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Perfluorooctane Sulfonate: Current Summary of Human Sera, Health and Toxicology Data 3M January 21,1999 l 000014: January 21, 1999 TABLE OF CONTENTS EXECUTIVE SUMMARY............................................................................................................................. 3 I. INTRODUCTION.......................................................................................................................................6 II. HISTORICAL REVIEW AND CURRENT FINDINGS OF FLUOROCHEMICALS IN HUMAN SERA................................................................................................................................................................11 III. SUMMARY OF MEDICAL SURVEILLANCE AND EPIDEMIOLOGY STUDIES.................. 22 IV. SUMMARY OF TOXICOLOGY DATA ON PFOS..........................................................................30 V. RISK CHARACTERIZATION.............................................................................................................. 64 VI. CURRENT AND PLANNED RESEARCH......................................................................................... 69 VII. REFERENCES......................................................................................................................................81 APPENDIX..................................................................................................................................................... 86 2 000015 January 21, 1999 Executive Summary 3M has prepared this document to summarize the data related to the biological effects of perfluorooctane sulfonate (PFOS). It also 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. Routes of exposure to PFOS or precursor molecules are not well understood at this time. PFOS is an example of an "organic" fluorine molecule. Human serum has been known to contain organic fluorine molecules for over 30 years. The primary constituent of 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 fraction of this 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 surveillance of exposed workers in 1992. Detection limits have been lowered to allow the more recent analysis of serum 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 3 000016 January 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 of increased 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. Atrophy of 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 early 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 early rhesus monkey studies, and death at 1100 ppm and above. More complete quantitative absorption, distribution and excretion data for PFOS is being obtained. 4 00001*7 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 this research is to explore the potential for chronic and reproductive effects, understand toxic mechanisms and obtain a better understanding of absorption, distribution, metabolism and excretion. The plan is to make as much use as possible of observational data in exposed workers and to establish no effect levels in both rats and primates for endpoints of importance. 000018 5 January 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 of OF present are not identified, it now appears that a compound called perfluorooctane sulfonate constitutes a significant fraction. Recent data provide evidence that PFOS is present at tens of parts-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 group of products is developed: F I CF3(CF2)6C - S 0 2- F I F POSF 6 0 0 0 0 19 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 CF3(CF2)6 C - S 0 2- R I F POSF derived molecule PFOS may result from the loss of 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 I CF3(CF2)6c - S03I F PFOS Most POSF that is produced is used in 2-(N-ethylperfluoroctanesulfonamido)-ethyl alcohol (N-Et-FOSE) and 2-(N-methylperfluoroctanesulfonamido)-ethyl alcohol (NMe-FOSE) based products. Figure 1.1 shows the chemical structure of N-Et-FOSE and metabolites that have been found in rat serum. All except compound VIII have been verified to metabolize further to PFOS. (Missing Roman numerals represent hypothesized intermediates not shown in this figure.) 7 000020 Figure LI N-EtFOSE(*) Metabolites Identified in Rat Serum ch2ch3 / C7F,7-S02-N \ ch2- ch2- o - h 000021 H / r - so2n \ ch2- coo' V ili c7F]7- S03" XII (PFOS) 8 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 samples of pooled human sera. A single 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 part of very large molecules that act as protective chemicals on fabrics, leather and rugs. Outside of the occupational setting, routes of human exposure to PFOS or its metabolic precursors are not understood, but are the subject of intense 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 of employees and was reported in general population sera samples in 1976 (Taves). This is perfluorooctanoic acid, or PFOA: 9 000022 January 21, 1999 CF3(CF2)6COOH 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 PFOS. It is likely that PFOA was misidentified as a major fraction of organic fluorine in the 1976 Taves paper. The evidence for this is discussed in Section II. The purpose of this report is 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 II. 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 0 0 0 0 2 3 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 of organic 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 evidence of significant change in PFOS concentration in serum samples taken over the last two to three decades. The advancement of analytical chemistry technology has had a significant influence on our knowledge of fluorocarbons 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 of a rapid analytic technique in the late 1970's decreased analytic time to under an hour, allowing large scale medical surveillance of production 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 advancement of 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 validity over a period of several months. 11 0 0 0 0 2 4 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 of a PFOS 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) a brief summary of the historical information regarding organic fluorine in human sera, 2) data from 3M employees involved in fluorochemical production, 3) data from a small group of non-occupationally exposed 3M employees, 4) data from commercially available human pooled serum samples, 5) data from pooled sera 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 forms of fluorine 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 (1968b) showed that the non exchangeable 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", i.e. covalently bound to carbon (Taves et. al., 1976). Using NMR spectroscopy, these authors tentatively identified a component of the organic fluorine as perfluorooctanoic acid (PFOA). There was some variation in the observed spectra from an authentic sample of PFOA, however, leading the authors to suggest that branching, or the presence of a sulfonate, was possible. A number of studies over the past 25 years reported levels of organic fluorine in human blood serum. Table II. 1 presents the study author, level measured, population studied and methods of analysis. The variety of methods used for determination of 12 0 0 0 0 2 5 January 21, 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. Table II. 1. Historical Findings of Serum Organic Fluorine Levels Year 1972 1975 1976 1978 1979 1980 1980 1981 1989 Author Guy Venkateswarlu Guy, Taves Belisle Singer Paez Ubel Belisle Yamamota OF* I'ncb'l 30 36 25 20 45 85 45 11 32 N 65 2 106 9 264 pooled 4 8 11 Method** ash O bomb ash O bomb ash ash mod O bomb O bomb LOPA Source US US US US US Argentina US China Japan * Organic fluorine, specific identities not provided. ** Varied methods were used to measure organic fluorine. See papers for details. Occupationally Exposed Employees 3M has produced PFOA (the ammonium salt) by electrochemical fluorination since the early 1950's. It is a surfactant used in fluoropolymer production. The company began medical monitoring of employees involved in PFOA production in 1976, by measuring serum levels of organic fluorine (OF) and performing medical assessments. Employee monitoring was expanded significantly in 1980 following the development of a more 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 consists of 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 13 0 0 0 0 2 6 January 21, 1999 as measurement of serum levels of OF. Among employees with six or more measurements, OF levels averaged 2.9 to 6.5 ppm from 1981 to 19921(Figure II. 1). With the introduction of high performance liquid chromatography-mass spectrometry, serum PFOS was measured in 1994 and 1997 (see also Figure II. 1). 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 III, 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. It is also the primary site for PFOA production. 1We are aware of one occasion in 1979 where the serum of 5 Decatur employees was measured for PFOS by electron capture gas chromatograph and microwave plasma detection methods [Central Analytical Laboratory, 1979], Total serum organic fluorine levels for these five employees were 10.1, 5.7, 9.4, 11.8 and 4.1 ppm. The percent of PFOS found was 60%, 70%, 80%, 55% and 65% of the total serum organic fluorine levels, respectively. 14 0 0 0 0 2 7 Figure 11.1. Mean Levels (ppm) of Total Serum Organic Fluorine (solid black or Perflurooctane Sulfonate (striped bars) in Decatur Who Had at Least Six Measurements From 1981 - 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 Year 15 000028 Non-Occupationally Exposed 3M Employees A total of 31 3M employees were tested for PFOS in their serum in 1998 (Table II.2). All were corporate staff or 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 to 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% of the variance in PFOS levels. There was no gender-related difference if age was considered. Only four employees had PFOA measured above the detection limit of 10 ppb. The average of 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 (R2 = .94) in the range of quantification (Figure A1 in the Appendix). 16 0 0 0 0 2 9 Table II.2. Summary of Mean and Range of PFOS (ppb) Levels in Current and Historical Human Populations, All Data Analyzed in February - April, 1998 Mean* Populations Description of Sample (ppb) A. Current Populations (blood collected in 1998) 1. Non-occupationally exposed 3M employees 31 individuals 47 2. Commercial pooled serum samples Intergen Laboratory 3 samples each with > 100 donors 44 Sigma Laboratory 3 samples each with > 100 donors 33 3. Pooled serum from 21 separate U.S. blood banks (see Figure 1 for more detail) 3 to 6 pooled samples per location with 5 to 10 donors per sample 30 B. Historical Samples (chronological order) 1. Korean War era U.S. military recruits, 1948 to 1951 10 pooled samples with 10 donors per sample N.D.** 2. Swedish samples, 1957 10 individual samples 2 3. Michigan Breast Cancer Study, 1969-1971 5 individual samples 33 4. Swedish samples, 1971 10 individual samples 1 5. MRFIT pooled calibration samples, 1976 (Multiple Risk Factor Intervention Trial) 6 pooled samples with unknown number of donors per sample 31 6. MRFIT pooled calibration samples, 1980 3 pooled samples with unknown number of donors per sample 23 7. China samples (Linxian, rural province), 1984 8. MRFIT individual samples, 1985 9. China samples (Shandong, rural province), 1994 * Rounded to nearest ppb ** Not detected 6 individual samples 3 individual samples 6 individual samples N.D. 31 N.D. Range* (ppb) 28-96 43-44 26-45 9-56 N.D. N.D.-2 N.D. - 59 N.D. - 1 14-56 14-41 N.D. N.D. - 44 N.D. 00003 17 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 levels of 43, 44 and 44 ppb (Table 2A.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 minimum of 200 donors. The three Sigma pools contained PFOS at levels of 26, 28 and 45 ppb. 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 II.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 (see 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) of the 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 of historic organic fluorine measurements from published literature is 37.6 ppb. If this were all PFOS, a PFOS sera level would have been 57.9 ppb (1.54 x 37.6). 