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Perfluorooctane Sulfonate: 

Current Summary of Human Sera, 

Health and Toxicology Data 

3M 

January 21,1999 

I 1 000014 
January 2 1. 1999 

TABLE OF CONTENTS 

EXECUTIVE SUMMARY ........................................................................................................................... 3 

I . INTRODUCTION .................................................................................................................................... 6 

I1 . HISTORICAL REVIEW AND CURRENT FINDINGS OF FLUOROCHEMICALS IN HUMAN 

SERA ....................................................................................... .................................................................... 11 

111 . SUMMARY OF MEDICAL SURVEILLANCE AND EPIDEMIOLOGY STUDIES .................. 22 

IV . SUMMARY OF TOXICOLOGY DATA ON PFOS ........................................................................ 30 

V . RISK CHARACTERIZATION ............................................................................................................ 64 

VI . CURRENT AND PLANNED RESEARCH ....................................................................................... 69 

VI1 . REFERENCES ................................................................................................................................... 81 

APPENDIX .................................................................................................................. 86 

2 
January 2 1,1999 

Executive Summary 

3M has prepared this document to summarize the data related to the biological effects of 

perfluorooctane sulfonate (PFOS). It also presents current thinking on human health risk 

related to PFOS and includes information about future study plans. 3M Medical 

Department scientists and physicians, in consultation with outside experts, are the 

authors. 

PFOS has been found at tens of parts per billion levels in serum samples of 

nonoccupationally exposed employees, in commercially available human serum and in 

pooled samples from multiple blood banks. PFOS is an eight-carbon molecule that is 

perfluorinated except for the sulfonate group on the terminal carbon. 3M has 

manufactured PFOS and molecules that may be metabolic precursors to it since 1948. 

Routes of exposure to PFOS or precursor molecules are not well understood at this time. 

PFOS is an example of an “organic” fluorine molecule. Human serum has been known to 

contain organic fluorine molecules for over 30 years. The primary constituent of this 

organic fluorine fraction was tentatively identified as another molecule 

(perfluorooctanoate) in 1976. Current analysis of stored sera samples fiom a variety of 

sources are more consistent with PFOS being a major fraction of this organic fluorine. 

Improved analytic techniques allowing a relatively rapid analysis at low levels of 

detection make the current analyses possible. These analytic techniques were first 

available for use in medical surveillance of exposed workers in 1992. Detection limits 

have been lowered to allow the more recent analysis of serum from those without 

occupational exposure. 

Medical surveillance has been done among 3M employees occupationally exposed to 

PFOS precursors for over 20 years. To date, no adverse health effect associated with 

PFOS exposure has been found in these employees. This conclusion applies at sew 

levels up to 6 parts per million, about 100 times higher than levels seen in the general 

3 
January 2 1, 1999 

population. PFOS has a long residence time in the human body. In three retirees, the 

half-life in human sera ranges from 1 100 to 1500 days. A mortality study at the U.S. 

plant primarily involved with production of PFOS related materials has found no 

significantly elevated standardized mortality ratios (SMRs). 

Toxicology studies show that PFOS is well absorbed orally and distributes primarily in 

the serum and liver. It does not appear to be further metabolized. Some enterohepatic 

circulation of PFOS occurs, based on the observation of increased excretion in rats given 

cholestyramine. Elimination from the body is slow and occurs via both urine and feces. 

Mutagenicity testing is negative in five salmonella species. It is not genotoxic in a mouse 

bone marrow micronucleus assay. The acute LD50 in rats is 250 mg/kg (moderately 

toxic). It does not produce dermal or ocular toxicity. 

Subchronic studies have been done in rats and primates. PFOS causes liver enzyme 

elevations and hepatic vacuolization in rats, and hepatocellular hypertrophy at higher 

doses. Higher doses also cause other GI toxicity, hematological abnormalities, weight 

loss, convulsions, tremors and death. Monkeys show anorexia, emesis, diarrhea, 

hypoactivity and at higher doses prostration, convulsions and death. Atrophy of exocrine 

cells in salivary glands and the pancreas, and lipid depletion in the adrenals is found at 

high doses in the monkey. 

The serum levels at which these compound related effects occurred in these early rhesus 

monkey studies are unknown. In a recently completed rangefinder study in cynomolgus 

monkeys the first observed biological effect was a decrease in serum cholesterol, first 

observed at a serum level of 72 ppm in one of the two monkeys in the high dose group. 

Using the relationship between cumulative dose and serum level found in this study, it 

can be estimate that significant toxicity occurred at 700 to 800 ppm in the early rhesus 

monkey studies, and death at 1 100 ppm and above. More complete quantitative 

absorption, distribution and excretion data for PFOS is being obtained. 

4 
January 2 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. The purpose of this research is to explore the potential for chronic 

and reproductive effects, understand toxic mechanisms and obtain a better understanding 

of absorption, distribution, metabolism and excretion. The plan is to make as much use 

as possible of observational data in exposed workers and to establish no effect levels in 

both rats and primates for endpoints of importance. 

5 
January 2 1, 1999 

I. INTRODUCTION 

Evidence that organic compounds containing the element fluorine covalently bonded 

to carbon (organic fluorine compounds, OF) can be found in human sera has been 

available for 30 years. Although all of the specific compounds contributing to the 

total amount of OF present are not identified, it now appears that a compound called 

perfluorooctane sulfonate constitutes a significant fraction. Recent data provide 

evidence that PFOS is present at tens of parts-per-billion (ppb) levels in serum 

samples from the general population, averaging 30 ppb in blood bank samples from 

diverse locations in the U.S. Single digit parts per million (ppm) levels 

(approximately 100 times greater) are found in individuals occupationally exposed to 

PFOS and its precursors, averaging 2.0 ppm among participating employees at the 

primary U.S. manufacturing location for these compounds. 

3M produces perfluorinated molecules by mixing anhydrous HF and hydrocarbon 

feed stock in an electrochemical cell (electrochemical fluorination). Perfluorooctane 

sulfonyl fluoride (POSF) is the cell product from which a group of products is 

developed: 

I 

CF3(CF2)6 C - SO2 - F 

I 

F 

POSF 

6 
January 2 1, 1999 

Other moieties (“R”) are added to the sulfur, which leads to the creation of materials 

that may be polymerized or esterified. The vast majority of POSF produced is used in 

this way. 

F 

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 hrther metabolized. Some PFOS is 

produced and sold directly into industrial applications as a surfactant. This, however, 

amounts to only a small fraction of total POSF production. 

F 

I 

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. 1 shows the chemical structure of N-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 intermediates not shown in this figure.) 

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In vitro studies in both rat and human hepatocytes lead to identification of the same 

compounds and hypothesized intermediates. Compound VI11 has been detected in 

some samples of pooled human sera. A single dose absorption, distribution, 

metabolism and excretion study of N-Et-FOSE in cynomolgus monkeys is through the 

in life phase and tissue analysis is pending. N-Et-FOSE is esterified to produce larger 

molecules that are used on paper and packaging for oil and water repellency. 

It is presumed that N-Me-FOSE, in which a methyl group replaces the ethyl group on 

the nitrogen, has a similar metabolism. N-Me-FOSE becomes part of very large 

molecules that act as protective chemicals on fabrics, leather and rugs. 

Outside of the occupational setting, routes of human exposure to PFOS or its 

metabolic precursors are not understood, but are the subject of intense study. 

Exposure could occur from environmental releases of PFOS or its precursors at the 

Decatur, Alabama and Antwerp, Belgium manufacturing sites. It could occur from 

the environmental or biological degradation of products to PFOS or molecules 

metabolized to PFOS. Products also contain small amounts (generally less than a few 

percent) of residuals, such as N-Et-FOSE and other molecules found in Figure I. 1 , 

which are known or suspected metabolic precursors to PFOS. These residuals 

represent a source of PFOS that would not require environmental or biological 

degradation of large molecules. Downstream industrial users of POSF based products 

are also potential sources of environmental releases of PFOS or its precursors. The 

relative contribution of these various sources to population exposure is currently 

unknown. 

Another surfactant is known to be found in the sera of employees and was reported in 

general population sera samples in 1976 (Taves). This is perfluorooctanoic acid, or 

PFOA: 

9 
January 2 1, 1999 

PFOA is also made by electrochemical fluorination. It is mentioned here because 

following Taves' report in 1976 it was presumed to be a significant fraction of the 

total organic fluorine found in the sera samples analyzed by Taves and others. This 

report was a stimulus for investigation and subsequent medical surveillance of 

employees in fluorochemical production, including those producing POSF based 

materials. It should be recognized, however, that PFOA is a different and unrelated 

compound. It does not metabolize to or become PFOS. It is likely that PFOA was 

misidentified as a major fraction of organic fluorine in the 1976 Taves paper. The 

evidence for this is discussed in Section 11. 

The purpose of this report is to describe the data on PFOS levels in human sera, and 

to discuss the potential for those levels to affect health based on current scientific 

knowledge. A review of current findings and historical information on PFOS levels 

in sera is presented in Section 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 of the serum findings in light of the available health effects 

data. 

3M is actively developing further human health and toxicological information. 

Section VI outlines the current research agenda. 

10 080023 
January 2 1, 1999 

11. HISTORICAL REVIEW AND CURRENT FINDINGS OF 

FLUOROCHEMICALS IN HUMAN SERA 

The data on fluorochemicals in human sera is presented in this section. The presence 

of organic forms of fluorine in human serum was observed 30 years ago, and PFOA 

was thought to account for most of this fraction. 3M finds little current evidence to 

support this view. Evidence is presented below that PFOS is more likely to be 

consistently found in sera. Based on the limited data provided by historical samples, 

there is no evidence of significant change in PFOS concentration in serum samples 

taken over the last two to three decades. 

The advancement of analytical chemistry technology has had a significant influence 

on our knowledge of fluorocarbons in human sera. The techniques developed and 

used by researchers in the 1960's and 1970's were time intensive, requiring hours for a 

single analysis. The methods were also nonspecific, measuring organic fluorine 

(fluorine covalently bonded to carbon) rather than specific molecules. The 

development of a rapid analytic technique in the late 1970's decreased analytic time to 

under an hour, allowing large scale medical surveillance of production employees at 

higher detection limits (about 0.5 parts per million organic fluorine) that were 

adequate for the levels found in occupationally exposed individuals. The 

advancement of chromatographic/mass spectroscopy technology enabled rapid 

analysis of specific fluorochemicals from small volumes of sera in the early 1990's. 

This technology was first used in medical surveillance in 1992. Detection limits for 

PFOS were lowered to 50 parts per billion by 1997. The first report to 3M of PFOS 

in commercially available pooled sera occurred in late summer of 1997, prompting 

more research into the technique, and confirming its validity over a period of several 

months. 

11 

I 

000024 
January 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% of the molecular weight of 

PFOS. The contribution of a PFOS value to organic fluorine, in ppb, will therefore be 

[0.65 x (PFOS value in ppb)]. Conversely, if the measured organic fluorine is 

entirely PFOS, the value of PFOS in ppb will be [1.54 x (OF level in ppb)]. 

This section presents 1) a brief summary of the historical information regarding 

organic fluorine in human sera, 2) data from 3M employees involved in 

fluorochemical production, 3) data from a small group of non-occupationally exposed 

3M employees, 4) data from commercially available human pooled serum samples, 5) 

data from pooled sera from 18 regional blood banks and 6) data from current analysis 

of stored serum samples. 

Historical Finding of the Organic Form of Fluorine in Blood 

Taves (1 96Sa) described two forms of fluorine in serum, one that was exchangeable 

with radioactive fluorine- 18 and one that was not. Pothapragada et. al. (1 97 1) 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-exchangeable or nonionic fluorine was “organic”, 

i.e. covalently bound to carbon (Taves et. al., 1976). Using NMR spectroscopy, these 

authors tentatively identified a component of the organic fluorine as perfluorooctanoic 

acid (PFOA). There was some variation in the observed spectra from an authentic 

sample of PFOA, however, leading the authors to suggest that branching, or the 

presence of a sulfonate, was possible. 

A number of studies over the past 25 years reported levels of organic fluorine in 

human blood serum. Table 11.1 presents the study author, level measured, population 

studied and methods of analysis. The variety of methods used for determination of 

12 00002s 
January 2 1, 1999 

fluorine suggests that some caution be used in interpreting results. All reported 

means were in the tens of part per billion levels. The average of reported values from 

United States sources is 37.6 ppb. 

Table 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 0 bomb us 

1976 Guy, Taves 25 106 ash us 

1978 Belisle 20 9 0 bomb us 

1979 Singer 45 264 ash us 

1980 Ubel 45 4 modObomb US 

1980 

1981 Belisle 11 8 0 bomb China 

1989 Y amamota 32 11 LOPA Japan 

Paez 85 pooled ash Argentina 

* Organic fluorine, specific identities not provided. 

** Varied methods were used to measure organic fluorine. See papers for details. 

Occupationally Exposed Employees 

3M has produced PFOA (the ammonium salt) by electrochemical fluorination since 

the early 1950’s. It is a surfactant used in fluoropolymer production. The company 

began medical monitoring of employees involved in PFOA production in 1976, by 

measuring serum levels of organic fluorine (OF) and performing medical assessments. 

Employee monitoring was expanded significantly in 1980 following the development 

of a more rapid test for organic fluorine. Measured serum levels of OF in these 

employees averaged less than 10 parts per million (ppm). 

As noted earlier, PFOS is a surfactant used as a wetting and foaming agent in 

industrial and commercial processes. Certain fluorochemicals that may transform 

metabolically to PFOS have been produced since the early 1950’s by electrochemical 

fluorination. Since 1980 this has occurred at primarily one location in the United 

States (Decatur, Alabama). This 3M site consists of a fluorochemical plant and a film 

plant that are physically separate entities. Chemical plant employees have been 

offered a medical monitoring program that includes standard medical testing as well 

13 0006926 
January 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 of high performance liquid chromatography-mass spectrometry, 

serum PFOS was measured in 1994 and 1997 (see also Figure 11.1). In these two 

years, mean PFOS levels were 2.44 ppm (range 0.25 - 12.83 ppm) and 1.96 ppm 

(range 0.10 - 9.93 ppm), respectively. 

Among employees with six or more 

Another 3M plant in the United States where PFOS has been measured in employees' 

serum is Cottage Grove, Minnesota. Some PFOS is manufactured at this plant. In 

1997, the mean serum level of PFOS among 74 Cottage Grove fluorochemical 

production employees was 0.82 ppm (range 0.05 - 6.25 ppm). Outside the United 

States, 3M manufactures PFOS related materials at its Antwerp, Belgium plant. 

PFOS levels were measured in Antwerp employees in 1995 (mean = 1.9 ppm, range 

0.0 - 9.9 ppm) and in 1997 (mean 1.5 ppm, range 0.1 - 4.8) ppm. 

The cross-sectional stratified analysis presented in section 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 facility was not included because little PFOS related product is 

manufactured there. It is also the primary site for PFOA production. 

We are aware of one occasion in 1979 where the serum of 5 Decatur employees was measured 

for PFOS by electron capture gas chromatograph and microwave plasma detection methods 

[Central Analytical Laboratory, 19791. Total serum organic fluorine levels for these five employees 

were 10.1, 5.7, 9.4, 11.8 and 4.1 ppm. The percent of PFOS found was 60%, 70%, 80%, 55% 

and 65% of the total serum organic fluorine levels, respectively. 

