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# Perfluorooctane Sulfonate: Current Summaryof

# Human Sera, Health and Toxicology Data

3M January 

# 21,1999

I 1 

## 000014January2 1. 1999



### TABLE OF CONTENTS

EXECUTIVE SUMMARY

## ........................................................................................................................... 3 I

### . INTRODUCTION

## .................................................................................................................................... 6 I1

## . HISTORICALREVIEWAND CURRENT FINDINGS OFFLUOROCHEMICALS IN HUMAN SERA

## ....................................................................................... .................................................................... 11 111

### . SUMMARY OF MEDICAL SURVEILLANCE AND EPIDEMIOLOGY STUDIES

## .................. 22 IV

## . SUMMARY OF TOXICOLOGYDATA ON PFOS 

## ........................................................................ 30 V

## . RISK CHARACTERIZATION 

## ............................................................................................................ 64 VI

## . CURRENTAND PLANNED RESEARCH

## ....................................................................................... 69 VI1

## . REFERENCES

## ................................................................................................................................... 81 APPENDIX

## .................................................................................................................. 86

2January 2 1,1999

Executive Summary


### 3M has prepared this documentto summarize the data related to the biological effects of perfluorooctane sulfonate 

### (PFOS). It also presents current thinking 

### onhuman health risk related to PFOS and includes information about future study plans. 

### 3M Medical Department scientistsand physicians, in consultation with outside experts, are the authors.


### PFOS has been found at tens ofparts per billion levels in serum samples of nonoccupationally exposed employees, in commercially available human serum and in pooled samplesfrom 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 fluorinemolecules for over 30 years. The primary constituent ofthis organic fluorinefraction was tentatively identified as another molecule (perfluorooctanoate) in 1976. Current analysis of stored sera samples fiom a variety of sources aremore consistent with 

### PFOS being a major fraction of this organic fluorine. Improved analytic techniques allowing arelativelyrapid 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 analysisof serum from those without occupational exposure.
Medical surveillancehas been done among 

### 3M employees occupationally exposed to 

### PFOS precursors for over 20 years. 

### To date, no adversehealth effect associatedwith 

### PFOS exposure has been found in these employees. This conclusion applies at sew levels up to 6 parts per million, about 100 times higher than levels seenin the general

3January 2 1, 1999 population. PFOS has a long residence time in the humanbody. In three retirees, the half-life in human sera ranges from 

### 1100to 

### 1500days. A mortality study at the 

### U.S. plant primarily involved with production ofPFOS related materials has found no significantly elevated standardized mortality ratios (SMRs). Toxicology studies showthat 

### PFOS is well absorbed orally and distributes primarily in the serum and liver. It does not appear to be further metabolized. Someenterohepatic 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 compoundrelated effects occurred in these early rhesus monkey studies are unknown. In a recently completed rangefinder study in cynomolgus monkeysthe first observed biological effect was a decrease in serum cholesterol, first observed at a serum level of 72ppm in one ofthe two monkeysin 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 

### 700to 800ppm in the early rhesus monkey studies, and death at 

### 1100ppm and above. More complete quantitative absorption, distribution and excretion data for 

### PFOS is being obtained.

4January2 1, 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. Thepurpose ofthis 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. Theplan is to make as muchuse aspossible of observational data in exposed workers and to establish no effect levels in both rats and primates for endpoints of importance.

5January 2 1, 1999 

### I. INTRODUCTION Evidence that organic compoundscontaining 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 ofthe specific compoundscontributing 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 ofparts-per-billion (ppb) levels in serum samples from the general population, averaging 30ppb in blood bank samples from diverse locations in the U.S. Single digit parts per million (ppm) levels (approximately 

### 100times greater) are found in individuals occupationally exposed to PFOS and its precursors, averaging2.0 ppm among participating employees at the primary 

### U.S. manufacturing location for these compounds.


### 3Mproduces 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: I CF3(CF2)6 

### C - SO2 -F I 

### F POSF

6January 21, 1999 Other moieties 

### (“R”) are added to the sulfur, which leads to the creation ofmaterials that may be polymerized or esterified. The vast majority of

### POSF produced isused in this way. F I CF-j(CF& C 

### - SO2 

### -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 hrthermetabolized. SomePFOS is produced and sold directly into industrial applications as a surfactant. This, however, amounts to only a small fraction of total POSFproduction. 

### F I I F CF3(CF2)6 

### C -SO3-

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 

### I.1shows the chemical structure ofN-Et-FOSE and metabolites that have been found in rat serum. All except compound

### VI11 have been verified to metabolize further to 

### PFOS. (Missing Roman numerals represent hypothesized intermediatesnot shown in this figure.)

7 

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# ooosa1In vitro studiesin both rat and human hepatocytes lead to identification of the same compoundsand hypothesized intermediates. Compound 

### VI11 has been detected in some samples ofpooled human sera. A single dose absorption, distribution, metabolism and excretion study ofN-Et-FOSE in cynomolgus monkeys 

### is throughthe in lifephase and tissue analysis is pending. N-Et-FOSE is esterified to produce larger molecules that areused onpaper and packaging for oil and water repellency.
It is presumed thatN-Me-FOSE, in which a methyl group replaces the ethyl group on the nitrogen, has a similar metabolism. N-Me-FOSE becomes part ofvery large molecules that act as protective chemicals on fabrics, leather and rugs.
Outside of the occupational setting, routes 

### ofhuman exposure to PFOS or its metabolic precursors are 

### not understood,but are 

### the subject 

### ofintense study. Exposure could occur from environmental releases 

### ofPFOS or its precursors at the Decatur, Alabama and Antwerp, Belgium manufacturing sites. It could occur 

### from the environmental or biological degradationofproducts to PFOS or molecules metabolized to PFOS. Products also contain small amounts (generally less than a few percent) ofresiduals, such as N-Et-FOSEand other molecules found in Figure 

### I. 1

### , which are known or suspected metabolic precursors to PFOS. These residuals represent a source ofPFOS that would not require environmental or biological degradation of large molecules. Downstream industrial users ofPOSFbased products are also potential sources of environmental releases of

### PFOS or its precursors. The relative contributionofthese various sourcesto population exposure

### is currently unknown.

Another surfactantis known to be found in the sera of employees and was reported in general population sera samples in 

### 1976(Taves). This is perfluorooctanoicacid, or PFOA:

9January 2 1, 1999

PFOA is also madeby electrochemical fluorination. It is mentioned here because following Taves' report in 

### 1976it was presumed to be a significant fraction ofthe 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 likelythat PFOA was misidentified as amajor fraction of organic fluorine inthe 1976Taves paper. The evidence for this is discussed in Section 11.

Thepurpose ofthis report isto describethe data 

### on PFOS levels inhuman 

### 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 ispresented in Section 11. This is followed in Section I11 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 ofthe serum findings in light ofthe available health effects data.


### 3M is actively developing further human health and toxicological information. Section VI outlines the currentresearch agenda.

10 

## 080023January 2 1, 1999



### 11. HISTORICAL REVIEWAND CURRENT FINDINGS

### OF FLUOROCHEMICALSIN 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 

### ofthis 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'sand 1970's were time intensive, requiring hours for a single analysis. Themethods 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

### ofproduction employees at higher detection limits (about 0.5 parts per million organic fluorine) that were adequate for the levels found in occupationally exposed individuals. The advancement of chromatographic/mass spectroscopy technology enabled rapid analysis of specific fluorochemicalsfrom 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 

### 50parts per billion by 1997. The first report to 

### 3M of

### PFOS in commercially availablepooled sera occurred in late summer of 1997,prompting more research into the technique, and confirmingits validity over a period 

### of several months.

11

I 

## 000024January 2 1, 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% ofthe 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 ofnon-occupationally exposed 

### 3M employees, 

### 4) data from commercially availablehuman pooled serum samples, 

### 5) data from pooled sera from 18regional blood banks and 6)data from current analysis of stored serum samples. Historical Finding 

### of the Organic Form ofFluorine in 

### Blood Taves (1 96Sa) described two forms of fluorine in serum, one that was exchangeable with radioactive fluorine-18 and one that wasnot. Pothapragada et. al. 

### (1971) also described two 

### forms, ionic and nonionic. Taves (1968b) showed that the nonexchangeable fluorine was bound to albumin. This finding, along with results of extraction and precipitation and the need for ashing to release this form of fluorine, led to the conclusion that the non-exchangeableor 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 asperfluorooctanoic acid (PFOA). There was somevariation in the observed spectra from an authentic sample ofPFOA, 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 11.1 presents the study author, level measured, population studied and methods of analysis. The variety ofmethods used for determination of

12 

## 00002sJanuary 2 1, 1999 fluorine suggeststhat some caution be used in interpreting results. All reported means were in the tens of part per billion levels. The average ofreported values from United States sources is 37.6 ppb.
Table 11.1. Historical Findings of Serum Organic Fluorine Levels
Year Author OF* (Dpb) N Method** Source 1972 GUY 30 65 ash 

### us 1975 Venkateswarlu 36 2 0bomb 

### us 1976 Guy, Taves 25 106 ash 

### us 1978 Belisle 20 9 0bomb 

## us 1979 Singer 45 264 ash 

## us 1980 Ubel 45 4 modObomb US 1980 1981 Belisle 11 8 0bomb China 1989 Yamamota 32 11 LOPA Japan Paez 85 pooled ash Argentina


### * Organic fluorine, specific identities not provided. 

### **Varied methods were usedto measure organic fluorine. See papers for details. Occupationally Exposed Employees 

### 3M has produced 

### PFOA (the ammoniumsalt)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. Employeemonitoring 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 10parts per million (ppm).
As noted earlier, 

### PFOS is a surfactantused 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’sby electrochemical fluorination. Since 1980 this has occurred at primarily one location inthe 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 

## 0006926January 2 1, 1999

as measurement of serum levels of OF. measurements, OF levels averaged 2.9 to 6.5 ppm from 1981 to 1992' (Figure 11.1). With the introduction 

### ofhigh performance liquid chromatography-mass spectrometry, serum 

### PFOS was measured in 1994and 1997 (see also Figure 11.1). In these two years, mean PFOS levels were 2.44 ppm (range 0.25 - 12.83ppm) and 1.96ppm (range 0.10 -9.93 ppm), respectively. Among employees with six or more

Another 3Mplant in the United States where PFOS has been measured in employees' serum is Cottage Grove, Minnesota. SomePFOS is manufactured at this plant. In 1997,the mean serum level of

### PFOS among 74 Cottage Grove fluorochemical production employeeswas 

### 0.82 ppm (range 

### 0.05 

### - 6.25 ppm). Outside the United States, 3Mmanufactures PFOSrelated materials at its Antwerp, Belgiumplant. PFOS levels were measured in Antwerp employees in 1995(mean = 1.9ppm,range 0.0 

### - 9.9 ppm) and in 1997 (mean 

### 1.5ppm, range 0.1 

### - 4.8) ppm.
The cross-sectional stratified analysis presented in section 111, 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 facilitywas not includedbecause little PFOSrelated product is manufactured there. It is also the primary site for PFOAproduction.