18 000031 January 21, 1999 Table II.3 PFOS (ppb) Levels from 18 Blood Banks Citv # of Pooled Samles Anchorage, AK 4 Billings, MT Jn> Cheyenne, WY Corpus Christi, TX -> Davenport, IA 3 East Orange, CA 3 Grand Rapids, MI 4 Greenville, SC J Kansas City, MO 3 Lafayette, LA n Las Vegas, NV J Meridian, MS 3 Minneapolis, MN 6 Newark, DE 5 Omaha, NE 5 Santa Barbara, CA 3 Santa Rosa, CA 3 Scottsdale, AZ n Overall mean = 29.7ppb Ranee 19-31 18-26 13-35 26-32 28-40 20-27 18-31 47-56 24-35 39-50 25-28 27-56 41-54 21-32 9-27 13-16 23-26 28-47 Averaee 24 23 23 29 32 24 26 52 30 46 23 39 46 24 17 14 24 37 Historical Samples Nine sets of historical samples have been analyzed for PFOS in 1998. These are summarized in chronological order below. Korean War era U.S. military recruits, 1948 to 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 of detection 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 to 59.4 ppb. The mean was 33.4 ppb. 19 0 0 0 0 3 2 January 21, 1999 Swedish samples, 1971. Ten individual samples ranged from below detection limit (1 ppb) to 2.8 ppb. Three were below detection limit. The mean was 1.13 ppb. MRF1Tpooled 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 number of donors per pool is unknown. Six pooled calibration samples ranged from 13.7 to 55.5 ppb. The mean was 30.9 ppb. MRFITpooled calibration samples, 1980. Three pooled calibration samples ranged from 13.8 to 40.5 ppb. The number of donors 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 of detection 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 limits of quantitation (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 limits of detection 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 20 January 21, 1999 Organic fluorine has been noted in human serum since the late 1960's. We have now identified PFOS as a part of this organic fluorine fraction. PFOS-related materials were not produced commercially prior to 1948, and only in small quantities for several years thereafter. Therefore, it is 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 distribution of this 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 be of 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. 21 0 0 0 0 3 4 III. SUMMARY OF MEDICAL SURVEILLANCE AND EPIDEMIOLOGY STUDIES Epidemiologic Investigation of Clinical Chemistries, Hematology and Hormones in Relation to Serum PFOS Levels in Male Fluorochemical Production Employees 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 analysis of 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).2 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 of variance 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 III. 1). (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, 2 Hematocrit, hemoglobin, red blood cells, white blood cells and platelet count, alkaline phosphatase, gamma glutamyl transferase, aspartate aminotransferase, alanine aminotransferase, total and direct bilirubin, blood urea nitrogen, creatinine, glucose, cholesterol, low density lipoproteins, high density lipoproteins and triglycerides, cortisol, dehydroepiandrosterone sulfate, estradiol, follicle stimulating hormone, 17-alpha 22 0 0 0 0 3 5 January 21, 1999 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 were below 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 III.2). When analyzed in aggregate, the two plant populations showed no consistent significant associations for both years between the clinical chemistries and hematology parameters and the employees' serum PFOS levels (Figure A l, 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 linear as well as non-linear transformations in order to maximize the possibility of finding associations between PFOS and the parameters of 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 sub sample of 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 up to 6 ppm. It was not possible to derive inferences from the few employees with serum PFOS levels > 6 ppm. Limitations of this study included its cross-sectional design, the voluntary participation rates and the few subjects with levels > 6 ppm. hydroxyprogesterone, luteinizing hormone, prolactin, sex hormone binding globulin, free testosterone, bound testosterone, and thyroid stimulating hormone. Q0OO36 23 Table III. 1 Distribution of Employees by Year, Location and PFOS Exposure Level (ppm) PFOS 0 - < 1 ppm 1 - < 3 ppm 3 - < 6 ppm > = 6 Dorn ______________________ 1995 Data_______________________ All Employees Antwerp Decatur N (% ) N (% ) N (% ) 45 25 34 39 11 12 91 51 32 36 59 66 35 20 19 22 16 18 74 3344 178 (100) 88 (100) 90 (100) _______________________ 1997 Data____________ All Employees Antwerp Decatur N (% ) N (% ) N 1%) 60 40 31 48 29 35 63 43 25 38 38 45 21 14 9 14 12 14 53 00 56 149 (100) 65 (100) 84 (100) 000037 24 Table III.2 Mean Values of PFOS, Demographic, Serum Chemistry and Hematologic Parameters for Antwerp and Decatur, 1995 and 1997 Examinations Variable PFOS (ppm) Age BMI 1995 Data Antwerp Decatur 1.93 2.44 32*** 45 23 9*** 29.2 1997 Data Antwerp Decatur 1.48 1.96 33*** 44 23.5*** 30.0 Cigarettes 4.7* 7.9 5.5 6.6 Alcohol Aik Phosphatase 25*** 0.2 97 1 1*** 2Q*** 0.1 87 GGT 41 48 26* 36 AST 26* 29 27 26 ALT 44 47 31 34 Total bilirubin 0.86*** 0.58 0.80*** 0.58 Direct bilirubin BUN Creatinine Glucose 0.22 12 o*** q 9*** gj *** 0.21 14.8 1.1 92 0.15*** 14.9 0.9** g^*** 0.12 14.1 1.0 98 Cholesterol 214 218 206 215 LDL 138 136 134 137 HDL Triglycerides 115*** 43 187 50*** Y\\ *** 42 192 000038 January 21, 1999 Table III.2 (Continued) Variable Hematocrit 1995 Data Antwerp Decatur 47*** 46 1997 Data Antwerp Decatur 46* 45 Hemoglobin 15.4 15.2 15.4 15.3 RBC 4.9 5.0 5.1 5.1 MCH MCHC MVC 22 Q*** y*** 30.7 33.4 91.8 30.5 33 3*** 91.6* 30.4 33.7 90.0 WBC 6.4*** 7.5 6.5 6.4 Platelets 224 229 237* 217 * p < .05; **p<.01; ***p<.001 (Antwerp compared to Decatur) 26 0 0 0 0 3 9 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 experience of these employees was significantly different from that which would be expected in a comparable population. The cohort was followed from March 1, 1961 through December 31, 1991. Table A2 (Appendix) provides a description of the 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 personyears experience, respectively (Table A3, Appendix). The majority of the male employees were hired between 1965 and 1974 (Table A4, Appendix) whereas the majority of the female employees were hired between 1975 and 1979 (Table A5, 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 III.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 duration of 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). None of these analyses resulted in significantly elevated SMRs. There were four deaths among female employees (SMR = 62.6; 95% Cl 17.1 - 160.4). Of these four deaths, three were from external causes (SMR = 213.4; 05% Cl 44.0 623.6). By examining the death certificates for the specific causes of death and 27 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 of Minnesota investigators have also followed the mortality experience of a cohort of employees 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 consisted of 2,788 male and 749 female workers employed between 1947 and 1983. There was no significantly increased cause-specific SMR for either men or women. In a proportional hazard regression model, ten years of employment in jobs associated with the chemical division was associated with a 3.3-fold increase (95% Cl 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 nonspecificity of the exposure index as well 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 et al., 1998). Current and proposed human health and exposure research initiatives are found in Section VI. 28 0 0 0 0 4 1 January 21, 1999 Table III.3. Selected Cause - Specific SMRs for Men by Comparison Population (n = 1,639) SMR (95% Confidence Interval) Cause of Death All Causes of Death Observed Deaths 70 All Malignant Neoplasms 15 Cerebrovascular Disease 1 All Heart Disease 17 Respiratory Disease 0 External Causes 25 U.S. 62.9 (49.0, 79.5) 68.4 (38.3, 112.8) 33.5 (0.8, 186.8) 59.1 (34.4, 94.7) Alabama 54.8 (42.7, 69.3) 60.9 (34.1, 100.4) 27.8 (0.7, 155.2) 53.2 (31.0, 85.1) AL Regional Counties 52.0 (40.5, 65.7) 59.9 (33.5,98.8) 26.5 (0.7, 147.5) 49.1 (28.6, 78.6) (0.0,91.2) 74.5 (48.2, 110.0) (0.0, 80.2) 59.0 (38.2, 87.1) (0.0, 73.9) 55.0 (35.6, 81.3) 29 000042 January 21, 1999 IV. SUMMARY OF TOXICOLOGY DATA ON PFOS Introduction 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 this information in risk 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, most of 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 intraspecies variability in pharmacokinetic handling of 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 II and the results of epidemiologic investigation as reported in Section III. Despite the current limitations in the ability to characterize risk based on the relationship of blood and tissue PFOS 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. 000043 30 January 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 of PFOS unless otherwise annotated. Studies on related compounds are considered outside the scope of this 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 summaries of rodent 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 results of rodent 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 descriptions of these studies can be found in the Appendix. Pharmacokinetics of PFOS The absorption, tissue distribution, potential metabolism and excretion of 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. 31 000044 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 elimination half-life after a single dose; appears to have extended elimination half 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% of a single oral dose averaging 4.2 mg/kg [14C]PFOS administered to two groups (24 hour and 48 hour sacrifice) of 3 male Charles River CD rats (248-315 g, mean = 285 g) was absorbed within 24 hours (Johnson and Ober, 1979). The radiochemical purity of the [14C]PFOS 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 free of hair as a suspension in water at 5,000 mg/kg covering 40% of the total body surface area of ten male and ten female rabbits. An impervious plastic sheet occluded the skin for 24 hours and was then removed. Animals were maintained and observed for a 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 32 0 0 0 0 4 5 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 under occlusion for 24-hours to an aqueous suspension of PFOS (0.06 %) at doses equal to 0, 0.003, 0.06, and 0.3 mg PFOS / 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 after a single iv dose of PFOS-l4C (mean dose, 4.2 mg/kg) six Charles River CD male rats (initial body weights 262-303 g, mean = 288 g) excreted a mean of 30.2% of the total carbon-14 via urine. Mean cumulative fecal excretion was 12.6%. At 89 days, mean tissue concentration of total carbon-14 expressed as pg PFOS-14C 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), or 2.0 mg/kg/day (one male and one female) (Thomford, PJ, 1998). The monkeys dosed with PFOS (0.02 and 2.0 mg/kg/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 slopes of the 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 33 0 0 0 0 4 6 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 end of 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 volume of distribution 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 is equivalent to 5.3 mg/L of serum (mg/L = ppm weight/volume); One (1) mg/kg given to 2.2 kg monkey results in a total dose of 2.2 mg; Vd (L) is calculated as 2.2 mg / 5.3 mg/L = 0.41 L; Normalizing this for body weight gives 0.41 L / 2.2 kg = 0.19 L / kg = Vd (L / kg); Vd for a 60 kg woman is estimated to be 0.2 L / kg x 60 kg = 12 L = Vd (L). Metabolism: Preliminary data from analysis of urine, feces and tissues of rats as well as the inherent stability of perfluorinated anions suggest that PFOS is not metabolized (Johnson et al., 1984). Exposure of primary human and rat hepatocytes to PFOS 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 [14C]PFOS in 0.9% NaCl were administered to male rats. By 89 days after dosing, 30.2% of the administered l4C had been excreted in the urine and 12.6% had been excreted in the feces. 34 000047 January 21, 1999 Whole body elimination in the male rat appeared to be biphasic. Initial redistribution from the plasma yielded a plasma elimination half-life of 14C of 7.5 days following single oral administration of [14C]PFOS (mean dose 4.2 mg/kg) to male rats (Johnson and Ober, 1979). In the aforementioned intravenous study, elimination of only 42.8 % of the dose through urine and feces after 89 days indicates that the half-life of elimination from the body is > 89 days in the male rat. Serum PFOS levels in three retired male 3M chemical workers have been followed for five and one-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 reported to any appreciable extent. Since urine and feces have not been followed, it is difficult to ascertain if this 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. 