Unclassified ENV/ JM/ RD( 2002) 17/ FINAL Organisation de Coopération et de Développement Economiques Organisation for Economic Co operation and Development 21 Nov 2002 ___________________________________________________________________________________________ English  Or. English ENVIRONMENT DIRECTORATE JOINT MEETING OF THE CHEMICALS COMMITTEE AND THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGY CO OPERATION ON EXISTING CHEMICALS HAZARD ASSESSMENT OF PERFLUOROOCTANE SULFONATE ( PFOS) AND ITS SALTS JT00135607 Document complet disponible sur OLIS dans son format d'origine Complete document available on OLIS in its original format ENV/

JM/

RD(

2002)

17/

FINAL Unclassified English

Or.

English ENV/ JM/ RD( 2002) 17/ FINAL 2 Preface In the margins of the ninth meeting of the Task Force on Existing Chemicals ( 29 30 May 2000) several Member countries agreed to informally work together to collect information on the environmental and human health hazards of perfluorooctane sulfonate ( PFOS) to produce a hazard assessment. The decision followed the announcement by a major US manufacturer   3M   to globally phase out the manufacture and use of these chemicals beginning in 2001. The US and the UK agreed to lead the activity with the Secretariat assisting by requesting readily available exposure information from Member countries as well as from non Member countries through IFCS.

An informal meeting was hosted by the US on 26 27 October 2000 ( Crystal City, Virginia, US) to:

  review the current status of assessment activities;   learn about actions being taken in other countries; and   identify planned or ongoing work on this issue.

In preparation for the meeting 3M circulated a draft initial assessment report including robust study summaries of key studies, together with exposure information.

At the 31st Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology ( 7 10 November 2000), it was agreed that, since this was a matter of sufficient interest to all Member countries, this activity should be undertaken under the existing Chemicals Programme, overseen by the Task Force. As PFOS is not an HPV Chemical, it was not dealt with under the HPV Chemicals Programme.

A draft hazard assessment was posted on the OECD web site for comment in December 2000. The OECD established an electronic discussion group to exchange comments and information. A special session on PFOS and its salts was held on 25 January 2001 in Orlando, USA, as part of the 11th SIAM meeting. At this session an overview of the draft hazard assessment was presented.

The draft hazard assessment was revised twice since December 2000 to incorporate comments that were received, as well as to incorporate newly completed studies. Comments were received from 3M, World Wildlife Fund, Health Canada, Environment Canada, and Australia.

At the 11th meeting of the Task Force on Existing Chemicals ( 27 28 May 2002), the revised hazard assessment was discussed. The Task Force agreed with the conclusions and recommendations of the hazard assessment. The Task Force also agreed that the Secretariat should gather information from governments and BIAC on risk management activities currently undertaken or planned in Member countries on PFOS.

At the 34th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology ( 5 8 November 2002), the final draft of the assessment was endorsed. The Joint Meeting recommended that this document be derestricted under the authority of the Secretary General.

This hazard assessment of perfluorooctane sulfonate ( PFOS) and its salts includes all information that was available by July 2002. A quantitative risk assessment was not conducted as this should entail regional exposure information. The hazard information on PFOS should be used with caution in evaluating the potential hazards of other perfluorinated compounds. The perfluorinated compounds represent a very unique chemistry whose toxicological properties are presently not well understood and clearly the presence of different length ( perfluorinated) carbon chains and functional groups are likely to influence toxicity. It is not clear at this time whether the hazard concerns of PFOS can be extrapolated to other perfluorinated compounds except under circumstances where the compound may degrade to PFOS.

Assessment activities on PFOS and its salts are also on going in other international fora, e. g. OSPAR. ENV/ JM/ RD( 2002) 17/ FINAL 3 TABLE OF CONTENTS Recommendations of the Hazard Assessment................................................................ 5 Summary and Conclusions of the Hazard Assessment.................................................. 5 1.0 Identity............................................................................................................................. 10 1.1 Physicochemical Properties ........................................................................................... 10 2.0 General Information on Exposure ................................................................................ 11 2.1 Production and Use of PFOS......................................................................................... 12 2.1.1 PFOS Based Surface Treatment Applications............................................................. 13 2.1.2 PFOS Based Paper Protection Applications ................................................................ 13 2.1.3 PFOS Based Performance Chemical Applications...................................................... 13 2.2 Environmental Exposure and Fate................................................................................ 15 2.2.1 Volatility .......................................................................................................................... 15 2.2.2 Combustion ..................................................................................................................... 15 2.2.3 Photolysis/ Oxidation....................................................................................................... 15 2.2.4 Biodegradation................................................................................................................ 15 2.3 Environmental Monitoring ............................................................................................ 15 2.4 Human Biomonitoring.................................................................................................... 17 2.4.1 Occupational Exposures................................................................................................. 17 2.4.2 Non occupational Exposures ......................................................................................... 19 3.0 Human Health Hazards.................................................................................................. 23 3.1 Metabolism and Pharmacokinetics ............................................................................... 23 3.1.1 Absorption....................................................................................................................... 23 3.1.2 Distribution ..................................................................................................................... 23 3.1.3 Elimination ...................................................................................................................... 23 3.1.4 Half life in Humans ........................................................................................................ 24 3.2 Acute Toxicity ................................................................................................................. 25 3.3 Mutagenicity.................................................................................................................... 26 3.4 Repeated Dose Toxicity .................................................................................................. 27 3.5 Carcinogenicity ............................................................................................................... 34 3.6 Developmental Toxicity.................................................................................................. 39 3.7 Reproductive Toxicity .................................................................................................... 43 3.8 Human Hazard................................................................................................................ 50 4.0 Hazards to the Environment.......................................................................................... 55 4.1 Effects on Fish, Invertebrates and Algae...................................................................... 56 4.1.1 Fish ................................................................................................................................... 57 4.1.2 Invertebrates ................................................................................................................... 64 4.1.3 Aquatic Plants ................................................................................................................. 69 4.2 Effects on Other Aquatic Organisms............................................................................ 73 4.2.1 Amphibians ..................................................................................................................... 73 4.2.2 Sediment Dwelling Invertebrates .................................................................................. 75 4.2.3 Bacteria ............................................................................................................................ 75 4.2.4 Activated Sludge Microorganisms................................................................................. 77 4.3 Effects on Terrestrial Organisms .................................................................................. 79 4.3.1 Soil dwelling Invertebrates ............................................................................................ 79 4.3.2 Terrestrial Plants ............................................................................................................ 79 4.3.3 Birds ................................................................................................................................. 79 4.3.4 Bees................................................................................................................................... 81 5.0 References........................................................................................................................ 83 ENV/ JM/ RD( 2002) 17/ FINAL 4 Annex 1 Ecological Studies ................................................................................................. 89 Annex 2 Robust Summaries of Key Ecotoxicology Studies ............................................. 91 Annex 3 Application of Equilibrium Partitioning Models to Determining Effect Concentrations for PFOS Salts in Soil and Sediment ..................................... 213 Annex 4 Summary of the Lowest Acceptable Effect Concentrations ........................... 216 Annex 5 Robust Summaries for Physical Chemical Properties and Environmental Fate Studies .............................................................................. 218 Annex 6 Robust Summaries of Toxicology and Human Biomonitoring Studies........................................................................................ 256 ENV/ JM/ RD( 2002) 17/ FINAL 5 RECOMMENDATIONS OF THE HAZARD ASSESSMENT Perfluorooctane sulfonate ( PFOS) is a candidate for further work.

Sufficient information exists to address hazard classification for all SIDS human health endpoints. PFOS is persistent, bioaccumulative and toxic to mammalian species. There are species differences in the elimination half life of PFOS; the half life is 100 days in rats, 200 days in monkeys, and years in humans. The toxicity profile of PFOS is similar among rats and monkeys. Repeated exposure results in hepatotoxicity and mortality; the dose response curve is very steep for mortality. This occurs in animals of all ages, although the neonate may be more sensitive. In addition, a 2 year bioassay in rats has shown that exposure to PFOS results in hepatocellular adenomas and thyroid follicular cell adenomas; the hepatocellular adenomas do not appear to be related to peroxisome proliferation. Further work to elucidate the species differences in toxicokinetics and in the mode of action of PFOS will increase our ability to predict risk to humans. Epidemiologic studies have shown an association of PFOS exposure and the incidence of bladder cancer; further work is needed to understand this association.

Sufficient information exists to address hazard classification for all SIDS environmental endpoints. PFOS is persistent in the environment and has been shown to bioconcentrate in fish. It has been detected in a number of species of wildlife, including marine mammals. Its persistence, presence in the environment and bioaccumulation potential indicate cause for concern. It appears to be of low to moderate toxicity to aquatic organisms but there is evidence of high acute toxicity to honey bees. No information is available on effects on soil and sediment dwelling organisms and the equilibrium partitioning method may not be suitable for predicting PNECs for these compartments. PFOS has been detected in sediment downstream of a production site and in effluents and sludge from sewage treatment plants.

Given the apparent widespread occurrence of PFOS, national or regional exposure information gathering and risk assessment may need to be considered. In addition, data on its toxicity to soil and sedimentdwelling organisms could be generated as a post SIDS activity. There is currently no information on effects on soil or sediment dwelling organsisms and PFOS has been detected in sediment and its presence in sewage sludge could lead to soil exposure if spread on agricultural land.

SUMMARY AND CONCLUSIONS OF THE HAZARD ASSESSMENT Perfluorooctane sulfonate ( PFOS) and its salts are fully fluorinated organic compounds. The number of production sites is not clear, but there is production in the US, Europe and Japan. In recent years ( to 2000), approximately 4,500 metric tons of PFOS related chemicals have been produced annually. The major global producer of PFOS intends to cease production by the end of 2002.

The majority of PFOS related chemicals are high molecular weight polymers in which PFOS represents a fraction of the total molecular weight. PFOS related chemicals are used in a variety of products, including as surface treatments of fabric for soil/ stain resistance, coating of paper as part of a sizing agent formulation and in specialised applications such as fire fighting foams.

PFOS has a solubility of approximately 550 mg/ l in pure water at 24 25 ° C. The solubility decreases significantly with increased salt content, for example the potassium salt of PFOS has a solubility in fresh water of 370 mg/ L and of 25 mg/ l in filtered sea water. Due to the surface active properties of PFOS, the Log Kow cannot be measured. The potassium salt of PFOS has a low vapour pressure, 3.31 x 10 4 Pa at 20 ° C. ENV/ JM/ RD( 2002) 17/ FINAL 6 Human Health In human blood samples, PFOS has been detected in the serum of occupational and general populations. In the U. S., the highest reported blood serum level of PFOS was observed in 1995 in a manufacturing employee in Decatur, Alabama ( 12.83 ppm). Mean PFOS levels have been dropping in that plant and a plant in Belgium since 1995, the most recent being 1.32 ppm and 0.80 ppm, respectively, in 2000. In the general population, serum collected from blood banks and commercial sources have indicated mean PFOS levels of 30 53 ppb. In individual serum samples obtained from adults and children in various regions of the U. S., mean PFOS levels were approximately 43 ppb.

Several occupational studies have been conducted on volunteers at the 3M plants in Decatur, Alabama and Antwerp, Belgium. Cross sectional studies, based on the results of a voluntary medical surveillance program for employees at each plant, did not report consistent associations between workers' PFOS levels less than 6 ppm and certain hematology, hormonal, and other clinical chemistry parameters in 1995 and 1997. In 2000 when the analysis included male employees from both plants, mean values for triglycerides, alkaline phosphatase, total bilirubin, and ALT were significantly ( p < .05) higher for workers with the highest PFOS serum levels ( 1.69   10.06 ppm). Serum triiodothyronine was significantly higher and thyroid hormone binding ratio was significantly lower in workers with the highest PFOS serum levels ( p < .05 for both). The association with T3 also remained significant and positive in multivariable regression analyses adjusted for potential confounders. A longitudinal analysis of these data did not reveal statistically significant associations over time between PFOS and cholesterol, triglycerides, and other lipid and hepatic parameters. Hormones were not included in these analyses. There are several limitations to both the cross sectional and longitudinal studies, such as the voluntary nature of the medical surveillance, the small number of employees participating across sampling periods, the different labs and analytical techniques used to measure serum PFOS, and the differences in PFOS levels, demographics, and clinical chemistries between employees in the Decatur and Antwerp plants.

In a mortality study, which followed workers for 37 years, mortality risks for most of the cancer types and non malignant causes were not elevated. However, a statistically significant risk of death from bladder cancer was reported. Three male employees in the cohort died of bladder cancer ( 0.12 expected), and all of them had been employed at the plant for more than 20 years. All of them had also worked in high exposure jobs for at least 5 years. In order to screen for morbidity outcomes, an " episode of care" analysis was undertaken for employees who had worked at the plant between 1993 and 1998. Many different types of cancer and other non malignant conditions were examined. Increased risks were not reported for most of the conditions or did not reach statistical significance. However, an increased risk of episodes was reported for neoplasms of the male reproductive system, the overall category of cancers and benign growths, and neoplasms of the gastrointestinal tract. These risk ratios were highest in employees with the highest and longest exposures to fluorochemicals.

Animal studies show that PFOS is well absorbed orally and distributes mainly in the serum and the liver. No further metabolism is expected. Elimination from the body is slow and occurs via the urine and feces. There are species differences in the elimination half life of PFOS. The half life in serum is 7.5 days in adult rats and 200 days in Cynomolgus monkeys. In humans, it appears to be quite longer. A recent halflife analysis was conducted on 9 retired 3M chemical workers. PFOS samples were collected over 4 time periods spanning 180 days, measured in triplicate with all time points from each subject analyzed in the same analytical run. The mean half life for PFOS was 8.67 years ( range 2.29   21.3 years, SD = 6.12).

PFOS has shown moderate acute toxicity by the oral route with a rat LD50 of 251 mg/ kg. A one hour LC50 of 5.2 mg/ l in rats has been reported. PFOS was found to be mildly irritating to the eyes and nonirritating to the skin of rabbits. PFOS has not been shown to be genotoxic in a variety of assay systems. ENV/ JM/ RD( 2002) 17/ FINAL 7 Numerous repeat dose oral toxicity studies on PFOS have been conducted in rats and primates. In general, exposure to PFOS results in hepatotoxicity and mortality; the dose response curve for mortality is very steep for rats and primates. Adverse signs of toxicity observed in 90 day rat studies included increases in liver enzymes, hepatic vacuolization and hepatocellular hypertrophy, gastrointestinal effects, hematological abnormalities, weight loss, convulsions, and death. These effects were reported at doses of 2 mg/ kg/ day and above. In a dietary 2 year bioassay in Sprague Dawley rats, hepatotoxicity, characterized by centrilobular hypertrophy, centrilobular eosinophilic hepatocytic granules, centrilobular hepatocytic pigment, or centrilobular hepatocytic vacuolation was noted in male and/ or female rats given 5 or 20 ppm. Hepatocellular centrilobular hypertrophy was also observed in mid dose ( 2 ppm) male rats. Significant increases in the incidence of cystic hepatocellular degeneration were found in all the male treated groups ( 0.5, 2, 5, or 20 ppm). Based on the pathological findings in the liver, the LOAEL was 5 ppm and the NOAEL was 2 ppm in female rats. In males, the LOAEL was 0.5 ppm, and a NOAEL was not established.

