Document G59MrZRoE8vN83qZ9XXqvR7ZV

Electronically Served 9/22/2017 10:30 PM Hennepin County, MN 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Expert Report of Jamie C. DeWitt, Ph.D. For the plaintiff In the m atter o f State o f M innesota v. 3M Com pany No. 27-cv-28862 Prepared for Covington & Burling LLP One CityCenter, 850 Tenth Street, N W W ashington, DC 20001-4956 Prepared by Jam ie C. DeW itt, P h D . September 22, 2017 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Table of Contents L ist o f F igu res.........................................................................................................................................................._iii L ist o f A cronym s.......................................................................................................................................................v I. Introduction...................................................................................................................................................... 1 II. Q ualifications................................................................................................................................................... 1 III. M aterials R elied On, E xhibits, R e s e rv a tio n s ........................................................................................ 3 IV. C o m p e n sa tio n .................................................................................................................................................3 V. Sum m ary o f P rocess...................................................................................................................................... 3 VI. Sum m ary o f P rim ary O pinions.................................................................................................................. 4 VII. B ackground...................................................................................................................................................... 5 VII. 1 B rie f Introduction to P F C s........................................................................................................... 5 V II.2 P rinciples o f T oxicology................................................................................................................8 V II.3 B rie f B ackground on P F C T oxicology and E pidem iology.............................................. 15 V II.4 P F C s in the E ast M etro P opulation.......................................................................................... 17 V II.5 C hronology o f 3M T oxicology Studies on P F C s.................................................................19 V II. 6 T oxicological E vidence R egarding H um an H e a lth ........................................................ 21 V II.6a 3M W o rk er Studies..................................................................................................... 21 V II.6b P P A R a A ctivation and P eroxisom e P roliferation ............................................ 22 VIII. D etailed Supported O pinions................................................................................................................... 23 V III. 1 B asis and M ethodology fo r R endering O pinions.................................................................23 V III.2 D etailed Supported O pinion - C a n c e r................................................................................... 24 V III.3 D etailed Supported O pinion - D evelopm ental T o x ic ity .................................................. 31 V III. 3a C urrent E vidence o f P F C -Induced D evelopm ental T oxicity......................31 V III3b H istoric E vidence o f P F C -Inducted T eratogenicity.......................................... 36 V III.4 D etailed Supported O pinion - Im m unotoxicity................................................................... 39 V III.5 D etailed Supported O pinion - O ther N o tab le T oxicological F indings.........................45 V III.5.a T hyroid H orm one D is ru p tio n ................................................................................. 45 V III.5.b L iv er T o x ic ity ..............................................................................................................47 V III.5.c M am m ary G land D e v e lo p m e n t..............................................................................48 VIII.6 Detailed Supported Opinion - Toxicities Associated with Areas o f High E xposure...........................................................................................................................................49 i CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN V III.7 D etailed Supported O pinion - O ther P F C s R elev an t in the State o f M in n e s o ta ......51 V III.7.a P F H xS ............................................................................................................................. 52 V III.7.b P F B A and P F B S ...........................................................................................................52 IX. U se o f T oxicology Studies to D evelop P rotective H ealth G uidelines.........................................56 X. A ffirm ation......................................................................................................................................................... 60 A ppendix A C urriculum V itae (C V ) o f Jam ie C. D e W itt........................................................................61 A ppendix B. E stim ating " Safe" P FO S D oses, an E xam ple and C o m m e n ts ...................................... 83 A ppendix C. L ist o f R eferences......................................................................................................................... 86 ii CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN List of Figures Figure 1 The general classification o f PFC s as outlined by B uck et al. (2011). Figure 2 Potential stages in the developm ent o f toxicity after exposure to an exogenous agent. From Gregus (2015). Figure 3 A representation of toxicokinetic and toxicodynam ic factors from exposure to adverse health outcom e (irreversible pathology). F rom H einrich-H irsch et al. (2001). Figure 4 A decision tree for determ ining hum an relevance of a M oA for carcinogenicity or toxicity observed in experim ental anim al m odels. From B oobis et al. (2006; 2008). Figure 5 Results o f the 2010 East M etro follow up study dem onstrating blood levels of PFCs in residents from the East M etro area in 2008 and 2010 and as compared to the blood levels o f PFCs in people from the general U.S. population. From M D H (2010). Figure 6 Results o f the 2014 East M etro follow up study dem onstrating blood levels of PFCs in residents from the East M etro area in 2014 and as compared to the blood levels of PFCs in people from the East M etro in previous years and from the general U.S. population. From M D H (2015). Figure 7 Summary table o f liver, testis, and pancreatic lesions observed in rats fed C8 or W Y for two years. N ote the statistically significant increases in hyperplasia (enlargem ent of organ/tissue) and neoplasia in the liver, testis, and pancreas from rats feed 300 ppm o f C8. R ed box added for em phasis. From B iegel et al. (2001). Figure 8 Summary table of lesions from various tissues observed in rats fed APFO for two years. N ote the statistically significant increases in Leydig cell adenom as in m ale rats fed 300 ppm of APFO. The findings of increased fibroadenom a in m am mary glands o f fem ale rats fed 300 ppm o f APFO was considered by the study authors to be w ithin the norm for background variation o f this lesion in SD rats. Red boxes added for em phasis. From B utenhoff et al. (2012a). Figure 9a Summary table of teratogenic outcomes observed in m ice exposed to PFO A from G D 1-17 o f gestation. R ed box added for em phasis. From Lau et al. (2006). Figure 9b Summary table of developmental landmarks observed in m ice exposed to PFO A from G D 1-17 o f gestation. R ed box added for em phasis. From Lau et al. (2006). F igure 10 Summary figure o f m otor activity in m ale rats orally exposed to PFOS from GD0 through PN D 20. R ed box added for em phasis. From B utenhoff et al. (2009b). in CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN F igure 11 F igure 12 F igure 13 F igure 14 F igure 15 F igure 16 Points w here transversal sections (a-h) w ould be m ade on a 21 day-old rat fetus, w ith B ouin's solution and W ilson's freehand method. M odified from B urdan et al., 2005. A schematic of the NTP system atic review process. From NTP (2015). A schematic demonstrating how levels of evidence were classified in the systematic review of PFO A and PFOS. From N TP (2015). A parallelogram model to illustrate the relationship between imm une suppression and increased risk of disease in rodent models and humans. From Selgrade (2007). A dverse outcom es o f im m unotoxicity. A dopted from D ietert et al. (2010). Summary o f studies that have evaluated m am mary gland developm ent in mice developm entally exposed to PFOA. From U.S. EPA, 2016c. iv CONFIDENTIAL SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN List of Acronyms ADME - absorption, distribution, metabolism, excretion AFFF - aqueous film forming foams APFO/C8 - ammonium perfluorooctanoate (the ammonium salt of PFOA) ATSDR - Agency for Toxic Substances and Disease Registry BMD - benchmark dose CAS - Chemical Abstracts Service CDC - Centers for Disease Control and Prevention ECU - East Carolina University EDCs - endocrine disrupting compounds DWEL - drinking water equivalent level GD - gestational day GI - gastrointestinal HAL - health advisory level or health advisory limit HBV - health based value HCDWG - Historical Control Data Working Group HDL - high density lipoprotein HED - human equivalent dose HRI - health risk index HRL - health risk level IARC - International Agency for Research on Cancer LD50 - lethal dose 50 LH - luteinizing hormone LOAEL - Lowest Observed Adverse Effect Level LOEL - Lowest Observed Effect Level v CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN MDH - Minnesota Department of Health MOA - mechanism of action MoA - mode of action MRL - Minimal Risk Level N-EtFOSE - N-ethyl-N-(2-hydroxyethyl)perfluorooctylsulphonamide NHANES - National Health and Nutrition Examination Survey NHEERL - National Health and Environmental Effects Research Laboratory NIEHS - National Institute of Environmental Health Sciences NIH - National Institutes of Health NJ DWOI - New Jersey Drinking Water Quality Institute NOAEL - No Observed Adverse Effect Level NOEL - No Observed Effect Level NRC - National Research Council NTP - National Toxicology Program PFAA - perfluoroalkyl acids PFASs - per- and polyfluoroalkyl substances PFBA - perfluorobutanoic acid PFBS - perfluorobutane sulfonate PFC - perfluorinated chemicals PFCA - perfluoroalkyl carboxylic acids PFDA - perfluorodecanoic acid PFDoA - perfluorododecanoic acid PFDS - perfluorodecane sulfonate PFESA - perfluoroethersulfonic acid PFHpA - perfluroheptanoic acid vi CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN PFHxA - perfluorohexanoic acid PFHxS - perfluorohexane sulfonic acid PFNA - perfluorononanoic acid PFOA - perfluorooctanoic acid/perfluorooctanoate PFOS - perfluorooctane sulfonate/perflurooctanesulfonate PFPeA - perfluoro-n-pentanoic acid PFSA - perfluoroalkyl sulfonic acids PFTA - perfluorotetradecanoic acid PFUnA - perfluroundecanoate PND - postnatal day POD - point of departure PPARs - peroxisome proliferator activated receptors (includes PPARa, PPARy, and PPARp/5) ppt - parts per trillion PTU - propylthiouracil RfD - reference dose RSC - relative source contribution SETAC - Society of Environmental Toxicology and Chemistry SD - Sprague-Dawley rat, a strain of rat SOT - Society of Toxicology SNUR - Significant New Use Rules STEM - Science, Technology, Engineering, and Mathematics SWDA - Safe Water Drinking Act T3 - triiodothyronine (a thyroid hormone) T4 - thyroxine (a thyroid hormone) TDAR - T cell-dependent antibody responses vii CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 T G F a - transform ing growth factor alpha TH - thyroid hormone(s) TIAR - T cell-independent antibody responses TSCA - Toxic Substances Control Act TSH - thyroid stimulating hormone U.S. EPA - U nited States Environm ental Protection Agency Vd - volum e of distribution W HO - W orld Health Organization W Y - W yeth-14,643 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN VIII CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN I. Introduction I have been retained by attorneys for the State o f M innesota to provide expert opinion, from a toxicological perspective, regarding w hether perfluorinated chemicals (PFCs), also known as per- and polyfluoroalkyl substances (PFASs) or highly fluorinated chemicals, pose a substantial present and potential hazard to hum an health. II. Qualifications I received Bachelor of Science (B.S.) degrees in Biology and Environm ental Science from M ichigan State U niversity and D octor o f Philosophy (Ph.D.) degrees in Environm ental Science and Neural Science from Indiana University-Bloom ington. I com pleted postdoctoral training in Environm ental and Ecotoxicology at Indiana University-Bloomington and in Immunotoxicology at the National Health and Environm ental Effects Research Laboratory (NHEERL) at the United States Environm ental Protection Agency (U.S. EPA ) through a cooperative training agreem ent w ith the U niversity o f N orth Carolina at Chapel Hill. D uring my postdoctoral training at Indiana University, I evaluated the developmental cardiotoxicity of polychlorinated biphenyls and dioxins in w ild passerine birds for a project funded by the U nited States Fish and W ildlife Service. A t the U.S. EPA, I evaluated the im m unotoxicity o f organotin com pounds used in polyvinylchloride pipes and o f perfluorooctanoic acid (PFOA) as an em erging contam inant in drinking w ater supplies. The U.S. EPA Office o f W ater funded both o f these projects. O f the eight papers that w ere published from my postdoctoral training at the U.S. EPA, three focused on PFOA, including a review of the imm unotoxicity of PFOA. D uring this time, I also was a co author on a paper concerning the developm ental toxicity o f perfluorooctane sulfonate (PFOS) in an avian model. In 2008 I joined the faculty of the Departm ent of Pharmacology and Toxicology at the Brody School o f M edicine o f East Carolina U niversity (ECU) as a tenure-track A ssistant Professor. In 2015 I was promoted to Associate Professor w ith tenure and currently serve in this capacity. I also am an adjunct Associate Professor in our Departm ent of Public Health and am affiliated with the Harriet and John W ooten Laboratory for A lzheim er's and N eurodegenerative Disease Research at ECU. I divide my tim e am ong research and scholarly activities (approximately 65%), teaching (approxim ately 25% ), and service (approxim ately 10%). M y didactic teaching activities are directed tow ard graduate (m asters and doctoral), medical, dental, and physician assistant students. M y graduate teaching activities include co-instruction in general and advanced toxicology, and I am the sole instructor for a biom edical statistics course. M y lectures to medical, dental, and physician assistant students focus on endocrine pharm acology, and I deliver general toxicology lectures to medical and dental students, as well as additional lectures to dental students on m anagem ent o f poisoned patients. In addition to classroom teaching, I teach and m entor students in my laboratory. I have hosted 23 high school students and 20 undergraduate students through various program s since com ing to ECU. I have m entored tw o m asters and three doctoral students through com pletion o f their degrees and am currently m entoring one doctoral student. M y departm ent lim its faculty to no m ore than two doctoral students at a time, so I am at the m axim um level o f graduate student m entoring productivity for my department. 1 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN I am actively engaged in professional, university, and comm unity service activities. O f particular relevance to the topic of PFCs, I helped to write or review several of the documents that I rely on throughout this report, including those from the U.S. EPA, the International Agency for Research on Cancer (IARC) o f the W orld Health Organization (W HO), and the U.S. National Toxicology Program (NTP). I was an external peer-review er for the U.S. E PA 's health effect docum ents for PFO A and PFOS in 2014. This process required being nominated, selected by the firm hired by the U.S. EPA to oversee the peer-review process, responding to specific charge questions posed by the U.S. EPA, and providing additional feedback on the docum ents based on m y peer-review. In 2014, I was part o f the M echanism s Subgroup for IARC Volum e 110. I was invited by the W orking Group for Volum e 110 in 2013 as this volum e included an assessm ent o f the evidence on carcinogenicity o f PFOA. As a m em ber of the M echanism s Subgroup, I helped to write the m echanism s sections for each o f the five chemicals included in Volum e 110 and as a m em ber of the W orking Group for Volum e 110, I was a full participant at the W orking Group m eeting in 2014 when the volum e w as drafted. Betw een 2013 and 2016, I reviewed the protocol and the draft report on the im m unotoxicity of PFO A and PFOS w ritten by the NTP. Similar to my role as a peer-review er for the U.S. EPA health effects documents, I responded to specific charge questions and provided additional feedback based on my peer-review. I currently am reviewing another docum ent concerning PFCs written by a U.S. federal governm ent agency, but due to a confidentiality agreement, cannot disclose the specific inform ation at this time. I also am part of a steering committee to organize a workshop in 2017 entitled "International W orkshop Supporting the Dialogue Between Science and Policy on Per- and Polyfluoroalkyl Substances (PFA Ss)." These roles all highlight that I am regarded as an expert on the toxicity o f PFC s by both the national and international scientific communities. The Society of Toxicology (SOT) and the Society of Environm ental Toxicology and Chemistry (SETA C) are professional societies for toxicologists and environm ental chem ists w ho are dedicated to the study o f agents that m ay induce adverse health outcom es in hum ans and wildlife. I support these societies through annual m em bership fees at both the national and regional (North Carolina SOT and Carolinas SETAC) levels, by presenting data from my laboratory at meetings, and through participation in leadership activities. For example, from 2013-2014 I served as the President of the N orth Carolina SOT, I currently serve as the V ice President o f the Imm unotoxicology Specialty Section o f the SOT, and am on the Program Planning Com m ittee for the 2017 annual SETAC meeting. I also serve my university through various committees such as the Institutional Animal Care and U se Com m ittee (2013-present), the Coastal M aritim e Council (2010-present), and the Brody Prom otion and Tenure Committee (2015-present). I also am dedicated to science education in the comm unity through Science, Technology, Engineering, and M athem atics (STEM ) program s and by serving as a docent at a local m useum dedicated to estuaries. M y research career has focused on synthetic compounds of industrial significance that are or m ight become environmental contaminants of concern. Beginning during my postdoctoral training at the U.S. EPA, the bulk o f my research focus has been on PFCs, their im m unotoxicity, and other associated toxicities. M y first PFC publication from m y training at the U.S. EPA was the first com prehensive dose-response assessm ent o f the im m unotoxicity o f PFOA. Since com ing to E C U in 2008, I have co-authored 11 prim ary research articles related to P F C toxicity (first or senior author of seven). These m anuscripts include assessments of imm unotoxicity, developmental imm unotoxicity, developm ental neurotoxicity, developm ental cardiotoxicity, 2 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN dosimetric anchoring, and epidemiological associations. I have co-authored three review articles and a c o m m e n t a r y on P F C toxicity, t w o b o o k chapters related to P F C immunotoxicity, and edited one of the first comprehensive texts on the toxicity of PFCs. M y other publications concern toxicological effects of environmental contaminants, including their impact on h u m a n diseases. Overall, I have co-authored approximately 60 scientific publications. Attached as Ap pendix A is a copy of m y current curriculum vitae, which contains a complete list of m y publications. I have not served as an expert witness at trial or in deposition in the past four years. III. Materials Relied On, Exhibits, Reservations M y expert opinion is based o n peer-reviewed scientific literature, including m y o w n scientific publications concerning P F C toxicity. Additionally, as m u c h of the publicly available peerreviewed scientific literature (including m y o w n scientific publications concerning P F C toxicity) has been reviewed and assessed in w h a t are termed "science assessments" b y governmental and quasi-governmental scientific organizations, including, but not limited to, the U.S. E P A , the U.S. N T P , the I A R C , and the U.S. A g e n c y for Toxic Substances and Disease Registry ( A T S D R ) , these assessments will be frequently referred to and relied upon. I cite various references in the text of m y report and include a list of references in A p p e n d i x C. I also cite various studies run or sponsored b y 3 M , but I do not necessarily rely on all of them. I m a y use as exhibits part or all of any of the papers or documents referenced in this report. I m a y also use as exhibits any graphs or tables d r a w n from data in any of those papers or documents, or any d o c u m e n t considered or cited or relied u p o n b y any other expert in this case. T h e opinions expressed in this report are m y o w n and are based o n the papers, data, documents, and facts available to m e at the time of writing. Should additional relevant information b e c o m e available, I reserve the right to supplement this report. I also reserve the right to respond to any opinions b y other experts in this matter, and to respond to any criticism or c o m m e n t o n m y opinions. IV. Compensation I a m being compensated at the rate of $250 per hour for m y time. M y compensation does not depend in any w a y o n the content of m y opinions or the ou t c o m e of this case. V. Summary of Process Faced with the general question presented, I undertook to do the following: Evaluate toxicity data for P F C s based on evidence from studies of experimental animal models involving exposure to P F C s b y various routes and durations, including an assessment of the types of effects that occur, the doses/serum/tissue concentrations at wh i c h these effects occur, and the relevance of these effects to exposed humans.3 3 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Evaluate toxicity data for PFC s based on evidence from studies o f prim ary cells, cell lines, and cells evaluated ex vivo, as well as studies done in silico to understand how PFCs interact w ith biomolecules, how PFCs may exert action on physiological processes, how PFCs partition w ithin living organisms, how /how rapidly PFCs are excreted from living organisms, how to estimate PFC toxicity, and how these data are relevant to understanding effects o f PFCs in exposed humans. Evaluate confirmatory findings in health effect data for PFCs based on evidence from epidem iological studies o f hum ans exposed to PFCs occupationally or environm entally, by various routes and durations, at any concentration, including an assessm ent of the types of adverse health outcomes that have been reported to occur in hum ans following exposure to PFCs. This is not a detailed evaluation o f the epidem iological studies, but rather is an assessm ent o f the health effects that have been reported in association with PFC exposures from epidemiological studies, as a directional check on my opinions. VI. Summary of Primary Opinions It is m y opinion, based on the w eight o f the toxicological evidence, w ith confirm ation from certain epidem iological evidence, that PFCs pose a substantial present and potential hazard to hum an health. The actual or potential toxicities resulting from exposure to PFCs that are supported by the greatest body o f evidence are (in no particular order): cancer, developm ental toxicity, and im m unotoxicity. W hile there is evidence that other toxicities also likely follow exposure -- including endocrine disruption, liver toxicity, and m am mary gland developm ent -- the body of evidence for cancer, developmental toxicity, and im m unotoxicity is particularly strong based on in vitro studies as well as studies o f experimental animal m odels and epidem iological studies of exposed humans. M y general opinion, stated above, is supported by extensive scientific w ork including assessm ents by the U.S. EPA, the NTP, the IARC, and the ATSDR. Based on these assessm ents and other relevant scientific studies, I hold in addition the following m ore specific prim ary opinions: As reflected in the 2016 IARC assessment, there is substantial evidence that PFO A is correlated w ith increased incidences o f cancer, leading to likely adverse health outcomes in populations o f exposed humans. By extension, other PFCs that induce sim ilar toxicities and w ork through similar m echanisms, such as PFOS, perfluorobutanoic acid (PFBA), perfluorobutane sulfonate (PFBS), and perfluorohexane sulfonic acid (PFHxS), are also likely to induce cancer in exposed humans. As reflected in the assessm ent o f PFO A and PFOS by the U.S. EPA, developing organisms are particularly sensitive to adverse health outcomes associated w ith exposure to these agents. There is substantial evidence that PFO A and PFOS are correlated w ith developm ental toxicity effects, leading to likely adverse health outcom es in populations of exposed humans. By extension, other PFCs that induce sim ilar toxicities and w ork through similar 4 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN m echanisms, such as PFBA, PFBS, and PFHxS, are also likely to induce developmental toxicity. As reflected in the assessm ent o f PFO A and PFO S by the U.S. NTP, the im m une system is sensitive to adverse health outcom es associated with exposure to these agents. There is substantial evidence that PFO A and PFOS are correlated w ith im m unotoxicity effects, leading to likely adverse health outcomes in populations of exposed humans. By extension, other PFCs that induce sim ilar toxicities and w ork through sim ilar m echanism s, such as PFBA, PFBS, and PFHxS, are also likely to induce immunotoxicity. As reflected in the assessm ent of PFCs by the ATSDR, certain highly exposed populations are at increased risk for adverse health outcomes associated with exposure to these agents. There is substantial evidence that in areas o f high PFC exposure, either due to occupational exposures or by living near fluorochem ical production and/or disposal facilities, there is particular hazard to hum an health, especially from effects on the liver and the im m une system and on developing organisms. The conclusions and observations in these assessm ents are supported by additional scientific w ork including my own extensive research experience w ith PFCs. This experience started with m y postdoctoral research experience and continues to present day. Thus, m y opinions also are supported by my knowledge of the published literature relating to PFCs that I have gained from my research endeavors, including writing and submitting scientific m anuscripts, review articles, book chapters, commentaries, and grant proposals, from review ing m anuscripts concerning the toxicity of PFCs submitted to scientific journals for publication, from serving as a peer-review er or participant for the EPA, NTP, and IARC assessment documents, and from presenting research findings and scientific opinions about the toxicity o f PFCs at various scientific meetings. M y opinions are based on a significant and grow ing body o f evidence from experimental animal m odels, and are supported by epidem iological findings in hum ans, as described in m ore detail below . VII. Background VII.1 BriefIntroduction to PFCs PFCs are a subset of highly fluorinated, synthetic substances that contain one or more carbon atoms on which the hydrogen substituents have been replaced by fluorine atoms so that they contain a perfluoroalkyl m oiety (Buck et al., 2011). PFC s are chem ically and therm ally stable and have both hydrophobic and lipophilic properties that m ake them com m ercially useful in surfactants and polym ers (Buck et al., 2011). As a result o f these characteristics, PFC s have been used in num erous applications, including processing aids for fluoropolym er m anufacture, stain, grease, and water resistant coatings, and aqueous film form ing foam s (AFFFs) used in fire suppression activities (B uck et al., 2011). A consequence o f their extensive use and resistance to degradation is their presence in the environment, including in tissues and blood o f wildlife and humans. The US National Health and Nutrition Exam ination Survey (NHANES) reported 5 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN detectable concentrations o f PFC s in the serum o f 97% o f surveyed individuals (Hu et al., 2016), indicating widespread hum an exposures. P F C s are enorm ously diverse (Figure 1) and can be distinguished by chain length and functional group. Jarnberg and van Bavel (2007) estimated from published literature that the total num ber o f described synthetic PFCs m ay exceed 10,000 individual compounds. These com pounds vary by the num ber o f carbons ("chain-length"), functional groups present (i.e., carboxylic acid, sulfonic acid, hexanoic acid, etc.), and other physicochem ical characteristics (Buck et al., 2011). N ot all o f these com pounds are produced for industrial or consum er uses; some are breakdow n products o f precursor com pounds (H outz et al., 2013), but nonetheless can contribute to environmental concentrations and to hum an exposures. It has been estim ated that approxim ately 3,000 PFCs have been intentionally produced (i.e., are not environm ental breakdow n products) and are being produced for various industrial purposes (W ang et al., 2017). Figure 1. The generalclassificationofPFCs asoutlinedby Buck etal.(2011) 6 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Both PFO A and PFOS are perfluoroalkyl acids (PFAAs), a subset o f PFCs, and have eight carbons each. PFO A has a carboxylic acid (-COOH) functional group w hereas PFOS has a sulfonic acid (-SO2OH) functional group. PFO A and PFOS thus differ by functional group (carboxylic versus sulfonic acid), but they have the same chain length, w hich is eight carbons. The U.S. EPA defines long-chain PFCs as perfluoroalkyl carboxylic acids (PFCAs) w ith eight or m ore carbons, w hich includes PFOA, as well as perfluoroalkane sulfonates (PFSAs) w ith six or m ore carbons, w hich includes PFOS and PFH xS (U.S. EPA, 2017b). The bulk of toxicological research efforts concerning PFCs has focused on PFO A and PFOS, presumably because they were widely produced and prevalently used. These compounds were produced for decades w ithin the US for the fluoropolym er and telom ere industry. PFOS m anufacture w ithin the US was voluntarily discontinued by the 3M com pany in 2002 (U.S. EPA, 2017b) and PFO A was phased out betw een 2006 and 2015 by eight m ajor U.S. m anufacturers through the 2010/15 PFO A Stewardship Program (U.S. EPA, 2017b). However, these compounds are still being produced in other countries, are used in the US through existing stocks, and are present in im ported item s (U.S. EPA , 2017b). Additionally, PFO A and PFOS may result from the breakdow n of other PFCs, generally term ed as "precursor compounds" (H outz et al., 2013). Alternative compounds currently are being produced to replace PFOA, PFOS, and other longchain com pounds phased out by the 2010/15 PFO A Stewardship Program. M any o f these fall under the Toxic Substances Control Act (TSCA) and the U.S. EPA has issued Significant N ew U se Rules (SNURs) for the m anufacture and process o f alternative com pounds (U.S. EPA, 2017b). H ow ever, not all PFC s are likely to be subject to TSC A guidelines as some are byproducts of production that may find their way into the environment. For example, in a recent report issued by the U.S. EPA (U.S. EPA, 2017a) concerning PFCs in the Cape Fear W atershed o f N orth Carolina, tw o perfluoroethersulfonic acids (PFESAs) containing seven carbons each were found in the river and were identified as byproducts of Nafion (another type of PFC fluoropolym er) production, presum ably from a Chemours production facility located in Fayetteville, NC. These long-chain PFCs have Chemical Abstracts Service (CAS) numbers, which are unique numerical identifiers for chemical substances (CAS, 2017), but no publicly available peer-review ed toxicological data are available for these PFCs (although toxicological data may exist as required under TSCA, it has not yet been published in the publicly available peer-review ed literature). Concentrations reported by the U.S. EPA w ere up to 73,900 ng/L (U.S. EPA, 2017a); these concentrations are orders o f m agnitude above the health advisory level (HAL) issued by the U.S. E PA for com bined w ater concentrations o f PFO A and PFO S (U.S. EPA, 2016a,b). The presence o f these byproducts indicates that long-chain PFCs beyond PFO A and PFOS exist in surface waters in the U.S. The alternative compounds being produced to replace PFOA, PFOS, and other long-chain com pounds are shorter chain PFC s (i.e., short-chain PFCs); such short-chain com pounds include PFB A and PFBS, for example. Short-chain PFCs m ay also be form ed from industrial synthesis, by m etabolism, and/or by environmental degradation of other PFCs (Butenhoff, 2012b). A lthough short-chain PFCs are thought to persist in the environm ent, they are believed by some to be less bioaccum ulative and less toxic in wildlife and hum ans than long-chain PFCs (U.S. EPA, 2017b). However, for many PFCs, especially the short-chain compounds, the num ber of publicly available peer-reviewed published studies are low compared to studies of PFO A and 7 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN PFOS. Beliefs about the low toxicity o f short-chain compounds are based on a relatively small database compared to PFOA and PFOS and some of the other long-chain compounds; however, em erging publications indicate that m any o f these short-chain com pounds elicit toxicities similar to the long-chain compounds, but along a different dose continuum. In a com m entary on PFCs that I co-authored and that was published in 2017, one recom m endation my co-authors and I had for improving the database on short-chain PFCs was to focus hum an biom onitoring studies on w orkers at production sites and populations downstream o f production sites to account for the relatively short exposure history to these alternative PFC s (W ang et al., 2017). A dditionally, we recom m ended that research is needed to understand PFCs as a group or as several subgroups to develop effect-oriented chemical and biological analyses and predictive m odels to evaluate the total burden o f sim ultaneous exposure to m ultiple PFCs, as the current hum an exposure paradigm is to m ultiple PFCs, rather than single PFC s (W ang et al., 2017). The toxicological inform ation generated for the long-chain compounds can therefore help to group alternatives and other less well-studied PFCs based on their toxicological profiles or putative toxicological profiles given sim ilarities (i.e., physiochem ical properties, persistence, bioaccum ulation potential, exposure routes, etc.). VII.2 Principles o f Toxicology Toxicology is the study o f the adverse health effects o f exogenous agents on living organisms. Toxicologists tend to classify them selves into sub-disciplines based on the types of potential adverse health outcom es they study. For example, im m unotoxicologists evaluate how exogenous agents affect the im m une system, neurotoxicologists assess effects o f exogenous agents on behavior or neurotransm itter levels, and developmental toxicologists determine how exogenous agents im pact developing organisms. These sub-disciplines can be further subdivided, but overall, regardless o f sub discipline, all toxicologists rely on the sam e key principles of toxicology to establish whether exposure to an exogenous agent is likely to cause harm to living organisms. W hile a detailed overview o f these principles is beyond the scope o f this report, some of them are outlined in detail in a basic prim er of toxicology for judges and lawyers (Eaton, 2003). Briefly, determ ining if toxic effects are induced by exposure to exogenous agents requires understanding concepts of dose, toxicokinetic and toxicodynamic factors, the exposure scenario (route, duration, and frequency of exposure), the chemical composition of the agent, factors associated w ith those exposed (age/lifestage, sex, genetic m akeup, health status), and mode (M oA) or m echanism of action (MOA). Dose. One key principle is dose m easurem ent and calibration. In other words, how m uch o f the exogenous agent is required to induce a toxic effect? As indicated in Figure 2, the dose o f the toxicant at the target Figure 2. Potentialstagesinthe development oftoxicityafterexposure to anexogenousagent.From Gregus (2015). 