Remarks: There are reasons to consider this a questionable result. No notation was made about the sensitivity of the analytical measurements in this study, but the quantification limit in the analysis in Part II was 0.625 µ g. The concentration range from impinger train 1 in Experiment # 3 was 0.07 0.075 µ g. Impinger concentrations ranged from 0 0.18 µ g in impinger train 2. These concentrations are near or below the quantification limit reported in Part II.

CONCLUSIONS No reliable conclusions can be made based on this study. The general observation is that this compound has very low volatility or a very low vapor pressure under ambient conditions.

Remarks: none DATA QUALITY Reliabilities: Klimisch ranking 3. There is no information on the validity of the test method for determining volatility of the test substance. This study lacks characterization of the purity of the test substance. There is no information on the validity of the analysis method.

REFERENCE 3M Environmental Laboratory. 1993. 3M Lab Request Number L3306, 3M Company, St. Paul, MN. ENV/ JM/ RD( 2002) 17/ FINAL 221 WATER SOLUBILITY STUDY Title: Solubility of PFOS in water TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1, 1, 2, 2, 3, 3, 4, 4, 5, 5, 6, 6, 7, 7, 8, 8, 8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: The test substance is a white powder. The sample was recrystallized from a production lot of FC 95, and assigned a test, control, and reference number TCR 00017 046. The purity was determined to be 97.9% by LC/ MS, 1H NMR, 19F NMR and elemental analysis techniques.

METHOD Method: Based on OECD 105, OPPTS 830.7840.

GLP: Yes Year study performed: 2001 Remarks: Water source is ASTM Type I water, Millipore. The definitive test consisted of placing an excess amount of test substance with the appropriate water in centrifuge tubes. The tubes were vortexed and shaken at 225 rpm at 30 ° C for 24, 48, or 72 hours followed by 24 hours of equilibration at 24 25 ° C. Following equilibration, samples were centrifuged and the supernatant was analyzed by high performance liquid chromatography with mass spectrometric detection ( LCMS).

RESULTS Value ( mg/ L) at temperature ° C: 680 mg/ L at 24 25 ° C Description of solubility: Slightly soluble pH value and concentration at temperature ° C: not stated pKa value at 25 ° C: not stated Remarks: The 24, 48, and 72 hour solubility concentrations were averaged to obtain the overall mean solubility concentrations.

CONCLUSIONS The overall mean solubility concentration of the test substance in pure water was 680 mg/ L.

Remarks: none DATA QUALITY Reliabilities: Klimisch ranking 1 ENV/ JM/ RD( 2002) 17/ FINAL 222 REFERENCE Ellefson, M. 2001c. Solubility of PFOS in Water. 3M Company, 3M Environmental Laboratory, Project Number E00 1716. ENV/ JM/ RD( 2002) 17/ FINAL 223 WATER SOLUBILITY STUDY Title: Solubility of PFOS in Natural Seawater and an Aqueous Solution of 3.5% Sodium Chloride TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1, 1, 2, 2, 3, 3, 4, 4, 5, 5, 6, 6, 7, 7, 8, 8, 8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: The test substance is a white powder. The sample was recrystallized from a production lot of FC 95, and assigned a test, control, and reference number TCR 00017 046. The purity was determined to be 97.9% by LC/ MS, 1H NMR, 19F NMR and elemental analysis techniques.

METHOD Method: Based on OECD 105, OPPTS 830.7840 GLP: Yes Year study performed: 2001 Remarks: The definitive test consisted of placing an excess amount of test substance with the appropriate water in centrifuge tubes. The tubes were vortexed and shaken at 150 rpm at 30 ° C for 24, 48, and 72 hours followed by 24 hours of equilibration at 22 24 ° C. Following equilibration, samples were centrifuged and the supernatant was analyzed by high performance liquid chromatography with mass spectrometric detection ( LCMS).

Water Sources: Natural Seawater = Ocean Scientific, lot # LN 58, salinity = 3.5% Sodium Chloride = EM Science, 99% pure, mixed with ASTM Type 1 water to achieve salinity of 3.5%

RESULTS Value ( mg/ L) at temperature ° C: Natural Seawater: 12.4 mg/ L at 22 23 ° C; 3.5% NaCl Solution: 20.0 mg/ l at 22 24 ° C Description of solubility: Slightly soluble pH value and concentration at temperature ° C: not stated pKa value at 25 ° C: not stated Remarks: The 24, 48, and 72 hour solubility concentrations were averaged to obtain the overall mean solubility concentrations for the natural seawater. The 24 hour values were not included in the mean solubility of the sodium chloride calculation because when the coefficient of variation was calculated for all of the replicate analyses for that day, it was > 15%.

CONCLUSIONS The overall mean solubility concentration of the test substance in natural seawater was 12.4 mg/ L. In a 3.5% NaCl solution it was 20.0 mg/ L. PFOS solubility decreases with increasing ionic strength of the medium. ENV/ JM/ RD( 2002) 17/ FINAL 224 Remarks: none DATA QUALITY Reliabilities: Klimisch ranking 1 Remarks: none REFERENCE Ellefson, M. 2001a. Solubility of PFOS in Natural Seawater and an Aqueous Solution of 3.5% Sodium Chloride. 3M Company, 3M Environmental Laboratory, Lab Project Number E00 1716. ENV/ JM/ RD( 2002) 17/ FINAL 225 SOLUBILITY STUDY Title: Solubility of PFOS in Octanol TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1, 1, 2, 2, 3, 3, 4, 4, 5, 5, 6, 6, 7, 7, 8, 8, 8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: The test substance is a white powder. The sample was recrystallized from a production lot of FC 95, and assigned a test, control, and reference number TCR 00017 046. The purity was determined to be 97.9% by LC/ MS, 1H NMR, 19F NMR and elemental analysis techniques.

