Remarks: 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.

CONCLUSIONS Remarks: Authors conclusions stated above in results. Reviewer agrees. ENV/ JM/ RD( 2002) 17/ FINAL 295 REFERENCE Goldenthal, E. I., D. C. Jessup, R. G. Geil and J. S. Mehring. 1978. Ninety day subacute rhesus monkey toxicity study. Study No. 137 092, International Research and Development Corporation, Mattawan, MI. ENV/ JM/ RD( 2002) 17/ FINAL 296 Title: Ninety day study in rats, 1978 TEST SUBSTANCE Identity: Potassium perfluorooctylsulfonate, CAS # 2795 39 3 Remarks: FC 95, purity not specified.

METHOD Method/ guideline followed: None Study duration: 90 days GLP ( Y/ N): No Year study performed: 1978 Species/ strain: CD rat Sex: Males and females Number of animals per dose group: 5/ sex/ group Route of administration: Diet Doses tested and frequency: 0, 30, 100, 300, 1000, 3000 ppm Equivalent to 0, 2, 6, 18, 60, 200 mg/ kg/ day Post observation period: None Statistical methods used: Body wts, hematological, biochemical and urinalysis and organ wts were compared by analysis of variance ( one way classification), Bartlett's test and the appropriate t test using Dunnett's multiple comparison tables to judge significance of differences.

Remarks: The males weighed 196 232 g and the females weighed 165 206 g at study initiation. The animals 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. 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. ENV/ JM/ RD( 2002) 17/ FINAL 297 RESULTS NOAEL ( dose and effect): None LOAEL ( dose and effect): 30 ppm ( 2 mg/ kg/ day) based on a significant increase in relative and absolute liver weights.

Toxic response/ effects by dose level: 3000 ppm   10/ 10 rats died between days 7 8. 1000 ppm   10/ 10 rats died between days 8 14. 300 ppm   5/ 5 male rats died between days 13 25; 5/ 5 female rats died between days 18 28. At 300, 1000 and 3000 ppm   histologic lesions in the primary ( thymus, bone marrow) and secondary ( spleen, mesenteric lymph nodes) lymphoid organs, stomach, intestines, muscle and skin. 100 ppm   2/ 5 males and 2/ 5 females died during week 5 and a third male died during week 11, mean body weights were reduced by 16.7% ( males) and 16.3% ( females) at study termination, food consumption significantly reduced, significant reduction in hematocrit ( males), erythrocyte ( males), hemoglobin ( males & females), leukocyte ( males), and reticulocyte ( females) counts, significant increase in absolute ( females) and relative ( males & females) liver weight and relative kidney weight. At 100, 300, 1000 and 3000 ppm   slight to marked focal necrosis of hepatocytes. 30 ppm   Significant reduction in food consumption ( males), significant increase in absolute and relative liver weight ( females). At all dose levels   very slight to slight cytoplasmic hypertrophy of hepatocytes in the centrilobular ro midzonal regions, especially in males.

Statistical results: 100 ppm   significant reduction in food consumption Remarks: 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.

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 ENV/ JM/ RD( 2002) 17/ FINAL 298 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. Decrease atrophy 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.

CONCLUSIONS Remarks: Authors conclusions stated above in results. Reviewer agrees.

REFERENCE Goldenthal, E. I., D. C. Jessup, R. G. Geil and J. S. Mehring. 1978. Ninety day subacute rat toxicity study. Study No. 137 085, International Research and Development Corporation, Mattawan, MI. ENV/ JM/ RD( 2002) 17/ FINAL 299 Title: 4 Week Capsule Toxicity Study with Perfluoroctane Sulfonic Acid Potassium Salt ( PFOS; T 6295) in Cynomolgus Monkeys. Unaudited Draft. 1998.

TEST SUBSTANCE Identity: Potassium Perfluorooctylsulfonate, CAS No.: 2795 39 3 Remarks: T 6295, purity not specified.

METHOD Method/ guideline followed: Range finding   Unaudited Draft report, sections missing Study duration: 28 days GLP ( Y/ N): Y, report contains GLP statement, but as submitted, unaudited draft report with sections missing Year study performed: 1998 Species/ Strain: Monkey/ Cynomolgus Sex: both Number of animals per dose: 2 per sex, 0 mg/ kg/ day; 3 per sex, 0.02 mg/ kg/ day; 1 per sex, 2.0 mg/ kg/ day Route of administration: Capsule in stomach Doses tested and frequency: 0 mg/ kg/ day; 0.02 mg/ kg/ day; 2.0 mg/ kg/ day Post observation period: none Statistical methods used: none Remarks: Monkeys were observed at least daily for general clinical signs and body weights were recorded twice 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. ENV/ JM/ RD( 2002) 17/ FINAL 300 RESULTS NOAEL ( dose and effect): None determined.

LOAEL ( dose and effect): None determined Toxic response/ effects by dose level: None determined Statistical results: None, high dose too few animals ( 1 male, 1 female Summary Hormone Analyses Data in Males on Females on Days   7 and 29 Dose Level Sex Day Estradiol pg/ mL Estrone pg/ mL Estriol pg/ mL TSH ØU/ mL Triiodothyronine ng/ dL Thyroxin Øg/ dL 0 Males  7 32.03 ± 4.002 28.23 ± 14.701 0.00 ± 0.000 2.82 ± 0.686 139.74 ± 29.232 5.43 ± 2.001 0 Males 29 32.09 ± 0.205 21.93 ± 4.30 0.00 ± 0.000 2.15 ± 2.008 186.35 ± 6.935 4.32 ± 1.407 0.02 Males  7 28.61 ± 4.874 17.32 ± 1.373 0.00 ± 0.000 2.11 ± 0.799 122.49 ± 48.610 4.28 ± 0.335 0.02 Males 29 34.25 ± 7.998 14.72 ± 4.767 0.00 ± 0.000 3.10 ± 0.584 188.34 ± 26.229 4.19 ± 0.612 2.0 Males  7 28.71 13.98 0.00 1.71 97.61 3.86 2.0 Males 29 18.16 20.12 0.00 0.95 90.73 3.18 0 Females  7 45.28 ± 0.212 28.77 ± 5.162 0.00 ± 0.000 4.37 ± 4.398 132.65 ± 56.434 4.88 ± 0.948 0 Females 29 53.47 ± 20.082 25.97 ± 2.128 0.00 ± 0.000 2.95 ± 0.530 162.70 ± 29.366 4.26 ± 0.438 0.02 Females  7 48.12 ± 21.124 30.40 ± 10.852 0.00 ± 0.000 2.51 ± 1.506 131.09 ± 56.434 4.76 ± 1.057 0.02 Females 29 58.72 ± 27.628 31.61 ± 0.921 0.00 ± 0.000 2.76 ± 1.764 180.58 ± 15.203 4.05 ± 1.108 2.0 Females  7 37.37 29.50 0.00 3.92 122.38 3.60 2.0 Females 29 19.82 25.85 0.00 90.17 3.38 CONCLUSIONS Estradiol, estrone, thyroid stimulating hormone ( TSH), thyroxin, and triiodothyronine levels were lower in the high dose animals at the end of the study. Since the numbers of tested animals are small ( one male and one female in the high dose group) and baseline levels are variable, it is not clear if these hormone level changes are treatment related.

Remarks: None REFERENCE Thomford, P. J. 1998. 4 Week Capsule Toxicity Study with Perfluoroctane Sulfonic Acid Potassium Salt ( PFOS; T 6295) in Cynomolgus Monkeys. Unaudited Draft. Study No. T 6295.6, for 3M, St. Paul, MN, by Covance Laboratories Inc., Madison, WI. ENV/ JM/ RD( 2002) 17/ FINAL 301 Title: 26 Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt ( PFOS; T6295) in Cynomolgus Monkeys, 2002 TEST SUBSTANCE Identity: Potassium perfluorooctylsulfonate, CAS # 2795 39 3 Remarks: FC 95, T 6295, purity not specified.

METHOD Method/ guideline followed: No guideline followed.

Study duration: 78 weeks, 26 week treatment period, followed by 52 weeks recovery.

GLP ( Y/ N): Y Year study performed: 2000 Species/ strain: Cynomolgus monkeys Sex: Males and females Number of animals per dose group: 6 animals/ sex/ dose group for groups 1, 3, and 4; 4 animals/ sex for group 2.

Route of administration: Oral capsule Doses tested and frequency: 0 mg/ kg/ day, 0.03 mg/ kg/ day, 0.15 mg/ kg/ day, or 0.75 mg/ kg/ day Post observation period: 52 weeks Statistical methods used: Levene's test for variance homogeneity: ANOVA, Dunnett's t test ANCOVA, covariate adjusted means, 5% two tailed probability level.

Remarks: 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 ENV/ JM/ RD( 2002) 17/ FINAL 302 mg/ kg/ day recovery animals were collected via biopsy and analyzed for PFOS levels.

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   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.

RESULTS NOAEL ( dose and effect): 0.15 mg/ kg/ day.

LOAEL ( dose and effect): 0.75 mg/ kg/ day. Death, liver effects, effect on cholesterol Toxic response/ effects by dose level: Death at 0.75 mg/ kg/ day; increased absolute liver weight, liver to body weight percentages, liver to brain weight ratios in females at 0.75 mg/ kg/ day; absolute and relative liver weight; Males and females in the 0.75 mg/ kg/ day dose group had lower total cholesterol and lower high density lipoprotein cholesterol, liver organ weights.

Statistical results: The difference in weight at the end of treatment between the control and the 0.75 mg/ kg/ day female treatment groups was statistically significant; the effect on total cholesterol and high density lipoprotein cholesterol in the 0.75 mg/ kg/ dose group was statistically significant; in males in the 0.75 mg/ kg/ day dose group the liver organ weights and the organ to body weight percentages were statistically significant and in females the liver weights, the organ to body weight percentage and the organ brain weight ratio were all significant. ENV/ JM/ RD( 2002) 17/ FINAL 303 Remarks:

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 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. 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. However, only the effect in the 0.75 mg/ kg group was statistically significant. 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 in males and females, respectively, 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 by 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 by again because of variation in the data none of these values were significantly different. Triiodothyronine values were notably 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 at recovery sacrifice indicating that the effects seen at terminal sacrifice may be reversible. ENV/ JM/ RD( 2002) 17/ FINAL 304 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.

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 ENV/ JM/ RD( 2002) 17/ FINAL 305 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.

CONCLUSIONS PFOS is toxic to cynamolgous monkeys at 0.75 mg/ kg/ day causing death, alterations in total cholesterol, and effecting liver weight and causing hepatocellular hypertrophy and vacuolation in both treated males and females. However, the effects on cholesterol and the liver appear to be reversible after a 52 week recovery period.

REFERENCE Thomford, PJ. ( 2000). 26 Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt ( PFOS; T6295) in Cynomolgus Monkeys. Unaudited Draft Final Report Prepared for 3M, St Paul, Minnesota by Covance Laboratories, Inc., Madison Wisconsin 53704 2595. April 12, 2000. 502 pp.

Seacat, AM. Analytical Laboratory Report from the 26 Week Capsule Toxicity Study with Perfluoroctanesulfonic Acid Potassium Salt ( T 6295) in Cynomolgus Monkeys on the Determination of the Presence and Concentration of Perfluorooctanesulfonate ( PFOS) in Liver and Serum Samples. 3M Medical Department Study: T 6295.7; Covance In Life Study:# 6329 223. Analytical Study; FACT TOX 030; 3M Laboratory Request No. U2279. ENV/ JM/ RD( 2002) 17/ FINAL 306 DEVELOPMENTAL TOXICITY STUDIES Title: Oral Teratology Study of FC 95 in Rats  Experiment No. 0680TR0008 TEST SUBSTANCE Identity: Potassium Perfluorooctylsulfonate, CAS No. 2795 39 3 Remarks: FC 95, Lot 640.

METHOD Method/ Guideline followed ( i. e., OECD 414, etc.): Actual guideline followed was not specified but appears to be similar in design to OECD 414.

GLP ( Y/ N): The procedure complies with the general recommendations of the FDA issued in January, 1966 (" Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use"). The study was conducted according to the 1978 Good Laboratory Practice regulations and Safety Evaluation Laboratory's Standard Operating Procedures.

Year study performed: 1980 Species/ Strain: Sprague Dawley rats Number of animals per dose: 22 Route of administration: Gavage Dosing regimen ( list all with units): Four groups of 22 time mated Sprague Dawley rats were administered Potassium Perfluorooctylsulfonate in corn oil by gavage on gestation days 6 15. Doses were adjusted according to the most recent recorded body weight.

