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AR 2%6-054% Perfluorooctane Sulfonate: Current Summary of Human Sera, Health and Toxicology Data 3M January 21, 1999 ) 000014 Jaary 21,1999 TABLE OF CONTENTS EXECUTIVE SUMMARY cscs I. HISTORICAL REVIEW AND CURRENT FINDINGS OF FLUOROCHEMICALS IN HUMAN 11, SUOM FMEDM ICALA SURVR EILLY ANCE AND EPIDEMIOLOGY STUDIES... 2 IV. SUMMARY OF TOXIDATCAOONPFLOS OGY cnr) V. RISK CHARACTERIZATION ccs VL. CURRENT AND PLANNED RESEARCH rvs 2 000015 Janay 21,1999 Executive Summary 3M has prepared this document to summarize the data related to the biological effects of perfluorooctane sulfonate (PFOS). Italso presents current thinking on human health risk related to PFOS and includes information about future study plans. 3M Medical Department scientists and physicians, in consultation with outside experts, are the authors. PFOS has been found at tens of parts per billion levels in serum samples of nonoccupationally exposed employees, in commercially available human serum and in pooled samples from multiple blood banks. PFOS is an eight-carbon molecule that is perfluorinated except for the sulfonate group on the terminal carbon. 3M has `manufactured PFOS and molecules that may be metabolic precursors to it since 1948. Routesof exposure to PFOS or precursor molecules are not well understood at this time. PFOS is an exampleofan "organic" fluorine molecule. Human serum has been known to contain organic fluorine molecules for over 30 years. The primary constituentof this organic fluorine fraction was tentatively identified as another molecule (perfluorooctanoate) in 1976. Current analysis of stored sera samples from a variety of `sources are more consistent with PFOS being a major fractionofthis organic fluorine. Improved analytic techniques allowing a relatively rapid analysis at low levels of detection make the current analyses possible. These analytic techniques were first available for use in medical surveillanceof exposed workers in 1992. Detection limits have been lowered to allow the more recent analysis ofserum from those without occupational exposure. Medical surveillance has been done among 3M employees occupationally exposed to PFOS precursors for over 20 years. To date, no adverse health effect associated with PFOS exposure has been found in these employees. This conclusion applies at serum levels up to 6 parts per million, about 100 times higher than levels seen in the general : 000016 nay 21,1999 population. PFOS has a long residence time in the human body. In three retirees, the half-life in human sera ranges from 1100 to 1500 days. A mortality study at the U.S. plant primarily involved with production of PFOS related materials has found no significantly elevated standardized mortality ratios (SMR's). Toxicology studies show that PFOS is well absorbed orally and distributes primarily in the serum and liver. It does not appear to be further metabolized. Some enterohepatic circulation of PFOS occurs, based on the observation ofincreased excretion in rats given cholestyramine. Elimination from the body is slow and occurs via both urine and feces. Mutagenicity testing is negative in five salmonella species. It is not genotoxic in a mouse bone marrow micronucleus assay. The acute LD50 in rats is 250 mg/kg (moderately toxic). It does not produce dermal or ocular toxicity. Subchronic studies have been done in rats and primates. PFOS causes liver enzyme elevations and hepatic vacuolization in rats, and hepatocellular hypertrophy at higher doses. Higher doses also cause other GI toxicity, hematological abnormalities, weight loss, convulsions, tremors and death. Monkeys show anorexia, emesis, diarrhea, hypoactivity and at higher doses prostration, convulsions and death. Atrophyof exocrine cells in salivary glands and the pancreas, and lipid depletion in the adrenals is found at high doses in the monkey. `The serum levels at which these compound related effects occurred in these carly rhesus `monkey studies are unknown. In a recently completed rangefinder study in cynomolgus `monkeys the first observed biological effect was a decrease in serum cholesterol, first observed at a serum level of 72 ppm in one of the two monkeys in the high dose group. Using the relationship between cumulative dose and serum level found in this study, it can be estimate that significant toxicity occurred at 700 to 800 ppm in the carly rhesus monkey studies, and death at 1100 ppm and above. More complete quantitative absorption, distribution and excretion data for PFOS is being obtained. 4 000037 January 21,1999 Available information therefore suggests that no identifiable health risk to humans would be expected to occur at the PFOS levels found in blood bank or commercial serum samples. Extensive further research, which includes epidemiological and laboratory studies, is planned or underway. The purpose of ths rescarch is to explore the potential for chronic and reproductive effects, understand toxic mechanisms and obtain a better understanding ofabsorption, distribution, metabolism and excretion. The plan is to make as much use as possibleof observational data in exposed workers and to establish no effect levels in both rats and primates for endpoints of importance. s 000018 Jamary 21,1999 I. INTRODUCTION Evidence that organic compounds containing the element fluorine covalently bonded to carbon (organic fluorine compounds, OF) can be found in human sera has been available for 30 years. Although all of the specific compounds contributing to the total amount ofOF present are not identified, it now appears that a compound called perfluorooctane sulfonate constitutesa significant fraction. Recent data provide evidence that PFOS is present at tensofparts-per-billion (ppb) levels in serum samples from the general population, averaging 30 ppb in blood bank samples from diverse locations in the U.S. Single digit parts per million (ppm) levels (approximately 100 times greater) are found in individuals occupationally exposed to PFOS and its precursors, averaging 2.0 ppm among participating employees at the primary U.S. manufacturing location for these compounds. 3M produces perfluorinated molecules by mixing anhydrous HF and hydrocarbon feed stock in an electrochemical cell (electrochemical fluorination). Perfluorooctane: sulfonyl fluoride (POSF) is the cell product from which a groupofproducts is developed: Fi CRA(CF; Ci = $0, -F F POSF s 000019 January 21, 1999 Other moieties ("R") are added to the sulfur, which leads to the creation of materials that may be polymerized or esterified. The vast majority of POSF produced is used in this way. F| CFy(CF, C| = S02 -R F POSF derived molecule PFOS may result from the lossof the "R" moiety through metabolic processes. Current evidence would indicate that PFOS is not further metabolized. Some PFOS is produced and sold directly into industrial applications as a surfactant. This, however, amounts to only a small fraction of total POSF production. F | CF3(CF2)e C1 - S05 F PFOS Most POSE that is produced is used in 2-(N-ethylperfluoroctancsulfonamido)-ethyl alcohol (N-Et-FOSE) and 2-(N-methylperfluoroctanesulfonamido)-ethyl alcohol (NMe-FOSE) based products. Figure LI shows the chemical structure of N-E-FOSE and metabolites that have been found in rat serum. All except compound VIII have been verified to metabolizefurtherto PFOS. (Missing Roman numerals represent hypothesized intermediates not shown in this figure.) 7 000020 Figure LI N-E(FOSE(*) Metabolites Identified in Rat Serum C7H,CH, CF =50,-N C\H= Cil;= 0-H 1LLEFOSE) ~ c4ry CF S0,-N< H IX /CH:CHy CF -50,-N J CiFip SO, ~NH, Cit - C00 " v R-SON 7 2 C\H, - C00 22 vin 8 x [ X11(PFOS) ' In vitro studies in both rat and human hepatocytes lead to identification of the same compounds and hypothesized intermediates. Compound VIII has been detected in `some samplesofpooled human sera. Asingle dose absorption, distribution, `metabolism and excretion study of N-Et-FOSE in cynomolgus monkeys is through the in life phase and tissue analysis is pending. N-Et-FOSE is esterified to produce larger `molecules that are used on paper and packaging for oil and water repellency. It is presumed that N-Me-FOSE, in which a methyl group replaces the ethyl group on the nitrogen, has a similar metabolism. N-Me-FOSE becomes partofvery large `molecules that act as protective chemicals on fabrics, leather and rugs. Outsideof the occupational setting, routes of human exposure to PFOS or its `metabolic precursors are not understood, but are the subjectofintense study. Exposure could occur from environmental releases of PFOS or its precursors at the Decatur, Alabama and Antwerp, Belgium manufacturing sites. It could occur from the environmental or biological degradation of products to PFOS or molecules `metabolized to PFOS. Products also contain small amounts (generally less than a few percent)of residuals, such as N-Et-FOSE and other molecules found in Figure 1.1, `which are known or suspected metabolic precursors to PFOS. These residuals represent a source of PFOS that would not require environmental or biological degradation of large molecules. Downstream industrial users of POSF based products are also potential sources of environmental releases of PFOS or its precursors. The relative contribution of these various sources to population exposure is currently unknown. Another surfactant is known to be found in the sera ofemployees and was reported in general population sera samples in 1976 (Taves). This is perfluorooctanoic acid, or PFOA: ' 000022 January 21,1999 CFy(CF2)COOH PFOA is also made by electrochemical fluorination. It is mentioned here because following Taves' report in 1976 it was presumed to be a significant fraction of the total organic fluorine found in the sera samples analyzed by Taves and others. This report was a stimulus for investigation and subsequent medical surveillance of employees in fluorochemical production, including those producing POSF based `materials. It should be recognized, however, that PFOA is a different and unrelated compound. It does not metabolize to or become POS. [tis likely that PFOA was misidentified as a major fractionof organic fluorine in the 1976 Taves paper. The evidence for this is discussed in Section Il `The purpose of this reporti to describe the data on PFOS levels in human sera, and to discuss the potential for those levels to affect health based on current scientific Knowledge. A review of current findings and historical information on PFOS levels in sera is presented in Section IL. This is followed in Section III with a description of 3M's epidemiology and medical monitoring database obtained from studies of its workers in plants in the United States and Belgium. The animal and other laboratory toxicology data available on PFOS are presented in Section IV. Section V offers a preliminary evaluation of the serum findings in light of the available health effects data. 3M is actively developing further human health and toxicological information. Section VI outlines the current research agenda. 10 000023 January 21,1999 II. HISTORICAL REVIEW AND CURRENT FINDINGS OF FLUOROCHEMICALS IN HUMAN SERA `The data on fluorochemicals in human sera is presented in this section. The presence oforganic forms of fluorine in human serum was observed 30 years ago, and PFOA `was thought to account for most of this fraction. 3M finds little current evidence to support this view. Evidence is presented below that PFOS is more likely to be consistently found in sera. Based on the limited data provided by historical samples, there is no evidenceof significant change in PFOS concentration in scrum samples taken over the last two to three decades. "The advancementofanalytical chemistry technology has had a significant influence on our knowledgeoffluorocarbons in human sera. The techniques developed and used by researchers in the 1960's and 1970's were time intensive, requiring hours for a single analysis. The methods were also nonspecific, measuring organic fluorine. (fluorine covalently bonded to carbon) rather than specific molecules. The development oaf rapid analytic technique in the late 1970's decreased analytic time to under an hour, allowing large scale medical surveillance ofproduction employees at higher detection limits (about 0.5 parts per million organic fluorine) that were adequate for the levels found in occupationally exposed individuals. The advancementof chromatographic/mass spectroscopy technology enabled rapid analysis of specific fluorochemicals from small volumes of sera in the early 1990's. `This technology was first used in medical surveillance in 1992. Detection limits for PFOS were lowered to 50 parts per billion by 1997. The first report to 3M of PFOS. in commercially available pooled sera occurred in late summer of 1997, prompting more research into the technique, and confirming its validityover a period of several `months. n 000024 January 21,1999 Since older published data described organic fluorine content rather than a specific molecule, it is useful to understand the relationship between PFOS levels currently observed and organic fluorine content. Fluorine is 65%of the molecular weight of PFOS. The contribution ofaPFOS value to organic fluorine, in ppb, will therefore be [0.65 x (PFOS value in ppb)]. Conversely, if the measured organic fluorine is entirely PFOS, the value of PFOS in ppb will be [1.54 x (OF level in ppb)]. "This section presents 1)abriefsummaryofthe historical information regarding organic fluorine in human sera, 2) data from 3M employees involved in fluorochemical production, 3) data from a small groupof non-oceupationally exposed 3M employees, 4) data from commercially available human pooled serum samples, 5) data from pooled scra from 18 regional blood banks and 6) data from current analysis. of stored serum samples. Historical Finding of the Organic Form of Fluorine in Blood `Taves (1968a) described two formsoffluorine in serum, one that was exchangeable with radioactive fluorine-18 and one that was not. Pothapragada et. al. (1971) also described two forms, ionic and nonionic. Taves (1968) showed that the nonexchangeable fluorine was bound to albumin. This finding, along with results of extraction and precipitation and the need for ashing to release this form of fluorine, led to the conclusion that the non-exchangeable or nonionic fluorine was "organic", ie. covalently bound to carbon (Taves et. al., 1976). Using NMR spectroscopy, these authors tentatively identified a componentofthe organic fluorine as perfluorooctanoic acid (PFOA). There was some variation in the observed spectra from an authentic: sample ofPFOA, however, leading the authors to suggest that branching, or the presence ofa sulfonate, was possible. A number of studies over the past 25 years reported levels of organic fluorine in human blood serum. Table ILI presents the study author, level measured, population studied and methodsofanalysis. The varietyofmethods used for determination of 2 000025 January21, 1999 fluorine suggests that some caution be used in interpreting results. All reported means were in the tens of part per billion levels. The average of reported values from United States sources is 37.6 ppb. Historical FindingsofTSaebrluem11O1raanic Fluorine Levels Y1e9a7r2 GAuutyhor 3O0Ftot) oNs 1199775 VeGnuky,atTeasvweasrlu 326s 1062 11997789 SBielnigselre a2s 2649 11998800 UPbaeclz a8 poole4d 9139801 BeYlaimsalmeota 2i i5 asMhethods ashbomb asOhbomb amsohd Obomb oboAm +** OVarrgiaendicmefltuhooridnse,wesrpeeciufsiecddteonmteiaessurneotorpgraonviidcedfl.uorine. See papers for details. SUosurce uuss uuss ArUgeSntina Cjhaipnaan Occupationally Exposed Employees 3M has produced PFOA (the ammonium salt) by electrochemical fluorination since the early 1950s. Itisa surfactant used in fluoropolymer production. The company began medical monitoringof employees involved in PFOA production in 1976, by `measuring serum levelsoforganic fluorine (OF) and performing medical assessments. Employee monitoring was expanded significantly in 1980 following the development ofamore rapid test for organic fluorine. Measured serum levels of OF in these: employees averaged less than 10 parts per million (ppm). As noted earlier, PFOS is a surfactant used as a wetting and foaming agent in industrial and commercial processes. Certain fluorochemicals that may transform `metabolically to PFOS have been produced since the early 1950's by electrochemical fluorination. Since 1980 this has occurred at primarily one location in the United States (Decatur, Alabama). This 3M site consistsof a fluorochemical plant and a film plant that are physically separate entities. Chemical plant employees have been offered a medical monitoring program that includes standard medical testing as well 000026 January 21,1999 as measurementofserum levels of OF. Among employees with six or more measurements, OF levels averaged 2.9 10 6.5 ppm from 1981 to 1992' (Figure IL1). `With the introduction of high performance liquid chromatography-mass spectrometry, serum PFOS was measured in 1994 and 1997 (see also Figure IL1). In these two years, mean PFOS levels were 2.44 ppm (range 0.25 ~ 12.83 ppm) and 1.96 ppm (range 0.10 -9.93 ppm), respectively. Another 3M plant in the United States where PFOS has been measured in employees' serum is Cottage Grove, Minnesota. Some PFOS is manufactured at this plant, In 1997, the mean serum level of PFOS among 74 Cottage Grove fluorochemical production employees was 0.82 ppm (range 0.05 - 6.25 ppm). Outside the United States, 3M manufactures PFOS related materials at its Antwerp, Belgium plant. PFOS levels were measured in Antwerp employees in 1995 (mean = 1.9 ppm, range 0.0-9.9 ppm) and in 1997 (mean 1.5 ppm, range 0.1 - 4.8) ppm. `The cross-sectional stratified analysis presented in section IIL, examining the relationship between PFOS sera level and various clinical chemistry and hormone parameters, was conducted at the Decatur, Alabama and Antwerp, Belgium plants. `The Cottage Grove facility was not included because little PFOS related product is `manufactured there. Its also the primary site for PFOA production. [foCWrenePtrFaaOrleSAnabawylayetrlieecacoltfroLoannbecoaropacttcouarrsyei.og1na8s7i5nc].h1r9o7Tm9oatwtahloegsrreearptuhhmeaonsrdegramuniimccroffolw5uaorDvieencepallteauvsremlesamfpdoelrtoteyhceteeisosenwfimavesethemomedpasls.ouyreeeds waendre6150%.1,of5.t7h,e 9to4t,al1s1e.8ruamndor4g.a1nipcpmf.luorTihnee lpeevreclse,ntreosfpePcFtiOvSelfyound was 60%, 70%, 80%. 55% * 000027 . . . .3 1 : 0 ts 32 8 77- vr 0 7 7 % % Non-Occupationally Exposed 3M Employees A total of 31 3M employees were tested for PFOS in their serum in 1998 (Table 11.2). All were corporatestaffor division managers. None had worked in fluorochemical production or in fluorochemical research and development. Samples were from five females and 26 males. Employees ranged in age from 37 10 62 years. All employees had measurable PFOS in their blood serum (mean = 47 ppb; range = 28 to 96 ppb). Age was significantly associated with increased serum PFOS and accounted for 24% ofthe variance in PFOS levels. There was no gender-related differenceifage was considered. Only four employees had PFOA measured above the detection limit of 10 ppb. The averageof these four PFOA measurements was 12.5 ppb. Twelve employees were re-tested eight weeks later to check for reliability of the analytical method. The findings suggested reliability (R" = .94) in the rangeofquantification (Figure Al in the Appendix). 1 000029 y Seventyaf etnsariofsPOo SgTyabed lel112.Cy r ad9H8ic HamanPopo, BeNo pttod eres bcmeasee ini Mor rae o 2000 2 Creo inporen rsnemtples Smad: dn 40 ou Stabs oFktesmnmr21ocpyr US.ods. Soma dn 53 s Sms dron 30 2008 ose sriKoalascnriiocr)s,(18m 051 dpoeirameici 10 [IE 2 Suiamps1957 oii pin : woz 5. Min rs Carr 60107 Sint ses wos 4Swe sng, 71 ovina , wot 5. (MRMFnpoolcFismeinmopoi, n07 soiatnessmmanon 31 ese g 6 MASTpositctipols,1n80 S nottaeobp 33 wa 23g ad2.A7.ClhaTn mmgileveiSihinaidgoo5vlier),o989i49 el GiSSiiidtioplss eliNmwbyops NNp xoae Commercial Pooled Serum Samples Six pooled sera samples, obtained from two commercial sources (Intergen and Sigma), were tested in 1998. No information was available about the donor pool, such as. age, sex or geographical location. Samples from Intergen came from donor pools consisting of approximately 500 individuals. Three Intergen pools showed PFOS levelsof43, 44 and 44 ppb (Table 24.2). Pools from another source, Sigma, were from an unknown number of donors. The pools from which the samples were drawn were 50 liters, suggesting a 45 ppb. minimum of 200 donors. The three Sigma pools contained PFOS at levels of 26, 28 and Blood Bank Pooled Sera Eighteen blood banks from various geographic areas across the continental United States and Alaska each donated three to six pooled samples that had from 5 to 10 donors per pool (Table 1.3). Altogether there were 68 total pools, representing 340 to 680 individual donors. All pools contained detectable levels of PFOS in tests performed in 1998. The range found in the pooled serum was 9 to 56 ppb of PFOS (sce table on next page). The location means ranged from 14 ppb in Santa Barbara, California to 52 ppb in Greenville, South Carolina. PFOA levels were detected in 20 (about one-third)ofthe pooled samples but quantifiable in only two samples (12 and 22 ppb). `The blood bank samples from the regional blood banks are not a statistically valid sample of the U.S. population, but do provide, to date, the best estimate of mean PFOS sera levels. The overall mean was 29.7 ppb of PFOS. If one wishes to make a comparison `with the organic fluorine levels in the published literature, this level of PFOS would contribute 19.3 ppb to the total serum organic fluorine level (0.65 x 29. 