1 

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Non-Occupationallv Exposed 3M Employees 

A total of 3 1 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 fluorochemical research and development. Samples were from five 

females and 26 males. Employees ranged in age from 37 to 62 years. All employees 

had measurable PFOS in their blood serum (mean = 47 ppb; range = 28 to 96 ppb). 

Age was significantly associated with increased serum PFOS and accounted for 24% 

of the variance in PFOS levels. There was no gender-related difference if age was 

considered. Only four employees had PFOA measured above the detection limit of 10 

ppb. The average of these four PFOA measurements was 12.5 ppb. Twelve 

employees were re-tested eight weeks later to check for reliability of the analytical 

method. The findings suggested reliability (R2 = .94) in the range of quantification 

(Figure A1 in the Appendix). 

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Commercial Pooled Serum Samples 

Six pooled sera samples, obtained from two commercial sources (Intergen and Sigma), 

were tested in 1998. No information was available about the donor pool, such as age, sex 

or geographical location. Samples from Intergen came from donor pools consisting of 

approximately 500 individuals. Three Intergen pools showed PFOS levels of 43,44 and 

44 ppb (Table 2A.2). Pools from another source, Sigma, were from an unknown number 

of donors. The pools from which the samples were drawn were 50 liters, suggesting a 

minimum of 200 donors. The three Sigma pools contained PFOS at levels of 26,28 and 

45 ppb. 

Blood Bank Pooled Sera 

Eighteen blood banks from various geographic areas across the continental United States 

and Alaska each donated three to six pooled samples that had from 5 to 10 donors per 

pool (Table 11.3). Altogether there were 68 total pools, representing 340 to 680 individual 

donors. All pools contained detectable levels of PFOS in tests performed in 1998. The 

range found in the pooled serum was 9 to 56 ppb of PFOS (see table on next page). The 

location means ranged from 14 ppb in Santa Barbara, California to 52 ppb in Greenville, 

South Carolina. PFOA levels were detected in 20 (about one-third) of the pooled samples 

but quantifiable in only two samples (12 and 22 ppb). 

The blood bank samples from the regional blood banks are not a statistically valid sample 

of the U.S. population, but do provide, to date, the best estimate of mean PFOS sera 

levels. The overall mean was 29.7 ppb of PFOS. If one wishes to make a comparison 

with the organic fluorine levels in the published literature, this level of PFOS would 

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

approximate mean of historic organic fluorine measurements from published literature is 

37.6 ppb. If this were all PFOS, a PFOS sera level would have been 57.9 ppb (1.54 x 

37.6). 

18 

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January 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 

Santa Barbara, CA 

Santa Rosa, CA 

Scottsdale, AZ 

# of Pooled Samples Range Average 

4 19-3 1 24 

3 18-26 23 

3 13-35 23 

3 26-32 29 

3 28-40 32 

3 20-27 24 

4 18-3 1 26 

3 47-56 52 

3 24-3 5 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 of historical samples have been analyzed for PFOS in 1998. These are 

summarized in chronological order below. 

Korean War era US. military recruits, 1948 to 1951. Ten pooled samples consisting of 

ten individual samples each were measured. All were below 1 ppb, the limits of 

detection. 

Swedish samples, 1957. Ten individual samples ranged from below limit of detection to 

4.1 ppb. Two were below detection limit (1 ppb). The mean was 1.98 ppb. 

Michigan Breast Cancer Study, 1969 - 1971. Five individual samples ranged from 11.8 

to 59.4 ppb. The mean was 33.4 ppb. 

19 000032 
January 2 1, 1999 

Swedish samples, 1971. Ten individual samples ranged from below detection limit (1 

ppb) to 2.8 ppb. Three were below detection limit. The mean was 1.13 ppb. 

MRFITpooled calibration samples, 1976. MRFIT (Multiple Risk Factor Intervention 

Trial) was a cardiovascular risk reduction program conducted in the 1970’s and early 

1980’s. The number of donors per pool is unknown. Six pooled calibration samples 

ranged from 13.7 to 55.5 ppb. The mean was 30.9 ppb. 

MRFITpooled calibration samples, 1980. Three pooled calibration samples ranged from 

13.8 to 40.5 ppb. The number of donors per pool is unknown. The mean was 25.5 ppb. 

China samples, 1984. Six individual samples from Linxian province in rural China were 

all below limits of detection or quantitation. The samples were from an NCI study on 

nutrition and cancer prevention. 

MRFIT individual samples, 1985. Three individual samples from participants in the 

MRFIT study were obtained. One was below limits of quantitation (5 ppb), the other two 

were 43.3 and 43.9. Assuming the LOQ for the low sample, the mean was 30.7. 

China samples, 1994. Six individual samples from Shandong province in rural China 

were all below limits of detection or quantitation. The samples were from an NCI study 

on nutrition and cancer prevention. 

In addition to the PFOS measurements described above, PFOA was also analyzed in these 

historical samples. PFOA was not found in any historical sample at a detection limit of 

10 ppb. 

Comment 

20 
January 2 1, 1999 

Organic fluorine has been noted in human serum since the late 1960's. We have now 

identified PFOS as a part of this organic fluorine fraction. PFOS-related materials were 

not produced commercially prior to 1948, and only in small quantities for several years 

thereafter. Therefore, it is not surprising that samples from 1948 to 195 1 show 

undetectable levels. There was clearly an increase 20 years later; however the very 

limited data shows no further upward trend despite steadily increasing production 

volumes since this time.' Due to limited knowledge on distribution of this chemical in the 

body it is uncertain that body burden is adequately reflected by serum levels. Information 

on distribution and kinetics is needed to shed further light. The available data on this 

topic will be presented in the toxicology section. It would also be of interest to measure 

total organic fluorine and PFOS in the same sample to determine how much PFOS 

currently contributes to the total organic fluorine content. 

PFOA may have been misidentified as a major component of organic fluorine in 1976 

(Taves, 1976). Although detectable in some samples, neither historic nor current samples 

confirm this as a major fraction, except in occupationally exposed employees. 

21 000834 
111. SUMMARY OF MEDICAL SURVEILLANCE AND EPIDEMIOLOGY 

STUDIES 

Epidemiologic Investigation of Clinical Chemistries, Hematology and 

Hormones in Relation to Serum PFOS Levels in Male Fluorochemical 

Production Employees 

Medical surveillance has been routinely performed on 3M fluorochemical production 

workers (in Decatur, Alabama and Antwerp, Belgium) with potential exposure to 

PFOS and/or to perfluorinated precursors that may metabolically degrade to PFOS. 

A recent study (Olsen et al., 1998) provided an analysis of hematology, clinical 

chemistries and hormonal parameters in relation to serum PFOS as determined by 

high performance liquid chromatography mass spectrometry methods. These 

relationships were assessed in fluorochemical production employees from two time 

periods, 1995 (N = 178) and 1997 (N = 149). 

the mean serum PFOS levels were 1.93 and 2.44 ppm, respectively. In 1997, the 

mean serum PFOS levels were 1.48 and 1.96 ppm, respectively. 

In 1995, for Antwerp and Decatur, 

Descriptive simple and stratified analyses, Pearson correlation coefficients, analysis 

of variance and multivariable regression were used to evaluate for possible 

associations between PFOS and each hematological and clinical chemistry test and 

hormonal assay. Age, body mass index, current alcohol consumption (drinks per 

day) and cigarette use (cigarettes smoked per day) were potential confounding factors 

that were considered in the analyses. 

Four categorizations of serum PFOS levels were assessed in relation to the response 

variables: 0 - < 1 ppm; 1 - < 3 ppm; 3 - < 6 ppm; and 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, 1.82 ppm, 4.12 ppm and 8.17 ppm, 

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

gamma glutamyl transferase, aspartate aminotransferase, alanine aminotransferase, total and direct bilirubin, 

blood urea nitrogen, creatinine, glucose, cholesterol, low density lipoproteins, high density lipoproteins and 

triglycerides, cortisol, dehydroepiandrosterone sulfate, estradiol, follicle stimulating hormone, 17-alpha 

22 
January 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 of the 

employees’ serum PFOS levels were below 6 ppm. The two plant populations 

differed by age, body mass index and alcohol consumption which resulted in 

differences, as expected, in several clinical chemistry parameters (Table 111.2). When 

analyzed in aggregate, the two plant populations showed no consistent significant 

associations for both years between the clinical chemistries and hematology 

parameters and the employees’ serum PFOS levels (Figure A1 , Appendix). Total 

bilirubin levels appeared to trend downwards but upon further analysis this was 

restricted to Decatur employees and the values were all within the reference range. 

Multivariable regression models were fitted with PFOS analyzed as a continuous 

variable using linear as well as non-linear transformations in order to maximize the 

possibility of finding associations between PFOS and the parameters of interest 

adjusting for potential confounders. No consistent associations were observed by 

plant and/or by year. In 1995, hormone values were also obtained from a subsample of employees with the higher PFOS measurements. After adjusting for age 

and body mass index, no significant associations were observed between hormones 

and serum PFOS levels. 

The findings from this study suggest that, among these Antwerp and Decatur male 

fluorochemical production employees, significant hematological, clinical chemistry 

and hormonal abnormalities were not associated with serum PFOS levels up to 6 

ppm. It was not possible to derive inferences from the few employees with serum 

PFOS levels 3 6 ppm. Limitations of this study included its cross-sectional design, 

the voluntary participation rates and the few subjects with levels >_ 6 ppm. 

hydroxyprogesterone, luteinizing hormone, prolactin, sex hormone binding globulin, free testosterone, 

bound testosterone, and thyroid stimulating hormone. 

23 000036 
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Table 111.2 

Mean Values of PFOS, Demographic, Serum Chemistry and Hematologic Parameters for 

Antwerp and Decatur, 1995 and 1997 Examinations 

1995 Data 1997 Data 

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 

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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*** 

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26" 

27 

31 

0.80*** 

0.15*** 

14.9 

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81*** 

206 

134 

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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 000038 
January 2 1, 1999 

Table 111.2 (Continued) 

1995 Data 1997 Data 

Variable Antwerp Decatur Antwerp Decatur 

Hematocrit 47" * * 46 46* 45 

Hemoglobin 15.4 15.2 15.4 15.3 

FU3C 4.9 5.0 5.1 5.1 

MCH 3 1.4""" 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 

Platelets 224 229 237" 217 

* p < .05; ** p < .01; ***p < .001 (Antwerp compared to Decatur) 

26 000039 
Mortalitv Study of Emplovees at the 3M Plant in Decatur, Alabama 

A retrospective cohort mortality study of 1,957 employees who worked at least one 

year at the 3M plant in Decatur, Alabama was conducted by epidemiologists at the 

University of Minnesota School of Public Health (Mandel and Johnson, 1995). The 

purpose of this study was to determine whether the mortality experience of these 

employees was significantly different from that which would be expected in a 

comparable population. The cohort was followed from March 1 , 196 1 through 

December 3 1 , 199 1. Table A2 (Appendix) provides a description of the final 

employment and 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. The two plants are physically distinct entities (approximately 300 yards 

apart) located at the Decatur site. The vital status of 99.7% of the cohort was 

identified. The male and female employees contributed 33,108 and 4,807 personyears experience, respectively (Table A3, Appendix). The majority of the male 

employees were hired between 1965 and 1974 (Table A4, Appendix) whereas the 

majority of the female employees were hired between 1975 and 1979 (Table A5, 

Appendix). Altogether there were 74 deaths (70 males and 4 females). Among 

males, SMRs were below the null value for all major causes of death regardless of 

the comparison population used to calculate the expected values (Table 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 conducted by duration of employment 

within the chemical plant. 

ever (n = 1 , 1 16) and only (n = 547) employed at the Decatur film plant (data not 

shown). None of these analyses resulted in significantly elevated SMRs. There 

were four deaths among female employees (SMR = 62.6; 95% 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 000040 
January 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 of Decatur plant employees be updated in 1998 which would extend the 

vital status search, via National Death Index records, through 1996. 

In addition to the Decatur mortality study, the University of Minnesota investigators 

have also followed the mortality experience of a cohort of employees associated 

with fluorochemical production at the 3M Cottage Grove (Gilliland and Mandel, 

1993). Unlike the Decatur plant, the greatest potential for exposure at Cottage 

Grove is to PFOA (perfluorooctanoic acid) although the plant does produce PFOS. 

The total cohort consisted of 2,788 male and 749 female workers employed between 

1947 and 1983. There was no significantly increased cause-specific SMR for either 

men or women. In a proportional hazard regression model, ten years of 

employment in jobs associated with the chemical division was associated with a 

3.3-fold increase (95% CI 1.02 - 10.60) in prostate cancer mortality compared to no 

employment in the chemical division. The authors suggested that this finding may 

be biologically plausible with exposure to PFOA as animal toxicology and human 

data had associated PFOA with reproductive hormone changes (Gilliland, 1992). 

However, the authors urged caution in the interpretation of this result due to the 

nonspecificity of the exposure index as well as the few subjects (n = 4) considered 

‘exposed.’ Subsequent research on the Cottage Grove male workforce involved 

with PFOA production has provided reasonable assurance that there are no 

significant hormonal changes associated with PFOA at the serum levels measured 

(Olsen et al., 1998). 

Current and proposed human health and exposure research initiatives are found in 

Section VI. 

28 000041 
January 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 of Death Deaths U.S. Alabama Counties 

All Causes of Death 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 8 68 0 42 
January 2 1, 1999 

IV. SUMMARY OF TOXICOLOGY DATA ON PFOS 

Introduction 

This section summarizes the existing database with respect to the potential 

mammalian toxicity of PFOS. Toxicology data are valuable in identifying potential 

health effects and provide a basis for risk characterization. The use of this information 

in risk characterization will be described in Section V. 

With few exceptions, past toxicology studies either did not characterize blood and 

tissue concentrations associated with exposure or were not completely specific in 

characterizing blood and tissue concentrations. For these past studies, blood and 

tissue concentrations can only be estimated based on limited pharmacokinetic data 

and results from more recent studies, most of which have been designed to include a 

pharmacokinetic component to obtain highly sensitive and specific identification of 

PFOS in blood and tissues. Also, the extent of potential interspecies and intraspecies 

variability in pharmacokinetic handling of PFOS is not presently well defined. 

Comparison of serurn concentrations of PFOS from toxicity studies to measured 

human serum concentrations may be an interesting and convenient comparison; 

however, body burden and/or target organ tissue concentration may be more 

meaningful in characterizing risk. The available body of animal toxicity data cannot 

be related quantitatively and without uncertainty and speculation to the concentrations 

of PFOS in human blood reported in Section I1 and the results of epidemiologic 

investigation as reported in Section 111. 

Despite the current limitations in the ability to characterize risk based on the 

relationship of blood and tissue PFOS concentrations from toxicity studies to human 

blood concentrations of PFOS, the existing toxicology database on PFOS does 

provide valuable insight into potential health hazards and forms a foundation for risk 

characterization. 
January 2 1, 1999 

It should be mentioned that the specific sources of PFOS identified in the serum of 

non-occupationally exposed humans are currently unknown. The PFOS molecule 

itself may not be the primary source. PFOS in the serum of non-occupationally 

exposed humans may derive from metabolism of higher molecular weight molecules 

present in industrial, commercial and consumer products or manufacturing 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 of PFOS unless otherwise annotated. Studies on 

related compounds are considered outside the scope of this discussion and will only 

be mentioned when they provide valuable insight into the toxicity of PFOS. Some 

data will be discussed from studies which are currently in progress or incomplete. 