Doses: 0, 1, 5, and 10 mg/ kg/ day Statistical methods used: The animals will be assigned cages according to a computer generated random numbers table. The statistical methods to be used for analysis of the data are: Dunnett's t test for dam and pup weights, number of fetuses, number of resorption sites, number of implantation sites and number of corpora lutea; Chi square for percent abnormalities.

Remarks   Detail and discuss any significant protocol parameters and deviations: Potassium Perfluorooctylsulfonate was administered in corn oil by gavage to four groups of 22 time mated Sprague Dawley rats weighing 175 261g, at doses of 0, 1, 5, and 10 mg/ kg/ day PFOS on days 6 15 of gestation ( Gortner, 1980). Purina Laboratory Chow and water were available ad libitum. The animals were dosed according to a constant dose volume of 5 ml/ kg of body weight and observed daily from day 3 through day 20 of gestation for abnormal clinical signs. Body weights were recorded on days 3, 6, 9, 12, 15, and 20 of gestation and the rats. All animals were sacrificed on day 20 by cervical dislocation and the ovaries, uteri and contents were examined for the number of corpora lutea, number of viable and non viable fetuses, number of resorption sites, and number of implantation sites. Fetuses were weighed and sexed and subjected to external gross necropsy. Approximately one third of the fetuses were fixed in Bouin's solution and examined for visceral abnormalities by free hand sectioning by the Wilson technique. The remaining fetuses were subjected to a ENV/ JM/ RD( 2002) 17/ FINAL 307 skeletal examination using alizarin red.

RESULTS NOAEL ( dose and effect)   maternal and developmental: A NOAEL of 5 mg/ kg/ day for maternal toxicity was indicated. No signs of maternal toxicity were reported at doses of 5 mg/ kg/ day and below. A NOAEL for developmental toxicity could not be established; signs of developmental toxicity were evident at all doses.

LOAEL ( dose and effect)   maternal and developmental: A LOAEL of 10 mg/ kg/ day for maternal toxicity was indicated based on significant reductions in mean body weights during gestation day 12 20 at the high dose group of 10 mg/ kg/ day. A LOAEL of 1 mg/ kg/ day for developmental toxicity was indicated based on abnormalities of the lens of the eye.

Toxic response/ effects by dose level  maternal: Significant reductions in mean body weights during GD 12 20 at the high dose group of 10 mg/ kg/ day.

Toxic response/ effects by dose level  developmental: Unusually high incidences of developmental variations and abnormalities of the lens of the eye were observed in all dose groups.

Statistical results: At 10 mg/ kg/ day, mean maternal body weights were statistically significantly lower than controls ( Dunnett's test p < 0.05). Mean litter data and pup weights were not significantly different from controls ( Dunnett's t test p< 0.05). Number of fetuses with gross findings were not significantly different from controls ( Chi square p< 0.05). Number and percent of fetuses with skeleton findings were not significantly different from controls ( Chisquare p< 0.05). Number and percent of fetuses with internal findings   developmental lens abnormalities with secondary lens aberrations were significantly higher than controls ( Chi square p< 0.05).

Remarks   Additional information to adequately assess the data: Signs of maternal toxicity consisted of significant reductions in mean body weights during GD 12 20 at the high dose group of 10 mg/ kg/ day. No other signs of maternal toxicity were reported. Developmental toxicity evident at doses of 10 mg/ kg/ day consisted of reductions in the mean number of implantation sites, corpora lutea, resorption sites and the mean numbers of viable male, female, and total fetuses, but the differences were not statistically significant. In addition, unusually high incidences of unossified, assymetrical, bipartite, and missing sternebrae were observed in all dose groups; however, these skeletal variations were also observed in control fetuses at the same rate and therefore were not considered to be treatment related. The most notable sign of developmental toxicity observed in all dose groups consisted of abnormalities of the lens of the eye, which was not seen in controls. The proportion of fetuses with the lens abnormality in one or both lenses was significantly higher in the high dose group. All eye abnormalities appeared to be localized to the area of the embryonal lens nucleus, although a variety of morphological appearances were present within that location. According to the authors, this abnormality appeared to be an arrest in development of the primary lens fibers forming the embryonal lens nucleus. Secondary lens fiber development progressed normally except immediately surrounding the abnormal embryonal nucleus. An amendment to the results and discussion section concludes that the gross finding of a lens cleft was an artifact created by freehand sectioning and the range of gross lens observations and the differences among the dose group incidences were due to the maner and frequency in which the lens cleft artifact was created by free hand sectioning and the limitations inherent in visualizing the embryonal nucleus. Additionally, a subsequent study ( Wetzel, 1893) of similar design was not able to repeat this finding. ENV/ JM/ RD( 2002) 17/ FINAL 308 CONCLUSIONS Given the explanation that the eye abnormalities reported at doses as low as 1 mg/ kg/ day in this study may have been due to an artifact of sectioning and that these findings could not be repeated in a second study of similar design, it is entirely plausible that the lens defect observed in this study is not treatment related.

REFERENCE Gortner, E. G. 1980. Safety Evaluation Laboratory and Riker Laboratories, Inc. Experiment Number: 0680TR0008, December, 1980. " Oral Teratology Study of FC 95 in Rats". ENV/ JM/ RD( 2002) 17/ FINAL 309 Title: Rat Teratology Study T 3351 Final Report   Project No. 154 160 TEST SUBSTANCE Identity: Potassium Perfluorooctylsulfonate, CAS No. 2795 39 3 Remarks: T 3351, Lot No. 80275 METHOD Method/ Guideline followed ( i. e., OECD 414, etc.): Actual guideline followed was not specified but appears to be similar in design to OECD 414.

GLP ( Y/ N): Quality Assurance inspections of the study and review of the final report were conducted according to the standard operating procedures of the Office of Quality Assurance and according to the general requirements of the Good Laboratory Practice regulations that were issued on December 22, 1978, by the Food and Drug Administration for compliance on and after June 20, 1979.

Year study performed: 1983 Species/ Strain: Sprague Dawley rats Number of animals per dose: 25 Route of administration: Gavage Dosing regimen ( list all with units): Four groups of 25 pregnant Sprague Dawley rats were administered Potassium Perfluorooctylsulfonate in corn oil by gavage on gestation days 6 15. Doses were adjusted according to the most recent recorded body weight.

Doses: 0, 1, 5, and 10 mg/ kg/ day Statistical methods used: Statistical methods used for analysis of the data : Dunnett's t test for control vs. compound treated group mean comparisons. If the variances were proved to be homogeneous, the data were analysed by one way classification analysis of variance ( ANOVA). Mean fetal body weights per litter were statistically analysed as follows: Bartlett's test for homogeneity of variances was performed by oneway classification of covariance ( ANCOVA).

If ANCOVA was significant, control vs. treatment group comparisons were analysed using the Games and Howell modification of the Tukey Kramer honestly significant difference test. Tests for homogeneity of variances, ANOVA, and ANCOVA were evaluated at the 5% one tailed probability level. Control vs. compound treated group mean comparisons of the above data were evaluated at the 5% two tailed probability level. Percent fetal viability, percent fetal loss ( dead and resorbing fetuses), percent early, late, and total resorptions, and the number of dead fetuses were analysed by nonparametric one way ANOVA and the Terpstra Jonckheere test for trend. The litter was used as the experimental unit. Teratology data were analysed using the Cochran Armitage test for linear trend in proportions. If a significant trend was noted, the results of Fisher's " exact" test were evaluated at the one tailed, 5% level. If a significant trend was not observed, or if there was a significant trend with severe departure from it, the results of Fisher's " exact" test were evaluated at the two tailed, 5% level. ENV/ JM/ RD( 2002) 17/ FINAL 310 Remarks   Detail and discuss any significant protocol parameters and deviations: Potassium perfluorooctylsulfonate was administered in corn oil by gavage to four groups of 25 pregnant Sprague Dawley rats at doses of 0, 1, 5, and 10 mg/ kg/ day PFOS on gestation days ( GD) 6 15 ( Wetzel, 1983). Sexually mature Sprague Dawley rats, one per sex per cage, were paired until confirmation of mating or until two weeks had elapsed. Mating was confirmed by daily vaginal examinations for the presence and viability of sperm or the presence of a copulatory plug. The day of confirmation of mating was designated as day 0 of gestation. Purina Rodent Laboratory Chow 5001 and tap water were available ad libitum. A dose volume of 3 ml/ kg of body weight was administered and doses were adjusted according to the most recently recorded body weight measurements. Dams were observed twice daily for signs of mortality and moribundity and once daily for clinical signs of toxicity. Individual body weights and food consumption were recorded on GD 6, 8, 12, 16, and 20. Animals were sacrificed on GD 20 by CO2 asphyxiation and the fetuses were delivered by cesarean section on GD 20. A gross necropsy was performed on all dams. The uterus from each female was excised, weighed and examined for the number and placement of implantation sites, number and of live and dead fetuses, number of early and late resorptions, and any abnormalities and then weighed again after the contents were removed. The ovaries were examined for the number of corpora lutea. Each female was examined by gross necropsy. Each fetus was sexed, weighed, and examined externally. Approximately one third of the fetuses were fixed in Bouin's solution and examined for visceral abnormalities by the Wilson technique, with particular attention to the eyes, palate, and brain. The remaining fetuses were subjected to a skeletal examination that included evaluation of the skull, long bones, vertebral column, rib cage, extremities, and pectoral and pelvic girdles using alizarin red; bone alignment and degree of ossification were also evaluated.

RESULTS NOAEL ( dose and effect)   maternal and developmental: The NOAEL for maternal toxicity is 1 mg/ kg/ day. The NOAEL for developmental toxicity is 1 mg/ kg/ day.

LOAEL ( dose and effect)   maternal and developmental: The LOAEL for maternal toxicity is 5 mg/ kg/ day, based on clinical signs of toxicity, decreases in body weight and food consumption, decreases in uterine weights, and an increased incidence in gastrointestinal lesions.

The LOAEL for developmental toxicity is 5 mg/ kg/ day, based on decreased fetal body weight and increases in external and visceral anomalies and variations.

Toxic response/ effects by dose level  maternal: Clinical signs of toxicity, decreases in body weights and food consumption at 5 and 10 mg/ kg/ day; decreases in uterine weights, increased incidence in gastrointestinal lesions, and two deaths at 10 mg/ kg/ day.

Toxic response/ effects by dose level  developmental: Decreased fetal weight at 5 and 10 mg/ kg/ day; external and visceral anomalies and skeletal variations at 10 mg/ kg/ day.

Statistical results: Statistically significant differences between controls and treated were noted for the following maternal endpoints: mean body weight gain, mean total food consumption, and mean gravid uterine weight. Nonparametric analysis of the mean incidence of late resorptions, total resorptions, number of dead fetuses, and fetal loss did not indicate statistical significance; however, there was a significant linear trend towards an increased incidence in these data with respect to control. The primary trend component was contributed by the high dose group. Statistically significant treatment related increases in the incidences of visceral anomalies and skeletal variants were also observed. ENV/ JM/ RD( 2002) 17/ FINAL 311 Remarks   Additional information to adequately assess the data: Evidence of maternal toxicity, that was observed at the 5 and 10 mg/ kg/ day dose groups both during and following treatment and considered to be treatment related, consisted of hunched posture, anorexia, bloody vaginal discharge, uterine stains, alopecia, rough haircoat, and bloody crust. Significant decreases in mean body weight gains during GD 6 8, 6 16, and 0 20 were also observed at the 5 and 10 mg/ kg/ day dose groups. These reductions were considered to be treatment related since mean body weight gains were greater than controls during the post exposure period ( GD 16 20). Significant decreases in mean total food consumption were observed on GD 17 20 in the10 mg/ kg/ day dose group, and on GD 7 16 and 0 20 in both the 5 and 10 mg/ kg/ day dose groups. The mean gravid uterine weight in the 10 mg/ kg/ day dose group was significantly lower when compared with controls. The mean terminal body weights minus the gravid uterine weights were lower in all treated groups, with significant decreases at 5 and 10 mg/ kg/ day. High dose animals also exhibited an increased incidence in gastrointestinal lesions. No significant differences were observed in pregnancy rates, number of corpora lutea, and number and placement of implantation sites among treated and control groups. Two dams in the 10 mg/ kg/ day dose group were found dead on GD 17.