Adverse signs of toxicity observed in Rhesus monkey studies included anorexia, emesis, diarrhea, hypoactivity, prostration, convulsions, atrophy of the salivary glands and the pancreas, marked decreases in serum cholesterol, and lipid depletion in the adrenals. The dose range for these effects was reported between 1.5 300 mg/ kg/ day. No monkeys survived beyond 3 weeks into treatment at 10 mg/ kg/ day or beyond 7 weeks into treatment at doses as low as 4.5 mg/ kg/ day. In a 6 month study of Cynomolgus monkeys, low food consumption, excessive salivation, labored breathing, hypoactivity, ataxia, hepatic vacuolization and hepatocellular hypertrophy, significant reductions in serum cholesterol levels, and death were observed at 0.75 mg/ kg/ day. No effects were observed at doses of 0.15 or 0.03 mg/ kg/ day. No effects were noted in animals at any dose level following a 52 week recovery period. The average concentration of PFOS in the serum following 26 weeks of treatment was 11.1 + 1.52, 58.5 + 4.67 and 160 + 23.9 µ g/ ml for the females in the 0.03, 0.15 and 0.75 mg/ kg/ day groups, respectively; for males, the average concentrations were 15.9 + 5.54, 68.1 + 5.75 and 194 + 8.93 µ g/ ml in the 0.03, 0.15 and 0.75 mg/ kg/ day groups, respectively. After the 52 week recovery period, the serum levels were 21.4 + 2.01 and 41.4 + 1.15 µ g/ ml for the females in the 0.15 and 0.75 mg/ kg/ day groups, respectively; for males, the average concentrations were 19.1 + 0.805 and 41.1 + 25.9 µ g/ ml in the 0.15 and 0.75 mg/ kg/ day groups, respectively.

The potential carcinogenicity of PFOS has been examined in a dietary 2 year bioassay in Sprague Dawley rats. There was a significant increase in the incidence of hepatocellular adenomas in males and females at the highest dose of 20 ppm; the females at 20 ppm also had a significant increase in combined hepatocellular adenomas and carcinomas. In addition, there was a significant increase in thyroid follicular cell adenomas and combined thyroid follicular cell adenomas and carcinomas in the male recovery group at 20 ppm. There was no evidence of peroxisome proliferation in the livers of the treated animals.

Postnatal deaths and other developmental effects were reported at low doses in offspring in a 2 generation reproductive toxicity study in rats. At the two highest doses of 1.6 and 3.2 mg/ kg/ day, pup survival in the first generation was significantly decreased. All first generation offspring ( F1 pups) at the highest dose died within a day after birth while close to 30% of the F1 pups in the 1.6 mg/ kg/ day dose group died within 4 days after birth. As a result of the pup mortality in the two top dose groups, only the two lowest dose groups, 0.1 and 0.4 mg/ kg/ day, were continued into the second generation. The NOAEL and LOAEL for the second generation offspring ( F2 pups) were 0.1 mg/ kg/ day and 0.4 mg/ kg/ day, respectively, based on reductions in pup body weight.

The liver and serum from the F0 and F1 animals was analyzed for PFOS. Qualitatively, the results for the F0 animals indicate that all rats ( including controls) had detectable levels of PFOS in serum and livers. PFOS concentration increased with dose. PFOS concentrations were higher in the liver than in the serum, and males had greatly increased PFOS concentrations in serum and liver when compared with females of ENV/ JM/ RD( 2002) 17/ FINAL 8 the same dose group. Pooled liver samples from the F1animals sacrificed shortly after birth had lower PFOS concentrations than adults of the F0 generation of the same dose group.

Based on the results of the two generation reproductive toxicity study, a cross fostering study was conducted as a means of determining whether the reductions in pup viability were a result of in utero exposure to PFOS or as a result of exposure during lactation; thus the potential for a distinction to be made between prenatal and postnatal effects following continuous maternal treatment. Under the limited conditions of the study, the data appear to indicate that reduced pup survival is mainly a result of in utero exposure to PFOS and that post natal exposure via milk in conjunction with in utero exposure may also contribute to reduced pup survival. In contrast, exposure during lactation alone, through milk from exposed dams, does not appear to have any adverse effect on pup viability.

Several mechanistic studies are being conducted to understand the neonatal death ( 3M Company, 2001c). Preliminary results indicate that reductions in serum lipids and cholesterol synthesis do not appear to play a significant role in the death of the offspring.

Developmental effects were also reported in prenatal developmental toxicity studies in the rat and rabbit, although at slightly higher dose levels. Signs of developmental toxicity in the offspring were evident at doses of 5 mg/ kg/ day and above in rats administered PFOS during gestation. Significant decreases in fetal body weight and significant increases in external and visceral anomalies, delayed ossification, and skeletal variations were observed. A NOAEL of 1 mg/ kg/ day and a LOAEL of 5 mg/ kg/ day for developmental toxicity were indicated. In the same study, evidence of treatment related signs of maternal toxicity were also observed at doses of 5 mg/ kg/ day and above and mainly consisted of hunched posture, anorexia, bloody vaginal discharge, uterine stains, alopecia, rough hair coat, and bloody crust, as well as decreases in body weight gains and food consumption. Reductions in the mean terminal body weights minus the gravid uterine weights were also observed at doses > 5 mg/ kg/ day. A NOAEL of 1 mg/ kg/ day and a LOAEL of 5 mg/ kg/ day for maternal toxicity were indicated. In rabbits, significant reductions in fetal body weight and significant increases in delayed ossification were observed in the offspring of pregnant females administered PFOS during gestation at doses of 2.5 mg/ kg/ day and above. A NOAEL of 1.0 mg/ kg/ day and a LOAEL of 2.5 mg/ kg/ day for developmental toxicity were indicated. Maternal toxicity in the does was evident at doses of 1.0 mg/ kg/ day and above, and consisted of an increase incidence of abortions and scant feces, as well as significant reductions in mean maternal body weight gains and food consumption. A NOAEL of 0.1 mg/ kg/ day and a LOAEL of 1.0 mg/ kg/ day for maternal toxicity were indicated.

Environment There is currently little information on the life cycle steps that may lead to release of PFOS to the environment. However, PFOS has been detected in surface water and sediment downstream of a production facility and in wastewater treatment plant effluent, sewage sludge and landfill leachate at a number of cities in the US. Sampling of several wildlife species from a variety of sites across the United States has shown widespread distribution of PFOS and it was detected in the ppb range in the plasma of several species of eagles, wild birds, and fish. PFOS has been detected in marine mammals at a number of locations across the world.

PFOS is persistent in the environment. It does not hydrolyse, photolyse or biodegrade under environmental conditions and is not expected to volatilize, based on an air/ water partition coefficient of < 2 E 6 Pa. m3/ mol. PFOS has been shown to bioconcentrate in the tissues of bluegill sunfish and carp. In bluegill sunfish, BCF ( BCFK) values between 1124 and 4013 were determined and PFOS depurated slowly with estimated 50% clearance times of up to 116 days. In carp, BCF values were determined to be between 200 and 1500. ENV/ JM/ RD( 2002) 17/ FINAL 9 The substance shows moderate acute toxicity to aquatic organisms, the lowest LC50 for fish is a 96 hour LC50 of 4.7 mg/ l to the fathead minnow Pimephales promelas for the lithium salt. For aquatic invertebrates, the lowest EC50 for freshwater species is a 48 hour EC50 of 27 mg/ l for Daphnia magna and for saltwater species, a 96 hour LC50 value of 3.6 mg/ l for the Mysid shrimp Mysidopsis  bahia. Both tests were conducted on the potassium salt. For algae, the potassium salt gave a 96h NOEC of > 3.2 mg/ l with Skeletonema costatum.

Long term toxicity data is available for fish and aquatic invertebrates. The lowest NOEC for fish is a 42 day NOEC ( survival) of 0.3 mg/ l in an early life stage test with Pimephales promelas using the potassium salt. The lowest NOEC for aquatic invertebrates is a 35 day NOECreproduction of 0.25 mg/ l for Mysidopsis bahia using the potassium salt. For freshwater species, there is a 28 day NOECreproduction of 7 mg/ l for Daphnia magna, also using the potassium salt.

A growth inhibition test has been carried out on PFOS potassium salt with Lemna gibba ( Duckweed). The test gave a 7 day IC50 of 108 mg/ l for inhibition of frond production and a 7 day NOEC of 15.1 mg/ l based on the inhibition of frond production and evidence of sub lethal effects.

PFOS does not appear to be toxic to sewage sludge microorganisms. In an activated sludge respiration inhibition test, the 3 hour IC50 value for PFOS ( potassium salt) was > 905 mg/ l ( nominal concentration).

No data are available for effects on soil dwelling or sediment dwelling species. The use of equilibrium partitioning models to derive a PNEC for these compartments may not be applicable to this anionic surfactant.

PFOS has been tested on two species of bird, the Mallard duck, Anas platyrhynchos, and the Northern Bobwhite quail, Colinus virginianus. The lowest acute dietary LC50 value of 220 mg/ kg of food was determined in the test with the quail. The lowest NOEC of 37 mg/ kg of food for effects on body weight was, in contrast, obtained in the test with the duck.

There are data available from acute oral and contact toxicity tests on the Honey bee ( Apis mellifera) using PFOS potassium salt. These studies indicate moderate and high orders of toxicity of PFOS to bees when administered via these routes. The acute oral test yielded a 72 hour LD50 for ingestion of PFOS of 0.40 µ g/ bee and a 72 hour NOEL of 0.21 µ g/ bee. The contact test yielded a 96 hour LD50 of 4.78 µ g/ bee and a 96 hour NOEL of 1.93 µ g/ bee.

The results of an amphibian teratogenesis study carried out with Xenopus laevis ( African clawed frog) show PFOS potassium salt to be acutely toxic to ( 96 hour LC50 = 13.8 mg/ l), and cause malformations in ( 96 hour EC50 = 12.1 mg/ l), frog embryos. The minimum concentration that inhibited growth of the embryos was determined to be 7.97 mg/ l. A teratogenic index of 1.1 was determined from the ratio of the 96 hour LC50 to the 96 hour EC50, indicating a low potential for PFOS to be a developmental hazard in this species. ENV/ JM/ RD( 2002) 17/ FINAL 10 1.0 Identity Chemical Name: Perfluorooctane Sulfonate The perfluorooctane sulfonate anion ( PFOS) does not have a specific CAS number. The acid and salts have the following CAS numbers:

acid ( 1763 23 1) ammonium ( NH4 +) salt ( 29081 56 9) diethanolamine ( DEA) salt ( 70225 14 8) potassium ( K+) salt ( 2795 39 3) lithium ( Li+) salt ( 29457 72 5)

Molecular formula: C8F17SO3 Structural formula: CF3 CF2 CF2 CF2 CF2 CF2 CF2 CF2 S(= O)(= O)

OSynonyms 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro; 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro 1 octanesulfonic acid; 1 Octanesulfonic acid, heptadecafluoro; 1 Perfluorooctanesulfonic acid; Hepatadecafluoro 1 octanesulfonic acid; Perfluoro n octanesulfonic acid; Perfluorooctanesulfonic acid; Perfluorooctylsulfonic acid 1.1 Physicochemical Properties Due to the surface active properties of PFOS and the test protocol itself, PFOS forms three layers in octanol/ water and hence, an n octanol/ water ( Kow) partition coefficient cannot be determined. Consequently, the various physicochemical properties ( e. g., bioconcentration factor, soil adsorption coefficient), which can usually be estimated for conventional organic compounds utilizing Kow equations, cannot be estimated, and a calculated ( estimated) log Kow cannot be trusted. Even if the log Kow were known, it may not be appropriate for predictive purposes, e. g., bioconcentration. Studies on laboratory rats indicate that PFOS does not bioconcentrate in the lipid fraction. Instead, it tends to bind to certain proteins. In two studies, PFOS was reported to have a mean solubility of 519 mg/ L and 570 mg/ L in pure water at 24 25 ° C. Solubility decreases significantly with increased salt content ( 12.4 mg/ L in natural seawater at 22 23 ° C, and 20.0 mg/ L in a 3.5% NaCl solution at 22 24 ° C ( 3M Company, 2001a). In a related study, PFOS was reported to have a mean solubility of 56.0 mg/ L in pure octanol ( 3M Company, 2001b). These data suggest that any PFOS discharged to a water source would tend to remain in that medium, unless it is adsorbed onto particulate matter or assimilated by organisms. If PFOS does bind to particulate matter the material would ultimately end up in the sediment. Further study is underway to determine the presence of PFOS in sediments from various locations and the binding potential of PFOS to sediments.

The available physicochemical properties for the potassium salt of PFOS are as follows ( 3M Report, 1999):

Melting point: >= 400 ° C Boiling point: not calculable Vapor pressure: 3.31 x 10 4 Pa at 20 ° C ( 3.27 x 10 9 atm) ENV/ JM/ RD( 2002) 17/ FINAL 11 Air/ water partition coefficient in pure water: 0 (< 2 x 10 6 )

Solubility: pure water: 570 mg/ L Solubility: fresh water: 370 mg/ L Solubility: unfiltered seawater: 12.4 mg/ L Solubility: filtered seawater: 25 mg/ L The specific gravities ( water = 1) and pH values ( in parentheses) of the PFOS salts are as follows ( 3M Report, 1999):

DEA, ~ 1.1 (~ 7) NH4+ , ~ 1.1 (~ 7) Li+, ~ 1.1 ( 6 8) K+, ~ 0.6 ( 7 8)

The above water solubility values correspond to the following Henry's Law constants ( atm. m3/ mol), calculated herein, utilizing the vapor pressure of 3.27 x 10 9 atm given in the 3M Report ( 1999):

Pure water: 3.05 x 10 9 Fresh water: 4.7 x 10 9 Unfiltered seawater: 1.4 x 10 7 Filtered seawater: 2.4 x 10 8 As a point of reference, the Henry's Law constant for pure water at 20 ° C is 4.34 x 10 7 .