7). An approximate mean ofhistoric organic fluorine measurements from published literature is 37.6) 37.6 ppb. Ifthis were all PFOS, a PFOS sera level would have been 57.9 ppb (1.54 x i 000031 January 21,1999 Table IL3 PFOS (ppb) Levels from 18 Blood Banks [A[ nchorC age AKy[T~#o~fPo4 oledSam] ples | Rage 19-31 | Average | Billings, MT [sas [Cheyenne WY _ 175 sss | 23 5 J Corpus Christi. TX 3 Davenport, IA 3 2632 I 29 28-40 32 ] [EastOrange,CA 17 5 Grand Rapids MI [4 | 5027 | 2 7Tiss | 26 | Kansas City MO |1 775435| 30 [LasVegus NV 177 39-50 75 -- | osas| 46 | Meridian, MS 3 I 27:36 39 [MiNnenwaerakp,olDiEs MN| 6 | aise | 46 2 Omaha, NE [sey SontaBarbara. CA | 3| Santa Rosa, CA 3 i3l6 23:26 | 2wa] Scottsdale, AZ. Overall mean = 29.7ppb 28-47 37 Historical Samples Nine setsofhistorical samples have been analyzed for PFOS in 1998. These are summarized in chronological order below. Korean War era U.S. military recruits, 1945 10 1951. Ten pooled samples consisting of ten individual samples each were measured. All were below 1 ppb, the limits of detection. Swedish samples, 1957. Ten individual samples ranged from below limit ofdetection to 4.1 ppb. Two were below detection limit (1 ppb). The mean was 1.98 ppb. Michigan Breast Cancer Study, 1969-1971. Five individual samples ranged from 11.8 1.59.4 ppb. The mean was 33.4 ppb. 19 000032 Janay21,1999 Swedish samples, 1971. Ten individual samples ranged from below detection limit a ppb) t0 2.8 ppb. Three were below detection limit. The mean was 1.13 ppb. MRFITpooled calibration samples, 1976. MRFIT (Multiple Risk Factor Intervention Trial) was a cardiovascular risk reduction program conducted in the 1970's and early 1980's. The numberofdonors per pool is unknown. Six pooled calibration samples ranged from 13.710 55.5 ppb. The mean was 30.9 ppb. MRFIT pooled calibration samples, 1950. Three pooled calibration samples ranged from 13.810 40.5 ppb. The numberofdonors per pool is unknown. The mean was 25.5 ppb. China samples, 1984. Six individual samples from Linxian province in rural China were all below limits ofdetection or quantitation. The samples were from an NCI study on nutrition and cancer prevention. MRFIT individual samples, 1985. Three individual samples from participants in the MRFIT study were obtained. One was below limitsofquantitation (5 ppb), the other two were 43.3 and 43.9. Assuming the LOQ for the low sample, the mean was 30.7. China samples, 1994. Six individual samples from Shandong province in rural China were all below limitsofdetection or quantitation. The samples were from an NCI study on nutrition and cancer prevention, In addition to the PFOS measurements described above, PFOA was also analyzed in these historical samples. PFOA was not found in any historical sample at a detection limit of 10 ppb. Comment 000033 January21,1999 Organic fluorine has been noted in human serum since the late 1960's. We have now. identified PFOS as a part ofthis organic fluorine fraction. PFOS-related materials were not produced commercially prior to 1948, and only in small quantities for several years thereafter. Therefore, itis not surprising that samples from 1948 to 1951 show undetectable levels. There was clearly an increase 20 years later; however the very limited data shows no further upward trend despite steadily increasing production volumes since this time. Due to limited knowledge on distributionofthis chemical in the body it is uncertain that body burden is adequately reflected by serum levels. Information on distribution and kinetics is needed to shed further light. The available data on this topic will be presented in the toxicology section. It would also beof interest to measure total organic fluorine and PFOS in the same sample to determine how much PFOS. currently contributes to the total organic fluorine content. PFOA may have been misidentified as a major component of organic fluorine in 1976 (Taves, 1976). Although detectable in some samples, neither historic nor current samples. confirm this as a major fraction, except in occupationally exposed employees. \ 000034 III. SUMMARY STUDIES OF MEDICAL SURVEILLANCE AND EPIDEMIOLOGY EHpoirdmeomnioelsoignicReIlnavteisotnigtaotiSoenroufmClPinFiOcSalLCehveemlisstieniMcsa.leHeFmluaotroolcohgeymiacnadl Production Emplovees Medical surveillance has been routinely performed on 3M fluorochemical production workers (in Decatur, Alabama and Antwerp, Belgium) with potential exposure to PFOS and/or to perfluorinated precursors that may metabolically degrade to PFOS. A recent study (Olsen et al, 1998) provided an analysisof hematology, clinical chemistries and hormonal parameters in relation to serum PFOS as determined by high performance liquid chromatography mass spectrometry methods. These relationships were assessed in fluorochemical production employees from two time periods, 1995 (N = 178) and 1997 (N = 149). * In 1995, for Antwerp and Decatur, the mean serum PFOS levels were 1.93 and 2.44 ppm, respectively. In 1997, the mean serum PFOS levels were 1.48 and 1.96 ppm, respectively. Descriptive simple and stratified analyses, Pearson correlation coefficients, analysis ofvariance and multivariable regression were used to evaluate for possible associations between PFOS and each hematological and clinical chemistry test and hormonal assay. Age, body mass index, current alcohol consumption (drinks per day) and cigarette use (cigarettes smoked per day) were potential confounding factors that were considered in the analyses. Four categorizations of serum PFOS levels were assessed in relation to the response variables: 0- < 1 ppm; 1 - <3 ppm; 3 - <6 ppm; and > 6 ppm (Table ILL). (Note: other PFOS categorizations were used with comparable findings). In 1995, mean serum PFOS levels by category were 0.49 ppm, 1.82 ppm, 4.12 ppm and 8.17 ppm, ?`blgHoaeommdmatuaorgcelraiutnai,mtyho!egemrno,agnlscorfbeeiarnta,isnri,neaed,sbpglaloruoacdtoseceea,lmsci,hnoowltherisattnersofblel,roaolsdoe,wceadllelansnsaiinntedy aplmliiaptnoeopltreorttaencisonfuser,rta,hsieag,lhkaodletinnaseiatpnyhdolsdipiphroaeptcratostbeei,liinruabnind, wiglycerides, cortisol, dehydrocpiandroserone sulfa, estradiol, flit stimulating hormonc, 17-alpha 2 000035 J respectively. In 1997, mean serum PFOS levels by category were 0.52 ppm, 1.78 ppm, 3.87 ppm and 7.20 ppm, respectively. For both years, 95 percent of the employees' serum PFOS levels werebelow 6 ppm. The two plant populations differed by age, body mass index and alcohol consumption which resulted in differences, as expected, in several clinical chemistry parameters (Table II1.2). When analyzed in aggregate, the two plant populations showed no consistent significant associations for both years between the clinical chemistries andhematology parameters and the employees' serum PFOS levels (Figure Al, Appendix). Total bilirubin levels appeared to trend downwards but upon further analysis this was restricted to Decatur employees and the values were all within the reference range. Multivariable regression models were fitted with PFOS analyzed as a continuous variable using linearas well as non-linear transformations in order to maximize the possibility offinding associations between PFOS and the parametersof interest adjusting for potential confounders. No consistent associations were observed by plant and/or by year. In 1995, hormone values were also obtained from a subsampleof employees with the higher PFOS measurements. After adjusting for age and body mass index, no significant associations were observed between hormones and serum PFOS levels. `The findings from this study suggest that, among these Antwerp and Decatur male fluorochemical production employees, significant hematological, clinical chemistry and hormonal abnormalities were not associated with serum PFOS levels upto6 ppm. It was not possible to derive inferences from the few employees with serum PFOS level>s 6 ppm. Limitationsof this study included its cross-sectional design, the voluntary participation rates and the few subjects with levels > 6 ppm. [oySogeotonv, niangeborton. parooline rman binson, eens, 000036 | Tein | Disibionof EmplobyeYosr,LocsdatOSiExoponseLew pe) ]| BbOeSl NSAE6lb0in:NAempaSONMDoeanee a aa a Sih NoEangyinres Whdopvey sw, Warl a FE msLeeawmmmemo_3a1 4 m55 Bo3 x a9la wi TG wo 0 Gon, F8I x To 5 ony HoWowm oouu aWl o 5 aq% 822 g2 a Table 111.2 Mean Values of PFOS, Demographic, Serum Chemistry Antwerp and Decatur, 1995 and 1997 and Hematologic Examinations Parameters for Variable PFOS (ppm) 1995 Data Antwerp Decatur 1.93 244 1997 Data Antwerp Decatur 1.48 1.96 Age 37ene 4s ene a4 BMI 2390 292 235 300 Cigarettes ar 79 55 66 Alcohol 1300 02 Lise ol Alk Phosphatase 75%+* 97 700% 87 GGT a a8 26+ 36 AST 26+ 29 27 26 ALT a4 a 31 34 Total bilirubin 086*r oss 080% 058 Direct bilirubin~~ 0.22 021 01s 012 BUN 17.00 148 149 14.1 Creatinine 09% Ll 0.9% Lo Glucose siren 92 sree 9 Cholesterol 214 218 206 215 LDL 138 136 134 137 HDL Saree 43 00+ a Triglycerides Tse 187 iss 192 2 000038 January 21, 1999 Table IIL.2 (Continued) Variable 1995 Data Antwerp Decatur 1997 Data Anwerp Decatur Hematocrit arse 46 46* 4s Hemoglobin 154 152 154 153 RBC 49 50 51 51 MCH Sate 307 305 304 MCHC mvc 29M 334 957 918 33 37 96 90.0 WBC 64m 75 65 64 Platelets 24 229 237+ 217 *P<.05; **p<.01; ***p<.001 (Antwerp compared to Decatur) 2% 000039 Mortality Study of Employees at the 3M Plant in Decatur, Alabama A retrospective cohort mortality study of 1,957 employees who worked at least one year at the 3M plant in Decatur, Alabama was conducted by epidemiologists at the University of Minnesota School of Public Health (Mandel and Johnson, 1995). The purpose of this study was to determine whether the mortality experienceof these employees was significantly different from that which would be expected ina comparable population. The cohort was followed from March 1, 1961 through December 31, 1991. Table A2 (Appendix) provides a descriptionofthe final employment and vital status for the male (n = 1,639) and female (n=318) employees. These totals include both the Decatur chemical and film plant employees. The two plants are physically distinct entities (approximately 300 yards apart) located at the Decatur site. The vital status of 99.7% of the cohort was identified. The male and female employees contributed 33,108 and 4.807 person- years experience, respectively (Table A3, Appendix). The majorityof the male employees were hired between 1965 and 1974 (Table Ad, Appendix) whereas the majority of the female employees were hired between 1975 and 1979 (Table AS, Appendix). Altogether there were 74 deaths (70 males and 4 females). Among males, SMRs were below the null value for all major causes of death regardless of the comparison population used to calculate the expected values (Table [11.3). Data analyses were also restricted to examining the mortality experience of employees who ever (n = 1,050) and only (n = 485) worked in the Decatur chemical plant (Tables A6 and A7, Appendix). Neither analysis resulted in significantly (p < 05) elevated SMRs. There were no analyses conducted by durationof employment within the chemical plant. The data were also restricted to employees who were ever (n= 1,116) and only (n = 547) employed at the Decatur film plant (data not shown). Noneofthese analyses resulted in significantly elevated SMRs. There `were four deaths among female employees (SMR = 62.6; 95% CI 17.1 -- 160. 4). of these four deaths, three were from external causes (SMR = 213.4; 05% CI 44.0 623.6). By examining the death certificates for the specific causesof death and 7 000040 January 21,1999 circumstances surrounding the deaths, it was determined that the excess mortality among females due to external causes was not work-related. The only recommendation by the University of Minnesota investigators was that the mortality experience of Decatur plant employees be updated in 1998 which would extend the vital status search, via National Death Index records, through 1996. In addition to the Decatur mortality study, the University ofMinnesota investigators have also followed the mortality experience ofa cohort ofemployees associated with fluorochemical production at the 3M Cottage Grove (Gilliland and Mandel, 1993). Unlike the Decatur plant, the greatest potential for exposure at Cottage Grove is to PFOA (perfluorooctanoic acid) although the plant does produce PFOS. The total cohort consistedof2,788 male and 749 female workers employed between 1947 and 1983. There was no significantly increased cause-specific SMR for either `men or women. Ina proportional hazard regression model, ten years of employment injobs associated with the chemical division was associated with a 3.3-fold increase (95% CI 1.02 ~ 10.60) in prostate cancer mortality compared to no employment in the chemical division. The authors suggested that this finding may be biologically plausible with exposure to PFOA as animal toxicology and human data had associated PFOA with reproductive hormone changes (Gilliland, 1992). However, the authors urged caution in the interpretation of this result due to the `nonspecificityofthe exposure index as wel as the few subjects (n = 4) considered `exposed.' Subsequent research on the Cottage Grove male workforce involved with PFOA production has provided reasonable assurance that there are no significant hormonal changes associated with PFOA at the serum levels measured (Olsen etal. 1998). Current and proposed human health and exposure research initiatives are found in Section VI 2 000041 Janay21,1999 Table IIL3. Selected Cause - Specific SMRs for Men by Comparison Population (n = 1,639) Causeof Death All Causes of Death All Malignant Neoplasms Observed Deaths 70 15 Cerebrovascular Disease 1 All Heart Disease 17 Respiratory Disease 0 External Causes 23 SMR (95% Confidence Interval) us. 62.9 (49.0,79.5) 68.4 (383,112.8) 335 (08,1868) 59.1 - (344,947) (0.0,91.2) 74.5 (48.2, 110.0) Alabama 54.8 (42.7693) 60.9 (34.1,1004) 27.8 (0.7,1552) 532 -- (31.0,85.1) (0.0,80.2) 59.0 (38.2,87.1) `AL Regional Counties 520 (405,657) 59.9 (33.5,98.8) 26.5 (0.7, 147.5) 49.1 a (28.6, 78.6) (0.0,73.9) 55.0 (35.6. 81.3) 000022 January 21,1999 IV. SUMMARY OF TOXICOLOGY DATA ON PFOS This section summarizes the existing database with respect to the potential `mammalian toxicity of PFOS. Toxicology data are valuable in identifying potential health effects and provide a basis for risk characterization. The use of his information in isk characterization will be described in Section V. With few exceptions, past toxicology studies either did not characterize blood and tissue concentrations associated with exposure or were not completely specific in characterizing blood and tissue concentrations. For these past studies, blood and tissue concentrations can only be estimated based on limited pharmacokinetic data `and results from more recent studies, mostof which have been designed to include a pharmacokinetic component to obtain highly sensitive and specific identification of PFOS in blood and tissues. Also, the extent of potential interspecies and intraspecics variability in pharmacokinetic handlingof PFOS is not presently well defined. Comparison of serum concentrations of PFOS from toxicity studies to measured human serum concentrations may be an interesting and convenient comparison; however, body burden and/or target organ tissue concentration may be more meaningful in characterizing risk. The available body of animal toxicity data cannot be related quantitatively and without uncertainty and speculation to the concentrations of PFOS in human blood reported in Section If and the results ofepidemiologic. investigation as reported in Section IIL. Despite the current limitations in the ability to characterize risk based on the relationship of blood and tissue POS concentrations from toxicity studies to human blood concentrations of PFOS, the existing toxicology database on PFOS does provide valuable insight into potential health hazards and forms a foundation for risk characterization. 30 000013 sary 21,1999 It should be mentioned that the specific sources of PFOS identified in the serum of `non-occupationally exposed humans are currently unknown. The PFOS molecule itself may not be the primary source. PFOS in the serum of non-occupationally exposed humans may derive from metabolism of higher molecular `weight molecules present in industrial, commercial and consumer products or manufacturing by- products. The studies described in the following discussion reflect the toxic responses resulting from direct dosing with PFOS. The studies that are summarized in this section`were performed with the potassium salt ofPFOS unless otherwise annotated. Studies on related compounds are considered outside the scopeofthis discussion and will only be mentioned when they provide valuable insight into the toxicity of PFOS. Some data will be discussed from studies which are currently in progress or incomplete. `Tabular summariesofrodent repeated-dose toxicity studies, primate repeated-dose toxicity summaries and developmental and reproduction studies will be found at the end of this section (Table IV.9, IV.10, and IV. 11, respectively). In addition to these summary tables, Tables IV. 12 presents the resultsofrodent and primate 90-day sub- chronic studies organized by increasing nominal mg/kg dose level. A list of studies initiated in 1998 can be found at the end of this section in Table IV.8 and summary descriptionsof these studies can be found in the Appendix. Pharmacokinetics of PFOS `The absorption, tissue distribution, potential metabolism and excretionof PFOS has been studied most extensively in rats by both radiolabel and direct quantitation. Limited data relating oral dose to serum and liver concentrations of PFOS in the `cynomolgus monkey is available from a recent 28-day rangefinder capsule dosing study. In addition, serum PFOS concentrations in three retired male 3M chemical workers have been followed in an attempt to estimate an elimination rate constant for the human. * 000014 January 21, 1999 In general, PFOS: is well absorbed from the digestive system; has some limited potential for dermal absorption; distributes predominantly to the blood and liver, with liver concentrations being several times higher than serum concentrations; appears to have a serum half-life shorter than the eliminationhal -life after a single dose; appears to have extended eliminationhalf lives in rats, monkeys and humans; shows slight but consistent sex differences in serum and liver concentrations on repeated dosing in the rat but not the cynomolgus monkey; appears to undergo enterohepatic recirculation; does not appear to be metabolized, including conjugation reactions. Absorption: At least 95% ofa single oral dose averaging 4.2 mg/kg [/CJPFOS administered to two groups (24 hour and 48 hour sacrifice) of3 male Charles River CD rats (248-315 g, mean = 285 g) was absorbed within 24 hours (Johnson and Ober, 1979). The radiochemical purityofthe ['`CJPFOS used in this and the other radiolabel studies listed below was >99% (Johnson and Behr, 1979) PFOS was applied to skin that had been clipped freeof hair as a suspension in water at 5,000 mg/kg covering 40%ofthe total body surface areaoften male and ten female rabbits. An impervious plastic sheet occluded the skin for 24 hours and was then removed. Animals were maintained and observed foar 28-day period. Blood samples were obtained on days 1,7, 14, and 28. Analysis for total blood fluoride was performed on the day one and day 28 samples from a single male and single female. Total serum fluoride values for the male were 10.3 ppm for day one and 130 ppm for day 28. The respective values for the female were 0.9 ppm and 128 ppm. Although this study indicated some dermal absorption, it is limited in that the values from only 2 000045 January 21,1999 two animals were measured, and only from the day one and day 28 samples (O'Malley and Ebbens, 1980). Three male and three female albino rabbits per dose group were exposed dermally underocclusion for 24-hours to an aqueous suspension of PFOS (0.06 %) at doses equal 10.0, 0.003, 0.06, and 0.3 mg POS / kg and held for 28 days (Glaza, 1995) Liver samples taken at term were analyzed for total organic fluorine. No quantifiable organic fluorine could be detected in the 28-day livers by combustion or by electrospray mass spectroscopy (Johnson, 1995) Distribution: By 89 days aftear single iv dose of PFOS-'C (mean dose, 4.2 mg/kg) six Charles River CD male rats (initial body weights 262-303 g, mean = 288 g) excreted amean of 30.2%ofthe total carbon-14 via urine. Mean cumulative fecal excretion was 12.6%. At 89 days, mean tissue concentrationoftotal carbo1n4 expressed as pg PFOS-'"C equivalents/g were: liver, 20.6; plasma, 2.2; kidney, 1.1; lung, 1.1; spleen, 0.5; and bone marrow, 0.5. Lower concentrations (0.5) were `measured in adrenals, skin, testes, muscle, fat and eye. No radioactivity (<0.05) was detected in brain. The carbon-14 in liver and plasma represents 25 and 3 percent of the dose, respectively (Johnson etal, 1979). Serum PFOS concentrations were measured throughout a 28-day oral rangefinder study in which male and female cynomolgus monkeys weighing 2.1 to 2.4 kg were given capsules containing PFOS at either 0.0 mg/kg/day (two males and two females), 0.02 mg PFOS/kg/day (three males and three females),or2.0 mg/kg/day (one male and one female) (Thomford, PJ, 1998). The monkeys dosed with PFOS (0.02 and 2.0 me/ke/day) demonstrated an extremely linear (r-squared > 0.99) increase in serum concentration throughout the exposure period (28 days). There was no apparent sex difference and the individual slopesofthe cumulative PFOS dose versus serum PFOS concentration curve appeared to be virtually identical between the monkeys in the two dose groups. The average slope of the curve in the 0.02 mg/kg/day group (n = 6) was = 000046 January 21, 1999 5.22.0.74 ppm PFOS in serum per mg/kg cumulative dose, and for the two monkeys in the 2.0 mg/kg/day dose group, the average was 5.40 0.61 ppm PFOS in serum per mg/kg PFOS cumulative dose. At the endof the 28-day dosing period, serum concentration in the 0.02 mg/kg/day dose group reached approximately 3 ppm and in the 2.0 mg/kg/day dose group, serum concentrations reached approximately 300 ppm. "This data suggests a volumeofdistribution of 0.19 L/kg for continuous dosing over a dose range of two orders of magnitude for the male and female cynomolgus monkey. This is calculated as follows: One (1) mg/kg results in 5.3 ppm serum PFOS; "This isequivalent to 5.3 mg/L of serum (mg/L. = ppm weight/volume); One (1) mkg given to 2.2 kg monkey results in a total dose of2.2 mg; Va (L) is calculated as 2.2 mg/ 5.3 mg/L = 0.41 L; Normalizing this for body weight gives 0.41 L/22 kg = 0.19 L / kg = Va (L/ ke); Va for a 60 kg woman is estimated to be 0.2 L./ kg x 60 kg = 12 L = Va (L). Metabolism: Preliminary data from analysisofurine, feces and tissues of rats as well as the inherent stabilityofperfluorinated anions suggest that PFOS is not metabolized (Johnson etal., 1984). Exposure of primary human and rat hepatocytes to POS did not result in further metabolism (Gordon, 1998). Analysis by LC/MS of serum and liver samples from studies currently in progress have not revealed any evidence of metabolism. Excretion: In the previously mentioned study (Johnson etal, 1979) single intravenous doses (mean 4.2 mg/kg) of [*CJPFOS in 0.9% NaCl were administered to male rats. By 89 days after dosing, 30.2% ofthe administered "*C had been excreted in the urine and 12.6% had been excreted in the feces. 