.

.

While the OW characterization of the epidemiological data for PFOA/PFOS is, technically, true,

it also is somewhat misguided. Almost any epidemiological database will contain uncertainty regarding the routes, levels, and timing of exposures and will have confounding influences of other compounds. Very few epidemiological studies are free from these uncertainties, but w hen similar observations and conclusions are reached from multiple studies with these types of uncertainties, the database becomes useful for determining a candidate RfD or other value relevant to human health. What is particularly valuable about the PFOA/ PFOS database is that it is relatively extensive in that it includes data not only from occupationally

- exposed humans, but from people highly exposed to environmental concentrations of PFOA/PFOS and from people in the general population who have detectable concentrations of these compounds. Additionally, for establishing an RfD, do all of these uncertainties need to be absent? In other words, do

animal studies used to derive RfDs lack these uncertainties?

What is missing from the OW characterization of the epidemiological data is a thorough evaluation concerning hepatotoxicity and developmental toxicity reported in human populations and how these endpoints are relevant to or related to animal studies.

Jeffrey W. Fisher

The use of non

- human and human data is very important for interpreting exposure extrapolations from rats. I am not an epidemiologist so I cannot comment with authority on the epidemiology

data for dose

- response. Justify why human data are not suitable for use in the analysis of the

health hazards of PFOA and PFOS.

William L. Hayton

There are a number of epidemiological studies that have been based on large numbers of subjects chronically exposed (over decades in some studies) to the subject compounds over a broad range of intakes. Steady state serum concentrations have also been available for quantification of the systemic exposure. While the route, levels and timing of the exposures may hav e been uncertain, the long half

- lives of PFOA and PFOS in humans and the long periods of exposure to them indicate that 1) subject serum concentrations were generally at steady state, and 2) daily fluctuations in the amount and timing of the exposure would not produce much day

to

- day fluctuation in the serum concentration of PFOA/PFOS. These consequen ces of the long exposure period and long half

- life indicate that variability in the route and level of exposure would not have led to a measured serum concentration that was unrepresentative of the subjects’ long

- term average serum concentration. The serum concentration then should be relatively stable over time and it should reflect an integrated measure of the individual’s exposure to

PFOA and PFOS.

The serum concentration is a quantitative measure of s ystemic exposure to the subject chemicals, and is arguably a better metric of exposure than are intake rate. The over

- all rate of intake (R) that produces a particular steady state serum concentration (Css) can readily beExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

29 calculated from the clearance (CL ) of the chemicals, which is about 0.08 mL/d/kg body weight: R = Css x CL. The calculated rate of intake would represent all intake routes.

Confounding influences of other PFCs and indeed other chemicals and life

- style factors such as smoking, diet, a lcohol use, etc. would have to be considered, as is generally the case with epidemiological studies. Methodology exists for dealing with such influences.

Thus it appears that the epidemiological results should be used in the RfD determination. Their

str ength is that uncertainties associated with extrapolation from laboratory animal studies are avoided. Health effects that are positively associated with serum PFOA/PFOS concentration and that are observed in large populations of subjects should seriously be considered as potentially arising from PFOA/PFOS exposure. If mode of act ion studies in lab animals or in vitro studies support a cause

- effect relationship, then the threshold serum concentration could

inform the calculation of the RfD.

Matthew P. Lon gnecker

As noted in the General Impressions section above, the human studies with data on plasma or serum concentrations of PFOA and PFOS, especially for several categories of such levels, could

be used to estimate dose

- response information. However, there are ot her reasons why the human data may not be useful for setting the RfD (see above). Either PK or PBPK models might be useful for estimating the dose that human are exposed to; an advantage of a PBPK model is that it could incorporate information about route s and timing of exposure. Estimates of the contribution of various routes are available (e.g., Haug et al. 2011; Lorber & Egeghy 2011), and exposure trends could be assumed and evaluated in sensitivity analyses. Some occupational

studies had data that al lowed an estimate of serum levels, or measured them directly. Several reports show estimated exposure based on serum concentrations of PFOA or PFOS (Locissano et al. 2013; Lorber & Egeghy 2011; Thompson et al. 2010). With respect to confounding, the

ass essment of how likely this is could be informed by: 1) the correlation of serum concentration of PFOA, PFOS, and other compounds of this type in a particular study population (or in a series of studies), and 2) whether the other compound(s) has been associ ated with the particular outcome being considered. If the correlation is low or the other compound has not been associated with the outcome, concern about confounding may not be strongly justified. Without

additional consideration of data that address th ese points, it may be premature to assume confounding would be a problem.

Angela L. Slitt

Strengths of the studies : Several studies, which all demonstrate a positive association between

serum PFOA and/or PFOS and cholesterol or LDL levels are based on drinking water as a route of exposure. These studies are in agreement with Nelson et al., 2010, which was analyzing data

from the 2003

- 4 NHANES study. Steenland et al., 2009 ( Environ Health Perspect . Jul 2009; 117(7): 1083

– 1088) as part of the C8 Health Project collected data on 69,030 subjects with findings that serum PFOA was higher for males, those consuming local vegetables, and those using well water rather than public water, and lower for those using bottled water. The estimated response ra te for participants >20 years of age was 81% and mean serum PFOAExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

30 concentration was 83 ng/l. Subjects were eligible to participate in the C8 Health Project if they

had consum ed drinking water for at least one year before 3 December 2004 supplied by Little Hocking Water Association (Ohio), City of Belpre (Ohio), Tuppers Plains Chester Water District (Ohio), Village of Pomeroy (Ohio), Lubeck Public Service District (West Virginia), Mason County Public Service District (West Virginia), or private water sources within these areas that were contaminated with PFOA. Subjects were also eligible if they could document that they had either worked in a contaminated water district or wen t to school there for at least

one year. From this population, which the route of e xposure is considered to be primarily via drinking water, serum lipids were analyzed with regard to PFOA levels and a positive correlation was observed for all serum lipids except HDL. Frisbee further characterized this cohort, analyzing 12, 476 children and adolescents included in the C8 Health Projec t, finding an

increase in total cholesterol.

A recent epidemiology study (Fitz

- Simon et al., 2013), not included in the current documents,

described positive associations bet ween PFOA and PFOS in serum LDL c holesterol. This study

examined a s tudy population that consisted of 560 adults living in parts of Ohio and West Virginia where public drinking water had been contaminated with PFOA. They had participated

in a cross

- sectional study in 2005

- 2006, and were followed up in 2010, by which time exposure to PFOA had been substantially reduced. Overall, the findings demonstrate a positive

association between serum PFOA and PFOS levels and serum and LDL cholesterol

.

Weaknesses: The studies did not appear to analy ze PFOS or PFOA levels in drinking water from the participants analyzed and did not analyze data based on the length of exposure.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

31 Question 4

. Please comment on the transparency and characterization of the epidemiological data.

James V. Bruckner

PFOA

- specific comments

See comments a bove

.

PFOS

- specific comments The document’s authors have done a good job describing and summarizing the designs and findings of the epidemiology studies.

Deborah A. Cory

- Slechta

The PFOA document in particular and to some extent the PFOS document present all of the epidemiological studies but do not actually evaluate them; there is not a consistent indication of individual strengths and limitations of the studies, failures or not to adequately control potential confounding variables. Furthermore, there is no ‘power analysis’ type of evaluation, i.e., some of these studies included very small sample sizes and thus their power to actually detect effects may be limited, and yet they a ll appear to be weighted basically the same, i.e., studies with very small sample sizes with obviously extremely limited power to detect any effects appear to be considered the same as those with extremely large sample sizes. Studies with small sample siz es that nevertheless do find an effect of PFOA or PFOS actually suggest a robust type of effect.

The discarding of positive associations in human epidemiological studies because they do not produce frank clinical disease seems inappropriate and inconsist ent with other EPA documents. For example, p. 4

- 3 in the PFOS document states that only a small number of ALT values were outside the normal range making the results difficult to interpret in terms of health. Physiological changes that are moving in the w rong direction, even if sub

- clinical at the time, are still adverse effects. Are actual clinical diagnoses required for an adverse effect? This is especially the case given that the ranges of normal across populations are extremely broad.

The latter al so raises the question of the cumulative toxicity of PFOA and PFOS and whether any consideration is being given to this.

Another such example is in the PFOS document, where it actually refers to a statistically significant, but not toxicologically signif icant effect (p.4

- 38); what does that mean? Also, p. 5

- 4 appears to dismiss any changes in thyroid function since no evidence of clinical hypothyroidism actually occurred. This whole approach with the human studies seems quite inconsistent with

the relia nce on increased liver weight in the absence of clinical pathology as the endpoint in the

human studies.

Jamie C. DeWitt

It is not obviously or abundantly clear how the OW characterized the epidemiological data for

either PFOA or PFOS. The studies were well

- described, but the contribution of particular studiesExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

32 to the overall assessment was not. The results of studies described in the hazard characterization section (4.4) need to be better characterized. For example, in the PFOA risk assessment:

 An incre ase in serum lipids associated with PFOA/PFOS exposure in humans is discussed as a risk factor for cardiovascular disease in humans; however, no evidence of increased cardiovascular disease has been observed in human populations. Additionally, serum lipids typically are decreased in animal models after PFOA/PFOS exposure, which is thought to be associated with/typical of exposure to agents that activate PPAR  . If humans are known to respond to PPAR  activators (i.e., fibrate drugs), why would the results between humans

and animal models be discordant? This should be discussed.

 Several epidemiological studies reporting changes in liver enzymes clearly state that the clinical relevance of the changes i n enzymes is unknown. Therefore, stating that the human studies “suggest effects on the liver as indicated by increases in liver enzymes” amounts to a

mischaracterization of the data.

 No direct evidence of hepatotoxicity has been reported in epidemiologica l studies. This

should be discussed.

 More in

- depth characterizations are needed for the additional sections of the hazard characterization, with the exception of the thyroid section, which was well

- described.

For example, in the PFOS risk assessment: Simi larly to the PFOA risk assessment, the hazard

characterization section needs to better discussion differences and similarities between effects reported in humans and effects reported in animal models.

Jeffrey W. Fisher

I am not an epidemiologist, but it appears to be adequate. Better characterization of the pros and cons of the human analyses and interpretation of the outcomes would be helpful.

William L. Hayton

The characterization of the individual epidemiological studies presented seems to be adequate. Public comments have identified the need to distinguish positive and negative associations with statistical significance, which seems to be a fair criticism. As noted in the response to Question 2, there are relevant studies that have not been described in the health

- effects documents that ought to be considered and this includes some epidemiological studies. Most of the cited epidemiological studies have focused on healthy adults

– workers exposed occupationally,

residents of communities with or without contaminated water. These populations might be expected to be less sensitive to adverse effects than would early life stages and particular disease populations. Studies of potentially more sensitive populations would be desirable. The Frisbee et al. (2010) study of children 1

- 11.9 years and adolescents 12

- 17.9 years showed significant positive associations with serum lipid levels. Studies such as this one would be informative.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

33 Matthew P. Longnecker

Please see the long paragraph above, und er General Impressions, and some of the comments in response to item #2 above. Another point that the authors may want to consider is that studies that examine external exposure in relation to health outcomes may have special advantages in

the case of PFO A and PFOS. While in general it is considered best to have a measure of exposure that is based on a biomarker of internal exposure, this may be problematic for several outcomes for PFOA and PFOS, because of the possibility of confounding or reverse causal ity that would not be an issue if an external estimate of exposure were used. For example, in

Steenland K , Zhao L, Winquist A. A cohort study of workers exposed to PFOA. Occup Environ

Med. 2014 Jun;71 Suppl 1:A55, when an external estimate of exposure was used for the Washington Works employees, no association with elevated cholesterol was found. The Viera et al. (2013) results are based on external estimates of exposure, whereas the similar stu dy by Barry et al. (2013) are based on serum levels or estimates based on serum levels. The fact that association with kidney cancer is present in the Viera study decreases concern that the association was due to reverse causality. Steenland et al. 2012 u sed an external estimate of exposure to study cancer mortality and also found an association with kidney cancer. Lundin et al. (external estimate of exposure) had no cases of kidney cancer, though their study was also

small.

Angela L. Slitt

The epidemiol ogy data is well described and a thorough read. The data would be put in better context for the reader if there are average serum concentrations or ranges for the studies summarized in tables in addition to other key pieces of information.

A recent publication should be included in the document for consideration. Simon N, Fletcher T, Luster MI, Steenland K, Calafat AM, Kato K, Armstrong B. Epidemiology. 2013 Jul;24(4):569

- 76. doi: 10.1097/EDE.0b013e31829443ee.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

34 Question 5

. The OW has concluded tha t the cancer classifications for PFOA and PFOS are most consistent with respective classifications of suggestive evidence for carcinogenicity as described the EPA Guidelines for Carcinogen Risk Assessment (pp. 2

- 56, 2

- 57). Please comment on the strengths a nd weaknesses of this classification.

James V. Bruckner

PFOA

- specific comments I agree with EPA’s choice of “Suggestive Evidence for Carcinogenicity .” Epidemiological findings in occupationally

- exposed and general populations to date are equivocal. Increases in Leydig cell tumors and liver adenomas have been reported in high

- dose male rats. Increased incidences of pancreatic cell hyperplasia/adenomas an d ovarian stromal hyperplasia/adenoma

have been observed in female rats. More studies are necessary to confirm/expand these findings, and to assess carcinogenic po tential in other species. Most mutagenicity and genotoxicity assays have been negative. Thus, there is some, but not undue cause for concern about the human carcinogenic potential of PFOA.