35 0 0 0 0 4 8 January 21, 1999 Table IV. 1 Serum Elimination of PFOS in Retired 3M Chemical Workers. Serum [PFOS] in mg/L (ppm) Retiree 1 Retiree 2 Retiree 3 3/4/92 5.8 3/18/92 1.6 3/18/92 4.2 9/20/93 6.0 11/15/93 1.4 11/29/93 3.3 3/1/94 4.0 5/5/95 0.7 5/4/95 1.7 6/19/95 3.0 11/18/97 0.7 10/23/97 1.4 12/5/97 2.6 Serum Elim. - 0.0132/month - 0.0136/month - 0.0176/month Constant (A) Serum Elim. 52.5 mo/1575 d 50.9 mo/1527 d 39.4 mo/1182 d T 1/2 Fecal and total excretion of 14C were markedly increased in male rats administered cholestyramine (~ 2.7 g/kg/d) in their diet following single intravenous doses of [14C]PFOS. 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 retention of carbon-14 in liver, plasma, and red blood cells and increased the elimination of carbon-14 via feces after iv dosing with PFOS-14C. Groups of five rats (twelve-week old Charles River CD averaging 320 g) were dosed intravenously with PFOS-14C (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 excretion of 14C for cholestyramine-treated 36 0 0 0 0 4 9 January 21, 1999 rats were compared to mean control rat values. Mean cholestyramine-treated rat l4C concentrations in liver (9.4pg/g), plasma (0.9pg/ml), and red blood cells (0.3 pg/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 l4C elimination, as a result of the relatively high rate of fecal elimination of l4C was lower in cholestyramine-treated rats. The extent of total elimination of l4C (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). 000050 37 January 21, 1999 Acute Toxicity Acute Oral Toxicity: PFOS was administered by gavage in a 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. At 215 mg/kg, three of ten animals died. At 100 mg/kg, no deaths were observed. The combined acute oral LD50 in male and female rats is 251 mg/kg (95% C.I.: 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, fiyperactivity 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 samples of one 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: Groups of five 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 liter of air. The rats were observed hourly for the first four hours and daily thereafter for 14 days. All rats 38 0 0 0 0 5 1 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 staining of 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 number of control rats and thus may not be treatment-related. The nominally determined median lethal concentration (LC50) for a one-hour exposure to PFOS was determined to be 5.2 milligrams per liter with 95% confidence limits of 4.4 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 irritation 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 of a 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-irritating to the skin of albino rabbits when tested under conventional Draize procedures. No signs of 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 out of a highest possible score of 8.0 (Biesemeier 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 39 0 0 0 0 5 2 January 21, 1999 in a 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 each dose/ harvest time group. The mice were euthanized 24, 48 and 72 hours after dosing for extraction of 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 summary of these studies will be found at the end of this section in Table IV.9 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 signs of toxicity 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. At the 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 40 000053 January 21, 1999 slightly elevated blood glucose, and one male rat showed a slightly elevated serum alkaline phosphatase. At three months, one male rat had slight to moderate elevations in blood glucose, blood urea nitrogen and gamma-glutamyl 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 two of 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. Time of death was dose-related. Overt clinical observations of toxicity included emaciation, convulsions, altered posture, red material (right eye and/or mouth), yellow material in the ano-genital region, increased sensitivity to external 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 of a few rats sacrificed at termination of study from the 30 and 100 ppm groups. Morphological changes consisting of centrilobular to midzonal cytoplasmic enlargement (hypertrophy) of hepatocytes and necrosis of liver 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, 41 0000-54 January 21, 1999 bone marrow) and secondary (spleen, mesenteric lymph nodes) lymphoid organs, stomach, intestines, muscle and skin (Goldenthal et al, 1978a). 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 part of an 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 PFOS 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 1 through 5. Five animals/sex in Groups 1 through 5 were sacrificed during Week 4; livers were collected and weighed. A portion of the liver was prepared for PFOS analysis, a portion was shipped to Pathology Associates International for hepatocellular proliferation rate (PCNA) measurements, and a portion was used for determination of palmitoyl-CoA oxidase activity. At week 14, necropsies were performed on five animals/sex in Groups 1 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. 42 0 0 0 0 5 5 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 administration of the 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. 43 0 0 0 0 5 6 January 21, 1999 Table IV.2 Clinical chemistry and hematology in Male rats at week 14 (n=10) Average Value and Std Dev. by Dose Group (ppm PFOS in A N O V A Diet) Parameter Oppm 0.5 ppm 2 ppm 5 ppm 20 ppm Pr > F ALB 5 0.17 5 0.2 4.8 0.35 5 0.3 5 0.16 0.32 ALT (SGPT) 38 31 42 7 40 6 51 15 81 74 0.28 AST (SGOT) 109 31 121 14 139 14 137 10 159 7 7 0.34 CHOL 6 4 + 13 53 2 2 50 16 57 8 36 18* 0.13 GLU 103 8 104 13 83 15 93 8 92 14 0.062 N_SEG 1 0.4 1.2 0 .3 0.94 0.2 1.3 0 .4 6 1.7 0 .3 N A UN 13 2 14 2 14 2 14 0 17 1* 0.0007 PCoAO 4.6 1.34 4.8 3.3 5.4 3 1.8 1.8 5.4 1.9 0.14 * Mean significantly different (Dunnetts' T-test, p < 0.05) than the contro group value Table IV.3 Clinical chemistry and hematology in female rats at week 14 (n=10) Average Value and Std Dev. by Dose Group (ppm PFOS in Diet) Parameter 0 ppm 0.5 ppm 2 ppm 5 ppm 20 ppm ALB 5.5 0.25 6.1 0.5* 5.7 0.17 6.2 0.2* 6.1 0.24* ALT (SGPT) 34 2 39 11 30 5 36 5 33 5 AST (SGOT) 103 21 108 23 98 8 109 22 79 9 CHOL 78 19 79 14 70 12 68 19 65 16 GLU 102 16 100 12 105 3 90 8 100 7 N_SEG 0.8 0.36 0.9 0.4 0.53 0.13 0.56 0.1 0.64 0.1 UN 14 1 16 4 14 2 16 1.7 16 1.8 PCOAO 1.8 1.6 3.0 2.6 10.8 1.6 2.6 5 2.9 * Mean significantly different (Dunnetts' T-test, p < 0.05) than the control group value ANOVA Pr > F 0.0057 0.24 0.092 0.64 0.31 NA 0.26 0.10 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 44 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 administration of the 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 hypertrophy of hepatocytes 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 concentrations of 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. 45 0 0 0 0 5 8 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 Diet) n = 5 per sex per cose group Time Dose 0 ppm 0.5 ppm 2 ppm 5 ppm 20 ppm Interval Group 4 Weeks Male < MDL** 1.14 0.07 5.05 1.2 8.95 2.31 46.4 8.46 Female 0.04 0.003 1.90 0.22 7.50 0.053 14.3 1.85 59.4 8.46 14 Weeks Male < MDL** 4.22 0.83 17.9 1.23 45.6 5.73 152 13.3 Female 2.45 4.19* 6.65 1.08 26.9 2.26 62.9 6.19 216 21.9 *0 ppm female [PFOS] in serum at 14 weeks without one outlying value (n=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 Time Interval 4 Weeks 14 Weeks Liver [PFOS] in ppm by Dietary Dose Group (ppm PFOS in Diet) n = 5 per sex per dose group Dose 0 ppm 0.5 ppm 2 ppm 5 ppm Group Male 0.11 0.74 11 1.70 33 8.0 49 12 Female Male Female 0.11 0.05 0.44 0.05 1.58 2.1 8.6 0.57 26 4.4 19 3.4 25 6.2 77 7.5 68 3.0 80 17 387 33 362 26 20 ppm 295 45 357 43 600 116 618 50 90-Day Drinking Water Study with Lithium Perfluorooctane Sulfonate (LPOS) in Rats: The lithium salt of perfluorooctane 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. 46 000059 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 47 O O O O G O 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, 0.2 and 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 mg/kg dose group). With the exception of 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 finding of hepatic 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 consisting of two male and two female rhesus monkeys initially weighing between 2.75 and 4.10 kg were given daily doses of 0, 10, 30, 100 and 300 mg PFOS / kg by gastric intubation (gavage) as distilled water suspension. This study was terminated after 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 observations of toxicity were similar for all PFOS dose groups, and time of onset of toxic 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 48 000061 January 21, 1999 pathology revealed a yellowish-brown discoloration of the 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 PFOS. Congestion and lipid depletion of the adrenal cortex was seen in all dose groups and was considered agonal. The study was terminated after the death of 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, 1978b). 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 PFOS / 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 month of the study. The monkeys treated at the 4.5 mg/kg/day dosage level died or were sacrificed in extremis between weeks 5 and 7 of the study. These monkeys exhibited gastrointestinal symptoms including anorexia, emesis, black stool and dehydration from the first or second day of study. 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 beginning of the study to 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. 000002 49 January 21, 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 week of the 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 end of 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 months of 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/kg/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 atrophy of the serous alveolar cells. No statistically significant variations in sex group mean weights of organs occurred between the control and experimental groups. 50 0 0 0 0 ^ 3 January 21, 1999 Table IV.6 summarizes the individual cholesterol values for all dose groups. 0000^4 51 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 ( = 2 per sex per dose group) (Goldenthal etal, 1978b). Individual Serum Cholesterol (mg /100 ml) by Study Period and Cumulative Dose (CD in mg/kg) Dose Group Individual 0 mg/kg/day Dose Group Monkeys 3 (ID # 7355) 3 (ID # 7358) $ (ID # 7368) 9 (ID # 7372) Pre-Dosing CD = 192 174 155 204 1 Month CD = 0 212 184 188 208 3 Months % of PreDosing Value at Term ll 3 179 93.2 144 82.8 144 92.9 185 90.7 0.5 mg/kg/day Dose Group Monkeys 3 (ID # 7463) 3 (ID # 7483) 9 (ID # 7466) 9 (ID # 7504) CD = 182 161 208 208 CD = 15 208 192 230 211 CD = 45 160 143 178 160 87.9 88.8 85.6 76.9 1.5 mg/kg/day Dose Group Monkeys 3 (ID # 7462) 3 (ID # 7486) 9 (ID # 7500) 9 (ID # 7501) 4.5 mg/kg/day Dose Group Monkeys 3 (ID # 7484) 3 (ID # 7485) 9 (ID # 7502) 9 (ID # 7503) CD = 0 196 174 172 236 CD - 0 186 194 182 170 CD = 45 222 204 154 184 CD = 135 132 112 128 96 CD = 135 76 97 128 96 No Survivors - - - 67.3 64.4 74.4 40.7 40.9 50.0 70.3 56.4 0000^5 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 sixmonth 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 determination of serum 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 degree of 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 evidence of a significant decrease occurred between day 2 and day 7 for the female, with a day 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 2 to 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 IV.7. There were no other significant findings. 0000*76 53 January 21, 1999 Table IV.7: Longitudinal analysis of serum cholesterol and PFOS concentrations in male and female cynomolgus monkeys in = 1 per sex) during 28 days dosing with 2.0 mg/kg PFOS. Study Day -J_ 2 7 14 29 Cumulative Dose (mg/kg) 0 2 12 26 56 <$Serum Cholesterol (mg/dl) 150 151 137 132 91 6 [PFOS] in serum (ppm) 0.013 12.6 71.8 129 313 9 Serum Cholesterol (mg/dl) 141 136 117 107 62 $ [PFOS] in serum (ppm) 0.014 14.4 72.7 143 299 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 for a period of six months. Dose groups include six monkeys per sex per group with the exception of the 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 females of the high dose group. While serum PFOS concentrations have not yet been analyzed and reported, previous experience in the range-finder 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 summary of this 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) 54 0000^7 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 15 of gestation. 