4 000047 January 21, 1999 `Whole body elimination in the male rat appeared to be biphasic. Initial redistribution from the plasma yielded a plasma elimination hal-life of "*C of 7.5 days following single oral administrationof ["C]PFOS (mean dose 4.2 mg/kg) to male rats (Johnson and Ober, 1979). In the aforementioned intravenous study, eliminationofonly 42.8 9% of the dose through urine and feces after 89 days indicates that thehal-lfe of elimination from the body is > 89 days in the male rat. Serum PFO levels in three retired male 3M chemical workers have been followed for five andone-half years and suggest a mean serum elimination half-life of 1,428 days. Over that time period that these serum samples were taken and analyzed, the analytical method changed from thermo-spray mass spectrometry to electro-spray mass spectrometry, and the analytical laboratories changed. These changes should not have affected the values reportedto any appreciable extent. Since urine and feces have not been followed, i is difficult to ascertainifthis represents a true elimination half-life from the body; however, this is the closest value for elimination half-life applicable to humans in existence. The actual data from these three retired chemical workers is presented in Table IV.1 3s 0006048 January 21,1999 Table IV.1 Serum Elimination of PFOS in Retired 3M Chemical Workers. Serum (PFOS] in mg/L (ppm) Sap: [38 92093 [60 394 [40 61995 [30 12597 | 26 Serum Elim. | - 0.0132/month Constant (A) Serum Elim.| 52.5 mo/1575 d Tia 02 [16 11593 | 14 si595 [07 wis7 [07 0.0136/month 509 mo/15274 S82 [42 129093 [33 5495 [17 1023/97 [14 ~0.0176/month, 394 mo182d Fecal and total excretion of '*C were markedly increased in male rats administered cholestyramine (~ 2.7 g/kg/d) in their diet following single intravenous doses of [MCJPFOS. The results suggest that there was significant enterohepatic circulation of PFOS (Johnson and Gibson, 1980, 1984). Cholestyramine administered at 4% by weight in feed to male rats decreased the retentionof carbon-14 in liver, plasma, and red blood cells and increased the eliminationofcarbon-14 via feces after iv dosing with PFOS-'C. Groups of five rats (twelve-week old Charles River CD averaging 320 g) were dosed intravenously with PFOS-'C (mean dose, 3.4 mg/kg). Groups of five control rats were dosed similarly but were not treated with cholestyramine. Rats were sacrificed at 21 days post dose. The mean liver, plasma, and red blood cell concentration as well as fecal and urinary excretionof '*C for cholestyramine-treated. * 000019 January 21, 1999 rats were compared to mean control rat values. Mean cholestyramine-treated rat 'C concentrations in liver (9.4yg/g). plasma (0.9g/ml), and red blood cells (0.3 ug/g) represent a decrease from mean control rat concentrations of 3.8, 7.7, and 6.0 fold, respectively. Fecal elimination (75.9% with cholestyramine treatment) was increased 9.5 fold. The extent of urinary "*C elimination, as a resultofthe relatively high rate offecal elimination of 'C was lower in cholestyramine-treated rats. The extent of total elimination of '*C (urine plus feces) was higher in the cholestyramine-treated rats. Since cholestyramine is approved for use in humans as a cholesterol lowering agent, these results in rats support the concept of testing cholestyramine in humans to promote excretion of PFOS (Johnson et al, 1980), 5 0006050 January 21, 1999 Acute Toxicity Acute Oral Toxicity: PFOS was administered by gavage in 20: 80, acetone: com oil suspension to five male and five female rats at dose levels of 100, 215, 464, and 1000 mg/kg. Observations of toxicity were made over a 14-day period. All rats in the 1000 and 464 mg/kg dose groups died. The signs before death included hyperactivity, decreased limb tone and ataxia. At215 mg/kg, threeoften animals died. At 100 `mg/kg, no deaths were observed. The combined acute oral LDSO0 in male and female rats is 251 mg/kg (95% C.L: 199-318 mg/kg). PFOS is considered moderately toxic on acute oral administration under the conditions of this study (Dean et al., 1978), Acute Dermal Toxicity: Ten male and ten female albino rabbits were clipped free of hair and an aqueous suspension equivalent to 5 g/kg PFOS was placed over 40% of the total body surface area, occluded with an impervious plastic sheet and left in contact with the skin for 24 hours, then removed. Animals were maintained and observed for a 28-day period after which they were necropsied. Blood samples were obtained on days 1,7, 14 and 28. Day 0, 7, 14 and 28 body weights were recorded. Hyperactivity was noted in 5 of 10 males on day 6. All animals recovered by day 7 and remained asymptomatic throughout the study period. Weight gains were observed for all rabbits. No visible lesions were noted at necropsy. Analysis of total blood fluoride from day 1 and day 28 blood samplesofone male and one female indicated that PFOS was absorbed through the skin, reaching blood concentrations of approximately 130 ppm after 28 days (see section above on absorption). PFOS can be considered practically non-toxic on single dermal contact (O'Malley and Ebbens, 1980). Acute Inhalation Toxicity: Groupsoffive male and five female Sprague-Dawley rats were exposed by inhalation for one-hour to nominal exposure concentrations of 24,7.1,6.5,4.9,2.9, 1.9 and 0.0 milligrams PFOS per literofair. The rats were observed hourly for the first four hours and daily thereafter for 14 days. All rats * 000051 January 21,1999 exposed to 24 milligrams per liter died. Partial mortality was produced at nominal exposure concentrations of 7.1 (80%), 6.5 (80%), 4.9 (20%) and 2.9 (10%) milligrams per liter. Symptoms observed during the exposure and post-exposure periods were labored breathing, reduced activity, excessive salivation and lacrimation, mucoid and red nasal discharge, yellow stainingof the ano-genital fur, and dried red material on the facial area. The most frequent abnormal necropsy observations were of lung and liver discoloration. Lung discoloration was also observed in a high numberofcontrol rats and thus may not be treatment-related. The nominally determined median lethal concentration (LCS50) foar one-hour exposure to PFOS was determined to be 5.2 milligrams per liter with 95% confidence limitsof4.4 milligrams per liter and 6.4 milligrams per liter (Rusch and Rinehart, 1979). Primary Irritation Ocular Irritation: PFOS was found to be mildly irritating to the eyes of albino rabbits when tested according to standard Federal Hazardous Substances Act guidelines. The ocular iritation was limited to the conjunctivae in the six test rabbits. Irritation was noted at the 1, 24 and 48 hour post-instillation reading times. The `maximum irritation score was 9.3 out ofa highest possible score of 110 at the 24 hour reading. By 72 hours post-instillation all readings were zero (Biesemeier and Harris, 1974), Dermal Irritation: PFOS was found to be non-iritating to the skin of albino rabbits `when tested under conventional Draize procedures. No signsof dermal irritation were: observed in any of the test animals at any time during the study period. The primary skin irritation score was 0.0 outof a highest possible score of 8.0 (Bicsemeier and Harris, 1974). Genotoxicity Gene Mutation: PFOS was not mutagenic in Salmonella typhimurium strains TA1535, TA-1537, TA-1538, TA-98, TA-100 or in Saccharomyces cerevisiae strain D4 0006052 January 21, 1999 ina standard plate incorporation assay with or without metabolic activation. (Jagannath and Brusic, 1978) Chromosomal Effects: PFOS did not cause chromosomal aberrations in an in vivo mouse bone marrow micronucleus assay. PFOS in water was administered by oral gavage at 0,237.5, 475, and 950 mg/kg to five male and five female mice for cach dose harvest time group. The mice were euthanized 24, 48 and 72 hours after dosing for extractionof bone marrow. No increase in bone marrow polychromatic erythrocytes was observed (Murli, 1996). Repeated-Dose Toxicity A number of repeated-dose toxicity studies have been conducted with PFOS and will be discussed in this sub-section. A tabular summaryofthese studies will be found at the endofthis section in Table IV. for rodent studies and Table IV.10 for primate studies. In addition to these summary tables, Table IV.12 presents the results of rodent and primate 90-day sub-chronic studies organized by increasing nominal mg/kg dose level. 90-Day Dietary Study in Rats: PFOS was fed in the diet at levels of 0, 30, 100, 300, 1,000 and 3,000 ppm to groups of five male and five female Charles River CD rats for 90 days. These doses represent approximately 0, 2,6, 18, 60 and 180 mg/kg/day. The rats were observed twice daily for overt signsoftoxicity and mortality. Individual body weight and sex group food consumption were recorded `weekly. Hematological, biochemical and urinalysis studies were conducted prior to commencement of dosing, at one month and at study termination. Atthe 30 ppm (approximately 2 mg/kg/day) dosage level, no rats showed any `compound related changes in appearance or behavior. Mean body weights were slightly lower when compared to the controls. At one month, one female rat showed a " 006053 January 21,1999 slightly elevated blood glucose, and one male rat showeda slightly elevated serum alkaline phosphatase. At three months, one male rat had slight to moderate elevations in blood glucose, blood urea nitrogen and gamma-glutamy transpeptidase activity. At the 100 ppm (approximately 6 mg/kg/day) dosage level, mean body weight and food consumption was significantly lower than the control group. One male and two females died. Slight increases in creatinine phosphokinase (CPK) and serum alkaline phosphatase activity, slight to moderate increases in blood glucose and blood urea nitrogen, and slight to marked increases in plasma transaminase activities (AST and ALT) were seen at one month. At three months, all rats in the 100 ppm dose group had slight to moderate decreases in hemoglobin, hematocrit and erythrocyte counts, and slight to moderate increases in transaminase activities were seen for twoof the three surviving female rats. At dosage levels of 300, 1,000 and 3,000 ppm (approximately 18, 60 and 180 mg/kg/day) all rats died prior to scheduled termination of the study. Timeofdeath `was dose-related. Overt clinical observationsof toxicity included emaciation, convulsions, altered posture, red material (right eye and/or mouth), yellow material in the ano-genital region, increased sensitivity to extemal stimuli and reduced motor activity. Compound-related gross changes such as emaciation and areas of discoloration involving the stomach and liver were observed among treated rats that died prior to sacrifice. Similar changes were also observed in the liver ofa few rats sacrificed at terminationof study from the 30 and 100 ppm groups. Morphological changes consistionfg centrilobular to midzonal cytoplasmic enlargement (hypertrophy) of hepatocytes and necrosisofliver cells was present in all PFOS dose groups. The incidence and relative severity of the above lesions were more evident among male rats. In addition, rats from the 300, 1,000 and 3,000 ppm dosage levels displayed compound-related changes involving the primary (thymus, - 000054 January 21,1999 bone marrow) and secondary (spleen, mesenteric lymph nodes) lymphoid organs, stomach, intestines, muscle and skin (Goldenthal et al, 19784). Two-Year Dietary Study in Rats, Four and Fourteen Week Results: Results are available from four-week and fourteen-week interim sacrifices in Sprague-Dawley rats which are partofan on-going 104-week dietary study (Covance, 1998). In these sub-studies, groups of five male and five female rats (four and 14-week sacrifice groups) and ten male and ten female rats (14-week sacrifice groups) were exposed to either 0, 0.5, 2, 5, or 20 ppm PFO in the diet for four or 14 weeks. Clinical observations were made twice daily. Body weights and food consumption were measured once a week. Organ weights were measured at term. Samples were taken at both sacrifices for PCNA, liver palmitoyl CoA oxidase activity, serum and liver PFOS concentrations and at the 14-week sacrifice for hematology, clinical chemistry (including urinalysis), and histopatholgy. During weeks 4 and 14, blood and urine were collected for hematology, clinical chemistry, urinalysis, and urine chemistry tests from ten animals/sex in Groups I through 5. Five animals/sex in Groups 1 through 5 were sacrificed during Week 4; livers were collected and weighed. A portionofthe liver was prepared for PFOS analysis, a portion was shipped to Pathology Associates Intemational for hepatocellular proliferation rate (PCNA) measurements, and a portion was used for determinationofpalmitoyl-CoA oxidase activity. At week 14, necropsies were performed on five animals/sex in Groups | through 5. At necropsy, macroscopic observations were recorded, organ weights were obtained, and tissues were placed in fixative as specified by the protocol. In addition, liver samples were collected for PFOS analysis, hepatocellular proliferation rate `measurement, and palmitoyl-CoA oxidase determination. Microscopic examinations were done on selected tissues from animals necropsied during week 14. The tissues were adrenals, brain, eyes, kidney, liver, mesenteric lymph node, pancreas, spleen, testes, and ovaries. In addition, microscopic examinations were done on tissues from animals that died or were sacrificed due to poor health. @ 000055 January 21,1999 There were relatively few statistically significant or otherwise notable differences for clinical pathology results between the control and treated groups. Although none of the statistically significant differences for high dose animals were consistent at both testing intervals, it was considered likely that administrationofthe test material was associated with mildly higher urea nitrogen at Week 14 for males and females fed 20.0 ppm and moderately lower cholesterol and mildly higher alanine aminotransferase at Week 14 for males fed 20.0 ppm. There were no correlative `microscopic renal findings for the minor change in urea nitrogen. The findings for alanine aminotransferase and cholesterol were likely associated with the histopathological findings of hepatocellular hypertrophy and vacuolation. Of uncertain relationship to the test material was mildly, but statistically, higher absolute neutrophil count for males fed 20.0 ppm. Females were unaffected, and there were no correlative microscopic findings for this small difference. All other statistically significant differences for clinical pathology results between the control and treated groups were considered incidental. Lower glucose at Week 4 for `high-dose males and lower aspartate aminotransferase at Week 4 for high dose females were considered incidental because they were not present at Week 14. Higher albumin at Week 14 for high dose females was considered incidental because the low dose females had a similar, but higher, statistically significant difference for albumin. Table IV.2 and IV.3 summarize key clinical pathology results at 14 weeks for males and females, respectively. 000056 January21, 1999 Table IV.2 Clinical chemistry and hematology in Male rats at week 14 (n=10) rr 7 [1] `Average Value and Std Dev.Dibety)Dose Group (ppm PFOS in |ANOVA | [Parameter | 0Oppm |0.5ppm[2ppm| Sppm |20ppm |Pr>F | [ALT SGPT) AST (SGOT) CHOL 1452 |1440 46134 54%3 [18+18 [54+19 [014| * Mean significantly different (Dunnetts' Test,p < 0.05) than the control group value, Table IV.3 Clinical chemistry and hematology in female rats at week 14 (n=10) 1 `Average Value and Std Dev. by Dose Group (ppm PFOS in Dict) [Former| Spo T0Swom |2pom[Soom [ ppm [PF] 0.0057 [ALATSTGS(GSGPODT))[3ax2 [50+11 [30=5 [3635 [335 [021 0.092 | oer | [[GN LSUEG"Tioo08=0361096204[1[ 0020 12 [15 05233[|02 o506=280 01[101 0604203 71 [031 |NA | [PcO[A 30:G 26[_ 1:0_ 8 T 16:2i 6 [8 522= 9 [i 0i06| * Mean significantly different (Dunnetts T-test, p < 0.05) than the control group value, Body and Organ Weights: Terminal body weights at 14 weeks for the 2 ppm dose group were reduced in comparison to controls by 4.5% in the males and 11.7% in the females. Absolute and relative liver weights were significantly increased by `approximately 35% in the males given 20.0 ppm PFOS in the diet. In females given a 000057 January 21, 1999 20.0 ppm, the absolute liver weights were increased by approximately 30% over control values, but only the liver-to-body weight percentage was significantly increased. The absolute spleen weight was significantly decreased in the females given 20.0 ppm, as was the absolute lung weight in females given 2.0, 5.0, or 20.0 ppm. Spurious significant increases in left thyroid/parathyroid-to-body weight ratios were seen in females given 5.0 or 20.0 ppm. Macroscopic Observations: There were no macroscopic observations that could be attributed to the administrationofthe test material. Microscopic Observations: Test material related histomorphologic changes were limited to the liver in the males given 5.0 or 20.0 ppm and in the females given 20.0 ppm. The changes consisted of hypertrophyofhepatocytes in centrilobular areas, and midzonal to centrilobular hepatocytic vacuolation. The incidence and severity of the changes tended to be greater in the males. `There were no apparent test material-related lesions in the remaining tissues examined. `Serum and liver concentrationsof PFOS increased with dose and with length of exposure, and liver values were significantly higher than corresponding serum values. These analytical results are presented in Tables IV.4 and IV.5 for serum and liver, respectively. as 006058 January 21, 1999 `Table IV.4 Mean Serum PFOS Concentrations (ppm) After 4 and 14 Weeks Dietary Exposure Serum (PFOS] in ppm by Dietary Dose Group (ppm PFOS in Dict) n= 5persexper dose group Time Dose 0.5 ppm 5 ppm Interval _| Group 8.95231| 46.4846 [Female [0.040.0031.90022 |7.50%0.053 59.4+846 [14Weeks[Male |<MDL** 1422083 [17.9%123 [4565.73 [Fe [545+m 219"[6a 65 1.0l 8[269e 4226 [629619 [216+21.9 "0 ppm female [PFOS] in serum at 14 weeks without one outlying value (r=4) is 0.59 = 0.59 ppm. *#MDL = Method Detection Limit, which was 15 ppb. Table IV.5 Mean Liver PFOS Concentrations (ppm) After 4 and 14 Weeks Dietary Exposure Liver [PFOS] in ppm by Dietary Dose Group (ppm PFOS in Diet) n=5 per sexper dose group Time | Dose 05ppm | 2ppm Sppm 20 ppm Interval _|Group [fWeeekse| 011074 [11170 |3380 49+12 [Female [011z005 3162 [80:17 [35758 [14 Weeks |Male |0.4430.05 |26+4.4 77275 38733 | 600116 [Female [158%21 [19%34 68+3.0 36226 | 61850 90-Day Drinking Water Study with Lithium Perfluorooctane Sulfonate (LPOS) in Rats: The lithium saltofperfluorooctane sulfonate was administered to male and female Sprague-Dawley rats in drinking water for 90 consecutive days. With the exception of controls, male and female doses did not directly correspond and each dose group was either ten males or ten females. Female LPOS doses, expressed as mg/kg body weight, were: 0 (two separate controls, one added later as a concurrent control for the 0.6 mg/kg dose group); 0.02; 0.06; 0.2; and 0.6 mg/kg (added later). Male LPOS doses, also expressed as mg/kg body weight, were: 0; 0.3; 1.0; and 3.0. a6 006059 January 21, 1999 Clinical observations included weekly body weight, daily observation, eye examination (day 0 and one week prior to termination), and food and water consumption. Clinical chemistry was performed on blood samples taken at termination. Gross pathology and organ weights were noted at sacrifice and histopathology was performed on preserved tissues. All rats gained weight in the course of the study. There were no compound-related early deaths. Clinical pathology demonstrated effects on hematologic and serum chemistry `parameters in the mid-dose and high-dose males as well as the high-dose females Significant hematologic effects in male rats were: 1) decreased hemoglobin, hematocrit and RBC (0.3 and 1.0 mg/kg dose groups); 2) decreased mean corpuscular hemoglobin content (3.0 mg/kg dose group); and 3) higher eosinophil counts (0.3 mg/kg dose group). Significant hematologic effects in female rats were: 1) decreased hematocrit and RBC count (0.6 mg/kg dose group); decreased mean corpuscular volume (0.02 and 0.2 mg/kg dose groups); 3) decreased mean corpuscular hemoglobin and mean corpuscular hemoglobin content (0.02, 0.06 and 0.2 mg/kg dose groups); and 4) increased mean corpuscular hemoglobin and mean corpuscular hemoglobin content (0.6 mg/kg dose group). These hematologic effects did not have a consistent dose-related pattern. Significant serum chemistry effects in male rats were: 1) an elevation in alkaline phosphatase and blood urea nitrogen and decreased cholesterol (1.0 and 3.0 mg/kg dose groups); 2) increased total bilirubin (3.0 mg/kg dose group) and 3) decreased triglycerides (0.3, 1.0 and 