8 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN m olecule that is sufficient to alter the biological environm ent is frequently sought and measured. This value m ay differ from the am ount that is given, for example, to test organisms, or that may be present in one particular com ponent o f a biological organism (i.e., blood concentration). Although it is an im portant element, the dose is only one elem ent o f the toxicological landscape. For some agents, the occurrence o f adverse health outcom es does not always follow a "standard" dose-response curve where an increase in dose leads to an increase in response. Som e doseresponse curves are U-shaped or inverse-U-shaped, indicating that lower and higher doses produce responses that differ from m oderate doses. Therefore, the dose-response relationship is never as simple as the phrase "the dose m akes the poison" and adverse health outcom es that do not follow a clear dose-response pattern (i.e., an increase in outcom e w ith an increase in dose) should not be sum m arily discounted as spurious or lacking in toxicological relevance w ithout further experim entation and analysis. Toxicodynamic and toxicokinetic factors. Toxicodynam ic (processes by which exogenous agents produce effects in the body) and toxicokinetic (distribution of exogenous agents in the body, w hich includes absorption, distribution, m etabolism, and excretion or "A D M E") factors also play an im portant role in understanding how exogenous agents m ay induce adverse health outcomes. W hile there are m any possible hypotheses about how to specifically apply these factors to com plex problem s such as risk assessm ents, H einrich-H irsch et al. (2001) have established a simplified model to illustrate how these two factors are related to exposure and an adverse health outcom e (Figure 3). It is im portant to note that "internal dose" generally refers to the amount of an agent that can penetrate across various absorption barriers of an organism (Zartarian et al., 2006). "Toxicity" therefore is also a function of how much of a dose of an exogenous agent is at a target site (i.e., the cellular/subcellular Figure 3. A representationoftoxicokineticandtoxicodynamic factorsfromexposuretoadversehealthoutcome (irreversible pathology).From Heinrich-Hirschetal.(2001). site) integrated over tim e (Society of Toxicology, 2008). Toxicokinetic and toxicodynamic factors are particularly important for understanding the toxicity of PFCs, especially as related to the relevance of measured serum concentrations in experimental animal m odels and hum ans to adverse health outcomes. Serum concentrations in humans, w hether from the general population or from highly exposed populations (i.e., occupationally exposed or from areas o f w ith high environm ental concentrations o f PFCs) are used as biom arkers o f exposure as exposure m onitoring data are generally lacking (ATSDR, 2015). In other words, as data on the am ount o f PFCs ingested, inhaled, and/or dermally absorbed (see next section) by hum ans has not been precisely determined, confirm ation o f exposure is based on concentrations o f PFCs in serum. However, serum concentrations do not always reflect the true exposure scenario, i.e., the am ount o f PFCs taken in by an individual, or the true body burden, i.e., the sum total o f PFCs in the body that 9 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN may reach target sites to induce toxicity. Therefore, serum concentrations should be regarded as biom arkers o f exposure rather than biom arkers o f effect. Biom arkers o f exposure are measurem ents of compounds that reflect internal doses, biologically effective doses, or target doses whereas biomarkers of effect include changes on a cellular level or m arkers for early pathological changes in complex disease developm ent (Silins and Hogberg, 2011). Biom arkers o f susceptibility also have been defined, w hich is an individual's responses to specific exposures (Silins and Hogberg, 2011). In a 2004 review of the toxicity of PFO A written by a team of authors from DuPont, 3M, Covance (a contract research laboratory), Atofina, and Ineos (chem ical/petrochem ical com panies), som e o f the know n A D M E properties o f PFO A w ere highlighted (K ennedy et al., 2004). Greater than 90% o f a single oral dose o f PFO A was absorbed from the GI tracts o f male rats w ithin 24 hours of exposure and levels of PFO A m easured in the blood after inhalation or dermal exposure also indicated absorption o f PFO A following these routes o f exposure. In a study perform ed by D uP ont in 1982 and cited in the K ennedy et al. (2004) review , absorbed PFO A is m ostly excreted in the urine, but w ith som e fecal excretion. K ennedy et al. (2004) also noted that the 1982 D uPont study and num erous other published reports indicate a profound difference in blood clearance and urinary excretion between m ale and fem ale rats, w ith fem ale anim als clearing serum levels m ore rapidly than m ale anim als. K ennedy et al. (2004) also cited studies indicating sex differences in hamster, rabbits, mice, and cynomolgus monkeys, but sex differences w ere not always parallel w ith sex differences observed in rats. O nce in the blood, PFO A avidly, but reversibly, binds to plasm a proteins (proteins that serve as transporters for endogenous agents in an organism), especially album in, and distributes m ainly to the liver and kidneys, but also is detectable in lungs, heart, skin, testes, muscle, fat, and brain o f rats, ham sters, and m ice (Kennedy et al., 2004). Lau (2015) noted that the liver, kidney, and blood com partm ents can account for greater than h a lf o f the body burdens o f PFA A s. K ennedy et al. (2004) also addressed human biopersistence and stated that hum ans have the least ability to elim inate PFO A o f any species studied. W ith the exception o f sex differences, PFOS exhibits sim ilar A D M E properties (Lau et al., 2007). The relatively few studies on A D M E properties o f short-chain PFCs, such as PFBA and PFBS, indicate that these PFCs also are readily absorbed following oral exposure and are eliminated m ainly in the urine, but that they are eliminated from the serum m ore rapidly than the long-chain PFC s (Chan et al., 2008; O lsen et al., 2009). W hether the short-chain PFCs accum ulate in tissues similarly to the long-chain PFCs has yet to be determined, although a sm all-scale study o f tissues from hum an autopsies suggests that s shortchain PFC s do accum ulate in tissues (Perez et al., 2013). As a reminder, "internal dose" generally refers to the am ount of an agent that can penetrate across various absorption barriers o f an organism (Zartarian et al., 2006). W ith the exception of agents that induce local reactions at the site o f contact, i.e., such as burns from acidic or alkaline substances, exogenous agents have to be absorbed across m em branes associated w ith routes of exposure (ingestion, inhalation, dermal; see next section). Some ingested exogenous agents, for example, m ay be ingested and completely eliminated in feces w ithout ever being absorbed across membranes. They may produce local effects on the gastrointestinal (GI) tract, but if they do not absorb across m embranes, they cannot be distributed throughout the body and produce toxic effects at sites distant from the site o f absorption. Because PFCs are elim inated in the urine from all routes o f exposure (ingestion, inhalation, derm al), this is evidence that they are distributed 10 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN throughout the body via the blood, which increases the probability that they m ay interfere with processes distant from the site o f absorption. Route, duration, andfrequency o f exposure. The route o f exposure is the pathw ay through w hich an exogenous agent enters the body. For environm ental agents, this is via ingestion (oral), inhalation (lung), or dermal (skin) absorption. Occupational exposures m ay include injection directly into the bloodstream as well, such as in cases o f accidental needle sticks. Com m on ways by w hich the general hum an population is exposed via ingestion is through contam inated food and water. Food can be contam inated while it is growing, or during storage, processing, or cooking. W ater is typically contam inated at its source (groundw ater or surface w ater) by direct releases, accidental spills, or atm ospheric deposition, and contam inants m ay still be present in finished drinking water. Inhalation can occur if the exogenous agent is in air as a particle, aerosol, or vapor. D erm al absorption can occur if contam inated m edia (i.e., soil, sediment, w ater) get onto the skin and if the exogenous agent has physical-chem ical characteristics that allow it to m ove through the skin. Duration refers to how long the exposure occurs, a single time, over hours, days, weeks, months, or years. Short duration exposures that are associated w ith a single or a relatively short exposure period (days or weeks when the lifespan o f an organism is years) are classified as acute or interm ediate whereas exposure periods that constitute a m ajor portion o f an organism 's lifespan are considered chronic. Adverse health effects that occur following exposure to an exogenous agent also can be term ed as acute (occurring im m ediately after exposure) or chronic (occurring weeks, months, or years after exposure). Frequency is the num ber o f tim es per hour, day, week, month, or year the exposure occurs. Chemical composition. This toxicological principle is based on the physical-chem ical characteristics o f the exogenous agent. Briefly, this relates to w hether the agent is fat or w ater soluble, w hether it is positively or negatively charged or neutral in charge, w hether it is relatively large or small, acidic or basic, reactive or non-reactive, and how it is shaped. These characteristics affect an exogenous agent's ability to get into living organisms, move around inside organisms, ability to bind to various receptors within organisms, and excretion from organisms. Exogenous agents that appear to be "inert," in other words not chem ically reactive in that they do not readily m etabolize to a reactive intermediate, nor directly damage cells or m em branes, nor create m utations in DN A, for exam ple, can still interact w ith endogenous biom olecules to induce adverse physiological outcom es through a variety o f mechanisms. PFCs fall into this latter category; they are not chem ically reactive as far as w e know, but can interact with myriad endogenous biomolecules. Therefore, PFCs may be chemically "inert" in some respects, but are still reactive in that they can alter physiological processes through their interactions with endogenous biomolecules. Factors associated with those exposed. F actors associated w ith those exposed m ay have an effect on potential adverse health outcom es that m ay arise from exposure to exogenous agents. Infants, for example, have imm ature enzyme systems and m ay be unable to appropriately detoxify exogenous agents to w hich they are exposed. A classic exam ple is m ethem oglobinem ia (com m only know n as "blue baby syndrom e"), a situation that m ay occur in infants exposed to nitrates in their drinking water. N itrates are able to oxidize the iron in hem oglobin (the carrier o f oxygen in red blood cells) from one valence state to the other, w hich reduces its efficiency at carrying oxygen. In adults, the enzyme cytochrom e b5 m ethem oglobin reductase can reduce the oxidized iron, but in infants, this enzym e is inefficient and occurs at low er levels than in adults, 11 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN thus m aking infants at increased risk of m ethem oglobinem ia and the adverse health outcomes that arise from this condition. M any other examples exist to demonstrate that factors associated w ith age/lifestage, sex, genetic m akeup, health status, nutritional status, and others can alter the existence or severity of adverse health outcomes following exposure. Some of these factors can reduce the likelihood of adverse health outcomes; for example, some individuals m ay rapidly m etabolize certain exogenous agents and reduce or eliminate their toxicity. However, m any of these factors can render individuals or groups of individuals m ore susceptible to adverse health outcomes. One goal, therefore, o f regulatory guidelines that reduce exposure to exogenous agents that produce adverse health outcomes can be to protect susceptible subpopulations from adverse health outcomes that arise from exposure. M ode or mechanism o f action. "M echanistic" (M OA) refers to the particular reaction o f the exogenous agent w ith a target m olecule or m olecules to induce toxicity, i.e., an adverse health outcom e or outcomes (Gregus, 2015). "M ode o f action" (M oA) is the interaction o f an exogenous agent w ith the cell through functional and anatom ical changes that results in adverse health outcomes and is generally regarded as a less detailed understanding o f the m olecular basis o f a toxic effect (OECD, 2012). In general, toxicological studies to identify the M oA and M OA are undertaken once adverse health outcom es following exposure have been observed. These types o f studies are particularly valuable in that they can establish pathways betw een a m olecular-level change and a phenotypic (i.e., anatom ical or functional change) outcom e following exposure and im prove extrapolations among species. However, lack of M oA or M OA does not negate findings associated with an adverse health outcome, but rather indicates additional experimental studies are necessary to identify those m olecular-level events that lead to the adverse health outcome. As a general m atter, all o f the concepts ju st listed are taken into consideration w hen studying effects o f exogenous agents on living organisms. Toxicologists frequently rely, for ethical reasons, on experim ental animal m odels (e.g. m ice and rats) to assess toxicity follow ing exposure to exogenous agents. Therefore, studies in experimental animal m odels are generally considered valuable for understanding and predicting adverse health effects in humans. Certainly, hum ans and rodents are not exactly alike, but evaluation o f toxicity o f exogenous agents in experimental animal m odels is a cornerstone o f hum an safety evaluation and findings in animal toxicology studies generally are applicable to hum ans (NRC, 2004). U ncertainty factors and extrapolations based on body weight equivalence, for example, often are applied to values derived from experimental animal m odels to account for differences betw een these animal m odels and hum ans and to derive factors that are relevant to hum ans (NRC, 2004). In addition, toxicologists often also consider and rely on the w ork o f epidem iologists, scientists w ho link exposure to exogenous agents with the num ber(s) and type(s) of adverse health outcomes observed in hum an populations, to understand the extent o f effects arising from exposures. W hat distinguishes adverse health outcomes from non-adverse effects is not always unam biguous and often relies on professional judgm ent for identification. Several reports and publications exist to help toxicologists determ ine which outcom es are adverse versus non adverse. One such publication from 2012 (K eller et al., 2012) reflects a harm onized opinion o f a diverse group of scientists from chemical and pharm aceutical industries, governm ent agencies, and academ ic institutions. The publication was from a workshop held by a com m ittee form ed in 2009 by the International Life Sciences Institute (ILSI) Health and Environm ental Sciences 12 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Institute (HESI). K eller et al. (2012) defines adverse and non-adverse/adaptive responses in the following ways: "A dverse Effect: A change in m orphology, physiology, growth, development, reproduction, or life span o f a cell or organism, system, or (sub)population that results in an im pairm ent o f functional capacity, an im pairm ent o f the capacity to com pensate for additional stress, or an increase in susceptibility to other influences. N on-adverse/A daptive Response: In the context of toxicology, the process w hereby a cell or organism responds to a xenobiotic so that the cell or organism will survive in the new environm ent that contains the xenobiotic without im pairm ent of function." Once adverse effects are identified, they are typically evaluated for dose-response, but again, not all responses follow a sim ple dose-response relationship where an increase in dose leads to a concom itant increase in response. W ith this basic toxicological assessment, doses that are unlikely to be associated w ith adverse health outcom es can be distinguished from those that are. The dose that is unlikely to be associated w ith adverse health outcomes is termed the No Observed Adverse Effect Level (NOAEL) and is defined as the highest dose that is not statistically distinguishable from unexposed organism s (i.e., a control group or population). Effects m ay occur at the N O A EL dose, but they are not considered adverse or precursors to adverse effects. The Low est O bserved A dverse Effect Level (LOA EL) is the low est dose that produces statistically distinguishable increases in the severity or frequency of adverse health outcomes when compared to unexposed organisms. Toxicologists often distinguish the relative toxicity of compounds by comparing the N O A EL and the LOAEL and frequently public health officials for use the N O A EL for establishing protective health guidelines. The NO A EL and LOAEL ideally are based on studies of chronic duration as they indicate adverse health outcomes that occur over a lifetim e o f exposure. Relative toxicity also can be com pared from acute exposures that lead to death as an outcome. These studies typically result in "LD 50" values, or the dose o f an exogenous agent that leads to death in 50% o f a group o f experimental animal models. W hile LD50s can be used to determine or compare the toxic potency o f acute doses of exogenous agent, they are not particularly useful for protection o f public health as death is an adverse health outcom e that is obviously avoided. However, as previously described, the dose-response is only one concept when determ ining if adverse health effects are induced by exposure to exogenous agents and not all dose-response patterns follow the typical pattern of an increase in response with an increase in dose. The NO AEL or LOAEL can represent a point of departure (POD), or a particular point on a doseresponse curve that is the beginning o f extrapolation to low er doses and is an estim ated dose, in hum an-equivalent term s, near the low er end o f the observed dose-response range (U.S. EPA, 2012). By way o f example, one group o f agents that has been shown to challenge the "dose m akes the poison" concept in toxicology is endocrine disrupting compounds (EDCs), agents that disrupt hormones. M any o f these agents produce effects at low doses that are not predicted by effects at higher doses and have dose-response curves that are "nonm onotonic." V andenberg et al. (2012) 13 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN defines these types of dose-response curves as "a nonlinear relationship betw een dose and effect where the slope o f the curve changes sign somewhere w ithin the range o f doses exam ined." In other words, as the dose goes up, the response m ay go down. This concept is particularly im portant for PFCs given evidence that indicates they can disrupt hormones. One w ay that the U.S. EPA has responded to agents that lack a typical dose-response relationship is through the developm ent o f an approach known as the benchm ark dose (BMD). D evelopm ent of the BM D approach also was a response to lim itations associated with using the N O A EL to establish protective health guidelines. A ccording to the U.S. EPA (U.S. EPA, 2012), using NO A ELs for protective health guidelines is lim ited to doses used in the particular study containing the N O A EL and does not account for variability or the slope in a dose-response curve. The BM D m ethod, therefore, is an approach that uses m athem atical m odels to fit doseresponse data and derive a reference value that is associated w ith a PO D that m ay not be equivalent to the NO A EL or LO AEL as it takes into account the entire dose-response curve and not a singular point (i.e., the N O A EL or LOAEL). Briefly, the BM D approach used by the U.S. EPA (U.S. EPA, 2012) recom m ends the use o f the 95% low er bound on a BM D (known as the BM D L) as the PO D for non-cancer effects. A ccording to the U.S. EPA (2012), "U sing the low er bound accounts for the experimental variability inherent in a given study and assures (with 95% confidence for the experimental context) that the selected B M R [benchm ark response] is n ot e x c e e d e d ... B ecause o f the lim itations o f the N O A EL/LO A EL a p p ro a c h ., the BM D approach is preferred to the N O AEL/LO AEL approach. For instance, a BM D (or BM DL) can be estim ated even w hen all doses in a study are associated w ith a significant adverse response (i.e., w hen there is no NO A EL). N ote, however, that there are some instances in which reliable BM Ds cannot be estim ated and the N O AEL/LO AEL approach m ight be warranted. In particular, the available data m ay not be am enable to m odeling, for exam ple w hen all exposed groups exhibit a m axim um response. In such a case, the observed data provide very little inform ation across the full range o f response levels, and BM D m odels cannot provide reliable estimates w ithin that range (although in such a case, information from the LO AEL is limited, as w ell)." W hile the details o f this m ethod are beyond the scope o f this report, it is im portant to m ention this approach as it was the predom inant approach used by the U.S. EPA to derive reference doses (RfDs) for PFCs. One last m ajor principle o f toxicology is how determ inations on cause-and-effect relationships betw een exposure to a particular exogenous agent and adverse health outcom es are made. In 2006, the International Program m e on Chemical Safety (IPCS), an international group of experts, published a fram ework (Figure 4) for analyzing the relevance of a cancer mode of action for hum ans (Boobis et al., 2006) and in 2008, extended the fram ew ork to a non-cancer m ode of action for hum ans (Boobis et al., 2008). B oth o f these docum ents are based on the "Bradford Hill criteria," a set o f guidelines w ritten by Sir Austin Bradford Hill in 1965 that allowed for the leap from observation to causation (Hill, 2015). Briefly, the Bradford Hill criteria ask the follow ing questions about associations betw een tw o variables: 1) Strength, 2) C onsistency, 3) Specificity, 14 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN 4) Temporality, 5) Biological gradient, 6) Plausibility, 7) Coherence, 8) Experimental intervention, and 9) Analogy. Hill (2015) cautions that "None of my nine viewpoints can bring indisputable evidence for or against the cause-and-effect hypothesis and none can be required as a sine qua non. What they can do, with greater or less strength, is to help us to make up our minds on the fundamental question - is there any other way of explaining the set of facts before us, is there any other answer equally, or more, likely than cause and effect?" Hill's viewpoints were therefore used by the IPCS to draft a framework that would be applicable to both cancer and non-cancer endpoints and that would provide a means for "ensuring a transparent evaluation of the data, identification of key data gaps and of information that would be of value in the further risk assessment of the compound, such as on dose-response relationships, and recognition of potentially susceptible subgroups, for example, based on lifestage considerations" (Boobis et al., 2006). While definitive "yes" and "no" answers are included in the framework, the authors of the framework recognized the need for judgment regarding the sufficiency of weight of evidence (Boobis et al., 2006). Specifically, answers on the left side of the diagram (Figure 4) indicate that weight of evidence for the MoA would be insufficient to support relevancy in humans whereas answers on the right side of the diagram (Figure 4) would indicate that weight of evidence for the MoA would be sufficient to support relevancy in humans or that it would not be possible to reach a conclusion regarding likely relevance in humans given uncertainties in available information (Boobis et al., 2006). Is the weight ol evidence sufficient to establish a mode of action (MOA) in animals? NO Continue with risk assessment MOA nol relevant YES *-- i YES Cun human relevance of the MOA be reasonably excluded on the basis of fundamental, qualitative differences in key events between experimental animals and humans? J, NO MOA nol relevant YES Can human relevance of the MOA be reasonably excludeJ on the basis of quantitative differences in either kinetic or dynamic factors between experimental animals and humans? NO Continue with --> risk assessment Figure 4. A decisiontreefordetermininghuman relevanceofaMoA forcarcinogenicityor toxicityobservedinexperimentalanimalmodels.From Boobis etal.(2006; 2008). VII 3 B riefBackground on PF C Toxicology' and Epidemiology- Based on these concepts of toxicity of exogenous agents, toxicologists have determined that exposure to PFCs induces a wide variety of adverse health outcomes in experimental animal models. Epidemiological studies are supportive, showing similar adverse health outcomes in exposed human populations. These toxicological and epidemiological studies indicate that exposure to PFCs pose a substantial present and potential hazard to human health. Although unifying MoAs or MOAs for the various toxicities have not been conclusively identified, a 15 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN num ber o f plausible M oA s and M O A s have been uncovered in experimental animal m odels that have relevancy to exposed humans. These will be discussed in subsequent sections on evidence associated with specific toxicological outcomes. As stated previously, NH AN ES has reported detectable concentrations o f PFCs in the serum of 97% o f surveyed individuals in the U.S. (Hu et al., 2016), indicating w idespread hum an exposures. PFCs are synthetic agents that do not occur naturally in the environment, so levels in biological tissues and fluids of humans and other organisms indicate exposure from industrial releases to the environm ent or from the leaching o f PFCs from consum er products. Exposure in the general hum an population therefore occurs w hen humans ingest PFCs from contaminated water or food, breathe them in, often from house dusts, or dermally absorb them from water or other m aterials containing PFCs. As PFCs are used in the textile industry to confer stain and w ater repellency to textiles such as carpets, children m ay have high exposures via hand-to-m outh transfer from treated carpets relative to adults (ATSDR, 2015). Areas of high exposure have been recorded in hum an and wildlife populations (im pacts on wildlife populations are addressed in Dr. Ronald K endall's report and will not be addressed here) living near fluoropolym er manufacturing facilities and people who w ork in m anufacturing facilities generally have higher concentrations o f PFCs in their serum (ATSDR, 2015). Again, as stated previously, levels in serum are generally used as biom arkers o f exposure (ATSDR, 2015). For purposes o f my opinions, PFO A and PFOS will be the m ain PFCs o f focus due to the volum e o f toxicological data surrounding these compounds. However, data from studies of perfluorinated carbon chains consisting o f four to 16 carbons w ith sulfonate functional groups (PFOS, PFDS, PFHxS, and PFBS) or carboxylate functional groups (PFOA, PFTA, PFDoA, PFUnA, PFDA, PFNA, PFHpA, PFHxA, PFPeA, and PFBA) that are terminal products, m etabolites, or degradation products, m ay be included as supportive data. To determine the likelihood of harm to hum an health following exposure to environmental contam inants, regulatory agencies often use a "w eight-of-evidence" approach. U sing this approach, the quantity and quality of data related to a particular contam inant, including in vitro (outside o f living organisms, usually in cells or tissues), in vivo (in living organisms), in silico (com puter-based simulations and models), and epidem iological studies (direct studies o f exposed humans), are assessed to determine if they support a likelihood o f harm to hum an health following exposure. This approach often is used by, for example, the U.S. EPA to establish health advisories, guidelines, and regulatory standards, or the IARC to determ ine the carcinogenicity o f agents. However, this is an approach and is not a regulatory requirement. Given that only PFO A and PFOS have been the subjects of broad assessments, other compounds will be evaluated based on relevant (i.e., toxicological) studies published in publicly available peer-review ed literature. Three agencies, the U.S. EPA, the U.S. N TP, and the IARC, have com pleted such weight-ofevidence assessments, or "systematic reviews," for PFO A and/or PFOS. These systematic reviews are thorough evaluations o f the body o f toxicological evidence concerning PFO A and PFOS and their likelihood of harm to hum an health following exposure. Each of these reviews are comprehensive o f m ost o f the published studies (up to a specified cut-off date determ ined by the agency sub-com mittee charged with writing the document) and accum ulated knowledge about PFO A and PFOS. In these reviews, although they differ somew hat from agency to agency, 16 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN publicly available peer-review ed published literature provide the bulk o f the reference materials. In some instances, patent filings and industry reports may provide background and support, but generally do not contribute substantially to the reviews. In addition, A TSD R has published a draft toxicological profile for PFCs, which is a succinct characterization o f the toxicological and adverse health effects fo r 13 separate P F C s (A T SD R , 2015). E ach o f th e assessm ents by the U .S. EPA, the U.S. N TP, and the IA RC underw ent rigorous peer-review . The A T SD R profile is still only available online as a draft for public com m ent and has not been fully peer-review ed or released in its final form. M oreover, for the U.S. EPA, N TP, and A TSD R documents, com m ents from the public were and are being solicited, giving private citizens, trade organizations, industry scientists, and others the opportunity to offer suggestions and criticisms. This is an extremely important aspect of the developm ent of these documents as it represents transparency in the process by which conclusions were reached by these various agencies. The IARC does not include public comm entary in its m onographs, but scientific participants in the developm ent o f a particular m onograph form ally review one another's w ork through an established process o f internal peer-review and interested parties, such as representatives from comm unity and trade groups, can observe m eeting proceedings for a particular monograph. Such weight-of-evidence evaluations are a w idely accepted m ethodology in the toxicological and epidem iological scientific communities. In form ulating my opinions, I use a similar approach that integrates the evidence from various sources to assess the likelihood that exposure to PFCs poses a substantial present or potential hazard to hum an health. As these agencies (ATSDR, IARC, NTP, U.S. EPA ) have com pleted rigorous, peer-review ed science assessments o f PFCs, a large part o f m y opinions are supported by these sources. VII.4 PFC s in the E ast M etro Population The eastern Twin Cities m etropolitan region ("East M etro") o f M innesota includes sites o f high PFC concentration as they include the 3M Cottage Grove m anufacturing facility as well as large waste disposal facilities where PFC-bearing wastes were disposed (MDH, 2017d). Some of the municipal drinking water wells in the East M etro area also contain levels of PFCs greater than current health based values (HBVs) established by the M innesota Departm ent of Public Health (MDH). As a result of the contam ination and concerns over health risks induced by exposure to PFCs via drinking water, the M D H conducted three biom onitoring studies o f a small sample of residents from the East M etro area (M DH, 2008; 2010; 2015). In the 2008 study, blood from 196 adults from Oakdale, Lam e Elmo, and Cottage Grove, M N, was evaluated for levels of PFOA, PFOS, PFHxS, PFBA, PFBS, PFHxA, and PFPeA (MDH, 2008). PFOA, PFOS, and PFHxS w ere found in the blood o f all participants at levels higher than the general U.S. population, but comparable to or lower than levels found in studies of other PFC-contam inated communities (MDH, 2008). PFBA and PFBS were found in a smaller proportion of participants and PFH xA and PFPeA were not found in any participants (MDH, 2008). A 2010 follow-up study was perform ed to not only better understand hum an exposure to PFCs, but to determine if a public health intervention to reduce drinking water exposure to PFCs actually reduced levels of PFCs in the blood of participants (MDH, 2010). In the follow-up study, the same seven PFC s as w ere m easured in the 2008 study w ere m easured in 164 participants. Levels o f PFO A , PFO S, and PFH xS, the PFC s that w ere present in all participants 17 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN in 2008 declined by ~25%, 30%, and 22%, respectively, from 2008 to 2010 (Figure 5; MDH, 2010). However, even though levels of these three PFCs had declined in the East Metro region in this two year time period, levels of these PFCs in this subset of East Metro residents were still greater than the general U.S. population, by -64%, 46%, and 69% respectively, for PFOA, PFOS, and PFHxS (Figure 5; MDH, 2010). 