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 lens of 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 summary of 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: In a subsequent study, PFOS (suspended in com 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 55 0000C8 January 21, 1999 + 23 (S.D.) in the control group. Clinical signs in surviving dams included hunching, lower body weight, alopecia, rough haircoat, anorexia. Gastrointestinal and kidney lesions were noted in the high-dose dams. Treatment-related fetal effects that were attributed to maternal toxicity included: increased resorptions and fetal death, decreased fetal body weight, delayed skeletal ossification, cleft palate, subcutaneous edema and cryptorchism (undescended testicles). These effects occurred primarily in the high-dose group. The maternal and fetal NOAELs for this study were both 1 mg/kg/day. Oral Developmental Toxicity (Teratology) in Rabbits: A final draft report of an oral developmental toxicity study in rabbits was recently received (York, 1998). In this study, dose groups of 22 pregnant new Zealand white rabbits were dosed on days 7 through 20 of gestation 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 0000^9 56 January 21, 1999 gavage (Argus, 1998). In this study, groups of 35 male and 35 female rats were exposed to 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 % of control. There was no effect on the number of pregnancies. 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 group of ten dams per dose group was sacrificed on day 10 of gestation. No increases in resorptions occured, and there were no decreases in the number of implantations or number of live fetuses. At parturition in the 3.2 mg/kg high-dose group, the mean number of pups delivered was decreased compared to the control (10 versus 14) and the percent of pups delivered stillborn was increased significantly (24.9 % as compared to 2.2 %.) Survival of pups during days one through four of lactation 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 body-weight gains through day 21 of lactation (72.1 % of controls.) A series of follow-up studies are to be initiated in November, 1998 to gain a better understanding of the reduced perinatal survival at the high dose levels. These will include a complete cross-fostering study, and two pharmacokinetic studies with pregnant dams. 000070 57 January 21, 1999 The post-weaning Fj generation currently exhibits no signs of toxicity 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 pM demonstrate: 1) detergent type effects on mitochondrial membranes; 2) stimulation of mitochondrial respiration, and 3) fluidization of the inner mitochondrial membrane (Wallace and Starkov, 1998). Interference with Fatty Acid Binding and Transport: Interference of PFOS (1-10 pM) 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 ImM DAUDA and an IC50of 4.9 pM (Nabbefeld et al., 1998; Nabbefeld, 1998). Peroxisome Proliferation: Liver biochemical effects associated with peroxisome proliferation have been investigated in two published studies. Sohlenius et al. (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 cytosolic fractions; 4) glutathione transferase; 5) epoxide hydrolase and 6) DTdiaphorase, Q- and Q -1-hydroxylation (Sohlenius et al., 1993). 000071 58 January 21, 1999 Ikeda et al. (1987) exposed male rats were to 0.02% PFOS in the diet for 2 weeks. The prominent induction of peroxisome proliferation was demonstrated by electron microscopy. Activities of catalase, fatty acyl-CoA oxidizing system, carnitine acetyl transferase and cytochrome P450s responsible for the Q-hvdroxylation of lauric acid were increased (Ikeda et al., 1987). Hypolipidemia: The mechanism of the hypolipidemic effect of PFOS has been studied. Rats were fed 12 mg/kg/day for 7 - 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 synthesis of cholesterol from acetate, pyruvate and hydroxymethylglutarate but not from mevalonate, increased oxidation of palmitate and reduced fatty acid synthesis. Activities of liver hydroxymethyl glutaric acid-CoA reductase and acylCoArcholesterol 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 esterification of cholesterol together with enhanced oxidation of fatty acids in the liver (Haughom and 0ystein, 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 outlines of them can be found in the Appendix. 59 0 0 0 0 7 2 Table IV.8: Summary of Rodent Repeated-Dose Studies Study 90-Day Dietary (Goldenthal et al., 1978a) S p e cies (strain) Rats/CD 14-Week Dietary Rats (Part of on-going (Sprague 2-yr study) Dawley 90-Day Drinking Rats Water (Sprague (Limoges, 1995) Dawley) CO D ose (u n its) 0 (ppm) 30 100 300 1000 3000 0 (ppm) 0.5 2 5 20 0 (mg/kg/d) 0 0.02 0.06 0.2 0.6 0.3 1.0 3.0 n (M ale = M Fem ale = F) 5 M/ 5 F 5 M/ 5 F 5 M/ 5 F 5 M/ 5 F 5M/ 5F 5 M/5 F 10 M / 10 F 10 M / 10 F 10 M / 10 F 10 M / 10 F 10 M / 10 F 10 M / 10 F 10 F 10 F 10 F 10F 10F 10M 10M 10M C linical O bservations; C linical Pathology; G ross Pathology; H istopathology No effect | body wgt, t glutamate-pyruvate transaminase & glutamate-oxalacetate transaminase, liver discoloration 3 deaths; Tsensitivity to stimuli, red material around the eyes or mouth, i food consump., T CPK, alk phos, glucose & BUN, | hemoglobin, hematocrit, erythrocyte count, reticulocyte count (in females) and leucocyte count, liver enlargement, necrosis and hepatocellular hypertrophy and stomach discoloration and hemorrhage Death; emaciation, convulsions, stomach mucosal hyperkeratosis, bone marrow hypocellularity, thymic follicular atrophy, splenic lymphoid follicular atrophy, atrophy of mesenteric lymph nodes, atrophy of villi in small intestines, skeletal muscle atrophy and dermal acanthosis and hyperkeratosis. Death; hunched posture Death; hypoactivity No effect l body wgt (6 & 9), i cholesterol (d),t liver wgt (d & 9), enlarged & vacuolated liver cells, t Palmitoyl CoA oxidase activity No effect No effect No effect Tliver weight t liver weight T liver weight, hematology and serum chemistry effects f serum triglycerides t liver weight, J, serum cholesterol and triglycerides, hepatic vacuolization t liver weight, | serum cholesterol and triglycerides, hepatic vacuolization 0000 60 January 21, 1999 Table IV.9: Summary of Primate Repeated-Dose Studies Study 90-Day Gavage (Goldenthal et al., 1978) Species (strain) Rhesus Monkey 90-Day Gavage Rhesus (Goldenthal et al., Monkey 1978b) 28-Day Capsule Cyno. Range-finding Monkey Dose (units) 0 (mg/kg/d) 10 30 100 300 0 (mg/kg/d) 0.5 1.5 4.5 0 (mg/kg/d) 0.02 2.0 n (Male = M Female -F) 2 M/ 2 F 2 M/ 2 F 2 M/ 2 F 2 M/ 2 F 2M/ 2F 2 M/ 2 F 2 M/ 2 F 2 M/ 2 F 2 M/ 2 F 2 M/ 2 F 3 M/ 3 F 1M/ 1F Clinical Observations; Clinical Pathology; Gross Pathology; Histopathology No effect Death within 11-20 days, weight loss, weakness anorexia, 1 activity, emesis, diarrhea, tremors, prostration, congestion, hemorrhage and lipid depletion of adrenal cortex Death within 7-10 days; weight loss, marked weakness anorexia, hypoactivity, emesis, diarrhea, tremors, prostration, congestion, hemorrhage and lipid depletion of adrenal cortex All dead in 3 - 5 days, weight loss, marked weakness anorexia, hypoactivity, emesis, diarrhea, tremors, prostration, congestion, hemorrhage and lipid depletion of adrenal cortex All dead in 2 - 4 days, weight loss, marked weakness, anorexia, hypoactivity, emesis, diarrhea, tremors, prostration, congestion, hemorrhage and lipid depletion of adrenal cortex No effect Slight & intermittent J, activity in 3 of 4 monkeys Blood & mucous in stools, diarrhea, dehydr., tremors, | body wgt, | cholesterol, marked 4 activity marked i cholesterol Death within 7 weeks; black or bloody stool, dehydration, rigidity, convulsions, prostration, decreased serum cholesterol, diffuse lipid depletion of adrenals, atrophy of pancreatic exocrine cells and atrophy of submandibular salivary gland serous alveolar cells, decreased serum alkaline phosphatase, increased SGOT. No effect No Effect | cholesterol ( & 9) o o o q 61 January 21, 1999 Table IV. 10: Summary of Developmental and Reproductive Studies Study Teratology (Riker, 1980) Species (strain) Rat (CD) Teratology (Wetzel, 1980 & Wetzel, et al., 1983) Rat (CD) Two-Gen Reproduct. (in progress, 10/98) Rat (CD) Dose (units) 0 (mg/kg/d) 1 5 10 0 (mg/kg/d) 1 5 10 n (Male = M Female = F) 20 F (preg.) 20 F (preg.) 20 F (preg.) 20 F (preg.) 25 F (preg.) 25 F (preg.) 25 F (preg.) 25 F (preg.) 0 (mg/kg/d) 0.1 0.4 1.6 3.2 35M/35F 35 M/ 3 5 F 35 M/ 3 5 F 35 M/ 3 5 F 35 M/ 3 5 F Clinical Observations; Clinical Pathology; Gross Pathology; Histopathology No effect No effect No effect 4Body wgt gain No effect No effect 4 4 4maternal (?), body wgt gain, food consumption, thinness, hunching, rough hair coat, anorexia, pup weight 4 4T subcutaneous edema, cleft palate & cryptorchism, maternal ($), body wgt gain, food 4 4consumption, slight pups per litter, pup weight No effect No effect No effect 4t stillborn pups & pup survival, ($), body weight gain 4 4 4body wgt gain ($), slight body wgt gain (<?), t stillborn pups & pup survival oo o q CA 62 January 21, 1999 Table IV.ll: 90-Day Suh-Chronic Study Results Organized by Increasing Nominal mg/kg Dose Level Dose as mg/kg/day 0.02 0.06 0.20 0.30 0.50 0.60 1.0 1.5 2.0 3.0 4.5 6.0 10 18 30 60 100 180 300 Dosage form Species Drink. Water Drink. Water Drink. Water Drink. Water Gavage Drink. Water Drink. Water Gavage F rat F rat F rat M rat Monkey F rat M rat Monkey Dietary Rat Drink. Water Gavage M rat Monkey Dietary Rat Gavage Dietary Gavage Dietary Gavage Dietary Gavage Monkey Rat Monkey Rat Monkey Rat Monkey Outcome No effect (Limoges, 1995) No adverse effect; increased liver weight (Limoges, 1995) No effect; increased liver weight (Limoges, 1995) Reduced serum triglycerides (Limoges, 1995) anorexia, emesis, diarrhea and slightly decreased serum alkaline phosphatase. (Goldenthal et al., 1978b) Hematology and serum chemistry effects; increased liver weights (Limoges, 1995) Reduced serum cholesterol and triglycerides; hepatic vacuolization; increased liver weights (Limoges, 1995) hypoactivity, tremors, weight loss, decreased serum alkaline phosphatase and inorganic phosphate (Goldenthal et al., 1978b) Weight loss, elevated plasma glutamate-pyruvate transaminase, elevated plasma glutamate-oxalacetate transaminase and liver discoloration. (Goldenthal et al., 1978a) Reduced serum cholesterol and triglycerides; hepatic vacuolization; increased liver weights (Limoges, 1995) Death within 7 weeks; black or bloody stool, dehydration, rigidity, convulsions, prostration, decreased serum cholesterol, diffuse lipid depletion of adrenals, atrophy of pancreatic exocrine cells and atrophy of submandibular salivary gland serous alveolar cells, decreased serum alkaline phosphatase, increased SGOT. (Goldenthal et al., 1978b) 3 deaths; increased sensitivity to external stimuli, red material around the eyes or mouth, decreased food consumption, elevated plasma creatinine phosphokinase, alkaline phosphatase, blood glucose and blood urea nitrogen, decreased hemoglobin, hematocrit, erythrocyte count, reticulocyte count (in females) and leucocyte count, liver enlargement, necrosis and hepatocellular hypertrophy and stomach discoloration and hemorrhage (Goldenthal et al., 1978a). Death within 11- 20 days; clinical signs included weight loss, marked weakness anorexia, hypoactivity, emesis, diarrhea, tremors, prostration, congestion, hemorrhage and lipid depletion of adrenal cortex. (Goldenthal, 1978) Death; emaciation, convulsions, stomach mucosal hyperkeratosis, bone marrow hypocellularity, thymic follicular atrophy, splenic lymphoid follicular atrophy, atrophy of mesenteric lymph nodes, atrophy of villi in small intestines, skeletal muscle atrophy and dermal acanthosis and hyperkeratosis. (Goldenthal et al., 1978a) Death within 7 - 1 0 days; clinical signs included weight loss, marked weakness anorexia, hypoactivity, emesis, diarrhea, tremors, prostration, congestion, hemorrhage and lipid depletion of adrenal cortex (Goldenthal, 1978) Death; hunched posture. (Goldenthal et a!., 1978a) Death within 3 - 5 days; clinical signs included weight loss, marked weakness anorexia, hypoactivity, emesis, diarrhea, tremors, prostration, congestion, hemorrhage and lipid depletion of adrenal cortex (Goldenthal, 1978) Death; hypoactivity. (Goldenthal et al., 1978a) Death within 2 - 4 days; clinical signs included weight loss, marked weakness anorexia, hypoactivity, emesis, diarrhea, tremors, prostration, congestion, hemorrhage and lipid depletion of adrenal cortex. (Goldenthal, 1978) 00007 63 V. RISK CHARACTERIZATION The principal issue of concern is the potential health risk associated with the levels of PFOS identified in serum samples from non-occupationally exposed employees, commercially available serum and blood bank samples. The purpose of this section is to evaluate this risk using available human and animal data. Sources of exposure and their control are discussed in a companion document. As noted previously (Section III), 3M has conducted medical surveillance programs at manufacturing locations where employees are exposed to PFOS or precursor molecules. These employees have PFOS 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-occupationally 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 it is from pooled samples, does not reflect the variability and range one would expect to find among individuals. Analysis of data 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 measurement of 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 employees of the 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 64 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 University of Minnesota completed a mortality study of 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 III. 