Signs of developmental toxicity included a dose related trend toward an increased incidence of late resorptions, total resorptions, number of dead fetuses, and fetal loss, although, none of these effects were statistically significantly different from controls. Significant decreases in mean fetal weights for both males and females were observed in the 5 and 10 mg/ kg/ day dose groups. The percent of male fetuses was 52%, 54%, and 60% for 1, 5, and 10 mg/ kg/ day, respectively, compared to 44% in controls. Statistically significant increases in incomplete closure of the skull were observed in the low and high dose groups but not in the mid dose group. Statistically significant increases in the incidences in the number of litters containing fetuses with visceral anomalies, delayed ossification, and skeletal variations were observed in the high dose group of 10 mg/ kg/ day. These included external and visceral anomalies of the cleft palate, subcutaneous edema, and cryptorchsm as well as delays in skeletal ossification of the skull, pectoral girdle, rib cage, vertebral column, pelvic girdle and limbs. Skeletal variations in the ribs and sternebrae were also observed at this dose level.

CONCLUSIONS The developmental eye abnormalities that were seen in Gortner ( 1980) were not observed in the present developmental toxicity study even though the study design and doses were the same. Findings of abnormalities in eye development were initially thought to be treatment related but then later suggested as being artifacts of sectioning.

REFERENCE Wetzel, L. T. 1983. Hazelton Laboratories America, Inc. Project Number: 154 160, December 19, 1983. " Rat Teratology Study, T 3351, Final Report". ENV/ JM/ RD( 2002) 17/ FINAL 312 Title: ORAL ( STOMACH TUBE) DEVELOPMENTAL TOXICITY STUDY OF PFOS IN RABBITS   3M T 6295.10, ARGUS RESEARCH LABORATORIES STUDY NUMBER: 6295.10, 1999.

TEST SUBSTANCE Identity: Potassium Perfluorooctylsulfonate ( PFOS), CAS No. 2795 39 3 Remarks: PFOS   Lot 217, 98.4% pure ( SMD Analytical Request 53030) Analytical Documentation filed along with final report. Note: Same lot as used in two year rat PFOS carcinogenicity study ( T 6295, Covance 6329 183).

METHOD Method/ Guideline followed ( i. e., OECD 414, etc.): The requirements of the International Conference on Harmonization ( ICH) Harmonized Tripartite Guideline on Detection of Toxicity to Reproduction for Medicinal products, stages C and D of the reproductive process in a non rodent species were used as the basis for study design ( U. S. Food and Drug Administration, 1994. Federal Register, September 22, 1194, Vol. 59, No. 183).

GLP ( Y/ N): The study was conducted in compliance with the Good Laboratory Practice ( GLP) regulations of the U. S. Food and Drug Administration ( FDA), the Japanese Ministry of Health and Welfare ( MHW) and the European Economic Community ( EEC). There were no significant deviations from the GLP regulations that affected the quality or integrity of the study.

Year study performed: 1999 Species/ Strain: New Zealand White rabbits Number of animals per dose: 22 ( additional total of 19 rabbits, 3 5 per dose, were included in a satellite study to determine the concentrations of PFOS in maternal liver and serum.)

Route of administration: Gavage Dosing regimen ( list all with units): Four groups of 22 pregnant New Zealand White rabbits were administered Potassium Perfluorooctylsulfonate ( PFOS) in 0.5% Tween 80 by gavage on gestation days 7 20. A dose volume of 5 ml/ kg was administered, adjusted daily on the basis of individual body weights.

Doses: 0, 0.1, 1.0, 2.5, and 3.75 mg/ kg/ day ( In the satellite study the number of does was 3, 5, 3, 3 and 5 in the control, 0.1, 1.0, 2.5 and 3.75 mg/ kg/ day groups, respectively).

Statistical methods used: The animals will be assigned to individual housing on the basis of computer generated random units. The litter was the unit of measurement. Clinical observation and other proportion data were analysed using the Variance Test for Homogeneity of the Binomial Distribution. Continuous data ( e. g., maternal body weights, body weight changes, feed consumption values and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights, fetal anomaly data and fetal ossification site data) were analyzed using Bartlett's Test of Homogeneity of Variances and the Analysis of Variance. If the Analysis of Variance was significant, Dunnett's Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate, theKruskal Wallis Test was used. In cases, in which Kruskal Wallis Test was statistically significant ( p< 0.05), Dunn's Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. Count data obtained ENV/ JM/ RD( 2002) 17/ FINAL 313 at Caesarean sectioning were evaluated using the procedures described for the Kruskal Wallis Test.

Remarks   Detail and discuss any significant protocol parameters and deviations: Timed pregnant New Zealand White rabbits ( obtained from Covance Research Products, Inc.), 22 per group, were given doses of 0, 0.1, 1.0, 2.5 or 3.75 mg/ kg/ day PFOS in 0.5% Tween 80 by gavage on gestation days 7 20. A dose volume of 5 mL/ kg was administered, adjusted daily on the basis of individual body weights. The does were observed twice daily for viability, and clinical observations were recorded 1 hour prior to and after dosing during the treatment period and once daily during the post treatment period ( i. e. gestation days 20 29). Maternal body weights were recorded on gestation days 0 and 6 29; food consumption was recorded daily throughout the study. On gestation day 29, the does were euthanized; a gross necropsy of the thoracic, abdominal and pelvic viscera was conducted and the number of corpora lutea in each ovary was recorded. The uteri were examined for number and distribution of implantations, live and dead fetuses, and early and late esorptions. The fetuses were weighed, sexed and examined for external abnormalities. All fetuses were examined for visceral and skeletal abnormalities and the brain of one half of the fetuses were free hand cross sectioned and examined in situ. In the satellite study, the does were euthanized on gestation day 21, blood samples were collected, and the liver was weighed and sectioned. The fetuses were removed and examined for external abnormalities. Fetuses and placentae were pooled per litter. All samples were sent to the Sponsor ( 3M) for analysis. At this time, only the liver and serum analyses have been Reported ( 3M Environmental Laboratory, 2001).

RESULTS NOAEL ( dose and effect)   maternal and developmental: The NOAEL for maternal toxicity is 0.1 mg/ kg/ day. The NOAEL for developmental toxicity is 1.0 mg/ kg/ day.

LOAEL ( dose and effect)   maternal and developmental: The LOAEL for maternal toxicity is 1.0 mg/ kg/ day, based on abortions, incidences of scant feces, and decreases in body weight gains and food consumption. The LOAEL for developmental toxicity is 2.5 mg/ kg/ day, based on reductions in body weight and increased incidences in fetal alterations.

Toxic response/ effects by dose level  maternal: Maternal toxicity was evident at dose levels of 1.0 mg/ kg/ day and above and consisted of the following: abortions at 2.5 mg/ kg/ day and above occurring on GD 22 28; increased incidence of scant feces at 1.0 mgkgday and above; reductions in mean body weight and body weight gain at doses of 1.0 mg/ kg/ day and above; reductions in absolute and relative food consumption at 2.5 mg/ kg/ day and above.

Toxic response/ effects by dose level   developmental: Developmental toxicity was evident at doses of 2.5 mg/ kg/ day and above and consisted of the following: reductions in mean fetal body weight at 2.5 mg/ kg/ day and above; delayed ossification at 2.5 mg/ kg/ day and above.

Statistical results:

Maternal data: Statistically significant increases in abortions were observed at 3.75 mg/ kg/ day. Incidences of scant feces at 3.75 mg/ kg/ day reached statistical significance ( p< 0.01). Maternal body weight gains were reduced or body weight losses occurred in the 1.0, 2.5, and 3.75 mg/ kg/ day dosage groups at most measured intervals during dosing; these reductions were significant ( p< 0.05 or 0.01) in the 3.75 mg/ kg/ day dose group on GDs 10 13, 13 16, and 16 19. Dosage dependent, significant body weight reductions or body weight losses ( p< 0.05 or 0.01) occurred in the 1.0, 2.5, and 3.75 mg/ kg/ day dosage groups for the entire dosage period ( calculated as GD 7 21). Dosage dependent reductions in body weight gains occurred in the 2.5 and 3.75 mg/ kg/ day dosage groups for the entire period of gestation ( GD 0 29) ENV/ JM/ RD( 2002) 17/ FINAL 314 and for the gestation period after the initiation of dosing ( GD 7 29; significant at p< 0.01 in the 2.5 mg/ kg/ day dosage group). Average body weights were significantly reduced ( p< 0.05 or 0.01) on GD 17 24 in the 3.75 mg/ kg/ day dosage group. Absolute and relative food consumption values were significantly reduced ( p< 0.05 or 0.01) in the 2.5 and 3.75 mg/ kg/ day dosage groups for the entire dosage period ( GD 7 21), and the entire period after the initiation of dosage ( GD 7 29).

Fetal data: Fetal body weights ( total, male and female) were significantly reduced ( p< 0.05 and p< 0.01, respectively) in the 2.75 and 3.75 dosage groups. Significant delays ( p< 0.05 and 0.01) in litter and fetal averages for ossification were seen at both 2.5 and 3.75 mg/ kg/ day dosage groups.

Remarks: Maternal toxicity was evident at doses of 1.0 mg/ kg/ day and above. One doe in the 2.5 mg/ kg/ day group and nine does in the 3.75 mg/ kg/ day aborted. All abortions occurred on gestation days 22 28 and were considered treatment related by the study authors. There was a significant increase in the incidence of scant feces in the 3.75 mg/ kg/ day group. Scant feces were also noted in one and three does in the 1.0 and 2.5 mg/ kg/ day groups, respectively. Mean maternal body weight gains were significantly reduced in the 3.75 mg/ kg/ day group on gestation days 10 13, 13 16, 16 19 and 21 24. Mean body weight gains were also calculated for the treatment period ( days 7 21), post treatment period ( days 21 29) and duration of the study ( days 7 29). There was a significant reduction in mean maternal body weight gain during the treatment period in the 1.0, 2.5 and 3.75 mg/ kg/ day groups. Mean body weight gain for the entire study period was also significantly reduced in the 2.5 mg/ kg/ day group. Mean food consumption ( g/ kg/ day) was significantly reduced in the 2.5 mg/ kg/ day group on gestation days 16 19, 19 21 and 21 24, as well as for the entire study period ( days 7 29). Mean food consumption was significantly reduced in the 3.75 mg/ kg/ day group on gestation days 13 16, 16 19, 19 21 and 21 24, as well as the entire treatment period ( GD 7 21) and the entire period after the initiation of dosage ( GD 7 29). Pregnancy occurred in 20 ( 90%), 19 ( 86.4%), 19 ( 86.4%), 17 ( 77.3%), and 21 ( 95.4%) rabbits in each dosage group. Ceasarean sectioning observations on GD 29 were based on 20, 18, 19, 16, and 12 pregnant rabbits in each of the five respective dosage groups.

In the satellite study of does euthanized on gestation day 21, the liver and serum analyses were reported by 3M Environmental Laboratory ( 2001). All serum and liver samples ( including those from untreated controls) had detectable levels of PFOS; the values are presented below. Data from analysis of fetal and placenta tissues were not presented in the report.

Average Concentration of PFOS in Rabbit Liver and Serum by Dose Group: Dose group PFOS conc. PFOS conc. ( mg/ kg/ day) liver ( ug/ g) serum ( ug/ ml)

0.0 0.239 0.0690 0.1 13.1 2.73 1.0 133 23.8 2.5 317 45.8 3.75 416 88.9 Qualitatively, increasing concentrations of PFOS were found in samples of liver and serum as doses of PFOS increased. The levels of PFOS are much higher in the liver than in the serum.

These values should be viewed with caution. It was stated that because radio labeled reference material was not available, " it is not possible to verify true recovery of endogenous analyte from tissues." Matrix spike recovery indicates the accuracy of quantitation to be +/ 30%. It is also noted that liver concentrations may be biased high. The only conclusion presented in the laboratory report is that " PFOS ENV/ JM/ RD( 2002) 17/ FINAL 315 was observed in the liver and serum of all rabbits dosed with the test article."

Developmental toxicity was evident at doses of 2.5 mg/ kg/ day and above. The number of corpora lutea, resorptions, live/ dead fetuses, litter size and sex ratio were comparable among treated and control groups. Mean fetal body weight ( male, female and sexes combined) was significantly reduced in the 2.5 and 3.75 mg/ kg/ day groups. There was also a significant reduction in the ossification of the sternum ( litter averages) in the 2.5 and 3.75 mg/ kg/ day groups, and a significant reduction in the ossification of the hyoid ( litter averages), metacarpals ( litter averages) and pubis ( litter and fetal averages) in the 3.75 mg/ kg/ day group. Other fetal gross external, soft tissue and skeletal alterations ( malformations and variations) were considered unrelated to treatment because the incidences were not dosage dependent and/ or were within historical control range.