Tabular summaries of rodent repeated-dose toxicity studies, primate repeated-dose 

toxicity summaries and developmental and reproduction studies will be found at the 

end of this section (Table IV.9, IV.10, and IV.11, respectively). In addition to these 

summary tables, Tables IV. 12 presents the results of rodent and primate 90-day subchronic studies organized by increasing nominal mg/kg dose level. A list of studies 

initiated in 1998 can be found at the end of this section in Table IV.8 and summary 

descriptions of these studies can be found in the Appendix. 

Pharmacokinetics of PFOS 

The absorption, tissue distribution, potential metabolism and excretion of PFOS has 

been studied most extensively in rats by both radiolabel and direct quantitation. 

Limited data relating oral dose to serum and liver concentrations of PFOS in the 

cynomolgus monkey is available from a recent 28-day rangefinder capsule dosing 

study. In addition, serum PFOS concentrations in three retired male 3M chemical 

workers have been followed in an attempt to estimate an elimination rate constant for 

the human. 

31 
January 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 averaging 4.2 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). The radiochemical purity of the [14C]PFOS used in this and the other 

radiolabel studies listed below was >99% (Johnson and Behr, 1979). 

PFOS was applied to skin that had been clipped free of hair as a suspension in water 

at 5,000 mg/kg covering 40% of the total body surface area of ten male and ten female 

rabbits. An impervious plastic sheet occluded the skin for 24 hours and was then 

removed. Animals were maintained and observed for a 28-day period. Blood 

samples were obtained on days 1,7, 14, and 28. Analysis for total blood fluoride was 

performed on the day one and day 28 samples from a single male and single female. 

Total serum fluoride values for the male were 10.3 ppm for day one and 130 ppm for 

day 28. The respective values for the female were 0.9 ppm and 128 ppm. Although 

this study indicated some dermal absorption, it is limited in that the values from only 

32 
January 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 (initial body weights 262-303 g, mean = 288 g) 

excreted a mean of 30.2% of the total carbon-14 via urine. Mean cumulative fecal 

excretion was 12.6%. At 89 days, mean tissue concentration of total carbon-14 

expressed as pg PFOS-14C equivalentdg 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 which male and female cynomolgus monkeys weighing 2.1 to 2.4 kg were 

given capsules containing PFOS at either 0.0 mg/kg/day (two males and two females), 

0.02 mg PFOS/kg/day (three males and three females), or 2.0 mg/kg/day (one male 

and one female) (Thomford, PJ, 1998). The monkeys dosed with PFOS (0.02 and 2.0 

mg/kg/day) demonstrated an extremely linear (r-squared > 0.99) increase in serum 

concentration throughout the exposure period (28 days). There was no apparent sex 

difference and the individual slopes of the cumulative PFOS dose versus serum PFOS 

concentration curve appeared to be virtually identical between the monkeys in the two 

dose groups. The average slope of the curve in the 0.02 mg/kg/day group (n = 6) was 

33 0006546 
January 2 1, I999 

5.22 k 0.74 ppm PFOS in serum per mg/kg cumulative dose, and for the two monkeys 

in the 2.0 mg/kg/day dose group, the average was 5.40 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 of magnitude for the male and female cynomolgus monkey. 

This is calculated as follows: 

One (1) mg/kg results in 5.3 ppm serum PFOS; 

This is equivalent to 5.3 mg/L of serum (mg/L = ppm weightholume); 

One (1) mg/kg given to 2.2 kg monkey results in a total dose of 2.2 mg; 

Vd (L) is calculated as 2.2 mg / 5.3 mg/L = 0.41 L; 

Normalizing this for body weight gives 0.41 L / 2.2 kg = 0.19 L / kg = Vd (L / kg); 

Vd for a 60 kg woman is estimated to be 0.2 L / kg x 60 kg = 12 L = Vd (L). 

Metabolism: Preliminary data from analysis of urine, feces and tissues of rats as well 

as the inherent stability of perfluorinated anions suggest that PFOS is not metabolized 

(Johnson et al., 1984). Exposure of primary human and rat hepatocytes to PFOS did 

not result in further metabolism (Gordon, 1998). Analysis by LC/MS of serum and 

liver samples from studies currently in progress have not revealed any evidence of 

metabolism. 

Excretion: In the previously mentioned study (Johnson etal, 1979) single intravenous 

doses (mean 4.2 mg/kg) of ['4C]PFOS in 0.9% NaCl were administered to male rats. 

By 89 days after dosing, 30.2% of the administered 14C had been excreted in the urine 

and 12.6% had been excreted in the feces. 

34 080047 
January 2 1, 1999 

Whole body elimination in the male rat appeared to be biphasic. Initial redistribution 

from the plasma yielded a plasma elimination half-life of I4C of 7.5 days following 

single oral administration of [14C]PFOS (mean dose 4.2 mg/kg) to male rats (Johnson 

and Ober, 1979). In the aforementioned intravenous study, elimination of only 42.8 

% of the dose through urine and feces after 89 days indicates that the half-life of 

elimination from the body is > 89 days in the male rat. 

Serum PFOS levels in three retired male 3M chemical workers have been followed 

for five and one-half years and suggest a mean serum elimination half-life of 1,428 

days. Over that time period that these serum samples were taken and analyzed, the 

analytical method changed from thermo-spray mass spectrometry to electro-spray 

mass spectrometry, and the analytical laboratories changed. These changes should not 

have affected the values reported to any appreciable extent. Since urine and feces 

have not been followed, it is difficult to ascertain if this represents a true elimination 

half-life from the body; however, this is the closest value for elimination half-life 

applicable to humans in existence. The actual data from these three retired chemical 

workers is presented in Table IV. 1. 
January 21, 1999 

Table IV. 1 Serum Elimination of PFOS in Retired 3M Chemical Workers. 

Serum Elim. 

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 

I Tu2 I 

Retiree 2 

311 8/92 

11/15/93 

5/5/95 

1 111 8/97 

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 182 d 

Fecal and total excretion of 14C were markedly increased in male rats administered 

cholestyramine (- 2.7 g/kg/d) in their diet following single intravenous doses of 

[ 14C]PFOS. The results suggest that there was significant enterohepatic circulation of 

PFOS (Johnson and Gibson, 1980, 1984). Cholestyramine administered at 4% by 

weight in feed to male rats decreased the retention of carbon-14 in liver, plasma, and 

red blood cells and increased the elimination of carbon-14 via feces after iv dosing 

with PFOS-I4C. Groups of five rats (twelve-week old Charles River CD averaging 

320 g) were dosed intravenously with PFOS-14C (mean dose, 3.4 mg/kg). Groups of 

five control rats were dosed similarly but were not treated with cholestyramine. Rats 

were sacrificed at 21 days post dose. The mean liver, plasma, and red blood cell 

concentration as well as fecal and urinary excretion of 14C for cholestyramine-treated 
January 2 1, 1999 

rats were compared to mean control rat 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 control rat concentrations of 3.8,7.7, and 6.0 fold, 

respectively. Fecal elimination (75.9% with cholestyramine treatment) was increased 

9.5 fold. The extent of urinary I4C elimination, as a result of the relatively high rate 

of fecal elimination of I4C was 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). 

, 37 
January 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-day period. All rats in the 

IO00 and 464 mg/kg dose groups died. The signs before death included hyperactivity, 

decreased limb tone and ataxia. At 215 mg/kg, three of ten animals died. At 100 

mg/kg, no deaths were observed. The combined acute oral LD50 in male and female 

rats is 251 mg/kg (95% C.I.: 199-318 mg/kg). PFOS is considered moderately toxic 

on acute oral administration under the conditions of this study (Dean et al., 1978). 

Acute Dermal Toxicity: Ten male and ten female albino rabbits were clipped free of 

hair and an aqueous suspension equivalent to 5 g/kg PFOS was placed over 40% of 

the total body surface area, occluded with an impervious plastic sheet and left in 

contact with the skin for 24 hours, then removed. Animals were maintained and 

observed for a 28-day period after which they were necropsied. Blood samples were 

obtained on days 1 , 7, 14 and 28. Day 0,7, 14 and 28 body weights were recorded. 

Hyperactivity was noted in 5 of 10 males on day 6. All animals recovered by day 7 

and remained asymptomatic throughout the study period. Weight gains were 

observed for all rabbits. No visible lesions were noted at necropsy. Analysis of total 

blood fluoride from day 1 and day 28 blood samples of one male and one female 

indicated that PFOS was absorbed through the skin, reaching blood concentrations of 

approximately 130 ppm after 28 days (see section above on absorption). PFOS can be 

considered practically non-toxic on single dermal contact (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, 1.9 and 0.0 milligrams PFOS per liter of air. The rats were 

observed hourly for the first four hours and daily thereafter for 14 days. All rats 

38 
January 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. Symptoms observed during the exposure and post-exposure periods were 

labored breathing, reduced activity, excessive salivation and lacrimation, mucoid and 

red nasal discharge, yellow staining of the ano-genital fur, and dried red material on 

the facial area. The most frequent abnormal necropsy observations were of lung and 

liver discoloration. Lung discoloration was also observed in a high number of control 

rats and thus may not be treatment-related. The nominally determined median lethal 

concentration (LC50) for a one-hour exposure to PFOS was determined to be 5.2 

milligrams per liter with 95% confidence limits of 4.4 milligrams per liter and 6.4 

milligrams per liter (Rusch and Rinehart, 1979). 

Primary Irritation 

Ocular Irritation: PFOS was found to be mildly irritating to the eyes of albino 

rabbits when tested according to standard Federal Hazardous Substances Act 

guidelines. The ocular irritation was limited to the conjunctivae in the six test rabbits. 

Irritation was noted at the 1 , 24 and 48 hour post-instillation reading times. The 

maximum irritation score was 9.3 out of a highest possible score of 110 at the 24 hour 

reading. By 72 hours post-instillation all readings were zero (Biesemeier and Harris, 

1974). 

Dermal Irritation: PFOS was found to be non-irritating to the skin of albino rabbits 

when tested under conventional Draize procedures. No signs of dermal irritation were 

observed in any of the test animals at any time during the study period. The primary 

skin irritation score was 0.0 out of a highest possible score of 8.0 (Biesemeier and 

Harris, 1974). 

Genotoxicity 

Gene Mutation: PFOS was not mutagenic in Salmonella typhimurium strains TA1535, TA-1537, TA-1538, TA-98, TA-100 or in Saccharomyces cerevisiae strain D4 

39 0000512 
January 2 1, 1999 

in a standard plate incorporation assay with or without metabolic activation. 

(Jagannath and Brusic, 1978). 

Chromosomal Effects: PFOS did not cause chromosomal aberrations in an in vivo 

mouse bone marrow micronucleus assay. PFOS in water was administered by oral 

gavage at 0,237.5,475, and 950 mg/kg to five male and five female mice for each 

dose/ harvest time group. The mice were euthanized 24,48 and 72 hours after dosing 

for extraction of bone marrow. No increase in bone marrow polychromatic 

erythrocytes was observed (Murli, 1996). 

Repeated-Dose Toxicitv 

A number of repeated-dose toxicity studies have been conducted with PFOS and will 

be discussed in this sub-section. A tabular summary of these studies will be found at 

the end of this section in Table IV.9 for rodent studies and Table IV. 10 for primate 

studies. In addition to these 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 Study in Rats: PFOS was fed in the diet at levels of 0, 30, 100, 

300, 1,000 and 3,000 ppm to groups of five male and five female Charles River CD 

rats for 90 days. These doses represent approximately 0,2,6, 18, 60 and 180 

mg/kg/day. The rats were observed twice daily for overt signs of toxicity and 

mortality. Individual body weight and sex group food consumption were recorded 

weekly. Hematological, biochemical and urinalysis studies were conducted prior to 

commencement of dosing, at one month and at study termination. 

At the 30 ppm (approximately 2 mg/kg/day) dosage level, no rats showed any 

compound related changes in appearance or behavior. Mean body weights were 

slightly lower when compared to the controls. At one month, one female rat showed a 

40 0000-53 
January 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, mean body weight and 

food consumption was significantly lower than the control group. One male and two 

females died. Slight increases in creatinine phosphokinase (CPK) and serum alkaline 

phosphatase activity, slight to moderate increases in blood glucose and blood urea 

nitrogen, and slight to marked increases in plasma transaminase activities (AST and 

ALT) were seen at one month. At three months, all rats in the 100 ppm dose group 

had slight to moderate decreases in hemoglobin, hematocrit and erythrocyte counts, 

and slight to moderate increases in transaminase activities were seen for two of the 

three surviving female rats. 

At dosage levels of 300, 1,000 and 3,000 ppm (approximately 18, 60 and 180 

mg/kg/day) all rats died prior to scheduled termination of the study. Time of death 

was dose-related. Overt clinical observations of toxicity included emaciation, 

convulsions, altered posture, red material (right eye and/or mouth), yellow material in 

the ano-genital region, increased sensitivity to external stimuli and reduced motor 

activity. Compound-related gross changes such as emaciation and areas of 

discoloration involving the stomach and liver were observed among treated rats that 

died prior to sacrifice. Similar changes were also observed in the liver of a few rats 

sacrificed at termination of study fiom the 30 and 100 ppm groups. 

Morphological changes consisting of centrilobular to midzonal cytoplasmic 

enlargement (hypertrophy) of hepatocytes and necrosis of liver cells was present in all 

PFOS dose groups. The incidence and relative severity of the above lesions were 

more evident among male rats. In addition, rats from the 300, 1,000 and 3,000 ppm 

dosage levels displayed compound-related changes involving the primary (thymus, 

41 000054 
January 2 1, 1999 

bone marrow) and secondary (spleen, mesenteric lymph nodes) lymphoid organs, 

stomach, intestines, muscle and skin (Goldenthal et al, 1978a). 

Two-Year Dietary Study in Rats, Four and Fourteen Week Results: Results are 

available from four-week and fourteen-week interim sacrifices in Sprague-Dawley 

rats which are part of an on-going 104-week dietary study (Covance, 1998). In these 

sub-studies, groups of five male and five female rats (four and 14-week sacrifice 

groups) and ten male and ten female rats (14-week sacrifice groups) were exposed to 

either 0, 0.5,2, 5, or 20 ppm PFOS in the diet for four or 14 weeks. Clinical 

observations were made twice daily. Body weights and food consumption were 

measured once a week. Organ weights were measured at term. Samples were taken 

at both sacrifices for PCNA, liver palmitoyl CoA oxidase activity, serum and liver . 

PFOS concentrations and at the 14-week sacrifice for hematology, clinical chemistry 

(including urinalysis), and histopatholgy. During weeks 4 and 14, blood and urine 

were collected for hematology, clinical chemistry, urinalysis, and urine chemistry 

tests from ten animaldsex in Groups 1 through 5. Five animaldsex in Groups 1 

through 5 were sacrificed during Week 4; livers were collected and weighed. A 

portion of the liver was prepared for PFOS analysis, a portion was shipped to 

Pathology Associates International for hepatocellular proliferation rate (PCNA) 

measurements, and a portion was used for determination of palmitoyl-CoA oxidase 

activity. At week 14, necropsies were performed on five 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. The tissues 

were adrenals, brain, eyes, kidney, liver, mesenteric lymph node, pancreas, spleen, 

testes, and ovaries. In addition, microscopic examinations were done on tissues from 

animals that died or were sacrificed due to poor health. 