METHOD Method: Based on OECD 105, OPPTS 830.7840 GLP: Yes Year study performed: 2001 Remarks: Octanol Source: Aldrich Screen Test: Lot JU0873804, 99+% Definitive Test: Lot 06238CI, 99.9+% HPLC Grade The definitive test consisted of placing ~ 0.010 g test substance with ~ 10 mL octanol in centrifuge tubes. The tubes were shaken at ~ 150 rpm at ~ 30 ° C for 24, 48, or 72 hours followed by 24 hours of equilibration at 22 23 ° C. Following equilibration, samples were centrifuged and the supernatant was analyzed by high performance liquid chromatography with mass spectrometric detection ( LCMS).

RESULTS Value ( mg/ L) at temperature ° C:

24 hours: 56.9 mg/ L 48 hours: 55.7 mg/ L 72 hours: 55.4 mg/ L Mean solubility of PFOS in octanol = 56.0 mg/ L Description of solubility: not stated pH value and concentration at temperature ° C: not stated pKa value at 25 ° C: not stated Remarks: The 24, 48, and 72 hour solubility concentrations were averaged to obtain the overall mean solubility concentration. ENV/ JM/ RD( 2002) 17/ FINAL 226 CONCLUSIONS The overall mean solubility concentration of the test substance in pure octanol was 56.0 mg/ L.

Submitters' Remarks: Typically, the Column Elution Method is recommended for use with substances with solubility screening results of < 10 mg/ L. However, the shake flask method was utilized in this study because of the difficulty in obtaining tubing compatible with octanol and the possible explosion hazard posed by possible leaks of a flammable solvent in an incubator.

DATA QUALITY Reliabilities: Klimisch ranking 1 Remarks: none REFERENCE Ellefson, M. 2001b. Solubility of PFOS in Octanol. 3M Company, 3M Environmental Laboratory, Laboratory Project Number E00 1716. ENV/ JM/ RD( 2002) 17/ FINAL 227 SOIL ADSORPTION Title: Pilot Study on Soil Adsorption TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: The sample was radiolabeled ( isotope and labeling position not specified). The 3M production lot number was not noted. The test substance was a white powder of uncharacterized purity.

METHOD Method/ guideline followed: The study explored the use of autoradiography of Thin Layer Chromatography Plates for determining the soil mobility of PFOS. This method was developed by 3M while looking for alternatives for studying the adsorption/ desorption properties of the test substance.

GLP ( Y/ N): No Year study performed: 1978 Statistical methods: None Temperature: Not indicated Remarks: Little detail was available regarding the study method.

RESULTS Results: The study used TLC greenhouse soil plates. The radiolabeled spot due to PFOS was too faint to be visualized.

CONCLUSIONS No conclusion could be reached in this study.

DATA QUALITY Reliability: Klimisch ranking 3. These studies lacked sufficient detail regarding methodology. Isotope specific activity and chemical/ radiochemical purity of the test substance were not provided. The method for radiosynthesis was also not provided. The analytical methodology lacked validation and a means of identifying and quantifying potential degradation products.

REFERENCES Boyd, S. A. 1993. Review of Technical Notebook. Soil Thin Layer Chromotography. Number 48277, p30. Michigan State University. Mendel, A. 1978. Soil Thin Layer Chromatography   FC 95, FC 143, FM 3422. Excerpt from 3M Technical Notebook. October 13, 1978. Number 48277, p30. Project Number 9970612600. ENV/ JM/ RD( 2002) 17/ FINAL 228 BIODEGRADATION STUDY Title: Determination of Methylene Blue Active Substance  FC 95 TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: The 3M production lot was 158. The test substance was a white powder of uncharacterized purity.

METHOD Method/ guideline followed: EPA Method 425.1 Test Type: Methylene Blue Active Substance GLP ( Y/ N): No Year study performed: 1989 Contact time units: Not indicated Inoculum: Not indicated RESULTS Degradation % after time: Not indicated Results:

Kinetic ( for sample, positive and negative controls): Not indicated Breakdown products ( yes/ no): Not indicated Remarks: None CONCLUSIONS Reviewer's remarks: The test indicated that the test sample exhibited high activity in this MBAS study.

DATA QUALITY Reliability: Klimisch ranking 3. This study lacked information regarding methodology and testing 3M Lab Request Number MBAS Result G1512 1 835,000 mg/ kg G1827 2 831,000 mg/ kg G1828 2 730,000 mg/ kg ENV/ JM/ RD( 2002) 17/ FINAL 229 parameters. Test substance purity within the test sample was not sufficiently characterized. The sample description from Twin City Testing was " liquid", but FC 95 was a solid material. This may indicate that the FC 95 was sent in a solution and that the actual MBAS may be higher than the values cited.