2008 average 2010 average U.S. population average 2007 08 PFOS PFOA PFHxS Figure 5.Resultsofthe2010 EastMetro followup studydemonstratingbloodlevelsof PFCs inresidentsfromtheEastMetro areain2008 and2010 and ascompared totheblood levelsofPFCs inpeoplefromthegeneralU.S. population.From M D H (2010). An additional follow-up investigation in 2014 indicated that while serum concentrations of PFCs in long-term East Metro residents had declined further, they nonetheless remained higher than concentrations in the U.S. general population. (Figure 6: MDH, 2015). Therefore, even with a public health intervention hi place to reduce drinking water exposure to PFCs, long-term residents of the East Metro region still carried a disproportionately higher body burden of PFCs relative to the general U.S. population. Blood PFC levels in long-term East Metro residents 40 35 30 25 20 15 10 5 0 Type of PFC Figure 6.Resultsofthe2014 EastMetro followup studydemonstratingbloodlevelsofPFCs hiresidents fromtheEastMetro areain2014 andascomparedtothebloodlevelsofPFCs inpeoplefromtheEast Metro inpreviousyearsand fromthegeneralU.S.population. From M D H (2015). 18 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Concentrations of PFCs in some of the m unicipal drinking w ater w ells in the East M etro area contain levels of PFCs greater than current and past M D H HBVs. Therefore, long-term East M etro residents have been exposed to levels of PFCs in their w ater above levels that the M DH currently considers to be safe for hum an consumption, including sensitive subpopulations. The current H B V s for four PFC s are as follows: PFO A = 0.035 pg/L (M DH, 2017f); PFOS = 0.027 pg/L (MDH, 2017g); PFBA = 7 pg/L (MDH, 2011a); PFBS = 7 pg/L (MDH, 2011b); and PFHxS = 0.027 pg/L (the M D H is using the H B V for PFOS as a protective level for PFHxS; M DH, 2009b). These H B V s are discussed in m ore detail in section IX. W ell m onitoring data (Karls, 2017) from Oakdale, for example, indicated that average concentrations PFOA, PFOS, and PFHxS exceeded their respective HBVs in 2012 and that average concentrations PFO A and PFOS exceeded their respective HBVs in 2016. One approach that the M D H takes to address concurrent exposures to m ultiple chemicals, w hich is in agreem ent with, for example, the U.S. EPA, is to derive a health risk index (HRI; M DH, 2017i). The H R I is calculated by first determining the ratio o f the m easured concentration o f an individual chemical to its respective H B V and then summ ing the ratio for the group o f chemicals being evaluated. For example, in Oakdale, a region in the East M etro area that contains a Superfund site that includes PFC w astes (M D H, 2016), the H R I w as 17.6 in 2012 and 16.9 in 2016. The w eighted PFOS concentration for Oakdale in 2012 was 0.275 pg/L (Karls, 2017); its ratio is 0.275 pg/L divided by 0.027 pg/L = 10.2. The w eighted PFO A concentration for O akdale in 2012 w as 0.214 pg/L (Karls, 2017); its ratio is 0.214 pg/L divided by 0.035 pg/L = 6.1. The w eighted PFH xS concentration for Oakdale in 2012 was 0.032 pg/L (Karls, 2017); its ratio is 0.032 pg/L divided by 0.027 pg/L = 1.2. The H R I for these three PFC s = 10.2 + 6.1 + 1.2 = 17.5. W hen the ratios o f PFB A and PFBS are added in (0.095 and 0.002, respectively), the H R I for these municipal drinking w ater w ells in O akdale is 17.6. In 2016, the H R I for these five PFC s in O akdale w as 16.9, again, greater than one and an indication that PFC levels in these m unicipal drinking w ater wells were greater than the HBV. By early 2007, two municipal wells in Oakdale had granular activated carbon filtration systems; accordingly, concentrations discussed above likely do not represent levels of current exposure (MDH, 2007). However, prior to the installation of filtration systems, long-term residents w ould have been exposed to higher concentrations o f PFCs. For comparison, although present at low er levels in m unicipal drinking w ater wells, the HRI for C ottage G rove in 2012 w as 1.7 and in 2016 w as 1.5 (based on w ell m onitoring data provided by Karls, 2017). A H R I greater than one indicates that concentrations in the exposure source (i.e., municipal drinking w ater w ells) are greater than the HBV. In other words, people exposed to the combined concentrations of these five PFCs in their drinking w ater with these municipal drinking water wells as a source exceeded a level that the M D H considers to be safe for hum an consumption, including sensitive subpopulations. VII.5 Chronology o f 3 M Toxicology Studies on PFC s As evidenced by documents produced by 3M that I have reviewed, it was clear that the company had been studying PFC toxicology since at least the 1940s. Below is a brief chronology o f a few o f the num erous toxicological studies on PFCs perform ed by 3M or their subcontractors: 19 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN 1949: 3M conducted acute toxicity tests o f heptafluorobutyric acid in mice. The results noted " [i]mmediate deaths characterized by respiratory distress. Anim als dying later, died after a period of lethargy" (3M _M N05305661). 1963: By this point, 3M had determ ined the acute toxicity o f several PFCs, as indicated by a list o f LD50 for several o f their PFCs, including FC-95 (the potassium salt of PFOS). In a technical brochure, 3M described FC-95 as "m oderately toxic" (3M Company, 1963). By w ay o f example, phenobarbital and m orphine, have sim ilar LD50s as com pared to FC-95 (~100-1000 mg/kg). 1978: After being inform ed about the presence of organic fluorine in the blood of the general population and subsequently discovering organic fluorine in the blood o f its own workers, 3M launched a series o f 90-day subchronic toxicity studies in rats and monkeys using three PFCs. 3M concluded, based on these studies, that FC-95 was "considerably m ore toxic to m onkeys than anticipated." One o f 3 M 's studies w as aborted because all o f the dosed m onkeys died. PFO A and N-EtFO SE exposure also was associated with various adverse health effects (3M A00593073, 3M A01797217, 3M _M N01663513, 3M A10065004, 3M_MN01663537). 1980-83: 3M conducted several teratology studies o f PFC s in rats. Initial studies suggested that PFCs induced lens abnorm alities and other birth defects in rat fetuses. W hile further 3M studies suggested that the lens abnorm alities m ay have been caused by a sectioning artifact, as described in section VII.3.b, below, a com pound-related effect cannot definitively be ruled out. 1987-88: 3M com pleted tw o 2-year carcinogenicity studies o f PFO A and N -EtFO SE in rats. A lthough 3M concluded in these studies that neither o f the compounds w ere linked to elevated tum or incidence in rats, subsequent re-exam ination o f the study's findings have established that the conclusions in the reports about tum or incidence were incorrect-- i.e., both PFO A and N -EtFO SE did induce tum ors in rats (3M A 00027126, 3M A00593233, 3MA10025147). 2001-2002: 3M completed 6-month toxicity studies of PFO A and PFOS in cynomolgus monkeys. M ortality was observed in both studies. In both studies, m onkeys exhibited effects on thyroid horm ones and the liver. In addition, m onkeys dosed w ith PFOS exhibited lowered cholesterol and high density lipoprotein (HDL; 3M _M N02343342, 3M _MN03278834). 2001-2002: 3M com pleted 2-year carcinogenicity studies of N -EtFO SE and PFOS in rats. Both studies observed statistically significant hepatocellular combined adenomas and carcinom as in fem ale rats and statistically significant com bined thyroid follicular cell adenomas and carcinom as in m ale rats. (3M B00040027, 3M A00812492). 20 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN VII.6 Toxicological Evidence Regarding Human Health "Of questionable relevance to human health" or "mean serum concentrations in experimental animal models is several hundred times higher than those reported for humans in the U.S. general population" are phrases that often are included in publications authored by scientists from 3M. These phrases occur in publications that focus on liver toxicity, carcinogenicity, developmental toxicity, and endocrine disruption. In many instances, when experimental animal models are exposed to precise concentrations of PFCs on a daily basis for a proscribed amount of time, the resulting serum concentrations are higher than what has been measured in humans from the U.S. general population. While experimental animal models are not tiny humans, they are accepted models for human health in the toxicological sciences as well as other biomedical sciences. As stated previously, serum levels of PFCs in humans are biomarkers of exposure; technically, serum levels in experimental animal models also are biomarkers of exposure and are not biomarkers of effect. PFCs have toxicokinetic complexity - they bind to serum proteins and partition into blood, liver, and kidney, and demonstrate half-life differences among species. This toxicokinetic complexity makes it inappropriate to directly compare serum levels in experimental animal models with serum levels in humans. Additionally, as the human exposure pathways for PFCs have not been fully mapped, serum levels may underestimate exposures and do not capture differences in serum protein binding and storage in the liver and kidney that may exist between humans and experimental animal models. When adverse health outcomes are observed in experimental animal models in multiple systems/organs/tissues/cells/pathways, in multiple species and stains, across myriad exposure pathways, occurfor a range o fPFCs, and show concordance with epidemiologicalfindings, the results are highly relevan t to hum an health. VII. 6a 3M Worker Studies 3M has repeatedly suggested that the myriad findings that exposure to PFCs is associated with adverse effects in experimental animal models should be ignored or discounted. They claim that epidemiology studies conducted by 3M regarding its workers demonstrated that, although the workers were more heavily exposed than the average population, they did not suffer adverse health effects. I disagree, for several reasons. First, as I discuss in more detail in section VIII, numerous epidemiology studies performed by independent experts have identified statistically significant associations between increased exposure to PFCs and adverse health effects that are supported by results of toxicology studies in experimental animal models exposed to PFCs alone. These epidemiology studies include both worker and non-worker populations in the U.S. and around the world, including at levels of exposure within and below blood serum levels measured in East Metro residents. Second, contrary to 3M's claims, its studies did demonstrate PFC-related adverse health effects in its workers. 3M's worker studies have found statistically significant associations between PFC exposure and health endpoints indicative of adverse effects, including prostate cancer mortality (Gilliland and Mandel, 1993, 3MA00631764; Lundin and Alexander, 2007, 3MA02557439), cerebrovascular disease (Alexander, 2001a, 3MA00417473; Lundin and Alexander, 2007, 3MA02557439), bladder cancer mortality (Alexander, 2001b, 3MA01472304), cholesterol and triglyceride increases (Olsen et al., 2001, 3M_MN02483163; Olsen unpublished draft, 2008, 3MA02543911), self-reported history of gallbladder disease (Olsen unpublished draft, 2008, 21 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN 3M A 02543911), and endocrine m odulation (G illiland, 1992, 3M _M N 03112178; O lsen et al., 1998, 3MA10069722). Finally, as discussed in m ore detail by Dr. Philippe Grandjean, I understand that a num ber of 3M 's epidem iology studies o f its w orkers w ere flawed, underm ining the reliability o f their findings. VII. 6b PPARa Activation and Peroxisome Proliferation 3M also argues that toxicology studies dem onstrating that exposure to PFCs associated with specific adverse effects in rodents-- m ost notably in the liver-- do not translate to risks for hum ans on the basis that these adverse effects are m ediated by a process called peroxisom e proliferation that rodents are uniquely susceptible to. This is incorrect, in part, as several liver effects observed in PFC studies using experimental rodent m odels result from im pacts on processes other than peroxisom e proliferation. Additionally, hum ans respond to pharmacological agents that act through the receptors that are thought to m odulate PFC-associated peroxisom e proliferation in rodents. Peroxisom e proliferation is associated w ith a group of ligand-activated transcription factors know as peroxisom e proliferator-activated receptors (PPARs). Three sub-types are known to exist, PPA R a, PPARy, and PPA Rp/5, and each have slight differences in term s o f their affinity for ligands and the effects that they produce w hen activated (Tyagi et al., 2011). Overall, these receptors play a regulatory role in energy hom eostasis and m etabolic function and when activated, m ay have both protective and detrimental effects in dyslipidem ia, diabetes, adipocyte differentiation, inflammation, cancer, lung diseases, neurodegenerative disorders, fertility or reproduction, pain, and obesity (Tyagi et al., 2011). One consequence o f P P A R a activation is peroxisome proliferation, an increase in the am ount and activity of the peroxisomal enzymes associated w ith various physiological functions related to energy hom eostasis and m etabolic function. Peroxisom e proliferation was considered to be a possible mechanism associated with hepatocellular vacuolation, hypertrophy, and tum or production induced by exposure to PFO A and PFOS. However, as discussed in m ore detail in section VIII, these adverse effects have been demonstrated to occur via m echanism s other than peroxisom e proliferation. Studies have demonstrated that hepatocellular hypertrophy and nonneoplastic liver lesions occur in rodent models in which peroxisom e proliferation cannot occur because the rodent has been genetically altered to lack the receptors by w hich peroxisom e proliferation is m ediated. (Filgo et al. 2015) Although hum an P P A R a levels are thought to be about 10% of the levels in rodents (DeW itt et al., 2009b), hum ans are responsive to agents that act via PP A R a; the M O A for the fibrate class o f drugs designed to low er serum lipids is via P P A R a activation. Accordingly, the risk of adverse effects to hum ans associated w ith PPA R a/peroxisom e proliferation cannot be discounted. Further supporting this point, studies in pigs-- an animal model that, like hum ans, is less susceptible to peroxisom e proliferation than hum ans-- have shown that peroxisom e proliferators m ay nonetheless pose a risk o f carcinogenesis (Luci, et al.2007). 22 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN VIII. Detailed Supported Opinions VIII.1 Basis andMethodologyfor Rendering Opinions As m entioned above, several agencies w ithin the U.S. and on the international level have performed comprehensive science assessments to determine if exposure to PFCs is associated w ith an increased risk o f adverse health outcomes in humans. Three o f these assessments are particularly relevant for the questions presented here, and I was involved in each of them either in form ulating a decision or as part o f the external body o f peer-review ers asked to evaluate the assessm ents. Specifically, these are: (1) M onograph 110, Some Chem icals U sed as Solvents and in Polym er M anufacture, by the IARC o f the W HO, (2) the Health Effects Support D ocum ent for Perfluorooctanoic Acid (PFOA) and the Health Effects Docum ent for Perflurooctane Sulfonate (PFOS) written by the U.S. EPA, and (3) the Systematic Review o f Im m unotoxicity Associated with Exposure to Perfluorooctanoic Acid (PFOA) or Perfluorooctane Sulfonate (PFOS) by the U.S. NTP. Additional science assessm ents have been perform ed in other countries, but given the comprehensive nature of those three assessment, I do not undertake here any detailed review of assessm ents in other countries. I also rely here on the Toxicological Profile for Perfluoroalkyls by the ATSDR, which also is particularly relevant. The A TSD R is the part o f the Centers for Disease Control and Prevention (CDC) that serves the public by preventing harmful exposures and diseases related to toxic substances, and their assessm ents are issued after an opportunity for public commentary and peer-review. In addition to the three assessments and the A STD R profile just m entioned, my opinion that PFCs pose a substantial present or potential hazard to hum an health is inform ed by the fo llo w in g : 1. M y extensive research experience w ith P F C s starting w ith m y postdoctoral research experience and continuing to present day. 2. M y know ledge o f the published literature relating to PFC s that I have gained from my research endeavors, including w riting and submitting scientific manuscripts, review articles, book chapters, commentaries, and grant proposals. 3. F rom review ing papers concerning the to xicity o f P F C s subm itted to and published in publicly available peer-reviewed scientific journals. 4. From serving as a peer-review er or participant for the U.S. EPA, N TP, and IARC assessment documents. 5. F rom presenting research findings and scientific opinions about the toxicity o f PFCs at various scientific meetings. The m ethodology for rendering m y opinion is based on the principles o f toxicology detailed in the introduction and an evidence-based integrative approach used by toxicologists to determine the likelihood o f harm to hum an health follow ing exposure to environmental contaminants. 23 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN VIII.2 Detailed Supported Opinion - Cancer It is m y opinion, based on the w eight o f the toxicological evidence, as supported by epidemiological evidence, that PFCs pose a substantial present and potential carcinogenic hazard to hum an health. I address first IA R C 's M onograph 110. B y w ay o f background, IARC invites interdisciplinary working groups of expert scientists to review publicly available published studies and to evaluate the w eight-of-evidence to determine whether exposure to an exogenous agent can increase the risk of cancer (IARC, 2016). The principles, procedures, and scientific criteria that guide evaluations are described in the pream ble to the IARC Monographs. These interdisciplinary groups are divided into sub-groups and m em bers o f the sub-groups review one another's sections (internal peer-review), which are then subm itted to the entire group for review, discussion, and ultim ately consensus (although dissenting opinions can be noted) about the classification o f the agent u n d er discussion. A gents are classified as carcinogenic to hum ans (G roup 1), probably carcinogenic to humans (Group 2A), possibly carcinogenic to hum ans (Group 2B), not classifiable as to its carcinogenicity to hum ans (Group 3), or probably not carcinogenic to hum ans (Group 4). Classification is based on hum an data, experimental animal data, and m echanistic data. I served as a m em b er o f the m echanism sub-group o f the w orking group fo r Monograph 110 in 2013-2014 (As a reminder, "m echanistic" refers to the particular reaction of the exogenous agent w ith a target m olecule or m olecules to induce toxicity, i.e., an adverse health outcom e or outcomes (Gregus, 2015)). P F O A w as evaluated in Monograph 110, w hich w as published electronically in 2016 (http://m onographs.iarc.fr/EN G /M onographs/vol110/index.php) and is freely downloadable. PFO A was classified as Group 2B, possibly carcinogenic to humans, based on lim ited evidence in humans of a positive association for cancers of the testis and kidney and based on lim ited evidence in experimental animal models. Lim ited evidence o f carcinogenicity in hum ans means that a positive association has been observed betw een the exogenous agent and cancer for which a causal interpretation is considered credible but that chance, bias, or confounding could not be ruled out (IARC, 2006). Lim ited evidence o f carcinogenicity in experimental animal models m eans that the data suggest a carcinogenic effect but are lim ited by a small num ber of experiments, unresolved questions in study design, conduct, or interpretation, exposure increases in incidence of only benign neoplasm s or lesions of uncertain neoplastic potential, or evidence of carcinogenicity is restricted to studies that dem onstrate prom otion in only a narrow range o f tissues or organs (IARC, 2006). Put m ore plainly, the evidence so far tends to show a carcinogenic effect, but m ore evidence is necessary before that tendency can be proven to be causal. The IA R C w orking group fo r Monograph 110 (IA R C , 2016) evaluated data from occupational cohorts, comm unities of high exposure, and the general hum an population where PFO A serum concentrations were examined in relation to various types of cancer. The w orking group determined that there was some evidence o f an association betw een PFO A exposure and cancer o f the kidney (Steenland and W oskie, 2012) and bladder (Raleigh et al., 2014) in occupationallyexposed humans. In hum ans from com m unities o f high PFO A exposure, elevated risks o f kidney 24 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN and testicular cancer (V iera et al., 2013; Barry et al., 2013) and increased risks for prostate and breast cancer w ere observed in relation to estim ated PFO A serum concentrations (V iera et al., 2013). Two published studies of the general hum an population available to the w orking group dem onstrated risk ratios (the ratio o f the cum ulative incidence in the exposed group to the unexposed group) o f greater than one (indicating that cumulative incidence in the exposed group is greater than cumulative incidence in the unexposed group) for pancreatic cancer (Eriksen et al., 2009) and prostate cancer (Eriksen et al., 2009; H ardell et al., 2014). O verall, the w orking group concluded that findings published in peer-reviewed literature established an association betw een PFO A exposure and cancer, particularly with respect to cancers of the kidney and testis in exposed humans. The IARC w orking group for M onograph 110 used tw o studies in experimental rodent m odels as strong evidence that exposure to PFO A (specifically amm onium perfluorooctanoate, the amm onium salt o f PFOA) is carcinogenic in experim ental animal models. In both studies, male and fem ale (B utenhoff et al., 2012a) or m ale (Biegel et al., 2001) Sprague-D aw ley (SD) rats w ere given diets containing PFO A . D iets contained 0, 30 (B utenhoff et al, 2012a), or 300 (B utenhoff et al., 2012a; Biegel et al., 2001) parts per m illion (ppm ) o f PFO A for tw o years, which is considered a chronic or lifetim e exposure. In these rodent cancer bioassays, m ale rats exposed to 300 ppm o f PFO A had an increased incidence o f testicular Leydig cell adenoma relative to unexposed anim als. In the Biegel et al. (2001) study, an increased incidence o f hepatocellular adenom a and pancreatic acinar cell adenom a was observed. A re-evaluation o f the B u ten h o ff et al. (2012a) data using evaluative criteria from the B iegel et al. (2001) study, revealed an increased incidence o f pancreatic acinar cell hyperplasia in m ale anim als given 300 ppm (Caverly-Rae et al., 2014). The w orking group (IARC, 2016) also reported that tw o feeding studies in rainbow trout (B enningthoff et al., 2012), an experim ental anim al m odel used for hepatic carcinogenesis, demonstrated that dietary exposure to PFO A promotes hepatocarcinogenesis, which was used as additional supportive evidence o f carcinogenicity in experimental animal models. The B iegel et al. (2001) study w as co-authored by scientists from C ovance Laboratories, Inc., Pfizer, Inc., and the D uPont Haskell Laboratory for Toxicology and Industrial M edicine. It is not clear from the m anuscript where the studies were perform ed or which company perform ed which analysis. Covance Laboratories, Inc., is a contract research organization that perform s scientific studies for various organizations by contract. Pfizer, Inc., is a biopharm aceutical company, so their role in the project is unclear. DuPont Haskell Laboratory for Toxicology and Industrial M edicine w as a research laboratory that studied, in part, DuPont products for safety (it appears to now be named the Stine Haskell Research Center). Regardless, this particular study was performed to evaluate potential m echanism s behind tum or production associated with amm onium perfluorooctanoate (APFO or C8) and W yeth-14,643 (WY), a pharm aceutical compound developed to low er serum cholesterol (not used clinically). Both o f these compounds were thought to induce peroxisomal enzymes via the process know n as peroxisom e proliferation. Therefore, the B iegel et al. (2001) study evaluated the ability o f C8, a potential P P A R a activator, and W Y, a known PPA R a activator, to induce tum ors in a tw o-year feeding study in m ale CD rats. Additional endpoints w ere evaluated to posit hypotheses about other m echanism s o f tum or production. 25 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN The B iegel et al. (2001) study dem onstrated that exposure to 300 ppm o f C8 over tw o years w as sufficient to induce increased tum or incidence com pared to control anim als in the liver, testes, and pancreas (Figure 7). W hile Biegel et al. (2001) concluded that several m echanism s m ay be TABLE 2 Summary of Hyperplasia/Neoplasia Incidence in the Liver. Testes, and Pancreas from Rats Fed C8 or WY Control CP-C8 C8 300 ppm WY 25 ppm Lesion Incidence % Incidence % Incidence % Incidence /q Liver Adenoma 2/80 3 1/79 1 10/76 13* 15/67 22* Carcinoma 0/80 0 2/79 3 0/76 0 3/67 4 Adenoma''carcinoma combined 2/80 3 3/79 4 10/76 13- 17/67 25* Testes Leydig cell hyperplasia Leydig cell adenoma 11/80 14 26/78 33 35/76 46* 46/67 69* 0/80 0 2/7S 3 8/76 il* 16/67 24* Pancreas Acinar cell hyperplasia 14/80 18 8/79 10 30/76 39' 41/67 61* Acinar cell adenoma 0/80 0 1/79 1 7/76 9" 25/67 37* Acinar cell carcinoma 0/80 0 0/79 0 1/76 1 0/67 0 Adenoma^'carcinoma combined 0/80 0 1/79 1 8/76 11s 25/67 37* Note. Values given for incidence of lesions are from all scheduled and unscheduled deaths: * Significantly different from the ad libitum control group, p < 0.05. "Significantly different from the pair-fed control group,/? < 0.05. percent of control. Figure 7. Summary tableofliver,testis,andpancreaticlesionsobservedinratsfedC8 orW Y fortwo years. Note thestatisticallysignificantincreasesinhyperplasia(enlargementoforgan/tissue)and neoplasiaintheliver, testis,andpancreasfromratsfeed300 ppm ofC8. Red box addedforemphasis. From Biegeletal.(2001). working differently in each organ, their overall conclusion was that C8 and W Y exposure produced increases in endogenous growth factors that stim ulated growth o f tum or cells in these organs. The endogenous factors included an increase in peroxisom es in the liver, an increase of cholecystokinin in the pancreas, and an increase in serum estradiol that targeted Leydig cells of the testes. These latter tw o factors are hormones, supporting the idea o f C8 as an endocrine disrupting compound. H orm ones often stim ulate growth o f cells and as such, m ay be labeled as "prom oters," or chemical carcinogens that promote growth of cancerous cells (Klaunig, 2015). Evidence that PFO A acts as an endocrine disruptor, and potentially as a promoter, in hum ans was provided in a 1992 study o f 3M w orkers conducted by Dr. Frank Gilliland, which found "hormonal changes associated w ith PFO A in hum ans," including consistent findings of increased estradiol as w ell as low free testosterone, and unchanged LH [luteinizing horm one]" (Dr. G illiland's 1992 thesis, 3M _M N 03112178 at 3M _M N 03112393). The B u ten h o ff et al. (2012a) article w as co-authored by scientists from the 3M C om pany and the D uPont Company. The underlying study o f the m anuscript was conducted by R iker Labs betw een 1981 and 1983 at 3M 's request (3M A 00027126). The study report, w hich was drafted by Conrad King, a consultant hired by 3M, was not completed until five years later, in 1987 (3M A00027126). The study authors noted that the m anuscript was actually a sum m ary from this 1987 report that had only been available on the U.S. EPA public docket (Adm inistrative Record AR-226) and that "the increased attention given to the potential health hazards of APFO in the scientific literature in recent years has prom pted this detailed summary o f the study to m ake the key findings and conclusions o f the study m ore accessible" (B utenhoff et al., 2012a). 26 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN This particular study was an evaluation o f both chronic toxicity and carcinogenicity (i.e., a rodent cancer bioassay) in a tw o-year feeding study o f m ale and fem ale SD rats. The 1987 final report noted that the incidence o f Leydig cell adenom as in rats dosed at 300 ppm was statistically significantly greater than the control group (3M A00027126 at -151) and the 2012 published study reported that an increased incidence in testicular Leydig cell adenom as in m ale rats fed 300 ppm of C8 was the only neoplastic outcom e observed; liver and pancreatic neoplasm s were not observed (Figure 8). Table 8 Incidence of neoplastic microscopic findings for male and female rats in either control groups or groups fed 30 ppm or 300 ppm APFOin their diet for 2 years. Organ/lesion Dietary dose group (ppm APFO) Males Female 0 30 300 0 30 300 Adrenal Pheochromocytoma, benign Pheochromocytoma, malignant 2/49(4)* 0/49 (0) 4/50(8) 1/50(2) 4/50(8} 0/50(2) 2/50(4) 0/50 (0) 0/50 (0) 0/50 (0) 0/49(0) 1/49(2) Liver Hepatocellular adenoma Hepatocellular carcinoma 0/49(0) 3/49(6) 0/50(0) 1/50(2) 0/50 (0) 5/50(10) 0/50 (0) 0/50(0) 0/50 (0) 0/50 (0) 0/50(0) 1/50(2) Mammary gland Adenocarcinoma Adenoma Carcinoma Fibroadenoma Lymphangiosarcoma 7/46(15) 14/45(31) 5/44(11) - - - 3/46 (7) 0/45 (0) 0/44(0) - - - 1/46(2) 0/45 (0) 0'44/m - - - 10/46(22) 19/45(42) |21.144 (48)1 - - - 0/46(0) 0/45 (0) 1/44(2) Reevaluation by PWGb Adenocarcinoma Adenoma Fibroadenoma Fibroadenoma (multiple) 9/50(18) 1/50(2) 16/50(32) 2/50(4) 16/50(327 0/50 (0) 16/50(32) 6/50(12) 5/50(107 0/50(0) 20/50 (40) 3/50(6) Pituitary Adenoma 17/48(35) 17/47 (36) 13/46(28) 33/46(72} 39/47(83) 36/50 (72) Testes/epididymis Leydig cell adenoma Thyroid C-cell adenoma C-cell carcinoma 0/49 (0) 0/43 (0) 2/43(5) 2/50(4) 2/47(4) 0/47 (0) 7/50 (14)' 4/47 (9) 0/47 (0) - 1/50(2) 0/50(0) - 0/45 (0) 0/45 (0) - 0/41 (0) 0/41 (0) Bolded values are statistically significant. * Statistically significantly different from controls (p < 0.05). 3 Number observed/number examined {%). b Hardisty etal. (2010). c The incidence in the groups sharing this footnote were statistically significantly different from each other (p <0.01, Hardisty et al., 2010). Figure 8. Summary table of lesions fromvarious tissues observed in rats fed APFOfor two years. Note the statistically significant increases in Leydig cell adenomas in male rats fed 300 ppmof APFO. The findings of increased fibroadenoma in mammary glands offemale rats fed 300 ppmof APFOwas consideredby the study authors to be withinthe normfor backgroundvariation ofthis lesion in SDrats. Redboxes added for emphasis. FromButenhoffet al. (2012a). However, the 1987 report discounted this finding as w ithin the range o f historical control animal data. (Id.) Several aspects o f this study report are questionable based on consensus principles to guide the use o f historic control data o f proliferative lesions from chronic rodent bioassays by the Historical Control D ata W orking Group (HCDW G) under the Scientific and Regulatory Policy C om m ittee o f the Society for Toxicologic Pathology (K eenan et al., 2009). First, the H C D W G asserts that the concurrent control group is the m ost relevant com parator for determ ining treatm ent-related effects in a study. H istoric control data "m ay be useful in the interpretation of rare tumors, marginally greater incidences and/or severity of proliferative 27 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN changes in treated animals compared to controls, and unexpected increases or decreases o f tum or incidences in study control anim als" and historic control data "from the laboratory that conducted the study under review will likely be m ore com parable than [historical control data] com piled from several laboratories" (K eenan et al., 2009). Therefore, w hile it is som etim es appropriate to use historical control data for com parison where a dosed group does not exhibit a statistically significant different effect from the experimental control group, it is rarely, if ever, appropriate to use historical control data where a dosed group does exhibit a different effect from the experimental control group. Second, even if it w ere appropriate to use historical control data, it appears that the historical animal data used were inaccurate, as suggested in a m em o sent to 3M by a D uPont scientist shortly after the study report was issued (3M _M N02330325). M oreover, it is apparent that 3M m ay have been aware that the Leydig cell adenom a finding was a valid treatm ent-related finding. A phone conversation report betw een a 3M employee and the veterinary pathologist responsible for the study reveals that the pathologist viewed the effect in the testes to be associated with APFO treatm ent and "not w ithin normal biological variation" (3M A10014327). A draft version o f the report states that, w ith respect to the Leydig cell tum ors, an A PFO effect "cannot be ruled out" (3M A01405733). This language is om itted from the final report. A dditionally, given the difference in findings betw een the B iegel et al. (2001) and the B utenhoff et al. (2012a) studies, a subset o f authors from both studies (B utenhoff, Chang, Fram e, and Kennedy) and an additional author (Caverly-Rae) perform ed a re-review o f data from the B u ten h o ff et al. (2012a) and B iegel et al. (2001) studies (C averly-R ae et al., 2014). The re review reported that the pancreatic changes observed in the B utenhoff et al. (2012a) study w ere found to be consistent w ith the changes observed in the Biegel et al. (2001) study. Therefore, tw o types o f neoplastic changes actually w ere observed in the B uten h o ff et al. (2012a) study. The authors o f the B iegel et al. (2001), B u ten h o ff et al. (2012a), and C avalry-R ae et al. (2014) studies discount the applicability o f their findings to hum an health. In this respect, I disagree w ith their conclusions, which I find to be unsupported by the evidence. It is notable that the authors o f the C averly-R ae et al. (2014) study w ere scientists from the D uP ont H askell Global Centers, the 3M Company, and a consultant to the D uPont Company, and, as noted above, the B u ten h o ff et al. (2012a) study w as co-authored by scientists from the 3M C om pany and the D uP ont Com pany. The B iegel et al. (2001) study did not itself associate the observed changes in tum or incidence in anim als w ith a hazard to hum an health except to note that hum an Leydig cell adenomas and surrounding hyperplastic Leydig cells secrete large amounts o f estradiol. For their part, the B u ten h o ff et al. (2012a) and C averly-R ae et al. (2014) studies discounted their findings as evidence that C8 posed a hazard to hum an health. The B u ten h o ff et al. (2012a) study affirm atively cited epidem iological studies that reported no association betw een serum PFOA and liver, pancreatic, testicular, and prostate cancer as evidence that exposure to PFO A w as not associated w ith cancer-related outcom es. The C averly-R ae et al. (2014) study argued that the type o f pancreatic tum or observed in rats is distinctly different from tum ors comm only observed in hum ans and that because there is no known association betw een other types o f peroxisome proliferators and pancreatic tum ors in humans, the relevance of the rodent data to hum an health risk assessment was questionable. 