CONCLUSIONS Conclusions are summarized above and this reviewer agrees.

REFERENCE Christian, M. S., Hoberman, A. M., and York, R. G. 1999. Argus Research Laboratories, Inc. Protocol Number: 418 012, January 1999. " Oral ( Stomach Tube) Developmental Toxicity Study of PFOS in Rabbits".

3M Environmental Laboratory. 2001. Analytical Laboratory Report, FACT TOX 099, February 9, 2001. " Determination of the Concentration of Potassium Perfluorooctanesulfonate ( PFOS) in Rabbit Liver and Serum Samples." ENV/ JM/ RD( 2002) 17/ FINAL 316 REPRODUCTIVE TOXICITY STUDIES Title: COMBINED ORAL ( GAVAGE) FERTILITY, DEVELOPMENTAL AND PERINATAL/ POSTNATAL REPRODUCTION TOXICITY STUDY OF PFOS IN RATS   ARGUS RESEARCH LABORATORIES STUDY NUMBER: 6295.9, 1999.

TEST SUBSTANCE Identity: Potassium Perflurooctylsulfonate, CAS No. 2795 39 3.

Remarks: The test article, FC 95 ( lot 217), was received on May 20, 1998, and stored at room temperature. Prepared suspensions were stored at room temperature overnight. Information regarding the purity, identity, strength and composition of the test article is on file with the Sponsor.

METHOD Method/ Guideline followed ( i. e., OECD 414, etc.): This study was designed to evaluate ICH Harmonized Tripartite Guideline stages A F. A modification of the requirements of the U. S. Food and Drug Administration ( FDA) were used as a basis for the study design.

Type of study ( one generation, two generation, etc.): Two generation reproductive toxicity GLP ( Y/ N): The study was conducted in compliance with the Good Laboratory Practice ( GLP) regulations of the U. S. Food and Drug Administration ( FDA), the Japanese Ministry of Health and Welfare ( MHW) and the European Economic Community ( EEC). There were no significant deviations from the GLP regulations that affected the quality or integrity of the study. Quality Assurance Unit findings derived from the inspections during the conduct of this study have been documented.

Year study performed: 1999 Species/ Strain: Sprague Dawley rats Sex ( males/ females/ both): Both Number of animals per dose: 35 Route of administration: Gavage Dosing regimen ( list all with units): Five groups of 35 rats per sex per dose group were administered PFOS by gavage for six weeks prior to and during mating. Treatment in male rats continued until one day before sacrifice ( approximately 22 days total); female rats were treated throughout gestation, parturition, and lactation.

Doses: 0, 0.1, 0.4, 1.6, and 3.2 mg/ kg/ day Premating exposure period for males/ females ( P and F1, if appropriate): Six weeks for P; Nine and a half weeks for F1.

Statistical methods used: Proportion data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution. Continuous data ( body weights, body weight changes, and feed consumption) were analyzed using Bartlett's Test of Homogeneity of Variance and Analysis of Variance ( ANOVA). If the ENV/ JM/ RD( 2002) 17/ FINAL 317 ANOVA was significant ( p < 0.05), Dunnett's Test was used to identify the statistical significance of the individual groups. If the ANOVA was not appropriate, the Kruskal Wallis Test was used. In cases where the Kruskal Wallis Test was statistically significant ( p < 0.05), Dunn's Method of Multiple Comparisons was used to identify the statistical significance of the individual groups.

If there were greater than 75% ties, Fisher's Exact Test was used. Fisher's Exact Test was also used to evaluate necropsy data for the pups which were stillborn or found dead. Data obtained at Ceasareansectioning natural delivery, preweaning reflex/ physical developmental data and postweaning behavorial data involving discrete data ( number of corpora lutea, number of pups per litter, trials to a criterion) were evaluated by the Kruskal Wallis Test.

Remarks   Detail and discuss any significant protocol parameters and deviations:

F0 Generation:

Parental animals ( F0) were observed twice daily for clinical signs. Body weights and food consumption values were recorded weekly during the treatment period in male rats; and weekly during mating and then daily during gestation, and on lactation days 1, 4, 7, 10, 14, and at sacrifice in female rats. Each dosage group consisted of two sets of female rats. One set consisted of the first ten female rats with confirmation of mating; this group was dosed until gestation day ( GD) 10 and delivered via Caesarean sectioning. The remaining females comprised the second set which delivered naturally. During the 21 day lactation period, the dams were evaluated for clinical signs during parturition and length of gestation, and then each litter was evaluated at least twice daily for size and pup viability at birth. Pup observations during the 21 day lactation period included physical signs, body weights, nursing behavior, surface righting reflex, pinna unfolding, eye opening, acoustic startle response and air righting reflex. Pupil constriction was evaluated only on lactation day 21. On lactation day 4, litters were randomly culled to four male and four female pups. The remaining pups were sacrificed and necropsied. The F0 male rats were sacrificed and necropsied after the end of dosing at the time of parturition ( lactation day 1). The testes, epididymides, prostate, and seminal vesicles were weighed. Evaluations of sperm number, motility, and morphology were not included in the protocol. The F0 generation females that delivered by Caesarean sectioning were sacrificed on GD 10 and necropsied. Pregnancy status was confirmed, the ovaries were examined for the number and distribution of corpora lutea, implantation sites were determined, and embryos were examined for viability. The F0 generation females that delivered naturally were sacrificed on lactation day ( LD) 21 and necropsied. Ovaries were examined as above and the number and distribution of implantation sites was recorded. The liver from each parental rat was removed, weighed and analyzed. Blood samples were collected from 5 male rats that had mated and from 5 female rats on LD 21 for pharmacokinetic analysis; livers from the pups from the litters of these five dams were also collected for analysis.

F1 Generation:

Since F1 generation pup viability was significantly reduced in the 1.6 and 3.2 mg/ kg/ day dosages groups, only the 0.1 and 0.4 mg/ kg/ day dosage groups were carried into the second generation. Twenty five F1 generation rats per sex per dose group were administered PFOS by gavage at doses of 0, 0.1, and 0.4 mg/ kg/ day beginning on LD 22 and continuing through the day before sacrifice. At 24 days of age, one rat per sex per litter in each dosage group was tested in a passive avoidance paradigm. On LD 28, females were evaluated for the age of vaginal patency and on LD 34, male rats were evaluated for the age of preputial separation. One rat per sex per litter were evaluated in a water filled M maze on LD 70. Assignment to cohabitation within each dosage group began on LD 90. Females with evidence of mating were considered to be at GD 0 and assigned to individual housing for the remainder of the dosing period. The F1 generation male rats were sacrificed after mating, necropsied and evaluated as described in the F0 generation. All F1 generation females were allowed to deliver naturally. Dams that delivered litters were ENV/ JM/ RD( 2002) 17/ FINAL 318 sacrificed and necropsied on LD 21. All F2 generation pups were sacrificed, necropsied, and examined on LD 21 as previously described for the F1 generation pups.

RESULTS NOAEL ( dose and effect)   for F0, F1, and F2 ( as appropriate): The NOAEL for the F0 generation male and female parental animals = 0.1 mg/ kg/ day, the lowest dose tested. The NOAEL for the F1 generation male parental animals could not be determined since treatment related signs of toxicity were observed at 0.1 and 0.4 mg/ kg/ day; the NOAEL for the F1 generation female parental animals = 0.1 mg/ kg/ day. The NOAEL for the F1 generation offspring = 0.4 mg/ kg/ day. The NOAEL for the F2 generation offspring = 0.1 mg/ kg/ day.

LOAEL ( dose and effect)   for F0, F1, and F2 ( as appropriate): The LOAEL for the F0 generation male and female parental animals = 0.4 mg/ kg/ day, based on reductions in body weight gain and food consumption. The LOAEL for the F1 generation male animals = 0.1 mg/ kg/ day based on significant reductions in absolute food consumption; the NOAEL for the F1 generation female animals = 0.4 mg/ kg/ day based on reductions in body weight and food consumption. The LOAEL for the F1 generation offspring = 1.6 mg/ kg/ day, based on significant reductions in the number of implantation sites, litter size, pup viability, growth and survival. The LOAEL for the F2 generation offspring = 0.4 mg/ kg/ day, based on significant reductions in pup growth.

Toxic response/ effects by dose level   parental/ F1: Toxic effects in F0 generation animals: reductions in both body weight gains and in absolute and relative food consumption at the 1.6 and 3.2 mg/ kg/ day dosage groups during the pre mating period. Following mating, food consumption was significantly reduced in the 0.4. and 1.6 mg/ kg/ day dosage groups. Terminal body weights were also significantly reduced in the 1.6 and 3.2 mg/ kg/ day dose groups. Signs of reproductive toxicity in the F0 generation males were seen at the highest dose group of 3.2 mg/ kg/ day and included significant reductions in the absolute weights of the seminal vesicles ( with fluid) and the prostate. A significant increase in the number of males with brown liver at 3.2 mg/ kg/ day dose group was also reported. The only findings reported in the F0 dams occurred in the 0.4, 1.6, and 3.2 mg/ kg/ day dosage groups and included localized alopecia during pre mating, gestation, and lactation; and reductions in body weight and food consumption values observed during the pre mating period and continuing throughout gestation and lactation.

Toxic effects in F1 generation animals: F1 males; the only reported effects were significant reductions in absolute food consumption on postweaning days 1 8 occurring at the 0.1 and 0.4 mg/ kg/ day dose levels. F1 females; observations at the 0.4 mg/ kg/ day dosage group included, reductions in body weights on day 1 postweaning, significant losses in body weight on LDs 1 4, and significant reductions in food consumption on days 1 8 postweaning and during lactation.

Toxic response/ effects by dose level   offspring ( F1/ F2):

Toxic effects in the F1 generation pups consisted of reduced pup viability at the two highest dosegroups ( 1.6 and 3.2 mg/ kg/ day). The reductions in pup viability began to appear on LD 4 postculling in the 1.6 mg/ kg/ day dose group, with over 26% of the pups found dead between LD 2 4. In the 3.2 mg/ kg/ day dose group 45% of the pups were found dead on LD1; no pups survived beyond LD 1. Statistically significant increases were observed in the number of dams with stillborn pups at the 3.2 mg/ kg/ day dose group. Viability and lactation indices were significantly reduced in these same dosage groups ( viability index = 0% at 3.2 mg/ kg/ day and 66% at 1.6 mg/ kg/ day; lactation index = 94.6% at 1.6 mg/ kg/ day). Statistically significant reductions in pup body weights were also observed at the two highest dosage groups. Gestation ENV/ JM/ RD( 2002) 17/ FINAL 319 length was significantly reduced at 3.2 mg/ kg/ day. Significant reduction in the number of implantation sites followed by a concomitant reduction in litter size was observed at 3.2 mg/ kg/ say. Other adverse signs in the 3.2 mg/ kg/ day dose level associated with reductions in pup viability and maternal care included litters with pups that were not nursing or who had no evidence of milk in the stomach, as well as maternal cannibalization of pups that were stillborn or found dead.

Toxic effects in the F2 generation pups consisted of reductions in mean pup body weights ( on a per litter basis) observed at 0.1 mg/ kg/ day on LD 4 and 7. At 0.4 mg/ kg/ day, statistically significant reductions in mean pup body weights were observed on LDs 7 14.