PFOS

- specific comments The document’s authors have adequately and convincingly presented evidence for classifying PFOS as “suggestive of carcinogenicity

. ”

Deb orah A. Cory

- Slechta

The classification of both PFOA and PFOS evidence for carcinogenicity as suggestive seems consistent with the clear limitations in the available data bases. In addition, the animal studies are limited to one species and mutagenicity d oes not occur in response to PFOA.

Jamie C. DeWitt

This classification is appropriate for both PFOA and PFOS given the epidemiological evidence, which is somewhat limited for PFOA and quite limited for PFOS. For PFOA, there is an

association between kidney and testicular cancer, but there are limited data in animal models for these cancers and there is uncertainty that the mechanism of PFOA

- induced carcinogenicity in animal models is applicable to humans. Studies of PFOS have the same limitations, but epidemiological studies have failed to find an association between PFOS exposure and cancer.

Jeffrey W. Fisher

I did not review the cancer studies for PFOA and PFOS.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

35 William L. Hayton

The classification of “suggestive” is not unreasonable. The epidemiological studies, while showing apparent associations between PFOA exposure and cancer incidence in testicle and kidney as well as other tissues, do not provide a cau se

- effect relationship. However, they certainly do raise a concern about the carci nogenicity of the subject substances. Studies in animals have demonstrated conclusively that PFOA causes liver cancer in rats but the MOA that

involves PPAR activation is absent in humans and it has been concluded that PFOA and PFOS cannot be carcinogenic in humans via this mechanism.

An EPA SAB panel (2006) consideration of this question resulted in a majority of the panel members favoring a classification of “likely to be carcinogenic” for PFOA. Board members acknowledged the PPAR MOA argument against causation of cancer in humans, but also found evidence that liver cancer in rats administered PFOA may also have had a MOA independent of PPAR activation. Recent epidemiological studies have added to the weight of evidence for an

association between PFOA/ PFOS exposure and cancer. Therefore a classification of “likely” is also not unreasonable to this reviewer. Lacking expertise in the nuances of applying the EPA’s classification scheme, it is difficult for this reviewer to argue in favor of either “sugge stive” or “likely”.

Matthew P. Longnecker

The classification as “suggestive evidence for carcinogenicity” for both PFOA and PFOS is consistent with the guidelines put forth in the EPA Guidelines for Carcinogen Risk Assessment (2005). There are few perti nent data, including some suggestive but weak human evidence. There is clearly not enough evidence to classify these agents as likely human carcinogens.

Angela L. Slitt

Overall, the assessments for each PFOS and PFOA appear to be consistent with the EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a)

.

Strengths: Both classifications use evidence from human studies as guidance.

PFOS: The limited data that exist regarding PFOS and cancer were presented, the classif ication

for PFOS under t he EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) is currently consistent with the suggestive evidence of carcinogenic potential descriptor. This assessment is reasonable, given that it is based on two studies that show a slight increase in

adenomas that occurred in males and females.

PFOA: There is conflicting evidence regarding PFOA exposure and cancer risk. However, several human studies have found associations between PFOA exposure and elev ation of cancer

of the bladder and kidney. This is also supported by a chronic bioassay in rats, which demonstrated that PFOA was tumorigenic.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

36 Question 6

. Significant interspecies differences in pharmacokinetics exist for both PFOA and PFOS. Adjusting for interspecies differences was an important step in developing candidate RfDs given the totality of the human and animal data. Please comment on the strengths and weaknesses of the pharmacokinetic model adjustments to accommodate the impact of albumin binding and renal tubule transporters in determ ining average serum

values.

James V. Bruckner

PFOA

- specific comments The adjustments made to accommodate the influence of albumin binding and saturable renal tubular resorption of PFOA seem reasonable. I would defer, however, to someone with more experi ence in providing for these processes in PBPK models.

PFOS

- specific comments The PBPK model adjustments to estimate human equivalent doses appear to be appropriate. I defer to someone more qualified on the subject.

Deborah A. Cory

- Slechta

This falls outside my area of expertise and therefore no significant comments are provided. However, at the face

- to

- face meeting there was significant discussion regarding the modeling, including clarifications from Dr. Wambaugh that were found by those pa nel members with expertise to clarify these issues and these particular issues were considered adequately

addressed.

Jamie C. DeWitt

Several PK models have been reported in the literature for these compounds and are relatively

well described in the docu ments. The documents assert that the existing PK models do not consider the impact of renal tubule transporters and albumin binding; while, many of the existing models appropriately predict serum concentrations in humans and other species, but they are mos tly based on empirical models. Please explain the weaknesses of such empirical

models.

Additionally, numerous studies for both compounds report serum and tissue concentrations in humans and other species, which can be compared to existing models. Both doc uments present a revised model that amounts to a reanalysis of data from studies that report serum concentrations. A more thorough discussion of the improvements made by the reanalysis is needed to better understand if the improved model adequately estimat es or predicts the clearance rate and other parameters for which confidence is low. Alternatively, the publication (Wambaugh et al., 2013) that thoroughly describes the reanalysis could be referenced.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

37 Jeffrey W. Fisher

Serum protein binding: Both PFOA and PFOS are highly bound in serum proteins across species, thus model adjustments seem trivial for interspecies extrapolation. Steady

- state conditions can be assumed to estimate the free fraction ( e.g. , 2% ba sed on paper by Han et al.,

2003 for humans). I did not find a discussion about the half

- life of serum proteins, which may have some influence on the ‘apparent’ serum half

- life of PFOA and PFOS. The estimated fraction of free PFOA or PFOS is important for describing urinary and fecal el imination in rats (and other species) and the plasma concentrations of total PFOA and PFOS. Thus, the model predicts total PFOA and PFOS in serum or plasma, but the free fraction estimates drive the gradual clearance of total BPA from plasma or serum by de scribing clearance of free.

Renal reabsorption: The renal reabsorption hypothesis involving species specific and sometimes gender specific transporters to describe the pharmacokinetic data represents sound judgment. This departure from normal allometric scaling is suggestive of active transport processes. Few PBPK models explicitly describe transporters with drugs or chemicals, although the field is moving in this direction. Thus, the approach used for PFOA and PFOS is adequate, that is, a hypothesis was eval uated by employing empirical PK

- based kinetic analyses. Because the mechanistic details are missing for each species/gender, scaling of this biological phenomenon is not possible at this time. This is not a weakness, but represents the state of th e science.

William L. Hayton

A very important strength of the documents is the attempt to deal with the interspecies differences in pharmacokinetics so that adverse effects across species are compared on the basis of internal, systemic exposure to PFOA and PFOS, instead of basing comparisons on the administered mg/kg dosages. PFOA and PFOS have complicated pharmacokinetics that have proven difficult to model. While a relatively simple one

- compartment model appears adequate to analyze single, low doses, this model fails when it is extended to higher doses and repeated doses. Nonlinearities appear associated with saturable plasma protein binding and with saturation of transporters thought to be involved in the reabsorption of the compounds from

ren al filtrate.

A weakness of the pharmacokinetic model adjustments is the lack of robustness of the models. Despite the extensive efforts of talented pharmacokineticists, development of a model that scales across species and handles a range of dosages an d a variety of administration routes has proven elusive. The two compartment model of Andersen et al. (2006) has formed the basis of the model used in the draft documents. The model incorporates saturable resorption of PFOA and

PFOS from renal tubular fi ltrate. While protein binding is known to be saturable (fraction free increases with concentration), the model uses a species

- specific but constant free fraction. Model parameter values for mouse, rat and monkey were used to predict reasonably well

measu red serum concentrations after a fixed daily dosing regimen, Tables 5

- 6

– 5

- 8 for PFOA

and 5

- 8 and 5

- 10 for PFOS. This agreement between predicted and measured serum concentrations gives confidence that the model

- calculated AUC values and final serum

conc entrations associated with adverse health effects (or in the case of liver weight, biological marker of exposure) are realistic and a basis for estimation of RfD. While the model usedExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

38 appears adequate for the purpose, the model parameters that were used h ave some markedly

non

- physiological values. (Information subsequently provided at the reviewers meeting explained some of the departure from expected physiological values, as discussed in a following section.)

Matthew P. Longnecker

For PFOA and PFOA, the MCMC model results (predicted final serum value) were compared to the measured final serum values, and the agreement was fairly good. For PFOS, the MCMC model results were compared to those from Loccisano et al. (2012b) and were found to be similar, w hich is also reassuring. Because the PBPK models of PFOA and PFOS are empirical, and have been shown to give results that agree reasonably well with observed data, the adjustments to accommodate the impact of albumin binding and renal tubule transporters are not critical. More data on albumin binding and renal tubule transporters might allow improved understanding of the pharmacokinetics of these compounds, but may not necessarily cause substantial improvements in the empirical predictions from current mo dels.

Angela L. Slitt

The current weakness of the models is that data on species differences in PFOA and PFOA for various key transporters is limited and the document is also using mRNA data for various transport proteins to explain gender differences in urinary elimination. First, with regard to PFOS accumulation in the liver compartment, it is necessary to compare affinity of human

versus rat for OAT p mediated transport. This alone is tricky because of species differences in

OAT p s. If PFOS

- induced li ver effects are related to PFOS accumulation in liver, it is would be helpful to understand whether a lower affinity of human OAT p 1 b 1 and 1 b 3 compared to rat

O AT p1a1 predicts lower hepatic PFOS accumulation. More is known about PFOA, but a similar argumen t can be made for PFOA. In addition, more comprehensive, controlled assessment of renal transporter affinity for PFOA and PFOS is needed to better model the species difference in urinary elimination.

The document often speculates about PFOA or PFOS regul ation of transporter expression, but some papers cited (Cheng and Klaasen) do not have enough data at the protein level to support whether these differences in transporter expression are the drivers of toxicokinetic differences

between males and females.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

39 Question 7

. Table 5

- 5 in the PFOA document and Table 5

- 7 in PFOS document list the parameters used for the ORD pharmacokinetic models that provide the final serum and AUC values for calculating the internal dose point of departure for the RfD calcu lation. Please comment on the strengths and weaknesses of the selected parameters.

James V. Bruckner

PFOA

- specific comments Despite the complexities and unknowns involved in plasma protein binding and renal tubular functions (i.e., glomerular filtration, basolateral tubular excretion and resorption, and apical tubular excretion and resorption), it is necessary to: (a) simply model only for saturable tubular resorption; and (b) use a range, or distribution of parameter values consistent with exi sting kinetic data. Unfortunately, optimization sometimes results in selection of physiological parameters that are not biologically

- realistic, or plausible.

PFOS

- specific comments The parameters used in the modeling are biologically plausible.

Deborah A . Cory

- Slechta

This falls outside my area of expertise and therefore no significant comments are provided. However, at the face

- to

- face meeting there was significant discussion regarding the modeling, including clarifications from Dr. Wambaugh that were f ound by those panel members with expertise to clarify these issues and these particular issues were considered adequately

addressed.

One unclear component of Table 5

- 7 in the PFOA document is the column labeled Species/Strain Used for prediction, which in every case is the same as the column labeled Species/Strain and is not otherwise adequately explained.

Jamie C. DeWitt

It is not clear that the parameters in Table 5

- 5 i n the PFOA document and Table 5

- 7 in the PFOS

document are from the Andersen et al . 2006 PK model or if they are parameters used in the reanalysis of the data. This needs to be better explained in both documents. Additionally, all of the units in the tables need to be explained and re

- checked for accuracy.

Jeffrey W. Fisher

The author s should entertain the calculation of data derived AUC (e

. g. Table 5

- 6) to compare to

the model derived AUC

, just as was done with measurement of total PFOA in serum. This works for the animal studies. The choice of using the empirical model over the more recent physiological models may be a weakness and our understanding of transporters advance.

The evolution of chemical

- specific PBPK models for use in risk assessment and regulatory applications has repeated itself several times. This is, the first empir ical non

- physiological model(s) or PBPK models contain hypotheses generating ideas and later models test some ofExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

40 these hypotheses

, especially if additional experimental data become available. In the case of

PFOA and PFOS

, the EPA selected not to use the most recent PBPK models for PFOA and PFOS, but instead use a computational empirical based model (Andersen et al. 2006) that was the first attempt to quantitatively interpret the kinetics of PFOA and PFOS across species of laboratory animals. T he authors did publish their model (Wambaugh et al., 2013). The authors chose not to use a human model because a lack of information for Bayesian analyses. The justification for their extrapolatio n methods should be stated and the published reference for t he

mod el should be cited.

Model parameter distributions (Bayesian analyses) appear to be biologically implausible in some cases, covering many orders of magnitude. The authors should discuss this issue and check the units of model parameters in Tables.

Both model parameters tables need to include a description of what the parameter represents and cite a figure. The figures showing the Andersen et al. 2006 model do not show all the model parameters and have different nomenclature.

The Andersen et al. 2 006 paper is a critical paper offering a quantitative explanation for the

PFOA and PFOS kinetic data sets.

William L. Hayton

In the “Pharmacokinetic Model Approach” sections of the documents, it is not made sufficiently clear that the parameter values i n Table 5

- 5 (PFOA) and Table 5

- 7 (PFOS) were from re

- fitting the published data, rather than using parameter values from the original literature reports.

PFOA Table 5

- 5, p. 5

- 12 Body Weight and Cardiac Output values are reasonable and typical.

k
a values for mouse and monkey seem extremely large; absorption half

- lives would be on the order of 10 seconds, which is physiologically unrealistic. All of an oral dose would be absorbed within a minute, mimicking a rapid i.v. bolus dose. Serum concentration

- tim e profiles may not be sensitive to these values, however so they are not disconcerting for the intended use of the models. The rat values appear reasonable.