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. It 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 study itself documents that a decrease in cholesterol, an early biological effect in both rodents and primates, was observed at a PFOS serum level of 72 ppm in one of the two high dose animals. In the rhesus monkey studies, lowered cholesterol was observed after 90 days of oral 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 concentration of 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. 000078 65 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 FI 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 FI 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 aware of 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 or below 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 absence of detectable toxicity in production workers are that the workers' serum and tissue levels 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. Of these 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, 66 000079 January 21, 1999 because of 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 orders of 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 serum of occupationally or non-occupationally exposed employees, blood bank samples or commercially available serum. 67 0 0 0 0 8 0 January 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 evidence of 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 utility of the 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 of these effects is desirable to determine whether they can occur at serum and tissue levels relevant to those observed or estimated in humans. Section VI of this report describes additional 3M studies that will address these issues. Further follow up 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, represent a 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. 68 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 hazards of 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 toxicity of this class of chemicals which have the perfluorooctane sulfonyl moiety as a base. The goals of this research program are as follows: To understand the relationship between measured concentrations of PFOS in serum and potential adverse health effects; To understand the kinetics of increases in body burden of PFOS as reflected in serum PFOS measurements; To determine if a critical cumulative body burden exists and how this is rleated to toxicokinetics; To define the metabolic relationship of PFOS to other Perfluorooctanesulfonylbased chemicals; To understand the primary mechanism of toxicity responsible for early toxic effects; 000082 69 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 VI. 1 and VI.2, 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 degradation of perfluorooctanesulfonamide-based chemicals to PFOS. Descriptive summaries of many of these studies are included in the Appendix. 000083 70 January 21, 1999 Table VI. 1: PFOS Toxicity Studies Initiated in 1998 Study Title Start Date Completion Date (In-Life) Four-Week Capsule Range-Finder Study in Cynomolgus Monkeys Six-Month Capsule Feeding Study in Cynomolgus Monkeys 4/23/98 8/5/98 5/22/98 2/8/99 (histo.) 5/7/99 (recov.) Two-Year Dietary Study in Rats 5/20/98 4/24/2000 Hepatic Peroxisome Induction and Cell Proliferation Study 12/1/98 1/7/98 Biochem ical and Molecular B iology Mechanistic Studies 7/1/97 On-going Segment II Teratology in Rabbits 8/28/98 9/29/98 Two-Generation Reproduction Study in Rats 5/26/98 12/31/98 One-Generation Cross-Fostering Reproduction Study in Rats 11/1/98 2/15/99 One-Generation Reproduction PK Study in Rats (through Gestation) 11/7/98 1/30/99 One-Generation Reproduction PK Study in Rats (through Lactation) 11/23/98 3/1/99 Table VI.2: N-EtFOSE Toxicity Studies Initiated in 1998 Study Title Two-Year Dietary Study in Rats Single-D ose ADM E in Cynomolgus Monkeys Biochem ical and Molecular B iology Mechanistic Studies Segment II Teratology in Rats Segment II Teratology in Rabbits Two-Generation Reproduction Study in Rats Start Date Com pletion Date (InLife) 1/26/98 1/25/2000 5/14/98 10/1/98 7/1/97 On-going 8/11/98 9/4/98 8/28/98 9/29/98 6/8/98 12/31/98 71 0 0 0 0 8 4 January 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: 4/24/2000 The purpose of 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 indicator of 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 occurrence of 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 carcinogenicity of the test material, N-ethyl 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 72 0 0 0 0 8 5 January 21, 1999 protocol. Microscopic examinations were done on selected tissues from animals necropsied during Weeks 8 and 14. A recovery group of animals 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 days of treatment, 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 0 of recovery and after 6, 30, and 90 days of 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 will 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 days of treatment, and after 30, 60, and 90 days of recovery. Clinical chemistry will include urea nitrogen, cholesterol, triglicerides, alkaline phosphatase, alanine aminotransferase, bile acids, total and direct bilirubin, lipase, and pancreatic-specific amylase. Palmitoyl CoA 000086 73 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-EtFOSE, 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. Determination of ADME parameters in non-human primate is an appropriate model for human risk assessment, as opposed to the rat. Two groups of 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 animal/sex 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, 1, 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 with mass > 0.5 g will be analyzed for N-EtFOSE 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. 000037 74 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 of FC-807 in rats. FC-807 is the brand name of a 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 determine if 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 route group 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 the remainder of the animals were sacrificed 28 days post dose. The preliminary results indicated absorption of the 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 consisted of six 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 of 5 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, 75 0 0 0 0 8 8 January 21, 1999 30 and 100 ppm N-MeFOSE in the diet. After 4 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. Groups of 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, number of pups/ litter, pup viability index and lactation index. The FI 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 F0 (Parent) animals and analyzed for compound by 3M. Two-Generation Rat Reproduction Study with N-EtFOSE. Start Date: 6/8/1998. In life completion date: 12/31/1998. The purpose of this study is to test for toxic effects of N-EtFOSE 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. Groups of 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, number of pups/ litter, pup viability index and lactation 0000S9 76 January 21, 1999 index. The FI 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 5 male and 5 female rats/dose group from F0 (Parent) animals and analyzed for compound by 3M. Segment II Teratology in Rabbits with PFOS. In-life Start date: 8/28/1998. In life end date 9/29/1998. The purpose of this study is to detect adverse effects of PFOS on New Zealand White pregnant female rabbits and development of the embryo on 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 a 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 21 of gestation (the day following the last dosage) serum, liver, fetal and placental samples were analyzed for PFOS 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 II 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 effects of N-EtFOSE on presumed pregnant female rats and development of the embryo and fetus consequent to exposure of the dam from implantation to closure of the 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 group of female 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-EtFOSE and possible metabolites. Rats were Caesarian 000090 77 January 21, 1999 sectioned on day 20 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 II Teratology in Rabbits with N-EtFOSE. In-life Start date: 8/28/1998. In life end date 9/29/1998. The purpose of this study was to detect adverse effects of N-EtFOSE on New Zealand White pregnant female rabbits and development of 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 a 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 21 of gestation (the day following the last dosage) serum, liver, fetal and placental samples were analyzed for N-EtFOSE and possible metabolites. Rabbits were Caesarian section on day 29 of presumed gestation. The fetuses will be 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. 000091 78 January 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 in a 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. Analysis of the 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 nature of the medical surveillance program does not allow for a 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-methyl perfluoroctanesulfonamido ethanol. A sub-sample of employees will also be tested for total serum organic fluorine. A brief 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 Analysis of the Inpatient Claims Experience of 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 79 0 0 0 0 3 2 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. Flowever, neither of these studies utilized the employees' fluorochemical serum measurements to design job-, department- and calendar-year exposure matrices. The purpose of these 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.e., Standardized Mortality Ratios, for more than 50 causes of death. Vital status and cause of death will be ascertained through December 31, 1997. 5. Geographical Reliability Study. The purpose of this 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 II.3) to determine whether comparable serum levels are measured. 80 0 0 0 0 9 3 January 21, 1999 VII. 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. 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Induction of cytochrome P-450 and peroxisome proliferation in rat liver by perfluorinated octanesulfonic acid. In: Peroxisomes in Biology and Medicine, H.D. 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. 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Cholestramine-enhanced fecal elimination of carbon-14 in rats after administration of ammonium 000096 83 January 21, 1999 [14C]perfluorooctanoate or potassium [l4C]perfluorooctanesulfonate. Fund. Appl. Toxicol 4, 972-976. Mandel JS, Johnson RA (1995). Mortality study of employees 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 Effects of Fluorochemicals on Liver Fatty Acid-Binding Protein. Masters Thesis, University of Minnesota. Thesis research performed at and supported by 3M Nabbefeld D., Butenhoff J., Bass N. and Seacat A. 1998. Displacement of a 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 JM, Mandel JH, Zobel LR (1998). An epidemiologic investigation of clinical chemistries, hematology and hormones in relation to serum levels of perflurooctane sulfonate in male fluorochemical production employees. Unpublished report. St Paul:3M Company, April 22, 1998. Olsen GW, Gilliland FD, Burlew MM, Burris JM, Mandel JS, Mandel JH. An epidemiologic investigation of reproductive hormones in men with occupational exposure to 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. 