We are aware of one occasion in 1979where the serum of 5 Decatur employees was measured for PFOS by electron capturegas chromatograph and microwave plasma detection methods [CentralAnalytical Laboratory, 19791. Total serum organic fluorine levelsfor these five employees were 10.1,5.7, 9.4, 11.8and 4.1 ppm. The percentof PFOS found was 60%, 70%, 80%,55% and 65% of the total serum organic fluorine levels, respectively. 1

14 

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## 000828Non-Occupationallv Exposed 

### 3M Employees A total of 

### 31 3M employees were tested for PFOS in their serum in 1998 (Table 11.2). All were corporate staff or division managers. None had worked in fluorochemical production or in fluorochemicalresearch and development. Samples were from five females and 

### 26 males. Employees ranged in age from 37to 62 years. All employees had measurable 

### PFOS in their blood serum (mean =47 ppb; range =28 to 96 ppb). Age was significantly associated with increased serum PFOS and accounted for 

### 24% ofthe variance in PFOS levels. There was no gender-related difference if age was considered. Only four employees had PFOAmeasured above the detection limit of 10 ppb. The average of these four PFOAmeasurements was 12.5ppb. 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 

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### 2 2 0 03 d c-'CommercialPooled SerumSamples 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 anunknown number of donors. Thepools from which the samples were drawn were 50 liters, suggesting a minimum of 200 donors. Thethree Sigmapools 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 samplesthat had from 

### 5 to 10donors per pool (Table 11.3). Altogether there were 68 total pools, representing 340 to 680 individual donors. All pools contained detectable levels 

### ofPFOS 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 14ppb in Santa Barbara, California to 

### 52ppb 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).
Theblood 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 

### ofmean PFOS sera levels. The overall mean was 29.7 ppb 

### ofPFOS. If one wishes to make a comparison with the organic fluorine levels in the published literature, this level of

### PFOS would contribute 19.3ppb to the total serum organic fluorine level (0.65 x 29.7). 

### An approximate mean 

### ofhistoric organic fluorine measurements from published literature is 37.6 ppb. Ifthis were all PFOS, a 

### PFOS sera level would have been 57.9 ppb (1.54 x 37.6).

18

bJanuary 2 1, 1999



### city Anchorage, AK Billings, 

### MT Cheyenne, 

### WY Corpus Christi, 

### TX Davenport, IA East Orange, CA Grand Rapids, 

### MI Greenville, SC Kansas 

### City, MO Lafayette, LA Las Vegas, NV Meridian, 

### MS Minneapolis, 

### MN Newark, 

### DE Omaha,NE SantaBarbara, CA Santa 

### Rosa, CA Scottsdale, AZ # 

### of Pooled Samples Range Average 4 19-31 24 3 18-26 23 3 13-35 23 3 26-32 29 3 28-40 32 3 20-27 24 4 18-31 26 3 47-56 52 3 24-35 30 3 39-50 46 3 25-28 23 3 27-56 39 6 41-54 46 5 2 1-32 24 5 9-27 17 3 13-16 14 3 23-26 24 

### 3 28-47 37

Historical Samples Nine sets 

### ofhistorical samples have been analyzed for 

### PFOS in 1998. These are summarized in chronological order below.
Korean 

### Warera 

### US.military recruits, 1948 to 

### 1951. Tenpooled samples consisting of ten individual samples each were measured. All were below 1ppb, the limits 

### of detection. Swedish samples, 1957. Ten individual samplesranged from below limit of detection to 4.1 ppb. Two were below detection limit (1 ppb). The mean was 1.98ppb. Michigan Breast CancerStudy, 1969 

### - 1971. Five individual samples ranged from 11.8 to 59.4 ppb. The mean was 33.4 ppb.

19 

## 000032January 2 1, 1999 Swedish samples, 1971. Ten individual samplesranged from below detection limit (1 ppb) to 2.8 ppb. Three were below detection limit. The mean was 1.13 ppb. MRFITpooled calibrationsamples, 1976. MRFIT (Multiple Risk Factor Intervention Trial) was a cardiovascularrisk reductionprogram conducted in the 1970’sand early 

### 1980’s. The number of donors per pool is unknown. Six pooled calibration samples ranged from 13.7to 

### 55.5 ppb. The mean was 30.9 ppb. MRFITpooled calibrationsamples, 1980. Threepooled calibration samplesranged from 

### 13.8to 

### 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 anNCI study 

### on nutrition and cancer prevention.
MRFIT individual samples, 1985. Three individual samples from participants in the MRFIT study were obtained. One wasbelow limits 

### of quantitation (5ppb), the other two were 43.3 and 43.9. Assumingthe LOQ for the low sample,the meanwas 

### 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 anNCI 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 10ppb.

Comment

20January 2 1, 1999 Organic fluorine has been noted in human serum since the late 1960's. We have now identified 

### PFOS as a part 

### ofthis organic fluorine fraction. PFOS-related materials were not produced commercially prior to 1948,and only in small quantities for several years thereafter. Therefore, it is not surprisingthat samples from 1948to 195

### 1 show undetectable levels. There was clearly an increase 20 years later; however the very limited data shows no furtherupward trend despite steadily increasing production volumes since this time.' Due to limited knowledge on distribution 

### ofthis chemical inthe body it isuncertain that body burden 

### is adequately reflected by serum levels. Information on distribution and kinetics is needed to shed further light. The available data onthis topic will be presented in the toxicology section. It would also be ofinterest to measure total organic fluorine and 

### PFOS in the same sample to determinehow much 

### PFOS currently contributes to the total organic fluorine content.


### PFOA mayhave 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 

## 000834111. SUMMARY 

### OF MEDICAL SURVEILLANCEAND 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 chromatographymass spectrometry methods. These relationships were assessed in fluorochemical production employees from two time periods, 1995 (N = 178)and 1997 (N = 149). the mean serum PFOS levels were 1.93 and 2.44ppm, respectively. In 1997,the mean serum 

### PFOS levels were 1.48and 1.96ppm,respectively. In 1995, for Antwerp and Decatur,

Descriptive simple and stratified analyses, Pearson correlation coefficients, analysis ofvariance and multivariable regression were used to evaluate for possible associations between PFOS and each hematological and clinical chemistry test and hormonal assay. Age, body mass index, current alcohol consumption (drinks per day) and cigarette use (cigarettes smokedper 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 

### - < 1ppm; 1

### - <

### 3 ppm; 3 

### - <6ppm; and 

### 2 6 ppm (Table 111.1). (Note: other PFOS categorizations were used with comparable findings). In 1995, mean serum PFOS levels by category were 0.49 ppm, 1.82ppm, 4.12 ppm and 8.17 ppm, 

## * Hematocrit, hemoglobin, red blood cells, white blood cells and platelet count, alkaline phosphatase, gammaglutamyl 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

22January 2 1, 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 ofthe employees’ serum PFOS levels were below 

### 6ppm. 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 111.2). When analyzed in aggregate, the two plant populations showedno consistent significant associations for both years between the clinical chemistries and hematology parameters and the employees’ serum PFOS levels (Figure 

### A1 

### ,Appendix). Total bilirubin levels appeared to trend downwardsbut upon further analysisthis 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,hormonevalues were also obtained from a subsample of employeeswith 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 abnormalitieswere not associatedwith serum PFOS levels up to 

### 6 ppm. It was not possible to derive inferences from the few employees with serum PFOS levels 

### 36 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.

23 

## 000036n a 

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000037Table 111.2 Mean Values 

### ofPFOS, Demographic, SerumChemistry and Hematologic Parameters for Antwerp and Decatur, 1995 and 1997 Examinations 1995Data 1997Data Variable Antwem Decatur Antwerp Decatur 

### PFOS (PP@ Age 

### BMI Cigarettes Alcohol Alk Phosphatase GGT AST ALT Total bilirubin Direct bilirubin 

### BUN Creatinine Glucose Cholesterol LDL HDL Triglycerides 1.93 37*** 23.9"

## ** 4.7" 1.3*** 75*** 41 26" 44 0.86*** 0.22 17.0*** 0.9*** 

### 81*** 2 14 138 54*** 115*** 2.44 45 29.2 7.9 0.2 97 48 29 47 

### 0.58 0.21 14.8 

### 1.1 92 218 136 43 187 1.48 33*** 23.5*** 

### 5.5 1.1""" 70*

## * * 26" 27 31 0.80*** 0.15*** 14.9 0.9** 81*** 206 134 50""" 111*** 1.96 44 30.0 6.6 0.1 87 36 26 34 0.58 0.12 14.1 1

## .o 98 215 137 42 192

25 

## 000038January 2 1, 1999 Table 111.2 (Continued) 1995 Data 1997 Data Antwerp Decatur Antwerp Decatur Variable Hematocrit 47"

## * * 46 46* 45 Hemoglobin 15.4 15.2 15.4 15.3 FU3C 4.9 5.0 5.1 5.1 MCH 31.4""" 30.7 30.5 30.4 MCHC 32.9*

## * * 33.4 33.3

## **

## * 33.7 MVC 95.7*** 91.8 91.6" 90.0 WBC 6.4*** 7.5 6.5 6.4 217 Platelets 224 229 237" 

## * p <.05; 

## **p < 

### .01; ***p < .001 (Antwerp compared to Decatur)

26 

## 000039Mortalitv Study ofEmplovees at the 

### 3M Plant in Decatur, Alabama
A retrospective cohort mortality study of 1,957employees who worked at least one year at the 3Mplant in Decatur, Alabamawas conducted by epidemiologists at the University of Minnesota School ofPublic Health (Mandel and Johnson, 1995). The purpose ofthis study was to determine whether the mortality experience ofthese employees was significantly different from that which would be expected in a comparable population. The cohort was followed from March 

### 1

### , 196

### 1through December 3 

### 1

### ,1991. Table A2 (Appendix) provides a description ofthe final employmentand vital status for the male (n = 1,639)and female (n = 3

### 18) employees. These totals include both the Decatur chemical and film plant employees. Thetwo plants arephysically 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 ofthe male employees were hired between 1965 and 1974 (Table A4, Appendix) whereas the majority of the female employeeswere hired between 1975 and 1979 (Table A5, Appendix). Altogether there were 74 deaths (70 males and 4 females). Among males, SMRswere below the null value for all major causes of death regardless of the comparison population used to calculate the expected values (Table 111.3). 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 

### < .OS) elevated SMRs. There were no analyses conductedby duration of employment within the chemical plant. 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 amongfemale employees (SMR = 62.6; 95% 

### CI 17.1 - 160.4). Of these four deaths, three were from external causes (SMR =213.4; 05% 

### CI 44.0 

### - 623.6). By examining the death certificates for the specific causes of death and The data were also restricted to employees who were

27 

## 000040January 2 1, 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 ofDecatur plant employeesbe updated in 1998 which would extend the vital status search, via National Death Index records, through 1996.
In addition to the Decatur mortality study, the University ofMinnesota investigators have also followed the mortality experience of a cohort of employees associated with fluorochemical production atthe 3M Cottage Grove (Gilliland and Mandel, 1993). Unlike the Decatur plant, the greatest potential for exposure at Cottage Grove is to PFOA (perfluorooctanoic acid) althoughthe plant does produce PFOS. Thetotal cohort consisted of 2,788 male and 749 female workers employed between 1947and 1983. There was no significantly increased cause-specific 

### SMR for either men or women. 

### In a proportional hazard regression model, ten years of employment injobs associated with the chemical division was associated with a 3.3-fold increase (95% CI 1.02- 10.60) in prostate cancer mortality compared to no employmentin the chemical division. The authors suggested that this finding may be biologically plausible with exposureto PFOA as animal toxicology and human data had associated PFOA with reproductivehormonechanges (Gilliland, 1992). However, the authors urged caution in the interpretation ofthis result due to the nonspecificity 

### ofthe exposure index as well as the few subjects (n =

### 4) considered ‘exposed.’ Subsequentresearch 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 

## 000041January 2 1, 1999

Table 111.3. Selected Cause 

### - Specific 

### SMRs for Men by Comparison Population (n = 1,639) SMR 

### (95% Confidence Interval) Observed AL Regional Cause 

### ofDeath Deaths 

### U.S. Alabama Counties All Causes 

### ofDeath 70 62.9 54.8 52.0 (49.0,79.5) (42.7,69.3) (40.5, 65.7) (38.3, 112.8) (34.1, 100.4) (33.5, 

### 98.8) (0.8, 186.8) (0.7, 155.2) (0.7, 147.5) (34.4,94.7) (31.0, 85.1) (28.6, 78.6) 

### (0.0, 91.2) 

### (0.0, 80.2) (0.0,73.9) (48.2, 110.0) (38.2, 87.1) (35.6, 81.3) All Malignant Neoplasms 

### 15 68.4 60.9 59.9 Cerebrovascular Disease 

### 1 33.5 27.8 26.5 All Heart Disease 17 59.1 53.2 49.1



### --- --- --- Respiratory Disease 0 External Causes 25 74.5 59.0 55.0

29 

## 0

## 8680

## 42January 2 1, 1999

IV. SUMMARY

### OF TOXICOLOGY DATA 

### ON

### PFOS
Introduction This section summarizes the existing database with respect to the potential mammaliantoxicity of PFOS. Toxicology data are valuable in identifying potential health effectsand provide a basis for risk characterization.The use of this information in risk characterizationwill 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 thesepast studies, blood and tissue concentrations can only be estimatedbased on limited pharmacokinetic data and results from morerecent studies, most of which have been designed to include a pharmacokineticcomponentto obtain highly sensitive and specific identification

### of 

### PFOS inblood and tissues. Also, the extent ofpotential interspecies and intraspecies variability in pharmacokinetic handling of PFOS is not presently well defined. Comparison

### of serurn concentrations of 

### PFOS fromtoxicity studies to measured human serum concentrationsmay be an interesting and convenient comparison; however, body burden and/or target organ tissue concentration may be more meaningful in characterizing risk. The availablebody of animal toxicity data cannot be related quantitatively and without uncertainty and speculationto the concentrations of PFOS in humanblood reported in SectionI1 and the results of epidemiologic investigationas reported in Section 111. Despite the current limitations in the abilityto characterize risk based on the relationship of blood and tissue PFOS concentrations from toxicity studiesto human blood concentrationsof

### PFOS, the existing toxicology database 

### on PFOS does provide valuableinsight into potential health hazards and forms a foundation 

### for risk characterization.January2 1, 1999

It should be mentioned that the specific sources ofPFOS 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 ofnon-occupationally exposed humansmay derive from metabolism of higher molecular weight molecules present in industrial, commercial and consumer products or manufacturing byproducts.
The studies described in the following discussion reflect the toxic responses resulting from direct dosing with PFOS. The studies that are summarized in this section were performed with the potassium salt ofPFOS unless otherwise annotated. Studies on related compoundsare considered outside the scope ofthis 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 

### ofrodent 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 summarytables, Tables IV. 12 presents the results of rodent and primate 90-day subchronic studies organized by increasingnominal mg/kg dose level. A list of studies initiated in 1998can be found at the end of this section in Table 

### IV.8 and summary descriptions 

### ofthese studies can be found in the Appendix.
Pharmacokinetics of

### PFOS The absorption, tissue distribution, potential metabolism and excretion ofPFOS has been studied most extensivelyin rats by both radiolabel and direct quantitation. Limited data relating oral dose to serum and liver concentrations ofPFOS in the cynomolgus monkeyis 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.