V cc values appear reasonable. The total steady

- state volume of distribution value [V
ss = V cc x (1 + R v2:v1 )] compares favorably with one

- compartment V d values for CD1 mouse, but V
ss values for the other columns (species) appear too large, due to the large R v2:v1 values.

k 12 values vary a lot across the columns, suggesting that k 12 may be highly correlated with another parameter (e.g., R v2:v1 ).

R v2:v1 values also vary a lot across the columns.

T maxc values are consistent across the columns; expressed in Gm/hr, they seem very large. For example, 2032 Gm/hr (4.91 moles x 414 Gm/mole) for the CD1 m ouse. Even on a kg body weight basis could mouse renal tubules resorb 2 kg PFOA per hour? This maximum rate of resorption must far exceed the rate of filtration of PFOA at the glomerulus. (Clarification at the reviewers meeting explained this apparent d eparture from physiological reality. The

units had been mis

- specified in Tables 5

- 5 and 5

- 7. They were in fact micromole per hourExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

41 and micromolar for T
maxc and k T instead of molar based. Thus T
maxc mouse value was 2 mg/hr, which is physiologically plausib le.)

k T values are the concentration in glomerular filtrate that half saturates the resorption transporters. Expressed in mg/mL, they seem large, much larger than the urine concentration that would be expected; e.g., for CD1 mouse, k T is 15 mg/mL where fre e serum concentrations (Free x C
serum ) would be about 0.3 μg/mL with 10 mg/kg in the mouse. So the transporter would not become saturated except at extreme doses. The value used by Andersen et al. (2006) for monkey was 0.00001 mg/mL. Unit specified in T ables

5

- 5 and 5

- 7 should be  M, not M. Free fraction values measured in vitro are 0.01 or less at low PFOA serum concentrations (Table

3

- 1). The Free values for rat seem much higher than the measured values. Q filc is defined as a fraction of blood flow (r enal or cardiac output?) to the filtrate (bottom of p.

5

- 11) but has units of flow in Table 5

- 5.

V filc values are much smaller than the 0.01 L value used by Andersen et al. (2006), although Andersen et al. state that the model output is insensitive to th is parameter and that their

value was assumed.

PFOS Table 5

- 7, p. 5

- 15 Body Weight and Cardiac Output values are reasonable and typical.

k
a values for female mouse and monkey seem extremely large

– see comment above for PFOA.

V cc values appear reasonable. See comment above for PFOA.

k 12 values vary a lot across the columns, suggesting that k 12 may be highly correlated with another parameter.

R v2:v1 values appear reasonable and consistent with other reports of V
ss values for PFOS.

T maxc values are highly variable across the columns and seem much higher than physiological reality would allow. See comment above for PFOA.

k T values are physiologically unrealistic and highly variable across columns. See comment

above for PFOA.

Free fraction values have been measured in vitro and are 0.01 or less at low PFOS serum concentrations (Table 3

- 1, p. 3

- 3). The Free values in Table 5

- 7 are consistent with the

measured values. Q filc is defined as a fraction of blood flow (renal or cardiac out put?) to the filtrate (bottom of p.

5

- 14) but has units of flow in Table 5

- 7.

V filc values are much smaller than the 0.01 L value used by Andersen et al. (2006), although Andersen et al. state that the model output is insensitive to this parameter and th at their

value was assumed.

While the parameter values for the pharmacokinetic models predict reasonable serum concentrations that generally agree with measured values (Tables 5

- 6

– 5

- 8 for PFOA and

Tables 5

- 8 and 5

- 10 for PFOS), their high interspecies v ariability suggest that the models may be unreliable for prediction of internal exposures after other intake regimens and during a depuration phase.

Matthew P. Longnecker

Please see the answer to the previous question.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

42 Angela L. Slitt

The parameters inc luded appear to be appropriate, but this lies outside of my area of expertise.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

43 Question 8

. The volume of distribution (Vd) and half

- life values are critical in the derivation of the interspecies uncertainty factor applied in derivation of candidate RfDs from a NOAEL, LOAEL or a BMDL. The available data for both values are provided in Section 3.5.2 and 3.5.3 of both documents. Please comment the strengths and weaknesses of

the values selected.

James V. Bruckner

PFOA

- specific comments

The adult male rat data of Kemper (2003), from which the rat half

- life and clearance (CL) were obtained, appear to be solid. It is reasonable to select the human half

- life of 2.3 years reported by Bartell et al. (2010), as their study population included equal numbers of ma les and females. Division of the rat CL by the human CL to yield a value of 219 is fine. I did not examine the publication of Bartell et al. (2010) to evaluate their data or methodology used to derive a human

half

- life of 2.3 years. Therefore

, I am uncerta in about its accuracy.

PFOS

- specific comments I would again defer to someone with more expertise.

Deborah A. Cory

- Slechta

This falls outside my area of expertise and therefore no significant comments are provided. However, at the face

- to

- face meeting there was significant discussion regarding the modeling, including clarifications from Dr. Wambaugh that were found by those pa nel members with expertise to clarify these issues and these particular issues were considered adequately

addressed.

Jamie C. DeWitt

While the overview of the individual studies that calculated Vd and half

- life for each compound was detailed and complet e, the rationale and analysis concerning why particular values were selected were insufficient. Additionally, a s addressed in Charge Question 6, the rate of clearance/elimination likely contributes to the differences in half

- life that are not associated wi th differences in the Vd. Therefore, a 3

- fold uncertainty factor for species differences in pharmacodynamics (UF A ) was utilized for both compounds. What was the justification for using

a UF A of 3? The section on UF application needs a more thorough discuss ion regarding the choice of this value given differences in clearance. If the section on model adjustment (a suggestion in Charge Question 6 ) is better described, this comment may no longer be applicable.

Jeffrey W. Fisher

The use of this non

- compartmental method should be justified. Why not use a PBPK model? Assuming steady state in the humans does allow for calculation a human equivalent serum concentration associated with a laboratory animal concentration. In what regio n of the

exposure

- dose range would nonlinearity occur in humans? Some type of discussion is neededExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

44 about the assumptions of this methodology and why it was used. I would like to see statements about if the NOAEL, LOAEL, and a BMDL doses are in the linear r ange for kinetics.

The authors should use the Bayesian analysis for animal studies to inform the UF. Use percentiles to explore Vd and half

- life to support UF values. I did not see any attempt to use distribution information generated from the model bey ond the central tendency or mean values. Please state why this is the case. It seems that the distribution information generated from the Bayesian analysis could be used to support UF development.

William L. Hayton

PFOA

- specific comments For male rat, th e Kemper (2003) study appears to be the best source of pharmacokinetic parameter values, which were obtained by a model

- independent analysis of serum

concentration

- time data from rats that were dosed by oral gavage at dosages of 0.1, 1.0, 5.0 and 25 mg/kg. In addition, there was a 1.0 mg/kg dosage administered intravenously, and a 0.1 mg/kg oral gavage dose with an extended sampl ing time. Each treatment used four animals.

The CL and t 1/2 values appeared to be independent of dosage and route of administra tion. It would therefore be reasonable to average all 6 mean values for each parameter to give an over

all mean of 24 determinations. The average (n=24) values for male rat were CL = 0.0209 L/kg/d

and t 1/2 = 7.83 d. These values can be used to calculate a V d value (t 1/2 x CL / ln 2), which is 0.236 L/kg.

It is not apparent on p. 5

- 20 why a V d v alue of 0.17 was used with half

- life to calculate CL rat when Kemper (2003) reported CL values and not t 1/2 values. (At the peer review meeting

, it was clarified that the data of Kemper (2003) were re

- analyzed and as a result the parameter values in the health effects documents differ somewhat from those published with the data in the original reports.)

The CL

human value was taken to be 0.00014 L /kg/d. There are no direct measurements of this parameter. Thompson et al. (2010) assumed that the intake rate of PFOA for subjects using

PFOA

- contaminated water was 91% of the PFOA in 1.4 L/d of water. This intake rate was used along with a PFOA half

- l ife of 2.3 years to calculate a V d value of 0.17 L/kg. This is the same

va lue that was used in the health effects document for the rat (p. 5

- 20). The V d values available in mouse, rat and monkey are about 0.2 L/kg, so the V d,human set at 0.17 L/kg is not unreasonable but it lacks the certainty of the rat V d value.

The health effects document used a t 1/2 for PFOA in human of 839.5 d (2.3 years), which seems to be toward the low end of the range of values that have been reported. Along with Vd = 0.17

L/ kg one arrives a t CLhuman = ln 2 x 0.17 / 839.5 = 0.00014 L/kg/d.

The ratio CL rat / CL human calculated using the mean CL rat from Kemper ( 2003) would be 0.0209 /

0.00014 = 149, which is about twice the value calculated on p. 5

- 21. This difference aris es from the calculation of CL rat using the V d,human and a half life of 11.5 d instead of using the CL rat directly from Kemper (2003). The mean half life from Kemper (2003) was 7.8 d.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

45 The CL
mouse / CL human ratio is accurate, using Lou et al. (2009) data. A calculation for monkey is

not shown.

PFOS

- specific comments Chang et al. (2012) appears to be the best source of pharmacokinetic parameter values for mouse, rat and monkey. Butenhoff and Chang (2007) is given as the reference for a 48

- day half

life in rat; this is a final report, internal to 3M. The Chang paper gives half

- life values for male and female Sprague

- Dawley rat at 2 mg/kg and 15 mg/kg. The average V d for the four groups of three/group was 0.71 L/kg. This is higher than the 0.23 L/kg value used in the draft document (p. 5

- 23). The 0.71 L/kg value is also higher than values for mouse, monkey and human, which are closer to the 0.23 L/kg value used in the draft document. The draft document ought to acknowledge this difference; it may be that the value in the 3M report is lower than the published value; Chang was a co

- author for both sources. The Chang et al. (2012) paper gives

CL rat values that are 0.0051 L/h/kg for female (similar for 2 and 15 mg/kg doses) and for males,

0.022 and 0.011 L/h /kg for the 2 and 15 mg/kg doses. A single average value for CL rat would be 0.011 L/h/kg, about 3 times the value used for the UF A calculation in the draft document. The

male value is about 2

- 3 times the female value and it may be appropriate to calculat e a different

UF A value for each sex. Using the single CL rat averaged across two doses and both sexes (0.011 L/H/kg) would give a CL rat / CL human ratio of 0.011 / 0.000081 = 135 and a UF A = 407, substantially higher than the value of 123 in Table 5

- 15.

The UF A values calculated for mouse and monkey appear to be in line with the literature values for PFOS CL values in these species.

Matthew P. Longnecker

The PBPK model of Loccisano et al. 2013 (for humans) can be used to calculate a volume of distribution for PFOA of 177 ml/kg, which is very close to the value of 170 ml/kg based on Thompson et al.’s 2010 one

- compartment model . For PFOS, the corresponding value from the PBPK is 280 ml/kg, compared with the value of 230 ml/kg used in the Health Effects Document. This 22% difference could have an impact on some calculations. (Note: the PBPK model

- based volumes of distribution were calculated by Marc

- Andre Verner of the University of Montreal. He had calculated these values in the course of a separate project.)

For humans, the half

- life data all depend on the assumption that ongoing exposure is negligible compared to baseline exposure, a reasonable assum ption in most of the populations used to

estimate half

- life. While the Seals et al. (2011) gave estimates that were slightly different for PFOA in some cases, the methods employed in this study were not as strong as for Bartell et al. (2010) or the Burris et al. studies (2000; 2002). The agreement within species for the half

- life estimates for PFOS are reassuring. The animal data on the half

- life of PFOA are relatively sparse (2 rat studies that agreed reasonably well, 1 mouse study, 1 monkey study). Fo r PFOA,

the UF A s and RfD that were calculated based on the half

- lives (expressed as clearance) would not have been substantially altered by alternate choices for specific values. The same is true for

PFOS.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

46 Angela L. Slitt

Strengths of the available data is that for the several species thorough evaluated, the half

- life values are very consistent. For example, the several human studies cited report a range in

calculated PFOS half

- life in humans to be 4.1

- 8.67 years, two studies putting monkeys at 110

- 132 days, and rat generally has a narrow range with 3 out of 4 values provided ranging from

39.8

- 48.2 days for PFOS. An inconsistency is the Chang et al., 2012 describing a half

- life of females of 66.7 days when in general female rodents may have faster e limination of PFOS.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

47 Question 9

. A variety of endpoints and studies were used to compare points of departure and the resultant RfDs for both PFOA and PFOS. In addition, comparisons were provided across RfD outcomes based on the model outputs compared to those for the NOAEL, LOAEL and BMDL points of departure. The range of candidate RfDs derived from the different points of departure is fairly narrow. Please comment on the strengths, weaknesses

and t ransparency of this analysi s.

James V. Bruckner

PFOA

- specific comments The procedure used to calculate PODs adheres to standard EPA guidelines and policy. The presentation of their derivation is clear, concise and transparent. It is certainly interesting that the range of PODs and resulting candidate RfDs is so narrow. Nevertheless, as discussed previously, I do not agree with their selection.

PFOS

- specific comments See my comments under General Impressions.