REFERENCE Determination of Methylene Blue Active Substance  FC 95. 1989. Pace Analytical and Twin City Testing. Minneapolis, Minnesota   at the request of the 3M Company, 3M Lab Request numbers: G1512, G1827, and G1828. ENV/ JM/ RD( 2002) 17/ FINAL 230 SOIL ADSORPTION Title: Soil Adsorption/ Desorption Study of Potassium Perfluorooctane Sulfonate ( PFOS)

TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: The test substance was a white powder. The sample was recrystallized from a production lot of FC 95. The purity was determined to be 97.9% by LC/ MS, 1H NMR, 19F NMR, and elemental analysis techniques.

METHOD Method/ guideline followed: OECD 106 GLP ( Y/ N): Yes Year study performed: 2001 Statistical methods: Statistical analysis and plotting of the data was done according to OECD Method 106 using Microsoft Excel.

Temperature: Room temperature ( 19 30 ° C)

Stock and test solution preparation: The test concentrations and conditions were determined in a preliminary experiment. For the definitive experiment, test solutions were made by diluting a stock solution of unradiolabeled perfluorooctanesulfonate to a final test substance concentration of approximately 0.5 mg/ L in aqueous 0.01 M CaCl2. ENV/ JM/ RD( 2002) 17/ FINAL 231 Soil Characteristics 1Value is for pH in water, not pH in 0.1 M CaCl2 Test Conditions:

Adsorption kinetics: Replicate study samples containing the soils ( or sediments or sludges) were equilibrated by shaking for at least 12 hours at room temperature with 0.01M CaCl2. Study samples were dosed with the test substance at approximately 0.5 mg/ L and placed on an orbital shaker. Replicate sets of these study samples were removed at designated time points throughout a 48 hour time period. Study samples were then prepared and analyzed for the target analyte. The adsorption kinetics were determined using this data. The last set of study samples ( 48 hour) were saved and used for the desorption kinetics portion of the method.

Desorption kinetics ( one concentration): After the adsorption kinetics experiment, the 48 hour study samples were centrifuged and the aqueous phase removed. The volume of solution removed was replaced by an equal volume of 0.01 M CaCl2 without test substance. The new mixture was agitated until the desorption equlibrium was reached. During a 48 hour period, at defined time intervals, small aliquots of the aqueous phase were removed and analyzed for the target analyte. The desorption kinetics were determined using this data. Soil Class Clay Loam Clay Loam Sandy Loam River Sediment Domestic Sludge Source Agvise Laboratories, Northwood, ND Agvise Laboratories, Northwood, ND Agvise Laboratories, Northwood, ND Agvise Laboratories, Northwood, ND Agvise Laboratories, Northwood, ND NIST, from Denver, CO POTW Lot Number 00 2407 00 2404 00 2405 99 2564 00 2046 2781 Physical Description 1.00 mm airdried 0 6" deep 1.00 mm airdried 0 6" deep 1.00 mm airdried 0 6" deep 1.00 mm airdried 0 6" deep 1.00 mm airdried 0 6" deep 200 mesh, oven dried, sterilized % Organic Carbon 2.6% 4.9% 2.6% 2.8% 1.3% Not analyzed % Sand 16% 39% 21% 58% 39% Not analyzed % Silt 22% 50% 46% 22% 42% Not analyzed % Clay 62% 11% 33% 20% 19% Not analyzed CEC ( meq/ 100g) 54.5 23.9 24.7 23.3 17.5 Not analyzed pH in 0.01 M CaCl2 7.2 7.4 6.0 7.81 7.7 Not analyzed ENV/ JM/ RD( 2002) 17/ FINAL 232 RESULTS Adsorption Kinetics of PFOS, 1: 5 Soil: Solution Ratio, 48 hour Time Point Soil Type Average Distribution Coefficient, Kd, L/ g Percentage of Organic Carbon in Soil Average Organic Carbon normalized Adsorption Coefficient, Koc, L/ g Clay 0.0183 2.6 70.4 Clay Loam 0.00972 2.6 37.4 Sandy Loam 0.0353 2.8 126 River Sediment 0.00742 1.3 57.1 Domestic Sludge < 0.120 Not available Not calculable All matrices adsorbed the test substance strongly. The sludge demonstrated very strong adsorption (> 96%) and PFOS was not detected in the extracts. The data indicated that adsorption occurred within the first few hours of exposure and the test substance concentration did not vary significantly after 16 hours.

Apparent Desorption Kinetics of PFOS, 1: 5 Soil: Solution Ratio, 48 hour Time Point Soil Type Desorption Coefficient, Kdes, L/ g Barnes Loam 0.0000471 Clay Loam 0.0000158 Clay 0.0000349 River Sediment 0.0000100 Domestic Sludge < 0.000237 The test substance was poorly desorbed from the soil/ sediment/ sludge matrices during the 48 hour study period. The river sediment displayed the most desorption at 39% after 48 hours. The sludge samples did not desorb a detectable amount of test substance. Desorption that did occur was accomplished rather quickly; after the 8 hour time point the test substance concentration did not vary significantly.