31January 2 1, 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 averaging4.2 rng/kg [14C]PFOS administered to two groups (24 hour and 48 hour sacrifice) of 3 male Charles River 

### CD rats (248-3 15 g, mean =285 g) was absorbed within 24 hours (Johnson and Ober, 1979). Theradiochemical 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 often 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 onthe day one and day 28 samples from a single male and single female. Total serum fluoride values for the male were 10.3ppm for day one and 130ppm for day 28. The respective values for the female were 

### 0.9ppm and 128ppm. Although this study indicated some dermal absorption, it is limited in that the values from only

32January 2 1, 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-14C(mean dose, 4.2 mg/kg) six Charles River 

### CD male rats (initialbody weights 262-303 g, mean =288 g) excreted 

### amean 

### of 30.2% of the total carbon-14via urine. Mean cumulative fecal excretion was 12.6%. At 89 days, meantissue concentration oftotal carbon-14 expressed 

### as pg PFOS-14Cequivalentdg were: liver, 20.6; plasma, 2.2; kidney, 

### 1.1; lung, 

### 1.1; spleen, 0.5; and bone marrow, 0.5. Lower concentrations 

### (50.5)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 whichmale and female cynomolgusmonkeysweighing 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 exposureperiod (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 

### ofthe curve in the 0.02 mg/kg/day group (n = 

### 6) was

33 

## 0006546January 2 1, I999 5.22 k 0.74ppm 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 f 0.61 ppm PFOS in sew 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 ofmagnitude 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 equivalentto 5.3 mg/L of serum (mg/L =ppm weightholume); One (1) mg/kg givento 2.2 kg monkey results in a total dose 

### of2.2 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 

### 60kg = 12

### L =Vd 

### (L).

Metabolism: Preliminary data from analysis ofurine, feces and tissues 

### ofrats as well as the inherent stability ofperfluorinated anions suggest that PFOS is not metabolized (Johnson et al., 1984). Exposure ofprimary 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 currentlyin progress have not revealed any evidence of metabolism.
Excretion: In the previously mentioned study (Johnson etal, 1979) single intravenous doses (mean4.2 mg/kg) of ['4C]PFOS in 0.9% NaCl were administered to male rats. By 89 days after dosing, 30.2%of the administered 14Chad been excreted in the urine and 12.6%had been excreted in the feces.

34 

## 080047January 2 1, 1999 Wholebody elimination in the male rat appeared to be biphasic. Initial redistribution from the plasma yielded a plasma elimination half-life of I4Cof 

### 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 ofonly 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-halfyears 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 analyticalmethod 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 ifthis 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 ispresented 

### in Table 

### IV.1.January 21, 1999 Table IV. 1 SerumElimination of PFOS in Retired 3M Chemical Workers.

SerumElim. Serum [PFOS] in mg/L (ppm)

52.5 moll 575 d Retiree 

### 1 3/4/92 9/20/93 3/1/94 61

### 1 9/95 12/5/97 5.8 6.0 4.0 3.0 2.6 

# I - 0.0132/month Constant (A) 

# I I
ITu2 

# I Retiree 2 3118/92 11/15/93 5/5/95 

### 1

### 1118/97 1.6 1.4 0.7 0.7



### - 0.0136/month

50.9 moll 527 d Retiree 3 311 8/92 11/29/93 5/4/95 10123197 4.2 3.3 1.7 1.4



### - O.Ol76Imonth

39.4 moll 182d

Fecal and total excretion 

### of 14Cwere markedly increased in male rats administered cholestyramine (- 2.7 g/kg/d) in their diet following single intravenous doses of [14C]PFOS.Theresults 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-I4C. Groups of five rats (twelve-weekold 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 14Cfor cholestyramine-treatedJanuary 2 1, 1999
rats were comparedto mean controlrat values. Mean cholestyramine-treated rat 14C concentrations in liver (9.4pg/g), plasma (0.9pg/ml), and red blood cells (0.3 pg/g) represent a decrease from mean controlrat 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 ofurinary 

### I4Celimination, as a result ofthe relatively high rate of fecal elimination of I4Cwas lower in cholestyramine-treated rats. The extent of total elimination of 14C(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).

, 37January 2 1, 1999
Acute Toxicity Acute Oral Toxicity: PFOS was administered by gavage in a 20 :SO, acetone: corn 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-dayperiod. All rats in the 

### IO00 and 464 mg/kg dose groups died. The signsbefore 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 administrationunder the conditions ofthis 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, thenremoved. Animals were maintained and observed for a 28-day period after which they were necropsied. Blood samples were obtained on days 

### 1

### ,7, 14and 28. Day 0,7, 

### 14 and 28 body weights were recorded. Hyperactivity was noted in 5 of 10males 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 130ppm after 28 days (see section above on absorption). PFOS can be considered practically non-toxic on single dermal contact (0’ 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, 1.9and 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

38January 2 1, 1999 exposed to 24 milligrams per liter died. Partial mortality was produced at nominal exposure concentrations of 7.1 (SO%), 6.5 (SO%), 4.9 (20%) and 2.9 (10%) milligrams per liter. Symptomsobserved during the exposure and post-exposure periods were labored breathing, reduced activity, excessive salivation and lacrimation, mucoid and red nasal discharge, yellow staining ofthe 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. Thenominally determined median lethal concentration (LC50) for a one-hour exposureto 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 accordingto standard Federal Hazardous Substances Act guidelines. The ocular irritation was limited to the conjunctivae inthe sixtest 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 ofthe test animals at any time during the study period. Theprimary 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 

## 0000512January 2 1, 1999 in a standard plate incorporationassay with or without metabolic activation. (Jagannath and Brusic, 1978).
Chromosomal Effects: 

### PFOS did not cause chromosomal aberrations in an in vivo mousebone marrowmicronucleus 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 ofbone marrow.

### No increase in bone marrowpolychromatic erythrocytes was observed (Murli, 1996).

Repeated-Dose Toxicitv 

### A number ofrepeated-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 ofthis section in Table IV.9 for rodent studies and Table IV. 10for primate studies. In addition to these 

### surnmary 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 Studyin Rats: 

### PFOS was fed in the diet at levels of 0, 

### 30, 100, 300, 1,000 and 3,000ppm 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. Therats 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 commencementof dosing, at one month and at study termination.
At the 30 ppm (approximately2 mg/kg/day) dosage level, no rats showed any compoundrelated 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 

## 0000-53January 2 1, 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, meanbody weight and food consumption was significantly lower than the control group. One male and two females died. Slight increasesin creatinine phosphokinase 

### (CPK) and serum alkaline phosphatase activity, slightto 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 

### 100ppm dose group had slight to moderate decreases in hemoglobin, hematocrit and erythrocyte counts, and slight to moderate increasesin transaminase activities were seen for two ofthe three surviving female rats.
At dosage levels of 

### 300, 1,000and 3,000ppm (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 amongtreated rats that died prior to sacrifice. Similar changes were also observed in the liver of a fewrats sacrificed at termination of study fiom the 

### 30 and 

### 100ppm groups. Morphological changes consisting of centrilobular to midzonal cytoplasmic enlargement (hypertrophy)ofhepatocytes and necrosis 

### of liver cells was present in all PFOS dose groups. The incidence and relative severity of the above lesions were more evident amongmale rats. In addition, rats from the 300, 1,000 and 3,000 ppm dosage levels displayed compound-related changes involving the primary (thymus,

41 

## 000054January2 1, 1999 bone marrow) and secondary (spleen, mesenteric lymphnodes) 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 PFOSin the diet for four or 14weeks. 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 animaldsex in Groups 

### 1through 

### 5. Five animaldsex in Groups 1 through 5 were sacrificed during Week 4; livers were collected and weighed. A portion ofthe liver wasprepared for 

### PFOS analysis, a portion was shipped to Pathology Associates International for hepatocellular proliferation rate (PCNA) measurements, and a portion was used for determination 

### ofpalmitoyl-CoA oxidase activity. At week 14,necropsieswere performed on five animaldsex 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. Thetissues were adrenals, brain, eyes, kidney, liver, mesenteric lymphnode, pancreas, spleen, testes, and ovaries. In addition, microscopic examinations were done on tissues from animals that died or were sacrificed due to poor health.

42January 2 1, 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 atboth testing intervals, it was considered likely that administration ofthe test material was associated with mildly higher urea nitrogen at Week 

### 14for males and females fed 20.0 ppm and moderately lower cholesterol and mildly higher alanine aminotransferase at Week 

### 14for males fed 

### 20.0 ppm. There were no correlative microscopic renal findings for the minor change in urea nitrogen. The findings for alanine aminotransferaseand cholesterol were likely associated with the histopathological findings ofhepatocellularhypertrophy and vacuolation.
Ofuncertain 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 correlativemicroscopic 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 

### 14for 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 summarizekey clinical pathology results at 

### 14weeks for males and females, respectively.

43 

## 000056January 2 1, 1999

Average Value and Std Dev. by Dose Group (ppm PFOS in Diet) Table IV.2 Clinical chemistry and hematology in Malerats at week 14 (n=lO)

ANOVA

Parameter ALB ALT (SGPT) AST (SGOT) CHOL GLU N-SEG UN PCOAO 

## * Mean significantly different (Dunnetts' T-test, p 

### <0.05) than the control group value

Average Value and Std Dev. by Dose Group (ppm PFOS in Diet) ANOVA

0 ppm 0.5 ppm 2 PPm 5 PPm 20ppm Pr>F 5.5 k 0.25 6.1 k 0.5" 5.7 k0.17 6.2

## + 0.2" 6.1 k 0.24" 0.0057 34 k2 39+ll 30+5 36k5 33 +5 0.24 103

## +21 108

# +23 98

## + 8 109rir22 79+9 0.092 78+19 79+14 70rt12 68+ 19 65 k 16 0.64 102k 16 1OOk 12 105k3 90 k 8 100k 7 0.31 0.8f0.36 0.9k0.4 0.53rt0.13 0.56k0.1 0.64rt0.1 NA 14rt 1 16+4 1422 16k 1.7 16rt 1.8 0.26 1.8+ 1.6 3.0k2.6 1 k0.8 1.6

# +2.6 5

## + 2.9 0.10 Table IV.3 Clinical chemistry and hematology in female rats at week 14 

### (n=lO)

group value

Body and Organ Weights: Terminalbody weights at 14weeks for the 2 ppm dose group were reduced in comparisonto controlsby 

### 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. 

### Infemales given

44January2 1, 1999 

### 20.0 ppm, the absolute liver weights were increased by approximately 

### 30% over control values, but only the liver-to-bodyweight 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 thyroidparathyroid-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 

### ofthe 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 

### ofhypertrophy 

### ofhepatocytes in centrilobular areas, and midzonal to centrilobularhepatocytic vacuolation. The incidence and severity 

### ofthe 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 significantlyhigher than corresponding serum values. These analytical results are presented in Tables