3.0 mg/kg dose groups). There were no significant treatment-related serum chemistry effects in female rats. Gross pathology revealed the following organ-weight effects in male rats: 1) increases in absolute liver weights and liver weight relative to brain weight and liver weight relative to body weight (1.0 and 3.0 mg/kg dose group) and 2) decreased absolute heart weight, decreased heart weight relative to body weight and increased brain a 000050 January 21,1999 weight, kidney weight and testes weight relative to body weight (3.0 mg/kg dose group). Gross pathology revealed the following organ-weight effects in female rats: 1) increases in absolute liver weights and liver weight relative to body weight (0.06, 0.2and 0.6 mg/kg dose groups); 2) increased liver weight relative to brain weight (0.2 and 0.6 mg/kg dose groups); 3) decreases in absolute heart weight, heart weight relative to brain weight and heart weight relative to body weight (0.02, 0.06, 0.2 and 0.6 mg/kg dose groups); 4) increased brain weight relative to body weight (0.02 and 0.06 mg/kg dose groups) and 5) increased kidney weight relative to body weight (0.2 my/kg dose group). With the exceptionof liver, these organ weight changes were related to reduced body weight gain and were not associated with histopathologic alterations. The dose-related increases in liver weights and reductions in serum cholesterol and triglycerides are supported by the histopathologic findingofhepatic vacuolization in the 1.0 and 3.0 mg/kg male rats 90-Day Oral Gavage Study in Rhesus Monkeys: In an initial study, five groups consistingoftwo male and two female rhesus monkeys initially weighing between 2.75 and 4.10 kg were given daily dosesof0, 10, 30, 100 and 300 mg PFOS / kg by gastric intubation (gavage) as distilled water suspension. This study was terminated afer 20 days due to the death of all PFOS-dosed monkeys (Goldenthal, 1978). Time of death was dose-related. Monkeys in the 300 mg/kg/day dose group died between the second and fourth day. The 100 mg/kg dose group died between the third and fifth day. At 30 mg/kg, deaths occurred between the seventh and tenth day. The 10 mg/kg dose group died between days 11 and 20. All PFOS-treated monkeys lost weight. Clinical observationsoftoxicity were similar for all POS dose groups, and time of onsetoftoxic effects was dose-related. These toxic symptoms included anorexia, slight to severe decreased activity, frothy or food-like emesis, and occasional diarrhea. Prior to death, body and limb stiffening, general body tremors, convulsions and prostration were observed. The 10 mg/kg dose group included one `monkey who had black stools and one who developed facial erythema. Gross. " 000061 January 21,1999 pathology revealed a yellowish-brown discolorationofthe livers of several monkeys in the 100 mg/kg and 300 mg/kg dose groups. Organ weights appeared within normal limits; however, due to early deaths of PFOS-treated monkeys and the reuse of control monkeys on a follow-up study at lower dose levels, concurrent control data was not available. Histopathologic examination of tissues from treated monkeys did not reveal any consistent observations which could be directly related to an effect of POS, Congestion and lipid depletion of the adrenal cortex was seen in all dose groups and was considered agonal. The study was terminated after the deathof the last 10 mg/kg. dose-group monkey. After termination of the initial study after early deaths of all PFOS-treated monkeys, the control group monkeys were used in a follow-up study at lower doses (Goldenthal etal, 1978). In this study, five groups consisting of two male and two female rhesus `monkeys initially weighing between 2.55 and 3.75 kg were given daily doses of 0, 0.5, 1.5, and 4.5 mg POS / kg by gastric intubation (gavage) as distilled water suspension for 90 days. The monkeys were observed twice daily for general physical appearance, behavior and other clinical signs of toxicity. Body weights were recorded weekly. Hematological and biochemical studies and urinalysis were conducted once in the control period and at the end of the first and third monthofthe study. The monkeys treated at the 4.5 mg/kg/day dosage level died or were sacrificed in extremis between weeks 5 and 7ofthe study. These monkeys exhibited gastrointestinal symptoms including anorexia, emesis, black stool and dehydration from the first or second dayofstudy. These monkeys also exhibited decreased activity and showed marked to severe rigidity, convulsions, generalized body trembling, prostration and loss of body weight prior to death. The mean body weight decreased from 3.44 kg at the beginningofthe study 10 2.70 kg at week 5 of the study. All monkeys at the 4.5 mg/kg/day dosage level had decreased serum cholesterol values and serum alkaline phosphatase activity at one month. a 000052 January21, 1999 All monkeys at the 1.5 mg/kg/day dosage level survived to the end of the study. These monkeys exhibited slightly decreased activity from the first weekofthe study `which occasionally became moderate to marked. In addition, they occasionally had black stools, diarrhea, mucous in the stool and bloody stool and exhibited dehydration or general body trembling at the endof study. The monkeys from this group had a slight decrease in mean body weight. Slight decreases in serum alkaline phosphatase activity and serum inorganic phosphate concentrations were evident at the end of the. study in addition to a marked decrease in serum cholesterol. All monkeys at the 0.5 mg/kg/day dosage level survived to the end of the study. Monkeys at this dosage level exhibited an occasional soft stool, diarrhea, anorexia and emesis, all of which also occurred occasionally in the control group. Slightly decreased activity was noted intermittently in three monkeys at this dosage level. At three monthsof study a statistically significant decrease in the serum alkaline phosphatase activity was noted in the males as compared to control values; however, the toxicologic significance of this finding is questionable when compared to individual pre-dose values. The 0.5 mg/kg dose has been considered to present a LOEL by past reviewers. Recent evaluation of the study by 3M toxicologists lend to a conclusion that this dose more likely represents a NOAEL,if not a NOEL. No treatment-induced gross or microscopic pathological lesions were seen in tissues other than the adrenals, pancreas, and submandibular salivary glands of male and female rhesus monkeys at the 4.5 mg/ke/day dosage level. Microscopically, the adrenals from male and female monkeys at the 4.5 mg/kg/day dosage level had compound-related marked diffuse lipid depletion; the pancreas from male and female `monkeys at the 4.5 mg/kg/day dosage level had compound-related moderate diffuse atrophy of exocrine cells; the submandibular salivary glands from male and female monkeys had compound-related moderate diffuse atrophyofthe serous alveolar cells. No statistically significant variations in sex group mean weightsof organs occurred between the control and experimental groups. 0 000043 January 21, 1999 `Table IV.6 summarizes the individual cholesterol values for all dose groups. 51 003054 January 21,1999 `Table IV.6: Longitudinal analysis of serum cholesterol and PFOS concentrations of individual male and female rhesus monkeys during 90 days dosing with either 1.5 or 4.5 mg/kg PFOS (1 =2 per sex per dose group) (Goldenthal etal, 1978b). Individual Serum Cholesterol (mg /100 mi) by Study Period and Cumulative Dose (CD in mg/kg) Dose Group Individual PreDosing 1Month 3Months Do%sionfg PVrael-ue at_Term c-0 c-0 cD=0 0 SDHT7355) mgkgiday 8 (ID# 7358) 192 212 174 184 179 144 92 8238 Dose Group $ (#I73D68) 15s 188 144 929 Monkeys 9 (ID # 7372) 204 208 185 90.7 -0 cp-i1s CD=45 05 SDA) 182 208 160 879 mghg/day 3(ID #7483) 161 Dose Group (I#D 7466) 208 21902 114738 885868 Monkeys (ID # 7504) 208 21 160 769 =0 CD=4s CD=135 15 aaD#7e) 196 mg/kg/day 3 (I#D 7486) 174 m 204 132 n 673 644 Dose Group (I#D 7500) 2 Monkeys (ID # 7501) 26 154 184 128 9% 74 07 CD-0 CD=135 Ne 45 S(D# TAB) 186 76 Survi-vors 09 mg/kglday 3 (I#D 7485) 194 Dose Group (ID #7502) 182 7 128 - 500 03 Monkeys 9.(ID # 7503) 170 9% - S64 2 000055 January 21,1999 28-Day Range-Finding, Oral Capsule-Dosing Study in Cynomolgus Monkeys: Male and female cynomolgus monkeys weighing 2.1 to 2.4 kg were given capsules placed directly in the stomach that contained PFOS at either 0.0 mg/kg/day (two males and two females), 0.02 mg PFOS/kg/day (three males and three females), or 2.0 mg/kg/day (one `male and one female) for 28 days in a range-finding study to determine doses for a six`month chronic oral capsule-dosing study (Thomford, PJ, 1998). Blood was collected for clinical chemistry on study days -7 (baseline values) , 2,7, 14 and 29. In addition to standard hematologic parmeters and serum chemistry determinations, sex and thyroid hormones, cholecystokinin (CCK) and pancreatic amylase were measured. At the same time points and on day 3, blood was also obtained for determinationofserum PFOS concentration. Tissues were obtained at necropsy, weighed, fixed and prepared for histopathologic analysis. In addition to histopathologic samples, liver specimens were obtained for analysis for proliferating cell nuclear antigen (PCNA), determination of PFOS concentration, and determination of palmitoyl CoA oxidase activity. Serum PFOS concentrations increased with a high degreeof linearity at both dose levels, with no difference between males and females and at a linear rate of 5.3 ppm serum PFOS per mg/kg (for details please see subsection on toxicokinetics). At the end of the 28-day dosing period, serum PFOS concentration in the 0.02 mg/kg/day dose group reached approximately 3 ppm and in the 2.0 mg/kg/day dose group, serum concentrations reached approximately 300 ppm. The only treatment-related effect observed in the study was a dramatic reduction in serum cholesterol in the male and female that received a dose of 2.0 mg/kg/day. Serum cholesterol dropped from baseline values of 150 and 141 mg/dl for the `male and female, respectively, to 91 and 62 mg/dl at termination on day 29. The first evidenceof a significant decrease occurred between day 2 and day 7 for the female, with aday 2 value of 136 mg/dl and a day 7 value of 117 mg/dl. The male cholesterol value fell from to 151mg/dl on day 20 137 on day 7. The day 7 values for the male and female corresponded to a serum PFOS concentration of 72 ppm. This cholesterol data and corresponding cumulative dose and serum PFOS concentrations are summarized in Table 1V.7. There were no other significant findings. 000046 BE January 21,1999 TmaablleeaInVd.7f:emLaolnegictyundionmaollganuaslysmiosnokfesyesr(u7m=ch|olpeesrtesreoxl) adunrdiPngFO28Sdcaoynscednotsriantgiownisthin2.0 mg/kg PFOS. Study Day 2 2 2 14 29 | Comutive Dose (mg/kg) 0 2 22% 56 | 3 Serum Cholesterol (mg/dl) | 150| 151] 137 132 91 & [PFOS] in serum (ppm) | 0013] 126] 718 129 313 Serum Cholesterol (mg/dl|) 141 | 136| 117 | 2 [PFOS] in serum (ppm) oni) 14s] m7 107 143 62 299 Six-Month Oral (Capsule) Study in Cynomolgus Monkeys Unaudited clinical pathology and clinical observations through 90 days of dosing are available from an ongoing six-month oral (capsule) dosing study in cynomolgus `monkeys (Covance Study Number 6329-223, in progress). In this study cynomolgus `monkeys are being dosed by capsule with either 0, 0.03, 0.15, or 0.75 mg/kg/day PFOS foar periodofsix months. Dose groups include six monkeys per sex per group with the exceptionofthe 0.03 mg/kg/day dose group which includes four monkeys per sex. The only significant finding through 90 days is a reduction in serum total cholesterol in the males and femalesofthe high dose group. While serum PFOS concentrations have not yet been analyzed and reported, previous experience in the rangefinder for this study suggests that the serum concentrations are expected to center on approximately 350 ppm PFOS as 90 days for the high-dose monkeys. Reproductive and Developmental Toxicity `This sub-section discusses the available information on developmental and reproductive toxicity. A summaryofthis data in tabular form can be found at the end of this section in Table IV.11. Oral Developmental Toxicity (Teratology) in Rats: A rat PFOS oral teratology study was conducted at Riker Laboratories (Gortner etal, 1980). Dose levels (oral) st 0006057 January 21,1999 given to the pregnant rat dams were 0, 1, 5, and 10 mg/kg. Maternal toxicity (reduced weight gain) occurred at the high dose of 10 mg/kg on days 6 through 15ofgestation. Evidence of fetal toxicity was not found at any dose level. No skeletal and soft tissue teratogenic changes were found at any dose level with one exception. A change in the lensof the eye was found in all dose groups including the control but the incidence in high dose group was significantly higher. This change was reported out as a developmental eye abnormality and the summaryof the report states the compound was teratogenic. An outside consultant and teratology expert, Dr. E. Marshall Johnson from Jefferson Medical College, visited 3M and reviewed the rat pup eye specimens in question. He concluded that the eye/lens changes were, in fact, sectioning artifacts and not compound related teratology abnormalities. Thus, the weight of the evidence indicates that PFOS does not cause teratogenic effects in rats `when dosed at levels which are not maternally toxic. The lens change observed in rat pups in Riker Laboratories studies was a sectioning artifact and was not found upon repeat studies at independent laboratories. Oral Developmental Toxicity (Teratology) in Rats: Ina subsequent study, PFOS (suspended in corn oil) was administered on gestational days 6-15 by oral gavage to `groups of 25 pregnant Sprague-Dawley CD rats at doses of 0 (control), 1, 5, and 10 mg/kg/day (Wetzel, etal, undated). Severe maternal toxicity occurred in the 5 mg/kg and 10 mg/kg dose groups, as evidenced by significant reductions in mean body weight gain, terminal body weight minus gravid uterine weight and food consumption compared to control dams, actual losses in body weight on commencement of treatment among numerous dams and death in two dams in the 10 mg/kg dose group prior to gestational day 20. Mean body weight gains (days 0-20) at 5 and 10 mg/kg were 104: 35 (S.D,) and 34 73 (S.D.), respectively, as compared to 125 + 24 (S.D.) in the control group. Mean food consumption values (days 0-20) at 5 and 10 mg/kg were 363 60 (S.D.) and 264 + 90 (S.D.), respectively, as compared to 421 28 for the control group. Mean terminal body weight minus gravid uterine weight at 5 and 10 mg/kg was 293 28 (S.D.) and 241 + 60 (S.D.), respectively, as compared to 321 ss 000048 January 21,1999 23 (S.D.) in the control group. Clinical signs in surviving dams included hunching, Tower body weight, alopecia, rough haircoat, anorexia. Gastrointestinal and kidney lesions were noted in the high-dose dams. Treatment-related fetal effects that were tributed to maternal toxicity included: increased resorptions and fetal death, decreased fetal body weight, delayed skeletal ossification, cleft palate, subcutaneous edema and eryptorchism (undescended testicles). These effects occurred primarily in the high-dose group. The matemal and fetal NOAELS for this study were both 1 mg/kg/day. Oral Developmental Toxicity (Teratology) in Rabbits: A final draft reportofan oral developmental toxicity study in rabbits was recently received (York, 1998). In this study, dose groupsof 22 pregnant new zealand white rabbits were dosed on days 7 through 20 ofgestation with either 0, 0.1, 1.0, 2.5, or 3.75 mg/kg/day PFOS. Maternal toxic effects included: 1) decreased body weight at the highest three dose levels with a minimal effect at the 1.0 mg/kg dose; 2) decreased food consumption at the highest two doses; 3) frequent scant feces at the highest dose, and 4) increased abortions at the highest two doses. Fetal toxic effects included reduced fetal weight and an increase in delayed ossification at the highest two doses. No teratogenic events were observed in the study. Based on this draft final report, PFOS was not teratogenic under conditions of the study and the maternal and fetal NOELs are 0.1 mg/kg/day and 1.0 mg/kg/day, respectively. Two-Generation Reproductive Toxicity in Rats by Oral Gavage: Interim results are available from an on-going two-generation reproduction study in rats by oral " 000059 January 21, 1999 `gavage (Argus, 1998). In this study, groupsof 35 male and 35 female rats were exposed 10.0, 0.1, 0.4, 1.6 and 3.2 mg/kg by daily oral intubation six weeks prior to and during mating. For the females, treatment continued during gestation, parturition and lactation. A pre-mating reduction in mean body-weight gain as compared to controls occurred in females and possibly in the males at the high dose level. At the 3.2 mg/kg dose level, male body-weight gain was 97.2 %of control and female body-weight gain was 91.5 %ofcontrol. There was no effect on the number ofpregnancies. During gestation, females showed a reduction in mean body-weight gain at the three highest dose levels, reaching 86.9% of the control at 3.2 mg/kg. A groupof ten dams per dose group was sacrificed on day 10ofgestation. No increases in resorptions occured, and there were no decreases in the number of implantation or number of live fetuses. At parturition in the 3.2 mg/kg high-dose group, the mean numberof pups delivered was decreased compared to the control (10 versus 14) and the percentofpups delivered stillborn was increased significantly (24.9 % as compared t0 2.2 %.) Survivalof pups during days one through fouroflactation was severely affected at 1.6 and 3.2 mg/kg (66 % and 0% survival, respectively.) Most deaths at the high dose `occured within the first 24 hours after birth. Surviving pups in the 1.6 mg/kg dose `group showed severely depressed mean bodyweight gains through day 21 of lactation (72.1% of controls.) A seriesof follow-up studies are to be initiated in November, 1998 to gain a better understandingofthe reduced perinatal survival at the high dose levels. These will include a complete cross-fostering study, and two pharmacokinetic studies with pregnant dams. 5 000070 January 21, 1999 `The post-weaning F) generation currently exhibits no signsoftoxicity at 0.1 and 0.4 mg/kg/day. Mechanistic Studies The specific mechanisms relating to PFOS toxicity are not completely understood. Several studies provide clues to the potential operative mechanism(s)of toxicity. Effects on lipid and lipoprotein processing, cholesterol synthesis and bioenergetics have been studied. Mitochondrial Bioenergetics: Studies in isolated rat liver mitochondria at PFOS concentration in the range of 10 uM demonstrate: 1) detergent type effects on mitochondrial membranes; 2) stimulation of mitochondrial respiration, and 3) fluidizationof the inner mitochondrial membrane (Wallace and Starkov, 1998). Interference with Fatty Acid Binding and Transport: Interferenceof PFOS (1-10 1M) with endogenous fatty acid binding to carrier protein substrates, liver fatty acid binding protein (L-FABP) and albumin (BSA) has been investigated and show a 66% reduction of initial fluorescence when added to solutions containing ImM L-FABP and mM DAUDA and an ICso 0f 4.9 iM (Nabbefeld et al., 1998; Nabbefeld, 1998). Peroxisome Proliferation: Liver biochemical effects associated with peroxisome. proliferation have been investigated in two published studies. Sohlenius etal. (1993) exposed mice to 30 mg/kg/day PFOS for five days (0.05% in diet). In addition to weight loss, increases in each of the following hepatic parameters were observed: 1) relative liver weight (slight elevation): 2) mitochondrial and microsomal protein; 3) palmitoyl-CoA oxidation; 3)catalase in mitochondrial and eytosolic fractions; 4) glutathione transferase; 5) epoxide hydrolase and 6) DTdiaphorase, Q- and Q-1-hydroxylation (Sohlenius et al, 1993). El oeno71 January 21,1999 Ikeda et al. (1987) exposed male rats were to 0.02% PFOS in the diet for 2 weeks. The prominent inductionofperoxisome proliferation was demonstrated by electron microscopy. Activitiesof catalase, fatty acyl-CoA oxidizing system, camitine acetyl transferase and cytochrome P4505 responsible for the Q-hydroxylation of lauric acid were increased (Ikeda et a., 1987) Hypolipidemia: The mechanismof the hypolipidemic effectofPFOS has been studied. Rats were fed 12 mg/kg/day fo7r 14 days (0.02% in diet). Decreased body `weight, increased liver weight, increased liver triacylglycerol, increased liver free cholesterol, decreased liver cholesterol ester, decreased serum cholesterol and triacylglycerols were observed. Hepatocytes isolated from treated rats showed reduced synthesisof cholesterol from acetate, pyruvate and hydroxymethylglutarate: but not from mevalonate, increased oxidation of palmitate and reduced fatty acid synthesis. Activitiesofliver hydroxymethyl glutaric acid-CoA reductase and acylCoAscholesterol acyltransferase were reduced. These results suggest that the hypolipemic effect of PFOS may be due to impaired production of lipoprotein particles due to reduced synthesis and esterificationof cholesterol together with enhanced oxidation of fatty acids in the liver (Haughom and Gystein, 1992), On-Going Research Program A number of studies were initiated in 1998 to gain better insight into the potential health hazards of PFOS and to provide a strong foundation for risk characterization. `These studies are discussed in Section VI, and outlinesofthem can be found in the Appendix. 