28 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN However, as noted above and discussed in section VII.6b, evidence that carcinogenicity in a rodent model m ay be m ediated by peroxisom e proliferation is an insufficient basis upon which to discount the applicability o f findings to humans. In addition, as Dr. Grandjean discusses in more detail in his report, epidem iology studies have found statistically significant associations betw een PFC exposure and various cancers, and, as he concluded, demonstrate that PFC exposure has a substantial potential to cause cancer in humans, particularly with respect to kidney and testicular cancer, and that the same is highly likely w ith respect to prostate and bladder cancer and possibly with respect to breast cancer. I m ake additional points, below, which further support that peroxisom e proliferation is not the only m echanism by which various types of cancers can be induced by exposure to PFCs. M y opinion that these studies are indicative of carcinogenicity in hum ans is consistent w ith that of the IARC. The IARC w orking group for M onograph 110 reviewed the data from the Biegel et al. (2001), B u ten h o ff et al. (2012a), and C avalry-R ae et al. (2014) studies and deem ed the data from these studies to be evidence of the carcinogenicity of PFO A in experimental animal m odels. C onsidering these and other studies, the IARC concluded that the w eight o f all o f the evidence, considered together, tended to show a carcinogenic effect. M echanistic data linking PFO A exposure to cancer in hum ans and experimental animal models w as considered m oderate and not quite strong enough to m ove P F O A to G roup 2A or G roup 1. Stronger m echanistic data would have included greater evidence that PFOA is mutagenic or genotoxic, acts as a grow th prom oter in m ultiple cell types, or induces carcinogenicity through accepted and recognized pathways. The m echanism s considered in the m onograph included PPA R a activation and peroxisom e proliferation, activation of other nuclear receptors, oxidative stress (including production of reactive oxygen species, decreased antioxidant capacity, and m itochondrial dysfunction), modulation of inflamm atory pathways, and m odulation of hormone levels (IARC, 2016). A lthough the w orking group for M onograph 110 discussed some studies dem onstrating the ability o f P FO A to act as a cancer prom oter (A bdellatif et al. 1990, 1991, 2003; B enninghoff et al., 2012; N ilsson et al., 1991), an additional study in using the cell transform ation assay on Syrian ham ster embryo (SHE) cells further supports that PFO A acts as a cancer prom oter in the absence o f genotoxicity (Jacquet et al., 2011). The IARC w orking group for M onograph 110 also noted that modulation of reproductive horm ones by PFO A has been reported in hum an cells, rodents, and fish, including activation of estrogen receptors, interference w ith testosterone/estradiol balance, and induction of aromatase, the enzyme that, in part, converts testosterone to estrogen. Interference w ith these pathways can be associated w ith cancers o f reproductive tissues. Biegel et al. (1995) designed a series o f studies to test com ponents o f the proposed m echanism by w hich C8 induces Leydig cell tum ors in rats (originally reported in a study by C ook et al., 1992). Biegel et al. (1995) reported that serum estradiol was increased in rats treated w ith C8 and that this increase was likely due to induction o f arom atase in the liver and reduced m etabolism /excretion o f estradiol. In addition, increases in levels o f estradiol and transform ing growth factor alpha (T G Fa) w ere m easured in the interstitial fluid of the testes, which the authors interpreted as consistent w ith their hypothesis that estradiol m ay m odulate growth factor expression w ithin the testis (Biegel et al., 1995). T G F a is a ligand for the epidermal growth factor receptor, which activates a signaling pathw ay for cell proliferation, differentiation, and developm ent, and has been associated w ith m any types o f cancers in experimental animal m odels and hum ans (NCBI, 2017). Estradiol is a well-known 29 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN growth promoter that has been implicated in several types of cancers o f the female reproductive system, notably the endometrium and the ovary, and has been classified as carcinogenic to hum ans by the IARC (IARC, 2012). Estradiol also m ay play additional roles in the male reproductive system as evidence is em erging that supports estrogens as prostate cancer-causing agents (Nelles et al., 2011). A nother putative m echanism is inhibition by PFCs o f gap junctional intercellular com m unication (GJIC). GJIC is a process that plays a crucial role in the m aintenance o f normal cell grow th and w hen inhibited, m ay be a non-genotoxic m echanism o f carcinogenicity (Upham et al., 1998) and a response o f cells to agents that prom ote cell grow th (Upham et al., 2009). In tw o articles published by a single laboratory from M ichigan State University, PFOA, PFOS, PFOSA, PFHxS, and PFDA were reported to inhibit GJIC in a dose-dependent manner, with PFO S and PFD A having the m ost potent effects (Upham et al., 1998; H u et al., 2002). In a follow -up study published in 2008 (U pham et al., 2009), the authors reported on additional m olecular m echanism s associated w ith PFO A-induced inhibition o f GJIC. Dr. John Giesy, a co-author of studies on GJIC, and form er consultant to 3M, has testified that chemicals that block cell-to-cell interaction, which occurs through inhibition o f GJIC, are "potent cancer prom oters" (Giesy Dep. Tr. 68:13-18, 201:3-203:7). These studies w ith GJIC provide additional evidence that PFC s m ay induce carcinogenic outcomes via several mechanisms. Based on the assessment of PFO A by the IARC, the weight of evidence so far tends to show that PFO A can induce cancer as an adverse health outcom e in exposed humans. It is also notable that the U.S. EPA has concluded that there is suggestive evidence that PFO A is carcinogenic, and that there are sufficient data to derive a cancer slope factor for this com pound (U.S. EPA, 2016c). Evidence from studies perform ed by 3M and reviews from governm ental agencies also support the conclusion that PFOS and N -EtFO SE are carcinogenic. B etw een 1981 and 1983, 3M com pleted a 2-year carcinogenicity study o f N -EtFO SEin rats (3M A00593233). Conrad King, a 3M consultant, drafted the final report, w hich w as not com plete until 1988 (Id.). In this report, 3M concluded that although liver carcinom as were observed in fem ale rats at the highest adm inistered dose, they w ere " slightly outside" o f "reasonable historical control lim its" (Id.). Again, as I observed above, it is rarely, if ever, appropriate to use historical data to discount a statistically significant finding o f an adverse health effect in a treatm ent group. Further, upon a subsequent independent re-exam ination o f the tissues used in this study, a separate pathologist concluded that "the distinct increases in hepatocellular neoplasm s in high dose fem ales combined w ith increased hyperplastic nodules in both sexes are clear indicators that the test m aterial should be regarded as a liver carcinogen in rats" (3M A10025147). M oreover, in a subsequent study com pleted by Covance Laboratories at 3M 's request, statistically significant increases in combined liver adenomas and carcinom as were found in female rats at the highest administered dose, as well as a statistically significant increases in com bined thyroid follicular cell adenomas and carcinomas in male rats at the highest administered dose (3M A00812492). W ith respect to PFOS, 3M completed a 2-year carcinogenicity study o f PFOS in rats in 2002 (3M B00040027). This study found a statistically significant increase in com bined thyroid follicular cell adenom as and carcinom as in m ale rats from the highest adm inistered dose group, and a statistically significant increase in com bined hepatocellular adenom as and carcinom as in 30 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN fem ale rats from the highest tested dose group. In addition to this study, the U.S. E P A 's recent review o f PFO S concluded that there is suggestive evidence that PFO S is carcinogenic (U.S. EPA, 2016d). In addition, Dr. John Butenhoff, the form er m anager o f 3M 's corporate toxicology department and head of 3M 's fluorochemical research program agreed that he has characterized PFOS and N -EtFO SE as carcinogenic in rats (B utenhoff Depo. Tr. 193:23-25; 194:1-9). In short, the w eight o f the evidence supports the conclusion that PFOS and N -EtFO SE are also carcinogenic to humans. By extension, other PFCs that induce toxicities sim ilar to PFOA, PFOS, and N -EtFO SE and that w ork through similar m odes or m echanism s o f action, are likely to induce cancer as an adverse health outcom e in exposed hum ans as well. I agree w ith the assessm ent by the IARC that PFO A is possibly carcinogenic to hum ans based on evidence in experimental animal m odels that is supported by epidem iological findings in humans. In addition, the U.S. EPA classifies evidence of carcinogenicity for both PFO A and PFOS as suggestive of carcinogenic potential in humans (U.S. EPA, 2016c,d). Since the IARC assessment, an additional study o f an occupationally-exposed cohort by the C8 Science Panel (Steenland et al., 2015) dem onstrated a positive trend o f prostate cancer with serum PFO A levels. A study o f D anish wom en reported an increased breast cancer risk associated w ith PFO SA exposure (B onefeld-Jorgensen et al., 2014). Published studies o f other PFCs exist, but do not report positive associations betw een serum PFC levels and cancer outcomes. Evidence o f cancers of the m ale and fem ale reproductive system build support for a pathway to carcinogenicity that results from horm one modulation. VIII.3 Detailed Supported Opinion - Developmental Toxicity It is m y opinion, based on the w eight o f the toxicological evidence, as supported by epidem iological evidence, that PFCs pose a substantial present and potential developmental toxicity hazard to hum an health. VIII.3a Current Evidence o fPFC-Induced Developmental Toxicity The U.S. EPA has identified developm ental toxicity as a critical endpoint for PFO A and PFOS toxicity. B y w ay o f background, the U.S. EPA established risk assessm ent guidelines to assess potential health risks to hum ans following exposure to exogenous agents. See https://www.epa.gov/risk/risk-assessm ent-guidelines#tab-1. The Health Effects Support Docum ent for Perfluorooctanoic Acid (PFOA) and the Health Effects Docum ent for Perflurooctane Sulfonate (PFOS) w ere w ritten by the U.S. EPA (U.S. EPA, 2016c,d) to address the presence of PFO A and PFOS in drinking w ater as part of their regulatory requirem ents under the Safe W ater D rinking Act (SW DA). Both documents are available electronically and are freely downloadable (https://ww w.epa.gov/ground-w ater-and-drinking-water/supportingdocum ents-drinking-w ater-health-advisories-pfoa-and-pfos). These docum ents w ere both reviewed by scientists from w ithin the U.S. EPA and by scientists external to the U.S. EPA. Additionally, w hen these docum ents w ere released in draft form, any party (i.e., private citizen, industry representative, representative from another agency, trade group representative, etc.) was given the opportunity to provide evaluative comm ents for consideration in revised versions. 31 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN D uring external peer-review proceedings, these parties also could attend as observers. The process therefore ensured a docum ent that is transparent, responsive, and rigorously reviewed by scientific experts. H ow ever, the docum ents are still in draft form and cannot yet be quoted or cited as official U.S. EPA documents. In this report, they are used as support docum ents as they reflect a thorough assessm ent of the toxicological data associated with PFO A and PFOS. I served as an external peer review er to the draft form s released by the U.S. EPA in 2014. One goal of a science assessment that supports an assessm ent of risk to human health is to assist in deriving a RfD. As defined by the U.S. EPA (1993), a RfD is an estimate o f a daily exposure to the hum an population that is likely to be w ithout an appreciable risk of deleterious effects during a lifetim e and is expressed in m g/kg o f body weight/day (mg/kg/day). The RfD includes values for uncertainties in the estimate as well as factors for protecting sensitive subpopulations and is often derived from a N O A EL or LO A EL identified for a critical endpoint (U.S. EPA, 1993). In general, a critical endpoint is an adverse health outcom e exhibiting the low est N O A EL as this seeks to ensure that if that critical toxic effect is prevented, then other toxic effects also are prevented (U.S. EPA, 1993). However, as I addressed above, the U.S. EPA now often uses BM Ds, rather than NO AELs or LOAELs to establish RfDs. The U.S. EPA health docum ents w ere generated after review o f a large num ber o f relevant studies, and the weight of the evidence showed a correlation between PFO A /PFO S exposure and various adverse health effects. As discussed in the U.S. EPA health effects docum ents, a critical endpoint identified for both PFOA and PFOS was developm ental toxicity. Am ong the large num ber of studies that were considered and that are cited in the respective health effects support docum ents for PFO A and PFOS (U.S. EPA, 2016c,d), are studies that dem onstrate developm ental toxicity follow ing in utero exposure to PFO A or PFOS. In a tw o-generation study of PFO A in utero exposure was associated with a delay in sexual m aturation in both m ale and fem ale rats (B utenhoff et al. 2004). Studies w ith PFO S have dem onstrated th at in utero exposure leads to birth defects such as cleft palate, ventricular septal defect, and enlargem ent of the right atrium in rat pups (T hibodeaux et al. 2003), and significantly reduces the survival o f rat pups (Lau et al. 2003). In the end, tw o studies w ere used to develop R fD s for each com pound, but were supported by findings of other studies that reported adverse health outcomes at sim ilar doses and for similar exposure durations. For PFOA, reduced ossification o f bones and accelerated puberty (in m ales) was observed in CD-1 m ice exposed to a range o f PFO A doses from gestational day (G D ) 1 through GD 17 and w ere used as the endpoints for deriving the RfD (Lau et al., 2006). The LO A E L identified in this study w as 1 m g/kg given to pregnant dam s (Figures 9a,b); no N O A E L w as identified. All authors o f the Lau et al. (2006) study w ere m em bers o f the U.S. EPA and levels o f PFO A in the serum w ere reported for both dams and the offspring. A dditional endpoints reported in the study included 1) birth outcom es, such as tim e to birth, condition of the new borns, and num ber of live offspring; 2) offspring param eters, such as num ber of offspring and body weights throughout the lactational period, age of eye opening, and age of puberty (vaginal opening in females and preputial separation in males); and 3) teratological assessm ent o f offspring collected at GD18 for external abnorm alities, skeletal examination, and visceral evaluation. 32 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN TABLE 2 Mouse Reproductive Outcome and Fetal Teratology. Examined at Term PFOA dosage (mg/kgi 0 l 3 5 10 20 40 Dams examined (#} Dams with FLR (#) Dams with FLR (%) Implants (# per litter with FLR) Implants (# per live litter) Live fetuses (# per live litter) Prenatal loss per live litter) Fetal body weight (g) Notable skeletal findings in) Ossification in umber o f sites): Sterne brae Caudal vertebrae Metacarpal s Metatarsals Proximal phalanges (foreiimb) Proximal phalanges (hindlimbi Reduced ossification (% ): Calvaria Supraoccipital Unossifted hyoid Enlarged fontanel Notable visceral findings in) Tail defects (curly, bent) (%) Limb defects (club, bent) (% ) Microcardia (%) +1 os 45 3 6.7 7.0 4.0 12.9 0.4 12 5 0.4 4.1 + 1.4 1.05 0.02 13 5.9 0 .1 43 0.3 7.7 + 0,2 9.3 0.3 4.8 0.8 13.5 9.2 14.7 + 4.0 0 17.3 9.1 10 0 0 0 17 2 11.8 10.0 .3.0 3.1 0.4 3.0 0.4 1.0 0.7 0.98 0.03 6 6.0 0.1 4.1 0.1 7.3 0.3 8.9 0.4 1.8 1.0* 0.4 0.3* 62.5 15.5 33.3 10.5 0 66.7 21.1 6 0 0 0 17 1 5.9 13.0 11.6 0.9 10.8 0.9 7.4 2.5 1.03 0.04 7 6.0 0.1 4.0 0.2 7.6 + 0.2 9.1 0.3 2,2 0.9* 1.5 1.0 66.7 + 13.0* 28.6 8.5 0 53.6 15.8* 6 0 0 0 27 7 25.9* 11.6 1.2 11.5 0.5 11.1 0.4 2.4 0.8 1.03 0.04 11 5.5 0.3 4.3 0.3 6.6 0.5 8.2 0.6 2.9 0.9 2.8 0.9 22.7 10.4 27.3 9.2 0 18.2 9.6 11 20.5 5.7* 5.7 2.8* 0 26 12 46.1* 10.8 + 12 12.6 0.6 11.7 0.8 7.7 33 0.98 0.05 5 5.7 0 2 3.7 02 6.8 0,4 8.6 0.4 1.0 0.6* 1.0 0.6* 35.0 12.7 45.0 9.4* 0 45.0 20.0 5 5.0 5D* 0 5.0 5.0* 42 37 88.1* 115 0.6 102 2.1 72 20* 25.9 11.7* 0,86 0.11* 5 4 .0 1.1* 2.1 0.7* 5 2 1.4* 6 2 1.6* 0.0 0.0* 0.0 0.0* 55.0 20.0* 90.0 10.0* 26.7 19.4* 95,0 5.0* 5 11.7 7,3* 5.8 3.9* 30.0 IS.3* 9 9 100* 11.9 0.5 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- Note. Data represent means SE of litters examined as indicated. One-way ANOVA indicates significant differences (p < 0.05) in number of live fetuses and prenatal loss. Asterisks denote significant differences from controls (p < 0.05) by Fisher's exact test for full litter resorptions (FLR) and by Dunnett's /-test for other parameters. Figure 9a. Summary tableofteratogenicoutcomes observedinmice exposedtoPFOA from GD1-17 of gestation.Redbox addedforemphasis. From Lau etal.(2006). TABLE 5 Developmental Landm arks of Mouse Pups Mxposed to PPOA In U te r o Eye opening Vaginal opening First estrus Preputial separation PFOA (mg/kg) N Age (days) N Age (days) Body Weight (g) N Age (days) N Age (days) Body Weight (g) 0 27 14.8 0.1" 47 (20) 28.4 + 03"* 18.0 + 0.2" 47 (20) 29.9 + 0.4"4 56 (22) 30.5 + 0.2" 25.0 0.3" i 8 15.2+0.2" 21 (8) 27.4 05 4 18.2 0.5" 21 (8) 282 + 0.64 22 (8) 267 + 0.24 20.3 0.34` 3 8 15.5 0.1" 4 21 (7) 28.8 + 04"* 17.7 + 0.4"'' 21 (7) 302 + 0.4" 20 (7) 27.1 + o . r 19.4 + 0.64`J 5 17 16.0 0.24 43 (16) 29.9 0.4" 17.7 + 0.4"'' 43 (16) 31.8+0,5" 46 (16) 28.2 + 0.2" 18.3 +0.5""' 10 13 17.2 0.3" 27 (12) 29.3 03" 16.7 + 0.34 27 (12) 302 + 0.3" 28(11) 28.5 0.3" 17.5 + 0.7" 20 3 17.9 + 0.8" 8 (2) 31.3 03*' 19.3 0.4" 8 (2) 312+ 0.5" 4(2) 31.7 + l.f'' 20.8 + 1.24 Note. Data represent means SE of numbers of litters (for eye opening) or individual pups (for vaginal opening, first estrus, and preputial separation) examined as indicated. For eye opening, N = litter; for other landmarks. N = individual animal, numbers in parenthesis indicate litters represented. ANOVA indicate significant treatment effect in all parameters examined (p < 0.05). Significant differences ip < 0.05) between each dose group were determined by Duncan's multiple range test and are depicted by different letters (a, h, c, and d ) Figure 9b. Summary tableofdevelopmentallandmarksobservedinmice exposedtoPFOA from GD1-17 of gestation.Redbox addedforemphasis. From Lau etal.(2006). 33 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN For PFOS, increased m otor activity and decreased habituation were observed in male SD rats exposed to a range o f PFOS doses from GD 0 through postnatal day (PND) 20 (B utenhoff et al., 2009b). The LO A EL identified in the study w as 1.0 m g/kg and the N O A EL was 0.3 m g/kg (Figure 10). As both o f these studies (Lau et al., 2006 and B utenhoff et al., 2009b) measured serum P F O A /P F O S concentrations, the U.S. EPA was able to calculate hum an equivalent doses (HED) for the LOAEL and NOAEL (when applicable) and use the HED for the RfDs. The HED is the hum an serum PND 17 PND 21 PND 17 PND 21 co n cen tratio n , e stim ated Male Female from serum 0 mg/kg-d ^ 0.1 mg/kg-d H 0.3 mg/kg-d 1.0 mg/kg-d concentrations in experim ental animal m odels (in units o f m g o f Figure 10. Summary figureofmotoractivityinmaleratsorallyexposedto PFOS fromGD0 throughPND20. Red box added foremphasis. From Butenhoffetal.(2009b). PFC/L of serum), which would be associated with the N O A EL and/or LO A EL in the experim ental anim al studies. .Another w ay that the U.S. EPA (U.S. EPA, 2016d) explained the HED is that "The HED values are the predicted hum an oral exposures necessary to achieve serum concentrations equivalent to the NO A EL or LOAEL in the animal toxicity studies." In both the PFO A and PFOS documents, pharm acokinetic models were used to estimate the HEDs. The HEDs for the NO AELs calculated for PFOA was 0.0053 m g/kg/day and for PFOS was 0.00088 mg/kg/day. The U.S. EPA determ ined the RfD for PFOA, based on the HED calculated from the LOAEL identified in the Lau et al. (2006) study, to be 0.00002 m g/kg/day, w hich reflects uncertainty associated w ith intrahum an variability (i.e., sensitive subpopulations), toxicodynam ic differences betw een anim als and hum ans, and the use o f a LOAEL instead o f a NOAEL. The U.S. EPA determ ined the RfD for PFOS, based on the HED calculated from the N O A EL identified in the B utenhoff et al. (2009) study, to be 0.00003 m g/kg/day, w hich reflects uncertainty associated w ith intrahuman variability and toxicodynamic differences betw een animals and humans. Therefore, these were doses that, based on the evidence at the time, were believed not be associated w ith adverse health outcomes in exposed humans. 34 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Several other studies were considered supportive o f the RfDs determ ined for PFO A and PFOS, based on similarities in dose, exposure duration, and the identification of NO AELs and/or LOAELs. One o f the studies was my first postdoctoral paper from a project concerning PFO A (D eW itt et al., 2008), and another w as from w ork perform ed during m y postdoctoral training and as a faculty m em ber at E C U (D eW itt et al., 2016b). The U.S. EPA noted that these studies, as supported by epidem iology data, identified suppression o f an im m une system response as an area o f concern for hum ans and experim ental animal m odels exposed to PFOA. The U.S. EPA noted that although the D eW itt et al. (2008) study identified a N O A E L from w hich the H ED could be derived, the L au et al. (2006) study w as chosen as it, and sim ilar developm ental studies, indicated that lifetime consequences to PFO A exposure m ay occur for a less-than-lifetim e exposure. H ow ever, the R fD derived from the Lau et al. (2006) study w as supported by a R fD determ ined from the D eW itt et al. (2008) study and both had identical H E D s o f 0.0053 m g/kg/day. Therefore, the D eW itt et al. (2008) study played an im portant role in supporting the RfD ultim ately derived for PFOA. The L au et al. (2006) study w as co-authored by scientists from the U.S. EPA , w hereas the B u ten h o ff et al. (2009) study w as co-authored by scientists from the 3M C om pany as w ell as scientists from two different contract research facilities, Biotechnics, LLC and W IL Research Laboratories. W hile these tw o studies used different species o f rodents, different exposure doses and durations, and evaluated different endpoints, they both dem onstrated that developing organism s are sensitive to PFC exposure. The L au et al. (2006) authors did not attem pt to extend directly the results o f their study to comparisons w ith exposed hum ans, but noted that m ice may be a good model for hum an health risk assessm ent of PFO A and that developmental endpoints m ay be appropriate for understanding other PFC s. The B u ten h o ff et al. (2009) study authors noted that m ean serum concentrations of PFOS in the dams (pregnant fem ale rats) reported in a com panion article (Chang et al., 2009) w ere several hundred tim es higher than those reported for fem ales in the U.S. general population. However, the HED calculated by the U.S. EPA was based on a serum concentration o f 10.87 m g/L reported in the B utenhoff et al. (2009) study, w hich w as low er than m easured PFO S serum concentrations in, for example, a hum an developm ental epidem iological study o f maternal PFOS levels and m otor or mental developm ent in their offspring, w hich reported a m ean m aternal serum concentration o f 35 m g/L (Fei et al., 2008). Therefore, given uncertainties that exist between experimental animal responses and hum an responses, m easured serum concentrations in experimental animal models generally should not be directly compared to m easured serum concentrations in humans. In June 2017, a systematic review of the epidem iological literature concerning associations betw een PFC levels and developm ental outcom es and using a process sim ilar to the U.S. NTP (see D etailed Supported Opinion - Im m unotoxicity, section VIII.4), was published by scientists from the U.S. E PA (R appazzo et al., 2017). The authors observed consistent evidence for a positive association between PFCs and 1. A ltered serum lipids, 2. A ltered im m unity (vaccine responses and asthm a), 3. A ltered renal function, and 4. A ltered age at m enarche. 35 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN R appazzo et al. (2017) noted that w hile the above four endpoints had the strongest evidence for a positive association with PFCs, findings of developm ental perturbations associated with PFC blood levels were diverse and include cardiom etabolic effects, neurological, neurodevelopm ental and attention effects, and thyroid changes. They also noted that these early-life im pacts on physiological param eters have the potential to increase an individual's disease risk trajectory as they age into adulthood. Supportive o f these findings and in another critical review o f the literature surrounding exposure pathways for PFCs, the authors concluded that individuals experience a higher exposure intake to PFC s as infants and toddlers than during adulthood (W inkens et al., 2017). This conclusion was largely based not only on empirical measures o f higher PFC concentrations in infants and toddlers as com pared to adults, but on higher uptake rates per body w eight for infants and toddlers and differences in behavior (being breastfed, m outhing behavior, and m ore contact with the ground and accom panying dusts) betw een children and adults (W inkens et al., 2017). Thus em erging evidence supports the U.S. EPA assessm ent that developing organism s are a susceptible group. Based on the assessm ent of PFO A and PFOS by the U.S. EPA and on additional published materials, developing organisms are sensitive to adverse health outcomes associated with exposure to these agents. The w eight o f the evidence shows that that PFO A and PFO S can induce developmental toxicity as an adverse health outcom e in exposed humans. At least one study (Das et al. 2008) has also reported developm ental effects, including delayed eye opening and delayed sexual m aturation in m ice exposed in utero to PFBA. By extension, other PFCs that induce toxicities sim ilar to PFO A and PFOS and that w ork through sim ilar m echanism s, such as PFB A and PFHxS, are likely to induce developm ental toxicity as an adverse health outcom e in exposed hum ans as well. I agree w ith the assessm ent by the U.S. EPA that PFO A and PFO S are developm ental toxicants to hum ans based on evidence in experim ental animal m odels that is supported by epidemiological findings in humans. VIII.3b H istoric Evidence o f PFC -Induced Teratogenicity Since at least 1980, studies conducted or funded by 3M have shown that PFCs induce teratogenic and developm ental defects in laboratory rats. In a 1980 study (3M A00356530), SD rats w ere exposed to 0, 1, 5, or 10 m g/kg o f FC -95 (a m ixture o f P F C s) from G D 6-15. T eratogenic changes w ere noted in the eyes o f all FC -95 dose groups, but only the 10 m g/kg dose group had statistically higher abnorm alities relative to the control group (the num ber o f fetuses examined w ere v ery sm all for the control, 1, and 5 m g/kg groups, m aking statistically detectable differences betw een those groups unlikely). The abnorm ality was described as "an arrest in developm ent of the prim ary lens fibers form ing the embryonal lens nucleus, followed by secondary aberrations of the secondary lens fibers of the fetal nucleus." One lens abnorm ality w as observed in the control group and 66.7% , 57.1% , and 40% o f fetuses exposed to 1, 5, and 10 mg/kg, respectively, had eye abnorm alities. In this particular study, the eye abnorm ality observed in the control group was considered by the authors to be an artifact. In a 1981 study o f FM -3422, a long-chain PFC also know n as N -EtFO SE (3M A 00326722), teratogenic changes to the eyes of SD rat fetuses exposed from GD6-15 also were described as "an arrest in developm ent of the prim ary lens fibers form ing the embryonal lens nucleus 36 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN followed by secondary aberrations o f the secondary lens fibers o f the fetal nucleus. The proportions o f fetuses w ith the lens changes w ere significantly higher in all FM -3422 groups than in the control group." N o lens abnorm alities were observed in the control group and 64.3%, 56.9%, and 50.7% o f fetuses exposed to 25, 37.5, and 75 m g/kg, respectively, had eye abnorm alities (all statistically different from the control group). The discussion in this report was nearly identical to the discussion in the 1980 report. Besides lens abnormalities, rat fetuses exam ined in this study exhibited other m alform ations, including nonossification o f cranial bones, bipartite vertebrae, cleft palate, and blood in the kidney parenchyma. At least three other studies with two different PFC compounds (T-2999CoC and T-2998CoC) w ere perform ed in 1981 (3M A 01508403, 3M A10008766, 3M B00012976), either w ith SD rats or w ith Fisher-344 rats, to verify the findings o f the previous tw o studies. How ever, unlike the previous study, these additional studies reported lens abnorm alities in the control groups and concluded that the lens findings observed across all groups w ere not abnorm alities but artifacts. Regardless, there w ere still statistically significant increases in lens abnorm alities in the highest dose groups relative to the control groups, w hich the authors did not attem pt to explain. A study also was performed by the DuPont Haskell Laboratory in 1982 (3M A00248385) in response to information relayed to them by the 3M Company that exposure to C8 was possibly teratogenic because o f lens changes in the eyes o f near-term offspring rats. By way o f background, artifacts in sections of tissue evaluated histopathologically are not considered pathological findings, but findings associated with tissue handling, processing, fixation, storage, etc. (Chatterjee, 2014). Artifacts in tissues may include rips, tears, folds, forceps m arks, cloudiness, etc. (Chatterjee, 2014). In these three latter studies, the lens abnorm ality was reclassified as an artifact of freehand sectioning used as part of the W ilson technique. W ith this technique, soft tissues are typically fixed in a solution know n as B ouin's solution, w hich hardens soft tissues and decalcifies the bones (Burdan et al., 2005). Once the tissues are preserved, they are cut w ith a razor blade or other sharp cutting instrum ent. W ith an embryo, cuts are m ade at specific points (Figure 11) so that m icroscopic evaluation of internal organs can be h Figure 11.Pointswhere transversal sections(a-h)wouldbe made ona21 dayoldratfetus,withBouin'ssolutionand Wilson'sfreehandmethod. Modifiedfrom Burdan etal.,2005. performed. James G. W ilson designed, developed, and established this protocol in 1959 and with m inor m odifications, it is still w idely used in teratological assessm ents today (Seegm iller et al., 2012). Any laboratory that evaluates teratological outcom es will have technical staff w ho are highly skilled with this technique and who use it routinely to assess fetuses. Dr. John B utenhoff has testified that the sectioning artifact w as produced because the laboratory technicians responsible for sectioning the fetuses used the same razor to section the control groups and then the dosed groups. Thus, in his view, the razor becam e blunt by the tim e it was used for the fetuses of the dosed group, causing the purported sectioning artifact (Butenhoff Depo. Tr. 140:22-25; 141:1-16). 