### IV.4 and 

### IV.5 for serum and liver, respectively.

45January 2 1, 1999

Time Dose Interval Group 

### 4 Male Weeks 14 Weeks Male Female Female Table IV.4 Mean Serum

### PFOS Concentrations (ppm) After 4 and 14Weeks Dietary Exposure

Oppm 0.5 ppm 2 PPm 

### 5 PPm 20 PPm 0.11 k0.74 11 

### k 1.70 33 

### k 8.0 49k 12 295 

### k45 0.11

### k 0.05 8.6

### k0.57 25 k 6.2 80k 17 357 

### k43 0.44 

### f0.05 26 

### k4.4 77 

### k7.5 387 

### rt 33 

### 600k 116 1.58

### k2.1 19

### k 3.4 68 

### k 3.0 362 126 618 

### k 50 "0 ppm female [PFOS]in serum at 14weeks without one outlying value (n=4) is 0.59 

### k 0.59 ppm. **MDL =Method Detection Limit, which was 

### 15ppb.

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 mgkg dose group); 0.02; 0.06; 0.2; and 0.6 mgkg (added later). Male 

### LPOS doses, also expressed as mg/kg body weight, were: 0; 0.3; 1.0;and 3.0.

46January 2 1, 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 chemistrywas 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.O mg/kg dose groups); 

### 2) decreased mean corpuscular hemoglobin content (3.0mg/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 mgkgdose 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 

### 0.2mg/kg dose groups); and 

### 4) increased mean corpuscular hemoglobin and mean corpuscular hemoglobin content 

### (0.6mg/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.O and 

### 3.O mg/kg dose groups); 

### 2) increased total bilirubin (3

### .O 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.


### Grosspathology 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.O and 3.0 mg/kg dose group) and 2) decreased absolute heart weight, decreased heart weight relative to body weight and increased brain

47January 2 1, 1999 weight, kidney weight and testes weight relative to body weight 

### (3.0 mg/kg dose group). Gross pathologyrevealed 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). 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 supportedby the histopathologic finding of hepatic vacuolization in the 1.0and 3.0mg/kg male rats. With the exception of liver, these organ weight changes were

90-Day Oral Gavage Study in Rhesus Monkeys: In aninitial study, five groups consisting oftwo male and two female rhesus monkeysinitially weighing between 2.75 and 4.10 kg were given daily doses of 

### 0, 10,30, 100 and 

### 300mg

### PFOS / kg by gastric intubation (gavage) as distilled water suspension. This study wasterminated after 

### 20 days due to the death of all PFOS-dosed monkeys (Goldenthal, 1978). 

### ofdeath was dose-related. Monkeys in the 300 mg/kg/day dose group died between the second and fourth day. The 100mg/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 

### oftoxic effects was dose-related. These toxic symptomsincluded 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 10mg/kg dose group included one monkey who had black stools and one who developed facial erythema. Gross Time

48January 2 1, 1999 pathology revealed a yellowish-brown discoloration ofthe livers of several monkeys in the 100mg/kg and 300 mgkgdose 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 monkeysdid not reveal any consistent observationswhich 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 ofthe initial study afterearly 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 monkeysinitially weighing between 2.55 and 3.75 kg were given daily doses of 0, 

### 0.5, 1 

### S, 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 ofthe first and third month of the study. The monkeystreated 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 severerigidity, convulsions, generalizedbody trembling, prostration and loss ofbody weight prior to death. The mean body weight decreased fiom 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.

49January 2I, I999 All monkeys at the 

### I 

### .5 mg/kg/day dosage level survived to the end ofthe 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 grouphad 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 ofthe study by 

### 3M toxicologists lend to a conclusion that this dose more likely represents aNOAEL, 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 ofthe serous alveolar cells. 

### No statistically significant variations in sex group mean weights of organs occurred’ between the control and experimental groups.

50January 2 1, 1999

Table 

### IV.6 summarizes the individual cholesterol values for all dose groups.

51January 2 1, I999

Table 

### IV.6: Longitudinal analysis of serum cholesterol and PFOS concentrations of individualmale and female rhesus monkeys during 90 days dosing with either 1.5or 

### 4.5 mg/kg 

### PFOS (n=

### 2 per sex per dose group) (Goidenthal etal, 

### 1978b). Individual Serum Cholesterol (mg

### /I00ml) by Study Period and Cumulative 

### Dose (CD

Dose Grow Individual Pre-Dositin, CD = 0 0 

### d (ID # 7355) 192 mg/kg/day 

### 8 (ID # 7358) 174 Dose Group Q (ID # 7368) 155

Monkeys ? (ID # 7372) 204
CD=O 0.5 8 (ID # 7463) 182 mg/kg/dq 

### d (ID # 7483) 161 Dose Group 9 (ID # 7466) 208

MonkW 

### 9 (ID # 7504) 208

CD=O 1.5 

### 8(ID # 7462) 196 mg/kgday 

### 6 (ID # 7486) 174 Dose Group 9 (ID # 7500) 172

Monkeys 9 (ID # 7501) 236
CD=O 4.5 

### 6 (ID # 7484) 186 nzg/kg/day 

### 6 (ID # 7485) 194 Dose Group 9 (ID # 7502) 182

IMonkeYs 9 (ID # 7503) 170 in mg/ka)

1 Month CD = 0

212 184 188 208
CD = I5 208 192 230 21 1

CD = 45 222 2 04 154 184


### CD = I35 76 97 128 96 % of PreDosing Value 3 Months at Term

CD = 0 179 93.2 I44 82.8 144 92.9 185 90.7
CD =45 160 143 178 160

CD = I33 132 112 128 96



## - NO Survivors



### - 87.9 88.8 85.6 76.9

67.3 64.4 74.4 40.7

40.9 50.0 70.3 56.4

52January 2 I, I999 

### 28-Day Range-Finding, 

### Oral Capsule-Dosing Study in Cynomolgus Monkeys: Male and female cynomolgusmonkeys weighing 2.1 to 2.4 kg were given capsules placed directly inthe stomach that contained PFOS at either 0.0 mg/kg/day (two males and two females), 0.02 mg PFOS/kdday (three males and three females), or 

### 2.0mg/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 (Thornford, PJ, 1998). Blood was collected for clinical chemistry on studydays 

### - 7 (baseline values) ,2,7, 14and 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. Tissueswere 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 ofpalmitoyl 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 detailsplease seesubsection on toxicokinetics). At the end of the 28-day dosingperiod, serum

### PFOS concentration in the 

### 0.02 mg/kg/day dose group reached approximately 

### 3 ppm and inthe 2.0 mg/kg/day dose group, serum concentrationsreached approximately 

### 300 ppm. The only treatment-related effect observed in the study was a dramaticreduction in serum cholesterol in the male and female that received a dose of 2.0 mg/kg/day. Serumcholesterol dropped fiom baseline values of 150 and 14 1mg/dl for the male and female, respectively,to 

### 9 1 and 

### 62 mg/dl at termination on day 

### 29. The first evidenceof a significant decrease occurred between day 2 and day 

### 7 forthe female,with 

### a day 2 value of 136mg/dl and a day 

### 7 value 

### of 117mg/dl. The male cholesterol value fell from to 15lmg/dlon day 2 to 137on 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 cumulativedose and serum PFOS concentrationsare summarized in Table IV.7. There were no other significant findings.

53January 2 1, I999

Study Day Cumulative Dose (mgikg) 

### 6Serum Cholesterol (mgidl) 

### 8 [PFOS] in serum (ppm) 

### 0 Serum Cholesterol (mg/dl) 

### 9 [PFOS] in serum (ppm)

Table IV.7: Longitudinal analysis of serum cholesterol and 

### PFOS concentrationsin male and female cynomolgus monkeys (n= I per sex) during 28 days dosing with 2.0 mg/kg 

### PFOS.

- -7 - 2 - 7 

### - 14 

### - 29 0 2 12 

### 26 56 

### 150 151 137 132 91 0.013 12.6 71.8 129 3 13

141 136 I17 107 

### 62 0.014 14.4 72.7 143 299

Six-Month Oral (Capsule) Studv in Cvnomolgus Monkevs Unaudited clinicalpathology and clinical observations through 

### 90 days of dosing are available from an ongoing six-month oral (capsule) dosing study in cynomolgus monkeys (Covance StudyNumber 6329-223, in progress). In this study cynomolgus monkeys are being dosedby capsule with either 

### 0, 0.03, 0.15, or 0.75 mglkgiday 

### PFOS for a period of six months. Dose groups include six monkeys per sexper 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 ofthe 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 approximately350 ppm 

### PFOS as 90 days for the high-dose monkeys. Reproductive and Developmental Toxicitv 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 studywas conducted at Riker Laboratories (Gortner etal, 1980). Dose levels (oral)January 2 1, I999 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 10mgkg on days 6 through 15of 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 ofthe 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 specimensin question. He concluded that the eye/lens changes were, in fact, sectioning artifacts and

## not compoundrelated teratology abnormalities. Thus, the weight ofthe 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 Deveiopmental Toxicity (Teratology) in Rats: In a subsequent study, 

### PFOS (suspended in corn oil) was administered on gestational days 

### 6- 15by 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 10mg/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 10mg/kg dose group prior to gestational day 20. Mean body weight gains (days 

### 0-20) at 5 and 10mg/kg were 104 k 35 

### (S.D.) and 34 k73 (S.D.), respectively, as compared to 125 

### t24 

### (S.D.) in the control group. Mean food consumption values (days 0-20) at 

### 5 and 10 mg/kg were 363 5 

### 60 

### (S.D.) and 264 

### It 90 

### (S.D.),respectively, as compared to 421 i28 for. the control group. Mean terminal body weight minus gravid uterine weight at 5 and 10mg/kg was 293 

### k28 

### (S.D.) and 241 li: 

### 60 

### (S.D.), respectively, as compared to 321

55January 2 I, 1999


### k 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 1mg/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 of22 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.O 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 

### NOELSare 0.1 mg/kg/day and 

### 1.O 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 

## 0 130

## 0*5 

## 9 56January 2 1, 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 duringmating. 

### For the females, treatment continued during gestation, parturition and lactation.
A pre-mating reduction inmean body-weight gain as comparedto controls occurred in females and possibly in the males at the high dose level. At the 3.2 mgkgdose 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 

### ofpregnancies. During gestation, females showed a reduction in mean body-weight gain at the three highest dose levels, reaching 86.9% ofthe control at 3.2mg/kg.A group often dams per dose group was sacrificed

### on day 

### 10of gestation. 

### No increasesinresorptions occured, andthere were no decreases in the number of implantations or number of live fetuses. At parturition inthe 3.2 mg/kg high-dose group, the mean number of pups delivered was decreasedcompared to the control (10 versus 

### 14) andthe percent ofpups delivered stillborn was increased significantly (24.9 % as comparedto 2.2 %.) Survival ofpups during days one through four of lactation was severely affected at 1.6 and 3.2 mg/kg 

### (66 

### YO and 0% survival, respectively.) Most deaths at the high dose occured within the first 24hours after birth. Survivingpups in the 1.6mg/kg dose group showed severely depressed mean body-weightgains through day 

### 2 1 of lactation (72.1

### YO of controls.)
A series 

### of follow-up studies are to be initiated in November, 1998 to gain 

### a better understanding ofthe reduced perinatal survival at the high dose levels. These will include a completecross-fostering study, and two pharmacokinetic studies with pregnant dams.January 2 I, 1999 Thepost-weaning 

### FL generation currently exhibits no signs of toxicity at 0.I 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) oftoxicity. 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 10pM demonstrate: 

### 1) detergent type effects on mitochondrial membranes; 2) stimulation ofmitochondrial 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 ofinitial fluorescence when added to solutions containing 1 mM

### L-FOP and 1mM

### DAUDA and an 

### ICjo of4.9 

### IJ.M (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 ofthe 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 C2- 1-hydroxylation (Sohlenius et al., 1993).January 2I, I999 Ikeda et 

### al. 

### (I987) exposed male rats were to 0.02% 

### PFOS in the diet for 2 weeks. The prominent induction ofperoxisome proliferation was demonstrated by electron microscopy. Activities of catalase, fatty acyl-CoA oxidizing system, carnitine acetyl transferase and cytochrome P450s responsible for the Q-hydroxylation of lauric acid were increased (Ikeda et al., 1987).

Hypolipidemia: The mechanism ofthe hypolipidemic effect of 

### PFOS has been studied. Rats were fed 12mg/kg/day for 7 - 14days (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 ofpalmitate and reduced fatty acid synthesis. Activities of liver hydroxymethyl glutaric acid-CoA reductase and acylCoA:cholesterol acyltransferase were reduced. These results suggest that the hypolipemic effect 

### ofPFOS 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-GoingResearch Program 

### A number of studieswere 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.

59h F' 

### z 

# - '? 0 a. 30rnlnN0 m 0 

# z

I

n 

## - n on F(u OON 9 %I GO 0

### M G 

### Y 

# E 2 .- a f
9) > .- * a

a C tu tu 

# - * 

### 2 

# 2 a" 0 a, > 

# -

L 0 5 E a 2 m m 

## 2

a

.. 