2.0 General Information on Exposure The starting material for PFOS related chemicals is perfluorooctanesulfonyl fluoride ( POSF). POSF is manufactured through a process known as Simons Electro Chemical Fluorination ( ECF) in which an electric current is passed through a solution of anhydrous hydrogen fluoride and an organic feedstock of 1 octanesulfonyl fluoride. The ECF process replaces the carbon hydrogen bonds on molecules of the organic feedstock with carbon fluorine bonds. Perfluorination occurs when all the carbon hydrogen bonds are replaced with carbon fluorine bonds. The ECF process yields between 30 45 percent straight chain ( normal) POSF, along with a variable mixture of byproducts and impurities. The output of the ECF process is not a pure chemical, but instead a mixture of isomers and homologues including higher and lower straight chain homologues; branched chain perfluoroalkyl fluorides of various chain lengths; straightchain branched, and cyclic perfluroalkanes and ethers; and other byproducts ( 3M Company, 2000b).

According to information available to the U. S. Environmental Protection Agency ( USEPA), 3M Company is the dominant producer of POSF. In 1997, 3M reported the manufacture or importation into the United States of approximately 1,848 metric tons of POSF. For 2000, 3M forecasts a volume of 1,820 metric tons manufactured or imported into the United States. After accounting for 3M operations in Antwerp, Belgium, 3M estimates a total global POSF production of 3,665 metric tons for 2000 ( 3M Company, 2000d). Production of POSF by 3M is expected to decline to zero by the end of 2002 as 3M scales back the production POSF derived chemicals ( 3M Company, 2000d).

Since most POSF is incorporated into higher molecular weight polymers, it comprises only a portion of the mole fraction of the entire polymer in the final product. However, it should be noted that the secondary reactions used to produce POSF derivatives do not necessarily produce pure products. Typically, 1 2% of the final product is comprised of unreacted or partially reacted fluorinated starting materials or intermediates that are carried forward into the final product as impurities ( 3M Company, 2000b). ENV/ JM/ RD( 2002) 17/ FINAL 12 To date, 3M has not provided information on the total cumulative production volumes of POSF or PFOSrelated chemicals since initial commercialization over 40 years ago. Precise production volume information for manufacturers other than 3M outside the United States has also been difficult to obtain. 3M, however, has asserted that it is the dominant global producer of PFOS chemicals, responsible for the large majority of total global production volumes ( 3M Company, 2000e). The limited production volume information provided by OECD member countries for companies located outside the United States supports this conclusion.

Aside from the United States and Belgium, other OECD Member countries that reportedly have production capacity include Italy and Japan. There may also be some production in non OECD countries. Following are companies that have been identified in various chemical buyer's guides as offering PFOS related chemicals for sale ( Directory of World Chemical Producers, 2000; ChemSources USA, 2000; OPD Chemical Buyers Directory, 2000). This information has not been corroborated independently, except for Miteni S. p. A. of Italy and Dianippon Ink & Chemicals, Inc. of Japan.

OECD member countries ° Miteni S. p. A. ( Italy) ° EniChem Synthesis S. p. A. ( Italy) ° Dianippon Ink & Chemicals, Inc. ( Japan) ° Midori Kaguka Co., Ltd. ( Japan) ° Tohkem Products Corporation ( Japan) ° Tokyo Kasei Kogyo Company, Ltd. ( Japan) ° Fluka Chemical Co, Ltd. ( Switzerland) ° BNFL Fluorochemicals Ltd. ( United Kingdom) ° Fluorochem Ltd. ( United Kingdom)

Non OECD countries ° Milenia Agro Ciencias S. A. ( Brazil) ° Changjiang Chemical Plant ( China) ° Indofine Chemical Company, Inc. ( India) ° Scientific Industrial Association P & M Ltd. ( Russian Federation)

2.1 Production and Use of PFOS The majority of PFOS related chemicals are high molecular weight polymers in which PFOS represents a fraction of the total molecular weight. PFOS related chemicals are used in a variety of products, as shown in the figure following this section. These products can be divided into three main categories of use:

° Surface treatments, ° Paper protection, and ° Performance chemicals.

Each of these categories and the associated 3M production volume is described in more detail below. Note that in many cases the production volumes represent total solid metric tons of fluorochemical containing compound, not PFOS itself. Less than 91 metric tons of PFOS and its salts are commercialized as finished products ( 3M, 2000b).

In addition, varying amounts of fluorochemical residuals ( unreacted or partially reacted starting materials ENV/ JM/ RD( 2002) 17/ FINAL 13 or intermediates) are carried forward into final products at concentrations of 1 2% or less as a result of the ECF manufacturing process. These residuals also have the potential to degrade or metabolize to PFOS ( 3M, 2000b).

2.1.1 PFOS Based Surface Treatment Applications PFOS related chemicals produced for surface treatment applications provide soil, oil, and water resistance to personal apparel and home furnishings. Specific applications in this use category include protection of apparel and leather, fabric/ upholstery, and carpet. These applications are undertaken in industrial settings by customers such as textile mills, leather tanneries, finishers, fiber producers, and carpet manufacturers. PFOS related chemicals are also used in aftermarket treatment of apparel and leather, upholstery, carpet, and automobile interiors by the general public or professional applicators ( 3M Company, 2000b). In 2000, the global production volume of PFOS related chemicals for this use category is estimated at approximately 2,160 metric tons ( 3M Company, 2000b).

2.1.2 PFOS Based Paper Protection Applications PFOS related chemicals produced for paper protection applications provide grease, oil, and water resistance to paper and paperboard as part of a sizing agent formulation. Specific applications in this use category include food contact applications ( plates, food containers, bags, and wraps), as well as non food contact applications ( folding cartons, containers, carbonless forms, and masking papers). The application of sizing agents is undertaken mainly by paper mills and, to some extent, converters who manufacture bags, wraps, and other products from paper and paperboard ( 3M Company, 2000d). In 2000, the global production volume of PFOS related chemicals for this use category is estimated at approximately 1,490 metric tons ( 3M Company, 2000b).

2.1.3 PFOS Based Performance Chemical Applications PFOS related chemicals in the performance chemical category are used in a variety of specialized industrial, commercial, and consumer applications. This category includes various salts of PFOS that are commercialized as finished products. Specific applications in this category include fire fighting foams, mining and oil well surfactants, acid mist suppressants for metal plating and electronic etching baths, photolithography, electronic chemicals, hydraulic fluid additives, alkaline cleaners, floor polishes, photographic film, denture cleaners, shampoos, chemical intermediates, coating additives, carpet spot cleaners, and as an insecticide in bait stations ( 3M Company, 2000b; Boeing, 2000). In 2000, the global production volume of PFOS chemicals for this use category is estimated at approximately 831 metric tons ( 3M Company, 2000d). Of this volume, approximately 151 metric tons will be used in fire fighting foams. ENV/ JM/ RD( 2002) 17/ FINAL 14 Perfluorooctanesulfonyl fluoride ( PFOSF) chemical intermediate Perfluorooctanesulfonic acid ( PFOSA) chemical intermediate acid catalyst for photoresists K, Li, DEA, NH 4 Salts surfactant in fire fighting foam surfactant for alkaline cleaners emulsifier in floor polish mist suppressant for metal plating baths surfactant for etching acids for circuit boards pesticide active ingredient for ant bait traps N Alkylperfluorooctanesulfonamide ( FOSA) chemical intermediate pesticide active ingredient Alkoxylates Carboxylates antistatic agent in photographic paper Oxazolidinones waterproofing casts/ wound dressings Amides pesticide active ingredient Quaternary Ammonium Salts mist suppressant for metal plating baths Amines mist suppressant for metal plating baths Amphoterics water/ solvent repellency for leather/ paper Silanes N Alkylperfluorooctanesulfonamidoethanol ( FOSE) chemical intermediate Acrylates Adipates Phosphate Esters Fatty Acid Esters Alcohols Urethanes Copolymers Electro Chemical Fluorination ( ECF) Cells ( Octanesulfonyl fluoride + HF + electricity)

soil/ oil/ water repellency for: carpet fabric/ upholstery apparel leather metal/ glass oil/ water repellency for: plates food containers bags wraps folding cartons containers carbonless forms masking papers Polyesters Perfluorooctyl Sulfonates: Major Product Categories ENV/ JM/ RD( 2002) 17/ FINAL 15 2.2 Environmental Exposure and Fate PFOS does not hydrolyze, photolyze or biodegrade under environmental conditions and it is persistent in the environment.

2.2.1. Volatility PFOS is not expected to volatilize, based on an assigned air/ water partition coefficient of < 2 x 10 6 Pa. m3/ mol. This assignment was made by Prof. Donald Mackay based on experimental data generated by 3M. In that study, the Hc was too small to measure experimentally using pure water. The interpretation by Mackay states "[ PFOS] is thus essentially non volatile from aqueous solution. This is probably because of its ionic nature. The simple expedient is to assign it a Kaw of zero, i. e. is a type 2 involatile chemical in our nomenclature." This determination is in agreement with the Henry's Law constant of 3.05 x 10 9 calculated herein for pure water. Overall, the conclusion is that PFOS is a substance with very low and possibly negligible volatility.

2.2.2 Combustion To begin to understand the potential degradation pathways for perfluorinated chemicals, the incineration process was thermodynamically modeled. The most pertinent finding of the reported analysis is that the carbon sulfur bond in the perfluorooctane sulfonate molecule is a fairly weak bond. To validate the models prediction, laboratory combustion studies on a series of perfluoroalkyl compounds, including PFOS and two polymeric product formulations are being conducted. An assessment will be made of combustion by products through a range of temperatures. These data will be important to determine whether PFOS may enter the atmosphere as a result of incomplete combustion of waste.

2.2.3 Photolysis/ Oxidation PFOS does not appear to photolyze ( Hatfield, T. 2001). Screening studies on the aqueous photolytic degradation of PFOS, EtFOSE alcohol, MeFOSE alcohol, EtFOSA and MeFOSA as well as a surfactant and foamer product, all appeared to undergo indirect photolysis to FOSA, PFOA, a hydride and olefins; PFOS was not detected ( Hatfield, T. 2001, 3M Report No. W2775). One product, an aromatic perfluorooctane sulfonate, did photodegrade to form PFOS ( 3M Report, 1999).

2.2.4 Biodegradation The biodegradability of PFOS was examined in a MITI I test ( Kurume Laboratory ( 2002). No significant degradation of PFOS was observed in 28 days, either as net oxygen demand from degradation of test substance ( i. e., ultimate degradation), loss ot total organic carbon ( TOC; another way to measure ultimate degradation), or loss of parent compound identity ( primary degradation). Average percentage biodegradation after 28 days was observed to be 0% by oxygen demand; 6% by removal of total organic carbon; and 3% by liquid chromatography mass spectrometry ( LC MS; measures primary degradation). These values are indicative of no significant degradation, within the accuracy limits for this test. Validity of the test was confirmed by degradation of the control substance aniline, for which % of theoretical oxygen demand exceeded the pass criterion of 45% after 7 days and 60% after 14 days respectively ( 74% and 85% of theoretical at days 7 and 14, respectively).

2.3 Environmental Monitoring 3M's Multi City Study reported on PFOS concentrations from water, sludge, sediment, POTW effluent and landfill leachate samples taken in six cities ( 3M, 2001a). Four of the cities ( Decatur AL, Mobile AL, ENV/ JM/ RD( 2002) 17/ FINAL 16 Columbus GA, Pensacola FL) were supply cities that have manufacturing or industrial use of fluorochemicals; two of the cities ( Cleveland TN, Port St. Lucie FL) were control cities that do not have significant fluorochemical activities. Across all cities, POTW effluent concentrations ranged from 0.041 to 5.29 ppb. The POTW sludge ( dry wt.) range was less than 0.2 ug/ kg to 3,120 ppb; the drinking water range was non detect to 0.063 ppb; the landfill leachate range was non detect to 53.1 ppb; the surface water range was non detect to 0.138 ppb; the sediment range was non detect to 1.13 ppb ( dry wt.); and the quiet water range was non detect to 2.93 ppb. The control cities samples generally inhabited the lower end of the above ranges, except for the POTW effluent and sludge findings for Cleveland, which were intermediate in their ranges, and the quiet water samples at Port St. Lucie, which were the highest. 3M states that all the PFOS surface water concentrations are below laboratory derived NOEC ( No Observed Effect Concentrations) for aquatic organisms.

The Multi City Study also included a market basket sampling of PFOS residue in a total of over 200 samples taken from green beans, apples, pork muscle, cow milk, chicken muscle, chicken eggs, bread, hot dogs, catfish, and ground beef ( 3M, 2001a). Measurable quantities of PFOS, to 0.852 ng/ g, were found in four milk samples and one ground beef sample; one of the four milk samples was from a control city, the balance of the samples with measurable PFOS were from cities with fluorochemical manufacture or use.

Giesy reported the results of a global monitoring survey of PFOS in marine mammals; they were located in Florida, California and Alaskan U. S. coastal waters, the northern Baltic Sea, the Mediterranean Sea, the Arctic ( Spitsbergen), and Sable Island in Canada. PFOS was detected in the liver and blood of marine mammals from most locations. The largest liver concentration was 1520 ng/ g, wet wt., in bottlenose dolphin from Florida; the largest blood concentration was 475 ng/ mL in ringed seal from the northern Baltic Sea.

The plasma of piscivorous ( fish eating) birds was sampled in the late 1980' s and early 1990' s or obtained from the U. S. Fish and Wildlife Service ( date of liver samples was not listed), and these samples were later sent to 3M for analysis of PFOS levels ( 3M Company, 2000a). Species sampled for PFOS plasma levels included the bald eagle, albatross, and sea eagle. Plasma levels ranged from below the limit of detection ( 1 ppb) up to a maximum of 1047 ppb, present in a bald eagle. Levels in the livers sampled from six bird species ranged from below the limit of detection up to a maximum of 2055 ppb.

Giesy reported on PFOS in fish and fish eating water birds. Fish were sampled from the U. S., certain European countries, the North Pacific Ocean and Antarctic locations ( Giesy, 2001a). The highest concentration was 923 ng/ g, wet wt., in the muscle of fish from a Belgium estuary. The highest concentration of PFOS in U. S. Great Lakes fish was 297 ng/ g, wet wt., in the muscle of carp. Muscle tissue of fish from several inland lakes in Michigan U. S. did not contain detectable PFOS, nor did fish from the North Pacific and Antarctic oceans. Fish eating bird samples were collected from the U. S., including Midway atoll, the Baltic and Mediterranean Seas, Japanese and Korean coasts ( Giesy, 2001b). PFOS concentrations collected from the plasma of bald eagles in the midwestern USA ranged from not quantifiable to 2220 ng/ mL, with a mean of 330 ng/ mL. The largest PFOS concentration in liver samples from USA birds was 1780 ng/ g, wet wt. from a Brandts cormorant. PFOS concentrations in the sera of North Pacific Ocean albatrosses ranged from 3 to 34 ng/ mL. PFOS concentrations in blood or livers of birds from the Mediterranean and Baltic Seas were relatively less than those found in U. S. birds.