50 006072 ] TRV: SummofaRordye RepDeosaStcs | T i aDtyomiim RaThFTAmo , JS1 asmveB[Fr Toomsreomem crs eams ] [EE [hema ||| [SSE |CC3eOoRiahn mmpiiSanhiOd oiNnnleseodsieo l adehmee ye en r mo,o oo dcnn. Xn, brs. omc mes ct bore mr So1000 [SME |oera Woe olstiy mk hy of SM/SF mesentericlymphnodes,atrophyofvilli insmall intestines, skeletal muscleatrophyand aTtoeronDsoyny | [CamWe w[25e |lToM/oioh BeDeseiedatpo) Homer [RET 2(Po30 [[oFOwlNTeIe0rF | bbyan. ch 0.1 ers s6.1cred Swine ioatsrsi99) rBTarype [[0 ogre o[Lor w | he imo Noi te [oF [Noches 2 o0o3ww be [[T[ilEornir|ee[nw,e. ekedscmshi ics 2228 303 0 1M 10M 1 liver weight, serum cholesteroland triglycerides, hepatic vacuolization liverweight, 4 scrum cholesterol and triglycerides, hepatic vacuolization pSEnomeirtetinnen J 5 i5ci ] 1978) | = espo HeR wrr |BBemm Ee ST, remors,postration, congestion, hemorrhage and lpidepletionofadrenal cortex Rear 53: 2i . | ayn9 ---- = | r[een ly Sreen EE ares TSSfeaeanmna 0 G h TR B E H Ee e || Ts nn. mmm sn ci. ht co i Toot fo EE tTneathio, im it. rto a a3BEN[[RaEaa||NS et tpOnpe 322s a " | SE pe Janarby2l1e,1I99-915 90:Day Sub-Chronic Stu Organized by Increasing Nominalmg/kgDoseLevel aoe oink.Water |r Noacre clot, ese Terwegfimo 1005) Pose [oss rkWater Gavaze |Mo__ 1 Monkey Redescrum ihc (Limoges 9) amore,rss,dambesadlghlyderssdscr Sin pipe.(GOMESaLT5775 Foo ink.Water|rn Se 0 ink.Water |M at HReemdacteoldosgcyraunmcheolrenstcrholeamnisypepeeecrtisd,es:rheeapsatibcvecrowoeligshitosn(:Lnimeogeeds199e5r) weighsLogeTST EE i rreTm e ee [30 ikWater {Mat Reducedscumcholesterolandrighcorides,beat vocuolsion;nresedToewags(Limoges T5957 .I E | fm -omfaD E E E E 5erR ATE e s s CB ER E e e s TEE e rI eTEeB eEs JE CE -- Popp R mE L 5 EE FF ---------- V. RISK CHARACTERIZATION The principal issueofconcern is the potential health risk associated with thelevels of PFOS identified in serum samples from non-occupationally exposed employees. commercially available serum and blood bank samples. The purposeof this section is 0 evaluate this risk using available human and animal data. Sourcesof exposure and their control are discussed in a companion document As noted previously (Section 111), 3M has conducted medical surveillance programs at manufacturing locations where employees are exposed to PFOS or precursor molecules. These employees have POS serum levels that range from less than one PPM Up to 12 ppm in the most recent testing. The 1997 mean among participating employees at the Decatur, Alabama plant, the U. S. location where PFOS related `materials are principally produced, was 2.0 ppm. The best current estimate of serum levels in non-oceupationally exposed individuals comes from the pooled samples collected from regionally diverse blood banks. These values ranged from 14 to 56 ppb, and averaged 30 ppb. It should be noted that while this is our best estimate, it cannot be considered representative of the U.S. population since blood donors are not necessarily representative of the U.S. population. In addition this data, since itis from pooled samples, does not reflect the variability and range one would expect to find among individuals, Analysisofdata collected in 1995 and 1997 reveals that the serum concentrations of PFOS identified in production workers are not associated with alterations in the hematological or clinical chemistry parameters that were evaluated. This includes measurementof the parameters that would reflect the pathology found in high dose animal studies (see below). In addition, no differences from normal were detected in ten different hormone levels. These analyses included employeesofthe 3M Belgium manufacturing facility, where serum levels are slightly lower than Decatur. The findings apply for PFOS serum levels up to 6 ppm, approximately two orders of o 000077 January 21, 1999 magnitude above the upper end of the range seen in the U.S. regional blood bank samples. The mean serum concentration in participating Decatur plant employees in 1997 was 68 times the mean of these blood bank samples. In 1995, epidemiologists from the Universityof Minnesota completed a mortality studyof the Decatur employee cohort. No higher than expected mortality rates were found for any cancer or for any other cause of mortality. This study involved 1,957 employees who had worked at least one year at 3M's Decatur, Alabama plant, and their mortality experience was traced from 1961 through the end of 1991. Details of this study can be found in Section II. Animal toxicology data has been reviewed in Section IV. An older data set from rhesus monkeys exists and new data from a rangefinder study in cynomolgus monkeys is available. Serum PFOS concentrations were not measured in the older rhesus monkey studies, but can be estimated based on data from the cynomolgus monkey studies. is recognized that uncertainties are introduced by such estimates, and that derived numbers need to be confirmed in ongoing or planned studies. Data from the cynomolgus monkey rangefinder studyitself documents that a decrease in cholesterol, an early biological effect in both rodents and primates, was observed at PFOS serum levelof 72 ppm in one of the two high dose animals. In the rhesus monkey studies, lowered cholesterol was observed after 90 daysoforal dosing at 1.5 mg/kg/day. Severe toxicity, resulting in death, occurred at doses equal to and greater than 4.5 mg/kg/day, in seven weeks or less. Serum concentrations of PFOS that might have been achieved in the older rhesus monkey studies have been estimated from the cumulative dose - serum concentrationof PFOS relationship seen in the recent cynomolgus study. These estimates suggest that severe toxicity occurred at serum levels of 700 to 800 ppm and death at 1000 to 1200 ppm. 0000s 6s January 21, 1999 Other available toxicity data also provide valuable information. PFOS is not mutagenic in five different bacterial strains. It fails to cause chromosomal aberrations in the mouse bone marrow micronucleus assay. Developmental studies in rats show. that the compound causes adverse effects on fetal structural development only at doses that are clearly toxic to the mothers. Preliminary results from an ongoing two generation reproductive study in rats, through F1 weaning, show no interference with sexual maturation, mating behavior, fertilization, number of resorptions, or litter size. At the highest two dose groups (1.6 and 3.2 mg/kg/day) there was significant perinatal mortality in the F1 generation. "This effect was not seen at 0.4 mg/kg/day. The study cannot be used in risk assessment until it is completed and fully evaluated, but is discussed here because we are awareof these interim results. The doses observed to cause effect are in the range where toxicity is observed in 90 day rat studies. Several 90-day studies in rats, involving PFOS administration over a wide range of doses in the diet, in drinking water, and by stomach tube, reveal that no significant toxic effects are produced at orbelow a dose of 0.2 mg/kg/day. Above this dose adverse effects on the liver, body weight, and changes in several indicators of fat metabolism are demonstrable. Possible explanations for the absenceofdetectable toxicity in production workers are that the workers" serum and tissulevels of PFOS are significantly below those achieved in the animal studies, as estimates would indicate, or human beings are less sensitive to the effects of PFOS than are laboratory animals. Ofthese two explanations, the former seems more likely, because both primates and non-primates were shown to be susceptible to PFOS exposure in the animal studies, so there is no obvious reason to expect large interspecies differences in sensitivity. The second possible explanation -- that humans are less sensitive -- cannot be ruled out based on available data. It is also possible that the human studies were insufficiently powerful, 3 0060779 samary21, 1999 becauseof sample size limitations, to detect the toxicity of PFOS. This seems unlikely because the animal studies showing toxicity involved even fewer subjects, and, at a given dose, all or most animals responded in similar ways. Sample size `would not seem to be a significant factor for evaluating clinical, hematological, or `hormonal effects. Excess mortality, including that associated with cancer, has not been observed in production workers. The experimental data showing that PFOS is not mutagenic (Ames tests) and does not cause chromosomal aberrations (mouse bone marrow micronucleus assay) is consistent with these findings. Non-occupationally exposed populations appear to exhibit serum levels that are approximately two orders of magnitude lower than those exposed occupationally, `more than three ordersof magnitude below levels estimated to show minimum biological effects (cholesterol reduction) in primate studies and three to four orders of magnitude below levels associated with significant toxic effects. None of the health effects evaluated and found to be absent in the worker studies would be expected to occur in the non-occupationally exposed populations. The additional information provided by the two generation reproductive study in rats is not interpretable at this time, but the dose at what currently appears to be a no effect level is comparable to doses that produce no effect for other endpoints. The animal data revealing adverse effects are not yet fully useful for quantifying human risks, but estimates from current and ongoing studies suggest that PFOS serum levels achieved in those earlier animal studies with compound related effects must have been greater than those experienced by workers and therefore much greater than those experienced by non-occupationally exposed populations. The currently available evidence does not suggest there is health risk associated with the levels of PFOS found in the serumofoccupationally or non-occupationally exposed employees, blood bank samples or commercially available serum. 006080 Sania 21,1999 Several avenues of further investigation are needed. The long-term consequences of PFOS exposure require additional study. Although the available worker studies do not suggest a health problem or evidenceof increased mortality rates, animal data reflecting chronic exposures are not yet available. Such data are recommended because worker studies, involving relatively small populations, are generally not sufficiently powerful to detect small excess risks. A two year PFOS feeding study in rats was started in April of 1998. The utilityofthe available animal data is limited because serum levels associated with adverse events can only be estimated at this time. This limitation is being remedied by collecting additional animal data, including sufficient kinetic information to assess the relationship between `administered doses and serum and tissue levels, as cited above for cynomolgus `monkeys, as well as more definitive data on compound distribution and elimination. There is data suggesting that PFOS can, at sufficiently high doses, induce peroxisome proliferation in rodents and alter metabolic processes in laboratory models. Further study ofthese effects is desirable to determine whether they can occur at serum and tissue levels relevant to those observed or estimated in humans. Section VIof this report describes additional 3M studies that will address these issues. Further followup on worker mortality experience is also underway, including an attempt to better classify workers with regard to PFOS exposure. Finally, studies are underway to improve knowledge about possible sources of PFOS, and the pathways by which non-occupationally exposed individuals are exposed. Although there is no current evidence that the reported serum levels, and corresponding body burdens, representa health risk, it is appropriate to identify and act on possible ways to reduce exposure. To date this has been done through reduction of residual monomer levels in products and reduced`manufacturing emissions. @ cerngy VI. CURRENT AND PLANNED RESEARCH Toxicology Studies Fluorochemical Study Purposes and Outlines A number of studies were initiated in 1998 to gain better insight into the potential health hazardsof PFOS and to provide a strong foundation for risk characterization. These studies include those conducted with PFOS as well as studies which are conducted with N-Ethyl Perfluorooctane Sulfonamido Ethanol (N-EtFOSE), NMethyl Perfluorooctane Sulfonamido Ethanol (N-MeFOSE) and other related compounds which are presumed to degrade metabolically to PFOS. N-EtFOSE and N-MeFOSE represent major starting points for additional synthesis and are regulated by FDA as contaminants of indirect food additives. These compounds are also known to be absorbed well from the gastrointestinal tract and will produce PFOS as a major `metabolite. Therefore, it is appropriate to use these two compounds as models in further investigating the toxicityofthis classofchemicals which have the perfluorooctane sulfonyl moiety as a base. The goalsof this research program are as follows: + To understand the relationship between measured concentrationsof PFOS in serum and potential adverse health effects; Serum PFOS measurements; * To understand the kinetics of increases in body burdenof PFOS as reflected in + To determineif a critical cumulative body burden exists and how this is rleated to toxicokinetics; To define the metabolic relationship of PFOS to other Perfluorooctanesulfonyl- based chemicals; + To understand the primary mechanism of toxicity responsible for early toxic effects; 000082 ' January 21,1999 To establish reasonable and substantiated no significant risk levels for risk assessment. Studies initiated in 1998 with PFOS and N-EtFOSE are identified in Tables V1.1 and VL2, respectively. In addition to these, a 90-day dietary study with N-MeFOSE has been completed and studies have been undertaken to better understand the potential for metabolic degradationof perfluorooctanesulfonamide-based chemicals to PFOS, Descriptive summaries of manyofthese studies are included in the Appendix. 000053 0 Janay21,1999 "Table VLI: PROS Tosicity Studies Initiated in 1998 Study Title MFoounrk-eWyesek Capsule Range-Finder Suady in Cynomolges |7358 Six-Month Capsule Feeding Study in Cymomolgas Morkeys |959% Two-Year Dietary Study in Rats SSE Completion Date (In-Life) | 55298 | 2899 (Wate) 5/7/99 (recov.) | 4000 Hepatic Peroxisome Induction and Cell Proliferation Study Pr Nos Ve Sue Segment I Teratology Tn Rabbis Two-Generation Reproduction Study n Rats One Generation CrossFostering Reproduction Suady in Rats OGensetaGteinoenr)ation Reproduction PK Study Tn Rats (rough One-Generation Reproduction PK Study fn Rats (hrowgh Lactation) 12/1/98 1/7/98 [ovaons| En EC EFT | T/IG8 [271599 [1/798 | 130099 | 1/3798 | 37709 "Table V1.2: N-EtFOSE ToxSistituydy TStiuddeies Initiated in 1998 Two-Year Dictary Study in Fas SingleDose ADME fn Cynomolges Momkeys Biochemical and Molecular Biology Mechanistic Swdies Segment 1 Teratology in Rats Segment Il Teratology Tn Rabbis Two-Generation Reproduction Study Tn Rats Taos Sass | 77797 Es Tes EE CoDmaptlee(tiino:n Life) | VASE | 0s [mes Ea n 000054 Jamary 21,1999 104-week dietary carcinogenicity study with perfluorooctane sulfonic acid, potassium salt in rats. In-life Start date: 4/20/98; In-life completion date: 242000 `The purposeof this two year assay is to determine: carcinogenic potential, bioaccumulation with repeated doses, threshold effect levels, a no observable adverse effect level (NOAEL), and mechanisms of toxicity. Proliferating cell nuclear antigen (PCNA) will be measured at early time points in both liver and pancreas as an indicatorof preneoplastic lesions. There are six dose groups: 0, 0.5, 2.0, 5.0, 20 and 20 (recovery) ppm PFOS in the feed. Interim sacrifices at weeks 4, 14 and 52 will be performed to determine PCNA, palmitoyl Co-A oxidation, clinical chemistry, and histopathology. Animals in group 6 will be treated for 52 weeks, then treatment `will be discontinued and the animals will be observed for reversibility, persistence, or delayed occurrenceof toxic effects for at least 26 weeks post-treatment. Serum samples will be collected for PFOS detection from the scheduled sacrifices and in-life blood draws. Hematology, serum chemistry, urinalysis, urine chemistry, and serum sampling (In-life blood draws) will be done at weeks 14, 27 and 53. 104 -Week Dietary Carcinogenicity Study with Narrow Range (98.1%) N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats. In-life Start Date: 1/26/1998. `The purpose of this study is to assess the carcinogenicityof the test material, N-cthyl perfluorooctanesulfonamido ethanol (N-EtFOSE) when administered in the diet to rats for at least 104 weeks. The test material was administered at dose levels of 0, 1, 3,30, 100, and 300 ppm. Necropsies were performed during Week 4 and during. Week 14 for hepatocellular proliferation rate measurements, hepatic palmitoyl-CoA oxidase activity, and serum and liver PFOS levels. Due to the morbidity and `morbundity, the 300 ppm group was terminated at 8 weeks. During Week 8 and 14, blood and urine were collected for hematology, clinical chemistry, urinalysis, and urine chemistry. At necropsy, macroscopic observations were recorded, organ `weights were obtained, and tissues were placed in fixative as specified by the " 0006085 January 21,1999 protocol. Microscopic examinations were done on selected tissues from animals necropsied during Weeks and 14. A recovery groupofanimals in the 100 ppm dose `group will be treated for at least 52 weeks then treatment will be discontinued, and the animals will be observed for reversibility, persistence, or delayed occurrence of toxic effects for at least 26 week post-treatment. Six-Month Capsule Feeding Study in Cynomolgus Monkeys. In-life Start Date: 8/5/1998. In-life completion date: 5/7/1999. `The purpose of this study is to establish a no observable effect level for PFOS in the Cynomolgus monkey. PFOS is a known hepatic peroxisome proliferator (PP) in the rat. Non-human primates such as the Cynomolgus monkey respond similarly to humans with no to low hepatic response to peroxisome proliferators, and therefore are the appropriate human surrogate species. Blood hormone levels will be determined pretreatment and after 30, 60, 90, and 180 daysoftreatment, and after 30, 60, and 90 days of recovery. Samples will be analyzed for estradiol, estrone, estriol, thyroid stimulating hormone, and triiodothryonine (T3), thyroxin (T4), cortisol and testosterone. Serum and liver PFOS levels will be determined and correlated to blood chemistries. Urine and feces PFOS determinations will be made day 0ofrecovery and after 6, 30, and 90 daysof recovery to track elimination Kinetics. Four groups of four male and four female Cynomolgus monkeys will be orally dosed with PFOS triturated in lactose at 0, 0.03, 0.15 and 0.75 mg/kg/day daily by gelatin capsule. Dose levels were determined in a previously conducted rangefinder study. Two additional animals in the control, mid and high dose groups wil be designated as recovery animals for which treatment will be discontinued at 26 weeks, and the animals will be observed for reversibility, persistence,or delayed occurrence of toxic effects for at least 13 weeks post-treatment. Hematology and clinical chemistry will be determined once before initiation, after 30, 60, 90, and 180 daysoftreatment, and after 30, 60, and 90 days of recovery. Clinical chemistry will include urea nitrogen, cholesterol, riglicerides, alkaline phosphatase, alanine aminotransferase, bile acids, total and direct bilirubin, lipase, and pancreatic-specific amylase. Palmitoyl CoA 7 0006056 January 21, 1999 oxidase activity will be analyzed from liver tissue to determine peroxisome proliferation. Complete necropsy will be performed after at least 26 weeks of treatment, and after at least 13 weeks post treatment for the recovery groups. Histopathology and proliferative cell nuclear antigen (PCNA) will be performed on a subset of the specimens. Metabolism of N-EFOSE, in Cynomolgus Monkeys following administration of a single dose by Oral Gavage. In-life start date: 5/14/98. In-life end date: 10/1/98 The purpose of this study is to determine the absorption, distribution, metabolism and excretion (ADME) of N-Et FOSE and its metabolites. Determinationof ADME parameters in non-human primate is an appropriate model for human risk assessment, as opposed to the rat. Two groupsof Cynomolgus monkeys will be used. Group A will have five animals/sex and will be given 10 mg/kg single treatment by oral gavage. Tissues will be collected from one animal per sex per time-point at 0.5, 1,7, 14 and 28 days. Urine and feces will be collected at 0.5, 1, and daily up to 28 days. Group B will have one animalsex which will be bile duct cannulated and will be given 10 mg/kg single treatment by oral gavage. Bile will be collected at 0.5, I, and daily up to 28 days. Blood samples will be drawn for serum (and/or plasma) at pre-dose, 1,2, 4,6, 12,24, 36, 48 hours,7 days and 14, 21 and 28days for both `groups. Tissues withmass > 0.5 g will be analyzed for N-EFOSE and its `metabolites: Cerebrospinal fluid, skin, fat, urinary bladder, testis, epididymus, seminal vesicles, prostate, uterus, spleen, kidneys, liver, thymus, heart, lungs, diaphragm, salivary glands, trachea, esophagus, muscle, bone, bone marrow, pancreas, lymph nodes, tongue, eyes, brain, spinal cord, stomach, small intestine, large intestine, thyroid/parathyroid, aorta (~0.5 g), adrenal glands, gallbladder,and ovaries will be included 000087 January 21,1999 Mono N-EtFOSE Phosphate -- Absorption, Disposition, Metabolism, Excretion in Rats. In-life Start Date: 5/15/1998. In-life completion date: 6/15/1998, `The purpose of this study was to assess the potential for absorption and metabolism of the monophosphate ester ofFC-807 in rats. FC-807 is the brand name ofa fluorochemical based product sold by 3M and approved by the FDA for use as an oil and water repellant in paper and paperboard food packaging. The primary concern of this study was to determineif and to what extent the monoester is absorbed from the intestinal tract, and to what extent it can be metabolized ton N- ethyl (perfluorooctane)sulfonamido ethanol (N-Ethyl FOSE) and other metabolites, including PFOS, once in the blood stream. Both a time-course study following a single dose of compound, and a dose response study were conducted. In the time course study, rats were dosed orally, via gavage, or by intravenous (i.v.) injection, via the tail vein, with FC-807 monoester. One animal from each dosage routegroup `was sacrificed two hours after dosing. Urine and feces were collected daily for four days. On day 4, one animal from each group was sacrificed, and theremainder of the animals were sacrificed 28 days post dose. The preliminary results indicated absorptionofthe oral 50 mg/kg dose and metabolism at day 4, and day 28. The i.v doses of 0.5 mg/kg were also extensively metabolized and were deemed to be too high. The dose-response study consistedofsix dose groups. There was one control `group, three groups dosed orally at 0.01, 0.1, and 5 mg/kg, via gavage, and two groups that received tail vein i.v. injections at 0.01, and 0.1 mg/kg at a volume of5 ml/kg on day one of the study to each group. All groups were sacrificed on day 4 of the study. 