# -

e C m bI .C 

### I u

t I I I

I 1 I I I I



### si 

### 2 0 W uI

m W

C :

### 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 

### HI), 3Mhas 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 1997mean among participating employees at the Decatur, Alabama plant, the 

### U. S. location where PFOS related materials are principally produced, was 2.0 ppm. Thebest 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 

### 14to 

### 56 ppb, and averaged 

### 30 ppb. It should be noted that while this is our best estimate, it cannot be considered representative ofthe U.S.population since blood donors are not necessarily representative ofthe 

### U.S. population. In addition this data, since it is frompooled 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 ofthe 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 ofJanuary 2 I, 1999 magnitude above the upper end of the range seen in the 

### US.regional blood bank samples. The mean serum concentration in participating Decatur plant employees in 1997was 

### 68 times the mean ofthese 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 oneyear at 

### 3M's Decatur, Alabama plant, and their mortality experience was traced from 196I through the end 

### of 1991. Details of this study can be found in Section 111.
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 cynomolgusmonkeys 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 800ppm and death at 1000to 1200ppm.

65January 2 I, 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 F1weaning, 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 

### F 1generation. 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 ofthese 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 shownto 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,

66January2 I, 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 (Amestests) and does not cause chromosomal aberrations (mouse bone marrow micronucleus assay) is consistent with these findings. Non-occupationally exposed populations appearto exhibit serum levels that are approximately two orders of magnitude lower than those exposed occupationally, more than three orders ofmagnitude 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 ofthe 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 comparableto 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.

67January 2 1, I999

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 ofincreased 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 

### ofthese 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 followup on worker mortality experience is also underway, including an attempt to better classify workers with regard to PFOS exposure. Finally, studies are underway to improve knowledge about possible sources 

### of PFOS, and the pathways by which non-occupationally exposed individuals are exposed. Although there is no current evidence that the reported serum levels, and corresponding body burdens, represent a health risk, it is appropriate to identify and act onpossible ways to reduce exposure. To date this has been done through reduction ofresidual monomer levels in products and reduced manufacturing emissions.

68VI. CURRENT AND PLANNED RESEARCH Toxicoiom Studies Fluorochemical Studv Purposes and Outlines A number of studieswere initiated 

### in 1998to gain bett insight into the itential health hazards ofPFOS 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 (74-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 contaminantsofindirect food additives. These compounds are also known to be absorbed well fromthe gastrointestinal tract and will produce PFOS as a major metabolite. Therefore,it is appropriate to use these two compounds as models in hrther investigatingthe toxicity ofthis class of chemicals which have the perfluorooctane sulfonyl moiety as a base. The goals of this researchprogram 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 definethe metabolic relationship ofPFOS to other Perfluorooctanesulfonylbased chemicals; To understand the primary mechanism of toxicity responsible for early toxic effects; 

## 000082 69January 2 1, 1999 

### To establish reasonable and substantiated no significant risk levels forrisk 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 studieshave been undertaken to better understand the potential for metabolic degradation 

### of perfluorooctanesulfonamide-based chemicals to 

### PFOS. Descriptive summaries ofmany of these studies are included in the Appendix.

70January 2I, 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 lMonkeys Six-Month Capsule Feeding Study in Cynomolgus Monkeys
Two-Year Dietary Study in Rats Hepatic Peroxisome Induction and Cell Proliferation Study Biochemical and Molecular Biology Mechanistic Studies Segment I1 Teratology in Rabbits Two-Generation Reproduction Study in Rats One-Generation Cross-Fostering Reproduction Study in Rats One-Generation Reproduction PK Study in Rats (through Gestation) One-Generation Reproduction PK Study in Rats (through Lactation)

rable VI.2: N-EtFOSE Toxicity Studies Initiated in 1998 Study Title 4/23/98 5/22/98 8/5/98 2/8/99 (histo.) 5/7/99 (recov.) 5/20/98 412 412000 12/1/98 1/7/98

. 7/1/97 On-going 8128198 9/29/98 5126198 1 213 1/98 11/1/98 2115/99 11/7/98 1 /30199
11/23/98 3/1/99

Two-Year Dietary Study in Rats 1/26/98 Single-Dose ADME in Cynomolgus Monkeys J 1 12512000

Biochemical and Molecular Biology Mechanistic Studies 5/14/95
Segment I1 Teratology in Rats 10/1/98

Segment I1 Teratology in Rabbits 618198 Two-Generation Reproduction Study in-Rats 1213 1/98 Date (InLife)

7/1/97 

# 1 On-going 

# I
811 1/98 9/4/98 8/28/98 912919 8

71January 2 1, 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: 4/2412000 The purpose ofthis two year assay is to determine: carcinogenic potential, bioaccumuiation 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 ofpreneoplastic 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 oftoxic 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 14,27and 53.
104 -Week Dietary Carcinogenicity Study with Narrow Range (98.1

### YO) N-Ethyl Perfluorooctanesulfonamido Ethanol in Rats. In-life Start Date: 

### 1/ 26/1998. Thepurpose ofthis study is to assess the carcinogenicity of the test material, N-ethyl 

### perfluorooctanesulfonamido ethanol m-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 14for 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

72January 2 1, 1999 protocol. Microscopic examinations were done on selected tissues from animals necropsied during Weeks 

### S and 14.A recovery group of animals in the 100ppm 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 

### ofthis 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 lowhepatic 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 

### 1SO days oftreatment, 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 ofrecovery and after 

### 6,30,and 

### 90 days ofrecovery 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 oftoxic effects for at least 

### 13weeks post-treatment. Hematology and clinical chemistry will be determined oncebefore initiation, after 30, 60, 90, and 

### 1SO days oftreatment, and after 30,60, and 90 days ofrecovery. 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

71January 2 1, 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 

### I3weeks post treatment for the recovery groups. Histopathology and proliferative cell nuclear antigen (PCNA) will be performed on a subset ofthe specimens.
Metabolism of

### N-EtFOSE, in Cynomolgus Monkeys following administration of a single dose by Oral Gavage. In-lifestart date: 5/14/98. In-life end date: 10/1/98 Thepurpose ofthis study is to determine the absorption, distribution, metabolism and excretion (ADME) ofN-Et 

### FOSEand 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 10mg/kg single treatment by oral gavage. Tissues will be collected from one animal per sex per time-point at 0.5, 

### 1, 7, 

### 14and 

### 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 wilI 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 14,21and 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, thyroidparathyroid, aorta (-0.5 g), adrenal glands, gallbladder , and ovaries will be included.

74January 2 I, 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 ofthe monophosphate ester of FC-807 in rats. FC-807 is the brand name of a fluorochemical based product sold by 3rVl 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 (Lv.) 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 ofthe animals were sacrificed 28 days post dose. The preliminary results indicated absorption 

### ofthe oral 

### 50 mg/kg dose and metabolism at day 4, and day 28. The i.v doses 

### of 

### 0.5mg/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,O. 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 ofthe 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: 12/18/1998. The purpose ofthis studywas to develop sub-chronic toxicity data and the toxicokinetics for N-iMethyl 

### FOSE when administered in the diet to rats for at least 13weeks. The study design included 20 males and 

### 20 female animals/group fed 0, 3,

75January 2 I, 1999 30 and 100ppm 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-GenerationRat Reproduction Study with 

### PFOS. In-life Start Date: 5/26/1998.In-life completion date: 12/31/1998. The purpose ofthis study is to test for toxic effects ofPFOS 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, 1.6and 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 ofpups/ litter, pup viability index and lactation index. The 

### F1 generation pups at each dose level will be assessed for developmental objectives, undergo learning testing, and reproductive performance. Serum and liver samples will be collected at necropsy from five male and five female ratsldose group from 

### F, (Parent) animals and analyzed for compound by 

### 3M.
Two-GenerationRat Reproduction Studywith 

### N-EtFOSE. StartDate: 6/8/1998. In life completion date: 12/31/1998. Thepurpose ofthis study is to test for toxic effects ofN-EtFOSE onreproductive 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 rng/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 ofpups/ litter, pup viability index and lactation

76January 2 1, 1999 index. The 

### F1 generation pups at each dose level will be assessed for developmental objectives, undergo learning testing and reproductive performance. Serum and liver samples will be collected at necropsy from 

### 5 male and 5 female ratsldose group from 

### F, (Parent) animals and analyzed for compound by 3M. Segment I1 Teratology in Rabbits with 

### PFOS. In-life Start date: 8/28/1998. Inlife end date 9/29/1998. Thepurpose 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 ofthe 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 stomachtube. 

### A toxicokinetic satellite group offemale 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 ofpresumed 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.

SegmentI1 Teratology with N-EtFOSE in rats. In-life Start date: 9/4/1998. In life end date 9/11/1998. Thepurpose ofthis study was to detect adverse effects ofN-EtFOSE on presumed pregnant female rats and development ofthe embryo and fetus consequent to exposure ofthe dam from implantation to closure ofthe 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 18of gestation (the day following the last dosage) serum, liver, fetal and placental sampleswere analyzed for N-EtFOSE and possible metabolites. Rats were Caesarian

77January 2 I, I999 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. SegmentI1 Teratology in Rabbits with N-EtFOSE. In-life Start date: 8/28/1998. In life end date 9/29/1998. The purpose ofthis study was to detect adverse effects ofN-EtFOSE on New Zealand White pregnant female rabbits and development ofthe embryo and fetus consequent to exposure 

### of the doe from implantation to closure ofthe 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 ofpresumed 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.January 2 I, I999 Current and Pronosed 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. 3iv retirees from the Decatur and Cottage Grove plants will be asked to participate in a study to determinethe half-life of PFOA and PFOS. years for PFOS, PFOA and perfluorohexane sulfonate. Retirees’ serum will be analyzed semi-annually for the next five

2. plant have in the past voluntarily participated in a fluorochemical medical surveillance program. Analysis 

### ofthe surveillancedatahas not shown significant associations between the employees’ clinical chemistry and hematology tests and either total serum Decatur SerumExposure Assessment Study. Employees at the 3M Decatur chemical

organic fluorine or serum 

### PFOS Ievels. However,the voluntary nature ofthe medical surveillance program does not allow for a complete understandingof the distribution of employee fluorochemical serum levels. In order to address this issue, a random sample of approximately 

### 80 Decatur film plant and 

### 125Decatur chemicalplant employeeswill 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, andN-methyl perfluoroctanesulfonamido ethanol. A sub-sampleof employees will also be tested for total serum organic fluorine. A brief questionnairewill also be administered to each employee inquiring about current and past work history aswell as possible routes of oral ingestion.
3. An EpidemiologicAnalysis ofthe Inpatient Claims Experience 

### of 3M Decatur Employees, 1993- 1997. The purpose of this study is to examine the inpatient claims database, asmaintained by Corporate Health Strategies, from January 1, 1993 - December 31, 1997 ofDecatur plant employees. The data will be stratified by whether the employeesare in the chemical or filmplants. The study population will include full79January 2 I, 1999 time active employeeswho worked throughout the five year interval, all hll-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 eachplant population will be compared to an expected experience based on the 

### 3M normative database. Inpatient claims analysis will proceed in a sequential,hierarchical manner. First we will examine Medical Diagnosis Codes; next Diagnosis Related Groups, and finally selected ICD-9 codes within each DRG.
4. PFOS and PFOA Retrospective Cohort Mortality Studies of Employees at the 

### 3M Decatur and Cottage Grove Plants. Previous retrospective cohort mortality studies have been conducted at the Decatur and Cottage Grove plants. However, neither 

### ofthese studies utilized the employees' fluorochemical serum measurements to design job-, department- and calendar-year exposure matrices. The purpose ofthese two studies is to construct PFOS and PFOA exposure matrices based onpreviously 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 ofrisk, i.e., StandardizedMortality Ratios, for more than 50 causes of death. Vital status and cause of death will be ascertained through December 

### 3 1

### , 1997.


### 5. Geographical Reliability Study. The purpose 

### ofthis study is to determine whether there are geographical differences in human serum measurements in the United States. We will request serum samples from the 18blood banks that originally participated in the fluorochemical determination study (see Table 

### 11.3) to determine whether comparable serum levels are measured.

80January 2 I, I999



### VII. 

### REFERENCES 3M Commercial Chemicals Division. 19xx. FluoradBBrand 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 serumby LC/MS

. Unpublishedreport. Ithaca:Advanced Bioanalytical Services, Inc., August 25, 1997. Anderson DJ, Mulvana DE (1997b). Analytical report for the determination of perfluorooctanoate and perfluorooctanesulfonate in human serumby LC/MS

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### of selected Decatur employee serum for sulfonic and carboxylic fluorochemicals. Tech- Report No. 723Q, 3M Central Analytical Laboratory, St.Paul, 

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### No. 7230, October4, 1979. Covance (1998): Study No. 6329-1 83 “104-week Dietary Carcinogenicity Studywith Perfluorooctane SulfonicAcid Potassium Salt (PFOS; T-6295) in Rats,” (Study in progress). Covance StudyNumber 6329-223 (in progress).