37 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN This rationale is unpersuasive for several reasons. First, the practice described by Dr. B utenhoff would be inconsistent with good laboratory practices as the technicians doing the sectioning would have been blinded to the treatm ent groups, as would be consistent w ith good laboratory practices. Therefore, sectioning w ould have occurred in a random order and not w ould have occurred by treatm ent group. Secondly, artifacts in tissues prepared for histological examination m ay occur random ly or systematically depending on how samples from different groups are treated. For example, a good laboratory practice w ould ensure that representative sam ples from all groups are included in a processing or staining batch so that all samples from one group are not processed or stained in a different batch from another group. This would ensure that certain types of artifacts are spread evenly across all groups. If good laboratory practices are incorporated, it w ould be highly unusual for artifacts to be disproportionately spread across dose groups, as evidenced by the studies reporting lens findings in treated groups but not in control groups, even in those studies with lens findings in the control groups as (som e) treated groups had a statistically higher findings relative to the control groups. All o f the reports for the studies indicated that the experiments were conducted according to good laboratory practices. Thus, assum ing that good laboratory practices were followed in the studies m entioned above, there was no valid reason to discount eye abnorm alities as artifacts where there were statistically significant differences betw een the control group and the various dose groups. Finally, if such an artifact was produced by sectioning fetuses from the dosed groups w ith a dull razor, it seems likely that additional artifacts would have been observed throughout the dosed fetuses in a variety o f tissues. It seems highly unlikely that a dull razor w ould produce a similar artifact in a sim ilar location in m ultiple fetuses. In a 1981 m em o (3M A 00360639), E.G. Lam precht, DV M , PhD, a R esearch Veterinary Pathologist w ho had signed off on the previous tw o reports o f lens findings as well as the studies that reported the lens findings as artifacts, attem pted to summ arize the "incorrect interpretation" o f the studies w ith toxicological findings. Dr. Lam precht noted the similarity betw een the observed abnorm ality and the Fraser developmental lens abnormality, and claimed that, because the observed abnormality did not develop into a cataract postnatally, as w ould occur with the Fraser abnormality, it w as his view that labeling the finding as a teratogenic response was in error. B u t Dr. L am p rech t w as w rong. H e assum ed th at the findings w ere exactly the same pathology as the Fraser developmental lens abnormality. The F raser abnorm ality is thought to arise from genetic m utations (M uggleton-H arris et al., 1987) and progresses to a cataract as the animals age. Because in a follow-up study (Experim ent No. 0680TR0020), offspring raised to PND21 failed to demonstrate a lens finding, Dr. Lam brecht concluded that the findings in rat fetuses was an artifact and not a teratogenic change. However, one o f the six general principles o f teratology developed by W ilson in 1973 (the same W ilson w ho developed the W ilson technique) is: " The four m anifestations o f deviant developm ent are death, m alform ation, growth retardation, and functional deficit' (E m phasis added, from Rogers, 2015). Dr. Lam brecht had no evidence that the observed eye abnorm ality w as in fact the Fraser abnorm ality; in fact, the lens finding could just as likely have represented a delay in eye 38 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN developm ent in the exposed groups relative to the control group that corrected by the tim e the postnatal assessment was made. As data were not collected along a postnatal developmental trajectory, it was not possible to determ ine if this finding represented a developm ental delay, i.e., growth retardation. Similarly, as the vision o f the offspring at PND21 was not assessed, it also was not possible to determ ine if the lens finding was associated w ith a functional deficit. Therefore, given the lack o f consistent artifacts in control groups, the lack o f studies to determine if the finding was a delay in developm ent, and the lack o f functional vision assessments, it is an incorrect interpretation that the lens finding was ju st an artifact of the sectioning technique. Supportive o f an ocular toxicity are inhalation and dermal toxicity studies which have noted eye irritation in adult anim als (G riffith and Long, 1980; K ennedy et al., 1986) follow ing PFO A exposure, and developmental studies that have noted delays in eye opening following PFO A exposure (Lau et al., 2006; A bbott et al., 2007; W o lf et al., 2007), PFO S exposure (Lau et al., 2003), PFB A exposure (Das et al., 2008), or PFN A exposure (Das et al., 2015) exposure. VIII.4 Detailed Supported Opinion - Immunotoxicity It is my opinion, based on the w eight of the toxicological evidence, as supported by epidem iological evidence, that PFCs pose a substantial present and potential imm unotoxicity hazard to hum an health. An early suggestion that PFCs m ay affect the im m une system can be found in 1978 studies by 3M. In a 90-day study o f PFOS fed to rats, lesions to the hem atopoietic tissues (including the thymus, bone marrow, spleen, and m esenteric lym ph nodes) were observed (3M A00593073, 3M _M N01663537). A 90-day study o f APFO fed to m onkeys resulted in findings of hypocellularity o f bone m arrow and atrophy o f lym phoid follicles o f the spleen and lym ph nodes (3M A00593073, 3M A01797217). These tissues are associated with the body's immune response; changes in their weights and signs of histopathology in their cells are considered w arning signs o f im m unotoxic effects. Additional studies w ould be w arranted to determine the functional im plications o f such effects, i.e., the ability of the imm une system to m ount an appropriate response. The m ost recent review of the imm unotoxicity of PFCs was conducted by the NTP. By way of background, the U.S. N TP system atically reviews data on particular exogenous agents to determine their likelihood to pose a health hazard to humans. Briefly, through the Office of Health A ssessm ent and Translation (OHAT), NTP system atic reviews are a m ethod for answering specific research questions (Figure 12) and "use[] a predefined, m ultistep process to identify, select, critically assess, and synthesize evidence from scientific studies to reach a conclusion" (NTP, 2015). Therefore, the NTP systematic review process, much like the IARC and U.S. EPA processes, is an assessm ent o f the existing scientific literature designed to analyze the quantity and Figure 12. A schematicoftheNTP systematicreviewprocess.From NTP (2015). 39 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN quality o f scientific studies related to a particular question that is relevant to environm ental health. The assessment titled Systematic Review of Imm unotoxicity Associated w ith Exposure to Perfluorooctanic Acid (PFOA) or Perfluorooctane Sulfonate (PFOS) was undertaken by the U.S. NTP (NTP, 2016) to determine w hether exposure to either chemical is associated with im m unotoxicity in humans. The resulting review is available electronically and is freely dow nloadable (h ttp s://ntp.niehs.nih.gov/pubhealth/hat/nom s/pfoa/index.htm l). T his docum ent was evaluated by a team composed o f scientists from the NIEHS (the N IH section under which the NTP resides) and contract research organizations, and was reviewed by scientists external to the NIEH S/N TP during protocol developm ent and draft report development. It w as also reviewed by a team o f external peer-review ers prior to the final release. Additionally, prior to the external peer-review process, outside parties (i.e., private citizens, industry representatives, representatives from other agencies, trade group representatives, etc.) were given an opportunity to provide evaluative comments. D uring external peer-review proceedings, these parties also w ere perm itted to attend as observers. Like the IARC and U.S. EPA, the N TP follows a process that seeks to generate a final assessm ent that is transparent, responsive, and rigorously reviewed by scientific experts. In my report here, the N TP m onograph is used as a support docum ent as it reflects a thorough assessm ent of the imm unotoxicological data associated with PFO A and PFOS. I served as an external peer review er o f the N TP assessm ent during protocol developm ent and draft report development from 2013-2016. Based on a systematic synthesis and integration o f evidence from published studies, the NTP assessm ent concluded that: "PFO A and PFOS are presum ed to be im m une hazards to hum ans and to alter im m une function in humans. Exposures to PFO A and PFOS are associated with changes in m ultiple im m une outcomes in both experimental animal and epidem iological studies. The strongest bodies o f evidence to inform the evaluation of PFOA- and PFO S-associated im m unotoxicity are on the antibody response." (NTP, 2015). A s depicted in F igure 13, "presum ed" is a term that the N T P used to dem onstrate high levels o f evidence from studies in experimental animal m odels and m oderate levels o f evidence in studies of exposed humans. The NTP undertook to evaluate the im m unotoxicity of PFO A and PFOS because of widespread hum an exposure to PFO A and PFOS, and because of the availability of data concerning these PFCs in relation to imm unotoxicity in both hum ans and non-hum an anim als (NTP, 2015). An im portant com ponent o f this particular review w as the evidence synthesis, which included a quality assessm ent o f individual studies evaluated in the review as well as a confidence rating of the body of evidence for a particular im m unotoxicological health outcome. W hile this approach is detailed in the NTP m onograph, it adds som ew hat to the evidence syntheses perform ed in the IARC and U.S. EPA docum ents in that it assigns m easures o f risk o f bias to particular studies, as well as m easures o f confidence for the overall body o f data. Briefly, risk o f bias in particular 40 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN studies was based on, for High "Known" example, w hether study authors i were blind to experimental groups " Moderate offrer re le v a n t data m ay provid e strong support * fo in c re a s e haza rd ID "Suspected" y y o th e r releva nt v data m ay provid e stro n g su p p o rt to decrease hazard ID * __________ "Presumed" or if animals were distributed randomly to treatm ent and control groups. Overall confidence in the ^ quality of evidence for each im m une outcome was evaluated as "high," "m oderate," "low," or ** . _ r _________ ^ 5 Low or ( " Not classifiable" 1 "Suspected" g Inadequate * ________ "Presumed" "very low " and can be found in the OHAT Handbook for Conducting a Literature-Based Low or Inadequate Moderate High Level o f Evidencefor Health Effects in Non-Human Animal Studies Health Assessment (http://ntp.niehs.nih.gov/go/38673 Figure 13. A schematicdemonstratinghow levelsofevidence were classifiedinthesystematicreviewofPFOA andPFOS. From NTP (2015). , see STEP 5). M y research contributions represent a substantial part o f the NTP systematic review o f the potential hazard to hum an health following exposure to PFO A or PFOS. The NTP concluded that exposures to PFO A and PFOS were associated with changes in m ultiple im m une outcom es in both experimental animal m odels and humans. For PFOA, this conclusion was based on (a) a high level o f evidence from animal studies and m oderate evidence from hum an studies o f suppression o f antibody responses (a m easure o f the adaptive imm une system), as well as (b) a high level o f evidence from animal studies and a low level o f evidence from hum an studies o f increased hypersensitivity-related outcom es (NTP, 2015). In their assessm ent of the data for PFOA, the N TP also considered other imm une-related endpoints that had been reported in epidem iological studies or in studies o f experimental animal models. These included changes in disease resistance or infectious disease outcomes related to im m unosuppression, suppression o f natural killer cell activity (a m arker o f the innate im m une system), and autoim m unity-related effects and outcomes. Changes in disease resistance or infectious disease outcomes. The N TP regarded the database for these outcomes as inadequate due to a lack o f experimental animal studies on this endpoint and a lim ited num ber o f epidem iological studies evaluating this endpoint. Suppression o f natural killer cell activity. The N TP regarded the database for this outcom e as inadequate due to a lim ited num ber o f experimental animal studies on this endpoint and a lack of epidem iological studies evaluating this endpoint. Autoim m unity-related effects and outcomes. The NTP regarded the database for these outcomes as inadequate due to a lack o f experimental animal studies on this endpoint and a lim ited num ber o f epidem iological studies evaluating this endpoint. 41 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN For PFOS, this conclusion was based on a high level o f evidence from animal studies and m oderate evidence from hum an studies o f suppression o f antibody responses (NTP, 2015). Each of these conclusions were supported by a body of evidence indicating that these PFCs affect m ultiple aspects o f the im m une system (NTP, 2015). In their assessm ent o f the data for PFOS, the N TP also considered other im m une-related endpoints that had been reported in epidem iological studies or in studies o f experimental animal models. These included changes in disease resistance or infectious disease outcomes related to imm unosuppression, suppression of natural killer cell activity (a m arker o f the innate im m une system), and autoim m unity-related effects and outcomes. Changes in disease resistance or infectious disease outcomes. The N TP regarded the database for these outcomes as sufficient to classify PFOS as "suspected to be an im m une hazard to hum ans" (see Figure 11) based on a low level o f evidence in hum ans and a m oderate level of evidence in experim ental animal studies. Suppression o f natural killer cell activity. The N TP regarded the database for these outcom es as sufficient to classify PFOS as "suspected to be an im m une hazard to hum ans" (see Figure 11) based on an inadequate level o f evidence in hum ans and a m oderate level o f evidence in experim ental animal studies. Autoim m unity-related effects and outcomes. The N TP regarded the database for these outcom es as inadequate due to a lack o f experimental animal studies on this endpoint and only one epidem iological pilot study evaluating this endpoint. It is im portant to note here that while the N TP considered the w eight-of-evidence strongest for the ability of PFO A to suppress antibody responses and to induce hypersensitivity and of PFOS to suppress antibody responses, the entirety o f the database on im m une effects was considered in their system atic review. Therefore, lack o f evidence for particular im m une endpoints (i.e., disease resistance/infectious disease outcomes, suppression o f natural killer cell activity, and autoim m unity-related effects and outcom es) should not be interpreted as evidence against the occurrence of such outcomes, but that the evidence was insufficient to m ake a definitive classification as to the hazard. Seven m anuscripts that I co-authored w ere used in this NTP m onograph and four were selected as studies in experimental animal m odels supportive o f suppression o f antibody responses in which the NTP had a high level of confidence. The following discussion provides a brief overview o f these four studies: In D eW itt et al. (2016b), w e evaluated suppression o f T cell-dependent antibody responses (TDAR) and T cell-independent antibody responses (TIAR) in a wild type m ouse model and the TD A R in a PPA R a knockout model, which is a m ouse that had been genetically altered to lack a functional P P A R a. W e found that exposure to > 1.88 mg PFOA/kg suppressed the TIAR and 30 m g PFOA/kg suppressed the TDAR in both w ild type and knockout mice. The findings dem onstrate that exposure to PFO A induces adverse effects on the ability o f the im m une system to function an appropriate response, as by suppression of antibody responses, and that it does so in a m anner not mediated exclusively by PPA Ra. 42 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN In H u et al. (2010), w e sought to evaluate the developm ental effects o f PF O A on the TDAR. Pregnant w ild type m ice w ere exposed to 0, 0.5, or 1 m g PFO A /kg and offspring were examined. Litter weights were statistically decreased by 10% in the 1 m g/kg group. The TD A R did not differ in fem ale offspring by dose. W hile w e concluded that 0.5 and 1 mg PFO A/kg did not induce developmental imm unotoxicity, we noted that in a pilot study, 5 m g PFO A /kg induced high (75%) neonatal toxicity. W e therefore concluded that C57BL/6 m ice are more sensitive to overt developmental toxicity of PFO A and to potential developmental immunotoxicity. In D eW itt et al. (2009a), w e sought to evaluate the potential role o f elevated corticosterone production on effects on the TD A R reported in D eW itt et al. (2008). W e dosed adrenalectomized (adx) or sham-operated C57BL/6N fem ale mice at several dose levels ranging from 3.75 m g PFO A /kg to 30 m g PFO A /kg and evaluated the TD A R as it related to corticosterone levels and liver markers. The data demonstrated that the im m unosuppressive effects o f PFO A w ere not the result o f liver toxicity or a stressrelated corticosterone response, as had been argued in a publication by D uPont authors (Loveless et al., 2008). In D eW itt et al. (2008), w e sought to evaluate the effects o f PFO A exposure on both humoral and cellular imm unity and report the results of several experiments. O f note were the dose-responsive effects o f PFO A on the TDAR. W ild type m ice received 0 through 30 m g P F O A /kg for 15 days. F rom these data, w e identified a N O A E L o f 1.88 m g PFO A/kg, a LO A EL o f 3.75 m g PFO A /kg, and a BM D o f 3 m g PFO A /kg in relation to the effects o f PFO A exposure on the TDAR. This was the first peer-review ed publication, to our knowledge, on the dose-responsive effects of PFO A on the TD AR in a m ouse model. Antibody responses are accepted m easures of imm une function. In particular, suppression of the antigen-specific antibody response is regarded as a sensitive predictor o f im m unotoxicity as it requires cooperation o f m ultiple cells and signals w ithin the im m une system (Luster et al., 1992). For example, the U.S. EPA has suggested that this response be included in im m unotoxicological assessments o f pesticidal compounds and agents that m ay fall under the Toxic Substances Control A ct (i.e., U.S. E PA H ealth Effects Test G uidelines OPPTS 870.7800 Im m unotoxicity). The NTP likewise regards reduction in this response as an indicator of decreased imm une function or im m unosuppression that may indicate a greater risk of disease (NTP, 2015). The antigen-specific antibody response is fairly straightforward to evaluate in experimental animal m odels and in humans. In animal models, an antigen, which is an agent that stim ulates antibody production, in injected into the bloodstream and then antibodies against the antigen are m easured in the blood sera a specific am ount o f tim e later, usually at the peak o f antibody production. Both prim ary (IgM ) and secondary (IgG) antibody responses can be measured, although secondary responses generally require a booster injection before collection of blood for m easurem ent o f antibodies. The hum an analog is vaccinations; antibody responses to vaccines can be m easured as well. Several epidem iological studies have m easured anti-vaccine antibody responses in which the N TP had moderate confidence. This confidence rating was moderate, and not strong, based largely on possible effects from co-exposures to other agents, including other PFCs, which could have confounded the observed associations (NTP, 2015). Regardless, overall 43 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN evidence associated w ith im m unotoxicity following PFO A or PFOS exposure is strong given that sim ilar responses have been observed in both hum ans and experimental animal models. Suppression of imm une responses in rodents by exogenous agents is predictive of suppression o f im m une responses in hum ans; in other w ords, observations of suppression of imm une responses in rodents is translatable to the hum an condition following exposure to the same exogenous agents (Selgrade, 2007). This relationship is depicted in F igure 14. T he cellular and humoral imm une response to vaccination, for example, is regarded as a sensitive indicator of imm une suppression and can reflect susceptibility to infectious and neoplastic diseases (D eW itt et al., 2016a). Figure 14. Aparallelogrammodel to illustrate the relationshipbetween immune suppressionand increased risk of disease in rodent models and humans. From Selgrade (2007). Adverse outcomes of developmental and adult immunotoxicity Suppression o f im m une responses is not the only consequence that arises from Dysregulated inflammation Inflammatory disease and Autoimmunity tissue damage Allergic disease Norma immune response Categories ot importance for risk of chronic disease m odulation o f the im m une system. Im m unom odulation by exogenous agents can result in not only im m unosuppression, but also autoimmunity, dysregulation of inflammation leading to inflammatory disease and tissue damage, and allergic disease (Figure 15; D ietert et al., 2010). Im m unom odulation that occurs during developm ent is especially problematic. Increases in childhood imm une-based Immunosuppression diseases following exposure to exogenous Figure 15. Adverse outcomes of immunotoxicity. a ents have been docum ented throughout Adopted fromDietert et al. (2010). the literature and a growing body of evidence supports the hypothesis that chronic diseases such as asthma, have fetal origins (DeW itt and Keil, 2017). Therefore, agents, such as PFCs, that induce imm unom odulation in developing organisms, have the potential to increase the risk of later-life diseases and disorders. Several epidem iological studies have reported associations between serum PFC levels and suppression o f the TD A R, including G ranum et al. (2013), G randjean et al. (2012), and Stein et al. (2016). I understand that Dr. Grandjean addresses im m unological effects seen in PFC epidem iology studies in greater detail in his report. Based on the assessm ent of PFO A and PFOS by the U.S. NTP, the im m une system is sensitive to adverse health outcomes associated w ith exposure to these agents. The w eight o f the evidence illustrates that PFO A and PFOS can induce im m unotoxicity as an adverse health outcom e in 44 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN exposed humans. By extension, other PFCs that induce toxicities sim ilar to PFO A and PFOS and that w ork through sim ilar m echanisms, such as PFBA, are likely to induce im m unotoxicity as an adverse health outcom e in exposed hum ans as well. I agree w ith the assessm ent by the U.S. NTP that PFO A and PFO S are im m une hazards to hum ans based on evidence in experimental animal m odels that is supported by epidem iological findings in humans. VIII.5 D etailed Supported Opinion - Other N otable Toxicological Findings As discussed above, there is a large body of evidence that exposure to PFCs leads to adverse health outcomes in a variety of experimental animal models, w ildlife species, and humans. A few specific toxicological findings are o f special note as they have been discounted as toxicologically irrelevant by 3M scientists, but evaluation o f the actual findings suggest that they are not toxicologically irrelevant. Three particular toxicological outcom es associated w ith exposure to PFCs that have not yet been discussed are thyroid horm one disruption, liver toxicity, and m am m ary gland development. VIII.5.a Thyroid H orm one D isruption One of the findings of the C8 Science Panel was a probable link betw een PFO A exposure and thyroid disease. Specifically, the panel found a positive association betw een m easured PFO A exposure and m edically validated thyroid disease, w hich w as defined as hyperthyroidism in wom en and hypothyroidism in men (C8 Science Panel, 2012). Briefly, the proper regulation of thyroid hormones is crucial to the growth, developm ent, and m etabolic function o f organ systems in the body. H yperthyroidism is overproduction o f thyroid horm ones and hypothyroidism is underproduction of thyroid hormones. Thyroid horm one production is a complex interplay am ong the hypothalam us of the brain, w hich secretes thyrotropin releasing hormone, the anterior pituitary gland, w hich secretes thyroid stim ulating hormone (TSH), and the thyroid gland itself, w hich secretes the thyroid horm ones (TH) triiodothyronine (T3) and thyroxine (T4; D ong and Greenspan, 2015). Com plex feedback loops balance the production of these hormones as well as dietary iodide, w hich is necessary for the biosynthesis of thyroid hormones, and efficacy of a variety of enzymes involved in TH biosynthesis and peripheral conversion (Dong and Greenspan, 2015). TH is mostly bound to plasm a proteins (thyroxine-binding globulin, transthyretin, and album in) while in blood circulation; only 0.04% o f T4 and 0.4% o f T3 are free, but this free com ponent is the biologically active component (Cooper and Ladenson, 2011). The C8 Science Panel did not elucidate all o f the underlying causes o f thyroid disease, or the m echanism s o f disease, in their probable link report, yet found convincing correlations. Additional studies of hum an populations and the association betw een thyroid disease and/or TSH/TH and PFCs have been published and support a positive association between thyroid disease and PFO A and to an extent, PFOS. Although few published studies in experimental animal models have specifically evaluated TH following PFO A exposure, a handful have included m arkers of thyroid gland function following exposure to PFOS or other PFCs. Exposure to PFO S during adulthood has been associated w ith suppression o f TH in m onkeys (Seacat et al., 2002) and in rats and m ice (Thibodeaux et al., 2003), and w ith suppression o f T4 in rats developm entally exposed (Lau et al., 2003). O ther studies in rodents also have reported 45 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN decreases in serum T4 (Curran et al., 2008; Yu et al., 2009a,b) w ithout changes in T3 and/or TSH, which suggests possible interference with biosynthesis or peripheral conversion enzyme A study published in 2005 by Luebker et al., exam ined thyroid horm one effects in rat pups exposed in utero to PFOS (Luebker et al., 2005, 3M _M N03095624). The results showed a statistically significant decrease in free T4, as m easured using an analog assay, and an increase in TSH-- which is indicative o f hypothyroidism . The study authors discounted the free T4 observation, however, as resulting from a bias in the analog m easurem ent technique. As discussed below , this asserted bias in the analog m easurem ent technique is questionable. The study authors purported to correct this bias by m easuring free T4 using the equilibrium dialysis radioimm unoassay (ED-RIA) method. However, they only ran this ED -RIA assay for a lim ited num ber o f samples from the control and low est dose group. Thus, the study did not provide any data on whether, under the ED -RIA assay, free T4 was in fact significantly decreased at higher PFOS doses. Although, the m anuscript states that "the data . . . did not suggest a hypothyroid state in pups," this conclusion is not supported based on the fact that the analog assay demonstrated a significant decrease in free T4 and the study authors did not obtain sufficient m easurem ents of free T4 using the ED -R IA assay. In a study published in 2007 (Chang et al.), w hich included three co-authors from the 3M Com pany (S-C Chang, D.J. Ehresm an, and J.L. Butenhoff) as well as funding for the project, in part, by the 3M Company, the authors suggest that that a certain type o f assay used to evaluate serum TH has led to a negative bias in reported results concerning free levels of T4. In other w ords, according to C hang et al. (2007), an assay know n as the analog assay has led to erroneous reports of decreased T4 due to the ability of PFOS to produce binding interference in the assay. A ccording to C hang et al. (2007), PFO S binds to the sites in the assay intended for the binding o f unbound T4, thus reducing the apparent am ount o f T4. The authors (C hang et al., 2007) argue that an assay that combines direct equilibrium dialysis followed by radioimm unoassay (ED-RIA) will not produce negative biases. They perform ed a series of studies to test their hypothesis that decreases in T4 would not be evident w hen the ED -RIA assay m ethod was employed and generated data that they claim is supportive o f their hypothesis. The claim by Chang et al. (2007) is questionable for several reasons. First, it is generally understood by the scientific comm unity that changing a paradigm in science, for example discounting a m ethod generally accepted by the scientific comm unity as valid (methods comm only used in assessm ent o f TH status) requires confirmatory studies by several laboratories; one study is considered insufficient to discount a generally accepted method. Additionally, epidemiological data from the C8 Science Panel support an association between m edically diagnosed thyroid disease and serum PFO A and additional studies support associations betw een other PFCs and thyroid disease and TH status, thus providing strong supportive evidence that the thyroid gland and/or the hypothalam ic-pituitary-thyroid axis is adversely affected by exposure to PFCs. In addition, Chang et al. (2007) claim that results reported in previous studies, reduced serum free T4 without a concomitant increase in TSH, do not fit the clinical profile of hypothyroidism and are further evidence of a negative bias in assay techniques. However, low free T4 can exist w ithout concom itant increases in TSH in a condition known as central hypothyroidism. In this situation, levels o f TH are reduced but levels o f TSH m ay be normal, low, or slightly elevated 46 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN (Gupta and Lee, 2011). The putative etiologies o f central hypothyroidism include genetic defects, tumors, vascular dysfunction, inflamm ation, iatrogenic, and others (Gupta and Lee, 2011). In a study to further understand the T H effects o f PFO S exposure, Y u et al. (2011) gave propylthiouracil (PTU), an anti-thyroid drug used to treat hyperthyroid, alone and in combination with PFOS to determine if PFOS was acting at the level of TH biosynthesis (PTU blocks biosynthesis at the level o f the thyroid gland and blocks conversion o f T4 to T3 in the periphery). The authors reported that PFOS appears to be acting at the level of the periphery, by increasing m etabolism o f T4 rather than acting at the level o f TH biosynthesis (Yu et al., 2011). Therefore, at a very minimum, additional studies are necessary before TH /thyroid disease can be ruled out due to assay discrepancies. Some o f the thyroid horm one effects seen in laboratory anim als exposed to PFO A and PFOS have also been noted in laboratory animals exposed to PFBA. In a 28-day and 90-day studies of PFBA in rats, a statistically significant dose-dependent reduction in total T4 and free T4 in male rats resulting from exposure to PFB A w as seen at high doses. (B utenhoff 2012b). Thus, it is likely that the thyroid hormone effects seen in long-chain PFCs-- including potential evidence of hypothyroidism -- could be applicable to PFHxS and short-chain PFCs like PFBA and PFBS. VIII.5.b Liver Toxicity Liver enzym es are often m easured clinically to determ ine the functionality o f the liver. W hen liver enzyme levels are skewed, physicians must often carefully interpret the findings in term s of the characteristics of the patient and the appropriate reference ranges for that patient and possibly do additional diagnostic evaluations to determine if liver function is com prom ised (Giannini et al., 2005). Changes in liver enzym es can be transient and increases can be m ild and such changes do not necessarily indicate a serious liver problem, but elevations in liver enzymes can indicate inflam m ation or dam age to cells in the liver (Giannini et al., 2005). H ow ever, from a toxicological perspective, w hen concordance is observed am ong organisms, such as changes in liver enzymes in exposed hum ans and in exposed experimental animal models, even if the changes do not follow the same pattern, this is a signal o f a shared target path w ay /cell/tissu e/o rg an . Num erous epidemiological studies have m easured liver enzyme levels and provide evidence of a positive association betw een PFO A and several liver enzym es (U.S. EPA, 2016c). M yriad studies in experim ental animal m odels also report changes in liver enzym es (U.S. EPA, 2016c). These findings in experimental animal models support the findings in epidem iological studies. W hile the U.S. EPA assessm ent o f PFO A noted that no evidence o f liver dam age has been reported in hum an epidem iological studies, histological evaluations o f livers from experimental animal m odels reveal pathological changes (U.S. EPA, 2016c). Several studies have suggested that liver toxicity from PFO A exposure in experimental animal m odels is not relevant to hum ans as liver toxicity is m ediated by PPA R a. Hum an P P A R a levels are thought to be about 10% o f the levels in rodents (D eW itt et al., 2009), but hum ans are responsive to agents that act via PPA R a; the M O A for the fibrate class of drugs designed to low er serum lipids is via P P A R a activation. Additionally, studies w ith experimental animal m odels lacking a functional P P A R a also dem onstrate liver toxicity (D eW itt et al., 2016b). Therefore, by extension, other PFCs that induce toxicities sim ilar to PFO A and that w ork 47 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN through sim ilar mechanisms, such as PFBA, are likely to induce liver toxicity as an adverse health outcom e in exposed hum ans as well. VIII.5.c M am m ary G land D evelopm ent Low dose exposure to PFO A during developm ent has been reported to delay m am mary gland developm ent in mice. The U.S. EPA (U.S. EPA, 2016c) sum m arized the findings o f these studies and they are presented in F igure 16. Table 3-21. Studies of Pregnant CD-I Mice Following Administration of PFOA Dose (m g PFO A kgday) 0. 0.01. 0.1. 0.3. 1 0 ,5 0,5 20 0 ,3 ,5 0,5 0 ,0 .3 ,1.0,3 0. 0.01,0.1. 1 0 , 1,5 0 ,1 + 5 ppb in dnnking water to both groups 0 ,3 0. 0.01,0.1, 0 .3 ,1 Tim ing GD 1 to PND 21 G D 1-17, 8-17, or 12-17 G D 7-17, 1 0 -1 7 ,13-17, 15-17 G D 15-17 G D 1-17 Cross-fostered at birth G D 8-17 Cross-fostered at birth GD 1-17 GD 10-17 G D 1-17 GD 1-17 Drinking water started on G D 7 and continued to F2 generation G D 1-17 G D 1-17 Study included both CD -I and C57B L/6 mice E n d p o in ts R eference Liver histopathology; periportal inflamm ation; clinical chemistry: im pact o f postw eaning HFD Q uistetal. 2015 Body weight; m am m ary gland m orphology GD 18 (dam s) an d P N D s 10 a n d 20 (dam s, fem ale W hite et al. 2007 im ps) Body weight: developm ental landm arks and grow th to PN D 189; m am m ary gland m orphology o f fem ale pups up to 18 m onths W hite et al. 2009: W olf et al. 2007 Body weight; developm ental landm arks and grow th to PN D 245: m am m ary gland m orphology o f fem ale pups up to 18 m onths W hite et al. 2009; W hlfetal. 2007; M am m ary gland m orphology o f dam s and fem ale W hite et al. 2009 pups o n PN D s 1 , 3 , 5. and 10 Liver weight: m am m ary gland m orphology o f fem ale pups on PN D s 7 , 1 4 ,2 1 ,2 8 .42, 63, and 84 M acon et al. 2011 Body weight; reproductive parameters; m am m ary glan d m o rp h o lo g y o f F0, F 1, a n d F 2 fem ales W hite et al. 2011 W ild-type, PPA R a-null, andliP P A R a w /1 2 9 m ice: pup body weight at PN D s 14 and 20 plus num m ary gland structure B ody weight; net body weight; absolute and relative liver w eight on PN D s 21, 35, and 56; serum estradiol and progesterone; num m ary gland morphology' A lb rech t e t aL 2013 T ucker et al. 2015 Figure 16.Summary ofstudiesthathave evaluatedmammary glanddevelopmentin mice developmentallyexposedtoPFOA. From U.S. EPA, 2016c. In M arch o f 2017, the N ew Jersey D rinking W ater Quality Institute (NJ DW QI) recom mended health-based m aximum contaminant levels for PFO A in drinking water (NJ DW QI, 2017). The N J DW Q I (2017) asserted that delayed m am mary gland developm ent and increased liver weight were the m ost sensitive non-carcinogenic endpoints with sufficient data for dose-response modeling. Based on their model, the N J D W Q I derived a target human serum level, which they describe as analogous to an RfD but on a serum level basis, o f 0.8 ng/m L (NJ DW QI, 2017). The N J DW Q I (2017) chose mam mary gland developm ent as a sensitive endpoint as it represents a structural change in an organ that persists to adulthood, which m akes it a valid endpoint for risk assessment. The U.S. EPA (U.S. EPA, 2016c) did not select delayed m am m ary gland developm ent as the critical endpoint for derivation o f the RfD as pups nursing from afflicted dams did not suffer effects on body weight and afflicted dams did not appear to differ from controls during a lactational challenge assay (volum e of m ilk produced during a m easured nursing period). However, the U.S. EPA did regard studies o f delayed m am m ary gland developm ent as supportive of the RfD that was derived for PFO A from other developmental endpoints (U.S. EPA, 2016c). Therefore, delayed m am m ary gland developm ent represents an adverse health outcom e that occurs at low doses and that persists into adulthood. By extension, other PFCs that induce toxicities similar to PFO A and that w ork through sim ilar mechanisms, 48 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN such as PFBA, are likely to induce impacts on m am mary gland developm ent as an adverse health outcom e in exposed hum ans as well. VIII.6 D etailed Supported Opinion - Toxicity A ssociated w ith A reas o fH igh Exposure It is m y opinion, based on the w eight o f the toxicological evidence, as supported by epidemiological evidence, that PFCs pose a substantial present and potential hazard to hum an health in areas of high PFC exposure, either due to occupational exposures or by living near fluorochem ical production facilities, especially from effects on the liver and the im m une system and on developing organisms. As previously m entioned, in addition to the system atic reviews o f the IARC, the U.S. EPA, and the U.S. NTP, I also rely on the draft Toxicological Profile for Perfluoroalkyls by the ATSDR. A TSD R is the part o f the CDC that serves the public by preventing harmful exposures and diseases related to toxic substances (ATSDR, 2017), and their assessments include an opportunity for public comm entary and peer-review. The toxicological profile available through the A T SD R 's web site is still the 2015 draft version available for public com m ent (https://w w w .atsdr.cdc.gov/toxprofiles/tp.asp?id=1117& tid=237), but the public com m ent period ended on D ecem b er 1, 2015 (A T SD R , 2017). W hile the final docum ent could differ from the draft docum ent given the scope of public comm ents, the draft docum ent has undergone internal ATSDR reviews and w as reviewed by a panel of peers. Like the other documents discussed for cancer (IARC), developm ental toxicity (U.S. EPA ) and im m unotoxicity (U.S. NTP), the ATSD R docum ent also is a system atic review of the current literature. A dditionally, the docum ent is somewhat broader in scope than the other three documents as it includes "perfluoroalkyls," and does not focus exclusively on PFO A and PFOS. I did not serve as an external peer review er for this 2015 draft docum ent nor did I submit comm ents during the public com m ent period. M y decision to use this docum ent is based on the public health m ission o f the A TSD R and m y understanding o f their review process, w hich is rigorous and transparent. The A TSD R summ arized findings from epidem iological studies and determ ined the following health effects were consistently associated w ith PFO A and PFOS serum levels: 1. Increases in serum lipid levels. 