Giesy reported on PFOS in mink and river otter livers from the U. S. ( Giesy, 2001c). PFOS was found in all samples, with the largest concentration of 4800 ng/ g, wet wt., found in mink liver. PFOS concentrations in river otter livers from Washington and Oregon states ranged from 34 to 994 ng/ g, wet wt.

Giesy reported that PFOS was detected in oysters collected in the Chesapeake Bay and Gulf of Mexico of the U. S. coast at 51 of 77 sampling locations at a concentration range of < 42 to 1225 ng/ g, dry wt. ( Giesy, ENV/ JM/ RD( 2002) 17/ FINAL 17 2001d).

Giesy reported on the concentrations of PFOS in surface water, sediments, clams, and fish collected from locations upstream and downstream of the 3M facility at Decatur AL ( Giesy, 2001e). Of the three downstream sampling locations, the two closest to the 3M facility had PFOS surface water concentrations significantly greater than the two upstream sites ( means of 150 ug/ L and 82 ug/ L, vs. 0.009 ( est.) and 0.053 ug/ L); the nearest two locations had sediment concentrations significantly greater than the upstream sites ( wet wt. means 5930 ug/ kg, 1299 ug/ kg vs. 0.18( est.) and 0.98). Clam and fish samples were collected at two locations, one upstream and one downstream of the 3M facility. The average fish whole body PFOS concentration for the upstream location was 59.1 ug/ kg ( wet wt.), while that for the downstream location was 1332 ug/ kg. The average PFOS concentration in clams at the upstream location was 15.6( est.) ug/ kg; that for the downstream location was 14.1( est.) ug/ kg.

Hansen ( 2002) reported concentrations of PFOS measured from surface water samples taken from the Tennessee River up and downstream of the outfall from the fluorochemical manufacturing facility at Decatur AL ( the 3M facility mentioned above). There were 20 sampling sites above and 20 sites below the outfall location, spaced at approximately 2 mile intervals. Upstream of the facility the average concentration of PFOS was 32 + 11 ng/ L; the downstream concentrations were observed to increase at a point approximately six miles below the outfall; the average PFOS concentration from that point downstream was 114 + 19 ng/ L. The report states that the consistency of the PFOS concentrations within these two regions suggests the absence of either major environmental sinks or additional sources of PFOS in the areas sampled.

The 3M Environmental Lab reported on analyses of PFOS in naive rat livers, rat chow, and fish meal ( a component of rat chow). Rat livers from two of three suppliers had endogenous levels of PFOS above the limit of detection ( 15 ng/ g). PFOS levels in the male rats with measurable levels increased with age, while the levels in female rats showed no correlation with age. In the rat chow study, two of the four chows examined had PFOS above the 2 10 ng/ g level of quantitation, 18 and 12 ng/ g, respectively. The fish meal study analyzed PFOS levels in six types of fishmeal formulated from at least three types of fish. Three of the six types had PFOS levels above the limit of quantitation of 3.5 ng/ g; the maximum concentration was 15.7 ng/ g.

The 3M Environmental Laboratory also summarized PFOS analyses of avian and aquatic feed matrices. PFOS levels were not detected in the two daphnid feeds, and were below the limits of quantitation in the avian feed. Dry artemia ( brine shrimp) cysts used for fish feed had PFOS levels of 9 10 ug/ kg.

2.4 Human Biomonitoring For many years, PFOS has been measured in the serum of workers occupationally exposed to PFOS. It was also recently detected in the serum of the general population, but at much lower levels. The results of the most recent analyses of blood samples are described below and summarized in Table 1.

2.4.1 Occupational Exposures PFOS serum levels have been measured in workers involved in both the manufacturing of perfluorochemicals and the processing of these compounds into products, such as fire protection and surface protection products. Biomonitoring data are available from manufacturing plants in Alabama and Belgium, a processing facility in Japan, and corporate offices in Minnesota. ENV/ JM/ RD( 2002) 17/ FINAL 18 Fluorochemical Manufacturing Two 3M plants, located in Decatur, Alabama and Antwerp, Belgium, have produced POSF from electrochemical cell fluorination and then subsequently used POSF to produce various products through polymerization processes. These POSF based products degrade or metabolize to PFOS. A third manufacturing plant, located in Cottage Grove, Minnesota, has not produced POSF, but has manufactured some POSF based products. Serum PFOS levels were measured in manufacturing plant employees in 1995 ( n = 178), 1997 ( n = 149), and 2000 ( n = 521) using high performance liquid chromatography/ mass spectrometry ( Olsen et al, 1999; Olsen et al, 2001d; Olsen et al, 2001e; Olsen et al., 2001f). Employees volunteered to participate for medical surveillance. Arithmetic means and ranges were reported for all surveillance periods. Geometric means have only been reported in 2000. These data are provided in Table 1.

Mean serum PFOS levels have been declining at both plants since 1995. Decatur serum levels have been higher than Antwerp levels during every surveillance period. The PFOS levels in workers across both plants in 1995 ranged from 0.10 ppm to 12.83 ppm. The range of PFOS levels of workers in 1997 was 0.10  9.93 ppm. The largest number of employees at each plant participated in the 2000 medical surveillance program ( 521 out of 840 employees). The mean serum PFOS level at the Decatur plant was 1.32 ppm ( range = 0.06   10.06 ppm). Most of the employees at both facilities had PFOS serum levels that were < 6 ppm across all of the sampling periods.

Except for the year 2000, the participation rate of the total number of potentially exposed employees in the medical monitoring program from which these data were derived was not provided. It was reported that the employees were offered a medical monitoring program, but it is not clear how many of them actually volunteered to participate. In addition, age of the employees and duration of employment were not reported for most of the surveillance years, and it is unclear how many of the total number of employees worked at each plant. Therefore, it should be noted that these data only provide a snapshot of the serum levels of participating workers during each sampling period.

In order to address the voluntary nature of the monitoring program at the 3M plants, a random sample of employees from the Decatur plant was chosen to measure seven different fluorochemicals in employees' blood ( Olsen et al., 1999). Limited job information, years worked, and building location were collected to provide a better understanding of the distribution of fluorochemical serum levels in employees. The random sample consisted of 187 chemical plant workers ( 126 randomly chosen workers and 61 volunteers) and 76 film plant workers ( 60 randomly chosen and 16 volunteers). The levels in these employees, who were sampled in late 1998, were similar to those reported above. Mean PFOS levels were 1.505 ppm ( range, 0.091 10.600) for the random sample, 1.259 ppm for the volunteers, and 1.424 ppm for all participants. Geometric means were 0.944 ppm ( 95% CI 0.787 1.126) for chemical plant employees and 0.136 ppm ( 95% CI 0.114 0.161) for film plant employees. When compared by job description, workers in the chemical plant had higher PFOS levels than workers in the film plant. PFOS levels in males were positively, although modestly, associated with number of years worked in the chemical plant.

Fluorochemical Processing Biomonitoring data were submitted on workers in Japan processing perfluorinated chemicals into fire protection products and surface products ( Burris et al., 1999). In 1999, PFOS, PFOA and PFHS ( perfluorohexanesulfonate) were measured in employees' serum at the Sagamihara plant. Serum was drawn from both production employees ( n = 32) who regularly handle fluorochemicals and management employees ( n = 32) who are not regularly exposed. Serum was also drawn from management employees ( n = 30) at the Head Office in Tokyo. The highest PFOS level in 32 production employees was 0.628 ppm, with an arithmetic mean of 0.135 ppm. The highest PFOS level in either of the management groups was ENV/ JM/ RD( 2002) 17/ FINAL 19 0.0967 ppm, while the average level was approximately 0.04 ppm. The results of biomonitoring performed at the Sagamihara plant indicate lower levels of PFOS than at either of the manufacturing facilities cited above.

Although the exposures would not necessarily be considered " occupational" for either group, PFOS levels in corporate staff/ managers at a 3M plant in St. Paul, Minnesota were similar to those reported in the corporate managers in Japan ( 3M Report, 1999). Thirty one employees, none of whom had worked in fluorochemical production or research and development, were sampled in 1998. All of the participating employees had measurable levels of PFOS in their serum. The mean PFOS level was 0.047 ppm, with a range of 0.028 to 0.096 ppm. In this group of 3M employees, age was significantly associated with increased serum PFOS. No other data on these workers were provided.

2.4.2 Non occupational Exposures PFOS has been measured in the serum of the general U. S. population and in small numbers of historical samples ( dating as far back as 1957) from sources throughout the world. In 1998, PFOS levels in pooled serum from 2 commercial sources in the U. S. ranged from 43 45 ppb and 26  45 ppb ( 3M Report, 1999). There were approximately 500 donors in the first source and an estimated 200 donors in the second. No other data, such as geographic location or age of the donors, were provided. 3M also analyzed thirty five lots of individual or pooled human sera samples from U. S. chemical or biological supply companies in 1999 ( 3M Letter, 1999). These samples yielded an average of 35 ppb PFOS, ranging from 5 to 85 ppb.

Sera pooled from 18 regional blood banks in various geographic regions of the U. S. were analyzed for PFOS in 1998 ( 3M Report, 1999). There were 68 pools and an estimated 340 680 donors. The overall mean PFOS serum level across the pools was 29.7 ppb. The PFOS levels varied quite a bit depending on the geographic location of the blood bank. The range of the levels across geographic regions was 9 to 56 ppb, while the range of the averages was 14 to 52 ppb.

Pooled sera from blood banks in Belgium ( 6 pools), the Netherlands ( 5 pools), and Germany ( 6 pools) also have been analyzed for PFOS. Each pool had 15 20 donors. The Netherlands had the highest PFOS levels ( mean = 53 ppb) and Belgium had the lowest ( mean = 17 ppb).

Individual blood samples from 3 different age populations were recently analyzed for PFOS and other fluorochemicals using high pressure liquid chromatography/ electrospray tandem mass spectrometry ( HPLC/ ESMSMS) ( Olsen et al., 2002a, 2002b, 2002c). The studies' participants included adult blood donors, an elderly population participating in a prospective study in Seattle, WA, and children from 23 states participating in a clinical trial. Overall, the PFOS geometric means were similar across all 3 populations ( 34.9 ppb, 31.0 ppb, and 37.5 ppb, respectively). The geometric means and 95% tolerance limits and their upper bounds were comparable across all 3 studies. However, the upper ranges for the children and adults were much higher than for the elderly population. It is not clear whether this is the result of geographic differences in PFOS levels or some other factor. It should be noted that PFOS and PFOA were highly correlated in all three studies ( r = .63, r = .70, and r = .75). The details of each study are provided below.

Serum PFOS levels in 645 blood donors ( 332 males, 313 females), aged 20 69 years, were obtained from 6 American Red Cross blood banks ( Olsen et al., 2002a). The blood banks were located in: Los Angeles, CA; Minneapolis/ St. Paul, MN; Charlotte, NC; Boston, MA; Portland, OR, and Hagerstown, MD. Each blood bank was requested to provide approximately 10 samples per 10 year age intervals ( 20 29, 30 39, etc.) for each sex. The only demographic factors known for each donor were age, gender, and location.

The geometric mean serum PFOS level for all locations and gender was 34.9 ppb ( 95% CI, 33.3 36.5 ppb). ENV/ JM/ RD( 2002) 17/ FINAL 20 The range was < LLOQ ( 4.3 ppb) to 1656 ppb. Males had significantly higher ( p < .05) geometric mean PFOS levels than females. The geometric mean for all males was 37.8 ppb ( 95% CI, 35.5 40.3) and was 31.3 ppb for all females ( 95% CI, 30.0   34.3). Age was not an important predictor of adult serum fluorochemical concentrations. When stratified by geographic location, the highest geometric mean for PFOS was in the samples from Charlotte, NC ( 51.5 ppb, range: 19.3   166.0) and the lowest from Boston ( 28.0 ppb, range: 4.3   87.2).

The highest serum PFOS measurement in this sample was 1656 ppb from a male blood donor, 67 years old from Portland. The next highest donor level was 329 ppb from a male donor, 62 years old also from Portland. The next 8 highest serum PFOS values ( range 139   226 ppb) were measured in 4 females and 4 males representing Charlotte ( n= 4), Hagerstown ( n= 2), Los Angeles ( n= 1) and Minneapolis/ St. Paul ( n= 1). In bootstrap analyses, the mean of the 95% tolerance limit for PFOS was 88.5 ppb with an upper 95% confidence limit of 100.0 ppb.

Serum PFOS levels were reported for 238 ( 118 males and 120 females) elderly volunteers in Seattle participating in a study designed to examine cognitive function in adults aged 65 96 ( Olsen et al., 2002b). Age, gender and number of years' residence in Seattle were the only data available on the participants. Most of the participants were under the age of 85 and had lived in the Seattle area for over 50 years.

The geometric mean of PFOS for all samples was 31.0 ppb ( 95% CI, 28.8 33.4). The range was 3.4   175 ppb. There was no significant ( p < .05) difference in geometric means for males and females. In simple linear regression analyses, age was negatively ( p < .05) associated with PFOS in men but not in women. The mean of the 95% tolerance limit for PFOS was 84.1 ppb with an upper 95% confidence limit of 104.0 ppb.

A sample of 599 children, ages 2 12 years old, participating in a study of group A streptococcal infections, was analyzed for PFOS levels ( Olsen et al., 2002c). The samples were collected in 1994 1995 from children residing in 23 states and the District of Columbia. The geometric mean of PFOS for all of the participants was 37.5 ppb ( 95% CI, 33.3 36.5). The range was 6.7 to 515.0 ppb. Male children had significantly ( p<. 01) higher geometric mean PFOS levels than females: 40.1 ppb and 35.2 ppb, respectively. In bootstrap analyses, the mean of the 95% tolerance limit for PFOS was 88.5 ppb with an upper 95% confidence limit of 97.0 ppb. When stratified by age, the geometric mean tended to rise for each age group from age 2 ( 28.6 ppb) through age 9 ( 42.8 ppb) where it was highest, and then started to decrease gradually to 32.8 ppb at 12 years. In simple linear regression analyses, age was not significantly ( p < .05) associated with PFOS. Although the data were not provided, a graphical presentation of log PFOS levels for each state by gender were similar across the states, however, it is difficult to interpret these data given the limited sample size for each gender/ location subgroup.