Deborah A. Cory

- Slechta

While it is the case for both PFOA and PFOS that values from different points of departure are fairly narrow, the transparency of the analyses in neither case is clear. There is no rationale described even as to why these analyses were done on all of the studies, what was the primary study and how o thers related to that etc., i.e., this presentation does not follow the typical presentation format of IRIS documents in either its presentation of rationales and strategies, nor in the conclusions that it reaches. In both cases, it is only the single sent ence indicating that modeling from one particular study will be protective of effects at other studies using higher exposures. This section in both documents needs introductory paragraphs that describe the specific strategy, choices of studies and the rati onales for those choices.

As noted in response to Charge Q uestion 3, the rationale for discarding the human epidemiological studies is not sufficient and requires rationale other than that stated and therefore, the question of using the human data remain s open. As noted in response to Charge Q uestion 1, in this reviewer’s opinion, the increased liver weight can be justified as a departure point for assessment of RfDs, but as discussed at the face

- to

- face meeting, additional text supporting this choice is needed.

Jamie C. DeWitt

This particular section contained inadequate detail on why particular studies were or were not chosen. For example, immunotoxicity as an endpoint was not chosen for PFOS, based on “ in

vitro measures of immunocompetence on mice may not be relevant to the human experience and limited human data from epidemiology studies are inconclusive regarding the immunotoxicity of PFOS in humans”; however, the breadth of data from in vitro/ex vivo immunotoxicity studies

for PFOS were not thoro ughly discussed (please see Charge Question #2 for two additional in vitro studies).External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

48 For both compounds, an increase in absolute liver weight was selected as an endpoint as it was a

common effect [sic] in both short and long term studies. However, the toxi cological relevance of an increase in absolute liver weight was not discussed other than to indicate that it was a sign of altered homeostasis. Further, the co

- occurrence of increases in absolute liver weight with other toxicologically

- relevant endpoints ( i.e., immunotoxicity and/or reproductive/developmental toxicity) is not a toxicologically valid justification for the use of liver weight as an endpoint for an RfD. Therefore, the analysis was not sufficiently transparent to deduce its relative strengths

a nd weaknesses. Certainly, choosing an endpoint that occurs across species and occurs at relatively low doses will likely be protective of exposed humans; however, will it be a defensible endpoint? As currently written, the choice of this endpoint for an Rf D is not adequately defended.

Jeffrey W. Fisher

I did not review the toxicity data.

William L. Hayton

For PFOA, a 10% increase in liver weight was selected as the metric for effect, which was “... not made based on toxicity but on the desire to find a common denominator against which to

evaluate dose

- response across studies and justified by the fact that other adverse effects accompanied the LOAEL for increased liver weight in some cases .” The lowest serum

concentration associated with an increase in l iver weight was calculated for female mouse to be 20.33 mg/L (p. 5

- 16, PFOA document). These data are referenced to DeWitt (2008); this paper has only summary information on liver weights, all of which exceeded 20% weight gain, going as high as 70%; and i t is not apparent in PFOA document how these liver weight gains were used to estimate an LOAEL for 10% liver weight gain.

Many of the animal studies of hazard assessment were conducted under conditions where the duration of the exposure was relative ly s hort compared with the half

- life, and steady state had not been achieved. It is not apparent how the NOAEL and LOAEL values from such studies were adjusted to account for the non

- steady state situation. For example, the 20.33 mg/L PFOA

concentration asso ciated with a 10% increase in liver weight (Table 5

- 9) emanated from a 15 day drinking water exposure to 0.94 mg/kg/day that resulted in an average serum exposure of 20.33 mg/L (0

– 29.7 mg/L over 15 d, Tables 5

- 7, 5

- 9). For a fixed daily dose, the time t o 90% steady state for mouse would be about 63 days (3.3 x half life, which was 19 days), and after 15 days the serum concentration would only be about 15% of its steady

- state value. This seems to suggest that the RfD would have been over

- estimated by a f actor of 7, since the 0.94 mg/kg/day at steady state would have produced a serum concentration of about 150 mg/L, not 20.33 mg/L. This analysis is based upon the behavior expected from one

- compartment model pharmacokinetics. As discussed on p.5

- 9 of the PFOA document, the steady

- state serum
concentration of PFOA is achieved in a much shorter time than one

- compartment model kinetics would predict. Whether the target

- site steady

- state concentration of PFOA also occurs in a

much shorter time than one

- compar tment model kinetics would predict (3.3 x half life) is apparently unknown.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

49 Matthew P. Longnecker

This part of the document seemed especially strong and transparent. The agreement between methods was reassuring. The weaknesses and assumptions were well discussed. Please see the

minor editorial c omment on this issue given for Charge Q uestion 1, above.

Angela L. Slitt

The RfD Point of Departure was based on animal studies that include monkey and rat.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

50 Question 10

. The RfDs for PFOS and PFOA are derived from the modeled steady state serum concentrations and their association with effects that include short term and longer term exposures with associated diverse effects. The studies considered included effects due to exposure durations that ranged f rom 11 to 182 days, and occur at comparable human equivalent dose (HED) levels. The current, draft RfDs do not include an uncertainty factor for study duration because of the apparent concordance HEDs despite duration differences. Given this pattern of res

ponse, is it appropriate to conclude that the candidate RfDs are applicable to both short

- term and lifetime exposures?

James V. Bruckner

PFOA

- specific comments

I do not believe it is appropriate to conclude that the candidate RfDs are applicable to both sho rt

term and lifetime exposures. Steady

- state is apparently achieved in monkeys within 4

- 6 weeks (Butenhoff et al., 2002). Steady

- state likely takes considerably longer in hu mans. Thus, RfDs for shorter periods of exposure should be based upon results of studies of similar duration.

PFOS

- specific comments

I do not believe the candidate RfDs, as calculated, are applicable to different durations of

exposure.

Deborah A. Cory

- Sl

echta

While initially believing that it was appropriate conclusion for PFOA and PFOS, based on the correspondences in RfDs across short and longer term exposure, discussion at the face

- to

- face meeting made clear that this approach is not reasonable and re quires additional consideration.

Jamie C. DeWitt

This approach may be appropriate given the relative similarity of serum concentrations attained regardless of study duration, i.e., steady state in serum is attained after a relatively short period

of exposure. This appears to be consistent across studies with various species of animal models. However, the document authors might need to reconsider given what we may or may not know about liver hypertrophy. In the Hall et al. (2012) paper on liver hypertr ophy (discussed during the public meeting), increase in liver weight is an adaptive response that may not be adverse UNLESS weight increases >150% over a three month or longer period may. Following this large and prolonged increase in weight, the end resul t may be a hepatocarcinogenic response. However, none of the studies contained in the documents indicate that longer term exposures increase liver weight to this degree.

Jeffrey W. Fisher

The departure from K=CXT (Haber’s law) should be based on the toxi city endpoints of concern

and what is known about dose

- exposure kinetics/responses for these chemicals and other chemicals that target the same endpoint, not that the HED values are comparable. The NASExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

51 AEGL committee only considered primary irritation for inhaled chemicals as an endpoint that was independent of duration of exposure. There is an SOP if needed for reference. The time to steady state should be included in a table for the lab animals. Toxicity studies conducted for less than 30 days (perhaps? ) are not at steady state for the pharmacokinetics of

PFOA. Thus the measured serum levels would be different tha n at steady state. The shorter the duration of the toxicity test, the more impact this could have on extrapolation to chronic exposures in huma ns. My personal preference would be to use PBPK models for all species and consider only long term exposures for extrapolation to humans.

William L. Hayton

This depends in part on how quickly the PFOA/PFOS concentrations at sites of toxicity come to steady state. Since the Vd for these chemicals is small (~ 0.3 L/kg) it seems likely that the concentrations in tissues rise in pseudo equilibrium with the rise in serum concentration. That said, the half lives are relatively long due to the very small c learance (t 1/2 = ln2 x V d / CL). If

one

- compartment kinetics apply, then a guideline for time to 90% steady state is 3.3 t 1/2 . For studies that expose animals for a period of time shorter than 3.3 t 1/2 , the serum concentration would not be at steady state and the internal systemic exposure (serum concentration) would be less than what it would be if the exposure were longer than 3.3 t 1/2 . This effect would seem to lead to overestimation of the intake rate that was associated with a particular interna l exposure and associated biological endpoint. For example, the t 1/2 of PFOS in mouse is about 36 days and

3.3 t 1/2 is 120 days. Consider a 28

- day exposure using a fixed daily dose that produced an LOAEL of “X” mg/kg/day. On Day 28, the body level would only be 42% of the steady state level, and the average body level over the 28

- day period would be about 21% (approximating the increase as linear and not exponential). The true LOAEL would be 0.21 “X” mg/kg/day; i.e., intake of 0.21 “X” mg/kg/day would p roduce a body level at steady state that was the same as the average body level produced by X mg/kg/day administered over 28 days. The time to 90% steady state for a fixed intake rate is quite long; from the literature in the health effects documents, the times in the following table were calculated. From this line of reasoning, exposure times less than two half

- lives begin to significantly overestimate intake rates associated with particular endpoints. This analysis is based upon the behavior expected f rom

one

- compartment model pharmacokinetics. As discussed on p.5

- 9 of the PFOA document, the steady

- state serum concentration of PFOA is achieved in a much shorter time than one

compartment model kinetics would predict. Whether the target

- site steady

- stat e concentration

of PFOA also occurs in a much shorter time than one

- compartment model kinetics would predict (3.3 x half life) is apparently unknown.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

52 CL Vd t 1/2 Time to 90% steady state

[mL/d/kg] [mL/kg] [d] [d]

Species PFOA PFOS PFOA PFOS PFOA PFOS PFOA PFOS

Mouse 6.6 5 180 265 19 36 63 120

Rat

- Male 23 16 273 947 8.4 40 28 92

Rat

- Female 776 5.2 150 476 0.13 66 0.43 218

Monkey 6.3 1.4 190 238 27 121 89 400

Human 0.085 0.08 170 230 1378 2000 12.5 yr 18 yr

In addition, this line of reasoning may be incorrect if the assumption of one

- compartment
kinetics is incorrect. For multi

- compartment models the serum concentration and target organ/tissue could come to their pseudo steady state levels relatively quickly while slowly equilibrating (deep) sites slowly approached steady state. Simulation with PBPK models for PFOS and PFOA may help answer this question.

Associated with the uncertainty introduced by exposures that were shorter than the time to achieve steady

- state concentration at the target site is the exposure time required for the adverse effect to be expressed. While some adverse effects may occur immediately and directly in proportion to the concentration of PFOA o r PFOS at the target site, other adverse effects may be slow to become manifest. These “indirect adverse response” behaviors are well known in the drug action arena; e.g., certain antidepressant drugs require several weeks exposure to the target

site befo re the effect of the drug appears. This lag time is not associated with pharmacokinetics (time to steady state) but with indirect

- response pharmacodynamics. It could be argued that uncertainty factors are needed for both pharmacokinetics (pre

- steady stat e condition) and pharmacodynamics (or toxicodynamics) to account for possible indirect response behavior.

Matthew P. Longnecker

EPA might want to consider using an uncertainty factor for duration, for two reasons. First, the monkey data for PFOS used for the point of departure were from a study where the duration of exposure was relatively short

- term relative to the half

- life, and it appeared that duration of dose affected liver and other adverse outcomes detected at higher doses, and no monkey data were used in the POD for PFOA. Second, questions raised by Drs. Hayton and Fisher at the peer

review meeting made me less comfortable with the calculations that used average serum concentration derived from the AUC and duration of dosing to compare with humans, who are more likely to be near steady

- state.

Angela L. Slitt

Yes, but this lies out side of my area of expertise.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

53 Question 11

. In addition to using the average serum values from animal studies to calculate internal doses for humans, the animal to human extrapolation can be accomplished by dividing animal average serum values by the human to animal clearance ratios to project a human average serum point of departure in units of mg/L serum. Please provide recommendations for applying uncertainty factors to the extrapolated average human

serum values to determine serum

- based thresholds that are protective for humans. A

NO AEL expressed in average human serum units would be useful in interpreting NHANES population monitoring data.

James V. Bruckner

PFOA

- specific comments

No comment.

PFOS

- specific comments

No comment.

Deborah A. Cory

- Slechta

In initial response to charge questions, I found it difficult to understand specifically what this charge question was asking for a response to: Does this refer to the data in Table 5

- 10 for PFOA? Wouldn’t you include animal to human UF values at the least. Since the data for t he studies listed in the Table is not clear as to their duration (columns are needed for this information, or add to the Study box), it i s not clear whether a UF for study duration is warranted. It is not clear how sex differences are being accommodated in any of these.

At the face

- to

- face meeting, however, with some additional input from EPA, it was clear to all that there was no need to do such derivations from animal to human, which could instead be derived directly from the human data and thus presumab ly this is no longer an issue.

Jamie C. DeWitt

Would this approach take into account differences between animal studies that have a defined exposure duration and data from NHANES, where exposure duration is assumed to be continuous (although it may not be), if exposure duration does not appear significantly impact serum concentrations? Additionally, how would the half

- life estimations from the Seals et al. (2011) study, which contained two half

- life estimations based on concentration and time, impact

thi s approach?

Jeffrey W. Fisher

Again, is the system linear in the exposure/dose ranges of interest? I would try to determine an UF by exploring a range of predicted human serum levels. Attempt to use 5,50, and 95% for

animal serum concentrations with a 5,50, and 95% CL values in the animals and for the human perhaps us e two CL values representing a high and low. The idea is to use as much informationExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

54 as you can to determine the possible range of values. This will help guide the selection of uncertainty values.