2. D ecreases in birth weight. 3. Increases in u ric acid levels. 4. A lterations in biom arkers o f liver damage. 5. C arcinogenicity. In their summary o f findings from studies o f experimental animal models, the ATSDR determ ined the following health effects w ere prim ary effects: 1. L iv er toxicity. 2. D evelopm ental toxicity. 3. Im m unotoxicity. 49 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN The ATSDR toxicological profile also examined, summarized, and interpreted available toxicologic inform ation to determine levels o f exposure that present a significant risk to hum an health (ATSDR, 2015). One o f the values that the A TSD R derives is sim ilar to a RfD or HA L that the U.S. EPA calculates. The analogous value derived by the A TSD R is the m inim al risk level (MRL), which they define as "an estimate o f daily hum an exposure to a substance that is likely to be w ithout an appreciable risk of adverse effects (noncarcinogenic) over a specified duration of exposure." (ATSDR, 2015) Although the overall document addresses the toxicity of PFCs beyond PFOS and PFOA, the ATSDR chose to derive specific M RLs only for compounds for which they deemed a sufficiently broad database. W ith regard to PFCs, the A TSD R determ ined that PFO A and PFOS had a database o f evidence sufficiently rich enough to derive M RLs for the oral route of exposure. For both compounds, the A TSD R determined that adverse hepatic effects, adverse immunological effects, and adverse developm ental effects were sensitive endpoints associated w ith exposures in rodent (rat and m ouse) species. U sing an approach very sim ilar to the U.S. EPA approach for derivation o f RfDs, the A TSD R derived M RLs based on hepatic effects that were supported by LOAEL values for developmental toxicity and imm unotoxicity. For PFOA, the M RL was 0.00002 m g/kg/day and for PFOS, it was 0.00003 m g/kg/day. These M RLs are equivalent to the U.S. EPA RfDs for both PFO A and PFOS. The M R L and RfD values derived by the A TSD R and U.S. EPA, respectively, w ere calculated to protect the m ost sensitive subpopulations, i.e., those w ith the greatest risk o f exposure and/or those w ho are m ost vulnerable to health effects following exposures, from a lifetim e of exposures. In the state o f M innesota, residents o f the East M etro area have putatively higher exposures to PFCs than do m em bers o f the U.S. general population, given levels in municipal drinking w ater wells and levels m easured in blood. States may institute health based values for protection o f their residents that are m ore stringent than U.S. EPA health based values, for any exogenous agent to w hich their residents m ay be exposed. The U.S. EPA also notes that their H A Ls m ay change given new evidence (U.S. EPA , 2016a,b). In 2017, follow ing the release o f HA Ls by the U.S. EPA, the M D H released updated guidance values for PFO A and PFOS that were m ore protective than the U.S. EPA HA L to reflect the potential for m others to pass along PFCs to fetuses and nursing infants (M DH, 2017b). The M D H acknow ledged that w hile the U.S. EPA H A L is protective for m ost people, they w anted to take additional steps to protect the highly sensitive developing organism and so revised their HAL based on a newly developed toxicokinetic model (MDH, 2017b). Because part o f the m ission o f the A TSD R is to help public health professionals and others address the needs of persons living or w orking near hazardous w aste sites, their toxicological profile documents contain sections on the relevance of toxicity and toxicokinetic data to public health (ATSDR, 2015). A majority o f the docum ent dissects these health effects by type of health effect (death, systemic, imm unologic, and reproductive), by route of exposure, and by length of exposure (acute, intermediate, and chronic), and indicates w hether they are "less serious" or "serious" effects (ATSDR, 2015). 50 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN The A TSD R (2015) also identified populations that may be particularly susceptible to PFCs, including populations with high exposures, those w ith existing high serum cholesterol or other existing cardiovascular risk factors, hypertension, or comprom ised liver function. By way of example, the A TSD R (2015) identified such populations to include those w ho w ork at or are located near fluorochemical facilities and those involved in activities with prolonged use of perfluoralkyl-containing products, such as those involved in the application of protective coatings for textiles and papers. Based on the assessm ent o f PFCs by the ATSDR, certain populations are at increased risk for adverse health outcom es associated w ith exposure to these agents. I agree that in areas o f high PFC exposure, either due to occupational exposures or by living near fluorochem ical production facilities, increased health risks, especially those associated with effects on the liver and the im m une system and on developing organisms, likely exist. Some o f the adverse health outcom es have been associated w ith PFC serum concentrations in highly exposed hum an populations, m ost notably residents in the m id-Ohio Valley who were exposed via drinking w ater due to PFO A contam ination of water supplies by a DuPont production facility. These studies, known as the "C8 Health Project" established probable links between serum PFO A concentrations and the following adverse health outcom es: 1. C ardiovascular disease (high cholesterol), 2. Pregnancy and pregnancy outcom es (pregnancy-induced hypertension and preeclam psia), 3. A utoim m une disease (ulcerative colitis), 4. Thyroid disease, and 5. C ancer (kidney and testicular). These C8 Health Project studies thus support the conclusion that hum ans exposed to PFCs have an increased risk of adverse health outcomes. In addition, studies exploring m yriad aspects of PFC-associated toxicities in experimental animal models have been published. These include m ammary gland development, disruption of various hormones, reproductive endpoints, diabetes and other m etabolic disorders, serum lipids and cardiovascular diseases, liver and kidney function and disease, and behavioral outcomes. These toxicities support the body of evidence that exposure to PFCs leads to adverse health outcom es in a variety o f experimental animal models, wildlife species, and humans. VIII.7 D etailed Supported Opinion - Other PFC s Relevant in the State o fM innesota As m entioned above, Jarnberg and van Bavel (2007) estimated from published literature that the total num ber o f described synthetic PFC s exceeds 10,000 individual compounds. Industry is demonstrably capable o f devising new PFCs faster and in greater volume, and is doing so at a rate faster than the scientific com m unity can study them. Thus, there are not sufficient studies and data for a w ide-scale com prehensive assessm ent of all of these individual com pounds sim ilar to what has occurred for PFO A and PFOS. However, sufficient toxicological evidence does exist concerning three other PFCs of concern in M innesota-- PFHxS, PFBA, and PFBS-- that suggest that these compounds may pose a risk to hum an health. 51 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN VIII.7.a P F H xS PFH xS is a six-carbon chain PFC. It is considered by the U.S. EPA as a long-chain PFC, so is subject to the same guidelines and restrictions as PFO A and PFOS. Like PFO A and PFOS, it has a long serum half-life in hum ans, up to 8.5 years by som e estim ates (O lsen et al., 2007). Like many o f the PFCs other than PFO A and PFOS, peer-review ed publications are lim ited and are largely lim ited to studies conducted by or in cooperation w ith scientists from the 3M Company. An exploratory 28-day study o f PFBS and PFH xS in rats com m issioned by 3M in 1999 revealed that PFHxS exposure was associated w ith a decline in total cholesterol, increased liver weight, and liver hypertrophy in rats dosed at 10 m g/kg (3M A 00760311). In a 2006 report on the effects o f several PFCs in APOE3Leiden mice, a high dose o f PFHxS had to be stopped because of overt toxicity to the mice, as manifested by reduced food intake and decreased body weight (3M A01507211). Exposure to PFHxS was also associated w ith a robust decrease in cholesterol and triglycerides (3M A01507211). In the publicly available peer-reviewed scientific literature, a 2009 publication authored by scientists from the M edical D epartm ent o f the 3M Com pany (B utenhoff et al., 2009a), indicated that it was the first article to report on the biological response o f experimental animal models exposed to PFHxS, despite that studies were conducted on this com pound as early as 1999. In 2012, a peer-review ed paper on the pharm acokinetics o f PFHxS in rats, mice, and m onkeys was published by three of the four 3M Company scientists from the 2009 paper in collaboration with scientists from tw o U.S. universities and a laboratory in Sw eden (Sundstrom et al., 2012). In 2013, from a university laboratory in Sweden, a study indicating that a single neonatal exposure to PFHxS was sufficient to alter levels o f proteins in the brain critical for neuronal developm ent was published (Lee and Viberg, 2013). Epidem iological studies o f PFC -exposed hum ans have associated liver dam age (G leason et al., 2015) and decreased antibody responses to vaccines w ith PFH xS serum concentrations (G randjean et al., 2012). Given the long half-life o f PFH xS and the published toxicological findings (although relatively few in num ber in com parison to PFO A and PFOS), exposure to PFH xS also poses a present or potential hazard to hum an health. Dr. D eanna Luebker, Global R egulatory M anager o f 3M 's Separation and Purification Sciences D ivision stated that ..if they [chemicals] can readily degrade and break down, so things like carbon and oxygen, hydrogen, they typically d o n 't cause any problem s. So it's nice w hen they b reak dow n and kind o f go aw ay" (Luebker Depo. Tr. 35:11-14). The opposite supposition being that if chemicals do not readily degrade and break down, they do not go away, and do cause problems. VIII. 7.b PFB A a n d P F B S Studies o f PFB A and PFBS, while lim ited in num ber relative to PFO A and PFOS, show certain toxicities, which is particularly notew orthy given that certain o f these studies were undertaken by scientists funded by the 3M Company. In a study o f PFB A given to m ale and fem ale SD rats in drinking w ater for 28- or 90-days, authored by scientists from both the U.S. E PA and the 3M Com pany (B utenhoff et al., 2012b), the authors identified several statistically significant changes in the weights of various organs. The authors attempted to dismiss these results as "spurious" or not "toxicologically significant" as they did not show progression with dose, were not consistent betw een 28-day and 90-day studies, and lacked histological findings. For example, mean absolute (not corrected for body 52 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN weight of the animals) thyroid gland weights in m ale rats given 6 or 30 m g/kg/day of PFBA for 28-days were elevated by approxim ately 100% compared to the control group; a 150 m g/kg/day dose group did not have a statistical elevation in thyroid gland w eights, but w as approxim ately 55% greater in w eight compared to the control group. The authors stated that these weight changes were not toxicologically significant. M oreover, these studies dem onstrated that PFB A lowered total and free T4 in rats exposed for 28-days at a concentration of 150 m g/kg/d and 90days at a concentration of 30 m g/kg/d (Butenhoff 2012b). Similar thyroid hormone lowering effects have been observed in experimental animal model studies using PFO S and PFOA. But the statistically significant results should not be so dismissed. As m entioned above, for some agents, the occurrence o f adverse health outcom es does not always follow a "standard" doseresponse curve. Some dose-response curves are U-shaped or inverse-U-shaped, indicating that lower and higher doses produce responses that differ from moderate doses. M oreover, while a three-w eek recovery period (evaluated in separate groups o f rats that were treated and then euthanized three weeks after cessation of treatm ent) demonstrated that thyroid weights returned to control levels, the long-term effects o f a short-term increase in thyroid w eight cannot simply be discounted as spurious, especially when it occurred in tw o dose groups and had such a large effect size. Increases in thyroid gland w eight m ay reflect changes in circulating TH, w hich are exquisitely controlled via positive and negative feedback am ong the hypothalamus, anterior pituitary gland, and the thyroid gland. Increases in thyroid gland weight also may reflect inflam m ation in the thyroid gland. Some o f the m ale anim als w ith increased thyroid gland weights had signs o f thyroid follicular epithelial hypertrophy/hyperplasia, which often accompanies hepatocellular hypertrophy (NTP, 2017). H epatocellular hypertrophy also was observed in m ale animals given 30 m g/kg/day of PFBA. In a developm ental study o f PFB A in CD-1 m ice (Das et al., 2008), authored by scientists from both the U.S. EPA and the 3M Com pany, statistically significant delays in eye opening and onset of puberty in offspring were noted in several o f the PFBA dose groups. The authors concluded that while developmental effects were detectable, developm ental exposure to PFBA did not produce adverse developm ental effects at the same profound level that had been reported in this strain o f m ouse following exposure to PFO A or PFOS. However, the authors also noted that early pregnancy loss at high doses m ay represent a com m on feature o f PFAAs. It is also notable that delayed sexual m aturation is an effect also seen in offspring o f PFO A dosed fem ale rats, and that a delay in eye opening has been observed in offspring o f PFOS dosed fem ale rats. Therefore, while the authors believed that PFBA was not as developm entally toxic as PFO A or PFOS, PFB A still produced m eaningful adverse toxicological outcomes, some o f w hich have also been observed in PFO A and PFO S reproductive toxicity studies. The authors o f the last-m entioned study (Das et al., 2008) did not describe in great detail the statistically significant and toxicologically m eaningful findings o f delayed eye opening and onset o f puberty observed in their study. B ut there is reason to hypothesize that thyroid effects and developm ental effects are related. In experimental rodent models, thyroid horm ones play a role in reproductive tract development, maturation, and function; both hypo- and hyperthyroid (decreased and increased thyroid hormones) conditions have been associated w ith delayed vaginal opening and onset o f puberty (Choksi et al., 2003). Similarly, hypo- and hyperthyroid conditions have been associated w ith changes in reproductive system development, maturation, and function in hum ans (Choksi et al., 2003). Therefore, TH status should be evaluated when 53 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN changes are observed in reproductive tract developm ent, maturation, and function following exposure to an exogenous agent, especially w hen the database suggests such linkages. A recently published study (Feng et al., 2017) evaluated these linkages; however, it concerned PFBS rather than PFBA. The toxicological database of peer-reviewed published m anuscripts for PFBS contains even few er studies than for PFB A . In addition to the L ieder et al. (2009a) study in adult rats used by the M D H for the PFB S H R L, L ieder et al. (2009b) also published a study on the developm ental effects o f PFBS. Otherwise, no other peer-review ed published studies concerning PFBS toxicity appear to exist, although several unpublished reports are on the U.S. EPA public docket (York, 2003a,b). R egardless, the Feng et al. (2017) study noted reports o f effects o f PFO S and other PFCs on fetal growth, development, and adult reproductive function as well as thyroid hormone status and decided to investigate the linkages betw een PFBS exposure and these endpoints. The Feng et al. (2017) study dem onstrated that w hen given to pregnant IC R m ice from G D 1-20 at doses of 200 or 500 mg/kg/day, PFBS induced perm anent hypothyroid with deficits in perinatal growth, pubertal onset, and reproductive organ developm ent in fem ale mice. Thus, this particular publication adds to the body of evidence that short-chain compounds, such as PFBA and PFBS, pose a substantial potential hazard to hum an health, especially to susceptible developing organism s. PFBS and other short-chain compounds have been touted by industry and industry trade representatives as having "favorable toxicological profiles." For example, in a recent press release by the FluroCouncil, a Global Industry Council for FluoroTechnology, the language "im proved health and environm ental profiles" is used to compare the newer, "approved" chemistries to the "older" fluorinated chemistry (FluroCouncil, 2017a). In an earlier News Update, also by the FluroCouncil (FluroCouncil, 2017b), the following is provided: " .. .the current PF A S chem istries used in these applications, know n as short-chain fluorinated polymers, are well studied, and data from these studies have been provided to regulators globally as part of their chemical review processes. The science shows these new short-chain chemistries are not bioaccum ulative and offer significantly im proved toxicological profiles over the chem istries they replaced. Short-chain fluorinated polym ers have been approved for use by the EPA and other regulators around the world." I would like to dissect this paragraph with respect to short-chain compounds: 1) Short-chain com pounds are w ell studied and data from these studies have been provided to regulators globally as part of their chemical review processes. It m ay be true that industry scientists have performed copious studies w ith short-chain PFCs and provided the results of these studies to regulators across the globe. However, the reality is that if these industry studies are not part o f the publicly available peer-review ed literature, then they are likely lim ited to only those studies that are required for chemical review processes. These required studies do not evaluate all possible toxicological effects. For example, testing for developm ental im m unotoxicity is not a requirem ent under TSC A w ithin the U.S., but it is well accepted that the developing im m une system is highly susceptible to the disruptive effects o f chemical agents (Dietert and D eW itt, 2010). Therefore, the term "well studied" is 54 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN m isleading as it likely only refers to a lim ited num ber of required studies performed by industry scientists or research laboratories contracted by PFC-producing industries. This same situation occurred w ith the legacy PFCs, PFO A and PFOS. Studies evaluating the toxicity of PFO A and PFOS were perform ed for many years by industry scientists and their contractors or grantees. As described in section VII.5, above, 3M was conducting PFC toxicology studies by 1949 and as early as 1963, had sufficient inform ation on the basic toxicity of various fluorochemical surfactants to provide LD50s in adult rats and m ice in a technical bulletin along w ith a statement that "D ue care should be exercised in handling these m aterials until further inform ation is available on their physiological properties" (3M Company, 1963). A lthough one o f the earliest studies on the toxicity o f PFC s in experimental animal models was published in 1980 (Griffith and Long, 1980), published studies did not explode in num ber until the 2000s, m ore than tw o decades after these early studies. The growth in the num ber of studies occurred after the 3M Com pany announced that it was phasing out the perfluorooctanyl chemistry used to m ake PFOS and after PFOS and PFO A w ere reported as w idespread environm ental contam inants by various environm ental chemistry studies (for example Giesy and Kannan, 2001). W hile it is possible to speculate about the timeline, papers authored by 3M Com pany scientists on the toxicity of PFCs were published in the 2000s and beyond and w ere based, in some cases, on studies performed in the 1980s and 1990s. Therefore, the 3M Com pany had data about the toxicity of legacy compounds that may have been shared with regulators, but was not part of the publicly available peer-review ed literature that is vitally im portant for the advancem ent o f scientific know ledge and for regulators and policy m akers at the local and state level. The same m ay be true o f the short-chain chemistries. 2) These short-chain chemistries are not bioaccumulative. Bioaccum ulation is a term that describes the net uptake o f chem icals from the environm ent by any or all o f the possible routes and from any source w here the chem icals are present (Spacie et al., 1995). The presence o f a chemical in an organism alone is not sufficient to induce an adverse health outcome, but depends on the response of an organism to the presence of the chemical (Spacie et al., 1995). Therefore, judgm ents about adverse health outcom es m ust arise from w hat is know about dose-response relationships and the factors associated with toxicity rather than ju st the m agnitude o f the bioaccum ulation (Spacie et al., 1995). Short-chain PFC s enter living organism s from the environm ent via the sam e putative pathways as long-chain PFCs. The few publicly available studies on these short-chain PFCs, for exam ple PFB A and PFBS, indicate that these com pounds are excreted from hum ans in days rather than years (PFBA: 3 4 days; Chang et al., 2008. PFBS: ~25 days; O lsen et al., 2008). H ow ever, PFH xS, w hich has a shorter chain length than PFOS, is known to have a longer half-life than PFOS. Thus, it m ay not uniform ly be the case that a shorter chain length equates to a shorter half-life in serum. Therefore, while these short-chain PFCs do not rem ain in the body as long as the long-chain PFCs, because they are in the environment, they can, and do, bioaccumulate. In a study to evaluate the bioaccum ulation o f various P F C s from the environm ent, P erez et al. (2013) dem onstrated the presence o f 21 different PFC s o f varying chain lengths, including PFB A and PFBS, in five hum an tissues (bone, brain, kidney, liver, lung) collected from 20 subjects at autopsy in Catalonia, Spain. PFB A had the highest m ean concentration o f any PFC in the lung and the kidney and was approximately equivalent in concentration to PFO A in the liver (Perez et al., 2013). PFBS, although low relative to other PFCs, was detectable in all tissues but brain. A lthough this is a single study and did not evaluate potential differences 55 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN in exposure scenarios among subjects, it demonstrates that short-chain PFCs do indeed bioaccumulate. Further, to the extent that PFCs accum ulate in hum an tissue, m easuring the half-life of these compounds in serum may not fully account for the presence of PFCs in the hum an body and their elimination therefrom. 3) These new short-chain chemistries offer significantly im proved toxicological profiles over the chem istries they replaced. The phrase "significantly im proved toxicological profiles" is entirely unclear and subjective. Based on w hat toxicological m easures? D eath? Cancer endpoints? Organ dam age? By definition, toxicology is the study of the adverse health effects of exogenous agents on living organisms, therefore, the presence of a toxicological profile implies the presence of adverse health effects in living organisms. 4) Short-chain fluorinated polymers have been approved for use by the EPA and other regulators around the world. Approval for use does not imply safety. A pproved for use by regulatory agencies typically is associated w ith caveats such as use restrictions, waste capture requirem ents, lim its on em issions to environm ental media, etc. Approval for use in no way implies that the compounds in question are safe for hum an or wildlife exposure. Regarding this final point on approval for use, I note that according to R odricks and Levy (2013), who summ arized a 2009 National Research Council (NRC) advisory report on human health risk assessment (Science and Decisions: Advancing Risk Assessment), assum ptions that are not yet certain m ay be required in order to assess em erging problems. The authors assert that an implicit regulatory assumption that compoundsfo r which no significant toxicity data exist carry no risk is a troubling default assum ption fo r which solutions are critical. Therefore, the authors acknowledge that the toxicological comm unity m ust offer the best scientific support for m oving risk assessm ent forward, even where there is a degree of uncertainty. I agree w ith this assertion and further assert that while the scientific database on PFCs is far from complete, it w ould be unethical, particularly when there are proven toxicities, to move forward under the assum ption that there are no other toxicities beyond those that have been proven. It is better to assum e that sim ilar compounds are likely to show sim ilar toxicities, until that is disproven. Given the toxicities shown already in the lim ited studies concerning PFBA, PFBS, and other short-chain PFCs designed to replace the long-chain PFCs, given that they bioaccum ulate in hum an organs as shown by Perez et al., (2013), and given their sim ilar physicochem ical structure to better-studied PFCs such as PFO A and PFOS, it is my opinion that PFBA, PFHxS, and other short-chain PFCs pose a present or potential hazard to hum an health IX. Use o f Toxicology Studies to D evelop Protective H ealth G uidelines As described at various points in this report, toxicology studies are regularly utilized by regulators and governm ent agencies to develop protective hum an health guidelines for various chemicals, including PFCs. These values are developed through an evaluation of the body of established toxicological literature for a particular chemical at a particular point in time. As such, these protective health guidelines may not account for new or novel findings that post-date their establishment, and revisions in these health guidelines may lag behind the leading scientific research. This is likely to be especially true for chem icals like PFCs, for w hich the scientific literature is growing at a rapid pace. 56 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN W hile protective health guidelines are used to establish a level at which there is an acceptably low risk o f adverse health effects, they should not be understood to reflect a chemical concentration at w hich there is no risk. As the below description o f the developm ent o f PFC health-based levels by M innesota and the EPA illustrate, health guidelines considered to be adequately protective at one point in tim e may be revealed to be insufficiently protective as the scientific literature on a chemical advances. The M D H has developed and issued health risk limits (HRLs) for four PFCs commonly detectable in M innesota: PFO A (MDH, 2009a), PFOS (MDH, 2009c), PFB A (MDH, 2011a), and PFBS (M DH, 2011b). Based on its sim ilarity to PFOS and its long half-life in hum ans, they have also determined that the H R L for PFOS is appropriate for PFH sX (MDH, 2009b). These initial HRLs represented levels o f these chem icals in drinking w ater that the M D H then considered safe for people, including sensitive subpopulations, to consume over the course of a lifetime and were based on the available inform ation at the tim e of issuance. The HRL developed for PFO A and PFO S w as 0.3 pg/L and w as 7 pg/L for PFB A and PFB S. Concentrations in drinking w ater above the HRLs w ere believed to pose hum an health risks. The M D H HRL for PFO A (MDH, 2009a) was based on a RfD of 0.000077 mg/kg/day identified from a chronic non-cancer study o f C ynom ologus m onkeys published by B u ten h o ff et al. (2002). M onkeys w ere given oral doses o f APFO for six m onths and a LO A EL (although B utenhoff et al. refer to this as the low est observable effect level or L O E L ) o f 3 m g/kg/day w as identified based on increases in liver weight (no N O A EL w as identified). It is also notable that a m onkey dosed at 3 m g/kg/d becam e ill and w as hum anely sacrificed during the course o f the study, and a com pound-related cause o f death could not be definitively ruled out. U sing the BM D approach, the M D H identified a serum concentration o f 23 m g/L as the POD, w hich when adjusted to a HED, was 0.0023 m g/kg/day. The M D H applied a total uncertainty factor o f 30 based on an uncertainty factor o f three for interspecies extrapolation for potential differences in toxicodynam ics and 10 for intraspecies variability, w hich brought the R fD to 0.000077 m g/kg/day. The M D H applied standard m etrics for converting this to a drinking water consum ption rate to determ ine the 0.3 pg/L HRL. The M D H H RL for PFOS (MDH, 2009c) was based on a RfD o f 0.00008 m g/kg/day identified from a subchronic non-cancer study o f C ynom ologus m onkeys published by Seacat et al. (2002). M onkeys w ere given oral doses o f PFOS for 182 days and a N O A EL o f 0.15 m g/kg/day was identified even though, at the 0.15 m g/kg/d dose, decreases in serum cholesterol (as high density lipoprotein) and total triiodothyronine (T3), and increased thyroid stim ulating horm one (TSH) w ere observed. It is also notable that, at the next highest dose level, 0.75 m g/kg/d, tw o dosed m onkeys died o f com pound-related causes. Thus, in this study, there w as not a significant gap betw een the NO A EL and a level at which PFOS induced m ortality in monkeys. Using the BM D approach, the M D H identified a serum concentration o f 35 m g/L as the POD, which when adjusted to a HED, w as 0.0025 m g/kg/day. The M D H applied a total uncertainty factor o f 30 based on an uncertainty factor o f three for interspecies extrapolation for potential differences in toxicodynam ics and a 10 for intraspecies variability, w hich brought the R fD to 0.00008 m g/kg/day. The M D H applied standard m etrics for converting this to a drinking water consum ption rate to determ ine the 0.3 pg/L HRL. 57 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN The M D H H RL for PFB A (MDH, 2011a) was based on a RfD o f 0.0038 m g/kg/day identified from a subacute study o f SD rats perform ed for the 3M Com pany by N O TO X (2007a). Rats w ere given PFB A in drinking w ater for 28-days and using a BM D approach (provided by Dr. B utenhoff from the 3M Com pany to the M D H via email correspondence) 3.01 m g/kg/day was identified as the POD, w hich w hen adjusted to a HED, was 0.38 mg/kg/day. This value was based on the critical effect o f decreased cholesterol. The M D H applied a total uncertainty factor o f 100 based on an uncertainty factor o f three for interspecies toxicodynam ic differences, 10 for intraspecies variability, and three for database insufficiencies, w hich brought the RfD to 0.0.0038 mg/kg/day. The M D H applied standard m etrics for converting this to a drinking water consumption rate to determine the 7 gg/L HRL. The M DH calculated subchronic and chronic HRLs, also from N O TO X (2007b) studies o f rats given PFB A in drinking w ater for 90-days, w hich w ere 8 and 10 gg/L, respectively. A value 7 gg/L w as selected as the H R L for all exposure durations. The M D H H RL for PFBS (M DH, 2011b) was based on a RfD o f 0.0014 m g/kg/day identified from a chronic study o f SD rats perform ed by L ieder et al. (2009a). R ats w ere given PFB S via oral gavage for 90-days and a N O A EL o f 60 m g/kg/day w as identified for m ale rats based on decreased hemoglobin and hematocrit and histological changes in the kidney. Using the BM D approach, the M D H calculated a HED o f 0.42 m g/kg/day by dividing the N O A EL by a half-life adjustm ent factor o f 142 for extrapolation from m ale rats to humans. The M D H applied a total uncertainty factor o f 100 based on an uncertainty factor o f three for interspecies extrapolation for potential differences in toxicodynam ics, 10 for intraspecies variability, and three for database insufficiencies, which brought the RfD to 0.0042 mg/kg/day. The M D H applied standard m etrics for converting this to a drinking w ater consumption rate to determine the 7 gg/L HRL. The M DH calculated a subchronic HRL, also from L ieder et al. (2009a), w hich w as 9 gg/L. Similarly, the U.S. EPA established H A Ls for PFO A and PFOS o f 0.07 gg/L in 2016 (U.S. EPA, 2016a,b). R fD s for P F O A and PFO S w ere 0.00002 m g/kg/day based on the Lau et al. (2006) developm ental toxicity study for PFO A and a study o f the developm ental toxicity o f PFOS by L uebker et al. (2005). The PFO S R fD for the drinking w ater health advisory differs from the RfD used in the H ealth Effects D ocum ent for Perfluorooctane Sulfonate (U.S. EPA, 2016d), but only by 0.00001 m g/kg/day (0.00003 m g/kg/day com pared to 0.00002 m g/kg/day for the HAL). A ccording to the U.S. EPA, the goal o f the H A L was to protect the m ost sensitive populations, w hich they identified as fetuses during pregnancy and breastfed infants (U.S. EPA, 2016a,b). Even though the RfD values used by the M D H prior to 2017 and the U.S. EPA are similar (0.00008 m g/kg/day for the M D H and 0.00002 m g/kg/day for the U.S. EPA), the final values differ (0.3 gg/L for the M D H com pared to 0.07 gg/L for the U.S. EPA ) based on correction factors applied by the M D H to adjust m g to gg (i.e, a factor o f 1,000) and by the chronic intake rate (0.049 L/kg/day for the M D H and 0.054 L/kg/day for the U.S. EPA). In the spring of 2017, the M D H released HBVs for PFO A (MDH, 2017f), PFOS (MDH, 2017g), and PFB A (M DH, 2017e). According to the M D H (2017a), the release o f the 0.07 gg/L HA L for chronic exposure to PFO A and PFOS in drinking w ater by the U.S. EPA prom pted the M D H to reassess the M D H HALs issued in 2009. These updated guidance values apply to short periods of tim e during pregnancy and breastfeeding as well as over a lifetim e of exposure and are health recom m endations to local officials operating public w ater supplies and to private well owners in areas with PFCs in groundwater (MDH, 2017b). 