In another study, Olsen et al. ( 2001g) obtained samples from 31 cadavers ( 16 males and 15 females) over an 18 month period from the International Institute for the Advancement of Medicine ( IIAM). The average age of the male donors was 50 years ( SD 15.6, range 5 69) and the average age of the female donors was 45 years ( SD 18.5, range 13 74). The causes of death were intracranial hemorrhage ( n = 16 or 52%), motor vehicle accident ( n = 7 or 23%), head trauma ( n = 4 or 13%), brain tumor ( n = 2 or 6%), drug overdose ( n = 1 or 3%) and respiratory arrest ( n = 1 or 3%). Both serum and liver tissue were harvested from 23 donors; 7 donors contributed liver tissue only and 1 donor contributed serum only. Serum samples were obtained from 5 ml of blood; liver samples consisted of 10 g of tissue. Samples were frozen at IIAM and shipped frozen to 3M for analysis. The samples were extracted using an ion pairing extraction procedure and were quantitatively assayed using HPLC ESMSMS and evaluated versus an unextracted curve. Extensive matrix spike studies were performed to evaluate the precision and accuracy of the extraction procedure. The average fortified sample recovery of PFOS from human sera was 89% ( SD 21%) and the average fortified sample recovery of PFOS from human liver was 78% ( SD 24%). ENV/ JM/ RD( 2002) 17/ FINAL 21 Serum PFOS levels that were determined to be less than the limit of quantitation ( LOQ) were assigned a value midpoint between zero and the LOQ. The mean serum PFOS level for the 24 serum donors analyzed was 17.7 ng/ mL; the geometric mean for serum PFOS was 14.7 ng/ mL. Of the 30 PFOS liver samples analyzed, 15 ( 50%) were determined to be < LOQ. These samples were assigned a value midpoint between zero and the LOQ. The mean liver PFOS level was 18.8 ng/ g; the geometric mean for liver PFOS was 15.2 ng/ g. Mean PFOS levels for male and female donors were similar for both serum ( male = 18.2 ng/ mL; female = 17.2 ng/ mL) and liver ( male = 19.2 ng/ g; female = 28.4 ng/ g). Although the data were not shown, the report stated that no associations were observed between measured PFOS levels and age. The average PFOS serum and liver data for each of the 23 paired samples ( serum and liver from the same individual) showed a good correlation. The mean liver to serum ratio was 1.3: 1. Of the 13 male donors with paired samples, the mean liver to serum ratio was again 1.3: 1; the mean serum level was 18.2 ng/ mL and the mean liver level was 20.8 ng/ g. For the 10 female donors with paired samples, the mean liver to serum ratio was 1.3: 1 with mean serum levels of 16.9 ng/ mL and mean liver levels of 16.3 ng/ g, respectively. ENV/ JM/ RD( 2002) 17/ FINAL 22 Table 1. Mean PFOS serum levels in human populations Occupational Exposures Plant Location Mean ( ppm) Range ( ppm)

Decatur, Alabama 1995 ( n = 90) 1997 ( n = 84) 1998 ( n = 126) 2000 ( n = 263) 2.44 1.96 1.51 1.32 ( GM = 0.91)* 0.25  12.83 0.10  9.93 0.09   10.6 0.06   10.06 ( 33.3   36.5)** Antwerp, Belgium 1995 ( n = 93) 1997 ( n = 65) 2000 ( n = 258) 1.93 1.48 0.80 ( GM = 0.44)* 0.10  9.93 0.1  4.8 0.04   6.24 ( 0.38  0.51)** Building 236 ( n = 45) 0.182 < 0.037   1.036 Sagamihara, Japan ( 1999) ( processing PFOS) n = 32 0.135 0.0475  0.628 General Population Exposures Source Mean ( ppb) Range ( ppb)

Non occupational ( n = 31) ( corporate staff or managers) St. Paul, Minnesota ( 1998) 47 28   96 Non occupational ( 1999) ( plant management, Japan) Sagamihara n = 32 Tokyo n= 30 40.3 52.3 31.9  56.6 33  96.7 Commercial Sources, U. S. ( 1998) Intergen n = ~ 500 donors Sigma n = ~ 200 donors 44 33 43  44 26   45 Other Commercial Sources, U. S. ( 1999) n = 35 lots 35 5   85 U. S. Blood Banks ( 1998) n = ~ 340 680 donors 29.7 9   56 European Blood Banks ( 1999) Belgium ( 6 pooled samples)

Netherlands ( 5 pooled samples)

Germany ( 6 pooled samples) 17 53 37 4.9   22.2 39   61 32   45.6 U. S. Blood Banks ( 2000) American Red Cross ( ages 20 69) n = 645 34.9 ( geometric mean) 4.3   1656 Samples in U. S. children ( ages 2 12) ( 1995) ( n = 599) 37.5 ( geometric mean) 6.7   515 Samples in elderly in Seattle, WA ( ages 65 96) ( 1999) ( n = 238) 31.0 ( geometric mean) 3.4  175 * GM is geometric mean; ** 95% Confidence Interval of the GM ENV/ JM/ RD( 2002) 17/ FINAL 23 3.0 Human Health Hazards 3.1 Metabolism and Pharmacokinetics 3.1.1 Absorption PFOS is well absorbed following ingestion. After a single oral dose of PFOS 14C ( mean dose, 4.2 mg/ kg) in solution to groups of three male rats, at least 95% of the total carbon 14 is systemically absorbed at 24 hours ( Johnson, Gibson and Ober, 1979a). The digestive tract and contents contained on average, 3.45% of the dose. The mean fecal excretion is 1.55% of the dose at 24 hours and 3.24% at 48 hours. At 24 hours, the mean sum of total carbon 14 in feces and digestive tract plus contents is 5% of the dose. Some of this 5% likely represents systemically absorbed carbon 14 present either in the digestive tract tissues or in the digestive tract contents as a result of excretion. The data from the 48 hour post dose group of rats are consistent with the 24 hour post dose data. Thus, at least 95% of the PFOS 14C dose was absorbed from solution after administration to non fasted rats.

3.1.2 Distribution At 24 to 48 hours after a single oral dose of PFOS 14C ( 4.2 mg/ kg) in rats, approximately 86% of the radioactivity recovered was found in the carcass ( Johnson, Gibson and Ober, 1979a). The carcass data are not as reliable as the other tissue data since large volume homogenates were necessary and homogeneity of sample aliquots was difficult to assure. There is some excretion of total carbon 14 in urine ( 1 2%/ day). The spleens from the 24 hour and 48 hour post dose rats were analyzed for total carbon 14 content, and the percent of the dose in the whole organ was ~ 0.2%. The concentrations of total carbon 14 in red blood cells and plasma were compared. The mean ratio of red blood cell to plasma concentration at 24 and 48 hours is 0.25 and 0.39, respectively. Thus, at 24 and 48 hours after a single oral dose of FC 95 14C, there is no selective retention of carbon 14 in red blood cells.

At 89 days after a single intravenous dose of PFOS 14C ( 4.2 mg/ kg) in male rats, mean tissue C 14 concentrations above one ug equivalents/ g were as follows: liver, 20.6; plasma, 2.2; kidney, 1.1; and lung, 1.1 ( Johnson, Gibson and Ober, 1979b). Other tissues such as muscle, skin, bone marrow, and spleen had concentrations ranging from 0.2 to 0.6 ug/ g. There was a difference in C 14 content of subcutaneous fat ( 0.2 ug/ g) and abdominal fat (<= 0.08 ug/ g). Very little C 14 was found in whole eye ( 0.16 ug/ g) and no detectable C 14 was found in brain. Only liver and plasma contained a substantial percentage of dose at 89 days post dose, 25.21% and 2.81%, respectively. The low levels of radioactivity found for kidney, lung, testes, and spleen are due in part to blood still contained in these organs when homogenized.

There have been several studies conducted to examine the levels of PFOS in the dam, fetus and neonate. These studies are described in detail in sections 3.4 and 3.5. In general, the levels of PFOS are higher in the liver than in the serum of dams, fetuses and neonates, and placental transfer of PFOS has been demonstrated 3.1.3 Elimination Urinary excretion is the primary route of elimination for PFOS in the rat. By 89 days after a single intravenous dose of PFOS 14C ( 4.2 mg/ kg) in male rats, mean urinary excretion was 30.2+ 1.5% of total C 14 administered ( Johnson, Gibson, and Ober, 1979b). Mean cumulative fecal excretion was 12.6+ 1.2%. Radioactive content in feces was too low to measure after 64 days.

The half life for elimination of total PFOS carbon 14 from plasma after a single oral dose ( 4.2 mg/ kg) in ENV/ JM/ RD( 2002) 17/ FINAL 24 male rats is 7.5 days ( Johnson, Gibson and Ober, 1979a). This determination was based upon analysis of plasma samples from groups of three rats at 1, 2, 6, 12, 24, 48, 96, and 144 hours after the single oral dose.

There is evidence of enterohepatic circulation of PFOS. After 21 days of cholestyramine treatment, the mean percentage of PFOS 14C dose eliminated via feces ( 75.8 + 5.0) was 9.5 fold the mean percentage of dose eliminated via feces by control rats ( 8.0 + 0.8, Johnson, Gibson and Ober, 1984). After adjustment for the amount of carbon 14 excreted in urine ( 18% for controls and 5% for cholestyramine treated), the amounts of carbon 14 remaining to be excreted are 19% for cholestyramine treated rats and 74% for control rats. After PFOS 14C administration, the mean liver carbon 14 content at 21 days represents 11% and 40% of the dose for cholestyramine treated and control rats, respectively. Mean plasma and red blood cell carbon 14 concentrations are significantly lower after 21 days of cholestyramine treatment. The authors conclude that the high concentration of PFOS 14C in liver at 2 to 3 weeks after dosing and the fact that cholestyramine treatment enhances fecal elimination of carbon 14 by nearly 10 fold suggest that there is a considerable enterohepatic circulation of PFOS 14C.

3.1.4 Half life in Humans In order to determine the half life of PFOS, 3 retirees who worked in 3Ms Chemical Division were followed for five and a half years ( 3M Company, 2000c). Serum PFOS levels suggested a mean elimination half life ( t1/ 2) of 1,428 days ( approximately 4 years). A half life study on a larger group of retirees ( n = 27) is currently underway, in which serum samples are drawn every 6 months over a 5 year period. Two interim reports describing the results thus far have been submitted ( Burris et al, 2000; Burris et al., 2002). The first interim report suggested a serum half life of 139 640 days. However, there were several limitations to these analyses including: 1) the limited data available and the range of serum PFOS levels measured; 2) serum was analyzed after each collection period with only one measurement per time period on different days using slightly different analytical techniques; and 3) the reference material purity was not determined until after the first 3 samples had been analyzed. An effort was made to minimize experimental error, including systematic and random error in the analytical method, involving 9 of the original 27 subjects. Serum samples were collected from each of the subjects over 4 time periods spanning 180 days, measured in triplicate with all time points from each subject analyzed in the same analytical run. This would allow for statistical evaluation of the precision of the measurement and assure that all systematic error inherent in the assay equally affected each sample used for half life determination.

Of the 9 retirees included in this analysis, there were 7 males and 2 females, all from the Decatur plant. The average age of the retirees was 61 years, the mean number of years worked at Decatur was 27.7 years, and the average number of months retired was 18.9. Average BMI of this group was 27.9. The mean PFOS value at study initiation was 0.89 ppm ( range 0.11   3.53 ppm, SD = 1.07).

The mean serum half life for PFOS was 8.67 years ( range 2.29   21.3 years, SD = 6.12). Age, BMI, number of years worked or years since retirement were not significant predictors of serum half lives in multivariable regression analyses. This analysis has attempted to reduce experimental error in the determination of a half life for PFOS. However, several issues should be noted. First, the effect of continued non occupational, low level exposure on the half life is unknown. Second, PFOS is a metabolic product of other compounds found in the retirees' blood; therefore, PFOS is likely being produced in the body while the study was ongoing. Third, it is not known if there are interactions between PFOS and other fluorochemicals in the body. Fourth, this estimate is much higher than that reported in lab animals. However, it may not be appropriate to directly compare the results of the animal half life data with these data due to potentially different protein binding sites and affinities. Fifth, systematic error of the analytical method could be as high as +/ 20% and still satisfy the data quality criteria. ENV/ JM/ RD( 2002) 17/ FINAL 25 3.2 Acute Toxicity Four reports of acute studies of PFOS have been submitted, one inhalation toxicity of rats, two oral studies of rats, and one dermal and eye irritation study of rabbits.

In a study to determine the median lethal concentration ( LC50), Rusch et al. ( 1979) administered PFOS dust in air to Sprague Dawley rats, 5/ sex/ group, levels of 1.89 to 45.97 mg/ l PFOS to eight test groups. A Wright dust feed mechanism with dry air at a flow rate of 12 to 16 liters per minute was used to administer the PFOS dust. Rats were exposed for 1 hour. The test group rats weighed 201 299 g at study initiation. The control group rats weighed 203 263 g at study initiation. The test group rats were exposed to 1.89, 2.86, 4.88, 6.49, 7.05, 13.9, 24.09 or 45.97 g/ l. The control rats were exposed to dry air at a flow rate of 12 liters per minute. All other protocols were the same as the test group rats. The rats were observed for abnormal signs prior to exposure, at 15 minute intervals during the 1 hour exposure, at removal from the exposure chamber, hourly for four hours after exposure, and daily thereafter for 14 days. Individual body weights were recorded on Day 0 ( prior to exposure), Day 1, Day 2, Day 4, Day 7, and Day 14. It is reported that all animals dying spontaneously were necropsied as soon as possible after death. Blood samples were collected on Day 14 from all surviving animals, but analyses were not provided. The LC50 of 5.2 mg/ l and 95% confidence limits of 4.4 and 6.4 mg/ l were calculated using the method of Litchfield and Wilcoxon.

The highest dose group, 45.97 mg/ l, was not used in the LC50 calculations and terminated on Day 2. At that point, only 5 animals survived and blood samples were taken at termination. The 13.9 mg/ l group was also terminated early ( Day 1) because of a mechanical problem during exposure. These animals were also not used in the LC50 determination.

In the 24.09 mg/ l exposure group, all animals died by Day 6. At 7.05 and 6.49 mg/ l there was 80% mortality. At 4.88, 2.86, and 1.89 mg/ l there was 20%, 10%, and 0% mortality, respectively. The rats in all these groups showed signs of toxicity including emaciation, red material around the nose or other nasal discharge, yellow material around the anogenital region, dry rales or other breathing disturbances, and general poor condition. Abnormal in life observations were reported to be less frequent in the lower exposure groups.

At necropsy, the most common abnormality was discoloration of the liver and lung. Discoloration of the lung was also observed in control rats and therefore may not be treatment related. Therefore, the most significant treatment related abnormality was varying degrees of discoloration of the liver. Among animals that died prematurely, decreased body weight, discoloration of the lung, and discoloration and distention of the small intestine were also observed.