Adsorption Isotherms Soil Type Log Kads F Kads F ( 1) Regression constant, 1/ n Regression Constant, n Clay  1.2515 0.0560 0.884 1.13 Clay Loam  1.3762 0.0421 0.841 1.19 Sandy Loam  1.0369 0.0919 0.829 1.21 River Sediment  2.0261 0.0094 0.989 1.01 Domestic Sludge  1.246 0.0568 1.2581 0.795 ( 1) Freundlich adsorption coefficient ENV/ JM/ RD( 2002) 17/ FINAL 233 Desorption Isotherms Soil Type Log Kdes F Kdes F ( 1) Regression Constant, 1/ n Regression Constant, n Clay  0.653 0.222 0.935 1.07 Clay Loam  1.084 0.082 0.954 1.05 Sandy Loam  0.981 0.104 1.01 0.988 River Sediment  1.41 0.039 1.02 0.984 Domestic Sludge 1.47 29.5 0.327 3.06 ( 1) Freundlich desorption coefficient Freundlich adsorption isotherms were used to relate the amount of test substance adsorbed on the soil to the amount present in the aqueous solution at equilibrium. The values calculated for the regression constant indicate that the data obtained for the test substance over two orders of magnitude were slightly non linear.

CONCLUSIONS Perfluorooctanesulfonate ( PFOS) appeared to adsorb strongly to all of the soil/ sediment/ sludge matrices tested. PFOS would not be considered to be qualitatively mobile as per OECD Guideline 106 ( 1/ 21/ 00) as the Kd values are > 1 mL/ g. The test substance, once adsorbed, does not desorb readily, even when extracted with an organic solvent. In either case, adsorption or desorption, an equilibrium is achieved in less than 24 hours, with substantial adsorption (> 50%) occurring in some of the time 0 samples after approximately 1 minute of contact. The test substance exhibited low mobility in all of the adsorbants tested. The shape of the PFOS adsorption isotherm ( H type) indicated a very strong chemical/ adsorbent interaction. Because PFOS is a strong acid, it likely forms strong bonds with soils, sludge, and sediment via the mechanism of chemisorption.

DATA QUALITY Reliability: Klimisch ranking 1.

REFERENCES Ellefson, M. E. 2001d. Soil Adsorption/ Desorption Study of Potassium Perfluorooctanesulfonate ( PFOS). 3M Technical Report. Project Number E00 1311, Completion date June 4, 2001. ENV/ JM/ RD( 2002) 17/ FINAL 234 BIOCONCENTRATION ( PARTITION COEFFICIENT)

Title: Kow ( Solubility in Water, Natural Seawater, An Aqueous Solution of 3.5% Sodium Chloride, and n Octanol with Subsequent Calculation of the n Octanol Water Partition Coefficient ( Kow) of PFOS for each of the Aqueous Matrices)

TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: The test substance was a white powder. The sample was recrystallized from a production lot of FC 95, and assigned the internal reference number of TCR 00017 046. Purity was determined to be 97.9% by LC/ MS, 1H NMR, 19F NMR, and elemental analysis techniques.

METHOD Method/ guideline followed: Calculated from n octanol solubility and water solubility according to OPPTS 830.7550 and OECD 107.

Test Type: n Octanol/ Water partition coefficient ( at saturation)

GLP ( Y/ N): Yes Year study performed: 2001 Test temperature: n Octanol solubility value at 23 24 ° C Water solubility value at 24 25 ° C Remarks: The physical properties of PFOS did not allow a determination of the partition coefficient by the shake flask method per guidance provided in the OPPTS and OECD guidelines. Therefore, this study did not bring the two phases ( n octanol and water) into contact with PFOS at the same time, and this testing reflected the partition coefficient for the subject material at saturation only. The n Octanol/ Water partition coefficient was calculated by dividing the solubility of PFOS in n octanol by the solubility in water and expressing it as the logarithmic value.

RESULTS The calculated log Kow for PFOS was determined to be   1.08 at saturation ( log( 56 mg/ L in n octanol/ 680 mg/ L in water)).

CONCLUSIONS Remarks: No conclusions could be derived from this information. It applied only to a saturated system, which would not likely exist in the environment.

DATA QUALITY Reliability: Klimisch ranking 1 ENV/ JM/ RD( 2002) 17/ FINAL 235 Remarks: The solubility studies were conducted properly. However, the applicability of this data point is limited. Application of this value in a risk assessment has limited or no value as it only applies to saturated systems.

REFERENCES Kow ( Solubility in Water, Natural Seawater, An Aqueous Solution of 3.5% Sodium Chloride, and n Octanol with Subsequent Calculation of the n Octanol Water Partition Coefficient ( Kow) of PFOS for each of the Aqueous Matrices). 2001. 3M Company. St. Paul, Minnesota. Environmental Laboratory Project Number E00 1716. ENV/ JM/ RD( 2002) 17/ FINAL 236 BIODEGRADATION STUDY Title: Microbial Metabolism ( Biodegradation) studies of Perfluorooctane Sulfonate ( PFOS) II. Aerobic Soil Biodegradation TEST SUBSTANCE Identity: Perfluorooctanesulfonate ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt) CAS # 2795 39 3 Remarks: White powder, 86.9% purity, also called PFOS or FC 95 METHOD Method/ guideline followed ( experimental/ calculated): Method was designed by Springborn Laboratories, Inc.