0979ABO632. 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). Determination of 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, Bio/dynamics, Inc. 84 0 0 0 0 9 V January 21, 1999 Singer L and Armstrong WD (1959). Determination of fluoride in blood serum. Analytical Chem 31:105-109.. Singer L and Ophaug RH (1979). Concentrations of ionic, total, and bound fluoride in plasma. Clin Chem 25:523-525. Sohlenius, A-K., Eriksson, A.M., 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. Pharmacol Toxicol. 72, 90-93. Taves D (1968a). Evidence that there are two forms of fluoride in human serum. Nature 217; 1050-1051. Taves D (1968b). Electrophoretic mobility of 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 effect of perfluorinated arylalkylsulfonamides on bioenergetics of rat liver mitochondria. Dept of Biochemistry and Molecular Biology, University of MN School of Medicine. Duluth, MN 55812, USA. Supported by a grant from 3M Company. Wetzel, L.T., Burdock, G.A., Durloo, R.S. and Colpean, B.R. 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 forms of fluorine 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 000098 85 January 21, 1999 FIGURE A1. Reliability of PFOS Serum Measurements in 12 3M Employees 80 70 i 2nd T e st (A p ril, 1998) 60 -1 50 -I SI 40 - 30 - 20 J--------------------------------------------- n----------------------------- ;----------------------------- ,----------------------------- ;----------------------------20 30 40 50 60 70 80 1st T e s t (F e b ru a ry, 1998) 86 0 ()O O 3 9 Appendix - Table A1 Mean, Median, Standard Deviation (SD) of Mean and Range of PFOS, Demographic, Serum Chemistries and Hematological for Antwerp and Decatur Employees Combined, 1995 (N=178) and 1997 (N=147) PFOS* (ppm) Mean 1995 Data Median SD Range Mean 1997 Data Median SD 0 - < lppm 1 - < 3ppm 3 - < 6ppm > 6ppm 0.49 0.5 1.821 1.77 4.121 3.97 8.171 7.73 F value = 321.9, p < . 0001 0.27 0.58 0.81 2.52 PFOS (ppm) 000 - 0.90 1.00-2.91 3.00-5.80 6.06-12.83 0.52 0.52 1.781 1.64 3.871 3.59 7.201 6.68 F value = 367.6, p < .0001 0.27 0.56 0.70 1.59 0.10-0.97 1.02-2.89 3.09-5.30 6.05-9.3 Age fvrs) 0 - < 1 ppm 37 36 8 21-58 36 34 11 21 -62 1 - < 3 ppm 422 41 9 25-60 422 41 9 24-63 3 - < 6 ppm 40 40 7 26-55 41 42 5 32-54 > 6 ppm 45 43 7 37-56 42 45 9 29-52 F value = 3.7, p = .02 F value = 5.1, p = .002 000100 87 January 21, 1999 PFOS (ppm) 0 - < lppm 1 - < 3 ppm 3 - < 6 ppm > 6ppm 0 - < 1 ppm 1 - < 3 ppm 3 - < 6 ppm > 6 ppm 0 - < 1 ppm 1 - < 3 ppm 3 - < 6 ppm > 6 ppm Table Al (Continued) ______________ 1995 Data______________ _______________________ 1997 Data Mean Median SD Range Mean Median SD Alcohol (drinks/davl 0.8 0.6 0.5 0.1 1.2 0.3 0.7 0.0 F value = 4.0, p = .009 0.9 0.7 1.9 1.1 0.0-3.6 0.0-3.6 0.0-6.0 0.0-2.9 0.5 0.1 0.5 0.1 1.0 0.1 0.2 0.1 F value == 1.8, p = .15 0.8 0.8 1.8 0.2 BMI (kg/m2) 25.5 24.8 27.7 26.3 24.93 25.0 27.7 29.4 F value = 3.7, p = .02 4.2 5.8 3.8 4.2 17.9-38.7 19.6-60.7 17.9-32.5 20.6-33.0 26.0 24.9 27.7 26.4 27.3 27.9 30.8 29.7 F value = 2.1, p = .10 4.9 5.7 4.4 4.0 Cigarettes (per dav) 2.6 0.0 6.8 0.0 10.6 8.0 0.4 0.0 F value ==4.8, p = .003 6.4 11.3 12.4 1.1 0.0-25.0 0.0 - 40.0 0.0 - 40.0 0.0-3.0 4.7 0.0 8.2 0.0 4.1 0.0 6.0 0.0 F value = 15, p = .23 9.4 11.3 8.3 13.4 Range 0-4.3 0-5.0 0-7.1 0.1 -0.8 20.1 -41.7 18.1 -48.5 19.1 -36.0 26.1 -36.2 0-40 0-40 0-30 0-30 000101 88 January 21, 1999 PFOS (ppm) Mean 1995 Data Median SD 0 - < lppm 1 - <3 ppm 3 - < 6 ppm > 6 ppm 16.5 15 15.4 15.0 16.4 16.0 15.1 14.0 F value = 1.1, p = .36 3.5 4.0 3.7 4.2 0 - < lppm 1 - <3 ppm 3 - < 6 ppm > 6 ppm 1.0 1.0 1.0 1.0 0.9 0.9 1.1 1.2 F value = 2.3, p = .08 0.2 0.2 0.2 0.3 0 - < 1 ppm 1 - <3 ppm 3 - < 6 ppm > 6 ppm 85 85 86 86 84 83 84 83 F value = 0.9, p = .44 15 22 12 14 000102 Table Al (Continued) 1997 Data Range Mean Median SD BUN 11-26 8.0-26.0 10.0-23.0 10.0-21.0 14.5 14.0 14.2 14.0 15.0 15.0 13.8 12.0 F value ==0.5, p = 0.67 2.8 3.2 2.9 4.1 Creatinine 0.7- 1.6 0.7- 1.6 0.7- 1.2 0.6- 1.6 0.9 0.9 0.9 0.9 0.9 0.9 1.0 0.9 F value = 0.4, p = 0.78 0.1 0.1 0.1 0.2 Glucose 66-170 60 - 260 66-114 71 - 105 87 85 93 84 95 89 89 88 F value = 0.6, p = .59 17 39 25 7 Range 9.0-21.0 6.0-26.0 9.0 - 20.0 9.0-19.0 0.6- 1.2 0.7- 1.3 0.7- 1.1 0.8- 1.4 58-174 65 - 303 74 -192 80-97 89 January 21, 1999 PFOS (ppm) Mean 1995 Data Median SD 0 - < 1 ppm 1 - <3 ppm 3 - < 6 ppm > 6 ppm 80 78 89 89 86 85 88 85 F value = 1.3, p = .28 22 27 21 24 0 - < 1 ppm 1 - <3 ppm 3 - < 6 ppm > 6 ppm 43 31 47 36 40 39 43 33 F value = 0.5, p = .71 28 39 15 18 0 - < 1 ppm 1 - <3 ppm 3 - < 6 ppm > 6 i ppm 27 25 29 26 25 24 33 33 F value - 1.8, p = .14 13 12 5 6 000103 Table Al (Continued) 1997 Data Range Mean Median SD Alkaline Phosphatase 31 - 158 49 -191 32 - 124 63 - 136 77 83 76 88 73 17 79 23 74 22 84 18 F value = 1.17, p = .32 GOT 16-155 2-293 21-80 28-79 28 22 36 25 28 27 33 37 F value = 1-1, p = .34 20 33 14 12 AST 15-96 14-90 13-37 26-43 27 25 26 25 25 23 29 28 F value = 0.5, p = .67 7 7 7 Range 49- 132 41 - 163 29-120 65-114 10-142 10-179 13-71 17-48 13-53 15-56 14-43 26-34 90 January 21, 1999 ______________ 1995 Data PFOS (ppm)________Mean______ Median_____ SD 0 - < 1 ppm 1 - <3 ppm 3 - < 6 ppm > 6 ppm 48 43 46 42 42 41 51 49 F value = 1.0, p = .38 20 21 7 17 0 - < 1 ppm 1 - <3 ppm 3 - < 6 ppm > 6 ppm 0.88 0.70 0.662 0.60 0.642 0.60 0.76 0.70 F value = 4.4, p = .005 0.50 0.30 0.28 0.23 0 - < 1 ppm 1 - <3 ppm Oo 3 - < 6 ppm o > 6 ppm o 0.22 0.20 0.21 0.20 0.21 0.20 0.20 0.20 F value = 0.6, p = .58 0.05 0.06 0.04 0.02 Table Al (Continued) 1997 Data Range______Mean______ Median________ SD_____________Range ALT 27-118 18-183 30-59 29-82 31 30 33 29 34 31 41 45 F value = 0.9,p == .46 11 16 18 10 13-60 10-89 14-82 25-49 Total Bilirubin 0.40 - 2.90 0.20-1.50 0.20-1.40 0.50-1.20 0.77 0.60 0.612 0.60 0.63 0.50 0.58 0.50 F value = 2.9, p = .04 0.40 0.21 0.31 0.24 0.30-2.30 0.30-1.30 0.40-1.30 0.40-1.00 Direct Bilirubin 0.02 - 0.40 0.10-0.40 0.20 - 0.30 0.10 0.30 0.15 0.10 0.122 0.10 0.12 0.10 0.10 0.10 F value ==3.5, p = .02 0.07 0.04 0.04 0.00 0.10-0.40 0.10-0.20 0.10-0.20 0.10-0.10 91 January 21, 1999 ______________ 1995 Data PFOS (ppm)________Mean______ Median_____ SD 0 - <1 ppm 1 - <3 ppm 3 - <6 ppm > 6 ppm 219 215 216 213 214 214 213 221 F value = 0.1, p =.96 47 43 35 36 0 - <1 ppm 1 - < 3 ppm 3 - <6 ppm > 6 ppm 0 - <1 ppm 1- <3 ppm 3 - <6 ppm > 6 ppm il o to xs .11 -boo 140 134 137 142 F value 137 134 135 136 43 40 34 32 53 53 48 47 45 46 45 46 F value = 2.9, p = 04 13 13 11 9 000.1.05 Table Al (Continued) 1997 Data Range Mean Median SD Cholesterol 100 - 340 118-315 128 -278 160-251 198 197 216 219 2292 224 229 238 F value = 4.3, p = 006 40 42 29 26 LDL 29-261 44 - 234 65 - 190 95 - 178 HDL 31-94 26 -94 23-74 34-61 124 128 141 134 1482 142 145 156 F value = 3.7, p = 01 46 48 44 45 48 47 40 38 F value = 1.1, p =.34 34 38 24 26 11 10 10 4 Range 110-280 116-365 192- 321 186-250 50 - 205 61-290 111-196 103-164 19-74 28-69 32-69 37-45 92 January 21, 1999 ______________ 1995 Data PFOS (ppm)________Mean______ Median_____ SD 0 - <1 ppm 1- <3 ppm 3 - <6 ppm > 6 ppm 129 96 161 133 158 142 132 151 F value = 1.1, p =.35 98 107 88 45 0 - <1 ppm 1 - <3 ppm 3 - <6 ppm > 6 ppm 47 47 46 46 47 47 47 48 F value = 2.4, p = 07 2 3 2 2 0 - <1 ppm 1 - <3 ppm 3 - <6 ppm > 6 ppm 15.5 15.5 15.2 15.2 15.5 15.5 15.5 15.4 F value = 2.2, p =.10 0.7 1.0 0.8 0.7 0001-06 Table Al (Continued) 1997 Data Range Mean Median SD Triglycerides 41 - 622 41 -651 34-413 64-187 148 107 156 122 166 158 220 191 F value = 0.5, p =.67 162 108 92 83 Hematocrit 43-51 38-52 41 -52 44-49 46 46 45 46 46 44 45 44 F value = 2.1, p = .11 2 3 3 2 Hemoglobin 14.0-16.7 13.0-17.4 13.6-17.4 14.7-16.2 15.5 15.5 15.4 15.5 15.0 14.7 15.1 15.0 F value = 1.8, p =.15 0.8 0.9 1.0 0.7 Range 38 - 1209 44 - 534 45 - 394 149-352 40-52 39-53 39-50 42-47 13.5 - 17.0 13.3 - 17.3 12.5 - 16.7 14.1 - 16.2 93 January 21, 1999 ._________1995 Data PFOS (ppm)________Mean_____ Median______ SD 0 - <1 ppm 1 - <3 ppm 3 - <6 ppm > 6 ppm 5.0 5.0 4.9 4.9 5.0 5.0 5.0 4.9 F value = 0.4, p = .75 0.3 0.3 0.2 0.5 0 - <1 ppm 1 - <3 ppm 3 - <6 ppm > 6 ppm 31.3 31.2 30.9 30.9 31.2 31.3 31.2 30.5 F value = 0.9 , p =.45 1.6 1.4 1.5 2.8 0 - <1 ppm 1 - <3 ppm 3 - <6 ppm > 6 ppm 33.1 33.2 33.2 33.1 33.1 32.6 33.1 :>j.J F value = 0.2, p = .90 0.7 0.6 0.7 0.6 0001.07 Table Al (Continued) 1997 Data Range Mean 1Median SD RBC 4.3 - 5.7 4.3 -5.7 4.6- 5.5 4.0-5.7 5.1 5.2 5.0 5.1 5.0 5.0 5.0 4.9 F value = 1.4, p = .25 0.3 0.3 0.3 0.3 MCH 27.7 - 34.3 26.4 - 34.3 26.0-33.6 28.2 -36.9 30.4 30.3 30.6 30.5 30.2 31.2 30.5 30.5 F value = 0.6, p = .65 1.5 1.2 1.9 2.1 MCHC 31.9-34.7 31.7-34.5 31.3-34.3 32.2-34.0 33.6 33.6 33.6 J J.O 33.5 33.5 33.9 33.9 F value ==0.7, p = .56 0.5 0.5 0.7 0.5 Range 4.3 - 5.9 4.1 -5.5 4.4 - 5.7 4.7-5.5 27.6-34.1 26.7-33.8 26.2 -32.9 27.5 - 33.4 32.2 -34.9 31.8-34.6 32.0-34.4 33.4-34.6 94 January 21, 1999 PFOS (ppm) 0 - <1 ppm 1 - <3 ppm 3 - <6 ppm > 6 ppm 0 - <1 ppm 1 - <3 ppm 3 - <6 ppm > 6 ppm 0 - <1 ppm 1 - <3 ppm 3 - <6 ppm > 6 ppm Table Al (Continued) 1995 Data 1997 Data Mean Median SD Range Mean Median SD MCV 94 95 93 94 94 94 94 92 F value =^1.1, p = .35 5.2 4.3 4.8 9.7 85-106 80-104 81-104 85-115 90 90 91 91 90 91 90 91 F value = 0.6, p = .59 4.4 3.6 5.2 5.7 WBC 6.1 6.0 7.02 6.8 7.62 6.9 7.0 6.9 F value = 4.3, p -0 0 6 1.3 4.1 -9 .4 6.1 5.8 1.4 2.0 3.6-15.5 6.9 6.6 1.9 2.2 4.1 - 13.3 6.2 6.1 1.5 0.6 6.4-7.8 6.2 7.1 1.5 F value = 2.2, p =.09 Platelets 226 224 40 159-309 227 220 53 229 224 47 122-367 229 223 49 228 226 45 132-344 220 221 45 185 182 50 132-277 199 191 58 F value ==2.1, p =10 F value = 0.6, p = 0.60 Range 83-101 81-99 80-97 81-96 3.8-10.3 3.8-13.2 4.0-10.0 4.2-7.4 106-406 124-359 147-316 146-295 0001.08 95 January 21, 1999 Table A1 (continued) 1. Mean is significantly different (p < .05, Bonferroni (Dunn) test) than the mean of the other PFOS categories. 2. Mean is significantly different (p < .05, Bonferroni (Dunn) test) than the mean of 0 - < 1 ppm PFOS category. 3. Mean is significantly different (p < .05, Bonferroni (Dunn) test) than the mean of 1 - < 3 ppm category. * Sample Size 0 - <1 ppm 1 - <3 ppm 3 - <6 ppm > 6 ppm 1995 Data 45 91 35 7 1997 Data 60 63 21 5 60^000 96 Table A2. Description of Final Employment and Vital Status for Male (n = 1,639) and Female (n = 318) Employees Final Employment Status Male Employees Female Employees and Vital Status_________________ N_________ %__________ N___________% Currently Employed 810 49.4 141 44.3 Retired Alive Deceased Unknown 59 3.6 9 2.8 10 0.6 0 0.0 0 0.0 0 0.0 Terminated Alive Deceased Unknown 694 42.3 164 51.6 29 1.8 1 0.3 6 0.4 0 0.0 Died While Employed TOTAL 31 1,639 1.9 3 100.0* 318 0.9 100.0* *percentages may not add to 100 due to rounding 97 0 0 0 1 1 0 January 21, 1999 Table A3. Characteristics of Male (n = 1,639) and Female (n = 318) Employees Variable Number of Employees Number of Person-years Number of Deaths Average Age Started Work Average Year of Entry Average Age at Death Average Year of Death Males 1,639 33,108 70 25 1971 47 1984 Females 318 4,807 4 26 1977 28 1980 OOOlll 98 January 21, 1999 Table A4. Distribution of Men by Age and Year of Entry into Follow-up (n = 1,639) Year of Entry into Follow-up 1960 1965 1970 1975 1980 1985 1990 to to to to to to to Age 1964 1969 1974 1979 1984 1989 1991 TOTAL <20 20-24 18 34 86 114 238 301 31 96 4 28 2 18 0 175 J">) 798 25-29 64 139 65 37 21 14 5 345 30-34 21 58 33 19 8 10 4 153 35-39 22 25 9 15 6 5 1 83 40-44 19 18 4 10 8 2 0 61 45-49 3 3 4 5 0 2 0 17 50-54 0 1 0 0 2 3 0 6 55-59 0 1 0 0 0 0 0 1 60+ 0 0000 00 0 TOTAL 261 517 502 213 77 56 13 1,639 99 000112 January 21, 1999 Table A5. Distribution of Women by Age and Year of Entry into Follow-up (n = 318) Year of Entry into Follow-up 1960 1965 1970 1975 1980 1985 1990 to to to to to to to Age 1964 1969 1974 1979 1984 1989 1991 TOTAL <20 2 12 18 16 4 0 0 52 20-24 4 22 23 50 4 9 1 113 25-29 0 4 4 47 4 5 3 70 30-34 2 3 2 15 1 4 2 29 35-39 1 2 16y y JJ jy 2 30 40-44 0 0 0 8 - J 2 0 13 45-49 0005 110 7 50-54 000 30 0 0 y J 55-59 000 10 0 0 1 60+ 0000 0 0 0 0 TOTAL 12 43 50 161 20 24 8 318 ' o o o f 3 100 January 21, 1999 Table A6. Cause-Specific SMRs for Men Ever Employed in Chemical Department (s) Using the U.S. as Comparison Population (n = 1,050) 