13-week Dietary Toxicity Study with N-Methyl FOSE in Rats. In-life Start Date: 9/1/1998. In life completion date: 12/8/1998. `The purpose of this study was to develop sub-chronic toxicity data and the toxicokinetics for N-Methyl FOSE when administered in the diet to rats for at least 13 weeks. The study design included 20 males and 20 female animals/group fed 0, 3, 7s 000088 January 21, 1999 30 and 100 ppm N-MeFOSE in the diet. Afie4r weeks of treatment, 5 rats/sex/group were sacrificed for Palmitoyl CoA oxidase, PCNA, liver and sera PFOS analysis. `Two-Generation Rat Reproduction Study with PFOS. In-life Start Date: 5/26/1998. In-life completion date: 12/31/1998. `The purpose of this study is to test for toxic effects of PFOS on reproductive function of dosed male and female rats and to assess whether rat pups exposed to the compound in utero & via milk have any developmental, learning and reproductive effects. A secondary objective will be to assess compound and/or metabolite levels in serum and liver from parent animals. Groupsof 35 rats will be dosed daily via gastric gavage at dose levels of 0, 0.1, 0.4, 1.6 and 3.2 mg/kg/d. Dosing will start 4 weeks prior to mating, will continue during mating, and for females will continue during gestation and lactation. Reproductive performance will be assessed by fertility index, gestation index, numberofpups/ litter, pup viability index and lactation index. The F1 generation pups at each dose level will be assessed for developmental objectives, undergo learning testing, and reproductive performance. Serum and liver samples will be collected at necropsy from five male and five female rats/dose group from F, (Parent) animals and analyzed for compound by 3M. `Two-Generation Rat Reproduction Study with N-E{FOSE. Start Date: 6/8/1998. In life completion date: 12/31/1998. "The purpose of this study is to test for toxic effects ofN-EIFOSE on reproductive functionofdosed male and female rats and to assess whether rat pups exposed to the compound in utero & via milk have any developmental, learning and reproductive effects. A secondary objective will be 10 assess compound and/or metabolite levels in serum and liver from parent animals. Groupsof 35 rats will be dosed daily via gastric gavage at dose levels of 0, 1, 5, 10 and 15 mg/kg/d. Dosing will start six weeks prior to mating, will continue during mating, and for females will continue during gestation and lactation. Reproductive performance will be assessed by fertility index, gestation index, numberofpups liter, pup viability index and lactation % 000039 January 21, 1999 index. The F1 generation pups at each dose level will be assessed for developmental objectives, undergo learning testing and reproductive performance. Serum and liver samples will be collected at necropsy from male and 5 female rats/dose group from Fi (Parent) animals and analyzedfor compound by 3M. Segment II Teratology in Rabbits with PFOS. In-life Start date: 8/28/1998. Inlife end date 9/29/1998. The purposeofthis study is to detect adverse effects of PFOS on New Zealand White pregnant female rabbits and developmentof the embryo on fetus consequent to exposure of the doe from implantation to closureof the hard palate. Dose groups of 22 presumed pregnant female rabbits were dosed with 0, 0.1,2.0,2.5 and 3.75 mg/kg/day viaa stomach tube. A toxicokinetic satellite group of female rabbits (5 at the control and high dose levels plus three at the other dose levels) were sacrificed at day 21ofgestation (the day following the last dosage) serum, liver, fetal and placental samples were analyzed for POS and possible metabolites. Rabbits were Caesarian sectioned on day 29 of presumed gestation. The fetuses were examined for body weight, gross alterations and skeletal alterations, number and distribution of corpora lutea, implantation sites, live and dead fetuses and early and late resorptions. Segment IT Teratology with N-EtFOSE in rats. In-life Start date: 9/4/1998. In life end date 9/11/1998. `The purpose of this study was to detect adverse effectsof N-E(FOSE on presumed pregnant female rats and development of the embryo and fetus consequent to exposureof the dam from implantation to closureofthe hard palate. Dose groups of 25 presumed pregnant female rats were dosed with 0, 1, 5, 10 and 20 mg/kg/day by oral gavage. A toxicokinetic satellite groupoffemale rats (five at the control and high dose levels plus three at the other dose levels) were sacrificed at day 18 of gestation (the day following the last dosage) serum, liver, fetal and placental samples were analyzed for N-EFOSE and possible metabolites. Rats were Cacsarian " 0006090 January 21, 1999 sectioned on day 20 of presumed gestation. The fetuses were examined for body weight, gross alterations and skeletal alterations, number and distributionof corpora lutea, implantation sites, live and dead fetuses and early and lat resorptions. Segment Il Teratology in Rabbits with N-ECFOSE. In-life Start date: 8/28/1998. In life end date 9/29/1998. "The purposeofthis study was to detect adverse effects of N-E(FOSE on New Zealand White pregnant female rabbits and developmentof the embryo and fetus consequent to exposure of the doe from implantation to closure of the hard palate. Dose groups of 22 presumed pregnant female rabbits were dosed with 0, 0.1, 2.0, 2.5 and 3.75 mg/kg/day via stomach tube. A toxicokinetic satellite group of female rabbits (five at the control and high dose levels plus three at the other dose levels) were sacrificed at day 21ofgestation (the day following the last dosage) serum, liver, fetal and placental samples were analyzed for N-E(FOSE and possible metabolites. Rabbits were Cacsarian section on day 29 of presumed gestation. The fetuses will be examined for body weight, gross alterations and skeletal alterations, `number and distributionofcorpora lutea, implantation sites, live and dead fetuses and carly and late resorptions. 000091 mary 21, 1999 Current and Proposed Human Health and Exposure Research Initiatives At present, there are several ongoing research studies and proposals. These are briefly outlined below. 1. Half-life Fluorochemical Determination Study. 3M retirees from the Decatur and Cottage Grove plants will be asked to participate ina study to determine the half-life of PFOA and PFOS. Retirees' serum will be analyzed semi-annually for the next five years for PFOS, PFOA and perfluorohexane sulfonate. 2. Decatur Serum Exposure Assessment Study. Employees at the 3M Decatur chemical plant have in the past voluntarily participated in a fluorochemical medical surveillance program. Analysisofthe surveillance data has not shown significant associations between the employees' clinical chemistry and hematology tests and either total serum organic fluorine or serum PFOS levels. However, the voluntary natureof the medical surveillance program does not allow foar complete understanding of the distribution of employee fluorochemical serum levels. In order to address this issue, a random sample of approximately 80 Decatur film plant and 125 Decatur chemical plant employees will be asked to participate in a serum determination study for the following fluorochemicals: perfluorooctane sulfonate, perfluorooctane sulfonate amide, perfluorohexane sulfonate, perfluorooctanoic acid, N-ethyle perfluorooctnae sulfonamido ethanol and its acetate derivitive, and N-methylperfluoroctanesulfonamido ethanol. A sub-sampleof employees `will also be tested for total serum organic fluorine. Abrief questionnaire will also be administered to each employee inquiring about current and past work history as well as possible routes of oral ingestion. 3. An Epidemiologic Analysoifs the Inpatient Claims Experienceof 3M Decatur Employees, 1993-1997. The purpose of this study is to examine the inpatient claims database, as maintained by Corporate Health Strategies, from January 1, 1993 -- December 31, 1997 of Decatur plant employees. The data will be stratified by whether the employees are in the chemical or film plants. The study population will include full- " 0006092 January 21, 1999 time active employees who worked throughout the five year interval, all full-time active employees who quit, died or went on long-term disability within the five year interval, and all employees who have retired. Observed inpatient claims for each plant population will be compared to an expected experience based on the 3M normative database. Inpatient claims analysis will proceed in a sequential, hierarchical manner. First we will examine Medical Diagnosis Codes; next Diagnosis Related Groups, and finally selected ICD-9 codes within each DRG, 4. PFOS and PFOA Retrospective Cohort Mortality Studies of Employees at the 3M Decatur and Cottage Grove Plants. Previous retrospective cohort mortality studies have been conducted at the Decatur and Cottage Grove plants. However, neitherof these studies utilized the employees' fluorochemical serum measurements to designjob-, department- and calendar-year exposure matrices. The purposeofthese two studies is to construct PFOS and PFOA exposure matrices based on previously collected employee serum PFOS and PFOA measurements in conjunction with their plant work history experiences. Person-years will be allocated based on the exposure matrices to calculate the traditional measures of risk, i.., Standardized Mortality Ratios, for more than 50 causesofdeath. Vital status and causeofdeath will be ascertained through December 31, 1997. 5. Geographical Reliability Study. The purposeofthis study is to determine whether there are geographical differences in human serum measurements in the United States. We will request serum samples from the 18 blood banks that originally participated in the fluorochemical determination study (see Table I1.3) to determine whether comparable serum levels are measured. 000093 January 21, 1999 VIL REFERENCES 3M Commercial Chemicals Division. 19xx. Fluorad Brand Fluorochemical Surfactants FC-95 and FC-98. Technical Information, 3M Company, St. Paul, MN. 3M Industrial Chemical Products Division. 1992. FC-95 FLUORAD Brand Fluorochemical Surfactant, Material Safety Data Sheet, Document 10-3796-9, St. Paul, MN, Anderson DJ, Mulvana DE (1997a). Analytical report for the determination of perfluorooctanoate and perfluorooctanesulfonate in human serum by LC/MS. Unpublished report. Ithaca: Advanced Bioanalytical Services, Inc., August 25, 1997. Anderson DJ, Mulvana DE (19975). Analytical report for the determination of perfluorooctanoate and perfluorooctanesulfonate in human serum by LC/MS. Ithaca: Advanced Bioanalytical Services, Inc., September 22, 1997. 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Oral teratology study of FC-95 in rats. Expt. No. 0680TR0008, Riker Laboratories, Inc., St. Paul, MN. Guy, WS (1972). Ph.D. Thesis, University of Rochester, Rochester, NY. 000095 82 January 21,1999 Guy, WS, Taves, DR, Brey, Ir., WS (1976). Organic fluorocompounds in human plasma: prevalence and characterization. (In) Biochemistry Involving Carbon-Fluorine Bond, pages 117-134. Haughom, B. and @ystein, S. 1992. The mechanism underlying the hypolipemic effect of perfluorooctanoic acid (PFOA), perfluorooctane sulphonic acid (PFOSA) and clofibric acid. Biochim. Biophys. Acta. 1128, 65-72. Ikeda, T., Fukuda, K., Mori, I, Enomoto, M., Koma,T.and Suga, T. 1987. Induction of cytochrome P-450 and peroxisome proliferation in rat liver by perfluorinated octanesulfonic acid. In: Peroxisomes in Biology and Medicine, FLD. Fahimi and H. Sies, Eds. Springer Verlag, New York, 304-308. Industrial Hygiene Information System. 1994. 3M Industrial Hygiene Services, Saint Paul, MN. Jagannath, D.R. and Brusic, D. 1978. Mutagenicity evaluation of T-2014CoC in the Ames Salmonella/microsome plate test. LBI Project No. 20838, Litton Bionetics, Inc... Kensington, MD. Johnson J.D. Final report analytical study Single -dose dermal absorption/toxicity study of T-6049 in Rabbits 1995 3M Environmental Laboratory, Saint Paul, MN Johnson JD, Wolter JT, Colaizy GE, Rethwill PA, Nelson RM (1996). Quantification of perfluorooctanoate and perfluorooctanesulfonate in human serum using ion-pair extraction and high performance liquid chromatography - thermospray mass spectrometry with automated sample preparation. J Chromatography Biomed Appl. Unpublished report, St. Paul:3M Company. Johnson, J. D. and Ober, RF. 1 979. Absorption of FC-95-14C in rats after a single oral dose. Project No. 8900310200, Riker Laboratories, Inc., St. Paul, MN. Johnson, J.D. and Behr, F.E- 1979. Synthesis and Characterization of FC-95-1C. Project No. 8900310200, Riker Laboratories, Inc., St. Paul, MN, Johnson, J.D., Gibson, S.J. and Ober, R.E. 1979. Extent and route of excretion and Ntios.su8e.90d0is3t1r0i2bu0t0i,onRoifkteortaLlabcoarrabtoorni-e1s4, iInncr.a,tsSt.afPtaeuarl,sMinNg.le i.v. dose of FC-95-'C. Project Johnson, J.D., Gibson, S.J. and Ober, R.E. 1980. Enhanced elimination of FC.-95-'C aLanbdorFaCt-o1r4ie3s-,"*ICnc.i,n Srta.tsPawuilt,hMcNho.lestyramine treatment. Project No. 8900310200, Riker Johnson, J.D., Gibson, S.J. and Ober, R.E. 1984. Cholestramine-enhanced fecal eliminationofcarbon-14 in rats after administrationofammonium 000096 January 21, 1999 T[o"xCipceolr.fl4u,o9r7o2o-c9t7a6n.oate or potassium ['`C]perfluorooctanesulfonate. Fund. Appl. Mandel JS, Johnson RA (1995). Mortality studyofemployees at 3M Plant in Decatur, Alabama. Unpublished report. Minneapolis: University of Minnesota. Murli H. Mutagenicity test on T-6295 in an in vivo mouse micronucleus assay 1996. CHV study No.: 17403-0-455 Corning Hazelton Inc. (CHV) Vienna, VA. Nabbefeld D. 1998 An Investigation of the EffectsofFluorochemicals on Liver Fatty Acid-Binding Protein. Masters Thesis, University of Minnesota. Thesis research performed at and supported by 3M Nabbefeld D., ButenhoJf.f,Bass N. and Seacat A. 1998. Displacement ofa fluorescently labeled fatty acid analogue from fatty acid carrier proteins by wyeth-14,643, ammonium perfluorooctanoate, potassium perfluorooctane sulfonate and other known peroxisome proliferators. (SOT Abstract. Accepted, Toxicologist 1998). Olsen GW, Burris IM, Mandel JH, Zobel LR (1998). An epidemiologic investigation of clinical chemistries, hematology and hormones in relation to serum levels of preeprofrlt.uroSotctPaanuels:u3lMfoCnoatmepainnym,alAeprfilluo2r2o,ch1e9m9i8.cal production employees. Unpublished Olsen GW, Gilliland FD, Burlew MM, Burris JM, Mandel JS, Mandel JH. An epidemiologic investigation of reproductive hormones in men with occupational exposure 10 perfluorooctanoic acid. J Occ Env Med 1998:40:614-622. O'Malley, K.D. and Ebbens, K.L.. 1980. 28 Day percutaneous absorption study with FC95 in albino rabbits. Expt. No. 0979ABOG32. Riker Laboratories, Inc., St. Paul, MN. Paez, DM, deBianchi, LP, Gil BA, Dapas O, Coronato,RG (1980). Fluoride 13:65. Roach DE (1982). Fluorochemical Control Study. Unpublished report. St. Paul:3M Company, May 25, 1982. Pothapragada V, (1975). Determinationof total fluorine in serum and other biological materials by oxygen bomb and reverse extraction techniques. Analytical Biochem 68:512-521. Pothapragada V, Singer R, Armostrong WD (1971). Determination of ionic (plus ionizable) fluoride in biological fluids. Procedure based on adsorbption of fluoride ion on calcium phosphate. Anal Biochem 42:350-359. Rusch, G.M. and Rinehart, W.E. 1979. An acute inhalation toxicity study of T-2306CoC in the rat. Project No. 78-7185, Biodynamic, Inc. 5 000097 January 21, 1999 Singer Land Armstrong WD (1959). Determinationoffluoride in blood serum. Analytical Chem 31:105-109.. Singer Land Ophaug RH (1979). Concentrations of ionic, total, and bound fluoride in plasma. Clin Chem 25:523-525. Sohlenius, A-K., Eriksson, AM., Hogstrom, C., Kimland, M. and DePierre, J.W. 1993. Perfluorooctane sulfonic acid is a potent inducer of peroxisomal fatty acid B-oxidation and other activities known to be affected by peroxisome proliferators in mouse liver. Pharrnacol. Toxicol. 72, 90-93. Taves D (1968). Evidence that there are two forms of fluoride in human serum. Nature 217;1050-1051. Taves D (1968b). Electrophoretic mobilityof serum fluoride. Nature 220:582-583. Taves D, Guy W, Brey W (1976). Organic fluorocarbons in human plasma: Prevalence and characterization. In: Filler R, eds. Biochemistry Involving Carbon-Fluorine Bonds. Washington, DC:American Chemical Society, pages 117-134. Thomford, PJ (1998). Study No. 6329-222 "4-week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys," Final Report Ubel FA, Sorenson SD, Roach DE (1980). Health status of plant workers exposed to fluorochemicals, a preliminary report. Am Ind Hyg Assoc J. 41;584-589. Wallace K. B. and Starkov A. 1998. The effectofperfluorinated arylalkylsulfonamides on bioenergeticsofrat liver mitochondria. DeptofBiochemistry and Molecular Biology, University of MN School of Medicine. Duluth, MN 55812, USA. Supported by a grant from 3M Company. Wetzel, LT., Burdock, G.A., Durloo, RS. and Colpean, BR. 1983. Rat teratology study. Project No. 1 54-160, Hazleton Laboratories America, Inc., Vienna, VA. Yamamoto G, Yoshitake K, Sato T, Kimura T and Ando T (1989). Distribution and formsoffluorine in whole blood of human male. Analytical Biochem 182:371-376. York, R., Oral (Stomach Tube) Developmental Toxicity Study of PFOS in Rabbits, Argus Laboratories Study Number 418-012 final draft report dated December 17, 1998. APPENDIX o 000098 January 21,1999 FIGURE A1. Reliability of PFOS Serum Measurements in 12 3M Employees. w 0 a 2 a ES : Eo sa 3 a a a : 5 re"r e S -- ee no tat Test (Fobruary, 1950) 86 000099 Appendix Table AL Mesn, Media, StandforrdADnetvwieartpioannd(5DD)eofcMteaEnplaonydeResaComonbfignFeOdeS,,1D9e9m8og(r1a7ph8i)c, Se1m99C7h(eNi-s1t4i7)es ad Hematological prose 1995 Da 1997 Dua gm) Mew Mem sp Rage Mem Metin sn Range PEOS pm) 0-< ppm 049" 05 027 000-0.90 052! 052 021 1 -<3ppm 182! 177 038 100-291 178! 1.64 056 3-<6ppm. an 397 081 3.00-5.80 387" 359 070 26ppm. 8F17ral=u32e197 ,p< 0001 252 606-1283 2F200 r=o367l6,6i6<80c001 159 010-097 102-289 309-530 605-93 22 oa<pigmm oown a5 55 251A0-5g60es) 23 a1 u5 u2ea 28 m emmm o3w 0 77 27.55%5 "2 52 55 nBisni < Falue=37,p= 02 Falue =5.1,p= 002 o ry21,199 vGoems 0V-<aipppmm S26lpempm oL<ispem 2S5cmomm Op<impm 22 o<emmm 2 22 1995 Dua Men Metin sp 008s 0016 0097 0172 0030 1no Flic=40.9 009 22875 u20s3 4528 M7Y B3s0 a325 Fralue=37,p=.02 2668 0000 1ed3 0046 S00 024i Fale =48,p =003 Table Al (Contin) Range Mean Median Also rinks) 0000.-3366 0053 0or 00002690 0120 o0i Fuale=18,p1=5 BMI Gai) em9sewr7 wmeennos m2670 22694 0w8s 779 Fualue =21,p=.10 Cigaerrdisy) o0o0l2e5o0 4872 0000 0000.3000 4501 0000 Fualue= 15,p= 23 1907s sn ooss 012s 97 0a0 u94s 1554 Range 00-:4530 00.17-108 w18a10s5 w191i73e60 00-4a0 005500 Soman 21,1999 pormo)s 01-25<3p1ppmm 23-6<p6mpm 01--<31ppomm 32-6<p6mpm PR 22 smi<m emm 2 emm 8 1995Data Mem Median 116554 115s0 118614 M16o0 Fualue= 11,p= 36 1100 0000 010 0129 Fualie=23,p=.08 8s 86 88 58 5 Fualue <09,p=44 "Table AI (Continued) SD Range Mem Metin BUN 34s0 86 0-260 1w4s2 11400 i37 100.022300 n15s0 121500 Fualue=05.0p67Crstinine 0022 00771166 0099 0099 0023 0076:62 0190 0099 Fualue 04,9078 Glucose 25 wao 9ow 85s B Wo eTesow 5 Fualue=p0=65,9 1997Daa sp 2328 2a9 o011 0012 3" 27 Range 9600--221600 9900--129000 0067-:1123 0078.-1114 6s5-s30n3 7500-997 eetom TaAlClote Groos 95D 1597008 Men Mem Shes ven Mem ange vCipoe mmwmm7om oaAslkamlinoePh@omsphatoase now z"n Slope wo won wm own a 2ri01ee20 Somm PBua w 13,p2-8 oan ame wowFuae=117,pi-5s2 a oCaspme Gar 8wow wm wrems wNonm 03 i 0-1 SSoiomm soFueow=05%p= 71 uown mWRm x 3F0a e311p 34 ii fih 25 SUiSemmm 02 ast oxw nom hmwwo omxaosx 77 b15n5 23a oamm 5Fouex0:o18%x57 14 08 wnsm oXmFuaie=0a05%p- 67 73 6u0n0 etm abeAL (Conic) wpoos soma Men Nein SD 1700 hese vem Mim Rane oUcSigpmm W#o @Booma wmAamLwT ownw i0 CSo e wSFraoo1wh0p 38 h3 owWey n on Fatu e-098,p- 40 is Toul Bin ocigm om om ow oma om ow om 1-<3 ppm 0.66 0.60 030 020-150 061 0.60 021 Tm 3-<6ppm 00P6u%4ahe- 4po0.m60 05 0o2n8 owax 020-140 o0.6mF3 r20e0a.5pn0e04 0o3n1 Dit Bian ocigm 02 om oes omeow ox ow oo 3223 io1-<mm3ppam 00o2Fu31nahe=06,po0o--2mm508 0o.o0wm6 o0G10oi-0sn40 0oo1m2baie 30oo51plw0- 02 0oo.0ow4 2 iWien ne 030-230 030-130 frit 040-130 010.000 0SS1oo0s-so0t2a00 " ey. Table A (Cote) proms 1998 Dua Men Malm SD Rage 1970s Men ves Cholesterol CCoppee aome woasn oonw wweassw aw e wan @"0 1i0e-:230480 S3-o<m6mppm 2Fm1r4alioe=01m,52-194% 3ks% des 128-278 0o9mFal432,40p06 %29 15962--235210 LDL GCcimgm m mo wmedow adows womm ws a50.2305 oom 3-<6ppm W3 Frwat =021.3pm5e287 Eodm sam 65-190 1e 4F8aew =37.1p42m00 624 3111-6190% HDL 32Eem R am w Aow a"ooonn mwwm oE u Yis 0 iou 0 29-67 Bie ER Fiaie-29,p"-080% ua hFroe 1w-34 J ni " sy21,199 p(rpoms) 01-<tppppmm 2566ppmm 01-2<51ppppmm 36ppmm 5 22 ooppmm 2 e 6pm m 1995 Daa Men Medin SD 1961 9B % w 11528 B woaoss Fuale L1, p35 ias ia 32 aa E@S 22 Fualve=24,p=07 115552 155251007 115555 540081 Fualie =22,p-10 Table AT (Continued) 1997 Daa Range Mem Mefim sn iglesrides awleses1s 11207 101682 eaaamn oime i10s1 952 Fale =05,p=67 Hemost foaes pay WW" 23 aulsi iI) w"" 32 Fuaue=21,p.11 femogibin 0B-o1c6a7 11554s 15555 009s 1u6raes150 1W510 0i7o Fua=1l8,up~e15 Range 348.