Test Type ( aerobic/ anaerobic): Aerobic GLP ( Y/ N): No Year study performed: 2000 Contact time/ units: 20 weeks Inoculum: The inoculum sources were soils collected from a hardwood forest in Hanson, MA, a pine forest in Onset, MA, and a river bank in Bridgewater, MA; and sediments collected from brackish sites below the Wareham, MA wastewater treatment plant outfall and from the Narrows area in Wareham, MA. Biomass was determined on day 83 by both the fumigation/ extraction and standard plate count methods and was reported as 17.4 mg C/ 100 g soil and 6 x 105 cells/ g, respectively.

Remarks: Soil and sediment samples were air dried, 2.0 mm sieved, and mixed together in equal dry weight portions. A nutrient mixture was prepared by combining a sterile potting soil extract, a trace mineral solution, a yeast extract, and reagent water. The soil/ sediment mixture was adjusted to 75% of the water holding capacity using the above nutrient mixture. Soil moisture was monitored weekly during the study, and adjusted using reagent water as needed. The nominal test concentration was approximately 21.2 mg/ kg. The incubation temperature was 22 ! 3   C, and the test was conducted in the dark. Information on test solution agitation was not provided. Test vessels were 40 mL I Chem glass vials with silicone/ Teflonlined septum screw caps containing 10 g ( dry weight) soil/ sediment mixture. Each test flask received all components at test initiation. Samples were taken at days 7, 14, 21, 28, 35, 42, 49, 56, and 63. Entire samples were extracted with methanol via accelerated solvent extraction. Extracts passed through a 0.2 : m nylon filter prior to analysis. Samples were diluted as necessary in methanol and analyzed via LC/ MS. The stock solution used to dose the biodegradation test systems was prepared at a concentration of 1,060 mg/ L, which is approximately twice the water solubility of PFOS. No day 0 samples were taken to determine starting concentrations.

RESULTS Degradation % after time: Not specified Results: Essentially no PFOS metabolism occurred during the study. ENV/ JM/ RD( 2002) 17/ FINAL 237 Kinetic ( for sample, positive and negative controls): Not specified Breakdown products ( yes/ no): None indicated Remarks: None CONCLUSIONS Remarks: PFOS is recalcitrant in the activated soil/ sediment system.

DATA QUALITY Reliability: Klimisch ranking = 2. Study not conducted according to Good Laboratory Practices. The stock solution used to dose the systems was prepared at twice the water solubility of PFOS. No Day 0 samples were taken to determine starting concentrations.

REFERENCE The study was conducted at Springborn Laboratories, Inc., Wareham, Massachusetts, at the request of the 3M Company. Report completed 10/ 31/ 00. Lab Project number E01 0434. ENV/ JM/ RD( 2002) 17/ FINAL 238 BIODEGRADATION STUDY Title: Microbial Metabolism ( Biodegradation) studies of Perfluorooctane Sulfonate ( PFOS) III. Anaerobic Sludge Biodegradation TEST SUBSTANCE Identity: Perfluorooctanesulfonate ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt) CAS # 2795 39 3 Remarks: White powder, 86.9% purity, also called PFOS or FC 95 METHOD Method/ guideline followed ( experimental/ calculated): Method was designed by Springborn Laboratories, Inc.

Test Type ( aerobic/ anaerobic): Anaerobic GLP ( Y/ N): No Year study performed: 2000 Contact time/ units: 56 days Inoculum: The inoculum source was an anaerobic digestor at the Rockland, MD wastewater treatment plant.

Remarks: A mixture containing dried sludge extract ( from rotating biological contacter wastewater treatment, Bridgewater, MA), unspecified OECD mineral media, and resazurin indicator served as the test medium. The media were prepared under nitrogen purge to exclude oxygen. The nominal test concentration was approximately 20.8 mg/ L, and loading in test vessels was 300 mL anaerobic sludge per liter of medium. The test temperature was 35   C, and the test was conducted in the dark. Information on test solution agitation was not provided. Twenty 160 mL serum bottles containing 100 mL of test solution ( purged with nitrogen after filling) with crimped butyl rubber tops served as test flasks. Each flask received all components at test initiation. Samples were taken from the test bottle on days 7, 14, 21, 28, 35, 42, 49, and 56, and from inoculum control bottles on days 7 and 56. For analysis, an aliquot was removed from each bottle and centrifuged. Both the supernatant and the solid biomass portion were analyzed via LC/ MS. The stock solution used to dose the biodegradation test systems was prepared at a concentration of 1,060 mg/ L, approximately twice the water solubility of PFOS. No day 0 samples were taken to determine starting concentrations.

RESULTS Degradation % after time: Not specified Results: No apparent PFOS biodegradation occurred over the 56 day period. ENV/ JM/ RD( 2002) 17/ FINAL 239 Kinetic ( for sample, positive and negative controls): Not specified Breakdown products ( yes/ no): None indicated Remarks: None CONCLUSIONS Remarks: PFOS is recalcitrant in the anaerobic system.

DATA QUALITY Reliability: Klimisch ranking = 2. Study not conducted according to Good Laboratory Practices. The stock solution used to dose the systems was prepared at twice the water solubility. No Day 0 samples were taken to determine starting concentrations.