42 
January 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 at both 

testing intervals, it was considered likely that administration of the test material was 

associated with mildly higher urea nitrogen at Week 14 for males and females fed 

20.0 ppm and moderately lower cholesterol and mildly higher alanine 

aminotransferase at Week 14 for males fed 20.0 ppm. There were no correlative 

microscopic renal findings for the minor change in urea nitrogen. The findings for 

alanine aminotransferase and cholesterol were likely associated with the 

histopathological findings of hepatocellular hypertrophy and vacuolation. 

Of uncertain relationship to the test material was mildly, but statistically, higher 

absolute neutrophil count for males fed 20.0 ppm. Females were unaffected, and 

there were no correlative microscopic findings for this small difference. 

All other statistically significant differences for clinical pathology results between the 

control and treated groups were considered incidental. Lower glucose at Week 4 for 

high-dose males and lower aspartate aminotransferase at Week 4 for high dose 

females were considered incidental because they were not present at Week 14. 

Higher albumin at Week 14 for high dose females was considered incidental because 

the low dose females had a similar, but higher, statistically significant difference for 

albumin. 

Table IV.2 and IV.3 summarize key clinical pathology results at 14 weeks for males 

and females, respectively. 

43 000056 
January 2 1, 1999 

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

Diet) 

Table IV.2 Clinical chemistry and hematology in Male rats 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 k 0.17 6.2 + 0.2" 6.1 k 0.24" 0.0057 

34 k 2 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 8 100 k 7 0.3 1 

0.8f0.36 0.9k0.4 0.53rt0.13 0.56k0.1 0.64rt0.1 NA 

14rt 1 16+4 1422 16 k 1.7 16 rt 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: Terminal body weights at 14 weeks for the 2 ppm dose 

group were reduced in comparison to controls by 4.5% in the males and 1 1.7% in the 

females. Absolute and relative liver weights were significantly increased by 

approximately 35% in the males given 20.0 ppm PFOS in the diet. In females given 

44 
January 2 1, 1999 

20.0 ppm, the absolute liver weights were increased by approximately 30% over 

control values, but only the liver-to-body weight percentage was significantly 

increased. The absolute spleen weight was significantly decreased in the females 

given 20.0 ppm, as was the absolute lung weight in females given 2.0,5.0, or 20.0 

ppm. Spurious significant increases in left 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 of the test material. 

Microscopic Observations: Test material related histomorphologic changes were 

limited to the liver in the males given 5.0 or 20.0 ppm and in the females given 20.0 

ppm. The changes consisted of hypertrophy of hepatocytes in centrilobular areas, and 

midzonal to centrilobular hepatocytic vacuolation. The incidence and severity of the 

changes tended to be greater in the males. 

There were no apparent test material-related lesions in the remaining tissues 

examined. 

Serum and liver concentrations of PFOS increased with dose and with length of 

exposure, and liver values were significantly higher than corresponding serum values. 

These analytical results are presented in Tables IV.4 and IV.5 for serum and liver, 

respectively. 

45 
January 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 14 Weeks 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 k 45 

0.1 1 k 0.05 8.6 k 0.57 25 k 6.2 80k 17 357 k 43 

0.44 f 0.05 26 k 4.4 77 k 7.5 387 rt 33 600k 116 

1.58 k 2.1 19 k 3.4 68 k 3.0 362 126 618 k 50 

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

0.59 ppm. 

**MDL = Method Detection Limit, which was 15 ppb. 

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. 

46 
January 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 chemistry was performed on blood samples taken at 

termination. Gross pathology and organ weights were noted at sacrifice and 

histopathology was performed on preserved tissues. 

All rats gained weight in the course of the study. There were no compound-related 

early deaths. 

Clinical pathology demonstrated effects on hematologic and serum chemistry 

parameters in the mid-dose and high-dose males as well as the high-dose females. 

Significant hematologic effects in male rats were: 1) decreased hemoglobin, 

hematocrit and RBC (0.3 and 1 .O mg/kg dose groups); 2) decreased mean corpuscular 

hemoglobin content (3.0 mg/kg dose group); and 3) higher eosinophil counts (0.3 

mg/kg dose group). Significant hematologic effects in female rats were: 1) decreased 

hematocrit and RBC count (0.6 mgkg dose group); decreased mean corpuscular 

volume (0.02 and 0.2 mg/kg dose groups); 3) decreased mean corpuscular 

hemoglobin and mean corpuscular hemoglobin content (0.02,0.06 and 0.2 mg/kg 

dose groups); and 4) increased mean corpuscular hemoglobin and mean corpuscular 

hemoglobin content (0.6 mg/kg dose group). These hematologic effects did not have 

a consistent dose-related pattern. Significant serum chemistry effects in male rats 

were: 1) an elevation in alkaline phosphatase and blood urea nitrogen and decreased 

cholesterol (1 .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. 

Gross pathology revealed the following organ-weight effects in male rats: 1) increases 

in absolute liver weights and liver weight relative to brain weight and liver weight 

relative to body weight (1 .O and 3.0 mg/kg dose group) and 2) decreased absolute 

heart weight, decreased heart weight relative to body weight and increased brain 

47 
January 2 1, 1999 

weight, kidney weight and testes weight relative to body weight (3.0 mg/kg dose 

group). Gross pathology revealed the following organ-weight effects in female rats: 

1) increases in absolute liver weights and liver weight relative to body weight (0.06, 

0.2 and 0.6 mg/kg dose groups); 2) increased liver weight relative to brain weight (0.2 

and 0.6 mg/kg dose groups); 3) decreases in absolute heart weight, heart weight 

relative to brain weight and heart weight relative to body weight (0.02,0.06, 0.2 and 

0.6 mg/kg dose groups); 4) increased brain weight relative to body weight (0.02 and 

0.06 mg/kg dose groups) and 5) increased kidney weight relative to body weight (0.2 

mg/kg dose group). 

related to reduced body weight gain and were not associated with histopathologic 

alterations. The dose-related increases in liver weights and reductions in serum 

cholesterol and triglycerides are supported by the histopathologic finding of hepatic 

vacuolization in the 1.0 and 3.0 mg/kg male rats. 

With the exception of liver, these organ weight changes were 

90-Day Oral Gavage Study in Rhesus Monkeys: In an initial study, five groups 

consisting of two male and two female rhesus monkeys initially weighing between 

2.75 and 4.10 kg were given daily doses of 0, 10,30, 100 and 300 mg PFOS / kg by 

gastric intubation (gavage) as distilled water suspension. This study was terminated 

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

of death was dose-related. Monkeys in the 300 mg/kg/day dose group died between 

the second and fourth day. The 100 mg/kg dose group died between the third and 

fifth day. At 30 mg/kg, deaths occurred between the seventh and tenth day. The 10 

mg/kg dose group died between days 11 and 20. All PFOS-treated monkeys lost 

weight. Clinical observations of toxicity were similar for all PFOS dose groups, and 

time of onset of toxic effects was dose-related. These toxic symptoms included 

anorexia, slight to severe decreased activity, frothy or food-like emesis, and 

occasional diarrhea. Prior to death, body and limb stiffening, general body tremors, 

convulsions and prostration were observed. The 10 mg/kg dose group included one 

monkey who had black stools and one who developed facial erythema. Gross 

Time 

48 
January 2 1, 1999 

pathology revealed a yellowish-brown discoloration of the livers of several monkeys 

in the 100 mg/kg and 300 mgkg dose groups. Organ weights appeared within normal 

limits; however, due to early deaths of PFOS-treated monkeys and the reuse of control 

monkeys on a follow-up study at lower dose levels, concurrent control data was not 

available. Histopathologic examination of tissues from treated monkeys did not reveal 

any consistent observations which could be directly related to an effect of PFOS. 

Congestion and lipid depletion of the adrenal cortex was seen in all dose groups and 

was considered agonal. The study was terminated after the death of the last 10 mg/kg 

dose-group monkey. 

After termination of the initial study after early deaths of all PFOS-treated monkeys, 

the control group monkeys were used in a follow-up study at lower doses (Goldenthal 

etal, 1978b). In this study, five groups consisting of two male and two female rhesus 

monkeys initially weighing between 2.55 and 3.75 kg were given daily doses of 0, 

0.5, 1 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 of the first and third month of the study. 

The monkeys treated at the 4.5 mg/kg/day dosage level died or were sacrificed in 

extremis between weeks 5 and 7 of the study. These monkeys exhibited 

gastrointestinal symptoms including anorexia, emesis, black stool and dehydration 

from the first or second day of study. These monkeys also exhibited decreased 

activity and showed marked to severe rigidity, convulsions, generalized body 

trembling, prostration and loss of body weight prior to death. The mean body weight 

decreased 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. 

49 
January 2 I, I999 

All monkeys at the I .5 mg/kg/day dosage level survived to the end of the study. 

These monkeys exhibited slightly decreased activity from the first week of the study 

which occasionally became moderate to marked. In addition, they occasionally had 

black stools, diarrhea, mucous in the stool and bloody stool and exhibited dehydration 

or general body trembling at the end of study. The monkeys from this group had a 

slight decrease in mean body weight. Slight decreases in serum alkaline phosphatase 

activity and serum inorganic phosphate concentrations were evident at the end of the 

study in addition to a marked decrease in serum cholesterol. 

All monkeys at the 0.5 mg/kg/day dosage level survived to the end of the study. 

Monkeys at this dosage level exhibited an occasional soft stool, diarrhea, anorexia 

and emesis, all of which also occurred occasionally in the control group. Slightly 

decreased activity was noted intermittently in three monkeys at this dosage level. At 

three months of study a statistically significant decrease in the serum alkaline 

phosphatase activity was noted in the males as compared to control values; however, 

the toxicologic significance of this finding is questionable when compared to 

individual pre-dose values. The 0.5 mg/kg dose has been considered to present a 

LOEL by past reviewers. Recent evaluation of the study by 3M toxicologists lend to a 

conclusion that this dose more likely represents a NOAEL, if not a NOEL. 

No treatment-induced gross or microscopic pathological lesions were seen in tissues 

other than the adrenals, pancreas, and submandibular salivary glands of male and 

female rhesus monkeys at the 4.5 mg/kg/day dosage level. Microscopically, the 

adrenals from male and female monkeys at the 4.5 mg/kg/day dosage level had 

compound-related marked diffuse lipid depletion; the pancreas from male and female 

monkeys at the 4.5 mg/kg/day dosage level had compound-related moderate diffuse 

atrophy of exocrine cells; the submandibular salivary glands from male and female 

monkeys had compound-related moderate diffuse atrophy of the serous alveolar cells. 

No statistically significant variations in sex group mean weights of organs occurred’ 

between the control and experimental groups. 

50 
January 2 1, 1999 

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

51 
January 2 1, I999 

Table IV.6: Longitudinal analysis of serum cholesterol and PFOS concentrations of 

individual male and female rhesus monkeys during 90 days dosing with either 1.5 or 

4.5 mg/kg PFOS (n = 2 per sex per dose group) (Goidenthal etal, 1978b). 

Individual Serum Cholesterol (mg /I 00 ml) 

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) 23 6 

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 

20 8 

CD = I5 

208 

192 

23 0 

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 

52 
January 2 I, I999 

28-Day Range-Finding, Oral Capsule-Dosing Study in Cynomolgus Monkeys: Male 

and female cynomolgus monkeys weighing 2.1 to 2.4 kg were given capsules placed 

directly in the stomach that contained PFOS at either 0.0 mg/kg/day (two males and two 

females), 0.02 mg PFOS/kdday (three males and three females), or 2.0 mg/kg/day (one 

male and one female) for 28 days in a range-finding study to determine doses for a sixmonth chronic oral capsule-dosing study (Thornford, PJ, 1998). Blood was collected for 

clinical chemistry on study days - 7 (baseline values) ,2, 7, 14 and 29. In addition to 

standard hematologic parmeters and serum chemistry determinations, sex and thyroid 

hormones, cholecystokinin (CCK) and pancreatic amylase were measured. At the same 

time points and on day 3, blood was also obtained for determination of serum PFOS 

concentration. Tissues were obtained at necropsy, weighed, fixed and prepared for 

histopathologic analysis. In addition to histopathologic samples, liver specimens were 

obtained for analysis for proliferating cell nuclear antigen (PCNA), determination of 

PFOS concentration, and determination of palmitoyl CoA oxidase activity. Serum PFOS 

concentrations increased with a high degree of linearity at both dose levels, with no 

difference between males and females and at a linear rate of 5.3 ppm serum PFOS per 

mg/kg (for details please see subsection on toxicokinetics). At the end of the 28-day 

dosing period, serum PFOS concentration in the 0.02 mg/kg/day dose group reached 

approximately 3 ppm and in the 2.0 mg/kg/day dose group, serum concentrations reached 

approximately 300 ppm. The only treatment-related effect observed in the study was a 

dramatic reduction in serum cholesterol in the male and female that received a dose of 2.0 

mg/kg/day. Serum cholesterol dropped fiom baseline values of 150 and 14 1 mg/dl for the 

male and female, respectively, to 9 1 and 62 mg/dl at termination on day 29. The first 

evidence of a significant decrease occurred between day 2 and day 7 for the female, with 

a day 2 value of 136 mg/dl and a day 7 value of 117 mg/dl. The male cholesterol value 

fell from to 15lmg/dl on day 2 to 137 on day 7. The day 7 values for the male and female 

corresponded to a serum PFOS concentration of 72 ppm. This cholesterol data and 

corresponding cumulative dose and serum PFOS concentrations are summarized in Table 

IV.7. There were no other significant findings. 

53 
January 2 1, I999 

Study Day 

Cumulative Dose (mgikg) 

6 Serum 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 concentrations in 

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 clinical pathology and clinical observations through 90 days of dosing are 

available from an ongoing six-month oral (capsule) dosing study in cynomolgus 

monkeys (Covance Study Number 6329-223, in progress). In this study cynomolgus 

monkeys are being dosed by capsule with either 0, 0.03, 0.15, or 0.75 mglkgiday 

PFOS for a period of six months. Dose groups include six monkeys per sex per group 

with the exception of the 0.03 mg/kg/day dose group which includes four 'monkeys 

per sex. The only significant finding through 90 days is a reduction in serum total 

cholesterol in the males and females of the high dose group. While serum PFOS 

concentrations have not yet been analyzed and reported, previous experience in the 

range-finder for this study suggests that the serum concentrations are expected to 

center on approximately 350 ppm PFOS as 90 days for the high-dose monkeys. 

Reproductive and Developmental 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. 1 1. 

Oral Developmental Toxicity (Teratology) in Rats: A rat PFOS oral teratology 

study was 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 10 mgkg on days 6 through 15 of gestation. 

Evidence of fetal toxicity was not found at any dose level. No skeletal and soft tissue 

teratogenic changes were found at any dose level with one exception. A change in the 

lens of the eye was found in all dose groups including the control but the incidence in 

high dose group was significantly higher. This change was reported out as a 

developmental eye abnormality and the summary of the report states the compound 

was teratogenic. An outside consultant and teratology expert, Dr. E. Marshall 

Johnson from Jefferson Medical College, visited 3M and reviewed the rat pup eye 

specimens in question. He concluded that the eye/lens changes were, in fact, 

sectioning artifacts and not compound related teratology abnormalities. Thus, the 

weight of the evidence indicates that PFOS does not cause teratogenic effects in rats 

when dosed at levels which are not maternally toxic. The lens change observed in rat 

pups in Riker Laboratories studies was a sectioning artifact and was not found upon 

repeat studies at independent laboratories. 