William L. Hayton

This calculation is equivalent to dividing the animal dosage by the CLhuman, assuming that the animal serum concentration is at steady state (C ss,animal ) maintained by a constant dose rate (DR).

C ss,animal / CL

human  CL animal = CL animal * C ss,animal / CL human = DR / CL human

This calculation would give the steady

- state serum concentration in human that would be produced by the animal dose rate. (I will have to study this to understand the question; the

calculation does not make sense to me.)

At the peer review meeting, the aim of this calculation was clarified. Authors desired a way to calculate a steady

- state serum concentration (C ss,human ) that would result from the human equivalent dose rate (HED) administered until steady state. The appropriate calculation would

be:

C ss,human = HED / CL human

Matthew P. Longnecker

The proposed division by animal clearance ratios does not make sense to me. The average serum values from animal studies is already taking pharmacokinetic variability in blood levels during the observation period into account, and human blood levels will be relatively constant. Thus, it would make sense to directly compare the POD estimated average serum concentrations

from animal models to the blood levels in NHANES. With respect to uncertainty factors that would be need consideration for this approach, it seems that UF H , UF L (For LOAEL and

HED LOAEL ), UF D , and the component of UF A that takes pharmacodynamics into account would

all still be applicable.

Angela L. Slitt

This is outside of my area of expertise.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

55 Question 12

. Please describe any suggestions you have for improving the clarity, organization, and/or transparency of the draft documents.

James V. Bruckner

PFOA and PFOS

- specific questions See specific observations.

Deborah A. Cory

- Slechta

While the EPA authors are aware of updates in the IRIS process, it might be very helpful to look at some of the new documents coming through that process for guidance as to t he levels of critique and evaluation that are now included in these documents. They also include an introductory chapter focused specifically on the literature searches and literature that is included

vs. excluded.

The Executive summary does not provide s ufficient rationale and descriptions to lead a reader through the steps to what is concluded and reads more like an abstract than an Executive Summary. Since this may be the only section read by many reviewers, it is important that it provide a succinct j ourney through the process. Here again, the new IRIS documents (e.g., trimethylbenzene) could provide a useful template.

Chapters 3, 4 and 5 could each benefit from an opening paragraph describing what the section’s goals are, and integration and conclus ion sections at the end that establish the basis for the presentation in Chapter 5. Currently the Hazard Identification studies generally treat all studies as of equal strength/power, which is certainly not the case. These chapters should present that

ki nd of critical and transparent assessment as it ultimately serves as the basis for decisions that

are made.

The inclusion of sections on in Vitro data did not ultimately seem particularly relevant in the outcome for these compounds and could be significa ntly shortened to add more to Chapter 4 on study strengths and weaknesses. However, where pertinent, it would probably be more useful to break that section up and insert test where it follows an in vivo discussion.

Tables could be considerably improved a nd made far more useful to the reader for comparative assessments. As of now, they require going back and forth to the text to capture additional details of the studies, e.g., sample sizes, species etc. and could benefit the reader significantly

with thos e additions. For the human assessments, it is equally important to include these details in the chapter as well as a column of study strengths and limitations.

While charge questions ask whether the appropriate studies were chosen as key studies, this reviewer does not remember that that term was even used in the documents, certainly no explicit mention was made as to which studies were considered key studies. Th is would seem to be a section that should be included in Chapter 4 more explicitly. Chapter 5 of both documents, more so PFOA, are confusing as almost all studies are subjected to modeling, for reasons that areExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

56 never presented in sufficient detail and simp ly followed by statements that a selected study (not really well presented in Chapter 4 as a selected study) will protect against other adverse effects.

Jamie C. DeWitt

The documents lack a critical analysis of differences between findings of epidemiological studies and findings of animal models. As stated in the comments to Charge Question #3, what is particularly valuable about the PFOA/PFOS database is that it is relatively extensive in that it includes data not only from occupationally

- expo sed humans, but from people highly exposed to environmental concentrations of PFOA/PFOS, and from people in the general population who have detectable concentrations of these compounds.

Critical to this analysis is a discussion of concordance and lack of concordance between human data and animal model data. For example, immunotoxicological findings appear to be consistent between humans and rodent models whereas serum lipids are not. How do these differences impact the overall confidence in the database an d derivation of the RfD?

All of the sections related to the PK models developed by ORD need additional information for clarity and transparency. As written, it is not clear that the PK values presented throughout the document actually represent a reanalys is of existing data from studies that reported serum

concentrations

. The Wambaugh et al. (2013) study could be referenced to shorten this exercise as this publication provides details on the reanalysis of existing data

.

Justifications for choosing or not choosing particular values or endpoints need to be more thoroughly detailed throughout both documents, especially for endpoints that appear to occur in both experimental animal models and exposed humans (i.e., thyroid hormone disruption and immunotoxicity)

.

Jeffrey W. Fisher

These documents represent an enormous undertaking to describe studies with PFOA and PFOS. Keep the same writing style for reporting studies. This was very good. A synthesis of the most important studies is needed and some statements a bout why other studies are not used by EPA. It is easy to get lost in the document because of its size, but if there was an analysis or synthesis section for the key toxicity studies and another for PK modeling rationale, it would help readers.

William L

. Hayton

It would be helpful to use one set of units for test article amount and concentration. The draft documents use ng/mL,  g/mL,  g/L, ppb, ppm, and  M for PFOA/PFOS concentration in water, diet, and serum. It would be more straightforward to use one concentration term, preferably ng/mL, and perhaps  g/mL in addition as necessary. But making comparisons among ng/mL, ppm, and  M is a distraction.

In S ection 3 of both documents

, it would be helpful to include a summary table of primary pharmacokinetic parameter values fo r the species included in this s ection . Tables 3

- 17

– 3

- 20 inExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

57 the PFOS draft document are a good start. In the PFOA document, Table 3

- 23 lacks CL values,

and Tables 3

- 24 and 3

- 25 lack V d values. For the pharmacokinetic model analyses presented, primary parameters values could be limited to CL, Vd
ss , and half life (see table in response to question 10). The CL and V dss values should be normalized to body weight. Where there are multiple models for a species, there should be separate entries for each study. Where there are multiple dosages for a species, there should be separate entries for each dosage. For the PBPK models, V dss value s are not available and therefore should not be included. Such a table would be helpful to show consistency or lack thereof among studies and would facilitate selection of

the best available values for CL and V dss for use in a human PK model that would pr edict steady

- state serum concentration from intake (dosing) rate and, conversely, predict intake rate from steady

- state serum concentration. These predictions are probably the primary reason to include a pharmacokinetics section in the documents.

The pha rmacokinetic sections of both documents lack example graphs of serum concentration

time data on semilog coordinates for PFOA and PFOS. Inclusion of a few representative graphs would help the reader evaluate the consistency of the data used to generate the pharmacokinetic parameter values, and where model

- based equations have been fitted to the data, the scatter of

the measured concentrations around the model

- predicted line would be informative as to the goodness of fit and the validity of the model and its parameters.

Matthew P. Longnecker

I can see advantages to treating this more like a systematic review of the literature, where the specific search algorithm for included articles is laid out, as are the range of dates of publication to be considered, and any other selection criteria applied for articles considered. In these documents, while the review of earlier literature appears to be comprehensive, after some point there must have been some decision making about which of the more recent articles to include.

The EPA has many guidelines about how data like these are to be evaluated, yet in the document few, if any, references to these guidelines were cited. Because so many guidelines exist, it could help readers if the authors cited specific places in critical documents that provide guidance for specific decisions.

Angela L. Slitt

The document reads very well. Although not incl uded in the RfD determination, including a

table of the observed human effects alon g with serum concentrations in S ection 5.0 would put

Tables 5

- 2 and 5

- 3 into context. Some sort of layman explanation to help understand why only

non

- human exposures are being included would be helpful to the general public.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

58 V. SPECIFIC OBSERVATIONS

James V. Bruckner

Specific Observatio ns for Health Effects Docume nt for Perfluorooctanoic Acid Page Paragraph Comment or Question

3

- 11 5, lines 7

- 11 It is stated that the PFOA concentration in bile increased by a factor of 12.5 with the increase in PFOA dose from 12.5 to 25 umol/kg in

wild

- type mice and 19.5 in PPARα

- null mice. These factors should

be 2.8 for wild

- type and 6.1 for PPARα

- null mice. The document’s authors may want to rethink their interpretation of the data. The

results for the wild

- type mice do suggest saturation of tra nsport from liver to bile ducts, but the PPARα

- null results do not, indicating a role for PPARα in this process. In contrast to the foregoing, the findings of Lou et al. (2009) (p. 3

- 11, pgr. 2) indicate their highest dose of PFOA is cleared from the

blood of mice more rapidly than lower doses, suggesting saturation of hepatic and/or renal reuptake transporters. What is the relative importance of biliary and renal elimination of

PFOA?

3

- 12 3, lines 2

- 4 It should also be stated that upregulation of MRP3& 4 and the OATs may be beneficial , due to increased biliary excretion of bile acids, bilirubin, conjugated metabolites of toxic chemicals, etc.

3

- 14 1 & 2 It might be stated that the findings of Hinderliter (2004) support those of Han (2003), in regards to development of female rats.

3

- 14&

3

- 15 It is problematic to try to compare values in Table 3

- 14 with values referred to at the end of the second paragraph on p. 3

- 17. Whole pup and pup serum PFOA levels decrease between PND 1&18 for each dosage in the table. It would be preferable to include another table showing the PFOA levels with body weight taken into account.

Table 3

– 14 and other tables should include the species in the title. It would also be helpful to include some details of the experimenta l protocol in the footnotes.

3

- 20 It would be useful at the end of this section (Distribution During Pregnancy and Lactation) to summarize the primary findings, or conclusions that can be drawn from the data that were presented.

3

- 23 4, line 2 It should be emphasized that urinary excretion of PFOA was substantially higher in female than male rats.

3

- 28 2, line 4 Replace “receptors” with “transporters”.

3

- 28 6 Did 10 uM PFOA inhbit PAH and estrone uptake to a greater extent

than 100 uM PFOA?

3

- 29 3 & 4 It is not clear what Yang et al. (2009) concluded about the role of OATp1a1 in the uptake of PFOA from glomerular filtrate.

3

- 32 3 & 4 These two summary paragraphs are very helpful.

3

- 37 1, line 1 Should “adsorption” be “absorption”?External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

59 Specific Observatio ns for Health Effects Docume nt for Perfluorooctanoic Acid Page Paragraph Comment or Question

4

- 7 &

4

- 9 Tables

4

- 1 & 4

- 2 Tables 4

- 1 and 4

- 2 are quite helpful in integrating the results of

studies of occupationally

- exposed populations.

4

- 13 A concluding sentence should be added to summarize the findings of a lack of association of PFOA with diabetes, metab olic syndrome,

etc.

4

- 32 The NOAEL and/or LOAEL for this study should be stated at the end of the paragraph.

4

- 34 2 Is the LOAEL for liver effects 1 ppm in the study of Loveless et al. (2008)?

4

- 38 1 Include the meaning of the abbreviation “mPPARα”.

4

- 39 Inclusion of the table for Minata et al. (2010) would be useful to help readers better comprehend the study findings.

4

- 40 A table of short

- term LOAELs and NOAELs should be added here

or in Section 5.

4

- 47 2 It is hard to believe, judging from the slight difference in mean values and their standard deviations, that absolute and relative liver weights are significantly higher than controls in the 1 mg/kg/day

group.
4

- 67 2, line 5 Insert “absolute” before “liver weight”.

4

- 69 1, l ines

1 & 2 It mig ht be worthwhile to point out that the actual study by

Butenhoff et al. was conducted prior to 2004.

4

- 73 A summary sentence (or two) should be added at the end of the Mutagenicity and Genotoxicity section.

4

- 83 A summary paragraph should be included at the end of the Immunotoxicity section.

4

- 101 1, line 14 Insert the word “some” before “occupational studies”. In order to present a more balanced perspective of findings in occupational studies, the following sentences could be added at the

end of the paragraph: “Olson and Zobel (2007) examined groups of male workers at 3 fluorochemical production facilities. Serum PFOA concentrations were not associated with total cholesterol, LDL or

HDL in workers at these facilities.”

4

- 102 4 It should be stated tha t the increases in serum enzyme activity in workers were quite modest/small. The following sentence should be added at the end of the paragraph: “Emmett et al. (2006), however, found no association between serum PFOA and liver or renal enzymes”.

4

- 103 2, line 2 Change “apoptotic or necrotic damage of” to “apoptosis or necrosis of”. Apoptosis and necrosis are types of cell death, not damage/injury.

4

- 103 3, line 1 It is true that PFOA may interfere with the biliary excretion of other compounds that are transported by the same transporters. Upregulation of the genes for these transporters, however, may be beneficial in that the excretion of bile acids, bilirubin and conjugatesExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

60 Specific Observatio ns for Health Effects Docume nt for Perfluorooctanoic Acid Page Paragraph Comment or Question of toxic chemicals/metabolites may be hastened.

4

- 103 4, line 2 I would avoid the word “critical” until the section on Dose

- Response

Assessment.

4

- 103 4 Increases in absolute and relative liver weights were dose

- dependent (Cui et al., 2009; El combe et al., 2010; Wolf et al., 2008a)

4

- 103 5 It is important to distinguish between effects of PFOA on rough and smooth endoplasmic reticulum (RER and SER). RER content was diminished, but there was a proliferation of SER.

4

- 104 2, line 5 This last line should be amended to read “that PFOA has some effects of unknown toxicological significance that appear to be independent of PPARα activation.