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### G., Romig, G. and Powell, 

### D. 1975. Fluorad63 Fluorochemical Surfactant FC-95 acute oral toxicity (LD50) study in rats. ReportNo. 137-083. International Researchand Development Corporation,Mattawan. MI. Gabriel, 

### K.L. 1976. [Acuteoral toxicity in rats of T-13891. Biosearch, Inc., Philadelphia, PA. Gilliland FD, Mandel 

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### (1996). Serum perfluorooctanoic acid and hepatic enzymes, lipoproteins and cholesterol: a study of occupationally exposedmen. Am 

### J Ind Med 29:590-568. Gilliland FD. Fluorocarbons and human healthstudies in an occupational cohort. Minneapo1is:UniversityofMinnesota (Ph.D. dissertation), 1992. Glaza 

### S. M. Single -dose dermal absorptiodtoxicitystudy of T-6049 in Rabbits 1995. Laboratory Project identification:HWI 6329-130Hazelton Wisconsin Inc. Madison, WI. Goldenthal E.I. 1978. Letterto 

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### No. 137-093, International Research and Development Corporation, Mattawan, MI. Goldenthal, E.I., Jessup, D.C., Geil, R.G., Jefferson, N.D. and Arceo, R.J. 1978a. Ninetyday subacuterat study. Study

### No. 137-085, International Research and Development Corporation, Mattawan,MI. Goldenthal, E.I., Jessup, D.C., Geil, R.G. and Mehring, J.S. 1978b. Ninety-day subacute rhesus monkey toxicity study. Study

### No. 137-092, International Research and DevelopmentCorporation, Mattawan, MI. Gordon, 

### S. C. 1998.Personal communicationofresults from a comparative in vitro metabolism study ofPFOS using primary rat and human hepatocytes. Gortner, E.G., Lamprecht, E.G. and Case, M.T. 1980. Oral teratology study of FC-95 in rats. Expt. 

### No. 0680TR0008,Riker Laboratories, Inc., St. Paul, MN. Guy, WS 

### (1972). Ph.D. Thesis, UniversityofRochester, Rochester,

### NY.

82January 2I, I999 Guy, WS, Taves, 

### DR, Brey, Jr., WS (1976). Organic fluorocompoiinds in human plasma: prevalence and characterization. (In) Biochemistry Involving Carbon-Fluorine Bond, pages 117-134. Haughom,B. and 0ystein, 

### S. 1992. The mechanism underlying the hypolipemic effect of perfluorooctanoicacid (PFOA), perfluorooctane sulphonic acid (PFOSA) and clofibric acid. Biochim. Biophys. Acta. 1128, 65-72. Ikeda, 

### T.,Fukuda, 

### K., Mori, I., Enomoto, 

### M., Komai, T. and Suga, T. 1987. Induction of cytochrome P-450 and peroxisome proliferation in rat liver by perfluorinated octanesulfonic acid. In:Peroxisomes inBiology and ibfedicine,H.D. Fahimi and H. Sies, Eds. SpringerVerlag,New York, 304-308. Industrial Hygiene Information System. 1994.

### 3MIndustrial Hygiene Services, Saint Paul, MN. Jagannath, D.R. and Brusic, 

### D. 1978. Mutagenicity evaluation of

### T-2014CoC in the Ames Salmonelldmicrosomeplate test. LBI Project 

### No. 20838, Litton Bionetics, Inc., Kensington, MD. Johnson J.D. Final report analytical study Single -dose dermal absorptiodtoxicity study 

### ofT-6049 inRabbits 1995

### 3MEnvironmental Laboratory, Saint Paul, 

### MN. Johnson JD, Wolter 

### JT, Colaizy GE, Rethwill PA, Nelson 

### RM (1 996). Quantification of perfluorooctanoate and perfluorooctanesulfonate in human serumusing ion-pair extraction and high performance liquid chromatography

## - thermospray mass spectrometry with automated samplepreparation. J Chromatography Biomed Appl. Unpublished report, St.Paul:3M Company. Johnson,

### J. D. and Ober, R.F 

### . 1 979. Absorption of FC-95-14C in rats after a single oral dose. Project No. 8900310200,RikerLaboratories, Inc., St.Paul, 

### MN. Johnson, J.D. and Behr, F.E- 1979. Synthesis and Characterization of FC-95-"C. Project 

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### MN. Johnson, J.D., Gibson, S.J.and Ober, R.E. 1979. Extent and route 

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### of FC-95-"CC.Project 

### No. 8900310200,Riker Laboratories, Inc., St. Paul, 

### MN. Johnson,J.D., Gibson, S.J.and Ober, 

### R.E. 1980. Enhanced elimination ofFC.-95-14C and FC-143-l4Cin rats with cholestyramine treatment. Project 

### No. 8900310200,Riker Laboratories, Inc., St.Paul, 

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### R.E. 1984. Cholestramine-enhanced fecal elimination of carbon-14 in rats after administration 

### of ammonium

53January 2 I, 1999 [14~~perfluorooctanoate or potassium [lJ~~perfluorooctanes~i~~onate. 

### FZ~M~. ~ppi. Toxicol, 4, 972-976.

Mandel JS, Johnson 

### RA (1995). Mortality study of employeesat 

### 3M Plant in Decatur, Alabama. Unpublished report. Minneapo1is:University of Minnesota. Murli 

### H. Mutagenicity test 

### onT-6295 in an in vivo mouse micronucleus assay 1996. CHV study

### No.: 17403-0-455 Corning Hazelton Inc. (CHV) Vienna, VA. Nabbefeld D. 1998An Investigationofthe Effects of Fluorochemicals on Liver Fatty Acid-Binding Protein. iMastersThesis, University of Minnesota. Thesis research performed at and supported by 3Ivl Nabbefeld D., Butenhoff

### J., Bass 

### N. and SeacatA. 1998. Displacement of a fluorescently labeled fatty acid analogue from fatty acid carrier proteinsby wyeth-14,643, ammonium perfluorooctanoate,potassium perfluorooctane sulfonate and otherknown peroxisome proliferators. 

### (SOT Abstract. Accepted, Toxicologist 1998). Olsen GW, Burris JM, Mandel 

### JH, Zobel 

### LR (1

### 998). An epidemiologic investigation of clinical chemistries,hematology and hormones in relation to serum levels of perflurooctane sulfonate in male fluorochemicalproduction employees. Unpublished report. St Pauk3M 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. 0979AB0632. Riker Laboratories,Inc., St. Paul,

### MN. Paez, DM, deBianchi, LP, Gil BA, Dapas 

### 0,Coronato, 

### RG (1

### 980). Fluoride 13:65. Roach 

### DE (1982). Fluorochemical Control Study. Unpublished report. St. Paul:3M Company, May 25, 1982. Pothapragada V, (1975). Determination oftotal fluorine in serum and other biological materials by oxygenbomb and reverse extraction techniques. Analytical Biochem 68:512-521. Pothapragada V, SingerR, Annostrong WD (197I). Determination of ionic (plus ionizable) fluoride inbiological fluids. Procedure based 

### on adsorbption offluorideion on calciumphosphate. Anal Biochem 423 50-359. Rusch, 

### G.iM. and Rinehart, W.E. 1979. An acute inhalation toxicity study 

### of T-2306CoC in the rat. Project 

### No. 78-71 85, Bio/dynamics, Inc.

84 

### 000097January 2 1, I999
Singer

### L and Armstrong WD (1959). Determination of fluoride in blood serum. Analytical Chem 3 1:

### 105-109.. Singer

### L and Ophaug 

### RH (1979). Concentrationsof ionic, total, and bound fluoride in plasma. Clin Chem 25523-525. Sohlenius,A-K., Eriksson, A.M., Hogstrom, C., Kimland, 

### M. and DePierre,J.W. 1993. Perfluorooctanesulfonic acid is a potent inducer of peroxisomal fatty acid B-oxidation and other activities

### known to be affectedby peroxisome proliferators in mouse liver. Pharrnacol. 

### Toxicol.72, 90-93. Taves D (1968a). Evidence that there are two forms of fluoride in human serum. Nature 217;1050-105 

### 1. Taves D (1968b). Electrophoretic mobility of serum fluoride. Nature 220:582-583. Taves 

### D, Guy W, Brey 

### W (1976)

. Organic fluorocarbonsin human plasma: Prevalence and characterization. In: Filler R, eds. BiochemistryInvolving Carbon-FluorineBonds. Washington,DC:Arnerican Chemical Society, pages 1 17-134. Thomford,PJ 

### (1998). Study

### No. 6329-222 “4-week Capsule Toxicity Study with Perfluorooctane SulfonicAcid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys,” Final Report. Ubel FA, Sorenson 

### SD, RoachDE (1980). Health status ofplant workers exposed to fluorochemicals, a preliminary report. Am Ind Hyg Assoc 

### J. 41;584-589. Wallace 

### K. B. and StarkovA. 1998.The effect 

### ofperfluorinated arylalkylsulfonamides onbioenergetics ofrat liver mitochondria. Dept 

### of Biochemistry and Molecular Biology, University of

### MN School of Medicine. Duluth, 

### MN 55812, USA. Supportedby a grant from 

### 3M Company. Wetzel, 

### L.T., Burdock, G.A., Durloo, R.S. and Colpean,B.R. 1983. Rat teratology study. ProjectNo. 1 

### 54- 160,Hazleton Laboratories America, Inc., Vienna, VA. Yamamoto 

### G, Yoshitake

### K, Sat0 T, Kimura T and Ando 

### T (1989). Distribution and forms of fluorine in whole blood 

### ofhuman male. Analytical Biochem 

### 182571-376. York, 

### R., Oral (Stomach Tube) Developmental Toxicity Study of

### PFOS in Rabbits, Argus Laboratories StudyNumber 4 18-012 final draft report dated December 17, 199s. 

### APPENDIX

85January 2 1, 1999
FIGURE AI. Reliability 

### of PFOS Serum Measurements in 12 3M Employees

20 30 40 50 60 70 80 1stTest (February, 1998)

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### vvv 

# aa.9Table A2. Description 

### of Final Employment and Vital Status for Male (n = 1,639)and Female (n = 3 18)Employees Final Employment Status Male Emulovees Female EmDIovees and Vital Status N 

### YO N 

### YO Curre.ntlyEmployed 810 49.4 141 44.3 Ret iredAlive Deceased Unknown Terminated Alive Deceased 

### unknown 59 3.6 9 2.8 10 0.6 0 0.0 0 

### 0.0 0 

### 0.0
694 42.3 164 51.6

29 1.8 1 0.3

6 0.4 0 0.0 Died While Employed 31 1.9 3 

### 0.9 TOTAL 1,639 100.0" 318 100.0" *percentages 

### may not add to 100due to rounding

97January 2 1, 1999

Table A3. Characteristics 

### of Male 

### (n = 1,639)and Female (n = 3 18)Employees
Females Variab1 e Males Number 

### of Employees 1,639 318 Number 

### of Person-years 33,l08 4,807 Number 

### ofDeaths 70 4 Average Age Started Work 

### 25 26 Average Year 

### of Entry 1971 1977 Average Age at Death 47 28 Average Year of Death 1984 1980January 2 1, I999 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 I990

to to to to to to to Age 1964 1969 1974 1979 1984 1989 1991 TOTAL a0 18 34 86 31 4 2 0 175 20-24 114 238 301 96 28 18 3 798 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 

### I 0 0 2 3 0 

### 6 55-59 

### 0 

### 1 0 0 0 0 0 1 60+ 0 0 0 0 0 0 0 

### 0 

### TOTAL 26

### 1 517 502 213 

### 77 56 13 1,639

99January2 1, I999 

### Table A5. Distribution of Women by Age and Year 

### ofEntry into Follow-up 

### (n = 3 18)
Year 

### of 

### Entry into Follow-up
1960 1965 1970 1975 1980 1985 I990

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 4 22 23 50 4 9 1 113 3 4 4 47 4 