REFERENCE The study was conducted at Springborn Laboratories, Inc., Wareham, Massachusetts, at the request of the 3M Company. Report completed 10/ 31/ 00. Lab Project number E01 0434. ENV/ JM/ RD( 2002) 17/ FINAL 240 BIODEGRADATION STUDY Title: Microbial Metabolism ( Biodegradation) studies of Perfluorooctane Sulfonate ( PFOS) IV. Pure Culture Study TEST SUBSTANCE Identity: Perfluorooctanesulfonate ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt) CAS # 2795 39 3 Remarks: White powder, 86.9% purity, also called PFOS or FC 95 METHOD Method/ guideline followed ( experimental/ calculated): Two study types were used ( Pure Culture and Closed Vial Headspace) and Springborn Laboratories, Inc./ Betts et al., 1974 is cited.

Test Type ( aerobic/ anaerobic): Aerobic GLP ( Y/ N): No Year study performed: 2000 Pure Culture Studies Contact time/ units: 7 days Inoculum: Four separate pure cultures from the American Type Culture Collection ( ATCC) were tested: Cunninghamella echinulata var. echinulata ( fungi, ATCC # 9244) Mucor circinelloides f. griseocyanus ( fungi, ATCC# 1207a) Phanerochaete chrysosporium ( fungi, ATCC # 24725) Streptomyces griseus ( actinomycete, ATCC # 13273)

Remarks: The test medium was soybean grits glucose ( SGG) and test vessel loading was 6 mL of Stage II cultures into 60 mL media, with 24 hours of agitation on a shaker table at 250 rpm prior to the addition of PFOS. The nominal test concentration was approximately 20.9 mg/ L. The test temperature was 26   C. The test vessel type was not noted. Each test flask received all necessary components at test initiation. All work utilized strict aseptic technique until harvest at day 7. Samples were taken on days 0 and 7. Analysis of the broth and cells collected via centrifugation was performed by LC/ MS. The stock solution used to dose the biodegradation test systems was prepared at a concentration of 1,060 mg/ L, approximately twice the water solubility of PFOS.

Degradation % after time: Not specified Results: The studies with Cunninghamella, Mucor, and Streptomyces did not provide any indication of biotransformation of PFOS. Possible biotransformation of PFOS ( 90% mass balance) by Phanaerochaete was noted, and closed vial studies were performed to confirm this.

Kinetic ( for sample, positive and negative controls): Not specified ENV/ JM/ RD( 2002) 17/ FINAL 241 Breakdown products ( yes/ no): None indicated Remarks: None Closed Vial Headspace Study Contact time/ units: 3 days Inoculum: Phanerochaete chrysosporium ( fungi, ATCC # 24725)

Remarks: The test medium was soybean grits glucose ( SGG) medium at 1/ 10 and 1/ 100 strength plus resazurin. The test concentration was 0.2 mg/ L. The incubation temperature was 26   C, and a shaker table in an environmental chamber at 250 rpm was used in incubation. Sterile 22 mL vials served as test vessels. Initial inoculum test vessel loading details were not noted. Each test flask received all necessary components at test initiation. Headspace was purged with oxygen and vials were immediately crimped. Samples were taken once on day 3. Analysis of the broth and cells collected via centrifugation was performed by LC/ MS. The stock solution used to dose the biodegradation test systems was prepared at a concentration of 1,011 mg/ L, approximately twice the water solubility of PFOS. No initial measured concentrations were taken in this closed vial study. Difficulties were encountered in maintaining aerobicity and only 3 days of exposure were maintained.

RESULTS Degradation % after time: Not specified Results: The results indicated no significant biotransformation of PFOS by Phanaerochaete fungi.

Kinetic ( for sample, positive and negative controls): Not specified Breakdown products ( yes/ no): None indicated Remarks: None CONCLUSIONS Remarks: It did not appear that the four species were capable of metabolizing PFOS.

DATA QUALITY Reliability: Klimisch ranking = 2. The stock solution used to dose the systems was prepared at twice the water solubility. There were no initial measured concentrations taken in the closed vial study. These studies were not conducted in accordance with Good Laboratory Practices.

REFERENCE The study was conducted at Springborn Laboratories, Inc., Wareham, Massachusetts, at the request of the 3M Company. Report completed 11/ 3/ 00. Lab Project number E01 0434. ENV/ JM/ RD( 2002) 17/ FINAL 242 BIODEGRADATION STUDY Title: The 18 day aerobic biodegradation study of perfluorooctanesulfonyl based chemistries.

TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or C8F17SO3  K+. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS# 2795 39 3)

Remarks: PFOS is a white powder. The original 3M product lot number was not noted. The PFOS was HPLC purified and assigned 3M standard identification # TCR 00017 046. Upon receipt at the testing laboratory, the test article was given a test, control, and reference ( TCR) number CA TCR02 014. The submitter suggests that an Interim Certificate of Analysis reports the purity to be 97.9%. All results in the study were calculated assuming 100% purity. However, in the study report, the purity is noted as 86.4%. No explanation of this discrepancy was given.

METHOD Method/ guideline followed: Based on EPA guidelines OPPTS 835.3200 Test Type: Aerobic GLP: No Year study performed: 2001 Contact time: 18 days Inoculum: Activated sludge collected 7/ 31/ 00 from the aeration basin at the Metro Wastewater Treatment Plant, St. Paul, MN. The MLSS was determined to be 2,280 mg/ L when first collected. The MLSS was stored at 4 º C for approximately 5 weeks prior to being used for this study. The sludge was allowed to settle and the solids used for inoculum. The settled sludge constituted approximately 20% of the volume (~ 200 mL) of the MLSS used.