4

- 104 4, line 3

- 5 The meaning of the sentence is not clear. Has something been

omitted?

4

- 105 3, line 3 Add “of offspring” between “abilities” and “at 6 and 18”.

Include Fei and Olsen’s (2011) finding of no association between prenatal PFOA exposure and behavioral or coordination problems in children at age 7.

4

- 109 3 The species (i.e., mice) studied by Wh ite et al. (2009) and by Wolf et al. (2007) should be stated.

4

- 111 4, line 2 Replace “examine” with “determine whether there was”.

4

- 112 2, lines

1 & 2 The first sentence is misleading and should be rewritten.

Butenhoff et al. (2012) did not see a significant increase in liver

adenomas or carcinomas. Biegel et al. (2001) reported an increased incidence of hepatic

adenoma but not carcinoma.

4

- 112 2, line 13 What is hepatic cystoid degeneration?

4

- 114 2, line 3 Insert “ decreased” before “apoptosis”.

4

- 115 5, line 2 What is meant by “PRAR exposures”?

4

- 116 There is no mention of PFOA

- induced changes in expression of genes (e.g., cell cycle control, peroxisomes biogenesis, inflammation, etc.) that are PRARα

- dependent. There is no mention of the role of PRARα or peroxisomes in oxidative injury and carcinogenesis.

4

- 120 1, lines

11 & 12 Insert “these” between “that” and “hormones”.

4

- 121 3 It would be helpful to give the PFOA dosages of White et al. (2007) and one or two other studies, so the reader will have some idea of the magnitude of PFOA exposure required to alter mammary gland development.

5

- 1 RfD: Omit the word “wealth” from the bullet pertaining to epidemiology studies. There have be en relatively few epidemiology

studies of PFOA

- exposed populations.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

61 Specific Observatio ns for Health Effects Docume nt for Perfluorooctanoic Acid Page Paragraph Comment or Question

5

- 2 1, lines 2

- 6 Another obvious point should be made here, mainly that occupational exposures result in much higher plasma PFOA levels and body burdens than do environmental exposures. Thus, it would be anticipated that adverse effects would be more apparent in PFOA facility workers.

5

- 2 1, line 5 Include the words “in some instances” between the words “shown” and “between”. Otherwise, it appears from this paragraph the serum

PFOA conce ntrations are consistently/usually associated with the

various maladies.

5

- 2 3, line 8 Insert “failure to attain” between the words “with” and “developmental”.

5

- 7 2, line 4 Insert the word “rodent” between “between” and “species”

5

- 19 1, line 1 Insert “from some studies” between “data” and “have”.

Specific Observations for Health Effects Document for Perfluorooctane Sulfonate Page Paragraph Comment or Question

3

- 2 5, l ines

2 & 3 It is stated here that “the ratio of PFOS identified in serum and liver

tissue are similar”. Do the authors mean that PFOS concentrations in

the serum and liver are similar?

3

- 2 6 How does PFOS distribute between plasma lipoproteins and proteins/albumin?

3

- 5 1, lines

9& 10 How much lower were milk PFOS levels than serum levels?

3

- 7 1, line 2 Oral and gavage are redundant.

3

- 16 Figure 3

- 1 This figure nicely illustrates relative PFOS levels in dams and fetuses/pups over time.

3

- 19 1,line 3 Insert “groups” between “day” and “on”.

3

- 21 1, line 10 Substitute “longer” for “slower”.

3

- 23 2 It is not clear who conducted the human PBPK modeling nor which model they used.

4

- 21 2, lines 1

- 3 What did the 2nd monkey die from?

4

- 26 3, line 3 The word “concentrations” should be replaced by “doses”.

4

- 39 1 Does an increase in motor activity on PND 17, but no such effect on PND 13, 21 or 61, constitute a toxicologically

- significant effect?

4

- 56 2, lines

1 & 2 It is stated that “taken together, these studies suggest a PPARα

independent mechanism...” Of the studies reviewed to this point in the document, only that of Abbott et al. (2009) supports this premise. Qazi et al. (2009), Rosen et al. (2010) and other groups of investigators have reported other PPARα

- independent effects of

PFOS.

4

- 60 2, lines

15

- 17 Is oxidative damage likely to be operative to a significant extent at

lower PFOS doses?External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

62 Specific Observations for Health Effects Document for Perfluorooctane Sulfonate Page Paragraph Comment or Question

4

- 61 2, line 4 What is meant by “The concentration...”?

4

- 61 4, line 2 Change “dose of exposure is” to “levels of exposure are”.

4

- 62 1, lines

2 & 3 What did Olsen et al. (2003) find correlation between?

4

- 62 3, lines

4 & 5 Identify the species (i.e., rat) studied by Chang et al. (2009) and

Stein et al. (2012).

4

- 62 5 The liver of rats and monkeys was examined for histopathological changes, but the histological changes should not be considered lesions nor pathological.

4

- 68 4, lines

5 & 6 The elevated incidence of hepatocellular adenomas/ carcinomas was

almost entirely due to adenomas. Only 1 of 60 high

- dose female rats

exhibited carcinoma.

4

- 69 5, lines

3 & 4 It is stated here that there was no increase in hepatocellular

proliferation detected in the subchronic study of Seacat et al. (2003).

It is stated previously on page 4

- 69 that “the data for PFOS are adequate to suppor t some but not all key events...” I assume that cell prol iferation is thought to be a missing event. Seacat et al. (2003) reported that the average hepatocyte proliferation index was not increased, but that some animals exhibited mild increases. It is clear in the current document that PFOS is not as potent a PP ARα inducer

as PFOA.

5

- 4 2 & 5, line 7 Again the terms “histopathological” and “lesions” are misnomers.

5

- 4 3, line 9 What is meant by a “biologically significant decrease in survival” at 0.8 mg/kg?External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

63 Deborah A. Cory

- Slechta

Specific Observations for Health Effects Document for Perfluorooctanoic Acid Page Paragraph Comment or Question Chapter

5 The text of Chapter 5 in the PFOA document (and other places) continues to state that a 10% increase in liver weight would not be an adverse effect, but merely a denominator for loss of homeostasis. On what basis was this conclusion derived? What is the support for this statement? It appears that benchmark dosing was applied to studies that had liver weight as the common denominator, but does this accommodate the lowest NOAELS and LOAELS observed for any endpoint in the long duration studies? Use of just studies with the common denominator because they provide replication ignores the fact that some other effect may occur at lower lev els but simply hasn’t been evaluated in as many studies as focused on PPARa

- based targets. If this isn’t the case, then the text should clearly address this.

5

- 7 2 States that the BMDL 10 values all fall below the experimental LOAELs. So, what does that me an, is there some conclusion that is supposed to be reached from this? IF so, please state it.

5

- 13 1 States “Generally these values were similar.” What does similar

mean? What is acceptable in this context?

5

- 16 3 States that the half

- life value Bartell et al. (2010) was sued for half

- life because it seemed more relevant to scenarios where exposure result from ingestion of contaminated drinking water by members of the general population. This rationale does not app ear to consider the potential different strengths and weaknesses of the other potential studies. Is it necessarily the case that general population is more important than occupational

studies? The rationale needs to be described in greater detail. Virtual ly no rationale is provided for the choice of the Thompson et al. (2010) study

for a volume of distribution value.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

64 Specific Observations for Health Effects Document for Perfluorooctane Sulfonate Page Paragraph Comment or Question

1

- 1 4 There are similar concerns for the PFOS document. Loose terminology should be eliminated, e.g., what is a ‘finding of note’ as used in the executive summary for PFOS.

Executive

summary The PFOS executive summary is of limited utility; for many readers this may be as much of the document as they read; as currently written it is not clear or transparent nor does it sufficiently explain how it arrived at an RfD.

3

- 3 Table 3

- 1 Couldn’t a sentence essentially substitute for Table 3

- 1; it really

isn’t useful.

3

- 25 1 Loose terminology should be eliminated, e.g., what is “generally good”

3

- 26 Figure 3

- 7 Figure 3

- 7 has no explanation of what is the black vs. gray line.

All tables There is a need to improve all of the tables; they should always include study name/year, sample size and exposure duration information on them; this would make all of the comparisons easier to evaluate and not require the reader to continue to go back

and forth to the text.

4

- 4 Table 4

- 1 For example, table 4

- 1 has only study name and year, but what really matters is also exposure duration and sample sizes, because the comparisons of outcomes in the Table depend upon the power of the study to detect effects at the very least.

4

- 9 Table 4

- 2 The same comment applies to Table 4

- 2 and any others with this intended purpose.

4

- 11 Table 4

- 3 Table 4

- 3 needs sample sizes, exposure duration etc.

4

- 24 Table 4

- 7 Tables that summarize a significant amount of data from a single study (e.g., 4

- 7) should include the study authors and year in the

Table title so it doesn’t have to be searched for. In several instances in the PFOS document, adverse effects early that appear to be reversed at a later age are disco unted with the suggestion that they therefore do not matter; given our increasing understanding of the importance of early changes in terms of epigenetic changes, this is no longer appropriate and in fact, misleading.

5

- 16 Table 5

- 8 What do the parentheses signify?

5

- 17 Table 5

- 9 What do the parentheses signify?

Jamie C. DeWitt

Specific Observations for Health Effects Document for Perfluorooctanoic Acid Page Paragraph Comment or Question

4

- 102 2 & 3 DeWitt et al. 2009 also included data on triglyceride levels in C57BL/6 mice exposed to PFOA for 15 days; triglyceride levels were dose

responsively decreased.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

65 Jeffrey W. Fisher

No specific observations.

William L. Hayton

Specific Observations for Health Effects Document for Perfluorooctanoic acid Page Paragraph Comment or Question

1

- 2 Last, line 5 Delete “in”; should read “... in rats was analyzed ...”

3

- 2 1, lines 6

- 8 Assumption that fecal excretion represented unabsorbed PFOA is problematic; suggest rephrasing this sentence.

3

- 3 Table 3

- 1 Protein binding is important for PK modeling, where the fraction unbound (fup) is the important parameter, not the fraction bound. Suggest listing fup values rather than percent bound.

3

- 6 Last, line 3 “concentration” should be “dose rate”

3

- 8 2, line 4 In addition to liver, kidney, and blood, other tissues are prominent. E.G., Table 42 of Kemper shows that in male at 1 mg/kg, t=Tmax, GI tract, GI contents, muscle, bone and skin contained a greater percentage of dose than did th e kidney.

3

- 8 2, line 8 “Blood to kidney” should be “kidney to blood”

3

- 8 2, line 10

- 11 In Kemper, Tables 44

- 45, blood to kidney ratios are not 10 or higher

in males.

3

- 8 2 This paragraph reports both percent of dose found in tissues, and

concentrations found in tissues. But Tables 3

- 4 and 3

- 5 present only the former. When presenting tissue concentrations, please make it

clear that those data are not shown.

3

- 18 Last, line 3 “were” is repeated.

3

- 19 1, line 1 Technically incorrect to say that the lev el peaked at PND7; that was the earliest sample time. The peak may have occurred before PND7.

3

- 19 Table 3

- 15 The last dose was on GD17; strange that at 1 and 3 mg/kg the serum

concentration increases from PND7 to PND14.

3

- 22 4 Last sentence is garbled.

3

- 22 4, 5 Agree that biliary elimination is possible, but it could be that chloestyramine binds PFOA and PFOS in the GI tract lumen after they passively diffuse from the blood to the gut. There seems to be

no direct evidence of biliary elimination, e.g., bile collected from

treated animals.

3

- 23 Last, line 4 Should be Table 3

- 18.

3

- 34 Last, line 9 Should be “nonlinear least squares”

3

- 35 Table 3

- 23 Column 2, “Adsorption” should be “Absorption”

3

- 38

3

- 38 2 Figure 3

- 7 The arrow from Gut to Liver appears to point in the wrong direction;

it should represent biliary excretion of PFOA from Liver to Gut.

3

- 43 Last line “... indicating the absence of active excretion in human kidneys.”

This does not follow from the observation of renal clearance being about 0.001% of GFR. A plasma free fraction of 0.001 wouldExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

66 Specific Observations for Health Effects Document for Perfluorooctanoic acid Page Paragraph Comment or Question account for the CLr being 0.1% of GFR, and passive tubular reabsorption would make it 0.001% of GFR since urine flow is about 1% of GFR. Other scenarios are possible that do n ot invoke the absence or presence of active excretion.

3

- 44 Table 3

- 24 Should report all data values with three significant figures. For example, Lambda z values have only one sig. fig., while T 1/2 values

have 5

- 6.

3

- 46 2 This reviewer does not follow the derivation and use of a value for volume of distribution with regard to intake rate and serum concentration of PFOA. If the subjects were at steady state, the body burden would have to be known. At steady state, the serum concentration would be indepen dent of the volume of distribution, so any V value ought to match the intake rate to the steady state serum

concentration.

4

- 9 1 Log transformed concentration was 1.51 and 1.48 ng/mL

– are these the logarithms? IE, are the actual concentrations 10^1.51 = 32 and 10^1.48 = 30 ng/mL?

4

- 20 2, line 8 Anderson here is spelled Andersen in the reference list.

4

- 30 1, line 9 prostrate should be prostate.

4

- 31 4, line 10 decreased should be decrease.

4

- 112 1 It would be helpful to restate the serum concentrations for the Eriksen and Vieira studies, or refer reader to p. 4

- 29 where they are provided.

4

- 112 1, line 9 Delete “for”.

4

- 112 2, line 12 Delete “were”.