Oral Deveiopmental Toxicity (Teratology) in Rats: In a subsequent study, PFOS 

(suspended in corn oil) was administered on gestational days 6- 15 by oral gavage to 

groups of 25 pregnant Sprague-Dawley CD rats at doses of 0 (control), 1, 5, and 10 

mg/kg/day (Wetzel, etal, undated). Severe maternal toxicity occurred in the 5 mg/kg 

and 10 mg/kg dose groups, as evidenced by significant reductions in mean body 

weight gain, terminal body weight minus gravid uterine weight and food consumption 

compared to control dams, actual losses in body weight on commencement of 

treatment among numerous dams and death in two dams in the 10 mg/kg dose group 

prior to gestational day 20. Mean body weight gains (days 0-20) at 5 and 10 mg/kg 

were 104 k 35 (S.D.) and 34 k 73 (S.D.), respectively, as compared to 125 t 24 (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 i 28 for. 

the control group. Mean terminal body weight minus gravid uterine weight at 5 and 

10 mg/kg was 293 k 28 (S.D.) and 241 li: 60 (S.D.), respectively, as compared to 321 

55 
January 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 1 mg/kg/day. 

Oral Developmental Toxicity (Teratology) in Rabbits: A final draft report of an 

oral developmental toxicity study in rabbits was recently received (York, 1998). In 

this study, dose groups of 22 pregnant new zealand white rabbits were dosed on days 

7 through 20 of gestation with either 0, 0.1, 1.0, 2.5, or 3.75 mg/kg/day PFOS. 

Maternal toxic effects included: 1) decreased body weight at the highest three dose 

levels with a minimal effect at the 1 .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 NOELS are 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 13 0 0 *5 9 

56 
January 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 during mating. For the females, treatment continued during gestation, parturition 

and lactation. 

A pre-mating reduction in mean body-weight gain as compared to controls occurred 

in females and possibly in the males at the high dose level. At the 3.2 mgkg dose 

level, male body-weight gain was 97.2 % of control and female body-weight gain was 

91.5 % of control. There was no effect on the number of pregnancies. 

During gestation, females showed a reduction in mean body-weight gain at the three 

highest dose levels, reaching 86.9% of the control at 3.2 mg/kg. A group of ten dams 

per dose group was sacrificed on day 10 of gestation. No increases in resorptions 

occured, and there were no decreases in the number of implantations or number of 

live fetuses. 

At parturition in the 3.2 mg/kg high-dose group, the mean number of pups delivered 

was decreased compared to the control (10 versus 14) and the percent of pups 

delivered stillborn was increased significantly (24.9 % as compared to 2.2 %.) 

Survival of pups during days one through four of lactation was severely affected at 

1.6 and 3.2 mg/kg (66 YO and 0% survival, respectively.) Most deaths at the high dose 

occured within the first 24 hours after birth. Surviving pups in the 1.6 mg/kg dose 

group showed severely depressed mean body-weight gains through day 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 of the reduced perinatal survival at the high dose levels. These will 

include a complete cross-fostering study, and two pharmacokinetic studies with 

pregnant dams. 
January 2 I, 1999 

The post-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) of toxicity. 

Effects on lipid and lipoprotein processing, cholesterol synthesis and bioenergetics 

have been studied. 

Mitochondrial Bioenergetics: Studies in isolated rat liver mitochondria at PFOS 

concentration in the range of 10 pM demonstrate: 1) detergent type effects on 

mitochondrial membranes; 2) stimulation of mitochondrial respiration, and 3) 

fluidization of the inner mitochondrial membrane (Wallace and Starkov, 1998). 

Interference with Fatty Acid Binding and Transport: Interference of PFOS (1 - 10 

pM) with endogenous fatty acid binding to carrier protein substrates, liver fatty acid 

binding protein (L-FABP) and albumin (BSA) has been investigated and show a 66% 

reduction of initial fluorescence when added to solutions containing 1 mM L-FOP 

and 1mM DAUDA and an ICjo of 4.9 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 of the following hepatic 

parameters were observed: 1) relative liver weight (slight elevation); 2) mitochondrial 

and microsomal protein; 3) palmitoyl-CoA oxidation; 3) catalase in mitochondrial 

and cytosolic fractions; 4) glutathione transferase; 5) epoxide hydrolase and 6) DTdiaphorase, Q- and C2- 1-hydroxylation (Sohlenius et al., 1993). 
January 2 I, I999 

Ikeda et al. (I 987) exposed male rats were to 0.02% PFOS in the diet for 2 weeks. 

The prominent induction of peroxisome proliferation was demonstrated by electron 

microscopy. Activities of catalase, fatty acyl-CoA oxidizing system, carnitine acetyl 

transferase and cytochrome P450s responsible for the Q-hydroxylation of lauric acid 

were increased (Ikeda et al., 1987). 

Hypolipidemia: The mechanism of the hypolipidemic effect of PFOS has been 

studied. Rats were fed 12 mg/kg/day for 7 - 14 days (0.02% in diet). Decreased body 

weight, increased liver weight, increased liver triacylglycerol, increased liver free 

cholesterol, decreased liver cholesterol ester, decreased serum cholesterol and 

triacylglycerols were observed. Hepatocytes isolated from treated rats showed 

reduced synthesis of cholesterol from acetate, pyruvate and hydroxymethylglutarate 

but not from mevalonate, increased oxidation of palmitate and reduced fatty acid 

synthesis. Activities of liver hydroxymethyl glutaric acid-CoA reductase and acylCoA:cholesterol acyltransferase were reduced. These results suggest that the 

hypolipemic effect of PFOS may be due to impaired production of lipoprotein 

particles due to reduced synthesis and esterification of cholesterol together with 

enhanced oxidation of fatty acids in the liver (Haughom and 0ystein, 1992). 

On-Going Research Program 

A number of studies were initiated in 1998 to gain better insight into the potential 

health hazards of PFOS and to provide a strong foundation for risk characterization. 

These studies are discussed in Section VI, and outlines of them can be found in the 

Appendix. 

59 
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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), 3M has conducted medical surveillance programs at 

manufacturing locations where employees are exposed to PFOS or precursor 

molecules. These employees have PFOS serum levels that range from less than one 

ppm up to 12 ppm in the most recent testing. The 1997 mean among participating 

employees at the Decatur, Alabama plant, the U. S. location where PFOS related 

materials are principally produced, was 2.0 ppm. The best current estimate of serum 

levels in non-occupationally exposed individuals comes from the pooled samples 

collected from regionally diverse blood banks. These values ranged from 14 to 56 

ppb, and averaged 30 ppb. It should be noted that while this is our best estimate, it 

cannot be considered representative of the U.S. population since blood donors are not 

necessarily representative of the U.S. population. In addition this data, since it is 

from pooled samples, does not reflect the variability and range one would expect to 

find among individuals. 

Analysis of data collected in 1995,and 1997 reveals that the serum concentrations of 

PFOS identified in production workers are not associated with alterations in the 

hematological or clinical chemistry parameters that were evaluated. This includes 

measurement of the parameters that would reflect the pathology found in high dose 

animal studies (see below). In addition, no differences from normal were detected in 

ten different hormone levels. These analyses included employees of the 3M Belgium 

manufacturing facility, where serum levels are slightly lower than Decatur. The 

findings apply for PFOS serum levels up to 6 ppm, approximately two orders of 
January 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 

1997 was 68 times the mean of these blood bank samples. 

In 1995, epidemiologists from the University of Minnesota completed a mortality 

study of the Decatur employee cohort. No higher than expected mortality rates were 

found for any cancer or for any other cause of mortality. This study involved 1,957 

employees who had worked at least one year at 3M's Decatur, Alabama plant, and 

their mortality experience was traced from 196 I through the end of 199 1. 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 cynomolgus monkeys 

is available. Serum PFOS concentrations were not measured in the older rhesus 

monkey studies, but can be estimated based on data from the cynomolgus monkey 

studies. It is recognized that uncertainties are introduced by such estimates, and that 

derived numbers need to be confirmed in ongoing or planned studies. 

Data from the cynomolgus monkey rangefinder study itself documents that a decrease 

in cholesterol, an early biological effect in both rodents and primates, was observed at 

a PFOS serum level of 72 ppm in one of the two high dose animals. In the rhesus 

monkey studies, lowered cholesterol was observed after 90 days of oral dosing at 1.5 

mg/kg/day. Severe toxicity, resulting in death, occurred at doses equal to and greater 

than 4.5 mg/kg/day, in seven weeks or less. Serum concentrations of PFOS that might 

have been achieved in the older rhesus monkey studies have been estimated from the 

cumulative dose - serum concentration of PFOS relationship seen in the recent 

cynomolgus study. These estimates suggest that severe toxicity occurred at serum 

levels of 700 to 800 ppm and death at 1000 to 1200 ppm. 

65 
January 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 F 1 weaning, show no interference with sexual maturation, mating behavior, 

fertilization, number of resorptions, or litter size. At the highest two dose groups (1.6 

and 3.2 mg/kg/day) there was significant perinatal mortality in the F 1 generation. 

This effect was not seen at 0.4 mg/kg/day. The study cannot be used in risk 

assessment until it is completed and fully evaluated, but is discussed here because we 

are aware of these interim results. The doses observed to cause effect are in the range 

where toxicity is observed in 90 day rat studies. 

Several 90-day studies in rats, involving PFOS administration over a wide range of 

doses in the diet, in drinking water, and by stomach tube, reveal that no significant 

toxic effects are produced at or below a dose of 0.2 mg/kg/day'. Above this dose 

adverse effects on the liver, body weight, and changes in several indicators of fat 

metabolism are demonstrable. 

Possible explanations for the absence of detectable toxicity in production workers are 

that the workers' serum and tissue levels of PFOS are significantly below those 

achieved in the animal studies, as estimates would indicate, or human beings are less 

sensitive to the effects of PFOS than are laboratory animals. Of these two 

explanations, the former seems more likely, because both primates and non-primates 

were shown to be susceptible to PFOS exposure in the animal studies, so there is no 

obvious reason to expect large interspecies differences in sensitivity. The second 

possible explanation - that humans are less sensitive - cannot be ruled out based on 

available data. It is also possible that the human studies were insufficiently powerful, 

66 
January 2 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 

(Ames tests) and does not cause chromosomal aberrations (mouse bone marrow 

micronucleus assay) is consistent with these findings. 

Non-occupationally exposed populations appear to exhibit serum levels that are 

approximately two orders of magnitude lower than those exposed occupationally, 

more than three orders of magnitude below levels estimated to show minimum 

biological effects (cholesterol reduction) in primate studies and three to four orders of 

magnitude below levels associated with significant toxic effects. None of the health 

effects evaluated and found to be absent in the worker studies would be expected to 

occur in the non-occupationally exposed populations. The additional information 

provided by the two generation reproductive study in rats is not interpretable at this 

time, but the dose at what currently appears to be a no effect level is comparable to 

doses that produce no effect for other endpoints. The animal data revealing adverse 

effects are not yet fully useful for quantifying human risks, but estimates from current 

and ongoing studies suggest that PFOS serum levels achieved in those earlier animal 

studies with compound related effects must have been greater than those experienced 

by workers and therefore much greater than those experienced by non-occupationally 

exposed populations. 

The currently available evidence does not suggest there is health risk associated with 

the levels of PFOS found in the serum of occupationally or non-occupationally 

exposed employees, blood bank samples or commercially available serum. 

67 
January 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 of increased mortality rates, animal data 

reflecting chronic exposures are not yet available. Such data are recommended 

because worker studies, involving relatively small populations, are generally not 

sufficiently powerful to detect small excess risks. A two year PFOS feeding study in 

rats was started in April of 1998. The utility of the available animal data is limited 

because serum levels associated with adverse events can only be estimated at this 

time. This limitation is being remedied by collecting additional animal data, 

including sufficient kinetic information to assess the relationship between 

administered doses and serum and tissue levels, as cited above for cynomolgus 

monkeys, as well as more definitive data on compound distribution and elimination. 

There is data suggesting that PFOS can, at sufficiently high doses, induce peroxisome 

proliferation in rodents and alter metabolic processes in laboratory models. Further 

study of these effects is desirable to determine whether they can occur at serum and 

tissue levels relevant to those observed or estimated in humans. Section VI of this 

report describes additional 3M studies that will address these issues. Further 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 on possible ways to reduce exposure. To date this has been done through 

reduction of residual monomer levels in products and reduced manufacturing 

emissions. 

68 
VI. CURRENT AND PLANNED RESEARCH 

Toxicoiom Studies 

Fluorochemical Studv Purposes and Outlines 

A number of studies were initiated in 1998 to gain bett insight into the itential 

health hazards of PFOS and to provide a strong foundation for risk characterization. 

These studies include those conducted with PFOS as well as studies which are 

conducted with N-Ethyl Perfluorooctane Sulfonamido Ethanol (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 contaminants of indirect food additives. These compounds are also known 

to be absorbed well from the gastrointestinal tract and will produce PFOS as a major 

metabolite. Therefore, it is appropriate to use these two compounds as models in 

hrther investigating the toxicity of this class of chemicals which have the 

perfluorooctane sulfonyl moiety as a base. The goals of this research program are as 

follows: 

To understand the relationship between measured concentrations of PFOS in 

serum and potential adverse health effects; 

To understand the kinetics of increases in body burden of PFOS as reflected in 

serum PFOS measurements; 

To determine if a critical cumulative body burden exists and how this is rleated to 

toxicokinetics; 

To define the metabolic relationship of PFOS to other Perfluorooctanesulfonylbased chemicals; 

To understand the primary mechanism of toxicity responsible for early toxic 

effects; 

000082 

69 
January 2 1, 1999 

To establish reasonable and substantiated no significant risk levels for risk 

assessment. 

Studies initiated in 1998 with PFOS and N-EtFOSE are identified in Tables VI. 1 and 

VI.2, respectively. In addition to these, a 90-day dietary study with N-MeFOSE has 

been completed and studies have been undertaken to better understand the potential 

for metabolic degradation of perfluorooctanesulfonamide-based chemicals to PFOS. 

Descriptive summaries of many of these studies are included in the Appendix. 

70 
January 2 I, 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.) 

5/20/98 412 412 0 00 

12/1/98 1/7/98 . 

7/1/97 On-going 

812 819 8 9/29/98 

512619 8 1 213 1/98 

1 1/1/98 211 5/99 

1 1/7/98 1 /3 0199 

11/23/98 3/1/99 

Two-Year Dietary Study in Rats 1/26/98 

Single-Dose ADME in Cynomolgus Monkeys 

J 

1 12 512 00 0 

Biochemical and Molecular Biology Mechanistic Studies 

5/14/95 

Segment I1 Teratology in Rats 

10/1/98 

Segment I1 Teratology in Rabbits 

Two-Generation Reproduction Study in- Rats 61819 8 1213 1/98 

Date (InLife) 

7/1/97 1 On-going I 

811 1/98 9/4/98 

8/28/98 912 919 8 

71 
January 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: 

4/2 412 000 

The purpose of this 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 of preneoplastic lesions, There are six dose groups: 0, 0.5,2.0, 5.0, 20 

and 20 (recovery) ppm PFOS in the feed. Interim sacrifices at weeks 4, 14 and 52' 

will be performed to determine PCNA, palmitoyl Co-A oxidation, clinical chemistry, 

and histopathology. Animals in group 6 will be treated for 52 weeks, then treatment 

will be discontinued and the animals will be observed for reversibility, persistence, 

or delayed occurrence of toxic effects for at least 26 weeks post-treatment. 