In a study to determine the acute oral LD50, Dean et al. ( 1978) administered CD rats, 5/ sex/ group, doses of 0, 100, 215, 464, and 1000 mg/ kg PFOS by gavage. The powdered test material was suspended in a 20% acetone/ 80% corn oil mixture. All dose levels were administered as volumes of 10 ml/ kg body weight. The rats weighed 172 212 g at the beginning of the study immediately prior to dosing and weights were recorded at Day 7 and Day 14. The rats were observed for abnormal signs during the four hours after exposure, and daily thereafter for 14 days. It is reported that all animals dying spontaneously were grossly necropsied, as well as all rats that survived to the end of the 14 day study.

Acute oral LD50 values and 95% confidence limits were calculated for males ( 233 [ 160 339] mg/ kg), females ( 271 [ 200 369] mg/ kg) and combined male and female rats ( 251 [ 199 318] mg/ kg).

All rats in the 464 and 1000 mg/ kg dose groups died before the end of the study. Three animals in the 215 mg/ kg group died prematurely. It appears signs of toxicity most frequently observed included: ENV/ JM/ RD( 2002) 17/ FINAL 26 hypoactivity, decreased limb tone, and ataxia. At necropsy, observations included: yellow stained urogenital region, stomach distention and signs of irritation of the glandular mucosa, and lung congestion.

In a second oral study, Gabriel ( 1978; cited in Dean et al., 1978) administered two groups of Sherman Wistar albino rats ( 5 / sex/ group) two doses of PFOS by gavage in water. The rats weighed between 200  300 grams. It was determined the acute oral LD50 was greater than 50 mg/ kg and less than 1500 mg/ kg.

All of the rats administered 1500 mg/ kg died before the 14 day observation period ended, with the last rat dead on Day 9. None of the rats administered 50 mg/ kg died before the end of the study. It is stated that rats were observed for signs of toxicity and mortality but it is not clear how frequently they were observed and no individual animal data was provided. It is stated that the high dose group rats were " depressed" at 2 3 hours after dosing and " severely depressed or semi comatose" by 24 hours; staying in this state until death occurred. No individual pathology data were provided. The author states: " Gross pathologic examination revealed nothing remarkable."

Biesemeier and Harris ( 1974) examined the potential for PFOS to cause skin and eye irritation. Six albino rabbits had their hair clipped from their backs and flanks, and it appears five tenths of one gram ( 0.5 g; the procedure states either 0.5 g or 0.5 ml and the test material as supplied by 3M was a solid) of the test material was placed on abraded or intact prepared test sites, then covered with gauze patches. After 24 hours and 72 hours the coverings were removed and the degree of erythema and edema was recorded according to a standardized scale. No reference is provided for method or scoring of results. Concentration or total dose of test material was not provided. In all six rabbits, it is reported the primary skin irritation scores were 0; which indicates no reddening or swelling detected.

In the eye irritation study, six albino New Zealand White rabbits were placed in collars so they could not rub their eyes. It appears one tenth of a gram ( 0.1 g; the procedure states either 0.1 g or 0.1 ml was used and T117 was a solid as supplied by 3M) of the test substance was instilled in one eye, the other eye was left untreated as a control. The concentration or total dose of the test substance were not provided. It is reported that the reaction to the test material was read against a scale of damage to the cornea, iris, and the bulbar and palpebral conjunctivae at 1, 24, 48, and 72 hours after treatment. The scale criteria were not presented or referenced. Each time the eyes were scored, any accumulated discharge or residue of test material was flushed from the eye. There is no reference provided for the method. It appears that scores were maximal at 1 hour and 24 hours after treatment then decreased over the rest of the study ( Biesemeier and Harris, 1974).

3.3 Mutagenicity PFOS was tested for its ability to induce mutation in the Ames Salmonella/ Microsome Plate Test and in the D4 strain of Saccharomyces cerevisiae ( Litton Bionetics, Inc., 1979). It was also tested in: ( 1) a Salmonella   Escherichia coli/ Mammalian microsome reverse mutation assay ( Mecchi, 1999); ( 2) an in vitro assay for chromosomal aberrations in human whole blood lymphocytes ( Murli, 1999); and ( 3) an assay for unscheduled DNA synthesis ( UDS) in rat liver primary cell cultures ( Cifone, 1999). It was negative in all assays in which it was tested. PFOS does not induce reverse mutation at the histidine locus of S. typhimurium or at the tryptophan locus of E. coli when tested with or without metabolic activation. It does not induce chromosomal aberrations in human lymphocytes when tested with or without metabolic activation and does not induce UDS in primary cultures of rat hepatocytes. PFOS was also tested in an in vivo mouse micronucleus assay ( Murli, 1996). PFOS did not induce micronuclei in the bone marrow of Crl: CD 1 BR mice and is negative in the mouse bone marrow micronucleus assay.

T 2247 CoC which is a 50% by weight solution of the diethanolammonium salt of perfluorooctanesulfonate in water was tested for its ability to induce gene mutation in 5 strains of S. ENV/ JM/ RD( 2002) 17/ FINAL 27 typhimurium and also for its ability to induce recombination in S. cerevisiae strain D3. ( Simmon, 1978). The chemical was negative in all 5 strains of Salmonella and in S. Cerevisiae D3 when tested with and without metabolic activation. T 2247 CoC was tested with S. typhimurium strains TA98 and TA100 in a desiccator assay for the detection of volatile compounds. It was nonmutagenic when tested under these conditions.

3.4 Repeated Dose Toxicity Three 90 day subchronic studies of PFOS have been conducted, two gavage studies in rhesus monkeys and one dietary study in rats. In addition, a four week and a 26 week capsule study in cynomolgus monkeys have been conducted. Both reports were unaudited drafts at the time of this review.

In the rat subchronic study, Goldenthal et al. ( 1978b) administered CD rats, 5/ sex/ group, dietary levels of 0, 30, 100, 300, 1000 or 3000 ppm PFOS ( FC 95) for 90 days. The males weighed 196 232 g and the females weighed 165 206 g at study initiation. The dietary levels were equivalent to doses of 0, 2, 6, 18, 60 and 200 mg/ kg/ day. The rats were observed daily for general clinical signs; body weights and food consumption were recorded weekly. Hematological and clinical chemistry analyses and urinalysis were conducted at the beginning of the study and after 30 and 90 days of treatment. The rats were sacrificed after 90 days of treatment and a gross necrospy was conducted. At necropsy the heart, liver, adrenals, spleen, pituitary, kidneys, testes/ ovaries and brain were weighed. The thyroid/ parathyroid were weighed after fixation. Tissues were preserved in buffered neutral 10% formalin; the eyes were preserved in Russell's fixative. The following organs from control and all treated groups were examined microscopically: adrenals, aorta, brain, esophagus, eyes, gallbladder, heart ( with coronary vessels), duodenum, ileum, jejunum, cecum, colon, rectum, kidneys, liver, lung, skin, mesenteric lymph node, retropharyngeal lymph node, mammary gland, nerve ( with muscle), spleen, pancreas, prostate/ uterus, bone/ bone marrow ( rib junction), salivary gland, lumbar spinal cord, pituitary, stomach, testes/ ovaries, thyroid, parathyroid, thymus, trachea, tonsil, tongue, urinary bladder and vagina.

All of the rats in the 300, 1000 and 3000 ppm groups died. Death occurred between days 13 25 and days 18 28 for the males and females, respectively, in the 300 ppm group. At 1000 ppm, death occurred between days 8 14, and at 3000 ppm, the rats died between days 7 8 of treatment. The rats in all groups showed signs of toxicity including emaciation, convulsions following handling, hunched back, red material around the eyes, yellow material around the anogenital region, increased sensitivity to external stimuli, reduced activity and moist red material around the mouth or nose.

Three males and two females in the 100 ppm group died prior to scheduled sacrifice. Two of the males and the two females died during week 5 and the third male died during week 11 of the study. At study termination, mean body weights were reduced by 16.7% and 16.3% in the male and female groups, respectively. Average food consumption during the entire study period ( g/ rat/ day) was significantly reduced for males and females at 100 ppm. After 30 days of treatment, hematologic values were comparable among the control and 100 ppm groups. Clinical chemistry analyses at one month showed a significant increase in mean glucose in males, blood urea nitrogen values in males and females, and creatinine phosphokinase and alkaline phosphatase values for females. After 90 days of treatment at 100 ppm, the two surviving males had significantly reduced erythrocyte, hemoglobin, hematocrit and leukocyte counts; the three surviving females had significantly reduced hemoglobin and reticulocyte counts, as well as slightly lower erythrocyte, hematocrit and leukocyte counts. Two of the surviving females showed slight to moderate increases in plasma glutamic oxalacetic and pyruvic transaminase activities. Urinalysis results were comparable among treated and control groups at 30 and 90 days. Relative liver weight was significantly increased in the males and absolute and relative liver weights were significantly increased in the females. Relative kidney weights were significantly increased in both sexes. ENV/ JM/ RD( 2002) 17/ FINAL 28 All rats in the 30 ppm group survived until the end of the study. At study termination, mean body weights were reduced by 8.7 and 8% in the males and females, respectively. Average food consumption during the entire study period ( g/ rat/ day) was significantly reduced for the males at 30 ppm. Hematologic values were comparable among the control and 30 ppm group at 30 and 90 days. One female showed a slightly elevated glucose level and one male showed a slightly increased alkaline phosphatase level at 30 days. At 90 days, one male showed moderate increases in glucose, blood urea nitrogen and y glutamyl transpeptidase activity. The females had significant increases in absolute and relative liver weights. The males had significant decreases in absolute and relative adrenal weights, absolute thyroid/ parathyroid weight and absolute pituitary weight. The biological significance of the changes in male organ weights is unclear since similar changes were not noted in higher dose groups.

At necropsy, treatment related gross lesions were present in all treated groups and included varying degrees of discoloration and/ or enlargement of the liver and discoloration of the glandular mucosa of the stomach. Histologic examination also showed lesions in all treated groups. Centrilobular to midzonal cytoplasmic hypertrophy of hepatocytes and focal necrosis was observed in the liver; the incidence and relative severity were greater in the males. In addition, especially among rats in the 300, 1000 and 3000 ppm groups, treatment related histologic lesions were noted in the primary ( thymus, bone marrow) and secondary ( spleen, mesenteric lymph nodes) lymphoid organs, stomach, intestines, muscle and skin. In the thymus, this consisted of depletion in the number and size of the lymphoid follicles and in the bone marrow hypocellularity was noted. The spleen was slightly atrophied with a corresponding decrease in the size and number of lymphoid follicles and cells and a similar depletion was noted in the mesenteric lymph nodes. Mucosal hyperkeratosis and/ or acanthosis was observed in the forestomach and mucosal hemorrhages were noted in the glandular portion of the stomach. Decreases in the height and thickness of the villi were noted in the small intestine. Atrophy of the skeletal muscle was noted, as well as epidermal hyperkeratosis and/ or acanthosis was noted in the skin.

In the first rhesus monkey study, Goldenthal et al. ( 1979) administered rhesus monkeys, 2/ sex/ group, doses of 0, 10, 30, 100 or 300 mg/ kg/ day PFOS ( FC 95) in distilled water by gavage. The males weighed 3.05 3.80 kg at study initiation and the females weighed 2.75 4.10 kg. The monkeys were observed daily for general clinical signs and body weights were recorded weekly. Hematological and clinical chemistry analyses and urinalysis were conducted at the beginning of the study. The study was terminated after 20 days due to the death of the monkeys. At necropsy the heart, liver, adrenals, spleen, pituitary, kidneys, testes/ ovaries and brain were weighed. The thyroid/ parathyroid were weighed after fixation. Tissues were preserved in buffered neutral 10% formalin; the eyes were preserved in Russell's fixative. The following organs from control and all treated groups were examined microscopically: adrenals, aorta, brain, esophagus, eyes, gallbladder, heart ( with coronary vessels), duodenum, ileum, jejunum, cecum, colon, rectum, kidneys, liver, lung, skin, mesenteric lymph node, retropharyngeal lymph node, mammary gland, nerve ( with muscle), spleen, pancreas, prostate/ uterus, bone/ bone marrow ( rib junction), salivary gland, lumbar spinal cord, pituitary, stomach, testes/ ovaries, thyroid, parathyroid, thymus, trachea, tonsil, tongue, urinary bladder and vagina.

All of the treated monkeys died. The monkeys in the 300 mg/ kg/ day group died between days 2 4, the monkeys in the 100 mg/ kg/ day group died between days 3 5, the monkeys in the 30 mg/ kg/ day group died between days 7 10, and the monkeys in the 10 mg/ kg/ day group died between days 11 20 of treatment. The monkeys from all the groups showed similar signs of toxicity including decreased activity, emesis with some diarrhea, body stiffening, general body trembling, twitching, weakness, convulsions and prostration. At necropsy, several of the monkeys in the 100 and 300 mg/ kg/ day groups had a yellowishbrown discoloration of the liver; histologic examination showed no microscopic lesions. Congestion, hemorrhage and lipid depletion of the adrenal cortex was noted in all treated groups. No other lesions were noted. ENV/ JM/ RD( 2002) 17/ FINAL 29 In the second study, Goldenthal et al. ( 1978a) administered rhesus monkeys, 2/ sex/ group, doses of 0, 0.5, 1.5 or 4.5 mg/ kg/ day PFOS ( FC 95) in distilled water by gavage for 90 days. The males weighed 2.55 3.55 kg at study initiation and the females weighed 2.7 3.75 kg. The monkeys were observed daily for general clinical signs and body weights were recorded weekly. Hematological and clinical chemistry analyses and urinalysis were conducted at the beginning of the study and after 30 and 90 days of treatment. The monkeys were sacrificed after 90 days of treatment and a gross necrospy was conducted. At necropsy the heart, liver, adrenals, spleen, pituitary, kidneys, testes/ ovaries and brain were weighed. The thyroid/ parathyroid were weighed after fixation. Tissues were preserved in buffered neutral 10% formalin; the eyes were preserved in Russell's fixative. The following organs from control and all treated groups were examined microscopically: adrenals, aorta, brain, esophagus, eyes, gallbladder, heart ( with coronary vessels), duodenum, ileum, jejunum, cecum, colon, rectum, kidneys, liver, lung, skin, mesenteric lymph node, retropharyngeal lymph node, mammary gland, nerve ( with muscle), spleen, pancreas, prostate/ uterus, bone/ bone marrow ( rib junction), salivary gland, lumbar spinal cord, pituitary, stomach, testes/ ovaries, thyroid, parathyroid, thymus, trachea, tonsil, tongue, urinary bladder and vagina.