Test Medium: Test flasks were prepared using a mineral salts medium defined in EPA Guideline OPPTS 835.3200. Methanol ( 1 mL per liter) was added per liter of mineral medium. Fifty mL of settled sludge was added per liter of mineral salts medium. Mineral medium plus sludge was prepared 9/ 7/ 00, while fresh medium without sludge ( abiotic controls) was prepared 8/ 10/ 00.

Remarks: Three types of samples were prepared for this study: blank sludge controls ( mineral medium, inoculum), abiotic controls ( mineral medium, PFOS), and test substance samples ( mineral medium, inoculum, PFOS). The test vessels, sterile 125 mL Nalgene polycarbonate culture flasks containing 25 mL of media, were set in duplicate. Additional quality control samples ( blanks) were prepared and analyzed as appropriate.

The test concentration used was 2.455 mg/ L. The samples were agitated at ~ 200 rpm at a temperature of 25 ± 3 º C. The test vessels were spiked with 6 µ L of a 10,230 mg/ L solution of PFOS in methanol yielding 2.455 mg/ L. Sampling was done on days 0 and 18. The day 0 test vessels were prepared and immediately placed in a freezer that was maintained at   20 º C until analyzed. After 18 days, the test vessels were removed from the incubator and frozen until final sample preparation by solid phase extraction ( SPE). Following thawing, test vessel contents were adjusted to 1% acetic acid and then passed through a ENV/ JM/ RD( 2002) 17/ FINAL 243 conditioned SEP VAC C18 6cc SPE cartridge. Methanol was then added to the emptied culture flask, shaken vigorously and then passed through the SPE cartridge to extract adsorbed analytes. A second methanol wash was collected separately for analysis to ensure quantitative extraction.

Quantitative analysis was conducted on an HP1100 high performance liquid chromatograph with mass spectrometer detector ( HPLC/ MSD) system. The MSD was operated in electrospray ionization in negative ion mode using selected ion monitoring ( SIM) for quantitation.

In addition to PFOS, the additional compounds quantified are specified below. In the case of the compounds that are potassium or ammonia salts, only the concentration of the fluorochemical anion was quantified and reported.

Compound Name Acronym Chemical Formula 2( N ethyl Perfluorooctane sulfonamido) ethyl alcohol N EtFOSE Alcohol C8F17SO2N( C2H5) CH2CH2OH 2( N ethyl Perfluorooctane sulfonamido) acetic acid N EtFOSAA C8F17SO2N( C2H5)( CH2COOH)

2( Perfluorooctane sulfonamido) acetic acid M556 C8F17SO2NH( CH2COOH)

N Ethyl perfluorooctane sulfonamide N EtFOSA C8F17SO2NH( C2H5) Perfluorooctane sulfinate, potassium salt PFOSulfinate C8F17SO2  K+

Perfluorooctanoate, ammonium salt PFOA C8F15COO NH4 +

Perfluorooctane sulfonamide FOSA C8F17SO2NH2 No reference substance was used. However, when the results from an EtFOSE alcohol study conducted at the same time are compared to the previous EtFOSE alcohol 35 day study, the viability of the microbial inoculum is confirmed.

RESULTS Degradation % after time: 0 Results: After 18 days, the analytical results demonstrate that after exposure to municipal wastewater treatment sludge, 2.455 mg/ L PFOS was not measurably degraded biotically or abiotically. Mass balance for PFOS test vessels was excellent and ranged from 104 108%.

Breakdown products: No Remarks: None CONCLUSIONS No loss of PFOS was demonstrated. Mass balance was 104 108%. The results from this study confirm the results from other aerobic biodegradation studies of PFOS. Submitters' Remarks: The submitters suggested a Klimisch data quality ranking of 2 because the study was conducted as a non GLP study, but with the understanding that good data quality objectives be met. ENV/ JM/ RD( 2002) 17/ FINAL 244 REFERENCE Lange, C. 2001a. The 18 day aerobic biodegradation study of perfluorooctanesulfonyl based chemistries. Pace Analytical Services, Inc. Minneapolis, MN. 3M Company. Minneapolis, MN. ENV/ JM/ RD( 2002) 17/ FINAL 245 STABILITY IN WATER STUDY Title: Hydrolysis Reactions of Perfluorooctane Sulfonate ( PFOS)

TEST SUBSTANCE Identity: Perfluorooctanesulfonate potassium salt. May also be referred to as: PFOS, PFOS potassium salt, 1 perfluorooctanesulfonic acid potassium salt, or 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluorosulfonate potassium salt ( CAS # 2795 39 3).

Remarks: 3M production lot number was 171. Test substance is a light colored powder at 25 º C.

METHOD Method/ guideline followed: Based on OPPTS: 835.2110.

Test Type: Hydrolysis as a function of pH GLP ( Y/ N): No Year study performed: 2001 Test sample preparation: Test solutions consisted of 1.0 mL buffered aqueous solutions at 6 pH levels ( 1.5, 3.0, 5.0, 7.0, 9.0, 11.0). The resulting PFOS concentration in all test samples ( sample triplicates and matrix spike samples) was approximately 500 µ g/ L. Samples were shielded from light during incubation at 50 º C for periods of 0 to 49 days. Control samples and blanks addressed potential non hydrolytic degradation routes.