4

- 118 4 Delete “of actions” after MOAs

4

- 120 3 The broad range of half lives could also be due to person

- to

- person variability in the free fraction of PFOA in serum (fup). This is the case for highly bound drugs; e.g., warfarin.

5

- 1 3 Pharmacokinetic is misspelled.

5

- 1 5 Disagree

– exposure assessment based on the human data is feasible.

I n fact, the serum concentrations are a better measure of exposure than are intake measures as they reflect all intake pathways and eliminate bioavailability and pharmacokinetic influences on internal

exposure. 5

- 12 Last Table numbers should be 5

- 6, 5

- 7, a nd 5

- 8.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

67 Specific Observations for Health Effects Document for Perfluorooctane Sulfonate Page Paragraph Comment or Question

3

- 5 3 The low CSF : serum concentration ratio could also be due to an export transporter that pumps PFOS out of the CSF and/or to extensive serum protein binding, where only the free serum concentration of PFOS is in equilibrium with the free PFOS

concentration in the CSF.

3

- 22 2 The free fraction used for the model is much larger than that determined experimentally, Table 3

- 1; this should be pointed out in

the text.

3

- 22

3

- 23 2 Figure 3

- 5 The arrow from Gut to Liver appears to point in the wrong direction;

it should represent biliary excretion of PFOS from Liver to Gut.

3

- 24 4 Anderson should be Andersen.

4

- 26 4 “concentratio ns” should be “dosages”.

5.2 3 Should note for many of these studies, that steady state may not have been achieved due to the long half

- life of PFOS. Half

- life values from Section 3 are: mouse, 37 days; rat male, 40 days and female 64 days; monkey, 120 days. Using a one

- compartment PK model, the time to 90% steady state is 3.3 half lives.

5

- 5 3 The NOAEL for liver effects in rats of 0.072 mg/kg/day is not consistent with p. 5.4, para. 2, which states that lesions of the liver

were observed in male rats after 104 weeks at this dosage.

5

- 7 2 For female rat, the PFOS half life is about 60 d and the period of gestation is about 20 d or one

- third of a half life. If PFOS is administered to the dam only during gestation at a fixed daily dose,

the serum conce ntration of PFOS would rise from 0 to 21% of the steady

- state serum concentration that the fixed dose rate would produce at steady state. The exposure of the fetus during gestation would average only about 10% of the exposure that would have

occurred if t he dam had received PFOS for 4 half

- lives (240 days) prior to mating. BMDs based on such a fixed dose could be elevated by as much as a factor of 10 compared with the steady state situation. Steady state would be the relevant situation for humans.

For t he Luebker study (Table 5

- 3) the serum concentration during gestation would have increased from about 38% to 50% of the eventual steady state concentration.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

68 Matthew P. Longnecker

Specific Observations for Health Effects Document for Perfluorooctanoic Page Paragraph Comment or Question

3

- 28 1 st complete Should the end of the sentence be “increase the transporters” rather than “increase the receptors”?

3

- 30 2 nd complete L 3, would insert “transfected” between “OAT3” and “cells”

3

- 39 1 st complete Next to last sentence: I doubt that Olsen assumed the major source of exposure was drinking water in the occupational study

3

- 41 4 th complete In the first formula listed, the plus sign should be an equal sign

4

- 9 1 st complete L 3 from bottom: the values of 1.51 and 1.48 given are probably better described as geometric means.

4

- 16 2 nd complete L 3 from bottom: would insert “draw” after “blood”

4

- 21 2 nd complete L 5: the value of 6.78 ug/L is a water level, not a serum level; this

issue recurs on P 4

- 23, paragraph at bottom

4

- 30 1 st complete L 8: should read “exposure categories” rather than “cancer categories”?

4

- 37 Table Would note dose of PFOA somewhere in table or footnote

4

- 55 Last para L 3: should the “>” be a “<”?

4

- 79 Last para Last sentence: should “50 and 25” be “50 and 250”?

4

- 80 1 st complete The last sentence does not accurately describe the table. E.G., the CD4+CD8+ cells decreased at the 47.21 mg/kg/d dose

4

- 82 Next to last

para Last sentence: the 37.5 mg/kg/dose is not mentioned earlier, so this

is a little confusing.

4

- 85 Last para L 2: should “0.5” be “0.05”?; Same issue for L 5.

4

- 89 4 th para How long were the animals dosed?

4

- 110 3 rd complete

para L 5: should “serum” be “blood”?

4

- 113 3 rd complete

para L 1: insert “in” before “liver cells”

5

- 4 Last para Were the criteria for inclusion in Table 5.2 the same as for Table

5.1?

5

- 12 Para below

table 8

- 6, 8

- 7, and 8

- 8 should be 5

- 6, 5

- 7, and 5

- 8

5

- 16 Last line I do not see in the Thompson et al. (2010) study any mention of using exposure data from NHANES to calibrate the volume of distribution. Other sources of data were used, where the water had

been contaminated.

5

- 17 1 st formula “/day” should be deleted from “0.17 L/kg bw /day”

5

- 20 Table 5

- 12 The first th ree values in the UF total column need to be corrected; they should be 21900, 219000, and 21900

5

- 21 Paragraph

above table Last sentence: UF L should be UF H

5

- 21 Last

sentence UD S should be UF SExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

69 Specific Observations for Health Effects Document for Perfluorooctanoic Page Paragraph Comment or Question

5

- 27 Calculations The text says the body weight conversions should be based on the 3⁄4 power. If so, the HED formulas are incorrect, and the HED should be 1.99 x 0.0254 = 0.0506, the dosimetric adjustment factor should be 0.0254, and the CSF should be 1.57. All the figures here should be checked as should the paragraph on P 5

- 28. The HED is 2,530

- fold greater than the RfD, not 29,000.

Specific Observations for Health Effects Document for Perfluorooctane Sulfonate Page Paragraph Comment or Question

1

- 1 2 nd 1 st sentence: would revise for clarity. Do you mean uncertainties

exist about whether PFOS

- induced peroxisome proliferation is involved in causing PFOS

- induced hepatic lesions?

1

- 1 3 rd 1 st sentence: would revise for clarity; the occupational studies were done at PFOS production plants, but to my knowledge there are no residential populations that have been studied for health effects who lived near PFOS production plants. (Mid

- Ohio valley f actory was a source of PFOA.) In the 2 n d sentence, I do not believe that exposure was mainly through contaminated drinking water in any of these

studies.

4

- 66 2 nd The earlier summary of the Bloom et al. study (P 4

- 10) said the

results were not statistic ally significant, whereas here the interpretation appears to be that the study found an association. The interpretation does not seem consistent across the two sections.

5

- 17 Below table L 3: the word “terminal” should be deleted from this sentence

5

- 20 1 st formula The “/day” should come out of “0.23 L/kg bw/day”

5

- 26 L 2 from

bottom This should be 35 ug/L not 35 mg/L

Angela L. Slitt

No specific observations.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

A

- 1 APPENDIX A : PEER REVIEWER BIOSKETCHES

### External Peer Review Meeting on

EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

Crystal City Marriott at Reagan National Airport

1999 Jefferson Davis Highway Arlington, VA, 22202

- 3526

August 21 and 22, 2014

LIST OF PEER REVIEWERS

James Bruckner, Ph.D. University of Georgia William Hayton, Ph.D. The Ohio State University (Emeritus)

Deborah Cory

- Slechta, Ph.D. (chair) University of Rochester School of Medicine and Dentistry Matthew Longnecker, Sc.D

. , M.D

National Institute of Environmental

Health Sciences

Jamie DeWitt, Ph.D. East Carolina University Angela Slitt, Ph.D. University of Rhode Island

Jeffrey Fisher, Ph.D. U.S. Food & Drug Administration

BIOGRAPHICAL SKETCHES OF PEER REVIEWERS

James Bruckner, Ph.D. University of Georgia

Dr. Bruckner is Professor of Pharmacology and Toxicology at the U niversity of G eorgia College of Pharmacy . He is also Professor in the Department of Physiology and Pharmacology at the University of Georgia College of Veterinary Medicine. He received his Ph.D. in Toxicology from the University of Michigan in 1974. He has previously held faculty position s at th e University of Kansas and t he University of T exas Medical School at Houston. He is actively engaged in graduate education and in federally

- funded research projects. Dr. Bruckner’s research focus is on the toxicology and toxicokinetics of solvents, drug

- so lvent interactions at occupational exposure levels, and toxicokinetic bases for susceptibility of children to insecticides and other chemicals. Dr. Bruckner has published more than 200 journal articles, book cha pters,
and abstracts . He has also served on a variety of expert panels and committees for the EPA,

National Institutes of Health

, National Aeronautics and Space Administration

, Agency for Toxic Substances and Disease Registry

, Food and Drug Administration, and the National Academy of

Sciences.Debo rah Cory

- Slechta, Ph.D. (chair)

University of Rochester School of Medicine and Dentistry

Dr. Cory

- Slechta is a Professor in the Department of Environmental Medicine and the Department of Pediatrics at the University of Rochester School of Medicine and Dentistry, where

she also serves as co

- director of the Behavioral Sciences Facility Core and director of the Animal

Behavior Core. Dr. Cory

- Slechta received her Ph.D. from the University of Minnesota in 1977 and worked as a junior staff fellow of the National Center for Toxicological Research beginning in 1979. She was appointed to the faculty of the University of Rochester Medical Sc hool in 1982 and was appointed Chair of the Department of Environmental Medicine and Director of the NIEHS Environmental Health Sciences Center at the University of Rochester in 1998 . From

2000 to 2002, she was the Dean for Research and Director of the AAB In stitute for Biomedical

Sciences

. Following her appointment as Dean, she served from 2003 to 2007 as the Chair of the Department of Environmental and Occupational Medicine at Robert Wood Johnson Medical

School and as Director of the Environmental and Occ upational Health Sciences Institute, a joint

Institute of the Robert Wood Johnson Medical School and Rutgers. Dr. Cory

- Slechta ’s research has focused largely on environmental neurotoxicants as risk factors for behavioral disorders and neurodegenerative di sease. These research efforts have resulted in over 170 papers and book chapters to date. Dr. Cory

- Slechta has served on numerous national research review and advisory panels, including committees of the National Institutes of Health, the National Institut e of Environmental Health Sciences, the Food and Drug Administration, the National Center for Toxicological Research, the Environmental Protection Agency, the National Academy of Sciences, the Institute of Medicine, and the Agency for Toxic Substances and Disease Registry, Centers for Disease Control. In addition, Dr. Cory

- Slechta has served on the editorial boards of several journals including Environmental Health Perspectives

, Neurotoxicology

, Toxicology

, Toxicological Sciences

, Fundamental and Applied Toxicology

, Neurotoxicology and Teratology

, and American Journal of Mental Retardation . She has held the elected positions of President of the Neurotoxicology Specialty Section of the Society of Toxicology, President of the Behavioral Toxicology Society, a nd been named a Fellow of the American Psychological Association. Sh e also previously serve d on the EPA Science Advisory Board Perfluorooctanoic Acid Risk

Assessment Review Panel.

Jamie DeWitt, Ph.D. East Carolina University

Dr. DeWitt is an Assistant Professor in the Department of Pharmacology and Toxicology at the Brody School of Medicine at East Carolina University (ECU) . She is affiliated with The Harriet

and John Wooten Laboratory for Alzheimer’s and Neurodegenerative Diseas es Research and

holds an adjunct a ppointment in the ECU Department of Public Health. Dr. DeWitt received her

Ph.D. in Environmental Science and Neural Science from the School of Public and Environmental Affairs and Program in Neural Science at Indiana Univ ersity in 2004. She also completed postdoc toral training in Developmental Cardiotoxicity at Indiana University

Bloomington and in Immunotoxicology at EPA through a cooperative training agreement with the University of North Carolina at Chapel Hill. Dr. DeW itt’s main research focus is on how

toxicants found in the environment can lead to neurodevelopmental and neurodegenerative disorders via disruption of the developing immune system. Much of her past research hasinvolved the imm unotoxicity of perfluoroocta noic acid (PFOA) and related polyfluroalkyl

substances (PFASs). Dr. DeWitt has published seven peer reviewed research articles, three review papers and two book chapters that address the biological effects of P F OA, as well as one paper on the effects of P F OS on immune function. Her publications describe effects as well as underlying mechanisms following adult and developmental exposure. Her research experience and publication record (more than 25 peer reviewed manuscripts, 6 review articles, 9 book chapter s) extend beyond the effects of PFA A s and working with rodent models. She is currently editing a book on the general toxicity of PFASs and is a current member of the mechanistic working group for Monograph 110 of the International Agency for Research on Cancer, which

will include an assessment of PFOA. She is on the editorial boards of the Journal of Immunotoxicology and the Journal of Environmental Toxicology and Health and has reviewed grants for the Department of Defense and the National Institute of O ccupational Safety and

Health. She has also been manuscript reviewer for more than 20 journals. Dr. DeWitt is the current president of the North Carolina chapter of the Society of Toxicology and the Junior

Councilor for the Immunotoxicology Specialty Secti on of the Society of Toxicology. She also

was awarded the Outstanding Young Investigator Award from the Immunotoxicology Specialty

Section in 2013.