### 5 3 70 

### 1 2 3 16 3 3 2 30 0 0 0 8 3 2 0 13 

### 0 0 0 5 1 

### I 0 7 0 

### 0 0 3 0 

### 0 0 3 

### 0 0 

### 0 

### I 

### 0 0 0 1 60+ 

### 0 0 0 0 

### 0 0 0 0 20-24 25-29 30-34 35-39 40-44 45-49 50-54 

### 55-59 TOTAL 12 43 50 161 20 24 8 318 2 3 2 15 1 4 2 29

100January 2I, 1999 Table A6. Cause-Specific 

### SMRs for Men Ever Employed in Chemical Department (s) Using the U.S. as Comparison Population (n = 1,050) Observed Expected Cause 

### of Death All Causes ofDeath All Malignant Neoplasms

Cancer 

### ofBuccal Cavity and Pharynx Cancer 

### ofDigestive Organs and Peritoneum Cancer 

### ofEsophagus Cancer of Stomach Cancer 

### of Large Intestine Cancer of Rectum Cancer 

### ofLiver and Biliary Passages Cancer 

### ofPancreas Cancer ofAll Other Digestive Organs Cancer 

### ofLarynx Cancer 

### ofBronchus Trachea and Lung Cancer of Other Respiratory Organs Cancer 

### ofProstate Cancer 

### ofTestes and Other Male Genital Organs Cancer 

### ofKidney Cancer 

### ofBladder and Other Cancer 

### of Respiratory System Deaths 57 13 0 

### 1

0 0 7 

### 0 7 0 0 0 

### 0 1 Deaths 81.5 16.9 0.5 3.6 0.4 0.5 1.3 0.3 0.3 0.8 0.1 6.1 0.2 5.8 0.1 0.5 0.2 0.5 0.2 

### SMR 95% CI 70.0 (53.0, 90.6) 76.9 (40.9, 131.5) 

### --- (0.0, 800.6) 27.5 (0.7, 153.2)



### --- (0.0, 913.3) (0.0, 686.1) 76.9 (1.9, 428.5) 

## --- (0.0, 1,271.9) 

### --- (0.0, 1

### ,188.2) 

### ---



### --- (0.0, 480.6) (0.0,3,203.2) 

## --- 115.1 (463,237.1) 

## --- (0.0, 1,891.9) 120.7 (48.5,248.7) (0.0,4,214.1) 

## ---

--- (0.0, 805.9) 

## --- (0.0, 1,678.5)



### --- (0.0, 768.2) 415.5 (10.4,2,315.3) Urinary Organs

101January 2 1, 1999 Table A6. (Continued) Observed Expected Cause 

### of Death Deaths Deaths 

### SMR 95% CI

Cancer 

### ofBrain and Other 

### 1 0.9 117.2 (2.9.653.0) Central Nervous System Cancer ofThyroid and Other 0 0.1 

### --- Endocrine Glands Cancer 

### ofBone 0 0.1 

### --- Cancer 

### ofAll 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 

## ---
Diabetes Mellitus 0 1.1 

### --- Cerebrovascular Disease 1 2.3 43.4 All Heart Disease 11 22.5 48.8 Hypertension 0 0.1 

### --- Respiratory Disease 0 3.2 

### --- Ulcer 

### of Stomachand 0 0.2 

### --- Duodenum Cirrhosis 

### ofLiver 3 

### 3.O 100.2 Nephritis and Nephrosis 0 0.3 

### --- External Causes 20 22.2 90.2

All Other Malignant 

### 1 1.5 68.9 Neoplasms

9

Accidents 16 13.6 117.3 MotorVehicle Accidents 

### 8 7.8 

### 102.8 All Other Accidents 

### 8 5.9 135.7 Suicides 3 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 \* (0.0,5,113.2) (0.0,4,368.3) (1 1.2, 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) 

### (1 1.9, 168.6) 

### (0.8, 175.1) (31.2, 159.9)

102January 2 1, 1999 Table A7. Cause-Specific 

### SMRs for Men Only Employed in Chemical Department (s) Using the U.S. as Comparison Population (n =485)

Observed Expected Cause

### ofDeath Deaths Deaths SMR 95% CI 

### All Causes of Death 32 44.1 72.5 (49.6, 102.4) 

### All Malignant Neoplasms 9 9.6 93.6 (42.8, 177.8)

Cancer ofBuccal Cavity and 0 0.3 

### --- (0.0, 1,396.4) Pharynx Organs and Peritoneum (0.0, 175.2) Cancer 

### ofDigestive 

### 0 2.1 

### ---

Cancer 

### ofEsophagus 

### 0 0.2 

## --- (0.0, 1,577.7) 

### (0.0, 1,195.2) Cancer of Stomach 

### 0 0.3 

### --- Cancer 

### ofLarge Intestine 

### 0 0.8 

### --- (0.0,488.5) (0.0,2,175.6) Cancer 

### ofRectum 

### 0 0.2 

### --- Cancer ofLiver and Biliary 0 0.2 

### --- (0.0,2,072.6) Passages Cancer ofPancreas 0 0.5 

### --- Cancer 

### ofAll Other 0 0.1 

### --- (0.0, 5,692.4) Digestive Organs Cancer 

### ofRespiratory System 5 3.6 141.0 (45.8, 329.0)

Cancer 

### ofLarynx 0 0.1 

## --- (0.0, 3,198.4) Cancer 

### ofBronchus 5 3.4 147.8 (48.0, 344.9) Trachea, and Lung Cancer 

### of Other 

### 0 0.1 

### --- (0.0, 7,763.4) Respiratory Organs 

### (0.0, 827.2)

Cancer 

### ofProstate 0 0.3 

### --- (0.0, 1,202.0)
Cancer 

### ofKidney 

### 0 0.3 

## --- Cancer 

### ofTestes and Other 0 0.1 

### --- (0.0, 3,501.4) Male Genital Organs 

### (0.0, 1,365.0) Cancer 

### of Bladder and Other 1 0.2 669.9 (16.7, 3,732.7) Urinary Organs

103January 2I, 1999 Table 

### A7. (Continued)
Observed Expected Cause 

### of Death Cancer 

### of Brain and Other Central Nervous System Cancer 

### ofThyroid and Other Endocrine Glands Cancer ofBone Cancer ofAll Lymphatic and Hematopoietic Tissue Lymphoma Hodgkin’s Disease Leukemia and Aleukemia Cancer of Other Lymphatic and Hematopoietic Tissue Malignant Melanoma

### of Skin All Other Malignant Neoplasms Diabetes Mellitus Cerebrovascular Disease All Heart Disease Hypertension Respiratory Ulcer 

### of Stomach and Duodenum Cirrhosis 

### ofLiver Nephritis and Nephrosis External Causes Accidents Motor Vehicle Accidents 

### All Other Accidents Suicides Homicides and Other External Deaths 1 0 

### 0 2

0 0 

### 0 0 7 

### 0 0 0 

### 1 

### 0 10 9 

### 5 4 1 

### 0 Deaths 

### 0.5 0.0 0.0 1.2 0.2 0.2 0.4 0.4 

### 0.3 0.8 0.6 1.3 13.1 0.1 1.9 

### 0.1 1.6 0.2 10.5 6.5 3.6 2.9 2.5 

### 1.5 SMR 95% 

### CI 222.6 (5.6, 1,240.3)



### --- (0.0,9,544.3)



### --- (0.0, 8,697.5) 174.0 (21.1, 628.7)



## --- (0.0,2,466.1) 

## --- (0.0,2,346.1) 226.3 (5.7, 1,260.7) 249.5 (6.2, 1,390.1)


## ---

e-- 

## --- *-- 53.4



## --- 62.3 95.0 139.4 138.0 140.2 40.0 

### --- (0.0, 1,200.4) 

### (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) 

### (0.0, 247.4) (0.0,455.3)

(1.0, 223.1) Causes Residual Causes* 3 4.7 63.4 

### (13.1, 185.3) Unknown Causes & 2 *all other causes of death combined & no death certificate obtained; included only in all causes 

### of death category

104 

## 000117January 2 1, 1999

Rat 

### Oral (diet) Carcinogenicityon N-EtFOSE Study Outline Study Objectives: Main Objective

### -To determine the carcinogenicity ofN-EtFOSE upon chronic oral administration. Secondary Objectives -

### To determine chronic and subchronic toxicity of N-EtFOSE after 52 and 13weeks, 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 aftervarious time-points of exposure. 

### GLP Status: 

### A 

### GLP study with appropriate QA audits; a signed QUA statement to be included in the finalreport. 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 ppmrecovery group (receive compoundfor 52 weeks followed by 52 weeks without compound). Number per Group: 50 males and 50 femalesper group plus additional animals for interim sacrifices at 4, 

### 13 & 52 weeks. (Note the 100ppm recovery group has 

### 40 males and 40 females). Test Article: N-EtPFOS furnished by Sponsorwho is responsible for compound identity and purity. Compound Administration: Daily administration for up to two-years (104 weeks) admixedin the diet. Clinical Observations: Twice daily Body Weights: Once a week for the first 16weeks; once every four weeks thereafter. 

### Food Consumption: Once a week for the first 

### 16weeks; once every four weeks thereafter. Hematology and Clinical Chemistry: Ten rats/sex from 

### 0, 1,3,30 & 100ppm groups at weeks 14,52and 104. Urinalysis: Ten ratslsex from 0, 1,3,30 & 

### 100ppm groups atweeks 14,52 and 104.

105January 2 1, 1999
Interim Necropsies: Five ratshex from 0, 1, 3, 30, 100 & 300 ppm groups at 

### 4and 14 weeks for 

### PCNA, palmitoyl 

### CoA oxidase, and compoundlevel samples. (Note samples for organ weights and histopathology taken at 14weeks.) Tenratslsex from 0, 1, 

### 3, 30 &IO0 ppm groups at 

### 52 weeks 

### for compoundlevels, organ weight, histopathology samples. Organ Weights: Measured at 

### 14and 

### 52 week interim necropsies. Histopathology: Microscopic examination of selected tissues at 14 and 

### 52 week animals. Complete tissue examination 

### of0 (one control group), 

### 3, 30, and 

### 100ppm 

### 104 weeks animals. Selected tissue examination 

### of 100ppm recovery animals. Histopathologic results will indicate extend 

### oftissue examination 

### of 1pprn 

### and second control group.

106 

## 0803-19January 2 1, 1999 Rat Oral (diet) Carcinogenicityon 

### PFOS Study Outline Study Objectives: Main Objective

### -To determine the carcinogenicity ofPFOS upon chronic oral administration. and 13 weeks, respectively, of compoundadministration. 

### 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. Secondary Objectives -

### To determine chronic and subchronic toxicity of after 52



### GLP Status: A GLP studywith 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 20ppm recovery group (receive compound for 52 weeks followedby 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 furnishedby Sponsorwho isresponsible for compound identity and purity. Compound Administration: Daily administration for up to two-years 

### (104 weeks) admixedin the diet. Clinical Observations: Twice daily. Body Weights: Once a week for the first 16weeks; once every four weeks thereafter. Food Consumption:Oncea week for the first 16weeks; once every four weeks thereafter. Hematology and Clinical Chemistry: Ten rats/sex from 0,0.5,2,5, & 20 pprn groups at weeks 14,52and 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 14weeks for PCNA, palmitoyl CoA oxidase, and compoundlevel samples. (Note samples for

107January2 1, 1999 organs weights 

### and histopathology taken at 

### 14weeks.) Ten rathexfrom 0, 0.5,2, 

### 5, & 

### 20 ppm groups at 

### 52 weeks for compoundlevels, organ weight, histopathology samples. Organ Weights: Measured at 14and 

### 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 

### 104weeks animals. Selected tissue examination 

### of 20 ppm recovery animals. Histopathologic results will indicate extend 

### oftissue examination

### of 0.5 ppm group.



# .

108January 21, 1999 Rat TeratologyRangefinderon 

### 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 auditswill not be done Animals: Mated female Charles River 

### CDrats Dose Groups: Seven 

### - control, 

### 0 mg/kg; low-1, 1mg/kg; low-2,5 mgkg; mid-1, 10 mg/kg; mid-2,20 mgkg; high-l,25 mg/kg; high-2,35 mg/kg Number per Group: 

### 8 mated females per group TestArticle: 

### 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 compoundsuspended 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 onhow dosing preparations were made. Clinical Observations: Twice daily; once approximately one hour aAer dosing and then 

### 4to 

### 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. Thenumber of implanations, the number of live and dead fetuses, and the number 

### ofearly and late resorptions will be determined and recorded. The fetuses will be sexed, weighed and examined for any gross abnormalities. The ovarieswill examined for the number of corpora lutea.