All monkeys in the 4.5 mg/ kg/ day group died or were sacrificed in extremis between week 5 and 7 of the study. Beginning on the first or second day of the study, these monkeys exhibited signs of gastrointestinal tract toxicity including anorexia, emesis, black stool and dehydration. All of the monkeys had decreased activity and just prior to death showed marked to severe rigidity, convulsions, generalized body trembling and prostration. The mean body weight decreased from 3.44 kg at the beginning of the study to 2.7 kg at week 5. After 30 days of treatment, there was a significant reduction in serum cholesterol and a 50% reduction in serum alkaline phosphatase activity. At necropsy, mean organ weights were comparable among the control and treated monkeys. Histologic examination showed several treatment related lesions. All the male and females had marked diffuse lipid depletion in the adrenals. One male and two females had moderate diffuse atrophy of the pancreatic exocrine cells with decreased cell size and loss of zymogen granules. Two males and one female had moderate diffuse atrophy of the serous alveolar cells characterized by decreased cell size and loss of cytoplasmic granules.

All monkeys in the 1.5 mg/ kg/ day group survived until the end of the study. During the first week of the study, the monkeys had decreased activity. Signs of gastrointestinal tract toxicity were noted occasionally during the study and included black stool, diarrhea, mucous in the stool and bloody stool; at the end of the study, anorexia, dehydration or general body trembling were noted. Although statistical significance was not achieved, the mean body weight of the males dropped from 3.15 kg at the beginning of the study to 2.93 kg at the end of the study, and the mean body weight of the females dropped from 3.22 kg to 2.75 kg. One of the females had very low serum cholesterol and another had a reduction in inorganic phosphate. Necropsy revealed no treatment related lesions.

All monkeys in the 0.5 mg/ kg/ day group survived until the end of the study. Signs of gastrointestinal tract toxicity were noted occasionally during the study and included diarrhea, soft stools, anorexia and emesis. Occasionally, decreased activity was noted in three of the monkeys. Necropsy revealed no treatment related lesions.

Serum samples from monkeys in the 0, 0.5 and 1.5 mg/ kg/ day dose group were individually analyzed for PFOS levels; liver samples from the 0, 0.5, 1.5 and 4.5 mg/ kg/ day dose groups were also analyzed for PFOS levels. Except for the control animals where animal 7355M was used in both the serum and liver control groups, different animals were used for serum and liver determinations. PFOS levels in serum were developed by pyrolysis; precision is estimated to be ± 10   25%. ENV/ JM/ RD( 2002) 17/ FINAL 30 Levels of PFOS in serum Monkey # Dosage Group PFOS Levels in Serum ( ppm) 7355M 0 40 7358M 0 20 7368F 0 15 7460M 0.5 mg/ kg/ day 150 7466F 0.5 mg/ kg/ day 150 7462M 1.5 mg/ kg/ day 250 7500F 1.5 mg/ kg/ day 275 No explanation is offered for the relatively high serum levels in the controls. Sample 7355M was run on two separate days and gave the same results.

Levels of PFOS in liver Monkey # Dosage Level PFOS in Liver ( mg) PFOS in Liver ( ppm) 7355M 0 3000 50 7368F 0 1500 20 7463M 0.5 mg/ kg/ day 7000 100 7466F 0.5 mg/ kg/ day 8000 100 7462M 1.5 mg/ kg/ day 45000 650 7500F 1.5 mg/ kg/ day 40000 600 7484M 4.5 mg/ kg/ day 40000 650 7502F 4.5 mg/ kg/ day 80000 1000 Livers were stored refrigerated for several months prior to analysis. This resulted in the separation of some liquid from the samples. At the time of analysis, only partial samples were taken in which the ratio of solid to liquid was estimated visually. Recovery of PFOS from the liver samples was estimated from experiments where a known amount of PFOS was added to a control liver. Here as in the serum, control values were higher than expected. Because of this, a 0.133 g liver sample was analyzed for total fluorine. The fluorine value calculated as PFOS was equivalent to 11 ppm in the liver.

PFOS values in serum and liver are approximately the same at the 0.5 mg/ kg/ day level but are substantially higher in the liver at the 1.5 mg/ kg/ day level. Serum levels were not determined at the 4.5 mg/ kg level. That may be because only a single female survived until scheduled sacrifice in the 4.5 mg/ kg/ day dose group. From this study, there do not appear to be any differences in PFOS in either serum or liver between the sexes.

The values of PFOS in the liver and serum should be viewed with caution. There were a very limited number, one male and one female, of animals per group. Storage of the liver samples at refrigeration temperature for long periods of time was not ideal. Although PFOS is stable, the liver itself obviously underwent some deterioration as evidenced by the separation of a liquid phase. Sample size may also have been inaccurate since the visual estimation of solid to liquid phase is not a precise measurement.

In order to determine the dose range for a six month study, Thomford et al. ( unaudited draft, 1998) conducted a 4 week range finding study in cynomolgus monkeys. Male and female cynomolgus monkeys were administered doses of 0 ( 2/ sex/ group), 0.02 ( 3/ sex/ group), or 2.0 mg/ kg/ day ( 1/ sex/ group) PFOS in capsules placed directly into the stomach. The monkeys weighed 2.1 2.4 kg at study initiation. It appears the monkeys were observed at least daily for general clinical signs and body weights were recorded twice ENV/ JM/ RD( 2002) 17/ FINAL 31 weekly. Hematological and clinical chemistry analyses were conducted on samples collected before the beginning of the study at day  7 ( baseline values) and day 29. Additional blood samples for clinical chemistry were collected on study days 2, 7, and 14. Blood samples for serum PFOS concentrations were taken on days 7, 2, 3, 7, 14, and 29. In addition, samples from day  7 and day 29 were analyzed for levels of estradiol, estrone, estriol, thyroid stimulating hormone, triiodothyronine, and thyroxin. The study animals were terminated as scheduled at 30 days. At necropsy a sample of liver was collected from each animal for palmitoyl CoA oxidase activity analyses. Samples of liver, testes, and pancreas were collected for proliferation cell nuclear antigen evaluation. A sample of liver was also collected from each animal for PFOS concentration analysis. The following organs from control and all treated groups were examined microscopically: adrenals, eye, kidney, liver, lung, spleen, pancreas, femoral bone marrow, testes, and thymus.

None of the monkeys died before the study was ended. There were no test related effects on clinical observations, body weight, food consumption, body temperatures, hematology, or macroscopic or microscopic pathology findings. No test related effects were noted in the levels of estrone, estriol, thyroid stimulating hormone, and thyroxin. Cell proliferation, as measured by immunohistochemical detection of proliferating cell nuclear antigen, was not increased in the liver, testes, or pancreas of monkeys.

The monkeys in the 2.0 mg/ kg/ day group both showed estradiol levels that were less than their prestudy values and controls at day 29. Similarly, the monkeys in the 2.0 mg/ kg/ day group both showed triiodothyronine levels that were less than their prestudy values and controls at day 29. However, pretreatment the high dose monkeys also had triiodothyronine values lower than controls in baseline samples as well. Since the numbers of tested animals are small and baseline levels are variable, it is not clear if these hormone level changes are treatment related effects.

In the final study, Thomford ( 2002) administered PFOS to cynomolgus monkeys by oral capsule at doses of 0 ( 6 monkeys per sex), 0.03 ( 4 monkeys per sex), 0.15 ( 6 monkeys per sex), or 0.75 mg/ kg/ day ( 6 monkeys per sex) for 26 weeks. Two animals from the control, 0.15 and 0.75 mg/ kg/ day groups were assigned to a recovery group and were not treated for at least 52 weeks following the last administration of PFOS. Animals were observed twice daily for mortality and moribundity and were examined at least once daily for abnormalities and signs of toxicity; food consumption was assessed qualitatively. Ophthalmic examinations were done before initiation of treatment and during weeks 26 and 52. Body weight data were recorded weekly before the start of treatment, on Days   1 and 1 and weekly thereafter. Blood and urine samples were collected for clinical hematology, clinical chemistry, and urinalysis before the start of treatment and at specified intervals during treatment and recovery. Blood samples were also taken for hormone determinations. Samples of serum were collected at various time points during the study and sent to 3M for analysis of PFOS levels. The following organs were weighed at scheduled and unscheduled sacrifices; paired organs were weighed separately: adrenal ( 2), brain, epididymis ( 2), kidney ( 2), liver, ovary ( 2), pancreas, testis ( 2), and thyroid ( 2) with parathyroid. The following tissues were collected for histopathology: adrenals ( 2), aorta, brain, cecum, cervix, colon, duodenum, epididymis ( 2), esophagus, eyes ( 2), femur with bone marrow, gallbladder, heart, ileum, jejunum, kidneys ( 2), lesions, liver, lung, mammary gland, mesenteric lymph node, ovary ( 2), pancreas, pituitary, prostate, rectum, salivary gland [ mandibular ( 2)], sciatic nerve, seminal vesicle ( 2), skeletal muscle ( thigh), skin, spinal cord ( cervical, thoracic, and lumbar), spleen, sternum with bone marrow, stomach, testis ( 2), thymus, thyroid ( 2) with parathyroid, trachea, urinary bladder, uterus, and vagina. Liver specimens from the 0.15 and 0.75 mg/ kg/ day recovery animals were collected via biopsy and analyzed for PFOS levels.

Males weighed 3.3 3.4 kg and females weighed 2.8 2.9 kg at the beginning of the study. At the end of 26 weeks of treatment, males weighed 3.7, 3.8, 3.5, and 3.3 kg for the 0, 0.03, 0.15 and 0.75 mg/ kg/ day treatment groups respectively. Females weighed 3.1, 3.1, 3.1 and 2.8 kg for the 0, 0.03, 0.15 and 0.75 mg/ kg/ day treatment groups respectively. The difference between the control and the 0.75 mg/ kg/ day ENV/ JM/ RD( 2002) 17/ FINAL 32 female treatment groups was statistically significant. At the end of the recovery period, differences in weight between the control and treated animals were no longer obvious.

Two males from the 0.75 mg/ kg/ day group did not survive to the scheduled sacrifice. One animal died after dosing on Day 155 ( Week 23). Clinical signs noted in this animal included: constricted pupils, pale gums, few, mucoid, liquid and black colored feces, low food consumption, hypoactivity, labored respiration, dehydration, and recumbent position. In addition, the animal was cold to the touch. An enlarged liver was detected by palpation. Cause of death was determined to be pulmonary necrosis with severe acute inflammation. On day 179, the second male was sacrificed in a moribund condition. Clinical signs noted included low food consumption, excessive salivation, labored respiration, hypoactivity and ataxia. The cause of death was not determined.

Males and females in the 0.75 mg/ kg/ day dose group had lower total cholesterol and males and females in the 0.15 and 0.75 mg/ kg/ day groups had lower high density lipoprotein cholesterol during treatment. The effect on total cholesterol worsened with time. By day 182, mean total cholesterol for males and females in the high dose group were 68% and 49% lower, respectively, that than levels in the control animals. The effect on high density lipoprotein was greater than that seen with cholesterol. On day 182, the mean high density lipoprotein levels were 79% and 62% lower, respectively in males and females from the high dose group than they were in male and female control animals. Males in the high dose group also had lower total bilirubin concentrations and higher serum bile acid concentrations than males in either the control or other treatment groups. The effect on total cholesterol was reversed within 5 weeks of recovery and the effect on high density lipoprotein cholesterol was reversed within 9 weeks of recovery.

Estradiol values were lower in males given 0.75 mg/ kg/ day on days 62, 91, and 182 but because of variation only the day 182 value was significant. Estrone values were generally higher in the treated females on days 37, 62 and 91 but again because of variation in the data none of these values were significantly different from the controls. Except for males in the 0.15 mg/ kg/ day group, triiodothyronine values were significantly lower on days 91 and 182 in males and females given 0.15 and 0.75 mg/ kg/ day. There were other instances in which hormone values in treated groups were different from those of controls but these differences were not consistent over time or between sexes, were not clearly dose related and did not appear to be related to the administration of the test material. Apparent differences in the sexual maturity of both males and females used in the study complicates the interpretation of the hormone data.

At terminal sacrifice, females in the 0.75 mg/ kg/ day dose group had increased absolute liver weight, liverto body weight percentages, and liver to brain weight ratios. In males, liver to body weight percentages were increased in the high dose group compared to the controls. " Mottled" livers were observed in two high dose males and in one high dose female. Of the two males not surviving until the scheduled terminal sacrifice, one had a " mottled" and large liver. Three of 4 high dose males ( including those that did not survive to scheduled sacrifice) had centrilobular or diffuse hepatocellular hypertrophy that was also observed in all high dose females. Centrilobular or diffuse hepatocellular vacuolation occurred in 2 of 4 females and 2 of 4 males in the high dose group.

No PFOS related lesions were observed either macroscopically or microscopically at recovery sacrifice indicating that the effects seen at terminal sacrifice may be reversible.

Serum and liver specimens collected from test animals were sent to the 3M Laboratory and analyzed for the presence of PFOS. Serum was harvested from blood that was centrifuged within one hour of collection. Liver specimens were flash frozen in liquid nitrogen. Both liver and serum samples were stored in a freezer set to maintain specimens at  60 to   80oC until shipped to the 3M Lab. Samples were shipped frozen and on dry ice from Covance Laboratories to 3M periodically from August 1998 through March 2000 which covered the in life phase of the study. Once received at 3M specimens were stored in freezer at either   ENV/ JM/ RD( 2002) 17/ FINAL 33 55oC ± 10 20oC or   20oC ± 10oC.

During the first 26 weeks of the study a total of 550 serum specimens and 30 liver specimens were collected. Of the serum specimens, 151 were from Group 1, 99 from Group 2, 152 from Group 3 and 148 from Group 4. Eight liver samples were collected from Group 1, 8 from Group 2, 12 from Group 3 and 14 from Group 4. In the recovery Groups, 72 serum and 4 liver samples were collected from Group 1; 72 serum and 4 liver samples from Group 3 and 80 serum and 4 liver samples from Group 4.

Liver and serum samples were extracted using an ion pairing reagent and methyl tert butyl ether ( MtBE). Liver samples were homogenized prior to extraction. Sample extracts were analyzed using high pressure liquid chromatography electrospray/ tandem mass spectrometry ( HPLC ES/ MS/ MS) in the multiple response mode. PFOS levels were quantitated by external standard calibration.

Liver samples were homogenized in water. An aliquot of each liver homogenate and all serum samples were spiked with THPFOS and extracted using an ion pairing extraction procedure. An ion pairing reagent was added to the samples and the ion pairs were partitioned into MtBE. The extracts were evaporated until dry on a nitrogen evaporator and then were reconstituted in 1.0 mL of methanol and passed through a 0.2 µ m nylon filter.