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 Cancer of Stomach Cancer of Large Intestine Cancer of Rectum Cancer of Liver and Biliary Passages Cancer of Pancreas Cancer of All Other Digestive Organs Cancer of Respiratory System Cancer of Larynx Cancer of Bronchus Trachea and Lung Cancer of Other Respiratory Organs Cancer of Prostate Cancer of Testes and Other Male Genital Organs Cancer of Kidney Cancer of Bladder and Other Urinary Organs Observed Expected Deaths Deaths 57 81.5 13 16.9 0 0.5 SMR 70.0 76.9 --- 1 3.6 27.5 0 0.4 -- 0 0.5 -- 1 1.3 76.9 0 0.3 -- 0 0.3 0 0.8 -- 0 0.1 7 6.1 115.1 0 0.2 -- 7 5.8 120.7 0 0.1 -- 0 0.5 -- 0 0.2 -- 0 0.5 -- 1 0.2 415.5 95% Cl (53.0, 90.6) (40.9, 131.5) (0.0, 800.6) (0.7, 153.2) (0.0,913.3) (0.0, 686.1) (1.9, 428.5) (0.0, 1,271.9) (0.0, 1,188.2) (0.0, 480.6) (0.0,3,203.2) (46.3,237.1) (0.0, 1,891.9) (48.5, 248.7) (0.0, 4,214.1) (0.0, 805.9) (0.0, 1,678.5) (0.0, 768.2) (10.4, 2,315.3) 000114 101 January 21, 1999 Table A6. (Continued) Observed Expected Cause of Death Deaths Deaths SMR Cancer of Brain and Other 1 0.9 117.2 Central Nervous System Cancer of Thyroid and Other 0 0.1 -- Endocrine Glands Cancer of Bone 0 0.1 -- Cancer of All Lymphatic and 2 2.2 92.9 Hematopoietic Tissue Lymphoma 0 0.3 -- Hodgkin's Disease 0 0.3 -- Leukemia and Aleukemia 1 0.8 120.0 Cancer of Other Lymphatic 1 0.7 137.2 and Hematopoietic Tissue Malignant Melanoma of Skin 0 0.6 -- All Other Malignant 1 1.5 68.9 Neoplasms Diabetes Mellitus 0 1.1 -- Cerebrovascular Disease 1 2.3 43.4 All Heart Disease Hypertension 11 22.5 48.8 0 0.1 -- Respiratory Disease Ulcer of Stomach and 0 3.2 -- 0 0.2 -- Duodenum Cirrhosis of Liver Nephritis and Nephrosis _> 3.0 100.2 0 0.3 -- External Causes 20 22.2 90.2 Accidents 16 13.6 117.3 Motor Vehicle Accidents 8 7.8 102.8 All Other Accidents 8 5.9 135.7 Suicides 5.2 57.7 Homicides and Other External 1 3.2 31.4 Causes Residual Causes* 7 9.0 77.6 Unknown Causes & 2 *all other causes of death combined & no death certificate obtained; included only in all causes of death category 95% Cl (2.9, 653.0) (0.0, 5,113.2) (0.0, 4,368.3) (11.2, 335.5) (0.0, 1,341.4) (0.0, 1,163.3) (3.0, 668.8) (3.4, 764.5) (0.0, 614.7) (1.7, 383.7) (0.0, 323.9) (1.1,241.7) (24.4, 87.4) (0.0, 3,389.9) (0.0, 116.3) (0.0, 1,796.3) (20.7, 292.7) 0.0, 1,308.2) (55.1, 139.3) (67.0, 190.5) (44.4, 202.5) (58.6, 267.4) (11.9, 168.6) (0.8, 175.1) (31.2, 159.9) 000115 102 January 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 Cancer of Stomach Cancer of Large Intestine Cancer of Rectum Cancer of Liver and Biliary Passages Cancer of Pancreas Cancer of All Other Digestive Organs Cancer of Respiratory System Cancer of Larynx Cancer of Bronchus Trachea, and Lung Cancer of Other Respiratory Organs Cancer of Prostate Cancer of Testes and Other Male Genital Organs Cancer of Kidney Cancer of Bladder and Other Urinary Organs Observed Expected Deaths Deaths 32 44.1 9 9.6 0 0.3 0 2.1 0 0.2 0 0.3 0 0.8 0 0.2 0 0.2 0 0.5 0 0.1 5 3.6 0 0.1 5 3.4 0 0.1 0 0.3 0 0.1 0 0.3 1 0.2 SMR 72.5 93.6 -- -- -- -- -- -- 141.0 -- 147.8 -- -- -- -- 669.9 95% Cl (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) q O013-6 103 January 21, 1999 Table A7. (Continued) Observed Expected Cause of Death Deaths Deaths SMR Cancer of Brain and Other 1 0.5 222.6 Central Nervous System Cancer of Thyroid and Other 0 0.0 -- Endocrine Glands Cancer of Bone 0 0.0 -- Cancer of All Lymphatic and 2 1.2 174.0 Hematopoietic Tissue Lymphoma Hodgkin's Disease 0 0.2 0 0.2 -- -- Leukemia and Aleukemia 1 0.4 226.3 Cancer of Other Lymphatic 1 0.4 249.5 and Hematopoietic Tissue Malignant Melanoma of Skin All Other Malignant Neoplasms Diabetes Mellitus Cerebrovascular Disease 0 0.3 0 0.8 0 0.6 0 1.3 -- -- -- -- All Heart Disease Hypertension Respiratory Ulcer of Stomach and Duodenum 7 13.1 0 0.1 0 1.9 0 0.1 53.4 -- -- -- Cirrhosis of Liver Nephritis and Nephrosis 1 1.6 0 0.2 62.3 -- External Causes 10 10.5 95.0 Accidents 9 6.5 139.4 Motor Vehicle Accidents 5 3.6 138.0 All Other Accidents 4 2.9 140.2 Suicides Homicides and Other External 1 2.5 0 1.5 40.0 -- Causes Residual Causes* 4.7 63.4 Unknown Causes & 2 *all other causes of death combined & no death certificate obtained; included only in all causes of death category 95% Cl (5.6, 1,240.3) (0.0, 9,544.3) (0.0, 8,697.5) (21.1,628.7) (0.0, 2,466.1) (0.0, 2,346.1) (5.7, 1,260.7) (6.2, 1,390.1) (0.0, 1,200.4) (0.0, 455.3) (0.0, 589.0) (0.0, 274.4) (21.5, 110.1) (0.0, 5,834.4) (0.0, 195.3) (0.0,3,083.3) (1.6, 347.3) (0.0, 2,346.9) (45.6, 174.7) (63.7, 264.5) (44.8, 322.0) (38.2,359.0) (1.0, 223.1) (0.0, 247.4) (13.1, 185.3) 000117 104 January 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. Secondary Objectives - To determine chronic and subchronic toxicity of N-EtFOSE after 52 and 13 weeks, respectively, of compound administration. To determine compound's 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 at 4, 13 & 52 weeks. (Note the 100 ppm recovery group has 40 males and 40 females). Test Article: N-EtPFOS furnished by Sponsor who is responsible for compound identity and purity. 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. 105 000118 January 21, 1999 Interim Necropsies: Five rats/sex from 0, 1, 3, 30, 100 & 300 ppm groups at 4 and 14 weeks for PCNA, palmitoyl CoA oxidase, and compound level samples. (Note samples for organ weights and histopathology taken at 14 weeks.) Ten rats/sex from 0, 1, 3, 30 &100 ppm groups at 52 weeks for compound levels, organ weight, histopathology samples. Organ Weights: Measured at 14 and 52 week interim necropsies. Histopathology: Microscopic examination of selected tissues at 14 and 52 week animals. Complete tissue examination of 0 (one control group), 3, 30, and 100 ppm 104 weeks animals. Selected tissue examination of 100 ppm recovery animals. Histopathologic results will indicate extend of tissue examination of 1 ppm and second control group. 000119 106 January 21, 1999 Rat Oral (diet) Carcinogenicity on PFOS Study Outline Study Objectives: Main Objective - To determine the carcinogenicity of PFOS upon chronic oral administration. Secondary Objectives - To determine chronic and subchronic toxicity of after 52 and 13 weeks, respectively, of compound administration. To determine compound's 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: Six - 0, 0.5, 2, 5, & 20 ppm in diet plus a 20 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 at 4, 13 & 52 weeks. (Note the 20 ppm recovery group has 40 males and 40 females). Test Article: PFOS furnished by Sponsor who is responsible for compound identity and purity. 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, 0.5, 2, 5, & 20 ppm groups at weeks 14, 52 and 104. Urinalysis: Ten rats/sex from 0, 0.5, 2, 5, & 20 ppm groups at weeks 14, 52 and 104. Interim Necropsies: Five rats/sex from 0, 0.5, 2, 5, & 20 ppm groups at 4 and 14 weeks for PCNA, palmitoyl CoA oxidase, and compound level samples. (Note samples for 107 000.120 January 21, 1999 organs weights and histopathology taken at 14 weeks.) Ten rats/sex from 0, 0.5, 2, 5, & 20 ppm groups at 52 weeks for compound levels, organ weight, histopathology samples. Organ Weights: Measured at 14 and 52 week interim necropsies. Histopathology: Microscopic examination of selected tissues at 14 and 52 week animals. Complete tissue examination of 0, 2, 5, and 20 ppm 104 weeks animals. Selected tissue examination of 20 ppm recovery animals. Histopathologic results will indicate extend of tissue examination of 0.5 ppm group. 000121 108 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 GLP principles but since it is a rangefinder study QA audits will not be done 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 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 6 thru 17 of gestation. Analysis of 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 4 to 6 hours later. Body Weights: On days 0, 4, 6, 8, 10, 12, 14, 16, 18 and 20 of gestation Food Consumption: On days when body weights are obtained Cesarean Section: On day 20 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 number of early and late resorptions will be determined and recorded. The fetuses will be sexed, weighed and examined for any gross abnormalities. The ovaries will examined for the number of corpora lutea. 109 OO\ January 21, 1999 Rat Teratology Study on N-EtFOSE Study Outline Study Objective: To determine maternal and fetal toxicity and teratogenic potential of orally administered N-EtFOSE in pregnant rats GLP Status: A GLP study with appropriate QA audits; a signed QUA statement to be included in the final report. Animals: Mated female Charles River CD rats Dose Groups: Five - control, 0 mg/kg; low, (1 or 1.5 ?) mg/kg; mid-1, (5 ?) mg/kg; mid-2, (10 or 15 ?) mg/kg; high, (20 or 25 ?) mg/kg. Final dose levels will be adjusted after range finder study results are available. Number per Group: 25 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 intubation of compound suspened in water with 2% Tween 80 on days 6 thru 17 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 4 to 6 hours later. Body Weights: On days 0, 4, 6, 8, 10, 12, 14, 16, 18 and 20 of gestation Food Consumption: On days when body weights are obtained Cesarean Section: On day 20 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 number of early and late resorptions will be determined and recorded. The fetuses will be sexed, weighed and examined for any gross abnormalities. The ovaries will examined for the number of corpora lutea. Fetal Examinations: Approximately one-half of the fetuses from each litter will processed for visceral examination by the Wilson Technique for soft tissue development. The remaining fetuses will be eviscerated and processed for skeletal examination using Alizarin Red S staining method. 000123 110 January 21, 1999 Toxicokinetic Satellite Animals: Groups of mated females, five females at the low dose (1 or 1.5 mg/kg) and five females at the high dose (20 or 25 mg/kg) plus three females at other dose levels, will be dosed. On the day after the last dose (day 18 of gestation) 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 together as one sample; likewise for the fetuses) These samples will be frozen and shipped to the sponsor for possible analysis. ill 000124 January 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 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: Seven - control, 0 mg/kg; low-1, 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-EtFOSE 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 20 of gestation. Analysis of 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 4 to 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 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 be examined for the number of corpora lutea. 112 0 0 0 1 2 5 January 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-EtFOSE 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-EtFOSE will be furnished by the Sponsor who is responsible for compound identity and purity. Reserve sample to be retained. Compound Administration: Oral intubation of 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 4 to 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 implantations, 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 number of corpora lutea. 113 000126 January 21, 1999 Fetal Examinations: A mid-coronal slice will be made in the head of each fetus to evaluate the contents of the cranium. The internal organs of the thoracic and abdominal cavities of all fetuses will be examined in the fresh state using Staples' technique for internal abnormalities. At this time the sex of the each fetus will be determined. After removal of the viscera, the carcasses will processed for skeletal examination. Toxicokinetic Satellite Animals: Groups of mated females, five females at the low dose (1 ? 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 21 of gestation) 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 together as one sample; likewise for the fetuses) These samples will be frozen and shipped to the sponsor for possible analysis. 114 0001^7 January 21, 1999 Rat Two Generation Reproduction Study of N-EtFOSE Study Outline Study Objective: To evaluate the effect of oral administration of N-EtFOSE on the reproductive function of male and female rats (Fo generation) and on the development and reproductive capacity of the subsequent Fi generation which were exposed in utero and via lactation. 