Serum samples will be collected for PFOS detection from the scheduled sacrifices 

and in-life blood draws. Hematology, serum chemistry, urinalysis, urine chemistry, 

and serum sampling (In-life blood draws) will be done at weeks 14,27 and 53. 

104 -Week Dietary Carcinogenicity Study with Narrow Range (98.1 YO) N-Ethyl 

Perfluorooctanesulfonamido Ethanol in Rats. In-life Start Date: 1/ 26/1998. 

The purpose of this study is to assess the carcinogenicity of the test material, N-ethyl 

perfluorooctanesulfonamido ethanol 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 14 for hepatocellular proliferation rate measurements, hepatic palmitoyl-CoA 

oxidase activity, and serum and liver PFOS levels. Due to the morbidity and 

morbundity, the 300 ppm group was terminated at 8 weeks. During Week 8 and 14, 

blood and urine were collected for hematology, clinical chemistry, urinalysis, and 

urine chemistry. At necropsy, macroscopic observations were recorded, organ 

weights were obtained, and tissues were placed in fixative as specified by the 

72 
January 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 100 ppm dose 

group will be treated for at least 52 weeks then treatment will be discontinued, and 

the animals will be observed for reversibility, persistence, or delayed occurrence of 

toxic effects for at least 26 week post-treatment. 

Six-Month Capsule Feeding Study in Cynomolgus Monkeys. In-life Start Date: 

8/5/1998. In-life completion date: 5/7/1999. 

The purpose of this study is to establish a no observable effect level for PFOS in the 

Cynomolgus monkey. PFOS is a known hepatic peroxisome proliferator (PP) in the 

rat. Non-human primates such as the Cynomolgus monkey respond similarly to 

humans with no to low hepatic response to peroxisome proliferators, and therefore 

are the appropriate human surrogate species. Blood hormone levels will be 

determined pretreatment and after 30,60, 90, and 1 SO days of treatment, and after 30, 

60, and 90 days of recovery. Samples will be analyzed for estradiol, estrone, estriol, 

thyroid stimulating hormone, and triiodothryonine (T3)> thyroxin (T4), cortisol and 

testosterone. Serum and liver PFOS levels will be determined and correlated to blood 

chemistries. Urine and feces PFOS determinations will be made day 0 of recovery 

and after 6,30, and 90 days of recovery to track elimination kinetics. Four groups of 

four male and four female Cynomolgus monkeys will be orally dosed with PFOS 

triturated in lactose at 0, 0.03, 0.15 and 0.75 mg/kg/day daily by gelatin capsule. 

Dose levels were determined in a previously conducted rangefinder study. Two 

additional animals in the control, mid and high dose groups will be designated as 

recovery animals for which treatment will be discontinued at 26 weeks, and the 

animals will be observed for reversibility, persistence, or delayed occurrence of toxic 

effects for at least 13 weeks post-treatment. Hematology and clinical chemistry will 

be determined once before initiation, after 30, 60, 90, and 1 SO days of treatment, and 

after 30,60, and 90 days of recovery. Clinical chemistry will include urea nitrogen, 

cholesterol, triglicerides, alkaline phosphatase, alanine aminotransferase, bile acids, 

total and direct bilirubin, lipase, and pancreatic-specific amylase. Palmitoyl CoA 

71 
January 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 I3 weeks post treatment for the recovery groups. 

Histopathology and proliferative cell nuclear antigen (PCNA) will be performed on a 

subset of the specimens. 

Metabolism of N-EtFOSE, in Cynomolgus Monkeys following administration of 

a single dose by Oral Gavage. In-life start date: 5/14/98. In-life end date: 10/1/98 

The purpose of this study is to determine the absorption, distribution, metabolism 

and excretion (ADME) of N-Et FOSE and its metabolites. Determination of ADME 

parameters in non-human primate is an appropriate model for human risk 

assessment, as opposed to the rat. Two groups of Cynomolgus monkeys will be used. 

Group A will have five animals/sex and will be given 10 mg/kg single treatment by 

oral gavage. Tissues will be collected from one animal per sex per time-point at 0.5, 

1, 7, 14 and 28 days. Urine and feces will be collected at 0.5, 1, and daily up to 28 

days. Group B will have one animal/sex which will be bile duct cannulated and 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 14,21 and 28days for both 

groups. Tissues with mass > 0.5 g will be analyzed for N-EtFOSE and its 

metabolites: Cerebrospinal fluid, skin, fat, urinary bladder, testis, epididymus, 

seminal vesicles, prostate, uterus, spleen, kidneys, liver, thymus, heart, lungs, 

diaphragm, salivary glands, trachea, esophagus, muscle, bone, bone marrow, 

pancreas, lymph nodes, tongue, eyes, brain, spinal cord, stomach, small intestine, 

large intestine, thyroidparathyroid, aorta (-0.5 g), adrenal glands, gallbladder , and 

ovaries will be included. 

74 
January 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 

of the 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 of the 

animals were sacrificed 28 days post dose. The preliminary results indicated 

absorption of the oral 50 mg/kg dose and metabolism at day 4, and day 28. The i.v 

doses of 0.5 mg/kg were also extensively metabolized and were deemed to be too 

high. The dose-response study consisted of six dose groups. There was one control 

group, three groups dosed orally at 0.01,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 of the study to each group. All groups were sacrificed on day 4 of 

the study. 

13-week Dietary Toxicity Study with N-Methyl FOSE in Rats. In-life Start 

Date: 9/1/1998. In life completion date: 12/18/1998. 

The purpose of this study was to develop sub-chronic toxicity data and the 

toxicokinetics for N-iMethyl FOSE when administered in the diet to rats for at least 

13 weeks. The study design included 20 males and 20 female animals/group fed 0, 3, 

75 
January 2 I, 1999 

30 and 100 ppm N-MeFOSE in the diet. After 4 weeks of treatment, 5 rats/sex/group 

were sacrificed for Palmitoyl CoA oxidase, PCNA, liver and sera PFOS analysis. 

Two-Generation Rat Reproduction Study with PFOS. In-life Start Date: 

5/26/1998. In-life completion date: 12/31/1998. 

The purpose of this study is to test for toxic effects of PFOS on reproductive function 

of dosed male and female rats and to assess whether rat pups exposed to the 

compound in utero & via milk have any developmental, learning and reproductive 

effects. A secondary objective will be to assess compound and/or metabolite levels in 

serum and liver from parent animals. Groups of 35 rats will be dosed daily via 

gastric gavage at dose levels of 0, 0.1 , 0.4, 1.6 and 3.2 mg/kg/d. Dosing will start 4 

weeks prior to mating, will continue during mating, and for females will continue 

during gestation and lactation. Reproductive performance will be assessed by fertility 

index, gestation index, number of pups/ litter, pup viability index and lactation 

index. The 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-Generation Rat Reproduction Study with N-EtFOSE. Start Date: 

6/8/1998. In life completion date: 12/31/1998. 

The purpose of this study is to test for toxic effects of N-EtFOSE on reproductive 

function of dosed male and female rats and to assess whether rat pups exposed to the 

compound in utero & via milk have any developmental, learning and reproductive 

effects. A secondary objective will be to assess compound and/or metabolite levels in 

serum and liver from parent animals. Groups of 35 rats will be dosed daily via 

gastric gavage at dose levels of 0, 1 , 5, 10 and 15 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 of pups/ litter, pup viability index and lactation 

76 
January 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. 

The purpose of this study is to detect adverse effects of PFOS on New Zealand 

White pregnant female rabbits and development of the embryo on fetus consequent 

to exposure of the doe from implantation to closure of the hard palate. Dose groups 

of 22 presumed pregnant female rabbits were dosed with 0, 0.1,2.0,2.5 and 3.75 

mg/kg/day via a stomach tube. A toxicokinetic satellite group of female rabbits (5 at 

the control and high dose levels plus three at the other dose levels) were sacrificed at 

day 21 of gestation (the day following the last dosage) serum, liver, fetal and 

placental samples were analyzed for PFOS and possible metabolites. Rabbits were 

Caesarian sectioned on day 29 of presumed gestation. The fetuses were examined for 

body weight, gross alterations and skeletal alterations, number and distribution of 

corpora lutea, implantation sites, live and dead fetuses and early and late resorptions. 

Segment I1 Teratology with N-EtFOSE in rats. In-life Start date: 9/4/1998. In 

life end date 9/11/1998. 

The purpose of this study was to detect adverse effects of N-EtFOSE on presumed 

pregnant female rats and development of the embryo and fetus consequent to 

exposure of the dam from implantation to closure of the hard palate. Dose groups of 

25 presumed pregnant female rats were dosed with 0, 1, 5, 10 and 20 mg/kg/day by 

oral gavage. A toxicokinetic satellite group of female rats (five at the control and 

high dose levels plus three at the other dose levels) were sacrificed at day 18 of 

gestation (the day following the last dosage) serum, liver, fetal and placental 

samples were analyzed for N-EtFOSE and possible metabolites. Rats were Caesarian 

77 
January 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. 

Segment I1 Teratology in Rabbits with N-EtFOSE. In-life Start date: 8/28/1998. 

In life end date 9/29/1998. 

The purpose of this study was to detect adverse effects of N-EtFOSE on New 

Zealand White pregnant female rabbits and development of the embryo and fetus 

consequent to exposure of the doe from implantation to closure of the hard palate. 

Dose groups of 22 presumed pregnant female rabbits were dosed with 0, 0.1 , 2.0, 2.5 

and 3.75 mg/kg/day via a stomach tube. A toxicokinetic satellite group of female 

rabbits (five at the control and high dose levels plus three at the other dose levels) 

were sacrificed at day 21 of gestation (the day following the last dosage) serum, 

liver, fetal and placental samples were analyzed for N-EtFOSE and possible 

metabolites. Rabbits were Caesarian section on day 29 of presumed gestation. The 

fetuses will be examined for body weight, gross alterations and skeletal alterations, 

number and distribution of corpora lutea, implantation sites, live and dead fetuses 

and early and late resorptions. 
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 determine the 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 of the surveillance data has not shown significant associations 

between the employees’ clinical chemistry and hematology tests and either total serum 

Decatur Serum Exposure Assessment Study. Employees at the 3M Decatur chemical 

organic fluorine or serum PFOS Ievels. However, the voluntary nature of the medical 

surveillance program does not allow for a complete understanding of the distribution of 

employee fluorochemical serum levels. In order to address this issue, a random sample of 

approximately 80 Decatur film plant and 125 Decatur chemical plant employees will be 

asked to participate in a serum determination study for the following fluorochemicals: 

perfluorooctane sulfonate, perfluorooctane sulfonate amide, perfluorohexane sulfonate, 

perfluorooctanoic acid, N-ethyle perfluorooctnae sulfonamido ethanol and its acetate 

derivitive, and N-methyl perfluoroctanesulfonamido ethanol. A sub-sample of employees 

will also be tested for total serum organic fluorine. A brief questionnaire will also be 

administered to each employee inquiring about current and past work history as well as 

possible routes of oral ingestion. 

3. An Epidemiologic Analysis of the Inpatient Claims Experience of 3M Decatur 

Employees, 1993- 1997. The purpose of this study is to examine the inpatient claims 

database, as maintained by Corporate Health Strategies, from January 1, 1993 - 

December 3 1, 1997 of Decatur plant employees. The data will be stratified by whether 

the employees are in the chemical or film plants. The study population will include full79 
January 2 I, 1999 

time active employees who 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 each plant population 

will be compared to an expected experience based on the 3M normative database. 

Inpatient claims analysis will proceed in a sequential, hierarchical manner. First we will 

examine Medical Diagnosis Codes; next Diagnosis Related Groups, and finally selected 

ICD-9 codes within each DRG. 

4. PFOS and PFOA Retrospective Cohort Mortality Studies of Employees at the 3M 

Decatur and Cottage Grove Plants. Previous retrospective cohort mortality studies have 

been conducted at the Decatur and Cottage Grove plants. However, neither of these 

studies utilized the employees' fluorochemical serum measurements to design job-, 

department- and calendar-year exposure matrices. The purpose of these two studies is to 

construct PFOS and PFOA exposure matrices based on previously collected employee 

serum PFOS and PFOA measurements in conjunction with their plant work history 

experiences. Person-years will be allocated based on the exposure matrices to calculate 

the traditional measures of risk, i.e., Standardized Mortality Ratios, for more than 50 

causes of death. Vital status and cause of death will be ascertained through December 3 1 , 

1997. 

5. Geographical Reliability Study. The purpose of this study is to determine whether 

there are geographical differences in human serum measurements in the United States. 

We will request serum samples from the 18 blood banks that originally participated in the 

fluorochemical determination study (see Table 11.3) to determine whether comparable 

serum levels are measured. 

80 
January 2 I, I999 

VII. REFERENCES 

3M Commercial Chemicals Division. 19xx. FluoradB Brand Fluorochemical Surfactants 

FC-95 and FC-98. Technical Information, 3M Company, St. Paul, MN. 

3M Industrial Chemical Products Division. 1992. FC-95 FLUORAD Brand 

Fluorochemical Surfactant. Material Safety Data Sheet, Document 10-3796-9, St. Paul, 

MN. 

Anderson DJ, Mulvana DE (1997a). Analytical report for the determination of 

perfluorooctanoate and perfluorooctanesulfonate in human serum by LC/MS . 

Unpublished report. Ithaca: Advanced Bioanalytical Services, Inc., August 25, 1997. 

Anderson DJ, Mulvana DE (1997b). Analytical report for the determination of 

perfluorooctanoate and perfluorooctanesulfonate in human serum by LC/MS . Ithaca: 

Advanced Bioanalytical Services, Inc., September 22, 1997. 

Anon. 1979. Analysis of selected Decatur employee serum for sulfonic and carboxylic 

fluorochemicals. Tech- Report No. 723Q, 3M Central Analytical Laboratory, St. Paul, 

MN. 

Argus (1998): Protocol No. 41 8-008 “Combined Oral (Gavage) Fertility, Developmental 

and PerinataUPostnatal Reproduction Toxicity Study of PFOS in Rats,” (Study in 

progress). 

Belisle J, Hagan DF (1 978). Anal Biochem. 87, 545 (Note Error: In this report the blank 

was erroneously reported as 0.02 mg; it should be 0.02p.g.) 

Belisle, J (1981). Science 212, pp- 1509-1510. 

Biesemeier, J.A. and Harris, D.L. 1974. Report T-1117. WARF No. 4102871, WARF 

Institute, Inc., Madison, W 

Central Analytical Laboratory (1 979): Characterization of Decatur RF Values. 3M 

Project Number 91721 100, Report No. 7230, October 4, 1979. 