58 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN The M D H used the same basic m ethodology as they previously used for establishment of HRLs, but used a different toxicokinetic model (with input from an external peer review panel) designed to protect developing organisms (MDH, 2017a). In the updated toxicokinetic model, additional exposure parameters w ere included for placental transfer, breastm ilk transfer, and breastm ilk intake, to take into account exposure scenarios for form ula-fed and breastfed infants (MDH, 2017a). One key difference betw een this m odel and m odels used by the U.S. EPA in the creation of their HAL was that the M D H used different volume of distribution (Vd) values for both PFOA and PFO S for infants that included an early-life stage Vd adjustm ent factor to account for the higher water content in bodies of infants com pared to older children and adults (MDH, 2017a). In addition, the M D H considered tw o scenarios for reasonable m axim um exposures (RM Es; m axim um exposure reasonably expected to occur): 1) an infant fed exclusively w ith form ula reconstituted with contam inated w ater starting at birth and then followed by lifetime consum ption o f contam inated w ater and 2) an infant exclusively breastfed for 12 m onths and then followed by lifetime consumption of contam inated w ater (MDH, 2017a). Another key difference is that the M D H used a relative source contribution (RSC; the percentage o f a person's exposure attributed to drinking w ater) o f 0.5 w hereas the U.S. E PA used a value o f 0.2. A value o f 0.2 indicates that 20% o f a person's exposure to a particular chem ical com es from drinking w ater (MDH, 2017a). The U.S. EPA Exposure D ecision Tree allows for one o f three values to be chosen, the default 20% value, a value of 50%, or a m aximum value of 80% (Krishnan and Carrier, 2013). The value o f 0.5 (or 50% ) used by the M D H is based on evidence from M D H 's biom onitoring studies (M DH, 2008; 2010) that dem onstrated that drinking w ater in the East M etro area is a m ajor source o f PFCs. M D H 's use o f an RSC o f 50% is less conservative than the E PA 's decision to use a default value of 20%. U sing the revised toxicokinetic model, the value derived for short-term, subchronic, and chronic exposures for PFO A was 0.035 pg/L and for PFO S was 0.027 pg/L. The PFO A H B V was based on a R fD o f 0.0053 m g/kg/day derived from the L au et al. (2006) developm ental toxicity study o f PFOA. The PFOS H B V was based on a RfD o f 0.0051 m g/kg/day derived from the Luebker et al. (2005) developm ental toxicity study o f PFO S. W hile the critical studies identified for both compounds for derivation of the HBVs were based on developmental endpoints, the M D H determined that these HBV s also w ould be protective for health effects in the liver, imm une system, and thyroid gland, and for PFOA, effects on the kidneys. The M D H determ ined that due to the long half-lives o f these compounds, a single H B V w as m ost appropriate for short-term, subchronic, and chronic exposures. The M D H retained the value of 7 pg/L for the PFBA HBV (MDH, 2017e). The M D H also lists PFHxS in the hum an health-based w ater guidance table, band utilizes that H B V for PFOS as a surrogate. Based on the lim ited toxicological evidence available regarding PFHxS, and its long half-life in hum an beings, there appear to be sufficient sim ilarities between PFH xS and PFO S to support M D H 's decision. In summ ary, the M D H H B V s for PFC s are: PFO A = 0.035 pg/L. PFO S = 0.027 pg/L. PFB A = 7 pg/L. PFBS (HRL) = 7 pg/L. PFH xS = 0.027 ug/L (based on the PFO S HBV). In Ex. 148 (3M N _M N00042206, Estim ation o f " Safe" Reference Level o f PFOS in Plasm a), Dr. John B utenhoff goes through a series o f calculations to estimate a reference level for PFOS in 59 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN hum an plasma, w hich w ould be com parable to a RfD. A breakdow n and assessm ent o f Dr. B u tenhoffs approach is found in A ppendix B. Dr. B utenhoff estim ated a RfD o f 0.000005 m g/kg/day, based on a LO A EL o f 0.5 m g/kg/day in a 90-day Rhesus m onkey study w ith PFOS. The HA L or H B V that could be calculated from this RfD is about 0.035 ug/L (see Appendix B), which is on the order o f m agnitude as the HA L calculated by the U.S. EPA and the HBV calculated by the M DH. Dr. B utenhoff m ade some calculations to estimate that the hum an plasm a concentration associated w ith this exposure level w ould be about 1.05 pg/L. I w ould like to point out here that this is lower than average hum an concentrations for the general U.S. population for PFOS and lower than the average hum an concentrations measured in the East M etro study in 2014, which were just under 20 ug/L (MDH, 2015). As early as the late 1990s, a toxicologist working for the 3M Company estimated that safe hum an serum concentrations for PFOS were lower than reported hum an serum concentrations in the general U.S. population at the time and at the present. In June o f 2017, the M D H inform ed the City o f Cottage Grove, M N, a city in M N w here the 3M Com pany operates a production facility, o f a N otice o f Health Risk A dvisory for Cottage Grove W ell N os. 2-8 and 10. A t this 3M C om pany facility, PFC production began in the late 1940s and w as phased out at the end o f 2002 (ATSDR, 2005), but PFC s are still detectable in w aters in and around this facility. The M D H based this advisory on com bined levels o f PFOA, PFOS, PFBA, PFBS, and PFHxS m easured in Cottage Grove Com m unity W ells. A ccording M innesota Adm inistrative Rules section 4717.7880, when a com bination of PFOA, PFOS, PFHxS, PFBA, or PFBS are found in drinking water, a health risk index (HRI) is calculated to determine if the combined health risk exceeds a level o f concern (M DH, 2017c). The H R I calculated by first creating a ratio that is the groundw ater concentration o f the chemical to the H B V for that chemical and then adding the ratios of the individual chemicals together (MDH, 2017c). An HRI that is greater than one is considered an exceedance o f the HBV. This is a typical approach for site-specific characterization o f risks (U.S. EPA, 1989). Based on this approach, the M D H determ ined th at P F C concentrations in eight out o f the 11 com m unity w ells in C ottage G rove contained combined levels of PFCs that exceeded the HBVs or HRLs. The M DH recom mended that the City o f Cottage Grove take action to reduce the levels of PFCs to below the H B V for PFO A and/or the H R I o f 1.0 (M D H, 2017c). A lso in June o f 2017, the M D H inform ed individual well owners/users that (a) samples from their w ells indicated exceedances o f HBVs or HRIs for PFCs, (b) well w ater should not be used for drinking or cooking on a long-term basis, and (c) bottled w ater and installation o f a granular activated carbon filter system (or connection to city water, if available) were available at no cost (M DH, 2017h). These were, in my opinion, reasonable actions to take to protect the public from exposure to PFCs above the HBVs and HRLs. X. Affirmation I affirm under penalty o f perjury that the foregoing is a true and correct statem ent of my opinions in this m atter and the grounds for those opinions. Jam ie C. DeW itt September 22, 2017 60 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Appendix A Curriculum Vitae (CV) o f Jam ie C. DeW itt Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN 61 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN ________________ Jamie C. DeWitt_______________ Contact Details: Department of Pharmacology & Toxicology Brody School of Medicine East Carolina University, 6S-10 Brody Building, 600 Moye Blvd., Greenville, NC 27834 Phone: 252-744-2474 Email: dewittj@ecu.edu Environmental toxicologist who employs principals from immunotoxicology, neurotoxicology, and developmental toxicology to understand how exposure to emerging environmental contaminants, such as per- and polyfluoroalkyl substances (PFASs), geogenic dusts, and pharmaceutical and personal care product pollutants, affect health and contribute to human diseases and disorders. EDUCATION________________________________________________ Ph.D., Environmental Science and Neural Science School of Public and Environmental Affairs and Program in Neural Science Indiana University, Bloomington, IN, 2004 Concentrations: Environmental and developmental neurotoxicology and risk assessment Dissertation title: Developmental intoxication of dioxins and polychlorinated biphenyls in an avian model: Correlations of brain asymmetry, behavior, and related developmental effects B.S., Environmental Science and Biology Lyman Briggs College Michigan State University, East Lansing, MI, 1992 PROFESSIONAL EXPERIENCE_________________________________ Associate Professor of Pharmacology and Toxicology Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, NC. July 2015-present. Adjunct Associate Professor of Public Health Department of Public Health, Brody School of Medicine, East Carolina University, Greenville, NC. July 2015-present. Adjunct Assistant Professor of Public Health Department of Public Health, Brody School of Medicine, East Carolina University, Greenville, NC July 2012-July 2014. Affiliated Member The Harriet and John Wooten Laboratory for Alzheimer's and Neurodegenerative Disease Research, East Carolina University, Greenville, NC. July 2011-present. 62 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Assistant Professor of Pharmacology and Toxicology Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, NC. July 2008-July 2015. Postdoctoral Trainee in Immunotoxicology University of North Carolina at Chapel Hill in cooperation with the U.S. Environmental Protection Agency (Training Agreement CT829472), National Health and Environmental Effects Research Laboratory, Experimental Toxicology Division, Immunotoxicology Branch, Research Triangle Park, NC (Advisor: Dr. Robert Luebke). Evaluation of immune function and exploration of immunotoxic mechanisms, including use of knock-out models and molecular techniques, of various xenobiotics (organotins and perfluoroalkyl acids) in rodent models. June 2004-June 2008. Postdoctoral Research Associate in Environmental and Ecotoxicology Developmental Neurobiology and Environmental Toxicology Laboratory, School of Public and Environmental Affairs, Indiana University, in cooperation with the U.S. Fish and Wildlife Service Bloomington Ecological Services Field Office, Bloomington, IN (Advisors: Dr. Diane Henshel and Daniel Sparks). Cardiotoxic effects in wild passerine birds developmentally exposed to polychlorinated biphenyls. September 2003-May 2004. Research Assistant in Environmental and Ecotoxicology Developmental Neurobiology and Environmental Toxicology Laboratory, School of Public and Environmental Affairs, Indiana University, Bloomington, IN (Advisor: Dr. Diane Henshel). Toxicological effects of dioxin and polychlorinated biphenyls after developmental exposure in an avian model, wild birds, and wild fish. August 1995-August 2003. Field Assistant in Limnology Lake Lemon Conservancy District, Unionville, IN. Canada goose control, littoral zone revegetation, and monitoring of native and exotic aquatic plant populations. June 2000-May 2003. Field Assistant in Ecotoxicology U.S. Fish and Wildlife Service-Bloomington Ecological Services Field Office, Bloomington, IN. Wild bird and macroinvertebrate population monitoring in a metal-contaminated lake, including assessment of fertile eggs for embryonic abnormalities. April 1997-October 1997. Research Associate in Entomology Landscape Entomology Division, Department of Entomology, Michigan State University, East Lansing, MI. Research and efficacy tests for forest, ornamental and turf entomological studies for the Michigan Department of Agriculture and Turf Foundation. September 1992-August 1995. Research Assistant in Entomology Medical Entomology Division, Department of Entomology, Michigan State University, East Lansing, MI. Assessment of Lyme disease prevalence in deer and dog ticks collected from a Lyme disease endemic area in Michigan. May 1992-May 1993. 63 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN PUBLICATIONS Peer-Reviewed Manuscripts vonderEmbse AN, Hu Q, and DeWitt JC. 2017. Developmental toxicant exposure in a mouse model o f Alzheim er's disease induces differential sex-associated microglial activation and increased susceptibility to amyloid accumulation. Journal o f Developmental Origins o fHealth and Disease. 2:1-9. Meadows JR, Parker C, Gilbert KM, Blossom SJ, and DeWitt JC. 2017. A single dose of trichloroethylene given during development does not substantially alter markers of neuroinflammation in brains o f adult mice. Journal o fImmunotoxicology. 14:95-109. DeWitt JC, Buck BJ, Goossens D, Teng Y, Pollard J, McLaurin B, Gerads R, and DE Keil. 2017. Health effects following subacute exposure to geogenic dust collected from active drainage surfaces (Nellis Dunes Recreation Area, Las Vegas, NV). Toxicology Reports. 4:19-31. Rushing BR, Hu Q, Franklin JN, McMahen R, Dagnino S, Higgins CP, Strynar MJ, and DeWitt JC. 2017. Evaluation of the immunomodulatory effects of 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoate in C57BL/6 mice. Toxicological Sciences. Epub ahead o f print: doi: 10.1093/toxsci/kfw251. Keil DE, Buck B, Goossens D, Teng Y, Pollard J, McLaurin B, Gerads R, DeWitt JC. 2016. Health effects from exposure to atmospheric mineral dust near Las Vegas, NV, USA. Toxicology Reports. 3:785-795. Jusko TA, Oktapoda M, Murinova LP, Babjakova J, Verner M-A, DeWitt JC, Babinska, ThevenetMorrison K, Conka K, Drobna B, Thurston SW, Lawrence BP, Dozier AM, Jarvinene-Seppo KM, Patayova H, Trnovec T, Legler J, Hertz-Picciotto I, and Lamoree MH. 2016. Demographic, reproductive, and dietary determinants of perfluorooctane sulfonic (PFOS) and perfluooctanoic acid (PFOA) concentrations in human colostrum. Environmental Science and Technology. 50:7152-7162. DeWitt JC, Buck B, Goossens D, Hu Q, Chow R, David W , Young S, Teng Y, Leetham-Spencer M, Murphy L, Pollard J, McLaurin B, Gerads R, and Keil D. 2016. Health effects following subacute exposure to dusts from arsenic-rich sediment at the Nellis Dunes Recreation Area, Las Vegas, NV. Toxicology and Applied Pharmacology. 304:79-89. Leetham M, DeWitt J, Buck B, Goossens D, Teng Y, Pollard J, McLaurin B, Gerads R, and Keil D. 2016. Oxidative stress and lung pathology following geogenic dust exposure. Journal o fApplied Toxicology. 36:1276-1283. Keil D, Buck B, Goossens D, Teng Y, Spencer M, Murphy L, Pollard J, Eggers M, Gerards R, and DeWitt J. 2016. Immunotoxicological and neurotoxicological profile o f health effects following subacute exposure to geogenic dust from sand dunes at the Nellis Dunes Recreation Area, Las Vegas, NV. Toxicology and Applied Pharmacology, 291:1-12. DeWitt JC, Williams W, Creech NJ, and RW Luebke. 2016. Suppression o f antigen-specific antibody responses in mice exposed to perfluorooctanoic acid: Role o f PPA Ra and B cell targeting. Journal o f Immunotoxicology. 13:38-45. 64 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Jiang Q, Ma W , W u J, W ingard CJ, and DeWitt JC. 2016. Perfluorooctanoic acid-induced toxicity in primary cultures of chicken embryo cardiomyocytes. Environmental Toxicology. 31:1580-1590. Khalil N, Chen A, Lee M, Czerwinski SA, Ebert JR, DeWitt JC, and Kannan K. 2016. Association of perfluoroalkyl substances, bone mineral density, and osteoporosis in the US population in NHANES 2009-2010. Environmental Health Perspectives. 124:81-87. W ambaugh JF, Setzer W, Pitruzzello AM, Liu J, Reif D, Kleinstreuer N, Ching N, W ang Y, Sipes N, Martin M, Das K, DeWitt J, Strynar M, Judson R, Houck K, and Lau C. 2013. Dosimetric anchoring of in vivo and in vitro studies for perfluorooctanoate and perfluorooctane sulfonate. Toxicological Sciences. 136:308-327. Jiang Q, Lust R, and DeWitt JC. 2013. Perflurooctanoic acid induced-developmental cardiotoxicity: Are peroxisome proliferator activated receptor a (PPARa) and bone morphorgenic protein 2 (BMP2) pathways involved? Journal o f Toxicology and Environmental Health Part A. 76:635 650. Hu Q, Franklin JN, Bryan I, Morris E, W ood A, and DeWitt JC. 2012. Does developmental exposure to perflurooctanoic acid (PFOA) induce immunopathologies commonly observed in neurodevelopmental disorders? NeuroToxicology. 33:1491-1498. Jiang Q, Lust R, Strynar MJ, and DeWitt JC. 2012. Perflurooctanoic acid induces developmental cardiotoxicity in chicken embryos and hatchlings. Toxicology. 293:97-106. Fair PA, Stavros H-C, Mollenhauer MAM, DeWitt JC, Henry N, Kannan K, Mitchum G, Y SH, Bossart GD, Keil DE, and Peden-Adams MM. 2012. Immune function in female B6C3F1 mice is modulated by DE-71, a commercial polybrominated diphenyl ether mixture. Journal o f Immunotoxicology. 9:96-107. Hu Q, Strynar MJ, and DeWitt JC. 2010. Are developmentally exposed C57BL/6 mice insensitive to suppression o f TDAR by PFOA? Journal o fImmunotoxicology. 7:344-349. DeWitt JC, Copeland CB, and Luebke RW. 2009. Suppression o f humoral immunity by perfluorooctanoic acid is independent o f elevated serum corticosterone concentration in mice. Toxicological Sciences. 109:106-112. Peden-Adams MM, Stuckey JE, Gaworecki K, Berger-Ritchie J, Bryant K, Jodice PG, Scott TR, Boone S, McGuinn WD, DeWitt JC, and Keil DE. 2009. Developmental toxicity in white leghorn chickens following in ovo exposure to perfluorooctane sulfonate (PFOS). Reproductive Toxicology. 27:307-318. W halen MM, DeWitt JC, Luebke RW. 2008. Serum supplementation modulates the effects o f dibutyltin on human natural killer cell function. Toxicological Sciences. 104:312-319. DeWitt JC, Copeland CB, Strynar MJ, and Luebke RW. 2008. Perflurooctanoic acid-induced immunomodulation in adult C57BL/6J or C57BL/6N female mice. Environmental Health Perspectives. 116:644-650. DeWitt JC, Copeland CB, and Luebke RW. 2008. An organotin mixture found in polyvinyl chloride (PVC) pipe is not immunotoxic to adult Sprague-Dawley rats. Journal o f Toxicology and Environmental Health Part A. 71:276-282. 65 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN DeWitt JC, Copeland CB, and Luebke RW. 2007. Immune function is not impaired in SpragueDawley rats exposed to dimethyltin dichloride (DMTC) during development or adulthood. Toxicology. 232:303-310. Lim J, DeWitt JC, Sanders RA, Watkins JB III, and Henshel DS. 2007. Suppression o f endogenous anti-oxidant enzymes by 2,3,7,8-tetrachlorodibenzo-p-dioxin-Induced oxidative stress in chicken liver during development. Archives o fEnvironmental Contamination and Toxicology. 52:590-595. DeWitt JC, Copeland CB, and Luebke RW. 2006. Developmental exposure to 1.0 or 2.5 mg/kg o f dibutyltin dichloride does not impair immune function in Sprague-Dawley rats. Journal o f Immunotoxicology. 3:245-252. DeWitt JC, Millsap DS, Yeager RL, Heise SS, Sparks DW, and Henshel, DS. 2006. External heart deformities in passerine birds exposed to environmental mixtures of polychlorinated biphenyls during development. Environmental Toxicology and Chemistry. 25:541-551. DeWitt JC, Copeland CB, and Luebke RW. 2005. Immune responses in Sprague-Dawley rats exposed to dibutyltin dichloride in drinking water as adults. Journal o fImmunotoxicology. 2:151-160. DeWitt JC, Meyer EB, and Henshel DS. 2005. Environmental toxicity studies using chickens as surrogates for wildlife: Effects of vehicle volume. Archives o f Environmental Contamination and Toxicology. 48:260-269. DeWitt JC, Meyer EB, Watkins JB, and Henshel DS. 2005. Environmental toxicity studies using chickens as surrogates for wildlife: Effects o f day of injection. Archives o fEnvironmental Contamination and Toxicology. 48:270-277. Stanton B, DeWitt J, Henshel D, Watkins S, and Lasley B. 2003. Fatty acid metabolism in neonatal chickens (Gallus domesticus) treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3,3',4,4',5-pentachlorobiphenyl (PCB-126) in ovo. Comparative Biochemistry and Physiology C - Pharmacology and Toxicology, 136(1):73-84. Henshel DS, DeWitt JC, and Troutman, A. 2002. Using chicken embryos for teratology studies. In: Current Protocols in Toxicology (M.D. Maines, L.G. Costa, E. Hodgson, D.J. Reed, and I.G. Sipes, Eds.), Supplement 14, pp. 13.4.1-13.4.19. Henshel DS, Martin JW and DeWitt JC. 1997. Brain asymmetry as a potential biomarker for developmental TCDD intoxication: A dose-response study. Environmental Health Perspectives. 105:718-725. Henshel DS, Martin JW, Norstrom RJ, Elliot J, Cheng KM and DeWitt JC. 1997. Morphometric brain abnormalities in double-crested cormorant chicks exposed to polychlorinated dibenzo-p dioxins, dibenzofurans, and biphenyls. Journal o f Great Lakes Research. 23:11-26. W alker ED, Smith TW, DeWitt J, Beaudo DC, McLean RG. 1994. Prevalence o f Borrelia burgdorferi in host-seeking ticks (Acari: Ixodidae) from a Lyme disease endemic area in northern Michigan. Journal o fM edical Entomology. 31(4):524-8. 66 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Review Articles Wang Z, DeWitt JC, Higgins CP, and Cousins IT. 2017. A never-ending story o f per- and polyfluoroalkyl substances (PFASs)? Environmental Science & Technology. 51:2508-2518. Hessel EVS, Ezendam J, van Broekhuizen FA, Hakkert B, DeWitt JC, Granum B, Guzylack L, Lawrence BP, Penninks A, Rooney AA, Piersma AH, and van Loveren H. 2016. Assessment of recent developmental immunotoxicity studies with bisphenol A in the context of the 2015 EFSA t-TDI. Reproductive Toxicology. 65:448-456. Corsini E, Luebke RW, Germolec DR, and DeWitt JC. 2014. Perfluorinated compounds: emerging POPs with potential immunotoxicity. Toxicology Letters, 230:263-270. DeWitt J, Peden-Adams M, Keller J, and Germolec D. 2012. The immunotoxicity o f perfluorinated compounds: Recent developments. Toxicologic Pathology, 40:300-311. DeWitt J, Peden-Adams M, Keil D, and Dietert R. 2012. Current status o f developmental immunotoxicity: Early-life patterns and testing. Toxicologic Pathology, 40:230-236. Dietert RR, Dietert J, and DeWitt JC. 2011. Environmental risk factors for autism. Emerging Health Threats Journal (invited review). 4:7111. Dietert RR, DeWitt JC, Germolec DR, and Zelikoff JT. 2010. Breaking patterns o f environmentally influenced disease for health risk reduction: Immune perspectives. Environmental Health Perspectives 118:1091-1099. DeWitt JC, Shnyra A, Badr MZ, Loveless SE, Hoban D, Frame SR, Cunard R, Anderson SE, Meade BJ, Peden-Adams MM, Luebke RW, and Luster MI. 2009. Immunotoxicity o f perfluorooctanoic acid and perfluorooctane sulfonate and the role of peroxisome proliferator activated receptor alpha. Critical Reviews in Toxicology 39:76-94. Edited Books DeWitt JC (ed). 2015. Toxicity ofPerfluoroalkyl and Polyfluoroalkyl Substances. Springer Science + Business Media, LLC (Invited). Book Chapters DeWitt JC and Keil DE. 2017. Current issues in developmental immunotoxicity. In: Immunopathology in Toxicology and Drug Development (Parker GA, ed). Springer International Publishing, Switzerland. DeWitt JC, Germolec DR, Luebke RW, and Johnson, VJ. 2016. Associating changes in the immune system with clinical diseases for interpretation o f risk assessment. In: Current Protocols in Toxicology. 67:18.1.1-18.1.22. DeWitt JC, Peden-Adams MM, and Keil DE. 2015. Immunotoxic effects o f perfluoroalkylated compounds: Mechanisms o f action. In: M olecular Immunotoxicology (Corsini E and van Loveren H, eds). Wiley-VCH GmbH & Co., Weinheim. DeWitt JC and Dietert RR. 2014. Immunotoxicity in autism spectrum disorders. In: The Comprehensive Guide to Autism (Patel VB, Martin CR, Preedy V, and Preedy VR, eds). Springer Reference, New York, NY. 67 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Dietert RR, DeWitt JC, and Luebke RW. 2012. Reducing the prevalence o f immune-based chronic disease. In: Immunotoxicity, Immune Dysfunction, and Chronic Diseases (Dietert RR and Luebke RW, eds), Molecular and Integrative Toxicology, Springer Science + Business Media, LLC. pp 419-440. DeWitt J, Peden-Adams M, Keil D, and Dietert R. 2012. Developmental immunotoxicity (DIT): Assays for evaluating effects o f exogenous agents on development o f the immune system. In: Current Protocols in Toxicology. Chapter 18: Unit 18.15. Luebke RW, DeWitt JC, Germolec DR, Salazar KD, and Kerkvliet NI. 2012. Immunomodulation by persistent organic pollutants. In: Dioxins and Health, Including Other Persistent Organic Pollutants and Endocrine Disruptors, 3ndEdition (Schecter A, ed), John Wiley and Sons, Inc., Hoboken, NJ. pp 171-192. DeWitt JC and Dietert RR. Postnatal immune dysfunction and its impact on growth parameters. 2012. In: Handbook o f Growth and Growth Monitoring in Health and Disease (Preedy VR, ed), Springer, New York, NY. pp 741-755. DeWitt JC and Luebke RW. 2010. Immunological Aging. In: Comprehensive Toxicology, 2nd Edition, Volume 5 (Lawrence D, ed), Elsevier Limited, Oxford, UK. pp 455-465. Dietert RR and DeWitt J. 2010. Developmental immunotoxicity (DIT): The why, where and how of DIT testing. In: Immunotoxicity Testing: M ethods and Protocols (Dietert RR, ed), Methods in Molecular Biology. Humana Press, Inc., Totowa, NJ. 598:17-25. Luebke RW, Beamer CA, Bowman C, DeWitt JC, Gowdy K, Johnson VJ, Shepherd DM, and Germolec DR. 2009. Immunotoxicology (developmental immunotoxicology section). In: General and Applied Toxicology, 3rd Edition (Marrs T, Ballantyne B, Syversen T, eds.), John W iley & Sons, Ltd., Chichester, UK, pp 1561-1583. Other Scholarly Contributions Portier CJ et al. (90+ co-authors). 2016. Differences in the carcinogenic evaluation o f glyphosate between the International Agency for Research on Cancer (IARC) and the European Food Safety Authority (EFSA). Journal o fEpidemiology and Community Health. 70:741-745. DeWitt JC and Luebke RW. 2014. Immunological Aging. Online Reference Database Biomedical Science. Benbrahim-Tallaa L, Lauby-Secretan B, Loomis D, Guyton KZ, Grosse Y, El Ghissassi F, Bouvard V, Guha N, Mattock H, and Straif K on behalfo f the International Agencyfo r Research on Cancer Monograph Working Group (DeWitt JC, Mechanisms Subgroup Member). 2014. Carcinogenicity o f perfluorooctanoic acid, tetrafluoroethylene, dichloromethane, 1,2dichloropropane, and 1,3-propane sultone. The Lancet Oncology. 15:924-925. IARC. 2014 Perfluoro-octanoic acid, Tetrafluoroethylene, Dichloromethane, 1,2-Dichloropropane, and 1,3-Propane sultone. IA RC M onogr Eval Carcinog Risks Hum (DeWitt JC, Mechanisms Subgroup Member). Monograph 110. DeWitt JC and Dietert RR. 2011. Response to "Theoretical aspects o f autism: Causes - a review" by Ratajczak, HV (Journal o fImmunotoxicology 8:68-79, 2011). Journal o fImmunotoxicology. 8:195-197. 68 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Non-Refereed Articles "Toxicant induced brain asymmetry: More than just a bird-brained scheme?" Learned Discourses, SETAC Globe, Jan/Feb 2001 (invited). RESEARCH FUNDING Brody Brothers Endowment Foundation 1year Immunomodulatory Effects o fAqueous Film Forming Foam (AFFF): An Effective Fire Suppressant or a Persistent Environmental Contaminant with Unknown Health Consequences? Role on project: Principal Investigator Status: Award notification November 2016. Direct costs: $20,000 Center for Human Health and Environment at NCSU Pilot Project Program Discovery o f Biomarkers o f Effectfollowing Environmentally-Relevant Exposure to Pharmaceutical Pollutants Role on project: Co-Principal Investigator Status: Award notification August 2015. Direct costs: $25,000 1year Brody School of Medicine Internal Seed/Bridge Grant Program 1year Post-translational Modifications to Potassium Channels in Alzheim er's Disease: Triggers o f Onset and Progression? Role on project: Co-Principal Investigator Status: Award notification August 2015. Direct costs: $25,000. The Harriet and John Wooten Laboratory for Alzheimer's and 1year Neurodegenerative Disease Research Microglia as a Target o f Environment x Gene Interactions Part II: Digging into the Biochemistry o fA lzheim er's Disease Role on project: Principal Investigator Status: Award notification November 2014. Direct costs: $12,000 Interdisciplinary Research Collaboration Award (East Carolina University) Pharmaceutical and Personal Care Product Contaminants in Fresh Water Role on Project: Corresponding Faculty P I Status: Award notification August 2014. Direct costs: $23,000 6 months Alzheimer's North Carolina A Multidisciplinary Approach to Fight Senior Dementia Role on Project: Co-Investigator Status: Award notification February 2014. Direct costs: $50,000 1year East-West Research Collaboration Award (East Carolina University) Pharmaceutical and Personal Care Product Contaminants in Fresh Water Role on Project: Corresponding Faculty P I Status: Award notification December 2013. Direct costs: $23,000 6 months The Harriet and John Wooten Laboratory for Alzheimer's and 1 year Neurodegenerative Disease Research M icroglia as a Target o f Environment x Gene Interactions: Exacerbation o fA lzheim er's Pathology by Early-life Exposure to Lead Role on project: Principal Investigator 69 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Status: Award notification April 2013. Direct costs: $12,000 Bureau of Land Management Nellis Dunes Recreation Area Dust Exposure and Human Health Risk Assessment Role on project: Co-Principal Investigator Status: Award notification March 2011. Direct costs: $105,699 3 years Department of Defense 1 year Immunopathogenesis in autism: Regulatory T cells and autoimmunity in neurodevelopment Role on project: Principal Investigator Status: Award notification December 2009. Direct costs: $75,000. School of Public and Environmental Affairs, Indiana University. Ph.D. Student Travel Award and Graduate Student Organization Travel Award. 2001. Funded amount: $500.00. Ohio Valley Chapter of the Society of Environmental Toxicology and Chemistry. Student Travel Grant. 1997, 1998, 2001. Funded amounts: $300.00 each year. EDITORIAL BOARDS/AD HOC MANUSCRIPT REVIEWER Editorial Board Member, Environmental Health Perspectives. 2017- Series co-Editor (with Sarah Blossom), M olecular and Integrative Toxicology. 2016- Associate Editor, Toxicology and Applied Pharmacology. 2016- Editorial Board Member, Journal o f Toxicology and Environmental Health Part A. 2013- Editorial Board Member, Journal o f Immunotoxicology. 2010- A d Hoc Reviewer: Advances in Physiology Education Archives of Environmental Contamination & Archives of Toxicology Toxicology Chemosphere Chemical Research in Toxicology Drug and Chemical Toxicology Environmental Health Perspectives Environment International Environmental Pollution Environmental Research Environmental Science & Pollution Research Environmental Science & Technology Epidemiology Food and Chemical Toxicology GENE Human & Experimental Toxicology International Aquatic Research International Immunopharmacology Journal of Environmental Immunology & International Journal of Tropical Biology Toxicology Journal of Immunotoxicology Journal of Toxicology & Environmental Health NeuroToxicology Pharmacological Research PLoS One Regulatory Toxicology & Pharmacology Reproductive Toxicology Science of the Total Environment Southeastern Naturalist Toxicology & Applied Pharmacology Toxicology Letters Toxicology Reports Toxicological Sciences GRANT REVIEWER Department of Defense Congressionally Directed Medical Research Programs, 2013-2016 Graduate Women in Science Graduate Fellowships, 2011, 2014-2017 CDC-NIOSH, 2010, 2013, 2016 EXTERNAL REVIEWER ATSDR, 2017 (manuscript and Toxicological Profile) 70 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN New York State Department of Public Health, 2017 (Cancer incidence investigation: Village of Hoosick Falls, Rensselaer County, New York) ORAL PRESENTATIONS (Invited) "Not Seeing the Forest for the Trees: How the Complexity ofthe Immune System Challenges Policies for Environmental Health Protection." Department o fPathobiology Annual Retreat, Brown University, Providence, RI. 2017. "The Science behind GenX." Water Wednesday, Clean Cape Fear, Wilmington, NC. 2017. "Is it Possible to Untangle Underlying Developmental Susceptibilities from Exogenous Triggers in ASD?" Autism Think Tank, Autism Research Institute, Dallas-Fort Worth, TX. 2017. "Urgent Research Needs for Better Understanding the Toxicity of PFASs." Northeast Superfund Research Program Meeting, Northeastern University, Boston, MA. 2017. "Emerging Toxicological Knowledge and Data Gaps for "Novel" PFASs." Public Workshop on Perfluoroalkyl and Polyfluoroalkyl Substances (PFASs) in Carpets, Rugs, Indoor Upholstered Furniture, and Their Care and Treatment Products, Safer Consumer Products Program, Department of Toxic Substances Control, California Environmental Protection Agency, Sacramento, CA. 2017. "Pharmaceuticals and Personal Care Products as Emerging Pollutants in Coastal Waters (with Dr. Siddhartha Mitra). Science on the Sound Symposium, Coastal Studies Institute, Wanchese, NC. 2017. "Emerging Aquatic Contaminants and Health: Finding Solutions with Transdisciplinary Teams." Coastal Health Initiative, East Carolina University, Greenville, NC. 2016. "Water Pollution: Is seeing believing?" Love a Sea Turtle Second Annual Environmental Symposium, River Park North, Greenville, NC. 2016. "Developmental Immunotoxicology."Middle Atlantic Reproduction and Teratology Association, Covance Research Products, Inc., Denver, PA. 2015. "A Little Bit ofthis and a Little Bit ofthat...How do we Understand Risks of Agents injust a Drop of Water?" Love a Sea Turtle First Annual Environmental Symposium, River Park North, Greenville, NC. 2015. "Updates on Alzheimer's Disease Research in the DeWitt Lab at East Carolina University" (with Annalise vonderEmbse). Senior Services Community Health Program, Vidant Medical Center, Greenville, NC. 2015. "Immunomodulatory Effects of Perfluoroalkyl Substances in Rodents and Humans." Immunotoxicology in Food and Ingredient Safety Assessment: Approaches and Case Studies, SO T FDA Colloquia on Emerging Toxicological Science Challenges in Food and Ingredient Safety, Washington, DC. 2015. "Updates on Alzheimer's Disease Research in the DeWitt Lab at East Carolina University" (with Annalise vonderEmbse). Alzheim er's Professional Partnership-Greenville, Greenville, NC. 2015. "From Sink to Sea: Evaluating Health Impacts of Pills and Perfumes after we Wash them away" (with Krista MCoy). FaculTea Seminar, East Carolina University, Greenville NC. 2014. 71 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN "Better Living through Chemistry: A Tale of Two Toxicants." Department o f Chemistry Seminar, East Carolina University, Greenville, NC. 2014. "The Nuts and Bolts of Interdisciplinary Toxicological Research" (with Christie Sayes). Western Carolina University Department o f Biology Spring Seminar Series. Cullowhee, NC. 2014. "Alzheimer's Disease and Neurodegenerative Disorders Research at East Carolina University" (with Annalise vonderEmbse). A lzheim er's Professional Partnership-Goldsboro, Goldsboro, NC. 2015. "Endocrine Disruption o f the Neuro-immune Interface." The Collaborative on Health and the Environment Partnership Call (Teleseminar). 