The analyses were performed by monitoring one or more product ions selected from a single primary ion characteristic of the fluorochemical of interest using HPLC/ ES/ MS/ MS. Molecular ion 499, the primary ion for PFOS ( C8F17SO3 ) analysis, was fragmented to produce ion 99 ( FSO3 ). Ion 99 was monitored for quantitative analysis.

Although low levels of PFOS were often detected in the sera and liver of the control animals, these levels were significantly lower than those found in the low dose test animals. PFOS levels in the sera of test animals increased with dose during treatment from 21.0 ± 1.57 and 20.4 ± 2.71 µ g/ ml in the Group 4 males and females respectively at the end of Week 1 to 194 ± 8.94 and 160 ± 23.1 µ g/ ml in males and females respectively in Group 4 at the end of Week 27. During recovery, PFOS levels in serum samples decreased over time until they reached 41.1 ± 25.9 µ g/ ml in males and 41.4 ± 1.15 µ g/ ml in females from Group 4 at 79 weeks post treatment. Control values were < LOQ ( the limit of quantitation) at Week 4 in both males and females and 0.0215 ± 0.00296 and 0.0243 ± 0.00355 µ g/ ml in males and females respectively at the end of Week 79. The serum values for selected weeks of treatment and recovery are shown in the table below. There were no significant differences between PFOS levels in the sera of treated males and females. ENV/ JM/ RD( 2002) 17/ FINAL 34 Table 2. Average PFOS Concentrations ( µ g/ ml) in Serum of Monkeys for Selected Weeks During Treatment and Recovery Group 1 0.0 mg/ kg/ day Group 2 0.03 mg/ kg/ day Group 3 0.15 mg/ kg/ day Group 4 0.75 mg/ kg/ day Males Females Males Females Males Females Males Females Week 1 < LOQ < LOQ 0.869 ± 0.147 0.947 ± 0.110 4.60 ± 0.782 3.71 ± 0.455 21.0 ± 1.57 20.4 ± 2.71 Week 4 < LOQ < LOQ 3.20 ± 0.577 3.40 ± 0.291 17.8 ± 1.68 16.5 ± 1.87 95.3 ± 70.4 92.7 ± 39.6 Week 16 0.0407 ± 0.0110 0.0432 ± 0.0081 11.2 ± 2.44 10.5 ± 1.90 56.2 ± 5.84 42.1 ± 4.04 189 ± 15.9 162 ± 19.3 Week 27 0.0529 ± 0.0145 0.0416 ± 0.0148 15.9 ± 5.54 11.1 ± 1.52 68.1 ± 5.75 58.5 ± 4.67 194 ± 8.93 160 ± 23.9 Week 35 0.0459 ± 0.00303 0.0723 ± 0.00352 Not Determined Not Determined 84.5 ± 12.0 74.7 ± 9.53 181 ± 19.5 171 ± 10.1 Week 47 0.0355 ± 0.00221 0.0459 ± 0.00323 Not Determined Not Determined 48.3 ± 3.69 42.6 ± 6.70 124 ± 25.9 98.3 ± 8.32 Week 57 0.0327 ± 000526 0.0445 ± 0.00385 Not Determined Not Determined 30.2 ± 2.36 32.3 ± 1.34 78.0 ± 16.3 106 ± 3.84 Week 69 0.0406 ± 0.00313 0.0400 ± 0.00301 Not Determined Not Determined 26.4 ± 2.59 34.5 ± 3.46 84.0 ± 52.4 75.0 ± 5.25 Week 79 0.0215 ± 0.00296 0.0243 ± 0.00355 Not Determined Not Determined 19.1 ± 0.805 21.4 ± 2.01 41.1 ± 25.9 41.4 ± 1.15 LOQ = Lowest Observable Concentration Liver values behaved in a manner similar to serum values and increased over time. At Week 27 mean PFOS values on an RSD basis were 22.2 ± 0.0269 in Group 1 males and 16.8 ± 0.0178 in females in Group 1; 27.0 ± 4.66 and 9.73 ± 2.15 in males and females in Group 2; 33.1 ± 19.5 in males and 21.4 ± 14.9 in females in Group 3, and 6.03 ± 23.9 in males and 5.00 ± 13.6 in females in Group 4. At Week 79 values in the liver were 71.0 ± 33.4 in males and 21.4 ± 10.8 in females in Group 4. At Week 80, values were 14.9 ± 1.38 in Group 3 males and 23.5 ± 4.98 in Group 3 females.

3.5 Carcinogenicity The chronic toxicity and carcinogenicity of perfluorooctane sulfonic acid potassium salt ( PFOS; T 6295) have been studied in rats ( 3M, 2002). The results of the study show that PFOS is hepatotoxic and carcinogenic, inducing tumors of the liver, and of the thyroid and mammary glands. Based on the liver toxicity, the no observed adverse effect level ( NOAEL) for PFOS is considered to be 0.5 ppm in male rats and 2 ppm in female rats; the low observed adverse effect level ( LOAEL) is 2 ppm in male rats and 5 ppm in female rats.

In this study, groups of 40 70 male and female Crl: CD ( SD) IGS BR rats were given PFOS in the diets at concentrations of 0.5, 2, 5, or 20 ppm for 104 weeks. A control group was given diets containing acetone, the vehicle. A recovery group was given the test material at 20 ppm for 52 weeks and was observed till ENV/ JM/ RD( 2002) 17/ FINAL 35 death. Five animals/ sex in the treatment groups were sacrificed during weeks 4, 14 and 53; liver samples were collected for mitochondrial activity, hepatocellular proliferation rate, and determination of palmitoyl CoA oxidase activity. Serum and liver specimens were collected for analyses of the presence and concentration of PFOS in liver and serum during and at the end of the in life phase of the study.

There was a significant increased trend in survival that occurred in the males that was due to significant increases in survival in mid high ( 5.0 ppm) and high dose ( 20.0 ppm) groups as compared to that of the control group. None of the other treated groups in the males revealed any significant differences in survival. No significant trend was noted in survival in females. There was a significant decrease in survival in the mid dose ( 2.0 ppm) group and not in the mid high ( 5.0 ppm) and high dose ( 20.0 ppm) groups as compared to that of the control.

Males given 20 ppm had significantly lower mean body weights compared to animals in the control group during weeks 9 through 37. Females given 20 ppm had significantly lower body weights compared to animals in the control group during weeks 3 through 101. At week 105, mean body weights of the surviving males and females were not significantly different from the controls. Food consumption for males and females was similar in all treated groups compared to animals given the control material except for the high dose females which had statistically significantly lower food consumption during weeks 2 through 44.

At the week 14 and week 53 interim sacrifice, absolute and relative liver weights were significantly increased in the males given 20 ppm. In females given 20 ppm, only the liver to body weight percentage was significantly increased. Treatment related histomorphologic changes were seen in the liver in the males given 5 or 20 ppm and in the females given 20 ppm. The changes consisted of hypertrophy of hepatocytes in centrilobular areas in males and females, and midzonal to centrilobular hepatocytic vacuolation. The incidence and severity of the changes tended to be greater in the males. Dietary administration of PFOS for approximately 53 weeks was associated with mildly to moderately lower cholesterol for males and females fed 20 ppm; and mildly higher alanine aminotransferase for males fed 20 ppm. In the unscheduled sacrifices between Weeks 54 and 105, animals given 20 ppm had increased hepatocellular centrilobular hypertrophy, eosinophilic hepatocytic granules, and centrilobular hepatocytic pigment were noted. Increased hepatocellular centrilobular hypertrophy was seen in animals given 5 ppm.

At the terminal sacrifice, the livers of animals given 5 or 20 ppm exhibited a slight increase in macroscopic findings, including enlarged, mottled, diffuse darkened, or focally lightened. Hepatotoxicity, characterized by significant increases ( P< 0.05) in centrilobular hypertrophy, centrilobular eosinophilic hepatocytic granules, centrilobular hepatocytic pigment, or centrilobular hepatocytic vacuolation was noted in male and/ or female rats given 5 or 20 ppm. A significant increase ( P< 0.05) in hepatocellular centrilobular hypertrophy was also observed in mid dose ( 2 ppm) male rats. Significant increases in the incidence of cystic hepatocellular degeneration was found in all the male treated groups ( 0.5, 2, 5, or 20 ppm); however, this lesion is believe to be due to old age of the animals and is not considered to be treatment related.

Based on the pathological findings in the liver, the no observed adverse effect level ( NOAEL) for PFOS is considered to be 0.5 ppm in male rats and 2 ppm in female rats; the low observed adverse effect level ( LOAEL) is 2 ppm in male rats and 5 ppm in female rats.

There was no effect on hepatic palmitoyl CoA oxidase activity. There were also no statistically significant increases in cell proliferation as measured by proliferative cell nuclear antigen ( PCNA) at weeks 4 and 14, or by bromodeoxyuridine ( BrdU) at week 53.

For neoplastic effects, a significant positive trend ( P= 0.0276) was noted in the incidences of hepatocellular adenoma in male rats. This was due to a significant increase ( P< 0.05) in the high dose group ( 11.7%, 7/ 60) ENV/ JM/ RD( 2002) 17/ FINAL 36 over the control ( 0%, 0/ 60). A significantly increased incidence ( P< 0.05) was observed for thyroid follicular cell adenoma in the high dose recovery group ( 23.1%, 9/ 39) when compared to the control group ( 5%, 3/ 60). There was also a slight increase in the combined thyroid follicular cell adenoma and carcinoma in the high dose recovery group ( 25.6%, 10/ 39) as compared to that of the control group ( 10%, 6/ 60); the increase did not reach statistical significance relative to the control but did reach statistical significance relative to the high dose group ( 8.5%, 5/ 59).

In the females, significant positive trends were observed in the incidences of hepatocellular adenoma ( P= 0.0153) and combined hepatocellular adenoma and carcinoma ( P= 0.0057). These cases were due to significant increases in the high dose group ( 8.3%, 5/ 60, and 10%, 6/ 60) as compared to the control ( 0%, 0/ 60). A significant increase ( P= 0.0471) for combined thyroid follicular cell adenoma and carcinoma was observed in the mid high ( 5.0 ppm) group ( 6%, 3/ 50) as compared to the control group ( 0%, 0/ 60). Except for the high dose group ( which showed a slight decrease in incidences of mammary fibroadenoma/ adenoma and combined mammary fibroadenoma and carcinoma), increases in mammary tumors were observed in all treatment groups when compared to the controls. Significant increases ( P< 0.05) in mammary fibroadenoma/ adenoma ( 60%, 30/ 50) and combined mammary fibroadenoma/ adenoma and carcinoma ( 72%, 36/ 50) were observed in the low   dose ( 0.5 ppm) group as compared to the respective controls ( 38.3%, 23/ 60 and 48.3%, 29/ 60). The mid dose ( 2.0 ppm) group also exhibited a statistically significant ( P< 0.05) increase ( 64.6%, 31/ 48) in the incidence of combined mammary fibroadenoma/ adenoma/ carcinoma over the control group ( 43.8%, 29/ 60). Increases in mammary tumors in the mid high ( 5 ppm) dose group did not reach statistical significance relative to the control. ENV/ JM/ RD( 2002) 17/ FINAL 37 The carcinogenicity data of PFOS in rats are summarized in Table 3.

Table 3. Summary of carcinogenicity data of PFOS in rats.

Tumor incidence (%) Tumors Male 0 0.5 ppm 2 ppm 5 ppm 20 ppm 20 ppm recovery# Liver Hepatocellular adenoma* 0 ( 0/ 60) 6.0 ( 3/ 50) 6.0 ( 3/ 50) 2.0 ( 1/ 50) 11.7** ( 7/ 60) Thyroid Follicular cell adenoma Follicular cell carcinoma Combined 5.0 ( 3/ 60) 5.0 ( 3/ 60) 10.0 ( 6/ 60) 10.2 ( 5/ 49) 2.0 ( 1/ 49) 12.2 ( 26/ 49) 8.0 ( 4/ 50) 2.0 ( 1/ 50) 10.0 ( 5/ 50) 8.2 ( 4/ 49) 4.1 ( 2/ 49) 10.2 ( 5/ 49) 6.8 ( 4/ 59) 1.7 ( 1/ 59) 8.5 ( 5/ 59) 23.1** ( 9/ 39) 2.6 ( 1/ 39) 25.6 ( 10/ 39)

Female Liver Hepatocellular adenoma* Hepatocellular carcinoma Combined* 0 ( 0/ 60) 0 ( 0/ 60) 0 ( 0/ 60) 2.0 ( 1/ 50) 0 ( 0/ 50) 2.0 ( 1/ 50) 2.0 ( 1/ 49) 0 ( 0/ 49) 2.0 ( 1/ 49) 2.0 ( 1/ 50) 0 ( 1/ 50) 2.0 ( 1/ 50) 8.3** ( 5/ 60) 1.7 ( 1/ 60) 10.0** ( 6/ 60) Thyroid Follicular cell adenoma Follicular cell carcinoma Combined 0 ( 0/ 60) 0 ( 0/ 60) 0 ( 0/ 60) 0 ( 0/ 50) 0 ( 0/ 50) 0 ( 0/ 50) 0 ( 0/ 49) 0 ( 0/ 49) 0 ( 0/ 49) 4.0 ( 2/ 50) 2.0 ( 1/ 50) 6.0** ( 3/ 50) 1.7 ( 1/ 60) 0 ( 0/ 60) 1.7 ( 1/ 60) Mammary Fibroadenoma/ adenoma Carcinoma Combined 38.3 ( 23/ 60) 18.3 ( 11/ 60) 48.3 ( 29/ 60) 60.0** ( 30/ 50) 24.0 ( 12/ 50) 72.0** ( 36/ 50) 45.8 ( 22/ 48) 31.2 ( 15/ 48) 64.6** ( 31/ 48) 52.04 ( 26/ 50) 22.0 ( 11/ 50) 58,0 ( 29/ 50) 25 ( 15/ 60) 23.3 ( 14/ 60) 40.0 ( 24/ 60)

* Significant positive trend ( P < 0.03). ** Significantly increased over the control ( P < 0.05). # Recovery group; after 52 weeks of treatment.

Serum and Liver level of PFOS Under the conditions of the studies, PFOS was observed in the serum and liver of rats dosed with perfluorooctane sulfonic acid potassium salt ( PFOS T 6295). Trace levels of PFOS were often detected in the serum and liver of the control animals. Detailed specimen data are presented in Table 4 and Table 5. ENV/ JM/ RD( 2002) 17/ FINAL 38 Table 4. Summary of PFOS Concentration Serum ( ug/ mL)