Statistical results: F0 generation male animals: Significant reductions ( p < 0.05 or p< 0.01) in body weight gains at 0.4 mg/ kg/ day and higher. Absolute and relative food consumption values were significantly reduced ( p < 0.05 or p < 0.01) in the 1.6 and 3.2 mg/ kg/ day dosage groups. A significant increase ( p < 0.01) in the number of male rats in the 3.2 mg/ kg/ day dosage group with brown liver. The gross lesions of the liver were considered to be treatment related because the incidences were dosage dependent. Significant reductions ( p < 0.05 and p < 0.01) in terminal body weights were observed in the 1.6 and 3.2 mg/ kg/ day dosage groups. Significant reductions ( p < 0.05 or p < 0.01) in the absolute weights of the seminal vesicles with fluid and the prostate were observed in the 3.2 mg/ kg/ day dosage group. F0 generation female animals: Significant increases ( p < 0.05 or p < 0.01) in localized alopecia were observed in the 0.4, 1.6, and 3.2 mg/ kg/ day dosages groups. Significant reductions in body weight and body weight gains and food consumption ( p < 0.05 or p < 0.01) were observed in the 1.6 and 3.2 mg/ kg/ day dosage during premating and gestation and then in 0.4 mg/ kg/ day dosage group and above during lactation. Significant reductions ( p < 0.01) in gestation length, implantation sites, and litter size were observed at 3.2 mg/ kg/ day. F1 generation offspring: Pup viability was significantly reduced ( p < 0.05 or p < 0.01) in the 1.6 and 3.2 mg/ kg/ day dosage groups. Significant increases ( p < 0.05 or p < 0.01) were observed in the number of dams with stillborn pups, while significant reductions ( p < 0.05 or p < 0.01) were observed in the viability index, lactation index, and averages for surviving pups at 3.2 mg/ kg/ day. A dosage dependent pattern of reduced pup body weight was evident in each dosage group, with statistical significance ( p < 0.01) in the 1.6 and 3.2 mg/ kg/ day dosage groups. F1 generation adult animals: Males  Significant reductions ( p < 0.05 or p < 0.01) in absolute food consumption at 0.1 and 0.4 mg/ kg/ day; females  significant ( p < 0.05) body weight loss on lactation days 1 4 at 0.4 mg/ kg/ day; and significant reductions ( p < 0.05) in food consumption at 0.4 mg/ kg/ day on days 1 8 postweaning. F2 generation offspring: Pup body weights tended to be reduced in the 0.1 mg/ kg/ day dosage group on lactation day 4 and 7, but were comparable to controls by lactation day 14. Pup body weights in the 0.4 mg/ kg/ day dosage group tended to be reduced, though not significantly, on lactation days 4 21, with significant reductions ( p < 0.05 and p < 0.01, respectively) on lactation days 7 and 14, as compared to controls.

Remarks: In the F0 generation male rats, there were no treatment related clinical signs of toxicity, no mortality, and no effects on mating or on any of the fertility parameters evaluated in any dose group tested. Reported effects included reductions in both body weight gains and in absolute and relative food consumption at the 1.6 and 3.2 mg/ kg/ day dosage groups during the pre mating period. Following mating, food consumption was significantly reduced in the 0.4 and 1.6 mg/ kg/ day dosage groups. Terminal body weights were also significantly reduced in the 1.6 and 3.2 mg/ kg/ day dose groups. Body weights, body weight gains, absolute and relative food consumption were unaffected by treatment at the 0.1 mg/ kg/ day dosage group. Signs of reproductive toxicity in the F0 generation males were seen at the highest dose group of 3.2 mg/ kg/ day and included significant reductions in the absolute weights of the seminal vesicles ENV/ JM/ RD( 2002) 17/ FINAL 320 ( with fluid) and the prostate. A significant increase in the number of males with brown liver at 3.2 mg/ kg/ day dose group was also reported.

In the F0 generation female rats, no deaths were reported at any dose level. In dams sacrificed on GD 10 for Caesarean sectioning, there did not appear to be any effects on estrous cycling, mating and fertility parameters, the numbers of corpora lutea and implantations, or in the number of viable or non viable embryos. The only findings reported in the F0 dams occurred in the 0.4, 1.6, and 3.2 mg/ kg/ day dosage groups and included localized alopecia during pre mating, gestation, and lactation; and reductions in body weight and body weight gain and food consumption values observed during the pre mating period and continuing throughout gestation and lactation.

Reversible delays in reflex and physical development were observed in the F1 generation offspring. The ability to surface right was significantly delayed in the 1.6 and 3.2 mg/ kg/ day dosage groups on LDs 3 10 ( delays in the 3.2 mg/ kg/ day dose group were observed on LD 1, after which there were no surviving pups remaining for further observation). By the end of the observation period, however, all surviving pups in the 1.6 mg/ kg/ day dosage group had the ability to surface right. There were no delays observed in the ability to surface right in dose groups < 0.4 mg/ kg/ day. Similar responses were seen for pinna unfolding and eye opening. Although there were transient delays seen with these signs of physical development across all dose groups, by the end of the observation period responses in pups were similar to controls. The time of development of the acoustic startle reflex and the ability to air right were both significantly reduced in the 1.6 mg/ kg/ day dosage group. No effects on these reflexes were observed in the low dose group of 0.1 mg/ kg/ day and only a transient delay ( on LD 16 only) in the ability to air right was seen in the 0.4 mg/ kg/ day group. At the end of lactation ( LD 21), all live pups in all dose groups ( 0, 0.1, 0.4, and 1.6 mg/ kg/ day) had pupil constriction response.

The most significant finding reported in the offspring was that of reduced pup viability at the two highest dose groups. The reductions in pup viability began to appear on LD 4 postculling in the 1.6 mg/ kg/ day dose group, with over 26% of the pups found dead between LD 2 4. In the 3.2 mg/ kg/ day dose group 45% of the pups were found dead on LD1; no pups survived beyond LD 1. As a result, the viability index was greatly reduced in these dosage groups ( 0% at 3.2 mg/ kg/ day and 66% at 1.6 mg/ kg/ day). The lactation index was also significantly reduced ( 94.6%) in the 1.6 dosage group. In addition, gestation length was significantly reduced in the high dose group and there also was a significant reduction in the number of implantation sites followed by a concomitant reduction in litter size. Statistically significant reductions in pup body weights were also observed at the two highest dosage groups. Other adverse signs in the 3.2 mg/ kg/ day dose level associated with reductions in pup viability and maternal care included litters with pups that were not nursing or who had no evidence of milk in the stomach, as well as maternal cannibalization of pups that were stillborn or found dead. The percentage of male pups was comparable across all dosage groups.

Since F1 generation pup viability was significantly reduced in the 1.6 and 3.2 mg/ kg/ day dosages groups, only the 0.1 and 0.4 mg/ kg/ day dosage groups were carried into the second generation.

Clinical observations in the F1 generation male rats appeared unremarkable. No treatment related deaths were reported and no statistically significant differences were reported for any of the following parameters: body weights/ body weight gains, average day of preputial separation; values for learning, short term retention, long term retention or response inhibition as evaluated by performance in a passive avoidance or watermaze performance paradigm; mating or fertility parameters; necroscopic examinations; absolute or relative weights for the right or left testis, seminal vesicles, right epididymis, or prostate; and terminal body weights. The only reported effects were significant reductions in absolute food consumption on postweaning days 1 8 occurring at the 0.1 and 0.4 mg/ kg/ day dose levels. ENV/ JM/ RD( 2002) 17/ FINAL 321 Clinical observations for the F1 generation females were likewise unremarkable. Observations at the 0.4 mg/ kg/ day dosage group included, reductions in body weights on day 1 postweaning, significant losses in body weight on LDs 1 4, and significant reductions in food consumption on days 1 8 postweaning and during lactation. There were no statistically significant differences reported for any of the following parameters: values for learning, short term retention, long term retention or response inhibition as evaluated by performance in a passive avoidance or watermaze performance paradigm; mating and fertility parameters; gestation index; pregnancy rates; and necroscopic examinations.

Evidence of treatment related effects in the F2 generation pups consisted of reductions in mean pup body weights ( on a per litter basis) observed at 0.1 mg/ kg/ day on LD 4 and 7. Body weights were comparable to control levels by LD 14. At 0.4 mg/ kg/ day, statistically significant reductions in mean pup body weights were observed on LDs 7 14. Mean body weights on LD21 continued to remain lower than controls, although the difference was not statistically significant ( 46.5 g in 0.4 mg/ kg/ day dose group vs. 50 g in controls). Clinical and necroscopic observationsof the F2 generation pups were unremarkable. No other toxicologically significant effects were reported.

Liver and sera samples collected from the initial population of dosed animals ( F0) and their offspring ( F1) were analyzed for the presence of PFOS. The F0 results for the F0 animals were:

Dose group Average PFOS conc. Average PFOS conc. ( mg/ kg/ day) in serum ( ug/ ml) in liver ( ug/ g)

0.0: female 0.0307 female 0.171 male 0.0244 male 0.665 0.1: female 5.28 female 14.8 male 10.5 male 84.9 0.4: female 18.9 female 58.0 male 45.4 male 176 1.6: female 82 female 184 male 152 male 323 3.2: female NR* female NR* male 273 male 1360 * samples not received Qualitatively, the F0 results indicate 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 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. The average PFOS concentrations in pooled liver samples from F1 animals shortly after birth were 0.0511, 6.19, 57.6, and 70.4 ug/ g in the 0.0, 0.1, 0.4, and 1.6 mg/ kg/ day dose groups, respectively. These quantitative values for the PFOS concentration in the liver and serum should be viewed with caution. The accuracy of quantitation is + 30%, the purity of the analytical reference substance is unknown, and there were several uncorrected dilution errors.

CONCLUSIONS Conclusions stated above and this reviewer agrees. ENV/ JM/ RD( 2002) 17/ FINAL 322 REFERENCE Christian, M. S., Hoberman, A. M., and York, R. G. 1999b. Argus Research Laboratories, Inc. Protocol Number: 418 008, Sponsor Study Number: 6295.9, June 10, 1999. Combined Oral ( Gavage) Fertility, Developmental and Perinatal/ Postnatal Reproduction Toxicity Study of PFOS in Rats.

3M Environmental Laboratory. 1999b. Analytical Laboratory Report on the Determination of the Presence and Concentration of Potassium Perfluorooctanesulfonate ( CAS Number: 2795 39 3) in the Serum and Liver of Sprague Dawley Rats Exposed to PFOS via Gavage. As amended April 19, 2000. ENV/ JM/ RD( 2002) 17/ FINAL 323 CROSS FOSTERING/ DEVELOPMENTAL TOXICITY STUDY Title: ORAL ( GAVAGE) CROSS FOSTERING STUDY OF PFOS IN RATS   ARGUS RESEARCH LABORATORIES STUDY NUMBER 418 014, 3M T 6295.13, JULY 1999.

TEST SUBSTANCE Identity: Potassium Perfluorooctylsulfonate, CAS No. 2795 39 3.

Remarks: The test article, FC 95 ( Lot 217) was received on October 21, 1998, and stored at room temperature. Prepared suspensions were stored at room temperature. Information regarding the identity, strength, composition, and purity of the test article is on file with the sponsor.

METHOD Method/ Guideline followed ( i. e., OECD 414, etc.): For the cross fostering study: The requirements of the U. S. Food and Drug Administration ( FDA) were used as the basis of the study design, i. e., the International Conference on Harmonization: Guideline on detection of toxicity to reproduction for medicinal products. Federal Register, September, 22, 1994, Vol. 59. No. 183. For the PFOS bioanalysis: US FDA GLP Final Rule 21 CFR 58, with exceptions: Two separate study directors were assigned to the in life phase and the analytical phase of this study. The ABS final report does not have a Statement of Compliance. The QAU statement in the ABS final report indicates compliance with EPA 40 CFR Part 792, rather than FDA 21 CFR Part 58. Dose confirmation analyses were not conducted according to the GLP regulations; analytical method was not fully validated. Not all raw data were verified by the group leader or designee.

GLP ( Y/ N): The study was conducted in compliance with the Good Laboratory Practice ( GLP) regulations of the U. S. FDA, the Japanese Ministry of Health and Welfare, and the European Economic Community. There were no deviations from the GLP regulations that affected the quality or integrity of the study. Quality assurance Unit findings derived from the inspections during the conduct of this study are documented.

Year study performed: 1999 Species/ Strain: Sprague Dawley rats Number of animals per dose: 25 Route of administration: Gavage Dosing regimen ( list all with units): Two groups of 25 female rats were administered PFOS by gavage beginning 42 days prior to mating to untreated ( breeder) males, and continuing throughout gestation and into day 21 of lactation.

Doses: 0, 1.6 mg/ kg/ day Statistical methods used: Averages and percentages were calculated. Litter values were used where appropriate.