2014 "Contaminated Drinking Water: A Case Study o f Perfluorinated Compounds." Coastal Water Resources Center, East Carolina University, Greenville, NC. 2013. "Villains and Heroes in the Battle for Clean W ater" (with Siddhartha Mitra and Anthony Cannon). STEM at Starlight, Greenville, NC. 2013. "The Nuts and Bolts o f Alzheimer's Disease Research at East Carolina University." Senior Services Community Health Program, Vidant Medical Center, Greenville, NC. 2013. "Alzheimer's Disease and Neurodegenerative Disorders Research at East Carolina University." A lzheim er's Professional Partnership-Greenville, Greenville, NC. 2013. "A Neuroimmune Investigation o f an Endocrine-Disrupting Compound" . Department o f Biology Seminar, East Carolina University, Greenville, NC. 2013. "A Neuroimmune Investigation o f an Endocrine-Disrupting Compund: How Bisphenol A may Disrupt Learning and Memory through Immunomodulation." Endocrine Disrupting Chemicals Forum, Research Triangle Park, NC. 2013. "Undecafluoro-2-methyl-3-oxahexanoic Acid Versus Perfluorooctanoic Acid: Is Polyfluorination a Less Immunotoxic Option than Perfluorination?" Department o fEnvironmental and Molecular Toxicology Seminar, North Carolina State University, Raleigh, NC. 2013. "Early Life Triggers o f Developmental Immunotoxicity." Society fo r Toxicologic Pathology, Annual Meeting, Denver, CO. 2011. "Is the Pathway to Autism Paved with Environmental Chemicals?" Department o f Comparative Medicine seminar. East Carolina University, Greenville, NC. 2011. "PPAR Involvement in PFAA Immunotoxicity." U.S. EPA PFAA Days III Workshop, U.S. Environmental Protection Agency, Research Triangle Park, NC. 2010. "Are Environmental Contaminants (Developmental) Immunotoxicants? A Case Study o f a Fluorinated Compound." Department o fMicrobiology and Immunology Seminar, East Carolina University, Greenville, NC. 2010. "Developmental Immunotoxicity o f PFOA, an Emerging Contaminant." Department o f Biology Seminar, East Carolina University, Greenville, NC. 2009. 72 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN "The Immunotoxicity o f Perfluorooctanoic Acid (PFOA)." Department o fPhysiology Seminar, Brody School of Medicine, East Carolina University, Greenville, NC. 2008. "Immunotoxic Potentials o f PFOA." U.S. EPA PFAA Days II Workshop, U.S. Environmental Protection Agency, Research Triangle Park, NC. 2008. "Chasing Down the Mechanism of Perfluorooctanoic Acid-Induced Immunomodulation: Knock-outs and Adrenalectomies." National Health and Environmental Effects Research Laboratory Work in Progress, U.S. Environmental Protection Agency, Research Triangle Park, NC. 2007. "Wildlife Immunotoxicology." Immunotoxicology course. College o f Veterinary Medicine, North Carolina State University. 2006. "Immunotoxicity o f Individual Organotin Compounds in Sprague-Dawley Rats." Societyfo r Risk Analysis 25thAnnual Meeting, Orlando, FL. 2005. "Immune Function in Rats Exposed to Organotins as Adults or During Development." National Health and Environmental Effects Research Laboratory Work in Progress, U.S. Environmental Protection Agency, Research Triangle Park, NC. 2005. "Brain Asymmetry in Domestic Hatchling Chickens Developmentally Exposed to TCDD: A Histological Examination." Society o f Environmental Toxicology and Chemistry 24th Annual Meeting, Austin, TX. 2003. "Service Learning and Scientific Research." Indiana University Community Outreach and Partnerships in Service-Learning Workshop, Indiana University, Bloomington, IN. 2004. "Toxic Effects of Mercury." Clean Air Indiana Speak out on the Clear Skies Initiative, Indiana University, Bloomington, IN. 2003. "Environmental Health Concerns for Toxics in Indiana Superfund Sites." Indiana Public Interest Research Group (INPIRG) Teach-In on Indiana Superfund Issues, Indiana University, Bloomington, IN. 2002. "Introduction to Environmental Toxicology." Techniques in Environmental Science and Environmental and People courses. School o f Public and Environmental Affairs, Indiana UniversityBloomington. 2000, 2001, 2003. "Bioaccumulation and Biomagnification of Environmental Chemicals in Colonial Fish-eating Waterbirds." Introduction to Environmental Sciences course. School of Public and Environmental Affairs, Indiana University-Bloomington. 2000 and 2001. "Women in the Sciences: Abolishing Gender Apartheid." IU Skills for Leadership Conference, Office o f W om en's Affairs, Indiana University, Bloomington, IN. 1999. ORAL PRESENTATIONS "Perspectives from the AAMC Mid-Career Women Faculty Professional Development Seminar "MIDWIMS"). Brody Women Faculty Committee. Greenville, NC. 2017. 73 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN "Immunopathogenesis in Autism: Regulatory T Cells and Markers of Autoimmunity in Mice Developmentally Exposed to Perflurooctanoic Acid (PFOA). 2 7 hAnnual NeuroToxicology Conference, Annual Meeting, Durham, NC. 2011. "PFOA-induced Immunomodulation in mice: An Overview." Society o f Toxicology 48th Annual Meeting, Baltimore, MD. 2009. "Pathways o f PFOA-mediated Immunosuppression." Society o f Toxicology 48th Annual Meeting, Baltimore, MD. 2009. "Dose-response o f Perfluorooctanoic Acid-Induced Immunomodulation in Adult C57BL/6 Mice." Society o f Toxicology 46th Annual Meeting, Charlotte, NC. 2007. "Immune Function in Rats Developmentally Exposed to Dibutyltin Dichloride." Society o f Toxicology 45th Annual Meeting, San Diego, CA. 2006. "Neurotoxic Effects in Avian Species: Implications for Human and Ecological Health." School of Public and Environmental Affairs 2ndAnnual Young Researchers Conference, Indiana University, Bloomington, IN. 2002. "TCDD-Induced Brain Asymmetry and Behavior: What do Individual Chicks Have to Say?" Ohio Valley Chapter o f the Society o f Environmental Toxicology and Chemistry, Hueston Woods State Park, College Corner, OH. 2000. "Behavioral and Morphological Changes in Domestic Chicks Exposed to TCDD or PCB-126 at Embryonic Day 0 or Embryonic Day 4." Ohio Valley Chapter o f the Society o f Environmental Toxicology and Chemistry, Indiana University, Bloomington, IN. 1997. "Behavioral Changes in Domestic Hatchling Chicks Exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in ovo." Conference on Chlorine in the Environment, Massachusetts Institute of Technology, Boston, MA. 1996. "Behavioral Assessment o f Hatchling Chicks Exposed to TCDD in ovo: Preliminary Results." Great Lakes Bioeffects Workgroup. W right State University, Dayton, OH and Ohio Valley Chapter o f the Society o fEnvironmental Toxicology and Chemistry, Eastern Kentucky University, Richmond, KY. 1996. CONTINUING EDUCATION COURSES Stress as a Confounding Factor in Toxicology Studies Rodent Pathology (Immunopathology) Basic Embryology and Developmental Toxicology Grants 101: Professional Grant Writing Workshop Immunology for Toxicologists Risk Communication for the General Public Estrogen Mimics in Health and Disease Methods for Assessment of Neurotoxicity PROFESSIONAL ORGANIZATIONS 2009-Present Carolinas Society of Environmental Toxicology and Chemistry 2005-Present Society of Toxicology (SOT) 2005-Present North Carolina Chapter of the Society of Toxicology 74 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN 1997-Present 1996-2004 1996-2004 Society of Environmental Toxicology and Chemistry (SETAC) Ohio Valley Chapter of the Society of Environmental Toxicology and Chemistry Great Lakes Bioeffects Workgroup AWARDS AND HONORS The Faculty Mentor Award, East Carolina University Honors College. 2017. Outstanding Young Investigator Award, Immunotoxicology Specialty Section, Society of Toxicology. 2013. Outstanding Teaching Award, School o f Public and Environmental Affairs, Indiana University. 1999 and 2002. Future Faculty Teaching Fellowship, Preparing Future Faculty program, Indiana University. 2002. Marian Vinegar Award, Outstanding Student Presentation at the annual meeting, Ohio Valley Chapter of the Society of Environmental Toxicology and Chemistry. 2000. Outstanding Educational Volunteer, Monroe County Humane Association, Bloomington, IN. 2000. Outstanding Student Poster Award, Society o f Environmental Toxicology and Chemistry 19th Annual Meeting (3rd place). 1998. Teaching Excellence Recognition Award, School of Public and Environmental Affairs, Indiana University. 1998. PROFESSIONAL PRACTICE W orkshop Participant, Is the European Food Safety Authority (EFSA) standard for bisphenol A sufficiently protective of the immune system and should the Dutch government consider a different standard? Organized by representatives o f RIVM (National Institute for Public Health and the Environment), Amsterdam, The Netherlands. September, 2015. Provided immunotoxicological guidance for regulatory consideration of bisphenol A (BPA). C onsultant, CZR Incorporated, Wilmington, NC. May-July, 2014-present. Provided toxicological interpretation of stream water quality monitoring data for heavy metals. Technical Advisor, Office of Health Assessment and Translation (OHAT), National Toxicology Program, National Institute of Environmental Health Sciences. March 2013. March 2015. April 2016. Evaluated OHAT protocol for evaluation of PFOS-PFOA immunotoxicity. E xternal Peer Reviewer, U.S. Environmental Protection Agency, External peer review o f EPA's Draft Health Effects Documents for Perfluorooctanoic acid (PFOA) and Perfluorooctane Sulfonate (PFOS). 2014. Scientific peer reviewer of the health effects documents. Nominated and selected. W orking G roup M em ber, International Agency for Research on Cancer (IARC), IARC Monographs on the Evaluation o f Carcinogenic Risks to Humans, Volume 110: Perfluorooctanoic acid, Tetrafluoroethylene, Dichloromethane, 1,2-Dichloropropane, and 1,3-Propane sultone. 2014. Member of Mechanistic and Other Relevant Data Working Group for perfluorooctanoic acid and tetrafluoroethylene. Invited. C onsultant, Constella Group, LLC, Durham, NC. October 2004-December 2005. Summarized immunotoxicology o f atrazine for the National Toxicology Program's Report on Carcinogens. C onsultant, Henshel EnviroComm, Bloomington, IN. June 1999-May 2004. 75 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Representative of the Restoration Advisory Board for Jefferson Proving Ground (Department of Defense) through the Technical Assistance for Public Participation program. Interpreted risk assessment documents associated with base clean-up for the general public. Consultant, Dinosaur Inc., Bloomington, IN. June 2000-September 2000. Summarized the potential health and environmental effects o f land-applied paper mill sludge. Consultant, Integrated Pest Management in Schools and Childcare Centers, School o f Public and Environmental Affairs Information Clearinghouse, Bloomington, IN. April 2000-September 2000. Summarized the potential health effects of pesticides commonly used in schools and childcare centers. Co-Director, Summer Program fo r Exploration o f Complex Issues in Environmental Science fo r Teachers (SPECIES-Teachers) and Environmental Education 99, School of Public and Environmental Affairs, Indiana University, Bloomington, IN. Summer 1999 and 2001. Directed hands-on environmental science summer field workshop for Indiana teachers. Consultant, Brownstown Elementary Fourth Grade, Brownstown, IN. September 1999-May 2000. Served as environmental science expert during weekly videoconferences in a "students as environmental scientists" program. Associate Director, Research Experience fo r High School Students, College o f Arts and Sciences, Indiana University, Bloomington, IN. February 1999-October 1999. Mentored high school students participating in research in university laboratories, provided weekly counseling, and oversaw development of final research reports and presentations. Co-Director, Environment 98 and Environment 99, School of Public and Environmental Affairs, Indiana University, Bloomington, IN. Summer 1998 and 1999. Directed hands-on environmental science summer field workshop for Indiana students. Chemical Safety Assistant, Office o f Radiation, Chemical and Biological Safety, Michigan State University, East Lansing, MI. August 1993-June 1995. Developed Michigan State University's Chemical Hygiene Plan, performed university-wide laboratory safety inspections, and trained new science employees in chemical and laboratory safety. TEACHING EXPERIENCE_____________________________________ Instructor, East Carolina University Practical Problems in Biometry. Graduate (course director). General Toxicology. Graduate (co-course director). Advanced Toxicology. Graduate (co-course director) Medical Pharmacology. Graduate physician assistant students (topical: endocrine pharmacology, toxicology). Medical Pharmacology. Second year medical students (topical: endocrine pharmacology, toxicology). Pharmacology Mini-Course. First and second year dental students (topical: toxicology, management of poisoned patients, endocrine pharmacology). Foundations of Medicine/Problem Based Learning Mini-Course. First and second year medical students. 76 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Foundations o f Medicine/PirateMD. First and second year medical students. Instructor, Indiana University Analytical Problem Solving (statistics). Undergraduate, honors, 2 semesters. Environment and People. Undergraduate (co-instructor), 1 semester. Introduction to Statistical Techniques. Undergraduate, 1 semester. Introduction to Environmental Sciences. Undergraduate, 1 semester. Environmental Risk Analysis. Graduate, 1 semester. Outdoor Environmental Awareness (Public land management). Undergraduate recruitment course, 6 semesters. Introduction to Risk Assessment and Risk Communication. Undergraduate, 1 semester. Environmental Toxicology. Combined undergraduate/graduate, 1 semester. Techniques in Environmental Science (field/lab techniques). Undergraduate, 3 semesters. Teaching Assistant/Coordinator, Indiana University Aquatic Habitat Analysis (field techniques). Combined undergraduate/graduate, 3 semesters. Terrestrial Habitat Analysis (field techniques). Combined undergraduate/graduate, 3 semesters. Environmental Toxicology. Undergraduate and graduate, 3 semesters. Teaching Assistant and Undergraduate Teaching Intern Training Workshop. Combined undergraduate and graduate, 6 sessions. Laboratory Mentor, East Carolina University High school students Sunnie Li and Alex Reulbach, Summer Ventures Program (high school laboratory research). Project: Neuroinflammation in a rodent model o f Gulf W ar Illness. 2017. Matthew Clayton, high school laboratory research for the NC Science Fair. Project: Developmental effects of pharmaceutical pollutants in an avian model. 2016. Virginia Billings, Summer Ventures Program (high school laboratory research). Project: Developmental effects of pharmaceutical pollutants in an avian model. 2016. Jaein Yoon, Medical Honors Program (high school laboratory research). Project: Developmental effects of pharmaceutical pollutants in an avian model. 2015-2016. Chevonne Parker, Summer Ventures Program (high school laboratory research). Project: Neuronal T-cell infiltration following developmental trichloroethylene exposure. 2015. Catherine Taylor and Jessi Zhou, Medical Honors Program (high school laboratory research). Project: Microglial responses in an Alzheimer's mouse model developmentally exposed to lead. 2014-2015. Janelle Neal, Summer Ventures Program (high school laboratory research). Project: Microglial responses following inhalation exposure to natural dusts. 2014. Brian Alloway, Medical Honors Program (high school laboratory research). Project: Peroxisome proliferation in livers of C57BL/6 mice exposed to undecafluoro-2-methyl-oxahexanoic acid (U2M3-OHxA) gavage. 2013-2014. Kortney Wager, Summer Ventures Program (high school laboratory research). Project: Developmental effects of BPA on immune responses. 2013. Brian Ennis and Jonathan Reed, Medical Honors Program (high school laboratory research). Project: Teratogenicity of PFOS in early chicken embryos. 2012-2013. W illa Chen, Summer Ventures Program (high school laboratory research). Project: Developmental effects o f PFOA in primary cardiomyocyte cultures from chickens. 2012. Elizabeth Fox and Samantha Rouse, Medical Honors Program (high school laboratory research). Project: Teratogenicity of PFOS in early chicken embryos. 2011-2012. 77 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Pranavi Sanka, Summer Ventures Program (high school laboratory research). Project: Developmental effects of PFOS: T cell infiltration into mouse brains. 2011. Erin Morris and Andrew Wood, Medical Honors Program (high school laboratory research). Project: Developmental effects o f PFOA on T cell infiltration and myelin basic protein levels in mouse brains. 2010-2011. Jillian Loftis, Summer Ventures Program (high school laboratory research). Project: Developmental effects of PFOA on glycogen deposition in a chicken model. 2010. Clarissa Morrissey, Medical Honors Program (high school laboratory research). Project: Teratogenicity of PFOA in early chicken embryos. 2009-2010. Taylor Brundage, Summer Ventures Program (high school laboratory research). Project: Developmental teratogenic effects of PFOA in a chicken model. 2009. Ian Bryan, Medical Honors Program (high school laboratory research). Project: Developmental effects o f PFOA on liver glucocorticoid receptor levels, and pancreatic alpha and beta cells in a mouse model. 2008-2009. Undergraduate students Robert Strickland, General Studies/Program in Neuroscience (undergraduate laboratory research). Project: Neuroinflamamtion in a rodent model of G ulf W ar Illness. 2017 Chastity Ward, Summer Biomedical Research Program (undergraduate laboratory research). Project: Immunotoxic effects of AFFF in a rodent model. 2017. Christopher Hamby, Mutidisciplinary Studies Program in Neuroscience (undergraduate laboratory research/senior thesis advisor). Project: Microglial morphology in a rodent model o f G ulf War Illness. 2016-2017. Ishmael Gomez, Summer Biomedical Research Program (undergraduate laboratory research). Project: DAP12 microglial signaling in a rodent model o f Alzheimer's disease. 2016. Brianna Davidson, Multidisciplinary Studies Program in Neuroscience (undergraduate laboratory research). Project: Synaptic degeneration in a rodent model o f Alzheimer's disease. 2016. Samuel Vance, Mutidisciplinary Studies Program in Neuroscience (undergraduate laboratory research/senior thesis advisor). Project: Project: Post-translational modifications and Alzheim er's pathology. 2015-2017. W aeya Lin, Summer Biomedical Research Program (undergraduate laboratory research). Project: Exacerbation o f Alzheimer's pathology by prenatal exposure to lead; Dystrophic microglia. 2015. Giovana Fernanda Cosi Bento, Brazil Scientific Mobility Program (undergraduate laboratory research). Project: Exacerbation o f Alzheimer's pathology by prenatal exposure to lead; Synaptosomes. 2015. Zoe Hinton, Mutidisciplinary Studies Program in Neuroscience (undergraduate laboratory research/senior thesis advisor). Project I: Exacerbation o f Alzheimer's pathology by prenatal exposure to lead; Synaptosomes. 2015. Project II: Microglial morphology in a rodent model of G ulf W ar Illness. 2016-2017. Andrew Wood, Biology (undergraduate honors thesis advisor). Project: Exacerbation of Alzheimer's pathology by prenatal exposure to lead; Measurement o f amyloid beta. 2014-2015. Dakota Johnson, Biology (undergraduate honors thesis advisor). Project: Exacerbation o f Alzheimer's pathology by early-life exposure to lead; Measurement o f amyloid beta. 2013-2014. Sydney Henry, Summer Biomedical Research Program (undergraduate laboratory research). Project: Neurotoxic effects of dust collected from the Nellis Dunes Recreation Area. 2014. Andrew Wood, Biology undergraduate student. Project: Developmental effects o f BPA on serum IL-4 and IgG. 2013. Dominique Baldwin, Biology undergraduate student. Project: Neurotoxic effects of dust collected from the Nellis Dunes Recreation Area. 2013. 78 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Megan Biller, Summer Biomedical Research Program (undergraduate laboratory research). Project: Microglia in Alzheimer's-prone triple transgenic mice. 2013. Blake Rushing, Summer Biomedical Research Program (undergraduate laboratory research). Project: Blood distribution, urinary excretion, and T cell-dependent immunotoxicity of undecafluoro-2-methyl-oxahexanoic acid (U2M3-OHxA) in C57BL/6 mice exposed via gavage. 2012. Alvin-Ming-Yun Tsang, Psychology (undergraduate honors thesis advisor). Project: Developmental effects o f nanoparticles in a rodent model. 2011-2012. Nick Creech, Summer Biomedical Research Program (undergraduate laboratory research). Project: Immunological effects o f PFOA on a T-cell independent antigen (DNP-Ficcol) in an adult mouse model. 2010-2011. Hatel Patel, Biochemistry undergraduate student. Project 1: Developmental effects o f PFOA on liver peroxisomes proliferation in a chicken model. Project 2: Effects o f PFOA on myelin basic protein levels in the brains of developmentally-exposed mice. 2009. Ian Bryan, Biology and Chemistry undergraduate student. Project: Developmental effects o f PFOA and PROS in a chicken model, including early embryo teratogenesis and hatchling glycogen levels. 2009-2012. M a ster's students Carmen Davis, Environmental Health M aster's Student. Project: Developmental effects of Triclosan in an avian model. 2015-2016. Cory Boles, Biomedical Sciences M aster's Student. Project: Exacerbation o f Alzheimer's pathology by early-life exposure to lead. 2013-2015. Annalise vonderEmbse, Biomedical Sciences M aster's Student. Project: Exacerbation of Alzheim er's pathology by early-life exposure to lead; Effects on microglia. 2012-2014. Doctoral students Jacqueline Meadows, Pharmacology and Toxicology Ph.D. Student. Project: Developmental effects of pharmaceutical pollutants in an avian model. 2015 Annalise vonderEmbse, Pharmacology and Toxicology Ph.D. Student. Project: Exacerbation of Alzheim er's pathology by early-life exposure to lead; Effects on microglia. 2014-2017. Jason Franklin, Pharmacology and Toxicology Ph.D. Student. Project: Developmental neuroimmunotoxicity o f bisphenol a in a rodent model. 2010-2014. Qixiao Jiang. Pharmacology and Toxicology Ph.D. Student. Project: Developmental cardiotoxicity of perfluorinated compounds in an avian model. 2009-2013. Medical students Amie McPherson and Danesh Ghiassi, Medical students. Project: Isolation and stimulation of regulatory T cells from spleens of PFOA-exposed mice. 2009. Advisory, East Carolina University Khoa Do, Biomedical Sciences M aster's Student (Thesis committee; Advisor: Hu Huang). 2016 2017. John Atkinson, Biology M aster's Student (Thesis committee; Advisor: David Rudell). 2015-2016. Blake Rushing, Pharmacology and Toxicology Ph.D. Student (Dissertation committee; Advisor: Mustafa Selim). 2015 Ahmed Aldhafiri, Pharmacology and Toxicology Ph.D. Student (Dissertation committee; Advisor: Ken Soderstrom). 2014 Jason Hoggard, Biomedical Sciences M aster's Student (Thesis committee; Advisor: Lance Bridges). 2014-2015. 79 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Matthew Edwards, Biology M aster's Student (Thesis committee; Advisor: Krista McCoy). 2014 2015. Bevin Blake, Biology M aster's Student (Thesis committee; Advisor: Krista McCoy). 2013-2015. Anastasia Weeks, Microbiology and Immunology M aster's Student (Thesis committee; Advisor: Mark Mannie). 2013-2017. Samar Rezq, Pharmacology and Toxicology Ph.D. Student (Dissertation committee; Advisor: Abdel Abdel-Rahman). 2013-2016. Partha Nagchowdhuri, Pharmacology and Toxicology Ph.D. Student (Dissertation committee; Advisor: Brian McMillen). 2013 Suelen Demor, Biology Ph.D. Student (Dissertation committee; Advisor: David Chalcraft). 2013 2017. Tessa Holland, Pharmacology and Toxicology Ph.D. Student (Dissertation committee; Advisor: Ken Soderstrom). 2013 Samantha Sellers, Anatomy and Cell Biology Ph.D. Student (Dissertation committee). 2013. Alvin Ming-Yun Tsang, Psychology Undergraduate Student (Honors Thesis Advisor). 2011-2012. Abdullah Aldossari, Pharmacology and Toxicology Ph.D. Student (Dissertation committee; Advisor: Jared Brown). 2011-. Michael Smith, Biology M aster's Student (Thesis committee; Advisor: Xiaoping Pan). 2010-2011. Pranita Katwa, Pharmacology and Toxicology Ph.D. Student (Dissertation committee; Advisor: Jared Brown). 2009-2012. COMMUNITY SERVICE________________________________________ Scientific Community Member, Society of Toxicology Specialty Section Collaboration and Communication Group. 2017-present. Vice-President, Society o f Toxicology Immunotoxicology Specialty Section. 2017-present. Program Planning Committee, Volunteers Sub-committee, 2017 Annual Meeting, Society of Environmental Toxicology and Chemistry. 2016-2017. Vice-President Elect, Society o f Toxicology Immunotoxicology Specialty Section. 2016-2017. Program Committee Member, Society o f Toxicology Immunotoxicology Specialty Section. 2015 2016. Moderator for Toxicology, Epidemiology, and Human Health section, FLUOROS 2015 meeting, Golden, CO. 2015. Program Planning Committee, 2015 Annual Meeting, Society of Environmental Toxicology and Chemistry. 2014-2015. Senior Councilor, Immunotoxicology Specialty Section Society o f Toxicology. 2014-2015. Past-President, North Carolina Society o f Toxicology. 2014-2015. Junior Councilor, Immunotoxicology Specialty Section Society o f Toxicology. 2013-2014. President, North Carolina Society o f Toxicology. 2013-2014. Appointed Member, Research Funding Committee, Society of Toxicology. 2012-2014. Vice President, North Carolina Society of Toxicology. 2012-2013. Vice President-Elect, North Carolina Society o f Toxicology. 2011 -2012. Program Committee Member, Society o f Toxicology Immunotoxicology Specialty Section. 2010 2011. Councilor, North Carolina Society of Toxicology. 2009-2011. Workshop co-chair and organizer, "Is Modulation o f the Immune System by Perfluoroalkyl Acids a Human Health Concern?" Society o f Toxicology 48th Annual Meeting, Baltimore, MD. 2009. Symposium co-chair and organizer, "Immune Biomarkers in Alternative Species: Implications for Risk Assessment," Society o f Toxicology 46th Annual Meeting, Charlotte, NC. 2007. 80 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Platform session co-chair, "Immunotoxicology: Immune Modulation and Cell Specific Responses," Society o f Toxicology 46th Annual Meeting, Charlotte, NC. 2007. Postdoctoral Representative and Program Committee member, Immunotoxicology Specialty Section of the Society of Toxicology. 2005-2007. Mentor, Association o f Women in Science Program (WISP) Mentoring Project, Office of W om en's Affairs, Indiana University, Bloomington, IN. 2000-2002. Student Board Member, Ohio Valley Chapter o f Environmental Toxicology and Chemistry. 2000 2001. Workplace Community Department of Comparative Medicine Promotion and Tenure Committee, Brody School of Medicine, East Carolina University, 2017-present. Committee member, Coastal Strategic Planning Committee, East Carolina University, 2016-2017. BSOM Promotion and Tenure Committee, Brody School of Medicine, East Carolina University, 2015-present. Secretary/Treasurer, Brody Women Faculty Committee. 2015-2017. Committee member, Planning committee for the joint PhD program in Integrated Coastal and Marine Sciences, East Carolina University. 2015-present Committee member, School of Dental Medicine Admissions Committee, East Carolina University, 2014-present. Committee member, BSOM Sustainability Committee, Brody School of Medicine, East Carolina University, 2014-2016. Committee member, BSOM Research Committee, Brody School of Medicine, East Carolina University. 2014-2015. Five-Year Review Committee for Dean Paul Cunningham, Dean of the Brody School of Medicine (appointed by the Vice Chancellor for Health Sciences). 2014. Committee member, Institutional Animal Use and Care Committee. 2013-present. M1 Curriculum Committee member, Brody School of Medicine, East Carolina University. 2013 2016. Master Educator Committee member, Brody School of Medicine, East Carolina University. 2012 2016. Chair, Brody Women Faculty Committee. 2012-2013. Group member, Brody Vision, Innovation, Achievement (VIA) group. 2011-2015. Undergraduate Research and Creative Activity Biomedical Sciences Grant Review Committee. 2011-present. Chair-elect, Brody Women Faculty Committee. 2011-2012. Committee member, Coastal Maritime Council, East Carolina University. 2010-present. Five-Year Review Committee for Dr. David Taylor, Chair o f the Department o f Pharmacology and Toxicology (appointed by the Dean of the School o f Medicine). 2010. Committee member, Shared Resources Committee, Brody School of Medicine, East Carolina University. 2008-2012. Faculty of the Interdisciplinary Doctoral Program in Biological Sciences, East Carolina University. 2009-present. Committee member, Brody Women Faculty Committee, East Carolina University. 2008-present. Graduate Faculty, Division of Research and Graduate Studies, East Carolina University. 2008- present. Vice-President and at-large member, EPA RTP Networking and Leadership Training Organization, USEPA. 2004-2008. Organizing committee member, 2007 NIEHS Biomedical Career Fair. 2006-2007. 81 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Executive committee member/Vice-chair, Environmental Science program representative, Association of SPEA Ph.D. Students, School o f Public and Environmental Affairs, Indiana University. 2001-2002. Environmental Science program representative, Dean's Student Advisory Committee, School o f Public and Environmental Affairs, Indiana University. 1997-2002. Other Science Event Co-Coordinator, "ADdMe to Tox Town," Girl Scouts TechnoQuest Event. 2017. Science Event Co-Coordinator, "Biometry in Action," Brody G irl's STEM Day. 2016. Science Event Coordinator, "The Water Cycle," Youth Ocean Conservation Summit. 2016. STEM volunteer, various events, Love a Sea Turtle. 2015-present. Science Event Coordinator, "Marshmallow Genetics" and "DNA Necklaces," ECU G irl's STEM Day. 2014-2016. Scientific Expert, Alzheimer's North Carolina fundraising walk, W ashington, NC. 2014-2016. Uber Judge, Blue Heron Bowl, Regional Competition for the National Ocean Sciences Bowl. 2012. Judge, North Carolina Region 1 Science and Engineering Fair. 2010. 2011. North Carolina Estuarium Docent. 2009-present. 82 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Appendix B Estim ating " Safe" PFOS Doses, an Exam ple and Comments 83 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN Estimating "Safe" PFOS Doses, an Example and Comments. Dr. John B utenhoff derived a plasm a PFO S reference level o f 1.05 ppb (pg/L), to his best recollection, in 1997 or 1998 (3M _M N 00042160. B utenhoff Depo. Tr. O ctober 27, 2006; 59-70). In this derivation, Dr. B utenhoff did the following (adopted from Ex.148. 3M _M N00042206): LO A EL in Rhesus m onkeys = 0.5 m g/kg/day over 90 days. Safety factors: 10 for L O A E L to N O A E L 10 for sub-chronic to chronic 100 for interspecies extrapolation 10 for exposure o f children (Food Q uality Safety A ct and W ater Act) A pplying a 100,000-fold safety factor (product o f four safety factors, above) to the LO A EL = 0.5 m g/kg/day divided by 100,000 = 0.000005 m g/kg/day or 0.005 pg/kg/day. Dr. B utenhoff adjusted this value for the default 70 kg body w eight for a hum an calculate a value o f 0.35 pg/day. Com m ent - Reference doses typically are expressed in m g/kg/day as it is the LO AEL or N O A EL divided by uncertainty factors (U.S. EPA, 1989). Alternatively, a hum an equivalent dose (HED) can be used. For PFOS, the U.S. EPA (U.S. EPA, 2016d) scaled serum concentrations (typically in m g/L) for the N O A EL or LO A EL identified in an experimental animal study to clearance of PFOS in humans. The HED also expressed in m g/kg/day. It is therefore som ewhat confusing as to w hy Dr. B utenhoff m ultiplied the 0.005 pg/kg/day by the default hum an body w eight value. Dr. B utenhoff then adjusted this 0.35 pg/day value by 90 days, w hich was the duration o f the R hesus m onkey study that identified the LO A EL o f 0.5 pg/kg/day. H e expressed this as a total dose o f 31.5 pg, w hich appears to be his estim ate o f the potential total m ass o f PFO A in the plasm a o f the Rhesus m onkeys after 90 days o f exposure to 0.35 pg. Finally, Dr. B utenhoff adjusted the 31.5 pg value by 10% as this was his assum ption that 10% of this mass would be in the plasm a of the Rhesus monkeys. He then assum ed that an average hum an has 3 liters o f plasm a and calculated 1.05 pg/L (3.15 pg divided by 3 L). Com m ent - Again, this approach is som ew hat confusing as it does not exactly follow approaches used by the U.S. EPA to derive RfDs or H A Ls even though Dr. B utenhoff indicates that ..it follows the approach used by federal agencies" (Ex.148. 3M _M N00042206). As a reminder, RfDs are expressed in m g/kg/day whereas HA Ls are expressed in pg/L (or mg/L) of drinking w ater and reflect RfD s adjusted by body w eight and the drinking w ater intake (U.S. EPA, 2016d). Com m ent - If we use the LO AEL from the Rhesus m onkey study as a starting point and adjust it by the uncertainty factors used by Dr. Butenhoff, the RfD is 0.000005 m g/kg/day. U sing 84 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN form ulas consistent w ith the U.S. EPA for PFO S (U.S. EPA, 2016d), the follow ing calculations can be made: 0.000005 m g/kg/day = 9.3 x 10-5 m g/L (D W EL; drinking w ater equivalent level) 0.054 L/kg/day* *drinking w ater intake rate Lifetim e health advisory = DW EL x RSC = 9.3 x 105 m g/L x 0.2 (value used by U.S. EPA ) = 0.000016 m g/L or 0.016 gg/L If we do the same calculations, but use values o f the M D H for drinking w ater intake rate (0.049 L/kg/day) and RSC (0.5), the calculations are as follows: 0.000005 m g/kg/day = 1.02 x 10-4 m g/L (D W EL; drinking w ater equivalent level) 0.049 L/kg/day Lifetim e health advisory = DW EL x RSC = 1.02 x 10-4 m g/L x 0.5 (value u sed by U .S. E P A ) = 0.000051 m g/L or 0.051 gg/L Com m ent - The "safe" PFOS dose calculated by Dr. B utenhoff in the late 1990s was therefore m ore conservative than the U.S. EPA 2016 H A L (0.07 gg/L) and on the same order o f m agnitude as the M D H H BV (0.027 gg/L). 85 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Appendix C List of References Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN 86 CONFIDENTIAL - SUBJECT TO A PROTECTIVE ORDER ENTERED IN HENNEPIN COUNTY DISTRICT COURT, NO. 27-CV-10-28862 27-CV-10-28862 Filed in Fourth Judicial District Court 11/17/2017 7:13 PM Hennepin County, MN 3M Company. 1963. Technical Information for 3M Brand Fluorochem ical Surfactants. 3M _M N00042160. B utenhoff Depo. Tr. O ctober 27, 2006. 3M _M N01663513. Fluorad Fluorochemical Surfactant FC-95. 90-day subacute rhesus monkey toxicity study (International Research and D evelopm ent Corporation). D ated January 2, 1979. 3M _M N01663537. Fluorad Fluorochemical Surfactant FC-95. Ninety day subacute rat toxicity study (International R esearch and D evelopm ent C orporation). D ated N ovem ber 10, 1978. 3M _M N02330325. M em o regarding tw o-year toxicity/carcinogenicity study o f fluorochem ical FC-143 in rats from Charles Reinhardt (DuPont) to Greg Sykes. 3M _M N02343342. Final report: 26-week capsule toxicity study w ith amm onium perfluorooctanoate (APFO) in cynomolgus monkeys (Covance Study Number: 6329-231; A uthored by P eter J. Thom ford, PhD ). D ated D ecem ber 18, 2001 (as study com pletion date). 3M _M N 02483163. See O lsen et al., 2001a. 3M _M N03095624. See Luebker et al., 2005. 3M _M N03112178 at 3M _M N03112393. Fluorocarbons and hum an health: Studies in an occupational cohort. Dr. G illiland's 1992 doctoral thesis. 3M _M N03278834. 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