Covance (1998): Study No. 6329-1 83 “104-week Dietary Carcinogenicity Study with 

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81 000094 
January 2 1, 1999 

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53 
January 2 I, 1999 

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Laboratories Study Number 4 18-0 12 final draft report dated December 17, 199s. 

APPENDIX 

85 
January 2 1, 1999 

FIGURE AI. Reliability of PFOS Serum Measurements in 12 3M Employees 

20 30 40 50 60 70 80 

1st Test (February, 1998) 

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Table 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.ntly Employed 810 49.4 141 44.3 

Ret ired 

Alive 

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 100 due to rounding 

97 
January 2 1, 1999 

Table A3. Characteristics of Male (n = 1,639) and Female (n = 3 18) Employees 

Varia b 1 e Males Females 

Number of Employees 1,639 318 

Number of Person-years 33,l 08 4,807 

Number of Deaths 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 1980 
January 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 

99 
January 2 1, I999 

Table A5. Distribution of Women 

by Age and Year of Entry 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 

100 
January 2 I, 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 of Death 

All Malignant Neoplasms 

Cancer of Buccal Cavity and 

Pharynx 

Cancer of Digestive 

Organs and Peritoneum 

Cancer of Esophagus 

Cancer of Stomach 

Cancer of Large Intestine 

Cancer of Rectum 

Cancer of Liver and Biliary 

Passages 

Cancer of Pancreas 

Cancer of All Other 

Digestive Organs 

Cancer of Larynx 

Cancer of Bronchus 

Trachea and Lung 

Cancer of Other 

Respiratory Organs 

Cancer of Prostate 

Cancer of Testes and Other 

Male Genital Organs 

Cancer of Kidney 

Cancer of Bladder and Other 

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, 13 1.5) 

--- (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 

101 
January 2 1, 1999 

Table A6. (Continued) 

Observed Expected 

Cause of Death Deaths Deaths SMR 95% CI 

Cancer of Brain and Other 1 0.9 117.2 (2.9.653.0) 

Central Nervous System 

Cancer of Thyroid and Other 0 0.1 --- 

Endocrine Glands 

Cancer of Bone 0 0.1 --- 

Cancer of All Lymphatic and 2 2.2 92.9 

Hematopoietic Tissue 

Lymphoma 0 0.3 --- 

Hodgkin’s Disease 0 0.3 --- 

Leukemia and Aleukemia 1 0.8 120.0 

Cancer of Other Lymphatic 1 0.7 137.2 

and Hematopoietic Tissue 

Malignant Melanoma of Skin 0 0.6 --- 

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 Stomach and 0 0.2 --- 

Duodenum 

Cirrhosis of Liver 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 

Motor Vehicle 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) 

(3 1.2, 159.9) 

102 
January 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 of Death 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 of Buccal Cavity and 0 0.3 --- (0.0, 1,396.4) 

Pharynx 

Organs and Peritoneum 

Cancer of Digestive 0 2.1 --- (0.0, 175.2) 

Cancer of Esophagus 0 0.2 --- (0.0, 1,577.7) 

Cancer of Stomach 0 0.3 --- (0.0, 1,195.2) 

Cancer of Large Intestine 0 0.8 --- (0.0,488.5) 

Cancer of Rectum 0 0.2 --- (0.0,2,175.6) 

Cancer of Liver and Biliary 0 0.2 --- (0.0,2,072.6) 

Passages 

Cancer of Pancreas 0 0.5 --- 

Cancer of All Other 0 0.1 --- (0.0, 5,692.4) 

Digestive Organs 

Cancer of Respiratory System 5 3.6 141.0 (45.8, 329.0) 

Cancer of Larynx 0 0.1 --- (0.0, 3,198.4) 

Cancer of Bronchus 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 of Prostate 0 0.3 --- (0.0, 1,202.0) 

Cancer of Kidney 0 0.3 --- 

Cancer of Testes 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 

103 
January 2 I, 1999 

Table A7. (Continued) 

Observed Expected 

Cause of Death 

Cancer of Brain and Other 

Central Nervous System 

Cancer of Thyroid and Other 

Endocrine Glands 

Cancer of Bone 

Cancer of All 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 of Liver 

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 000117 
January 2 1, 1999 

Rat Oral (diet) Carcinogenicity on N-EtFOSE 

Study Outline 

Study Objectives: Main Objective - To determine the carcinogenicity of N-EtFOSE 

upon chronic oral administration. 

Secondary Objectives - To determine chronic and subchronic toxicity of N-EtFOSE after 

52 and 13 weeks, respectively, of compound administration. To determine compound’s 

potential for perixosme proliferation (palmitoyl-CoA oxidase activity) and hepatocyte 

proliferation (PCNA, proliferative cell nuclear antigen). To measure compound and/or 

metabolite levels in the liver and serum after various time-points of exposure. 

GLP Status: A GLP study with appropriate QA audits; a signed QUA statement to be 

included in the final report. 

Animals: Male and female Charles River CD rats 

Dose Groups: Eight - controls (two groups), 1,3,30, 100 & 300 ppm in diet plus a 100 

ppm recovery group (receive compound for 52 weeks followed by 52 weeks without 

compound). 

Number per Group: 50 males and 50 females per group plus additional animals for 

interim sacrifices at 4, 13 & 52 weeks. (Note the 100 ppm recovery group has 40 males 

and 40 females). 

Test Article: N-EtPFOS furnished by Sponsor who is responsible for compound identity 

and purity. 

Compound Administration: Daily administration for up to two-years (1 04 weeks) 

admixed in the diet. 

Clinical Observations: Twice daily 

Body Weights: Once a week for the first 16 weeks; once every four weeks thereafter. 

Food Consumption: Once a week for the first 16 weeks; once every four weeks 

thereafter. 

Hematology and Clinical Chemistry: Ten rats/sex from 0, 1,3,30 & 100 ppm groups 

at weeks 14,52 and 104. 

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

105 
January 2 1, 1999 

Interim Necropsies: Five ratshex from 0, 1, 3, 30, 100 & 300 ppm groups at 4 and 14 

weeks for PCNA, palmitoyl CoA oxidase, and compound level samples. (Note samples 

for organ weights and histopathology taken at 14 weeks.) Ten ratslsex from 0, 1, 3, 30 

& IO0 ppm groups at 52 weeks for compound levels, organ weight, histopathology 

samples. 

Organ Weights: Measured at 14 and 52 week interim necropsies. 

Histopathology: Microscopic examination of selected tissues at 14 and 52 week 

animals. Complete tissue examination of 0 (one control group), 3, 30, and 100 ppm 104 

weeks animals. Selected tissue examination of 100 ppm recovery animals. 

Histopathologic results will indicate extend of tissue examination of 1 pprn and second 

control group. 

106 0803-19 
January 2 1, 1999 

Rat Oral (diet) Carcinogenicity on PFOS 

Study Outline 

Study Objectives: Main Objective - To determine the carcinogenicity of PFOS upon 

chronic oral administration. 

and 13 weeks, respectively, of compound administration. To determine compound’s 

potential for perixosme proliferation (palmitoyl-CoA oxidase activity) and hepatocyte 

proliferation (PCNA, proliferative cell nuclear antigen). To measure compound and/or 

metabolite levels in the liver and serum after various time-points of exposure. 

Secondary Objectives - To determine chronic and subchronic toxicity of after 52 

GLP Status: A GLP study with appropriate QA audits; a signed QUA statement to be 

included in the final report. 

Animals: Male and female Charles River CD rats 

Dose Groups: Six - 0, 0.5,2,5, & 20 ppm in diet plus a 20 ppm recovery group (receive 

compound for 52 weeks followed by 52 weeks without compound). 

Number per Group: 50 males and 50 females per group plus additional animals for 

interim sacrifices at 4, 13 & 52 weeks. (Note the 20 ppm recovery group has 40 males 

and 40 females). 

Test Article: PFOS furnished by Sponsor who is responsible for compound identity and 

purity. 

Compound Administration: Daily administration for up to two-years (1 04 weeks) 

admixed in the diet. 

Clinical Observations: Twice daily. 

Body Weights: Once a week for the first 16 weeks; once every four weeks thereafter. 

Food Consumption: Once a week for the first 16 weeks; once every four weeks 

thereafter. 

Hematology and Clinical Chemistry: Ten rats/sex from 0,0.5,2,5, & 20 pprn groups 

at weeks 14,52 and 104. 

Urinalysis: Ten rats/sex from 0, 0.5,2, 5, & 20 ppm groups at weeks 14, 52 and 104. 

Interim Necropsies: Five rats/sex from 0, 0.5,2, 5, & 20 ppm groups at 4 and 14 weeks 

for PCNA, palmitoyl CoA oxidase, and compound level samples. (Note samples for 

107 
January 2 1, 1999 

organs weights and histopathology taken at 14 weeks.) Ten rathex from 0, 0.5,2, 5, & 

20 ppm groups at 52 weeks for compound levels, organ weight, histopathology samples. 

Organ Weights: Measured at 14 and 52 week interim necropsies. 

Histopathology: Microscopic examination of selected tissues at 14 and 52 week 

animals. Complete tissue examination of 0,2, 5, and 20 ppm 104 weeks animals. 

Selected tissue examination of 20 ppm recovery animals. Histopathologic results will 

indicate extend of tissue examination of 0.5 ppm group. 

. 

108 
January 2 1, 1999 

Rat Teratology Rangefinder on N-EtFOSE 

Study Outline 

Study Objective: Explore possible dose levels for an oral rat teratology study 

GLP Status: Study should be conducted under GLP principles but since it is a 

rangefinder study QA audits will not be done 

Animals: Mated female Charles River CD rats 

Dose Groups: Seven - control, 0 mg/kg; low-1, 1 mg/kg; low-2,5 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 

Test Article: N-EtPFOS will be furnished by the Sponsor who is responsible for 

compound identity and purity. No reserve sample will be required. 

Compound Administration: Oral intubation of compound suspended in water with 2% 

Tween 80 on days 6 thru 17 of gestation. Analysis of dosing preparation will not be 

done, however, records will be maintained on how dosing preparations were made. 

Clinical Observations: Twice daily; once approximately one hour aAer dosing and then 

4 to 6 hours later. 

Body Weights: On days 0,4, 6, 8, 10, 12, 14, 16, 18 and 20 of gestation 

Food Consumption: On days when body weights are obtained 

Cesarean Section: On day 20 of gestation the females will be anesthetized and the 

uterus and its contents will be removed and weighed. The number of implanations, the 

number of live and dead fetuses, and the number of early and late resorptions will be 

determined and recorded. The fetuses will be sexed, weighed and examined for any gross 

abnormalities. The ovaries will examined for the number of corpora lutea. 

109 
January 2 I, 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 on days 6 thru 17 of gestation. Samples of dosing preparations will be frozen 

and retained for possible analysis by the Sponsor. Records will be maintained on how 

dosing preparations were made. 

Clinical Observations: Twice daily; once approximately one hour after dosing and then 

4 to 6 hours later. 

Body Weights: On days 0,4, 6, 8, 10, 12, 14, 16, 18 and 20 of gestation 

Food Consumption: On days when body weights are obtained 

Cesarean Section: On day 20 of gestation the females will be anesthetized and the 

uterus and its contents wilI be removed and weighed. The number of implanations, the 

number of live and dead fetuses, and the number of early and late resorptions will be 

determined and recorded. The fetuses will be sexed, weighed and examined for any gross 

abnormalities. The ovaries will examined for the number of corpora lutea. 

Fetal Examinations: Approximately one-half of the fetuses from each litter will 

processed for visceral examination by the Wilson Technique for soft tissue development. 

The remaining fetuses will be eviscerated and processed for skeletal examination using 

Alizarin Ked S staining method. 

000123 

110 
January 2 1, 1999 

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

(1 or 1.5 mg/kg) and five females at the high dose (20 or 25 mg/kg) plus three females at 

other dose levels, will be dosed. On the day after the last dose (day 18 of gestation) the 

satellite females will be killed and following samples will be collected: serum and liver 

from the dam and placentas and fetuses from the uterus. (Note placentas 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. 

111 
January 2 I, 1999 

Rabbit Teratology Rangefinder on N-EtFOSE 

Study Outline 

Study Objective: Explore possible dose levels for an oral rabbit teratology study 

GLP Status: Study should be conducted under GLP principles but since it is a 

rangefinder study QA audits will not be done 

Animals: Mated female New Zealand White rabbits (Note the rabbits are to be mated at 

the testing laboratory not at the supplier and shipped as mated females to the testing 

laboratory.) 

Dose Groups: Seven - control, 0 mg/kg; low- 1, 1 mg/kg; low-2, 5 mg/kg; mid- 1 , 10 

mg/kg; mid-2,25 mg/kg; high-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 is responsible for 

compound identity and purity. No reserve sample will be required. 

Compound Administration: Oral intubation of compound suspended in water with 2% 

Tween 80 on days 7 thru 20 of gestation. Analysis of dosing preparation will not be 

done; however, records will be maintained on how dosing preparations were made. 

Clinical Observations: Twice daily; once approximately one hour after dosing and then 

4 to 6 hours later. 

Body Weights: On days 0,7, 10, 13, 16, 19,24 and 29 of gestation 

Food Consumption: On days when body weights are obtained 

Cesarean Section: On day 29 of gestation the females will be anesthetized and the 

uterus and its contents will be removed and weighed. The number of implanations, the 

number of live and dead fetuses, and the number of early and late resorptions will be 

determined and recorded. The fetuses will be weighed and examined for any gross 

abnormalities. The ovaries will be examined for the number of corpora lutea. 

112 

000125 
January 2 1, 1999 

Rabbit Teratology Study on N-EtFOSE 

Study Outline 

Study Objective: To determine maternal and fetal toxicity and teratogenic potential of 

orally administered N-EtFOSE in pregnant rabbits 

GLP Status: A GLP study with appropriate QA audits; a signed QUA statement to be 

included in the final report. 

Animals: Mated female New Zealand White rabbits (Note the rabbits are to be mated at 

the testing laboratory not at the supplier and shipped as mated females to the testing 

laboratory.) 

Dose Groups: Five - control, 0 mg/kg; low, (1 ?) mg/kg; mid-1 , (5 ?) mg/kg; mid-2, 

(10 ?) mg/kg; high, (25 ?) mg/kg. Final dose levels will be adjusted after range finder 

study results are available. 

Number per Group: 22 mated females per group 

Test Article: N-EtFOSE will be furnished by the Sponsor who is responsible for 

compound identity and purity. Reserve sample to be retained. 

Compound Administration: Oral intubation of compound suspended in water with 2% 

Tween 80 on days 7 thru 20 of gestation. Samples of dosing preparations will be frozen 

and retained for possible analysis by the Sponsor. Records will be maintained on how 

dosing preparations were made. 

Clinical Observations: Twice daily; once approximately one hour after dosing and then 

4 to 6 hours later. 

Body Weights: On days 0,7, 10, 13, 16, 19,24 and 29 of gestation 

Food Consumption: On days when body weights are obtained 

Cesarean Section: On day 29 of gestation the females will be anesthetized and the 

uterus and its contents will be removed and weighed. The number of implantations, the 

number of live and dead fetuses, and the number of early and late resorptions will be 

determined and recorded. The fetuses will be weighed and examined for any gross 

abnormalities. The ovaries will examined for the number of corpora lutea. 