Remarks   Detail and discuss any significant protocol parameters and deviations: Two groups of 25 ENV/ JM/ RD( 2002) 17/ FINAL 324 female Sprague Dawley rats were administered 0 and 1.6 mg/ kg/ day PFOS in 0.5% Tween 80 by gavage, beginning 42 days prior to mating to untreated ( breeder) males, and continuing throughout gestation and into day 21 of lactation. A dose volume of 5 mL/ kg was administered, adjusted daily on the basis of individual body weight. Parental females were observed twice daily for viability and clinical observations were recorded 1 hour prior to and after dosing during the treatment period. Maternal body weights were recorded once during the acclimation period and then daily during the treatment period and at sacrifice; food consumption was also recorded once during the acclimation period and then daily during gestation and on days 1, 4, 7, 10, and 14 of lactation. During parturition, females were continually evaluated for clinical signs and also for duration of gestation, length of parturition, litter sizes, and pup viability at birth. Maternal behavior was recorded daily throughout lactation. All maternal rats were sacrificed by carbon dioxide asphyxiation on day 22 of lactation and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed; any gross lesions were preserved for future analysis. In addition, the number and distribution of implantation sites were recorded. Rats that did not deliver a litter were not included in the cross fostering procedure and were sacrificed on lactation day 25, examined for gross lesions, and the uteri examined to confirm the presence/ absence of implantation sites. Dams with no surviving pups were sacrificed after the last pup was found dead, missing, or presumed cannibalized.

Following completion of parturition, litters were immediately removed from their respective dams and placed with either a control or PFOS treated dam for rearing. This cross fostering procedure resulted in four groups of 12 13 dams or pups as follows: A) control dams with litters from PFOS treated dams, i. e., in utero exposure only; B) control dams with litters from control dams, i. e., negative control; C) PFOStreated dams with litters from PFOS treated dams, i. e., both in utero and post natal exposure; and D) PFOS treated dams with litters from control dams, i. e., post natal exposure only.

On day 1 of lactation ( birth), each pup was individually weighed and each litter was evaluated twice daily during lactation for viability. Pups were observed once daily for clinical signs and gross external physical anomalies. Pup body weights were recorded on days 1, 4, 7, 14, and 21of lactation, and then at sacrifice. On day 4 of lactation, each cross fostered litter was culled to 5 males and 5 females. On day 21 of lactation, all pups were sacrificed via decapitation and examined for gross lesions. Pups found dead or sacrificed because of moribundity were examined for gross lesions and for the cause of death or the moribund condition. The lungs, liver, and any gross lesions were collected from selected pups at various timepoints ( for the F0 generation, blood, milk, and liver samples were collected on LD 14; milk was collected 2 6 hours post dosing; for the F1 generation offspring, blood was collected on LD 14) and preserved for possible future analysis. Liver samples were evaluated via electron microscopy. In addition, samples of blood, milk ( including the milk secreting glands), and liver were collected from selected maternal rats and pups ( blood and liver samples were pooled per litter) at various timepoints for analysis of PFOS concentration. Only the analysis of the sera samples are available at this time.

RESULTS Toxic response/ effects by dose level  maternal: Mean maternal body weight and body weight gains at 1.6 mg/ kg/ day were reduced compared to controls during premating and continuing throughout gestation. Mean maternal body weight changes in the treated group were comparable to those seen in control animals during lactation. During the premating period, and on into gestation and lactation, food consumption was reduced in treated animals as compared to controls. Reductions in gestation length, the average number of implantation sites, delivered sizes, and live litter size were observed in treated animals.

Toxic response/ effects by dose level   developmental: Pup mortality was observed in two of the crossfostered groups. On lactation days 2 4, approximately 19% of the pups in group C were either found dead or presumed cannibalized. Pup mortality was also observed in group A at a rate of 9%. In addition, the ENV/ JM/ RD( 2002) 17/ FINAL 325 number of live pups on day 4 of lactation, numbers of surviving pups per litter, and live litter sizes were also reduced in these two groups. Pup mortality in groups B and D during lactation days 2 4 were at 1.6% and 1.1%, respectively. Reductions in pup body weights were observed in groups A and C on day 1 of lactation. Pup body weights in group D were comparable to controls during that same period. From lactation day 4 21, pup body weights in groups A, C, and D were reduced when compared to group B ( negative control), with the reductions greatest in group C. Two litters in group A and one litter on group C did not nurse. Milk analysis of the stomachs of pups found no milk in the stomachs of 57%, 100%, and 87% of the pups found dead and necropsied in groups A, C, and D, respectively.

Electron microscopic examination of the liver revealed an increase in the number of peroxisomes in pups from dams treated with 1.6 mg/ kg/ day PFOS.

Statistical results: The method of statistical analysis consisted of calculation of averages ( mean + S. D.) and percentages. Statistical significance was not assessed. The data were presented according to day of gestation/ lactation and compared by dose and by cross fostered group. Litter values were used where appropriate. The data are too cumbersome to cite here.

Remarks   Additional information to adequately assess the data: All maternal rats survived to schedule sacrifice. Signs of clinical toxicity observed in the dams during the study period ( e. g., chromorhinorrhea, scaly tail, abrasion on the head, neck, tail and/ or forelimb, missing, broken and/ or misaligned incisors, and localized alopecia, among others) were not considered to be treatment related since they also occurred in the control animals. All pregnant animals delivered live offspring.

Following cross fostering on LD 1, live litter sizes were comparable between treated and control. Sex ratios and the lactation index were comparable among all groups. Signs of clinical toxicity were observed in pups, but were not considered to be treatment related since they also occurred in group B ( negative control) at the same rate. No significant differences were observed between group B and the other groups following examination of pup lungs.

PFOS concentrations in the serum of untreated dams ranged from below the limit of detection ( 0.05 ug/ ml) to 5.34 ug/ ml. Serum PFOS concentrations in the pups from untreated dams, fostered with untreated dams, were below the limit of detection. Serum PFOS concentrations in the pups from treated dams, fostered with untreated dams, ranged from 47.6 ug/ ml to 59.2 ug/ ml. PFOS concentrations in the serum of treated dams ranged from 59.2 ug/ ml to 157 ug/ ml. Serum PFOS concentrations in the pups from untreated dams, fostered with treated dams, ranged from below the limit of detection to 35.7 ug/ ml. Serum PFOS concentrations in the pups from treated dams, fostered with treated dams, ranged from 79.5 ug/ ml to 96.9 ug/ ml. These data indicate that exposure to PFOS can occur both in utero and via milk from treated dams.

CONCLUSIONS Pups from control dams that were cross fostered with PFOS treated dams ( post natal exposure only) had the same low mortality rate ( 1.1%) as pups from control dams cross fostered with control dams ( 1.6%; negative control). Mortality rates in the remaining two groups, however ( i. e. control dams with litters from PFOS treated dams, i. e., in utero exposure only; and PFOS treated dams with litters from PFOS treated dams, i. e., both in utero and post natal exposure), had higher mortality rates at 9.6 % and 19.2%, respectively. Under the conditions of the study, this limited 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 affect on pup viability. Additionally, analysis of PFOS concentration showed that PFOS was observed in the sera of F0 female rats exposed during the in life phase of the study. Additionally, PFOS was observed in sera ENV/ JM/ RD( 2002) 17/ FINAL 326 samples taken from F1 generation pups from female rats exposed to the test substance, and in F1 generation pups exposed via lactation, but not exposed in utero. These are the conclusions of the study authors and this reviewer agrees.

REFERENCE Christian, M. S., Hoberman, A. M., and York, R. G. 1999c. Testing Facility: Argus Research Laboratories, Inc., Protocol Number 418 014, Sponsor: 3M Corporate Toxicology, Study Number T 6295.13, July 23, 1999. Oral ( Gavage) Cross Fostering Study of PFOS in Rats.

3M Environmental Laboratory. 1999. Analytical Laboratory Report on the Determination of the Presence and Concentration of Perfluorooctanesulfonate ( PFOS) ( CAS Number: 2759 39 3) in the Serum of Sprague Dawley Rats Exposed to Potassium Perfluorooctanesulfonate via Gavage, Laboratory Report No. U2779, Requestor Project No. 3M Tox 6295.13. Study initiation date: June 10, 1999. Completion at signing ( 2/ 11/ 00). Sample analysis completion June 28, 1999. Study Identification Number: FACT Tox 108 ENV/ JM/ RD( 2002) 17/ FINAL 327 REPEAT DOSE DATA Title: 104 Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt ( PFOS; T 6295) in Rats TEST SUBSTANCE Identity: Perfluorooctane Sulfonic Acid Potassium Salt ( PFOS; T 6295).

Remarks: The test substance, Lot No. 217, is a light colored, free flowing powder, and is 86.9% pure. The impurities of the test substance were not indicated in the main body of the study report.

METHOD Method/ guideline followed: Guideline number not stated Study duration: Two years GLP ( Y/ N): Yes Year study performed: 1998 2000 Species/ strain: Sprague Dawley rat [ Crl: COBSR CD( SD) BR]

Sex: Male and female Number of animals per dose group: The control and high dose groups contained 70 rats/ sex and the low, mid, and mid high dose groups contained 60 rats/ sex. A high dose recovery group contained 40 rats/ sex.

Route of administration: Diet Doses tested and frequency: Control ( Group 1): 0 ppm ; low dose ( Group 2): 0.5 ppm ; mid dose ( Group 3): 2 ppm; mid high dose ( Group 4): 5 ppm; high dose ( Group 5) : 20 ppm.

Post treatment observation period: A high dose recovery group ( Group 6) was observed for 52 weeks after 52 weeks of treatment.

Statistical methods used: Levene's test was done to test for variance homogeneity. In the case of heterogeneity of variance at P< 0.05, transformations were used to stabilize the variance. One way analysis of variance [ ANOVA] was used to analyze body weights, body weight changes, food consumption, continuous clinical pathology values, palmitoyl CoA oxidase activities, and organ weight data. ANOVA was done on the homogeneous or transformed data. If the ANOVA was significant, Dunnett's t test was used for pairwise comparisons between treated and control groups. Group comparisions were evaluated at the 5.0%, two tailed probability level.

Remarks: The animals were observed twice daily ( a. m. and p. m.) for mortality and moribundity; findings were recorded as they were observed. At least once prior to treatment and weekly thereafter, each animal was removed from its cage and examined; abnormal findings or an indication of normal was recorded. Body weight data were collected weekly through Week 17, once every 4 weeks thereafter, and at Week ENV/ JM/ RD( 2002) 17/ FINAL 328 105. Food consumption data were collected weekly for the first 16 weeks and once every 4 weeks thereafter. During Weeks 4, 14, 27, and 53, blood and urine were collected for hematology, clinical chemistry, urinalysis, and urine chemistry tests from 10 animals/ sex in Groups 1 through 5. Blood was collected for cholesterol and triglyceride determinations from all animals prior to the terminal sacrifice during Week 105 ( Week 103 for females in Group 3) and the recovery sacrifice ( Group 6) during Week 106. Blood films were also prepared for animals at the terminal and recovery sacrifices. Five animals/ sex in Groups 1 through 5 were sacrificed during Week 4; livers were collected and weighed. [ The liver samples were collected for PFOS analysis, mitochondrial activity, hepatocellular proliferation rate measurements by proliferation cell nuclear antigen ( PCNA), and determination of palmitoyl CoA oxidase activity. In addition, liver samples were collected for PFOS analysis ( Weeks 14, 53, 103, 105, and 106), hepatocellular proliferation rate measurement [ by PCNA at Week 14 and by bromodeoxyuridine ( BrdU) immunohistochemistry at Week 53] and palmitoyl CoA oxidase determination ( Week 4 and 14).]

RESULTS Survival rates: 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.

Neoplastic effects:

The results of the study show that PFOS is carcinogenic, inducing tumors of the liver, and of the thyroid and mammary glands. ENV/ JM/ RD( 2002) 17/ FINAL 329 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.

Remarks: At terminal of the studies, 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. Except for the mid dose ( 2.0 ppm) female group, there were no significant decreases in survival in any other male and female treatment groups as compared to that of the control. Therefore, it appears that higher dose levels could have been tolerated by the high dose groups.

Nonneoplastic effects: NOAEL ( dose and effect): 0.5 ppm ( Male) 2 ppm ( Female) LOAEL ( dose and effect): 2 ppm ( Male) 5 ppm ( Female)

Hepatotoxicity, characterized by significant increases in centrilobular hypertrophy, centrilobular eosinophilic hepatocytic granules, centrilobular hepatocytic pigment, or centrilobular hepatocytic ENV/ JM/ RD( 2002) 17/ FINAL 330 vacuolation was noted in male and/ or female rats given 5 or 20 ppm. A significant increase 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 liver lesion is believed to be due to old age of the animals and is not considered to be treatment related. Therefore, based on the pathological findings in the liver, the noobserved 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 considered to be 2 ppm in male rats and 5 ppm in female rats.

Results of Statistical Analyses of Nonneoplastic Lesions in Male Rats.