-5123040 1405-339542 W5 052 F39Y50 11353-0117703 114215-0116627 soma 21,1999 (oFpOm)S 10--<31 ppppmm 32-6<p6pmppm 01--<31pppomm 32-6<p6ppmpm 2 22 o-capm N 2 Sp pm 26pm 1995Data Mem Medi SD 4590 5a0 0033 5500 4590 0025 Fualue 04, p=.75 330193 0M921614 33122 0M53 2u8s Fralue=09,p=45 3n12 B Ba 0607 nl 2607 31 Fralue = BI 02,p=90 06 TableAl (Continued) 1997 Daa Range Mean Median sp RC 4433.5s7 5sa0 5512 0033 4406.-55337 5500 +590 0033 Fralue= 14, p=25 men 22674.-3333 228620..33669 30046 330053 112s 330032 330132 2119 Fralue 06, p=65 Mate N 9-M7 7 336 33366 0[3 N33 2230 Bs 3 3s 339 01 0s Fualue=07, p=.56 Range. 4413--5559 4474-.5557 22667--334318 7252--333249 32128--334496 320-344 354-346 o a1,1m Table AT (Continue) Pros 195 Dus 199700 Gm) Mem Meds SD Mme vem voli Range yey 0T-chtmpm ou 56pm 9% saos sax smisioow oma ow a w5o mmoeem a 52 mee Somm F5ralie=L1, p35 ar wns omFras=06,pea.59 57 mos wae oacpimm 1a10 i5s0 2B0o0 vaeess 6a 5sso v15o aassins: S3-o<o6pmpm Fra2u7e06=43, p=0i66o9 022 4e1a-r13s3 662 Fualoe=22,76p1 eo i1s5 aw 40-100 2 amma En 22 s1-o3mppem o2m9 o24m 2 em 8s in @ Fualue=2.1, p=10 Plaselets w mem am ED Sao 00 mmoou a2m9 mom mw p2i3r fo 5 w6-a B e14n -359 S$ ems Fvalue = 0.6, p=0.60 " samary21, 1999 Table A (continued) 21.. MMeeaanns issigsinginfiifcicaannttllyy ddiiffffrernetn(ptp <<..0055,, BBoonnffeerrroonnii ((DDuunnn)) eets))tthhaann tthheemmeaenaooffnth0e- <theprpPmROPSROcSatceagtoergioersy. 3. Meanissignificantly diferent (p< 05, Bonferroni (Dunn) test) than the mean of1 - <3 ppm category. 0"-S<mtplpeoSmize 31--<36 ppppmm 26pm 1a99s5Dun 1997Data o3n7 2&5 2222 28 Table A2. Descriptionof Final Employment and Vital Status for Male (n = 1,639) and Female (n = 318) Employees Final Employment Status and Vital Status Male Employees N % [Female Employees N %. Currently Employed Retired Alive Deceased Unknown 810 49.4 141 44.3 59 36 9 238 10 0.6 0 0.0 0 0.0 0 0.0 Terminated Alive Deceased `Unknown 694 423 164 516 29 18 1 03 6 04 0 0.0 Died While Employed TOTAL 3 1,639 19 100.0% 3 318 09 100.0% *percentages may not add to 100 due to rounding 7 000110 January 21, 1999 Table A3. Characteristics of Male (n = 1,639) and Female (n = 318) Employees Variable Males Number of Employees 1,639 Number of Person-years 33,108 Numberof Deaths 70 Average Age Started Work 25 Average Year of Entry 1971 Average Age at Death 47 Average Year of Death 1984 Females 318 4.807 4 26 1977 28 1980 0 00011. 1 Jamary 21,1999 Table Ad. Distribution of Men by Age and Yearof Entry into Follow-up (n = 1,639) Age <20 20-24 25-29 30-34 35-39 40-44 45-49 50-54 55-59 60+ TOTAL Wo1960 1964 18 114 64 21 22 19 3 0 0 0 261 1965 1969 34 238 139 58 25 18 3 1 1 0 517 Year of Entry into Follow-up 1970 1975 01980 1985 1974 1979. 1984 1989 86 31 4 2 301 96 28 18 65 37 21 14 33 19 8 10 9 15 6 Ss 4 10 8 2 4 5 0 2 0 0 2 3 0 0 0 0 0 0 0 0 502 213 77 56 ow1990 1991 0 3 Ss 4 1 0 0 0 0 0 13 TOTAL 175 798 345 153 83 61 17 6 1 0 1,639 000112 January21,1999) `Table AS. Distribution ofWomen by Age and YearofEntry into Follow-up (n = 318) YearofEntry into Follow-up 1960 1965 1970 1975 1980 1985 1990 to to to to to Age 1964 1960 1974 1979 1984 1989 1991 TOTAL 20 2 [Hl 15 16 a 0 0 52 20:24 4 2 23 50 4 9 1 113 2529 3 4 4 a7 4 5 3 70 3034 2 3 2 1s 1 4 2 29 35:39 1 2 3 16 3 3 2 30 40-44 0 0 0 8 3 2 0 13 45-49 000s 1 1 0 7 50-54 0 00 3 0 0 0 3 5559 000 1 00 0 1 60+ 0 00 0 0 0 0 0 TOTAL 12 43 50 161 20 24 8 318 100 rreeof(3 January 21, 1999 E`vTeabrleEmAp6l.oyCeadusien-CShpeecmiifciacl SDMepRasrtfmoernMte(n5) Using the U.S. as Comparison Population (n = 1.050) Cause of Death Observed Expected Deaths Deaths SMR `All Causes of Death All Malignant Neoplasms 57 13 31s 700 169 769 CancerofBuccal Cavity and Pharynx 0 05 - Cancer of Digestive Organs and Peritoneum 1 36 275 `CancerofEsophagus Cancer ofStomach 0 0 04 0s -- Cancer of Large Intestine ~~ 1 Cancer of Rectum 0 13 03 769 - CancerofLiver and Biliary 0 Passages 03 - Cancer of Pancreas CancerofAll Other 0 08 - 0 ol - Digestive Organs Cancer of Respiratory System 7 Cancerof Larynx 0 6.1 02 1s. -- Cancerof Bronchus `Trachea and Lung. 7 58 1207 Cancer of Other Respiratory Organs 0 ol - Cancer Cancer of Prostate ofTestes and Other ~~ 0 0 0s 02 -- - Male Genital Organs CancerofKidney 0 Cancer ofBladder and Other 1 0s 02 415.5 Urinary Organs 95% CL (55.0,90.6) (409, 131.5) (0.0,300.6) 07,1532) (00,9133) (0.0, 686.1) (1.9,428.5) (00,1271.9) (0.0,1,188.2) (0.0, 480.6) (0.0,3,203.2) (463,237.1) (0.0,1,891.9) 48.5,248.7) (0.0,4214.1) (0.0,805.9) (0.0, 1,678.5) (0.0,768.2) (104,2,3153) wi 000134 January 21, 1999 Table A6. (Continued) Cause of Death Observed Expected Deaths Deaths SMR `CanofcBreairn and Other 1 09 17.2 Central Nervous System CanofcTheyrorid and Other 0 Endocrine Glands 01 - CancerofBone 0 Cancer of All Lymphatic and 2 ol 22 929 Hematopoietic Tissue Lymphoma 0 03 - Hodgkin's Disease 0 Leukemia and Aleukemia | 03 03 1200 CancerofOther Lymphatic 1 and Hematopoietic Tissue 07 1372 Malignant Melanoma of Skin 0 06 - All Other Malignant Neoplasms 1 15 68.9 Diabetes Mellitus CerebrovascularDisease 0 11 - 1 23 434 All Heart Disease Hypertension 1 25 488 0 ol - Respiratory Disease UlcerofStomach and 0 32 - 0 02 - Duodenum Cirrhosisof Liver Nephritis and Nephrosis 5 50 1002 0 03 -- External Causes Accidents 2 22 902 16 136 173 Motor Vehicle Accidents ~~ 8 All Other Accidents 3 78 59 1028 1357 Suicides 3 52 57.7 Homicides and Other Extemal 1 Causes 32 314 Residual Causes* Unknown Causes & 7 90 776 2 "all other causesof death combined & no death death certificate category obtained; included only in all causes of 95% CI (29,653.0) 00,5132) (00,4,368.3) (11.2,3355) 0.0, 1,341.4) (0.0,1,1633) (3.0,668.8) (3.4,764.5) (0.0,614.7) (17,383.7) 00,3239) (11,2417) (244,874) (0.0,3,389.9) (00,1163) (0.0,1,796.3) (20.7,292.7) 00,1,308.2) (55,1393) (67.0,190.5) (44.4,202.5) (586,267) (11.9, 168.6) 0.8,175.1) (12,1599) 000115 102 Jamar 21, 1999 Table A7. Cause-Specific SMRs for Men Only Employed in Chemical Department (s) Using the U.S. as Comparison Population (n = 485) Cause of Death All Causes of Death All Malignant Neoplasms Cancer of Buccal Cavity and Pharynx Cancer of Digestive Organs and Peritoneum Cancer of Esophagus Cancerof Stomach Cancer ofLarge Intestine Cancerof Rectum PCaasnscaegresof Liver and Biliary Cancer of Pancreas Cancerof All Other Digestive Organs CancerofRespiratory System Cancer of Larynx Cancerof Bronchus Trachea, and Lung Cancer of Other Respiratory Organs Cancer of Prostate Cancerof Testes and Other Male Genital Organs. CancerofKidney Cancerof Bladder and Other Urinary Organs Observed Deaths 32 9 0 Expected Deaths 44.1 9.6 03 0 21 0 02 0 03 0 08 0 02 0 02 0 05 0 0.1 5 3.6 0 0.1 $ 34 0 0.1 0 03 0 0.1 0 0.3 1 02 SMR. 72.5 93.6 --- - w -- - --- n- -- --- 141.0 -- 147.8 -- --- -- -- 669.9 95% C1 (49.6, 102.4) (42.8,177.8) (0.0, 1,396.4) (0.0,175.2) (0.0,1,577.7) (0.0,1,195.2) (0.0, 488.5) (0.0,2,175.6) (0.0,2,072.6) (0.0,827.2) (0.0, 5,692.4) (45.8,329.0) (0.0,3,198.4) (48.0,344.9) (0.0, 7,763.4) (0.0, 1,202.0) (0.0,3,501.4) (0.0, 1,365.0) (16.7,3,732.7) 03 000116 January 21, 1999 Table A7. (Continued) Cause of Death Observed Expected Deaths Deaths SMR. `CanofcBreairn and Other Central Nervous System T0s 226 Cancer of Thyroid and Other Endocrine Glands 0 00 - Cancerof Bone 0 00 - Cancer ofAll Lymphatic and Hematopoietic Tissue 2 a2 1740 Lymphoma Hodgkin's Disease [I - 0 02 - Leukemia and Aleukemia Cancer ofOther Lymphatic 104 263 104 2495 and Hematopoietic Tissue Malignant Melanomaof Skin 0 03 - All Other Malignant Neoplasms Diabetes Mellitus 0 08 0 06 - Cerebrovascular Disease All Heart Disease 013 711 sa Hypertension ool - Respiratory [I] - Ulcer of Stomach and Duodenum ~~ 0 0.1 - Cirrhosis of Liver Lo16 623 Nephitis and Nephrosis Extemal Causes 0 02 -- 10105 950 Accidents 965 1394 Motor Vehicle Accidents 536 1380 All Other Accidents Suicides 4 29 1402 125 40.0 Homicides and Other External ~~ 0 15 - Causes Residual Causes* 30 a7 63.4 Unknown Causes & 2 "all other causesof death combined & no death death certificate category obtained; included only in all causes of 95% CL (56,12403) (0.0,9,544.3) (0.0, 8,697.5) (11,6287) (0.0,2,466.1) (0.0,2,346.1) (57,1260.7) (62,1390) (0.0,1,200.4) (0.0,455.3) (0.0,589.0) (00,2744) 15, 110.1) (0.0, 5,834.4) (00,1953) (0.0,3,083.3) (16,3473) (0.0,2,346.9) (45.6,174.7) (637,264) (48,3220) (38.2,359.0) (1.0,223.1) (0.0,247.4) (13.1, 185.3) - 000117 Samay 21, 1999 Rat Oral (diet) Carcinogenicity on N-EtFOSE Study Outline Study Objectives: Main Objective ~ To determine the carcinogenicity of N-EtFOSE upon chronic oral administration. 5S2ecaonndda1r3ywOebejkesc,tirveesspe--ctTivoeldye,teorfmcionmepcohuronndicadamnidnissutrbacthiroonn.icTtooxdiecitteyrmoifnNe-cEotmFpOoSuEnda'ftser potential for perixosme proliferation (palmitoyl-CoA oxidase activity) and hepatocyte proliferation (PCNA, proliferative cell nuclear antigen). To measure `compound and/or `metabolite levels in the liver and serum after various time-points of exposure. GLP Status: A GLP study with appropriate QA audits; a signed QUA statement to be included in the final report. Animals: Male and female Charles River CD rats Dose Groups: Eight ~controls (two groups), 1, 3,30, 100 & 300 ppm in diet plus a 100 ppm recovery group (receive compound for 52 weeks followed by 52 weeks without compound). Number per Group: 50 males and 50 females per group plus additional animals for interim sacrifices at4, and 40 females). 13 & 52 weeks. (Note the 100 ppm recovery group has 40 males TaensdtpuArrittiyc.le: N-EtPFOS furnished by Sponsor who is responsible for compound identity Compound Administration: Daily administration for up to two-years (104 weeks) admixed in the diet. Clinical Observations: Twice daily. Body Weights: Once a week for the first 16 weeks; once every four weeks thereafter. Food Consumption: Once a week for the first 16 weeks; once every four weeks thereafter. Hematology and Clinical Chemistry: Ten rats/sex from 0, 1, 3, 30 & 100 ppm groups at weeks 14, 52 and 104. Urinalysis: Ten rats/sex from 0, 1, 3, 30 & 100 ppm groups at weeks 14, 52 and 104. 108 0001.18 January 21,1999 IwneteekrsifmorNePcCroNpAs,iepsa:lmFitiovyelraCtos/Aseoxxfidraosme,0,an1,d3c, o3m0,po1u0n0d&le3v0el0 spapmpmlegsr.ou(pNsoatte4saamnpdle1s4 for organ weights 100 ppm groups and histopathology taken at at 52 weeks for compound 14 weeks.) Ten rats/sex from 0, 1, 3, levels, organ weight, histopathology 30 samples. Organ Weights: Measured at 14 and 52 week interim necropsies. aHniismtaolpsa.thCoolmopgly:eteMitcirssousecoepxiacmienxaatmiionnatoifon(oofnseelceocnttreodltgirsosuueps),at3,1430a,nadn5d21w0e0epkpm 104 weeks animals. Histopathologic Selected tissue examination of 100 ppm recovery results will indicate extend oftissue examination animals. of 1 ppm and second control group. 106 co0a0t119 January 21, 1999 Rat Oral (diet) Carcinogenicity on PFOS Study Outline Study Objectives: Main Objective chronic oral administration. ~ To determine the carcinogenicity of PFOS upon and 13 wSeeeckosn,darresypeOcbtjievcetliyv,eosf--cToompdoeutenrdmiadnmeicnhisrtornaitcioann.d subchronic toxicityofafter To determine compound's 52 p`prootleinfteiraaltifoonr p(ePrCiNxAo,smperoplriofleirfaetriavteiocnel(lpnaulcmlietaoryaln-tCigoeAn)o.xidTaosemeacatsiuvritey)coamndpohuenpadtoacnydt/eor `metabolite levels in the liver and serum after various time-pointsofexposure. GLP Status: A GLP study included in the final report. with appropriate QA audits; a signed QUA statement to be Animals: Male and female Charles River CD rats cDoosmepoGurnodupfsor:52Siwxe--e0k,s0.f5o,l2lo,w5e,d&by2502pwpemekisn dwiietthpoluutscao2m0popupnmd)recovery group (receive Number per Group: interim sacrifices at 4, 50 13 males and 50 & 52 weeks. females per group (Note the 20 ppm plus additional recovery group animals for has 40 males and 40 females). `Test Article: purity. PFOS furnished by Sponsor who is responsible for compound identity and aCdommipxoedunindtAhdemdiineti.stration: Daily administration for up to two-years (104 weeks) Clinical Observations: Twice daily. Body Weights: Once a week for the first 16 weeks; once every four weeks thereafter. Food Consumption: thereafter. Once a week for the first 16 weeks: once every four weeks Hematology and Clinical Chemistry: at weeks 14, 52and 104, Ten rats/sex from 0, 0.5, 2, 5, & 20 ppm groups Urinalysis: Ten ratsisex from 0, 0.5, 2, 5, & 20 ppm groups at weeks 14, 52 and 104. fIonrtePrCiNmAN,ecpraolpmsiiteoysl: CFoivAeorxaitdsa/ssee,x farnodmc0o,m0p.o5,un2,d5l,e&vel2s0amppplmes.gr(oNuposteatsa4mapnldes1f4orweeks is 06120 January 21,1999 o2r0gpanpsmwgerioguhptss aatnd52hiwseteokpsatfhoorlcogoymtpaokuenndatle1v4elwse,eokrsg.a)n Tweenigrha,ts/hsiesxtofpraotmho0l,o0g.y5,sa2m,p5l,es&. Organ Weights: Measured at 14 and 52 week interim necropsies. Histopathology: Microscopic examination animals. Complete tissue examinationof 0, of 2, selected tissues at 5, and 20 ppm 104 14 and weeks 52 week animals. Sinedlieccatteedetxitsesunedoefxtaimsisnuaetieoxnamoifn2a0tipopnomfr0e.c5ovperpymangirmoaulps.. Histopathologic results will os 000121 January 21, 1999 Rat Teratology Rangefinder on N-EtFOSE Study Outline Study Objective: Explore possible dose levels for an oral rat teratology study. GLP Status: Study should be conducted under rangefinder study QA audits will not be done GLP principles but since itis a Animals: Mated female Charles River CD rats Dose Groups: Seven- control, 0 mg/kg; low-1, 1 mg/kg; low-2, 5 mg/kg; mid-1, 10 mg/kg; mid-2, 20 mg/kg; high-1, 25 mg/kg; high-2, 35 mg/kg Number per Group: 8 mated females per group `Test Article: N-EtPFOS will `compound identity and purity. be fumished No reserve by the sample Sponsor who is responsible will be required. for `CTowmeepnou80ndonAddamyisni6stthrrauti1o7no:fgOersatlatiinotnb.atAinoanlyosfiscoofmpdoousinndgspurseppearnadteidoninwiwlaltenrotwibteh 2% done, however, records will be maintained on how dosing preparations were made. Clinical Observations: 4106 hours later. Twice daily; once approximately one hour after dosing and then Body Weights: On days 0,4,6,8, 10, 12, 14, 16, 18 and 20 ofgestation Food Consumption: On days when body weights are obtained uCteesrausreaanndStesctcioonnte:ntOsnwidlalyb2e0roefmgoevsetadtainodn twheeigfheemda.lesThweillnubmebaenresotfheitmipzleadnaatnidontsh,e the dneutmebremrinoefdlaivned arnedcodredaedd. feTtuhseesf,eatunsdesthweilnlubmebeseroxefd,eawreliygahnedd laanted reexsaomripnteiodnfwoirlalnbyegross abnormalities. The ovaries will examined for the number of corpora lutea. 109 cr 0O0P 0122 January 21, 1999 Rat Teratology Study on N-EtFOSE Study Outline Study orally Objective: To determine maternal and administered N-EXFOSE in pregnant rats fetal toxicity and teratogenic potential of GLP Status: A GLP study included in the final report with appropriate QA audits; a signed QUA statement to be Animals: Mated female Charles River CD rats Dose Groups: Five - control, 0 mg/kg; mid-2, (10 or 15?) mg/kg; high, (20 or low, 25?) (1 or 1.5 mg/kg. %) m/kg; Final dose mid-1, levels (5 2) me/kg; will be adjusted after range finder study results are available. Number per Group: 25 mated females per group `Test Article: N-EXPFOS will compound identity and purity. be furnished by the Reserve sample to Sponsor who be retained. is responsible for TCwoemepnou80ndonAddamyisni6sttrhrauti1o7no:f Ogersatlatiinotn.ubaStiaomnplofescoofmdpoosuinndg spurseppaernaetdioinnswwaitlelrbweitfhro2ze%n and retained for possible analysis by the Sponsor. dosing preparations were made. Records will be maintained on how Clinical Observations: 4106 hours later. Twice daily; once approximately one hour after dosing and then Body Weights: On days 0,4,6,8, 10, 12, 14, 16, 18 and 20 ofgestation Food Consumption: On days when body weights are obtained uCteesrausreaannd Sietsctcioonnt:entOsnwidlalyb2e0orfegmeosvteadtiaonnd twheeigfheemda.lesThweillnubmebaenresotfheitmipzleadnaatnidontsh,ethe dneutmebremrionfedliavnedarnedcodredaedd.feTtuhseesf,eatunsdesthweilnlubmebseerxoedf,eawreliygahnedd laanted reexsaormpitnieodnsfwoirlalnbyegross abnormalities. The ovaries will examined for the numberof corpora lutea. pFreotcaelsEsxeadthfiornavtiiscoenrsa:l eAxpapmrionxaitmiaotneblyy tohneeW-ihlasolonf Ttheechfneituqsuees fforrosmofetatcihssluietedrewvielllopment. The remaining Alizarin Red S fetuses will be eviscerated staining method. and processed for skeletal examination using 1o 000123 January21, 1999 `(TLooxric1o.k5imnegt/ikcg)SaatenldliftieveAnfiemmaalless; atGrthoeuhpisgohfdmoastee(d20feomral2e5s,mgf/ivkeg)fepmlaulsestharteethfeemlaolwesdoaste soattheelrlidteosfeemlaelveelss,wiwlilllbebekidlolseedd.andOnfotlhleowdianygasfatmerpltehes wlaisltldboeseco(lldeacyte1d8:ofsgeersutmatainodn)litvheer flirtotemr ctahendbaemstaonrdedptloagceetnhtearsaasnodnfeetsuasmepslfer;olmiktehweiusteerfuosr.th(eNofteteuspelsa)ceTnhtaessefrsoammpalseisngwliell be frozen and shipped to the sponsor for possible analysis. nm 000124 January21, 1999 Rabbit Teratology Rangefinder on N-EtFOSE Study Outline Study Objective: Explore possible dose levels for an oral rabbit teratology study GLP Status: Study should be conducted under rangefinder study QA audits will not be done GLP principles but since it is a Animals: the testing Mated female laboratory not New Zealand White rabbits a the supplier and shipped (Note the rabbits are to be mated as mated females to the testing at laboratory.) Dose Groups: Seven- control, 0 mgkg; low-1, 1 mgkg; low-2, mg/kg; mid-1, 10 mg/kg; mid-2, 25 mg/kg; high-1, 50 mg/kg; high-2, 75 mg/kg Number per Group: 5 mated females per group Test Article: N-EtFOSE will `compound identity and purity. be furnishedby the No reserve sample Sponsor who is wil be required. responsible for `CTowmeepnou80ndonAddamyisni7sttrhrauti2o0n:ofOgersatlatiinotnu.baAtnioanloyfsicsoofmdpoosuinndgspurseppearnadteidoninwiwlaltenrotwbieth 2% done; however, records will be maintained on how dosing preparations were made. Clinical Observations: 4106 hours later Twice daily; once approximately one hour after dosing and then Body Weights: On days 0,7, 10, 13, 16, 19, 24 and 29ofgestation Food Consumption: On days when body weights are obtained CuteesraurseaanndSietsctcioonnt:entOsnwidlalyb2e9roefmgoevsetadtainond twheeigfheemda.lesThwiellnubmebaenreosfthiemtpilzaendaatnidontsh,ethe d`neutmebremrinoefdlaivnedarnedcodredaedd.feTtuhseesf,etaunsdesthweilnlubmebewreoigfheeadrlaynadnedxlaamtienreedsorfoprtiaonnys gwriollssbe `abnormalities. The ovaries will be examined for the number of corpora lutea. 2 000125 January 21,1999 Rabbit Teratology Study on N-ECFOSE Study Outline Study Objective: To determine maternal and fetal toxicity and teratogenic potential of orally administered N-EtFOSE in pregnant rabbits GLP Status: A GLP study included in the final report. with appropriate QA audits; a signed QUA statement to be Animals: the testing Mated female laboratory not New Zealand White rabbits at the supplier and shipped (Note the rabbits are to be mated as mated females to the testing at laboratory.) Dose Groups: Five - control, 0 mg/kg; low, (1?) mg/kg; mid-1, (5 2) mg/kg: mid-2, (10 2) mg/ke; high, (25 2) mgke. study results are available. Final dose levels will be adjusted after range finder Number per Group: 22 mated females per group `Test Article: N-E(FOSE will compound identity and purity. be furnished by the Reserve sample to Sponsor who be retained. is responsible for Compound Administration: Oral intubation of compound suspended in water with 2% a`Tnwdereenta8i0neodnfdoarypsos7sitbhlreu a2n0aolfysgiesstbaytitohne. SpSoanmspolre.soRfecdoorsdisngwiplrlebpearmaatiionntsaiwnieldl boen fhroowzen dosing preparations were made. Clinical Observations: Twice daily; once approximately onehourafter dosing and then 410 6 hours later. Body Weights: On days 0,7, 10, 13, 16, 19, 24 and 29 of gestation Food Consumption: On days when body weights are obtained Cesarean Section: On day 29ofgestation the females will be anesthetized and the nutuemrbuseraonfdliitvsecoanntdendtesawdilfletbuesesr,emaonvdetdheanndumwbeiegrohefd.earTlhyeanndumlbateerroefsoirmptpiloannstawtililonb,e the determined and recorded. The fetuses will be weighed and examined for any gross abnormalities. The ovaries will examined for the number ofcorpora lutea. us oee1ze January 21, 1999 eFveatlaulaEtexatmhiencaotnitoenntss:ofAthmeidc-rcaoniruomn.al Tslhieceiwnitlelrnbael omragdaensionftthheehethaodroafciecacahndfaetbudsotmoinal icnatveirtniaelsoafbnaolrlmafleittuiseess.wilAltbtehiesxtaimmientehde isnextohfetfhreesheastcahtefeutsuisnwgiSltlabpeledse'tteercmhinnieqdu.e Affotrer removalofthe viscera, the carcasses will processed for skeletal examination. T(o1x?imcogk/iknge)tiacndSaftievlelifteemAalneismaatlst:he Ghrigohupdsoosefm(2a5te?dmgfe/mkagl)esp,lufsivtehrfeeemafleemsalaetsthate oltohwerdose fdeomsaelleesvewlisl,lwbiellkibleleddosaendd. foOlnlotwhiendgasyaamfptleersthweilllasbtedcooslele(cdtaeyd:21seorfugmeastnadtiloinv)erthferosamtetlhleite sdtaomreadntdogpeltahceerntaassoannedsfaemtpulsee;s flirkoemwitshee fuotrertuhse. f(etNuostees)plTahceensteassafmrpolmesa wsiilnlglbeelfitreorzecnananbde shipped to the sponsor for possible analysis. 