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: Five - control, 0 mg/kg; low, 1 mg/kg; mid-1, 5 mg/kg; mid-2, 10 mg/kg; high, 15 mg/kg. Number per Group: 35 males and 35 females per group Test Article: N-EtFOSE will be furnished by the Sponsor who is responsible for compound identity and purity. Reserve sample to be retained. Compound Administration: Daily oral intubation of compound suspended in water with 2% Tween 80. Compound administration will start 4 week prior to mating. Compound administration will continue through mating and in the females through gestation and lactation. The Fi pups will not receive any doses via gastric intubation. Samples of 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 4 to 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: After 4 weeks of compound administration, within a dose group one male will be co-inhabited with one female. A female will be determined as mated upon the 000128 115 January 21, 1999 presence of sperm positive vaginal smear. The day of mating shall be considered as day 0 of gestation. Early Gestation Evaluation: 10 Fo females per group will killed on day 10 of gestation. Pregnancy will be determined as well as the number of implanations and the number of early and late resorptions. 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. Male Sacrifice: One week after mating all Fo male rats will be killed after a terminal body weight has been obtained. The animals will be necropsied 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. (Note: testis will be fixed in Bouin's solution; other tissues in 10% buffered formalin). At necropsy serum and liver samples will be taken from five of the 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 litters. The offspring (Fi 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 litters of 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 of eye opening, pinna detachment, surface righting, testes descent, and vaginal opening will be recorded for each litter. Fi Dosing: After weaning at day 21 of lactation, the Fi pups will receive compound by daily oral (gastric intubation) dosing. Dose level on a mg/kg basis will be the same as the pup's dam dose level. Female Sacrifice: One or two days after weaning of the litter on day 21 of lactation, all Fo female rats will be killed after a 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 0 0 0 1 2 9 January 21, 1999 FI Pup Neurological Testing: During the 4th week postpartum, the Fi pups will be evaluated in a passive avoidance test for learning and short term memory retention. During the 10th week postpartum, the Fi pups will be evaluated in a water-filled maze for neuromuscular coordination, learning, and longer term more complex memory. Fi Growth and Reproduction: The Fi pups will be weaned after 21 days of lactation and litters will be culled to one of each sex. The pups will then allow to grow and body weights and food consumption will be recorded weekly. The Fi 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 F2 pup through 21 days of lactation. 117 000130 January 21, 1999 Rat Two Generation Reproduction Study of PFOS Study Outline Study Objective: To evaluate the effect of oral administration of PFOS on the reproductive function of male and female rats (Fo generation) and on the development and reproductive capacity of the subsequent Fi generation which were exposed in utero and via lactation. 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: Five - control, 0 mg/kg; low, 0.1 or 0.2 mg/kg; mid-1, 0.5 or 1.0 mg/kg; mid-2, 2 or 3 mg/kg; high, 5 or 8 mg/kg. Number per Group: 35 males and 35 females per group Test Article: PFOS will be furnished by the Sponsor who is responsible for compound identity and purity. Reserve sample to be retained. Compound Administration: Daily oral intubation of compound suspended in water with 2% Tween 80. Compound administration will start 4 week prior to mating. Compound administration will continue through mating and in the females through gestation and lactation. The Fi pups will not receive any doses via gastric intubation. Samples of 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 4 to 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: After 4 weeks of compound administration, within a dose group one male will be co-inhabited with one female. A female will be determined as mated upon the 118 000131 January 21, 1999 presence of sperm positive vaginal smear. The day of mating shall be considered as day 0 of gestation. Early Gestation Evaluation: 10 females per group will killed on day 7 of gestation. Pregnancy will be determined as well as the number of implanations and the number of early and late resorptions. Male Sacrifice: One week after mating all Fo male rats will be killed after a terminal body weight has been obtained. The animals will be necropsied 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. (Note: testis will be fixed in Bouin's solution; other tissues in 10% buffered formalin). At necropsy serum and liver samples will be taken from five of the 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 litters. The offspring (Fi 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 litters of 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 of eye opening, pinna detachment, surface righting, testes descent, and vaginal opening will be recorded for each litter. Fi Dosing: After weaning at day 21 of lactation, the Fj pups will receive compound by daily oral (gastric intubation) dosing. Dose level on a mg/kg basis will be the same as the pup's dam dose level. Female Sacrifice: One or two days after weaning of the litter on day 21 of lactation, all Fo female rats will be killed after a 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. 119 000132 January 21, 1999 FI Pup Neurological Testing: During the 4th week postpartum, the Fi pups will be evaluated in a passive avoidance test for learning and short term memory retention. During the 10th week postpartum, the Fi pups will be evaluated in a water-filled maze for neuromuscular coordination, learning, and longer term more complex memory. Fi Growth and Reproduction: The Fj pups will be weaned after 21 days of lactation. The pups will then allow to grow and body weights and food consumption will be recorded weekly. The Fj 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 F2 pup through 21 days of lactation. 120 000133 January 21, 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 laboratory not at 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 intubation of compound suspended in water with 2% Tween 80 on days 7 thru 20 of gestation. Analysis of 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 4 to 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 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. 121 0 0 0 1 3 4 January 21, 1999 Rabbit Teratology Study on PFOS Study Outline Study Objective: To determine maternal and fetal toxicity and teratogenic potential of orally administered PFOS 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, 0.1 mg/kg; mid-1, 1 mg/kg; mid-2, 5 mg/kg; high, 10 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: FOS will be furnished by the Sponsor who is responsible for compound identity and purity. Reserve sample to be retained. Compound Administration: Oral intubation of 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 4 to 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 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. 122 January 21, 1999 Fetal Examinations: A mid-coronal slice will be made in the head of each fetus to evaluate the contents of the cranium. The internal organs of the thoracic and abdominal cavities of all fetuses will be examined in the fresh state using Staples' technique for internal abnormalities. At this time the sex of the each fetus will be determined. After removal of the viscera, the carcasses will processed for skeletal examination. Toxicokinetic Satellite Animals: Groups of mated females, five females at the low dose (0.1 mg/kg) and five females at the high dose 10 mg/kg) plus three females at other dose levels, will be dosed. On the day after the last dose (day 21 of gestation) 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 together as one sample; likewise for the fetuses) These samples will be frozen and shipped to the sponsor for possible analysis. 123 0 0 0 1 3 6 January 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 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: Seven - control, 0 mg/kg; low-1, 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 20 of gestation. Analysis of 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 4 to 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 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. 124 January 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 intubation of 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 4 to 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 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. 125 000138 January 21, 1999 Fetal Examinations: A mid-coronal slice will be made in the head of each fetus to evaluate the contents of the cranium. The internal organs of the thoracic and abdominal cavities of all fetuses will be examined in the fresh state using Staples' technique for internal abnormalities. At this time the sex of the each fetus will be determined. After removal of the viscera, the carcasses will processed for skeletal examination. Toxicokinetic Satellite Animals: Groups of mated females, five females at the low dose (1 ? 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 21 of gestation) 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 together as one sample; likewise for the fetuses) These samples will be frozen and shipped to the sponsor for possible analysis. 126 0001-39 January 21, 1999 Corporate Toxicology Study Outline Title: 1) Effect of Perfluorochemicals on Bioenergetic Metabolism: Phase II Research Plan (Restricted Grant) 2) Mitochondrial Interactions of Peroxisome Proliferators (Unrestricted Grant) Purpose: 1) Compare mitochondrial bioenergetics between mitochondria from rat, guinea pig and human (Ca++ 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, markers of 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 mechanism of toxicity; b) Identify potentially useful metabolic biomarkers of exposure; c) 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 indicative of cell and peroxisome proliferation) to: a) Identify molecular biomarkers of exposure; b) Facilitate species extrapolation; c) Discriminate between proliferation of peroxisomes and cell proliferation (ongogenic response) to test dogma; d) Assess the relevance of rat tumorigenic response to potential human cancer response; e) Provide biomarkers for potential use in monitoring and interpreting traditional toxicity studies. Significance: 1) Comparing sensitivity between species will allow forjudging the most appropriate species for predicting human health outcomes following exposures to compounds and has relevance to establishing safety factors and putting human cancer risk in perspective; 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 127 000140 January 21, 1999 relationship between metabolic and oncogenic effects of these compounds in different species. Objective(s): 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 effects of test compounds on hepatocyte bioenergetics and cell proliferation in culture. Protocol: 1) Establish stable primary hepatocyte cultures from several species including rat, guinea pig and primate; 2) Develop molecular probes for compound-initiated transcription of specific genes related to mechanistic endpoints: a) ID genes of interest b) ID sequences of high homology between species using Gene Bank c) Design primers to 1.5 - 3.0 kbp sequences (Oligo) d) PCR amplify sequences from rat-liver cDNA template e) Separate, bands on low melting point gels f) Purify bands (band-stab) g) Restriction digest to confirm identity (MacDNAsis to obtain restriction site map for each probe) h) Label probe with 32P-dCTP by random priming (High Prime) 3) Treat sample (cells or flash frozen tissue) a) Isolate total RNA by triazole method b) Separate on denaturing HCOH agarose gels c) Transfer to nylon d) Hybridize with 32P probe e) Wash and expose to X-ray film to detect complementary mRNA 4) MRNA probes suggested for peroxisome proliferation a) CPT1 b) ACoAO c) PPARa d) FABP e) 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 of exposing cells in culture on induction of peroxisomal metabolism and stimulation of cell proliferation using biochemical markers and molecular markers (e.g., mitochondroial enzyme activities and cytochrome content, Adenine 128 0 0 0 1 4 1 January 21, 1999 nucleotides, AcoAO activity, PCNA, apoptosis (morphometric and TUNEL), CyQuant cell proliferation analysis, CPT1, AcoAO, PCNA, CDK) Timeline: 31 months beginning December 1, 1998 and ending June 30, 2001 Report: Quarterly progress reports 129 0001^2