113 
January 2 1, 1999 

Fetal Examinations: A mid-coronal slice will be made in the head of each fetus to 

evaluate the contents of the cranium. The internal organs of the thoracic and abdominal 

cavities of all fetuses will be examined in the fresh state using Staples’ technique for 

internal abnormalities. At this time the sex of the each fetus will be determined. After 

removal of the viscera, the carcasses will processed for skeletal examination. 

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

(1 ? mg/kg) and five females at the high dose (25 ? mg/kg) plus three females at other 

dose levels, will be dosed. On the day after the last dose (day 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 of N-EtFOSE 

Study Outline 

Study Objective: To evaluate the effect of oral administration of N-EtFOSE on the 

reproductive function of male and female rats (Fo generation) and on the development and 

reproductive capacity of the subsequent 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, 1 mg/kg; mid- 1 , 5 mg/kg; mid-2, 10 

mg/kg; high, 15 mg/kg. 

Number per Group: 35 males and 35 females per group 

Test Article: N-EtFOSE will be furnished by the Sponsor who is responsible for 

compound identity and purity. Reserve sample to be retained. 

Compound Administration: Daily oral intubation of compound suspended in water 

with 2% Tween 80. Compound administration will start 4 week prior to mating. 

Compound administration will continue through mating and in the females through 

gestation and lactation. The 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: Weekly except when male and females are eo-inhabited during mating. 

Food Consumption: Weekly except when male and females are co-inhabited during 

mating. 

Estrus Cycles: Daily vaginal smears will be taken from females two weeks prior to 

mating to determine estrus cycle information. 

Mating: After 4 weeks of compound administration, within a dose group one male will 

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

115 
January 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 10 of gestation. 

Pregnancy will be determined as well as the number of implanations and the number of 

early and late resorptions. At necropsy serum and liver samples will be taken from five 

of the females in each dose group. These samples will be frozen and shipped to the 

sponsor for possible analysis. 

Male Sacrifice: One week after mating all Fo male rats will be killed after a terminal 

body weight has been obtained. The animals will be necropsied and any gross lesions 

will be described and recorded. The testes, epididymis, prostate and seminal vesicles will 

be weighed and fixed for possible histologic examination. (Note: testis will be fixed in 

Bouin’s solution; other tissues in 10% buffered formalin). At necropsy serum and liver 

samples will be taken from five of the males in each dose group. These samples will be 

frozen and shipped to the sponsor for possible analysis. 

Female Parturition: The remaining 25 females will continue until term and be allow to 

deliver their litters. The offspring (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 of the litter on day 21 of lactation, all 

Fo female rats will be killed after a terminal body weight has been obtained. The animals 

will be necropsied and any gross lesions will be described and recorded. The ovaries will 

be weighed and fixed for possible histologic examination. (Note: ovaries will be fixed in 

10% buffered formalin). At necropsy serum and liver samples will be taken from five of 

the females in each dose group. These samples will be frozen and shipped to the sponsor 

for possible analysis. 

116 000.129 
January 2 1, 1999 

F1 Pup Neurological Testing: During the 4'h week postpartum, the F1 pups will be 

evaluated in a passive avoidance test for learning and short term memory retention. 

During the 1 Oth week postpartum, the FI pups 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. The pups will then allow to grow and body 

weights and food consumption will be recorded weekly. The F1 pups will undergo 

behavioraUfimctiona1 testing. At sexual maturity, within a dose group, one male will be 

mated with one female (sibling mating to be avoided). The females will be allowed to 

litter and raise the F2 pup through 21 days of lactation. 

117 

0140130 
January 2 1, 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 of the subsequent FI generation which were exposed in utero and 

via lactation. 

GLP Status: A GLP study with appropriate QA audits; a signed QUA statement to be 

included in the final report. 

Animals: Male and female Charles River CD rats 

Dose Groups: Five - control, 0 mg/kg; low, 0.1 or 0.2 mg/kg; mid-1, 0.5 or 1.0 mg/kg; 

mid-2,2 or 3 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 Sponsor who is responsible for compound 

identity and purity. Reserve sample to be retained. 

Compound Administration: Daily oral intubation of compound suspended in water 

with 2% Tween 80. Compound administration will start 4 week prior to mating. 

Compound administration will continue through mating and in the females through 

gestation and lactation. The 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: Weekly except when male and females are co-inhabited during mating. 

Food Consumption: Weekly except when male and females are co-inhabited during 

mating. 

Estrus Cycles: Daily vaginal smears will be taken from females two weeks prior to 

mating to determine estrus cycle information. 

Mating: After 4 weeks of compound administration, within a dose group one male will 

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

118 
January 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 females per group will killed on day 7 of gestation. 

Pregnancy will be determined as well as the number of implanations and the number of 

early and late resorptions. 

Male Sacrifice: One week after mating all Fo male rats will be killed after a terminal 

body weight has been obtained. The animals will be necropsied and any gross lesions 

will be described and recorded. The testes, epididymis, prostate and seminal vesicles will 

be weighed and fixed for possible histologic examination. (Note: testis will be fixed in 

Bouin’s solution; other tissues in 10% buffered formalin). At necropsy serum and liver 

samples will be taken from five of the males in each dose group. These samples will be 

frozen and shipped to the sponsor for possible analysis. 

Female Parturition: The remaining 25 females will continue until term and be allow to 

deliver their litters. The offspring (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 of the 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 1 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 of the litter on day 21 of lactation, all 

Fo female rats will be killed after a terminal body weight has been obtained. The animals 

will be necropsied and any gross lesions will be described and recorded. The ovaries will 

be weighed and fixed for possible histologic examination. (Note: ovaries will be fixed in 

10% buffered formalin). At necropsy serum and liver samples will be taken from five of 

the females in each dose group. These samples will be frozen and shipped to the sponsor 

for possible analysis. 
January 2 I, 1999 

F1 Pup Neurological Testing: During the 4'h week postpartum, the F1 pups will be 

evaluated in a passive avoidance test for learning and short term memory retention. 

During the loth week postpartum, the FI pups will be evaluated in a water-filled maze for 

neuromuscular coordination, learning, and longer term more complex memory. 

F1 Growth and Reproduction: The FI pups will be weaned after 21 days of lactation. 

The pups will then allow to grow and body weights and food consumption will be 

recorded weekly. The FI pups will undergo behavioral/functional testing. At sexual 

maturity, within a dose group, one male will be mated with one female (sibling mating to 

be avoided). The females will be allowed to litter and raise the F2 pup through 21 days of 

lactation. 
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 female New Zealand White rabbits (Note the rabbits are to be mated at 

the testing laboratory not at the supplier and shipped as mated females to the testing 

laboratory.) 

Dose Groups: Seven - control, 0 mg/kg; low-1,O.l mg/kg; low-2, 1 mg/kg; mid-l,2.5 

mg/kg; mid-2,5 mgkg; high-1, 10 mg/kg; high-2,20 mgkg 

Number per Group: 5 mated females per group 

Test Article: N-EtPFOS will be furnished by the Sponsor who is responsible for 

compound identity and purity. No reserve sample will be required. 

Compound Administration: Oral intubation of compound suspended in water with 2% 

Tween 80 on days 7 thru 20 of gestation. Analysis of dosing preparation will not be 

done; however, records will be maintained on how dosing preparations were made. 

Clinical Observations: Twice daily; once approximately one hour after dosing and then 

4 to 6 hours later. 

Body Weights: On days 0,7, 10, 13, 16, 19,24 and 29 of gestation 

Food Consumption: On days when body weights are obtained 

Cesarean Section: On day 29 of gestation the females will be anesthetized and the 

uterus and its contents will be removed and weighed. The number of implanations, the 

number of live and dead fetuses, and the number of early and late resorptions will be 

determined and recorded. The fetuses will be weighed and examined for any gross 

abnormalities. The ovaries will examined for the number of corpora lutea. 

121 
January 2 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 female New Zealand White rabbits (Note the rabbits are to be mated at 

the testing laboratory not at the supplier and shipped as mated females to the testing 

laboratory.) 

Dose Groups: Five - control, 0 mg/kg; low, 0.1 mg/kg; mid-1 , 1 mg/kg; mid-2,5 

mg/kg; high, 10 mg/kg. Final dose levels will be adjusted after range finder study results 

are available. 

Number per Group: 22 mated females per group 

Test Article: FOS will be furnished by the Sponsor who is responsible for compound 

identity and purity. Reserve sample to be retained. 

Compound Administration: Oral intubation of compound suspended in water with 2% 

Tween 80 on days 7 thru 20 of gestation. Samples of dosing preparations will be frozen 

and retained for possible analysis by the Sponsor. Records will be maintained on how 

dosing preparations were made. 

Clinical Observations: Twice daily; once approximately one hour after dosing and then 

4 to 6 hours later. 

Body Weights: On days 0, 7, 10, 13, 16, 19,24 and 29 of gestation 

Food Consumption: On days when body weights are obtained 

Cesarean Section: On day 29 of gestation the females will be anesthetized and the 

uterus and its contents will be removed and weighed. The number of implanations, the 

number of live and dead fetuses, and the number of early and late resorptions will be 

determined and recorded. The fetuses will be weighed and examined for any gross 

abnormalities. The ovaries will examined for the number of corpora lutea. 

122 
January 2 1, 1999 

Fetal Examinations: A mid-coronal slice will be made in the head of each fetus to 

evaluate the contents of the cranium. The internal organs of the thoracic and abdominal 

cavities of all fetuses will be examined in the fresh state using Staples' technique for 

internal abnormalities. At this time the sex of the each fetus will be determined. After 

removal of the viscera, the carcasses will processed for skeletal examination. 

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

(0.1 mg/kg) and five females at the high dose 10 mag) 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. 

123 
January 2 1, 1999 

Rabbit Teratology Rangefinder on N-EtFOSE 

Study Outline 

Study Objective: Explore possible dose levels for an oral rabbit teratology study 

GLP Status: Study should be conducted under GLP principles but since it is a 

rangefinder study QA audits will not be done 

Animals: Mated female New Zealand White rabbits (Note the rabbits are to be mated at 

the testing laboratory not at the supplier and shipped as mated females to the testing 

laboratory.) 

Dose Groups: Seven - control, 0 mg/kg; low- 1, 1 mg/kg; low-2, 5 mg/kg; mid- 1 , 10 

mg/kg; mid-2,25 mgkg; high-1,50 mg/kg; high-2,75 mg/kg 

Number per Group: 5 mated females per group 

Test Article: N-EtPFOS will be furnished by the Sponsor who is responsible for 

compound identity and purity. No reserve sample will be required. 

Compound Administration: Oral intubation of compound suspended in water with 2% 

Tween 80 on days 7 thru 20 of gestation. Analysis of dosing preparation will not be 

done; however, records will be maintained on how dosing preparations were made. 

Clinical Observations: Twice daily; once approximately one hour after dosing and then 

4 to 6 hours later. 

Body Weights: On days 0,7, 10, 13, 16, 19,24 and 29 of gestation 

Food Consumption: On days when body weights are obtained 

Cesarean Section: On day 29 of gestation the females will be 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 of early and late resorptions will be 

determined and recorded. The fetuses will be weighed and examined for any gross 

abnormalities. The ovaries will examined for the number of corpora lutea. 

124 
January 2 1, 1999 

Rabbit Teratology Study on N-EtFOSE 

Study Outline 

Study Objective: To determine maternal and fetal toxicity and teratogenic potential of 

orally administered N-EtPFOS in pregnant rabbits 

GLP Status: A GLP study with appropriate QA audits; a signed QUA statement to be 

included in the final report. 

Animals: Mated female New Zealand White rabbits (Note the rabbits are to be mated at 

the testing laboratory not at the supplier and shipped as mated females to the testing 

laboratory.) 

Dose Groups: Five - control, 0 mgkg; low, (1 ?) mgkg; mid-1, (5 ?) mgkg; mid-2, 

(1 0 ?) 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 funished by the Sponsor who is responsible for 

compound identity and purity. Reserve sample to be retained. 

Compound Administration: Oral intubation of compound suspended in water with 2% 

Tween 80 on days 7 thru 20 of gestation. Samples of dosing preparations will be frozen 

and retained for possible analysis by the Sponsor. Records will be maintained on how 

dosing preparations were made. 

Clinical Observations: Twice daily; once approximately one hour after dosing and then 

4 to 6 hours later. 

Body Weights: On days 0,7, 10, 13, 16, 19,24 and 29 of gestation 

Food Consumption: On days when body weights are obtained 

Cesarean Section: On day 29 of gestation the females will be anesthetized and the 

uterus and its contents will be removed and weighed. The number of implanations, the 

number of live and dead fetuses, and the number of early and late resorptions will be 

determined and recorded. The fetuses will be weighed and examined for any gross 

abnormalities. The ovaries will examined for the number of corpora lutea. 

125 
January 2 1, 1999 

Fetal Examinations: A mid-coronal slice will be made in the head of each fetus to 

evaluate the contents of the cranium. The internal organs of the thoracic and abdominal 

cavities of all fetuses will be examined in the fresh state using Staples’ technique for 

internal abnormalities. At this time the sex of the each fetus will be determined. After 

removal of the viscera, the carcasses will processed for skeletal examination. 

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

(1 ? 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 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. 

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January 21, 1999 

Corporate Toxicology Study Outline 

Title: 

1) Effect of Perfluorochemicals 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 characterization based on animal data; 

2) Compare the metabolic response ([ATP], respiration rate, mitochondrial membrane 

potential, markers of peroxisome Proliferation, et al.) of rat, guinea pig and primate 

hepatocytes to this class of compounds with particular emphasis on mitochondrial and 

peroxisomal metabolism to: 

a) Further test the proposed mechanism of toxicity; 

b) Identify potentially useful metabolic biomarkers of exposure; 

c) Help validate relative differences between species (assess markers of peroxisome 

proliferation across species); 

3) Compare molecular response (gene expression 3 mRNA) of rat, 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 of peroxisomes and cell proliferation 

(ongogenic response) to test dogma; 

Assess the relevance of rat 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 for judging the most appropriate 

species for predicting human health outcomes following exposures to compounds and 

has relevance to establishing safety factors and putting human cancer risk in 

perspective; 

Species differences in metabolic and molecular response will reveal important insight 

into mechanisms responsible for compound-induced peroxisome proliferation and/or 

tumorigenesis which is also vital to identifying valid biomarkers to assess exposures 

and potential risks; 

Comparing the enhanced transcription and expression of genes associated with 

peroxisome versus cell proliferation will allow opportunities to evaluate the 

127 
January 2 1, 1999 

relationship between metabolic and oncogenic effects of these compounds in different 

species. 

0 b j ec tiv e(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 on hepatocyte 

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 of high homology between species using Gene Bank@ 

c) Design primers to 1 S - 3.0 kbp sequences (Oligo@) 

d) PCR amplify sequences from rat-liver cDNA template 

e) Separate, bands on low melting point gels 

f) Purify bands (band-stab) 

g) Restriction digest to confirm identity (MacDNAsisB to obtain restriction site map 

h) Label probe with 32P-dCTP by random priming (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 of peroxisomal metabolism and 

for each probe) 

4) MRNA probes suggested for peroxisome proliferation 

5) Probes for cell proliferation 

6) Establish benchmarks for assessing the effects of PF compounds on hepatocyte 

stimulation of cell proliferation using biochemical markers and molecular markers 

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

128 000141 
January 2 I, 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 ending June 30,2001 

Report: 

Quarterly progress reports