Group 1 Control 2 Low 3 Mid 4 Mid High 5 High 6 High Recovery Groups 5 vs. 6 Liver   Vacuolation, Hepatocellular Midzonal/ Centrilobular 0/ 65 0/ 55 6/ 55 10/ 55 19/ 55 9/ 40 p values .0000+** NA .1690 .0024+** .0001+** .4152+ 0.0060+**

Liver   Hypertrophy, Hepatocellular Centrilobular 0/ 65 2/ 55 4/ 55 22/ 55 42/ 565 3/ 40 p values .0000+** .2080+ .0415+* .0000+** .0000+** .0527+ .0000**

Liver   Granular Cytoplasm, Eosinophilic, Centrilobular 0/ 65 0/ 55 0/ 55 0/ 55 14/ 65 0/ 40 p values .0000+** NA NA BA .0139+* NA .0007**

Liver   Pigment Hepatocellular, Centrilobular 0/ 65 0/ 55 0/ 55 0/ 55 6/ 65 0/ 40  values .006+** NA NA NA .0139+* NA .0513 Liver   Necrosis, Individual Hepatocyte 5/ 65 4/ 55 6/ 55 5/ 55 14/ 65 4/ 40 p values .0106+* NA NA NA .0224+* .0699+ .1024 Liver   Degeneration, Cystic 5/ 65 15/ 55 19/ 55 17/ 55 22/ 65 15/ 40 NA p values .0007+** .0041+** .0003+** .0011+** .002+** .0002+** .4306+ ENV/ JM/ RD( 2002) 17/ FINAL 331 Results of Statistical Analyses of Nonneoplastic Lesions in the Female Rats.

1 2 3 4 Group Control Low Mid Mid High 5 High 6 High Recovery Groups 5 vs. 6 Liver  Infiltrate, Lymphohistiocytic 42/ 65 42/ 55 38/ 55 41/ 55 p values .0080 +** NA .3738+ NA 56/ 65 .0038 +** 32/ 40 .0709+ .2852 Liver   Hypertrophy, Hepatocellular, Centrilobular 2/ 65 1/ 55 4/ 55 16/ 55 p values .0000 +** NA .2641+ .0001 +** 52/ 65 .0000 +** 2/ 40 NA .0000 **

Liver  Granular Cytoplasm, Eosinophilic, Centrilobular 0/ 65 0/ 55 0/ 55 7/ 55 p values .0000 +** NA NA .0034 +** 36/ 65 .0000 +** 1/ 40 .3810+ .0000 **

Liver  Pigment, Hepatocellular, Centrilobular 0/ 65 0/ 55 0/ 55 1/ 55 p values .0000 +** NA NA NA 36/ 65 .0000 +** 0/ 40 NA .0000 **

Liver  Necrosis, Individual Hepatocyte 7/ 65 6/ 55 6/ 55 6/ 55 p values .0359 +* NA NA NA 15/ 65 .0500 +* 3/ 40 .4254 .0329 *

Liver   Degeneration, Cystic 0/ 65 1/ 55 1/ 55 2/ 55 p values .0187 +* NA NA .2080+ 4/ 65 .0596+ 1/ 40 .3810+ .3660 Liver  Hypertrophy, Hepatocellular, Periportal 12/ 65 10/ 55 9/ 55 4/ 55 p values .0026 ** .5796 .4778 .0614 3/ 65 .0127 * 7/ 40 NA .0344 +*

Liver  Infiltrate, Macrophage, Pigmented 2/ 65 3/ 55 5/ 55 6/ 55 p values .0000 +** NA .1567+ .0889 23/ 65 .0000 +** 7/ 40 .0147 +* .0383 * ENV/ JM/ RD( 2002) 17/ FINAL 332 List of statistically different non neoplastic effects ( increased compared with controls, p < 0.05):

Males ( 0.5ppm): Liver  Degeneration, Cystic Males ( 2 ppm): Liver  Degeneration, Cystic Liver  Hypertrophy, Hepatocellular Centrilobular Males ( 5 ppm): Liver  Degeneration, Cystic Liver  Hypertrophy, Hepatocellular Centrilobular Liver  Vacuolation, Hepatocellular Midzonal / Centrilobular Males ( 20 ppm): Liver  Degeneration, Cystic Liver  Hypertrophy, Hepatocellular Centrilobular Liver  Vacuolation, Hepatocellular Midzonal / Centrilobular Liver  Granular Cytoplasm, Eosinophilic, Centrilobular Liver  Pigment Hepatocellular, Centrilobular Liver  Necrosis, Individual Hepatocyte Females ( 0.5 ppm): None Females ( 2 ppm): None Females ( 5 ppm): Liver  Hypertrophy, Hepatocellular Centrilobular Liver  Granular Cytoplasm, Eosinophilic, Centrilobular Liver   Infiltrate, Macrophage, Pigmented Females ( 20 ppm): Liver  Hypertrophy, Hepatocellular Centrilobular Liver  Granular Cytoplasm, Eosinophilic, Centrilobular Liver  Infiltrate, Macrophage, Pigmented Liver  Pigment Hepatocellular, Centrilobular Liver  Necrosis, Individual Hepatocyte Liver  Infiltrate, Lymphohistiocytic Liver  Hypertrophy, Hepatocellular, Periportal Remarks:

The cyctic degeneration of the liver is believed to be due to old age of the animals and is not considered to be treatment related. However, A significant increase in hepatocellular centrilobular hypertrophy was also observed in mid dose ( 2 ppm) male rats. Therefore, the NOAEL for the male rat is considered to be 0.5 ppm. The author concluded that the NOAEL for both male and female rats are 2 ppm ( which we do not agree).

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. ENV/ JM/ RD( 2002) 17/ FINAL 333 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.

Summary of PFOS Concentration Serum ( ug/ mL)

a Not corrected for purity of the standard material. b LOQ Limit of Quanfitation = 0.00910 pg/ mL c LOQ Limit of Quanfitation = 0.0457 pg/ mL d C92987F sample spilled during extraction, no sample remaining for analysis. It is not possible to verify true recovery of endogenous analyte from tissues without radio labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicated ( that the sera data are accurate to ± 30%; liver data are accurate to ± 50%. Group 6 Timepoint Sex 0 ppm 0.5 ppm 2 ppm 5 ppm 20 ppm High Recovery Average ± SD Average ± SD Average ± SD Average ± SD Average ± SD Average ± SD Male < LOQb ( n= 5) 0.907 ± 0.0619 ( n= 5) 4.33 ± 1.16 ( n= 5) 7.57 ± 2.17 ( n= 5) 41.8 ± 7.92 ( n= 5) Week 0 Female 0.0259 ± 0.00663 ( n = 5) 1.61 ± 0.207 ( n = 5) 6.62 ± 0.499 ( n = 5) 12.6 ± 1.73 ( n = 5) 54.0 ± 7.34 ( n = 5)

Male < LOQc ( n = 5) 4.04 ± 0.801 ( n = 5) 17.1 ± 1.22 ( n = 5) 43.9 ± 4.90 ( n = 5) 148 ± 13.8 ( n = 5) Week 14a Female 2.67 ± 4.58 ( n = 5) 6.96 ± 0.993 ( n = 4d) 27.3 ± 2.34 ( n = 5) 64.4 ± 5.48 ( n = 5) 223 ± 22.4 ( n = 5)

Male 0.0249 ± 0.0182 ( n = 5) 146 ± 33.5 ( n = 4) Week 53 Female 0.395 ± 0.777 ( n = 5) 220 ± 44.0 ( n = 5)

Male Day 719 Female 20.2 ± 13.3 ( n = 9)

Male 0.0118 ± 0.0104 ( n = 11) 1.31 ± 1.30 ( n = 10) 7.60 ± 8.60 ( n = 17) 22.5 ± 23.5 ( n = 25) 69.3 ± 57.9 ( n = 22) Week 105 Female 0.0836 ± 0.134 ( n = 24) 4.35 ± 2.78 ( n = 15) 75.0 ± 45.7 ( n = 15) 233 ± 124 ( n = 25)

Male 2.42 ± 5.09 ( n = 10) Week 106 Female 9.51 ± 8.70 ( n = 17) ENV/ JM/ RD( 2002) 17/ FINAL 334 Summary of PFOS Concentration Liver ( ug/ g)

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Timepoint Sex Control Low Mid Mid High High High Recovery Average ± SD Average ± SD Average ± SD Average ± SD Average ± SD Average ± SD Male 0.104 ± 0.0673 ( n = 5) 11.0 ± 2.31 ( n = 5) 31.3 ± 5.84 ( n = 5) 47.6 ± 12.5 ( n = 5) 282 ± 45.3 ( n = 5) Week 0 Female 0.107 ± 0.0486 ( n = 5) 8.71 ± 0.552 ( n = 5) 25.0 ± 6.11 ( n = 5) 83.0 ± 14.1 ( n = 5) 373 ± 44.1 ( n = 5)

Male 0.459 ± 0.0573 ( n = 5) 23.8 ± 3.45 ( n = 5) 74.0 ± 6.16 ( n = 5) 358 ± 28.8 ( n = 5) 568 ± 107 ( n = 5) Week 10 Female 12.0 ± 22.4 ( n = 5) 19.2 ± 3.77 ( n = 5) 69.2 ± 3.46 ( n = 5) 370 ± 22.3 ( n = 5) 635 ± 49.0 ( n = 5)

Male 0.635 ± 1.04 ( n = 10) 435 ± 96.9 ( n = 9) Week 53 Female 0.923 ± 1.77 ( n = 10) 560 ± 180 ( n = 10)

Male Day 719 Female 55.1 ± 31.5 ( n = 9)

Male 0.114 ± 0.148 ( n = 11) 7.83 ± 7.34 ( n = 10) 26.4 ± 20.4 ( n = 17) 70.5 ± 63.1 ( n = 25) 189 ± 141 ( n = 22) Week 105 Female 0.185 ± 0.184 ( n = 24) 12.9 ± 6.81 ( n = 15) 131 ± 61.4 ( n = 15) 381 ± 176 ( n = 25)

Male 3.12 ± 5.97 ( n = 10) Week 106 Female 12.9 ± 10.4 ( n = 17)

  It is not possible to verify true recovery of endogenous analyte from tissues without radio labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicated that the sera data are accurate to ± 30%; liver data are accurate to ± 50%.

CONCLUSIONS The study results are summarized as follows: 1. Treatment related changes were found more commonly in males than in females of each of the treatment groups, which were supported by earlier pharmacokinetic studies demonstrating a higher retention of the compound by males than females. 2. The test material was considered to be carcinogenic in the rat, inducing tumors of the liver and the thyroid gland in the males and tumors of the liver and of the thyroid and mammary gland in females. 3. 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 observedadverse effect level ( LOAEL) is considered to be 2 ppm in male rats and 5 ppm in female rats. ENV/ JM/ RD( 2002) 17/ FINAL 335 4. 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) or by bromodeoxyuridine ( BrdU). 5. 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.

REFERENCE 3M, ( 2002). 104 Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt ( PFOS; T 6295) in Rats. Final Report, 3M T 6295 ( Covance study no.: 6329 183), Volumes I IX , 4068 pages, January 2, 2002. 3M, St. Paul, Minnesota.

Seacar, A. M., Thomford, P. J., and Butenhoff, J. L. Terminal observations in Sprague Dawley rats after lifetime dietary exposure to potassium perfluorooctanesulfonate. Toxicol. Sci./ Toxicologist, 66 ( 1 S): 185, 2002. ENV/ JM/ RD( 2002) 17/ FINAL 336 EPIDEMIOLOGIC DATA Title: Identification of Fluorochemicals in Sera of American Red Cross Adult Blood Donors TEST SUBSTANCE Identity: PFOS and 6 other fluorochemicals Remarks: The results reported are preliminary. The final report is expected November 2001.

METHOD Study design: Cross sectional Manufacturing/ Processing/ Use: N/ A Hypothesis tested: To determine the levels of PFOS in the serum of American Red Cross blood banks in 6 regions of the U. S.

Study period: 2000 Setting: N/ A Total population: Serum pooled from 6 ARC blood banks in various geographic regions in the US: Los Angeles, CA; Minneapolis/ St. Paul, MN; Charlotte, NC; Boston, MA; Portland, OR, and Hagerstown, MD.

Subject selection criteria: Unknown Total # of subjects in study: 652 donors, age 20 69 years Comparison population: N/ A Participation rate: N/ A Subject description: No information was provided on the individuals from whom the sera samples were taken.