14 000127 January 21,1999 Rat Two Generation Reproduction Study of N-E(FOSE Study Outline SretpurdoyduOcbtjievcetifuvnec:tiTonooefvamlaulaeteanthdefeefmfaeclteorfaotrsa(lFoadgmeinneirsattriaotni)oannodfoNn-tEh{eFdOeSvEeloonpmtehent and. reproductive via lactation capaciotfy the subsequent Fy generation which were exposed in utero and GLP Status: A GLP study included in the final report. with appropriate QA audits; a signed QUA statement to be Animals: Male and female Charles River CD rats Dose Groups: Five - control, me/kg; high, 15 mg/kg. 0 mg/kg; low, | mg/kg; mid-1, 5 mg/kg: mid-2, 10 Number per Group: 35 males and 35 females per group TceosmtpAorutnicdlei:denNt-itEy{aFnOdSpEuriwtiyl.l bReesfuerrnvieshseadmpblyetthoe bSepornetsaoirnewdh.o is responsible for wCiotmh p2o%unTwdeAednmi8n0.istCroamtpioonu:ndDaaidlmyinoirsatlrianttiuobnawtiiloln sotfarct o4mwpeoeukndprsiuosrpteonmdaetdinign.water gCeosmtaptoiuonnadnaddmliacntiasttiroan.tiTohnewiFlyl pcuonptsiwniulel tnhortoruegcheimvaetianngy adnodseisnvtihaegfaesmtrailcesintthubraotuigonh. aSnaamlpylsiessobfy tdhoesiSnpgonpsroerp.araRteicoonrsdosnwwilelebke2maainndta8iwnieldl obne fhroowzednoasnidngrpertaeipnaerdatifoornspowsesrieble made. Clinical Observations: 410 6 hours later. Twice daily; once approximately one hour after dosing and then Body Weights: Weekly except when male and females are co-inhabited during mating. Food Consumption: mating. Weekly except when male and females are co-inhabited during Estrus Cyeles: Daily vaginal smears will be taken from females two weeksprior to `mating to determine estrus cycle information. Mating: Afie4r weeksof compound administration, within a be co-inhabited with one female. A female will be determined dose group one as mated upon male the will 000128 us January 21,1999 presence of sperm of gestation. positive vaginal smear. The day ofmating shall be considered as day 0 Early Gestation Evaluation: 10 Fy females per group will killed on day 10 ofgestation. eParrelgynaanndcylawtiellrebsoerpdteitoenrs.minAetd naescwreolplsyassethreumnuamnbdelrivoefrismapmlpalneastiwoinlls abnedtatkheennfurmobmerfiovfe of the females in cach dose group. sponsor for possible analysis. These samples will be frozen and shipped to the Male Sacrifice: One week after mating all Fo male rats will be killed aftear terminal body weight has been obtained. The animals will be necropsied and any gross lesions bwiellwebiegdheesdcrainbdedfiaxnedd rfeocroprodsesdi.blTehheistteosltoegs,icepeixdaimdiynmaitsi,onp.ro(stNaottee:antdestsiesmwiinlallbveesfiicxleedsiwnill Bouin's solution; other tissues in 10% buffered formalin). At necropsy serum and liver samples will be taken from fiveofthe males in each dose group. These samples will be frozen and shipped to the sponsor for possible analysis. Female Parturition: The remaining 25 females will continue until term and be allow to deliver their liters. The offspring (F generation) will be counted, sexed and weighed on postnatal days 0, 4, 7,14 and 21. Offspring will be randomly culled to four males and four females on day 4 postnatal, Milk Samples: On day 4 postnatal when litters are culled to eight pups, milk curds will be collected from the stomach of the discarded pups from five littersof each dose groups. `The milk curds from the pups in a litter are to be combined as a single sample. Pup Examinations: Pups will be observed daily for moribundity/mortality. Developmental landmarks consisting ofeye opening, pinna detachment, surface righting, testes descent, and vaginal opening will be recorded for each litter. Fy Dosing: After weaning at day 21 of lactation, the Fy pups will receive compound by daily oral (gastric intubation) dosing. Dose level on a mg/kg basis wil be the same as the pup's dam dose level Female Sacrifice: One or two days after weaningofthe litter on day 21oflactation, all Fo female rats will be killed aftear terminal body weight has been obtained. The animals will be necropsied and any gross lesions will be described and recorded. The ovaries will be weighed and fixed for possible histologic examination. (Note: ovaries will be fixed in 10% buffered formalin). At necropsy serum and liver samples will be taken from five of the females in each dose group. These samples will be frozen and shipped to the sponsor for possible analysis. 116 000129 January 21, 1999 F1 Pup Neurological Testing: During the 4" week postpartum, the Fy pups will be evaluated in a passive avoidance test for learning and short term memory retention. During the 10" neuromuscular week postpartum, the Fi pups will coordination, learning, and longer be evaluated in a water-filled term more complex memory. maze for Fi Growth and Reproduction: The F; pups will be weaned after 21 daysoflactation and litters will be culled to oneof each sex. The pups will then allow to grow and body weights and food consumption will be recorded weekly. The Fy pups will undergo behavioral/functional testing. At sexual maturity, within a dose group, one male will be `mated with one female (sibling mating to be avoided). The females will be allowed to litter and raise the F pup through 21 daysof lactation. ur 0001.30 January 21, 1999 Rat Two Generation Reproduction Study of PFOS Study Outline Study Objective: To evaluate the reproductive functionof male and effectoforal administration of PFOS female rats (Fo generation) and on the on the. development and reproductive via lactation. capacityofthe subsequent F) generation which were exposed in utero and GLP Status: A GLP study included in the final report. with appropriate QA audits; a signed QUA statement to be Animals: Male and female Charles River CD rats Dose Groups: Five - control, 0 mg/kg; low, mid-2,2 or 3 mg/kg; high, 5 or 8 mg/kg. 0.1 or 0.2 mg/kg; mid-1, 0.5 or 1.0 mg/kg; Number per Group: 35 males and 35 females per group TideesnttiAtrytiacnlde:purPitFy.OSRweislelrbvee fsuarmnpilsehetdobbye trheetaSinpeodn.sor who is responsible for compound Compound Administration: Daily oral intubationof compound suspended in water with 2% Tween 80. Compound administration will start 4 week prior to mating gCeosmtaptoiuonnadnaddmliacntiasttiroan.tiTohnewiFlyl pcuopnstiwniuleltnhortoruegcheimvaetianngyadnodseisnvtihaegfaesmtrailcesintthurboatuigohn. Samplesof dosing preparations on week 2 and 8 will be frozen and retained for possible analysis by the Sponsor. Records will be maintained on how dosing preparations were made. Clinical Observations: Twice daily; once approximately one hour after dosing and then 410 6 hours later. Body Weights: Weekly except when male and females are co-inhabited during mating. Food Consumption: Weekly except when male and females are co-inhabited during mating. Estrus Cycles: Daily vaginal smears will be taken from females two weeks prior to `mating to determine estrus cycle information. Mating: Afier 4 weeksof compound administration, within a dose group one male will be co-inhabited with one female. A female will be determined as mated upon the ns 000131 January 21,1999 presence of sperm positive vaginal smear. ofgestation. The dayofmating shall be considered as day 0 Early Gestation Evaluation: 10 females per group will killed on day 7 ofgestation. Pregnancy will be determined early and late resorptions as well as the number of implanations and the number of Male body Sacrifice: weight has One been week after obtained. mating all Fy male rats will be The animals will be necropsied killed aftera terminal and any gross lesions will be described and recorded. The testes, epididymis, prostate and seminal vesicles will be weighed and fixed for possible histologic examination. Bouin's solution; other tissues in 10% buffered formalin). (Note: testis will be fixed in At necropsy serum and liver samples wil be taken frozen and shipped to from five of the males in each dose the sponsor for possible analysis. group. These samples will be dFeleimvaelrethPeairrtluirttiertsi.onT:heThoeffrseprmianigni(nFyg g2e5nefreamtailoens) wwiillllcboentcionuuneteudn,tisletxeerdmaannddwbeeigahleldowotno postnatal days 0, 4, 7,14 and 21. four females on day 4 postnatal Offspring will be randomly culled to four males and bMeilcoklSleacmtpeldefsr:omOtnhedasyto4mapocshtnoaftatlhewdhiesncalridteedrspaurpescfurlolemdftioveeilgihtterpsoufpse,amcihlkdocsuerdgsrowuilpls. "The milk curds from the pups in a litter ar to be combined as a single sample. Pup Examinations: Pups will be observed daily for moribundity/mortality. Developmental landmarks testes descent, and vaginal consisting ofeye opening, pinna detachment, opening will be recorded for cach litter. surface righting, dFaiilDyosoiranlg:(gaAsfttriecr iwnetaunbaitnigona)t ddoasyin2g1.ofDolascetalteivoenl, othneaF,mgp/ukpgs bwaislilsrweiclelivbeectohmepsoaumnedasbythe pup's dam dose level, Female Sacrifice: Fy female rats will One or two days after weaningofthe be killed after aterminal body weight litter on has been day 21 of obtained. lactation, all The animals will be necropsiedandany gross lesions will be described and recorded. The ovaries will b1e0w%ebiugfhfeedreadndforfmiaxleidn)f.or pAotssniebclreophissytosleorguimc eaxnadmilnivaetriosna.mpl(eNsotwei:llovbaeriteaskewnilflrboemffiixveedoifn the females in each dose group. for possible analysis These samples will be frozen and shipped to the sponsor 1s 000132 January 21,1999 eFv1alPuuaptedNeiunraoplaosgsiicvael aTveostiidnagn:ceDeusrtinfogrtlheear4n"inwgeaenkdpsohsotrptatrteurmm,mtehemoF,rypurpetsenwtiiloln.be Dneuurrionmgutshceul10a%r cwoeoredkinpaotsitopna,rtlueamr,nitnhge,Fa,npdulposnwgielrltbeermevmaolruaetcedomipnlaexwatmeerm-ofrilyl.ed maze for Fi Growth and Reproduction: "The pups will then allow to grow The and F pups will be weaned body weights and food after 21 daysof lactation, consumption will be recorded weekly. The Fy pups will undergo behavioral/functional testing. At sexual mbaetauvroiitdye,d)w.ithTihnea fdeomsaelgersowuipl,lobneeamlalolweewdiltlo bliettmerataendd wraiitshe otnhee Ff:empaulpet(hsriobluignhg2m1atdianygstoof lactation. 120 00133 January21,1999 Rabbit Teratology Rangefinder on PFOS. Study Outline Study Objective: Explore possible dose levels for an oral rabbit teratology study GLP Status: Study should be conducted under GLP principles but since it is a rangefinder study QA audits will not be done Animals: Mated female New Zealand White rabbits (Note the rabbits are to be mated at the testing laboratorynotat the supplier and shipped as mated females to the testing laboratory.) Dose Groups: Seven - control, 0 mg/kg; low-1,0.1 mg/kg; low-2, 1 mg/kg; mid-1,2.5 mg/kg; mid-2, 5 mg/kg; high-1, 10 mg/kg; high-2, 20 mg/kg Number per Group: 5 mated females per group Test Article: N-EtPFOS will be furnished by the Sponsor who is responsible for compound identity and purity. No reserve sample will be required. `Compound Administration: Oral intubationof compound suspended in water with 2% Tween 80 on days 7 thru 20ofgestation. Analysisof dosing preparation will not be done; however, records will be maintained on how dosing preparations were made. 4Cl1i0ni6cahlouOrbsselartvera.tions: Twice daily; once approximately one hour after dosing and then Body Weights: On days 0,7, 10, 13, 16, 19, 24 and 29 of gestation Food Consumption: On days when body weights are obtained Cesarean Section: On day 29 ofgestation the females will be anesthetized and the uterus andits contents will be removed and weighed. The number of implanations, the numberoflive and dead fetuses, and the number of early and late resorptions will be determined and recorded. The fetuses will be weighed and examined for any gross abnormalities. The ovaries will examined for the number of corpora lutea. wr 000134 January 21,1999 Rabbit Teratology Study on PFOS Study Outline Study orally Objective: administered To determine maternal and PFOS in pregnant rabbits fetal toxicity and teratogenic potential of GLP Status: A GLP study included in the final report. with appropriate QA audits; a signed QUA statement to be Animals: Mated female New Zealand White rabbits (Note the rabbits are to be mated at the testing laboratory laboratory.) not at the supplier and shipped as mated females to the testing mDgo/skeg;Grhoiugphs,:10Fimvge/k-gc.ontFrionla,l 0domsge/klegv;ellsowwi,ll0.b1emagd/jkugs;tedmiadft-e1r,ra1 nmgge/fkign;demrisdt-u2d,y5results are available. Number per Group: 22 mated females per group `Test Article: FOS will be furnished by the Sponsor who is responsible for compound identity and purity. Reserve sample to be retained. `CTowmeepnou80ndonAddamyisni7sttrhrauti2o0no:fOgersatlatiinotn.ubatSiaomnpolfescoofmdpoosuinndgspurseppaernadteidonisn wwialtlebrewfirtohze2n.% and retained for possible analysis dosing preparations were made. by the Sponsor. Records will be maintained on how Clinical Observations: 410 6 hours later. Twice daily; once approximately one hour after dosing and then Body Weights: On days 0,7, 10, 13, 16, 19, 24 and 29ofgestation Food Consumption: On days when body weighs are obtained uCteesraurseaanndSietsctcioonnt:entOsnwidlalyb2e9roefmogevsetdatainodn twheeigfheemda.lesThwiellnubmebaenresotfheitmipzleadnaatnidontsh,e the dneutmebremrionfeldiavnedarnedcodredaedd.feTtuhseesf,etaunsdesthweilnlubmebewreoifgheeardlyanadndexlaatmeirneesdorfpotrioannsywgirlolssbe abnormalities. The ovaries will examined for the numberof corpora lutea. " 000135 January 21,1999 eFveatlaulaEtexatmhiencaotnitoenntss:ofAthmeidc-rcaoniruomn.al Tslhieceiwnitlelrnbale omragdaensoinftthehehetahodorafceiac cahndfaetbudsotmoinal cianvtiertniaels aobfnaolrlmafeltiutsieess.wilAltbtehiesxtaimmientehde isnextohefftrheesheastcahtefeutsuisngwiSltlabpleeds'etteremcihnneidq.ue Afforter removalof the viscera, the carcasses will processed for skeletal examination. `(0T.o1ximcgo/kiknge)tiacndSaftievlelifteemAalneismaaltst:heGhrioghupdsoosfe m1a0tmegd/kfgem)aplleuss, tfhirveeeffeemmaalleessatatthoethleorwdodsoese lfeevmealls,eswiwlilllbebedoksiellde.d aOnndtfhoeldloawyianfgtesramthpelelasstwidlolsbee(cdoalyle2c1teodf:gseestrautmioann)dtlhievsearteflrloitmethe dstaomreadntdogpeltahceerntaassoannedsfaemtpulsee;s from the likewise uterus. for the f(etNuostees)plTahceesnteassafmrpolmesa single will be litter can be frozen and shipped to the sponsor for possible analysis. 123 000136 Janay 21,1999 Rabbit Teratology Rangefinder on N-EtFOSE Study Outline Study Objective: Explore possible dose levels for an oral rabbit teratology study GLP Status: Study should be conducted under GLP principles but since it is a rangefinder study QA audits will not be done Animals: Mated female NewZealand White rabbits (Note the rabbits are to be mated at the testing laboratory not at the supplier and shipped as mated females to the testing laboratory.) Dose Groups: Seven - control, 0 mg/kg; low-1, | mg/kg; low-2, 5 mg/kg; mid-1, 10 mg/kg; mid-2, 25 mg/kg; high-1, 50 mg/kg; high-2, 75 mg/kg Number per Group: 5 mated females per group Test Article: N-EtPFOS will be furnished by the Sponsor who is responsible for compound identity and purity. No reserve sample will be required. Compound Administration: Oral intubation of compound suspended in water with 2% "Tween 80 on days 7 thru 20ofgestation. Analysisof dosing preparation will not be done; however, records will be maintained on how dosing preparations were made. Clinical Observations: Twice daily; once approximately one hour after dosing and then 410 6 hours later. Body Weights: On days 0,7, 10, 13, 16, 19, 24 and 29 of gestation Food Consumption: On days when body weights are obtained Cesarean Section: On day 29 of gestation the females will be anesthetized and the uterus and its contents will be removed and weighed. The number of implanations, the number of live and dead fetuses, and the numberofearly and late resorptions will be determined and recorded. The fetuses will be weighed and examined for any gross abnormalities. The ovaries will examined for the numberofcorpora lutea. 126 0001.37 Jay 21,1999 Rabbit Teratology Study on N-EtFOSE Study Outline Study Objective: To determine maternal and fetal toxicity and teratogenic potential of orally administered N-EtPFOS in pregnant rabbits GLP Status: A GLP study with appropriate QA audits; a signed QUA statement to be. included in the final report. Animals: Mated female New Zealand White rabbits (Note the rabbits are to be mated at the testing laboratory not at the supplier and shipped as mated females to the testing laboratory.) Dose Groups: Five - control, 0 mg/kg; low, (1 ?) mg/kg; mid-1, (5 ?) mg/kg; mid-2, (10 ?) mg/kg; high, (25 ?) mg/kg. Final dose levels will be adjusted after range finder study results are available. Number per Group: 22 mated females per group `Test Article: N-EtPFOS will be furnished by the Sponsor who is responsible for compound identity and purity. Reserve sample to be retained. Compound Administration: Oral intubationof compound suspended in water with 2% `Tween 80 on days 7 thru 20 of gestation. Samples of dosing preparations will be frozen and retained for possible analysis by the Sponsor. Records will be maintained on how dosing preparations were made. Clinical Observations: Twice daily; once approximately one hour after dosing and then 4106 hours later. Body Weights: On days 0,7, 10, 13, 16, 19, 24 and 29 of gestation Food Consumption: On days when body weights are obtained Cesarean Section: On day 29of gestation the females will be anesthetized and the uterus and its contents will be removed and weighed. The numberof implanations, the number of live and dead fetuses, and the number of early and late resorptions will be determined and recorded. The fetuses will be weighed and examined for any gross abnormalities. The ovaries will examined for the numberof corpora lutea. 12s 001.38 January 21,1999 Fetal Examinations: A mid-coronal slice will be made in the headofeach fetus to evaluate the contentsofthe cranium. The intemal organsofthe thoracic and abdominal cavities of all fetuses will be examined in the fresh state using Staples' technique for internal abnormalities. At this time the sexofthe each fetus will be determined. Afier removalof the viscera, the carcasses will processed for skeletal examination. Toxicokinetic Satellite Animals: Groupsof mated females, five females at the low dose (12 mg/kg) and five females at the high dose (25 ? mg/kg) plus three females at other dose levels, will be dosed. On the day after the last dose (day 21ofgestation) the satellite females will be killed and following samples will be collected: serum and liver from the dam and placentas and fetuses from the uterus. (Note placentas from a single litter can be stored togethaesr one sample; likewise for the fetuses) These samples will be frozen and shipped to the sponsor for possible analysis. 126 000139 Juay 21,1999 Corporate Toxicology Study Outline Tite: 1) E(RfefsetcrtoicftPeedrGfrlaunotr)ochemicals on Bioenergetic Metabolism: Phase II Research Plan 2) Mitochondrial Interactions of Peroxisome Proliferators (Unrestricted Grant) Purgaser 1) Compare mitochondrial bioenergetics between mitochondria from rat, guinea pig and human (CaTM loading capacity, uncoupling potential, et al.) to help set appropriate safety factors for human risk characterization based on animal data; 2) Compare the metabolic response ([ATP], respiration rate, mitochondrial membrane potential, markersof peroxisome proliferation, et al.) of rat, guinea pig and primate hepatocytes to this class of compounds with particular emphasis on mitochondrial and peroxisomal metabolism to: a) Further test the proposed mechanismof toxicity; b) Identify potentially useful metabolic biomarkersof exposure; ) Help validate relative differences between species (assess markers of peroxisome proliferation across species); 3) Compare molecular response (gene expression > mRNA) of rat, guinea pig and primate hepatocytes exposed to these chemicals (genes indicativeof cell and peroxisome proliferation) to: a) Identify molecular biomarkers of exposure; b) ) DFiacsiclriitmatienastpeecbieetsweexetnrap[rpoolliafteiroant;ion of peroxisomes and cell proliferation (ongogenic response) to test dogma; d) ASsessepsosnstehe relevanceofrat tumorigenic response to potential human cancer ) Provide biomarkers for potential use in monitoring and interpreting traditional toxicity studies. Significance: 1) Comparing sensitivity between species will allow for judging the most appropriate `species for predicting human health outcomes following exposures to compounds and phearsspreeclteivvaen;ce to establishing safety factors and putting human cancer risk in 2) Species differences in metabolic and molecular response will reveal important insight into mechanisms responsible for compound-induced peroxisome proliferation and/or tumorigenesis which is also vital to identifying valid biomarkers to assess exposures and potential risks; 3) Comparing the enhanced transcription and expression of genes associated with peroxisome versus cell proliferation will allow opportunities to evaluate the wr 000120 January 21, 1999 relationship between metabolic and oncogenic effectsofthese compounds in different species. Objectives): 1. Establish hepatocyte cell cultures from different species as models for assessing the `metabolic and mitogenic effects test compounds; 2. Develop molecular probes for assessing compound-induced transcription of genes related to peroxisome metabolism and cell proliferation; 3. Establish benchmarks for assessing the effectsoftest compounds on hepatocyte bioenergetics and cell proliferation in culture. Protocol: 1). Establish stableprimaryhepatocyte cultures from several species including rat, guinea pig and primate; 2) Develop molecular probes for compound-initated transcription of specific genes related to mechanistic endpoints: a) ID genes of interest b) ID sequencesofhigh homology between species using Gene Bank ) Design primers to 1.5 ~3.0 kbp sequences (Oligo) d) PCR amplify sequences from rat-liver cDNA template ) Separate, bands on low melting point gels ) Purify bands (band-stab) ) Restriction digest to confirm identity (MacDNASIs to obtain restriction site map for each probe) 3) Thr)eaLtasbealmpplreob(ecelwlisthor*f*lPa-shdCfTrPozbeny irsasnudeo)m priming (High Prime) 2) Isolate total RNA by triazole method b) Separate on denaturing HCOH agarose gels ) Transfer to nylon d)) HWyabsrhidainzde weixtphos*e?PtoprXo-breay film to detect complementary mRNA 4) MRNA probes suggested for peroxisome proliferation a) CPTI b) ACoAO ) PPARa 4) FABP ) Also, possibly Catalase, LPL, Aromatase, HMG-CoA synthetase 5) Probes for cell proliferation a) PCNA b) CDK 6) Establish benchmarks for assessing the effects of PF compounds on hepatocyte bioenergetics and cell proliferation in culture: a) Effect ofexposing cells in culture on induction ofperoxisomal metabolism and stimulation of cell proliferation using biochemical markers and molecular markers (e.g, mitochondroial enzyme activities and cytochrome content, Adenine 128 000141 sonia 21,1999 nucleotides, AcoAO activity, PCNA, apoptosis (morphometric and TUNEL), CyQuant cell proliferation analysis, CPT, AcoAO, PCNA, CDK) Timeline: 31 months beginning December 1, 1998 and ending June 30, 2001 Report: Quarterly progress reports 129 000142