Jeffrey Fisher, Ph.D. U.S. Food & Drug Administration

Dr. Fisher is a Research Toxicologist at the U.S. F ood and Drug Administration (FDA), National Center for Toxicological Researc h. He was formerly a Professor in the Department of Environmental Health Science, College of Public Health at the University of Georgia (UGA). He joined UGA in 2000 and served as Department Head of the Department of Environmental Health

Sci ences from 2000 to 2006 and Director of the Interdisciplinary Toxicology Program from

2006

- 2010. Prior to joining UGA, h e spent most of his career at Wright Patterson Air F orce Base ( AFB ) , where he was Principal Investigator and Senior Scientist in the Toxics Hazards Division and Technical Advisor for the Operational Toxicology Branch. Dr. Fisher's research interests are in the development and application of biologically bas ed mathematical models to ascertain health risks from environmental, food

- borne and occupational chemical exposures. Dr. Fisher’s modeling experience includes working with chlorinated and non

- chlorinated solvents, fuels, pesticides, perchlorate and bisphen ol A. He has developed physiologically

- based pharmacokinetic ( PBPK ) models for use in cancer risk assessment, estimating lactational transfer of solvents, understanding in utero and neonatal dosimetry, quantifying metabolism of solvent mixtures and develop ing biologically motivated models for the hypothalamic

- pituitary

- thyroid

axis in rodents and humans. D r. Fisher has published over 14 0 papers on pharmacokinetics and PBPK modeling in laboratory animals and humans. He has served on several national panels a nd advisory boards for the DoD, ATSDR, USEPA and non

- profit organizations. He was a U.S. delegate for the North Atlantic Treaty Organization. Dr. Fisher served on the International Life Sciences Institute Steering Committee, which evaluated chloroform and dichloroacetic acid using EPA

- proposed Carcinogen Risk Guidelines. He is Past President of the Biological Modeling Specialty Section of the Society of Toxicology, reviewer for several toxicology journals, and was Co

- Principal Investigator on a National Ins titutes of Health (NIH)

- supported workshop on Mathematical Modeling at the University of Georgia in the fall of 2003. Dr. Fisher

was also a member of the National Academy of Sciences subcommittee on Acute ExposureGuideline Levels (AEGLs) from 2004

- 2010 an d Science Advisory Board (SAB) for the US EPA

(2007

- 2010). He is an ad hoc EPA SAB member for dioxin and perchlorate. Dr. Fisher is a

F ellow of the Academy of Toxicological Sciences

, an associate edi tor for Toxicological Sciences, and on the editorial bo ard of Journal of Environmental Science and Health Part C Environmental Carcinogenesis & Ecotoxicology Reviews

.

William Hayton, Ph.D. The Ohio State University (Emeritus)

Dr. Hayton is a Professor Emeritus in the College of Pharmacy at The Ohio State University . Dr. Hayton received a Ph.D. in Pharmaceutics from the State University of New York at Buffalo in

1967. He was a member of the Washington State University College of Pharmacy faculty for 19

years , rising to Chair of the Pharmacology/Toxicology Graduate Program in 1982 and Acting Dean at the College of Pharmacy in 1987. In 1990, he transferred to the Ohio State University as Chair of the Division of Pharmaceutics, where he later served as Associate Dean for the Graduate Programs and Research unt il his retirement in 2010. Dr. Hayton’s expertise is pharmacokinetics, particularly construction and validation of mathematical models that describe or explain the kinetics of complex biological systems. One recent research interest is

characterization of the Fc receptor

- mediated transport and catabolism of albumin and IgG in wild type and FcR knockout mice. A second recent project is the quantitative modeling of the female hypothalamus

- pituitary

- gonad (HPG) axis in the female rainbow trout (Oncorhynchus my kiss). The model is based on and integrates the biology of gonadotropin, estrogen, androgen and maturational hormone signaling systems, and it includes key intermediate steps in the signaling pathways; viz., gonadotropin and sex steroid synthesis, hormone receptors and their corresponding mRNA levels. Dr. Hayton’s expertise extends to interspecies scaling of pharmacokinetic model parameter values and xenobiotic metabolism. Dr. Hayton is author or co

author of over 100 peer

- reviewed scientific publications a nd has held peer

- reviewed grant support from the National Institutes of Health , EPA, Air Force Office of Scientific Research

, Food and Drug Administration , and U

. S . Fish and Wildlife Service

. He previously serve d on the EPA Science Advisory Board Perfluorooctanoic Acid Risk Assessment Review Panel.

Matthew Longnecker, Sc.D., M.D

National Institute of Environmental Health Sciences

Dr . Longnecker , M.D., Sc.D., is the head of the Bio marker

- based Epidemiology Group at the National Institute of Environmental Health Sciences (NIEHS). Dr. Longnecker received a n
M

. D

. from Dartmouth Medical School and completed a residency in internal medicine at Temple University Hospital in Philadelphia. After receiving a Sc

. D

. in Ep idemiology from Harvard School of Public Health in 1989, he served as an Assistant Professor in the Department of Epidemiology at the University of California , Los Angeles, School Of Public Health . Since 1996, Dr. Longnecker has served as Adjunct Professor / Associate Professor in the Department of Epidemiology, School of Public Health, University of North Carolina at Chapel Hill. He came to

the NIEHS Epidemiology Branch in 1995, as a tenure

- track investigator. Dr. Longnecker's research program is focused o n the health effects of persistent organic pollutants (e.g., the DDT metabolite p,p'

- DDE, and polychlorinated biphenyls). He is particularly interested in the effects of intrauterine exposure to persiste nt organic pollutants in relation to intrauterine gro wth,preterm birth, birth defects, neurologic findings at birth, growth, neurodevelopment, intelligence,

and hearing. Recently, Dr. Longnecker has completed and has ongoing a series of studies on perfluorinated alkyl substances in relation to reproductive and pediatric outcomes

. In addition, he has begun studying the effects of early, low

- level exposure to the nonpersistent pollutants, bisphenol A and organophosphate pesticides. Dr. Longnecker’s research efforts have resulted in over 180 papers and book ch apters to date. He has served as a leader for numerous national and international committees, such as for the Society for Epidemiologic Research and the International Society for Environmental Epidemiology, and has been on numerous national and

internation al scientific advisory boards, including the EPA Science Advisory Board for the

Perfluorooctanoic Acid Risk Assessment Review.

Angela Slitt, Ph.D. University of Rhode Island

Dr. Slitt is an Associate Professor in the Department of Biomedical and Pharmaceutical Sciences at the University of Rhode Island. Dr. Slitt received her Ph.D. in Pharmacology and Toxicology from the University of Connecticut in 2000, and then served until 2004 as a postdoctoral fellow at the University of Kansas Medical Cent er

. Dr. Slitt has been a faculty member at the University of Rhode Island since 2006 . Dr. Slitt’s graduate and postdoctoral training was heavily focused on liver biology and health, with a focus in the area of toxicology, and included research

in nuclear receptors, biotransformation, and transporter expression. Her current research

interests focus on how 1) expression of drug transporters affects chemical disposition and toxicity, 2) nutrition and intake of dietary antioxidants affects the expression of d rug transporters, 3) liver disease (i.e.

, diabetes, cholestasis, and ethanol cirrhosis) affects transporter expression and chemical disposition, and 4) transporter expression affects cholesterol transport and susceptibility to gallstone formation. She has also recently investigated the effect of PFOS

on caloric restriction in mice. Dr. Slitt is presently on the Editorial Board of BMC Pharmacology and Toxicology, Journal of Biochemical and Molecular Toxicology

, and Toxicology Methods

and Mechanism , and is a n ad

- hoc reviewer for numerous other journals. She is author or co

author of over 50 peer

- reviewed scientific publications , and was recently awarded the University of Rhode Island Early Career Faculty Research Excellence Award

.External Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

B

- 1 APPENDIX B : MEETING AGENDA*Time for breaks and lunch are approximate and at the Chair’s discretion. 

### AGENDA

External Peer Review Meeting on

EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

Crystal City Marriott at Reagan National Airport

1999 Jefferson Davis Highway Arlington, VA, 22202

- 3526

August 21, 2014

Day 1

– PFOA Health Effects Document

8:3 0 AM Meeting Sign

- In Begins

9:0 0 AM Welcome, Goals of Meeting, and Introductions

David Bottimore, Versar, Inc.

9 :15 AM Welcome by EPA and Overview of PFOA /PFOS Health Effects Document s

Eliza beth Doyle

, Chief, EPA/OST/OW/HECD

9:30 AM Chair’s Introduction and Review of Charge

Deborah Cory

- Slechta

, Chair

10:0 0 AM Discussion Session

– “Round Table” General Overview Comments

10:45 AM Break *

11:00 AM PFOA Discussion Session

– Responses to Ch arge Questions:

Question 1: S trengt hs, Weaknesses, and Characterization of the S tudies Selected Question 2: Additional References for Consideration Question 3: Conclusions on Human Epidemiology Data Question 4: Transparency and Characterization of Ep idemiologic D ata

12: 15 P M Lunch *

1 : 15 PM PFOA Discussion Session

– Response to Charge Questions:

Question 5: Cancer Classification Question 6: Pharmacokinetic Model Adjustments Question 7: Selected Parameters for Pharmacokinetic Model Question 8: Vo lume of Distribution (Vd) and Half

- life Values

2:45 PM Break *

3:00 PM PFOA Discussion Session

– Response to Charge Questions:

Question 9: Points of Departure and RfDs Question 10: RfDs and Applicability to Short

- and Long

- term Exposures Question 11: Uncertainty Factors Question 12: Other Suggestions for Improving the Document

5 :0 0 PM Adjourn*Time for breaks and lunch are approximate and at the Chair’s discretion. 

### AGENDA

External Peer Review Meeting on

EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

Cryst al City Marriott at Reagan National Airport

1999 Jefferson Davis Highway Arlington, VA, 22202

- 3526

August 22, 2014

Day 2

– PFOS Health Effects Document

9:0 0 AM Recap of Day 1 and Agenda for Day 2

David Bottimore, Versar, Inc.

9:1 5 AM Chair’s Review of Charge for Day 2

Deborah Cory

- Slechta, Chair

9 :3 0 AM Discussion Session

– “Round Table” General Overview Comments

10: 00 AM Break*

1 0 : 15 AM PFOS Discussion Session

– Responses to Charge Questions:

Question 1: S trengt hs, Weaknesses, and Characte rization of the S tudies Selected Question 2: Additional References for Consideration Question 3: Conclusions on Human Epidemiology Data Question 4: Transparency and Characterization of Epidemiologic D ata

1 1 : 30 P M Lunch Break*

1 2 : 3 0 PM PFOS Discussion Session

– Response to Charge Questions:

Question 5: Cancer Classification Question 6: Pharmacokinetic Model Adjustments Question 7: Selected Parameters for Pharmacokinetic Model Question 8: Volume of Distribution (Vd) and Half

- life Values

2: 15 PM Bre ak*

2:30 PM PFOS Discussion Session

– Response to Charge Questions:

Question 9: Points of Departure and RfDs Question 10: RfDs and Applicability to Short

- and Long

- term Exposures Question 11: Uncertainty Factors Question 12: Other Suggestions for Imp roving the Document

5:0 0 PM AdjournExternal Peer Review of EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

C

- 1 APPENDIX C : MEETING ATTENDEE LIST

### LIST OF OBSERVERS

ATTENDING MEETING IN PERSON

August 21 and 22, 2014



## Ext ernal Peer Review Meeting on

EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

Name Affiliation

Janet Anderson , Ph.D. U

. S

. A ir Force

Robert Bilott Taft Stettinius & Hollister LLP

Norman Birchfield U.S. EPA

John Butenhoff , Ph.D. 3M Company

Sue Chang, Ph.D. 3M Company

C

- H Selene Chou , Ph.D. Agency for Toxic Substances and Registry

Steven Chranowski Chemistry Coun cil of New Jersey

Joyce Donohue U.S. EPA

Elizabeth Doyle U.S. EPA

Colleen Flaherty U.S. EPA

Maria Hegstad Inside EPA

Gerald Kennedy DuPont

La Rae Landers Dept of the Navy BRAC PMO

Willington Lin Public

Angela Lynch, Ph.D. American Chemistry Council

Geary Olsen, Ph.D. 3M Company

Gloria Post, Ph.D. New J ersey Department of Environmental Protection

Resha Putzrath, Ph.D. Navy and Marine Corps Public Health Center

Jennifer Seed U.S. EPA

Shalene Thomas AMEC Environment & Infrastructure

Steve Via American Water Works As sociation

Anthony Walters United Science, LLC

Carol Wood Oak Ridge National Laboratory

### LIST OF OBSERVERS

ATTENDING VIA PHONE August 21 and 22, 2014



## Ext ernal Peer Review Meeting on

EPA’s Draft Health Effects Documents for Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

Name Affiliation

Matthew Bailor Gilmore & Associates, Inc.

Victoria Binetti U.S. EPA

Stuart Cagen, Ph.D. Shell Health

Andrea Candara New York State Department of Health

Tom Cleveland Decatur Utilities

Jason Conder ENVIRON International Corporation

Michelle Dev eau Health Canada

Stiven Foster U.S. EPA

Helen Goeden Minnesota Department of Health

Christopher Lau, Ph.D. U.S. EPA

Anita Meyer, DABT U.S. Army Corps of Engineers

Jonathan Naile, Ph.D. Shell Health

Jessica Nelson Minnesota Department of Health

Bridget O'Brien U.S. EPA

Ramasamy Santhini U.S. EPA

Robert Rickard U.S. EPA

John Wambaugh U.S. EPA

Carol Rowan West, Ph.D. Massachusetts Department of Environmental Protection

Linda Wilson New York State Of fice of the Attorney General

Virginia Yingling Minnesota Department of Health

Tsedash Zewdie Massachusetts Department of Environmental Protection