Health effects studied: PFOS levels in blood Data collection methods: Blood sera samples were analyzed using high pressure liquid chromatography/ electrospray tandem mass spectrometry ( HPLC/ ESMSMS).

Details on data collection: No information was provided as to how the blood was drawn, stored, etc.

Exposure period: Unknown PFOS serum levels used as surrogate for exposure.

Description/ delineation of exposure groups/ categories: N/ A Measured or estimated exposure: N/ A ENV/ JM/ RD( 2002) 17/ FINAL 337 Exposure levels: N/ A Statistical methods: Arithmetic means, ranges, geometric means and 95% confidence intervals were calculated. Central tendency and distribution of the data by age, gender, location and their respective interaction terms will be done in the final report. A reliability assessment is also being analyzed.

Other methodological information: N/ A RESULTS Describe results: The mean serum PFOS level was 43.7 ppb. The range was 4.27 to 1656 ppb. Analyses stratified by age, gender, and geographic location will be forthcoming in the final report.

Study strengths and weaknesses: These data are cross sectional data used to determine PFOS levels in the general population. No other descriptive information about the subjects is available in this preliminary report. The sample size is relatively small. Blood donors cannot be considered representative of the general population of the US.

Research sponsors: 3M Medical Department, Corporate Occupational Medicine Consistency of results: Mean PFOS levels reported in this study are similar to those reported earlier in pooled blood samples, although they are on the higher end of the range.

CONCLUSIONS N/ A REFERENCE Olsen, GW, Burris, JM, Lundberg, JK, Hansen, KJ, Mandel, JH, Zobel, LR. Identification of fluorochemicals in sera of American Red Cross adult blood donors. Interim report. June 25, 2001. ENV/ JM/ RD( 2002) 17/ FINAL 338 EPIDEMIOLOGIC DATA Title: An Epidemiologic Analysis of Episodes of Care of 3M Decatur Chemical and Film Plant Employees, 1993 1998 TEST SUBSTANCE Identity: POSF based chemicals used at the Decatur plant Remarks: Episodes of care analyses are not often used in occupational epidemiologic studies.

METHOD Study design: Episode of care comparison Manufacturing/ Processing/ Use: The 3M Decatur, Alabama plant began production in 1961. It is made up of the film plant and the chemical plant. The 3 major product groups in the chemical plant are protective chemicals, performance chemicals, and fluoroelastomers. Perfluorooctanesulfonyl fluoride ( POSF) is the major sulfonate fluorochemical manufactured at Decatur and is used as the precursor to the production of a variety of perfluorinated amides, alcohols, acrylates, and other fluorochemical polymers.

Hypothesis tested: To use episodes of care methodology as a screen for morbidity outcomes associated with long term, high exposure to POSF based production at the 3M facility in Decatur, Alabama.

Study period: Episodes of care experience of 652 chemical employees and 659 film plant employees were analyzed for workers at the plant who were employed for at least 1 year between January 1, 1993 and December 31, 1998.

Setting: 3M plant in Decatur, Alabama.

Total population: 1311 workers were eligible for the cohort ( at least 1 year of employment at the plant). The total worker population was not reported.

Subject selection criteria: All workers employed at the Decatur plant for at least 1 year between Jan. 1, 1993 and Dec. 31, 1998. Episodes of care were limited to their Decatur time of employment for employees hired, terminated, or died during the study period. However, records of employees on Medicare, long term disability or who chose HMO coverage were not in the database and would not be included in the episodes of care for that employee.

Comparison population: Chemical and film plant employees were analyzed separately and then compared to each other. Employee comparison groups were defined according to their potential workplace exposure to POSF fluorochemical production. Group A: all chemical plant employees and all film plant employees eligible for the cohort. Group B: all chemical plant employees who worked solely in the chemical plant and all film plant employees who worked exclusively in the film plant. Group C: all chemical plant employees with high fluorochemical exposures compared to their job counterparts in the film plant. Group D: all plant workers with high fluorochemical exposure for at least 10 years prior to the study onset compared to their job counterparts in the film plant.

Participation rate: 97% of Decatur employees were eligible for participation in the study. ENV/ JM/ RD( 2002) 17/ FINAL 339 Subject description: 82% of the employees in the cohort were male ( 530 in the chemical plant and 558 in the film plant). The mean age was 45.1 in the chemical plant and 48.6 in the film plant. Sixty percent of the chemical plant employees had worked only in the chemical plant and a similar percentage of film plant workers had worked exclusively in the film plant. Seventy six percent of the chemical plant workers had high exposure jobs.

Health effects studied: Morbidity. Based on animal data and epidemiologic studies on PFOA and PFOS, certain episodes of care were considered a priori. They included: liver and bladder cancer, endocrine disorders involving the thyroid gland and lipid metabolism, gastrointestinal disorders of the liver and biliary tract, and reproductive disorders.

Data collection methods: The Clinical Care Groups episode of care software developed by Ingenix, Inc. was used to provide a comprehensive grouping of all visits ( inpatient and outpatient), procedures, ancillary services, and prescription drugs used in the diagnosis, treatment and management of more than 400 diseases or conditions. The software code constructs an episode of care around the index eligible record by searching backward and forward in time for the health claims records that are related to the disease or condition on the index record. The index record consists of either procedure codes indicative of a face toface encounter or a pharmacy record for a delineating drug.

Exposure period: The episodes of care that were included in the study were those experienced between Jan. 1, 1993 to Dec. 31, 1998.

Description/ delineation of exposure groups/ categories: Workers were placed into groups according to potential workplace exposures: workers who were employed solely in the chemical or film plants, those who had high exposure jobs, and those who worked at least 10 years in jobs with high potential for fluorochemical exposure.

Measured or estimated exposure: estimated based on job history information.

Exposure levels: Not measured. Employees were placed into exposure categories based on job description.

Statistical methods: A risk ratio episode of care ( RREpC) provided the estimate of risk between the observed to expected episodes of care for chemical plant employees compared to the observed to expected episodes of care among film plant employees. The expected number of episodes of care for both the film and chemical plant employees was calculated from health claims data of the 3M manufacturing population in the U. S. Because the chemical and film plant cohorts had slightly different age and gender structures, an adjusted ratio was calculated and compared to the unadjusted risk ratio. In most cases, the risk ratios were comparable. Therefore, 95% confidence intervals were only calculated for the unadjusted risk ratios.

Other methodological information: It should be noted that from an epidemiologic perspective, an episode of care could represent any and all incident cases, prevalent cases, and/ or misclassified cases ( both false positive and false negative). In addition, types and counts of episodes of care may differ by the software used, and it is possible that 2 different diagnoses may be assigned to the same episode. Certain services, such as lab procedures and prescriptions may not be reported for the episode. Also, the endpoint of an episode may vary among software programs. The clinical flexibility of the algorithm may differ depending on the software program.

RESULTS Describe results: The only increased risk of episodes for the conditions of a priori interest were for ENV/ JM/ RD( 2002) 17/ FINAL 340 neoplasms of the male reproductive system and for the overall category of cancers and benign growths ( which included cancer of the male reproductive system). There was an increased risk of episodes for the overall cancer category for all 4 comparison groups. The risk ratio was greatest in the group of employees with the highest and longest exposures to fluorochemicals ( RREpC = 1.6, 95% CI = 1.2   2.1). Increased risk of episodes in long time, high exposure employees also was reported for male reproductive cancers ( RREpC = 9.7, 95% CI = 1.1  458). It should be noted that the confidence interval is very wide for male reproductive cancers and the sub category of prostate cancer. Five episodes of care were observed for reproductive cancers in chemical plant employees ( 1.8 expected), of which 4 were prostate cancers. One episode of prostate cancer was observed in film plant employees ( 3.4 expected). This finding is important because an excess in prostate cancer mortality was observed in the Cottage Grove plant mortality study. However, the update of the study did not confirm this finding.

There was an increased risk of episodes for neoplasms of the gastrointestinal tract in the high exposure group ( RREpC = 1.8, 95% CI = 1.2 3.0) and the long term employment, high exposure group ( RREpC = 2.9, 95% CI = 1.7   5.2). Most of the episodes were attributable to benign colonic polyps. Similar numbers of episodes were reported in film and chemical plant employees.

In the entire cohort, only 1 episode of care was reported for liver cancer ( 0.6 expected) and 1 for bladder cancer ( 1.5 expected). Both occurred in film plant employees. Only 2 cases of cirrhosis of the liver were observed ( 0.9 expected), both in the chemical plant. There was a greater risk of lower urinary tract infections in chemical plant employees, but they were mostly due to recurring episodes of care by the same employees. It is difficult to draw any conclusions about these observations, given the small number of episodes reported.

Chemical plant employees in the high exposure, long term employment group were 2 ½ times more likely to seek care for disorders of the biliary tract than their counterparts in the film plant ( RREpC = 2.6, 95% CI = 1.2  5.5). Eighteen episodes of care were observed in chemical plant employees and 14 in film plant workers. The sub categories that influenced this observation were episodes of cholelithiasis with acute cholecystitis and cholelithiasis with chronic or unspecified cholecystitis. Most of the observed cases occurred in chemical plant employees.

Risk ratios of episodes of care for endocrine disorders, which included sub categories of thyroid disease, diabetes, hyperlipidemia, and other endocrine or nutritional disorders, were not elevated in the comparison groups. Conditions which were not identified a priori but which excluded the null hypothesis in the 95% confidence interval for the high exposure, long term employment group included: disorders of the pancreas, cystitis, and lower urinary tract infections.

Study strengths and weaknesses: See " other methodological information" section for limitations of episodes of care software. The results of this study should only be used for hypothesis generation. Although the episode of care design allowed for a direct comparison of workers with similar demographics but different exposures, there are many limitations to this design. Episodes of care are reported, not disease incidence; therefore, this parameter cannot be interpreted in any other manner. The data are difficult to interpret because a large RREpC may not necessarily indicate high risk of incidence of disease. In addition, many of the risk ratios for episodes of care had very wide confidence intervals. The analysis was limited to 6 years. Also, the utilization of health care services may reflect local medical practice patterns. Individuals may be counted more than once in the database because they can be categorized under larger or smaller disease classifications. Episodes of care may include the same individual several times. Not all employees were included in the database, such as those on long term disability.

Research sponsors: 3M Company ENV/ JM/ RD( 2002) 17/ FINAL 341 Consistency of results: No other morbidity studies have been conducted on fluorochemicals.

CONCLUSIONS This study should only be used for hypothesis generation regarding workers employed at the Decatur plant who are employed in jobs with high exposure to POSF based fluorochemicals.

REFERENCE Olsen, GW, Burlew, MM, Hocking, BB, Skratt, JC, Burris, JM, Mandel, JH. An epidemiologic analysis of episodes of care of 3M Decatur chemical and film plant employees, 1993 1998. Final Report. May 18, 2001. ENV/ JM/ RD( 2002) 17/ FINAL 342 EPIDEMIOLOGIC DATA Title: Identification of Fluorochemicals in Sera of Children in the United States TEST SUBSTANCE Identity: PFOS and 6 other fluorochemicals Remarks: The results reported are preliminary. The final report is expected November 2001.

METHOD Study design: Cross sectional.

Manufacturing/ Processing/ Use: N/ A Hypothesis tested: To determine the serum concentrations of selected fluorochemicals in a sample of children to provide a more specific understanding of the distribution of these compounds in children.

Study period: Child sera samples were collected from January 1994 to March 1995. The sera samples were analyzed in Spring 1999.

Setting: N/ A Total population: Not reported Subject selection criteria: The sera samples were provided to 3M by the University of Minnesota Department of Pediatrics. They were obtained from a large clinical trial on Group A streptococcal infections in children. The children were residents of 23 states in the US. These children presented with signs and symptoms of acute onset pharyngitis. All of the children had positive throat cultures at the initial visit.

Total # of subjects in study: n = 599 children, age 2 12 years Comparison population: N/ A Participation rate: N/ A Subject description: No information was provided on the children from whom the sera samples were taken.

Health effects studied: PFOS serum levels in blood, as well as 6 other fluorochemicals.

Data collection methods: Blood sera samples were collected using high pressure liquid chromatography/ electrospray tandem mass spectrometry ( HPLC/ ESMSMS). The samples were collected from equal numbers of male and female children residing in 23 states.

Details on data collection: No information was provided as to how the blood was drawn, stored, etc.

Exposure period: N/ A ENV/ JM/ RD( 2002) 17/ FINAL 343 Description/ delineation of exposure groups/ categories: Blood sera samples were collected from children 2   12 years old.