109January 2I, 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 ?) mgkg; high, (20 or 25 ?) mg/kg. after range finder study results are available. Final dose levels will be adjusted 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 ondays 6 thru 

### 17ofgestation. Samples of dosing preparations will be frozen and retained for possible analysisby the Sponsor. Records will be maintained onhow 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, 18and 20 

### of gestation Food Consumption: On days whenbody weights are obtained Cesarean Section: On day 20 of gestation the females will be anesthetized andthe uterus and its contents wilI be removedand weighed. Thenumber 

### of implanations, the number of live and dead fetuses, and the number of early and late resorptions will be determined andrecorded. 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 

### ofthe 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 Ked S staining method. 

## 000123 110January 2 1, 1999

Toxicokinetic Satellite Animals: Groups ofmated females, five females at the low dose 

### (1 or 

### 1.5mg/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 

### 18of 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 

### fiom 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.

111January2I, 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 femaleNewZealand 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, 1mg/kg; low-2, 5 mg/kg; mid- 1

### , 10 mg/kg; mid-2,25mg/kg; high-l,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 isresponsible 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 

### 7thru 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, 19,24and 29 

### of gestation 

### Food Consumption: On days whenbody 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 

## 000125January 2 1, 1999 Rabbit TeratologyStudy 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 (Notethe 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-EtFOSEwill be furnished by the Sponsor who is responsible for compound identity and purity. Reserve sample to be retained. CompoundAdministration: Oral intubation of compoundsuspended in water with 2% Tween 

### 80 on days 

### 7 

### thru20 of gestation. Samples of dosing preparations will be frozen and retained for possible analysisby 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 

### 6hours later. Body Weights: On days 0,7, 

### 10, 13, 

### 16, 19,24and 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 ofearly 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.

113January 2 1, 1999

Fetal Examinations: A mid-coronal slice will be made in the head of each fetus to evaluate the contents ofthe 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 ofthe each fetus will be determined. After removal ofthe 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 

### 2 1 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.January 2 1, I999 Rat 

### Two Generation Reproduction Study 

### ofN-EtFOSE Study Outline Study Objective: To evaluate the effect of oral administration of N-EtFOSE on the reproductive function ofmale and female rats 

### (Fo generation) and on the development and reproductive capacity ofthe subsequent

### F, 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, 1mg/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. Compoundadministration will start 4 weekprior to mating. Compound administration will continue through mating and in the females through gestation and lactation. The F1 pups will not receive any doses via gastric intubation. Samples of dosing preparations onweek 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 eo-inhabited during mating. Food Consumption: Weekly exceptwhen 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 compoundadministration, within a dose group one male will be co-inhabited with one female. A female will be determined as mated upon the

115January 2 1, 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 10of 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 ofthe 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: Theremaining 25 females will continue until term and be allow to deliver their litters. The offspring (F1 generation) will be counted, sexed and weighed on postnatal days 0,4, 7,14 and 2

### 1. 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. F1 Dosing: After weaning at day 21 of lactation, the F1 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 

### ofthe 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 

## 000.129January 2 1, 1999



### F1 Pup NeurologicalTesting: During the 

### 4'hweek postpartum, the 

### F1 pups will be evaluated in a passive avoidancetest for learning and short term memoryretention. During the 

### 1 Othweek postpartum, the 

### FIpups will be evaluated in a water-filled maze for neuromuscular coordination, learning, and longer term more complex memory. 

### F1 Growth and Reproduction: The F1 pups will be weaned after 21 days of lactation and litters will be culled to one of each sex. Thepups will then allow to grow and body weights and food consumption will be recorded weekly. The 

### F1 pups will undergo behavioraUfimctiona1testing. At sexual maturity, within 

### a dose group, onemale 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 oflactation.

117 

## 0140130January 21, I999 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 

### ofthe 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 mgkg; high, 5 or 

### 8 mg/kg. Number per Group: 35 males and 35 females per group Test Article: 

### PFOS will be furnished by the Sponsorwho 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. Compoundadministration will start 

### 4 weekprior to mating. Compound administration will continue through mating and in the females through gestation and lactation. The F1 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: Weeklyexcept 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 compoundadministration, within a dose group onemale 

### will be co-inhabited with one female. A female will be determined as mated upon the

118January 2 1, 1999
presence of sperm positive vaginal smear. The day of mating shall be considered as day 0 

### of gestation. Early Gestation Evaluation: 10females 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 ofthe males in each dose group. These samples will be frozen and shipped to the sponsor for possible analysis. Female Parturition: Theremaining 

### 25 females will continue until term and be allow to deliver their litters. The offspring 

### (F1 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 ofthe discarded pups fiom 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. 

### F1 Dosing: After weaning at day 

### 2 1of lactation, the 

### F1 pups Will receive compoundby 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 ofthe 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.January2I, 1999



### F1 PupNeurological Testing: During the 

### 4'h week postpartum, the F1 pups will be evaluated in a passive avoidance test for learning and 

### short term memoryretention. During the loth week postpartum, the 

### FIpups will be evaluated in a water-filled maze for neuromuscular coordination, learning, and longer term more complex memory. 

### F1 Growth and Reproduction: The 

### FIpups 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 

### FIpups 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.January 2 1, 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 femaleNew Zealand White rabbits (Notethe 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,O.l mg/kg; low-2, 

### 1mg/kg; mid-l,2.5 mg/kg; mid-2,5 mgkg; high-1, 10mg/kg; high-2,20 mgkg Number per Group: 

### 5 mated females per group Test Article: N-EtPFOSwill be furnished by the Sponsor who is responsible for compound identity and purity. 

### No reserve sample will be required. Compound Administration: Oral intubation ofcompound 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, 19,24and 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 

### ofimplanations, the number of live and dead fetuses, and the number of early and late resorptions will be determined and recorded. The fetuseswill be weighed and examined for any gross abnormalities. The ovarieswill examined for the number of corpora lutea.

121January2 1, 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 femaleNew 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

### , 1mg/kg; mid-2,5 mg/kg; high, 10mg/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 furnishedby the Sponsor who is responsible for compound identity and purity. Reserve sample to be retained. CompoundAdministration: Oral intubation 

### of compoundsuspended inwater with 2% Tween 

### 80 

### on days 

### 7thru 

### 20 

### of gestation. Samples of dosing preparations will be frozen and retained for possible analysisby the Sponsor. Records will be maintained onhow 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, 19,24and 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.

122January2 1, 1999

Fetal Examinations: 

### A mid-coronal slice will be made in the head of each fetus to evaluate the contents ofthe 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 

### ofthe each fetus will be determined. After removal of the viscera, the carcasses will processed for skeletal examination. Toxicokinetic Satellite Animals: Groups ofmated females, fivefemales at the low dose 

### (0.1 mg/kg) and five females at the high dose 10mag) plus three females at other dose levels, will be dosed. On the day after the last dose (day 

### 2 1 of gestation) the satellite females will be killed and following samples will be collected: serum and liver fromthe 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.

123January 2 1, 1999 Rabbit Teratology Rangefinderon 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 femaleNew Zealand White rabbits (Notethe 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, 1mg/kg; low-2, 5 mg/kg; mid- 1

### , 10 mg/kg; mid-2,25 mgkg; high-1,50 mg/kg; high-2,75 mg/kg Number per Group: 

### 5 mated females per group TestArticle: N-EtPFOSwill be furnished by the Sponsor who 

### is responsible for compound identity and purity. 

### No reserve sample will be required. Compound Administration: Oral intubation ofcompound suspended in water with 2% Tween 80 ondays 

### 7thru 20 ofgestation. Analysis of dosing preparation will not be done; however, records will be maintained onhow 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, 19,24and 

### 29 

### of gestation 

### Food Consumption: Ondays whenbody weights 

### are obtained Cesarean Section: On day 29 of gestation the females will be anest,etized 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 ofearly 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 ofcorpora lutea.

124January 2 1, 1999 Rabbit TeratologyStudy 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 NewZealand White rabbits (Notethe 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 mgkg; low, (1 ?) mgkg; mid-1, 

### (5 ?) mgkg; mid-2, 

### (10 ?) mgkg; 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 funishedby the Sponsor who isresponsible 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 

### 7thru 

### 20 ofgestation. Samples of dosing preparations will be frozen and retained for possible analysisby the Sponsor. Records will be maintained onhow 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, 19,24and 29 of gestation 

### Food Consumption: On days whenbody 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. Thenumber 

### 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.

125January 2 1, 1999

Fetal Examinations: A mid-coronal slice will be made in the head of each fetus to evaluate the contents ofthe cranium. The internal organs ofthe 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 SatelliteAnimals: Groups ofmated females, five females at the low dose 

### (1 ? mgkg)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 andplacentas 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.

126January 21, 1999
CorporateToxicology Study Outline
Title: 1) Effect ofPerfluorochemicals on Bioenergetic Metabolism: Phase I1 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 characterizationbased on animal data; 

### 2) Comparethe metabolic response ([ATP], respiration rate, mitochondrial membrane potential, markers ofperoxisome Proliferation, et al.) ofrat, guinea pig and primate hepatocytes to this class of compoundswith particular emphasis onmitochondrial and peroxisomal metabolism to: a) Further test the proposed mechanismof toxicity; b) Identify potentially useful metabolic biomarkers 

### ofexposure; c) Help validate relative differencesbetween species (assess markers ofperoxisome proliferation across species); 3) Compare molecular response (gene expression 3 

### mRNA) ofrat, guinea pig and primate hepatocytes exposed to these chemicals (genes indicative of cell and peroxisome proliferation) to: Identify molecular biomarkers of exposure; Facilitate species extra[polation; Discriminate between proliferation ofperoxisomes and cell proliferation (ongogenic response) to test dogma; Assess the relevance ofrat tumorigenic response to potential human cancer response; Provide biomarkers for potential use in monitoring and interpreting traditional toxicity studies. Significance: Comparing sensitivity between species will allow forjudging the most appropriate species for predicting humanhealth outcomes following exposures to compoundsand has relevance to establishing safety factors and putting human cancer risk in perspective; Species differences in metabolic and molecular response will reveal important insight into mechanismsresponsible for compound-induced peroxisome proliferation and/or tumorigenesis which is also vital to identifying valid biomarkers to assess exposures and potential risks; Comparingthe enhanced transcription and expression of genes associated with peroxisome versus cell proliferation will allow opportunities to evaluate the

127January 2 1, 1999 relationship between metabolic and oncogenic effects of these compounds in different species. 

### 0

b

jective(s): 1. Establish hepatocyte cell cultures from different species as models for assessing the 2. Develop molecular probes for assessing compound-induced transcription of genes 3. Establish benchmarks for assessing the effects of test compounds onhepatocyte metabolic and mitogenic effects test compounds; related to peroxisome metabolism and cell proliferation; bioenergetics and cell proliferation in culture. Protocol: 

### 1) Establish stable primary hepatocyte cultures from several species including rat, guinea 

### 2) Develop molecular probes for compound-initiated transcription of specific genes pig and primate; related to mechanistic endpoints: a) ID genes of interest. b) 

### ID sequences 

### ofhigh homology between species using Gene

### Bank@ c) Designprimers to 

### 1 

### S 

### -3.0kbp sequences (Oligo@) d) PCR amplify sequences from rat-liver cDNAtemplate e) Separate, bands on 

### low melting point gels f) Purify bands (band-stab) g) Restriction digest to confirm identity (MacDNAsisBto obtain restriction site map h) Label probe with 32P-dCTP by randompriming (High Prime@) 3) Treat sample (cells or flash fiozen 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 a) CPTl b) ACoAO c) PPARa d) FABP e) Also, possibly Catalase,LPL, Aromatase, HMG-CoA synthetase a) PCNA b) 

### CDK bioenergetics and cell proliferation in culture a) Effect of exposing cells in culture on induction ofperoxisomal metabolism and for each probe)



### 4) MRNA probes suggested for peroxisome proliferation



### 5) Probes for cell proliferation 

### 6) Establish benchmarks for assessingthe effects ofPF compounds on hepatocyte
stimulation of cell proliferation using biochemical markers 

### and molecular markers (e.g., mitochondroial enzyme activities and cytochrome content, Adenine

128 

## 000141January 2I, 1999 nucleotides, AcoAO activity, 

### PCNA,apoptosis (morphometric 

### and TUNEL), CyQuant cell proliferation analysis,

### CPTl ,AcoAO, 

### PCNA, 

### CDK) Timeline: 31 months beginning December 

### I, 

### I998 and endingJune 

### 30,2001 Report: Quarterly progress reports