Analytical Procedures: Samples were analyzed by quantitative HPLC/ MS.

Remarks: This study was conducted at 50 º C in order to facilitate hydrolysis. Rates derived at 50 º C were extrapolated to 25 º C by dividing by a factor of 10, which is valid for reactions with Arrhenius heats of activation near 18 kcal/ mole.

RESULTS Degradation %: Not applicable; no degradation was reported.

Half life ( t1/ 2):          	 Breakdown products ( yes/ no): No Remarks: The analytical results indicate no degradation of PFOS or dependence on pH. The mean and standard deviation of all observed PFOS concentrations, pooled over the 6 observed pH levels, indicate that the pseudo first order hydrolytic half life of PFOS is greater than 41 years.

CONCLUSIONS The analytical results indicate no degradation of PFOS or dependence on pH. The study indicates that the hydrolytic half life of PFOS in water is greater than 41 years. ENV/ JM/ RD( 2002) 17/ FINAL 246 Submitters' Remarks: The authors assigned a Klimisch ranking of 2 for the reliability of this study. The authors also noted that the study was well conducted, but not under GLP.

Reviewer's Remarks: None REFERENCE Hatfield, T. 2001a. Hydrolysis Reactions of Perfluorooctane Sulfonate ( PFOS). 3M Environmental Laboratory and Pace Analytical Services. Lab request number W1878. Minneapolis, MN. ENV/ JM/ RD( 2002) 17/ FINAL 247 BIODEGRADATION STUDY Title: The 35 day aerobic biodegradation study of PFOS TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or C8F17SO3  K+. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS# 2795 39 3)

Remarks: PFOS is a white powder. The original 3M production lot number was not noted. The PFOS was HPLC purified. An Interim Certificate of Analysis reports the purity to be 97.9%. All results in this report are based on a purity of 86.4%, however. The lower purity value was associated with a standard the laboratory was using during this study.

METHOD Method/ guideline followed: Based on EPA Guidelines OPPTS 835.3200, OPPTS 835.3210, OPPTS 835.5045.

Test Type: Aerobic GLP: No Year study performed: 2001 Contact time: 35 days Inoculum: Activated sludge collected 9/ 18/ 00 from the aeration basin at the Metro Wastewater Treatment Plant, St. Paul, MN. The suspended solids were allowed to settle for approximately 2 days at 4 C and the settled sludge, approximately 20% of the volume, was used to prepare cultures for the biodegradation study. The mixed liquor suspended solids, MLSS, was not noted.

Test medium: Test flasks were prepared using a mineral salts medium defined in EPA Guideline OPPTS 835.3200. Fifty mL of settled sludge was added per liter of mineral salts medium.

Remarks: Three types of samples were prepared for this test: blank sludge controls ( mineral medium, inoculum), abiotic controls ( mineral medium, PFOS), and test substance samples ( mineral medium, inoculum, PFOS).

Test vessels, sterile 125 mL Nalgene polycarbonate culture flasks containing 25 mL media, were set in duplicate. Additional quality control samples ( blanks) were prepared and analyzed as appropriate.

The test concentration used was 2.582 mg/ L. The samples were agitated at 200 rpm at 25 ± 3 º C. Test vessels were spiked 6 µ L of 10,760 mg/ L solution of PFOS in methanol yielding a 2.582 mg/ L PFOS solution. Samples were taken on days 0, 2, 5, 7, 14, and 35.

The day zero test vessels were prepared and immediately placed in a freezer that was maintained at   20 ± 7 º C. Upon removal from the incubator, test vessels on other days were either immediately frozen, or prepared by solid phase extraction ( SPE). Following thawing, if needed, test vessel contents were adjusted to 1% acetic acid and then passed through a conditioned SEP VAC C18 6cc SPE cartridge containing a plug of quartz wool to deter plugging. Methanol was then added to the emptied culture flask, shaken ENV/ JM/ RD( 2002) 17/ FINAL 248 vigorously and then passed through the SPE cartridge to extract adsorbed analytes. A second methanol wash was then collected separately for analysis to ensure quantitative extraction.

Quantitative analysis was conducted on an HP1100 high performance liquid chromatograph with mass spectrometer detector ( HPLC/ MSD) system. The MSD was operated in electrospray ionization in negative ion mode using selected ion monitoring ( SIM) for quantitation.

In addition to the parent, PFOS, the compounds below were quantified. In the case of the compounds that are potassium or ammonia salts, only the concentration of the fluorochemical anion was quantified and reported.

Compound Name Acronym Chemical Formula 2( N ethyl Perfluorooctane sulfonamido) ethyl alcohol N EtFOSE Alcohol C8F17SO2N( C2H5) CH2CH2OH 2( N ethyl Perfluorooctane sulfonamido) acetic acid N EtFOSAA C8F17SO2N( C2H5)( CH2COOH)

2( Perfluorooctane sulfonamido) acetic acid M556 C8F17SO2NH( CH2COOH)

N Ethyl perfluorooctane sulfonamide N EtFOSA C8F17SO2NH( C2H5) Perfluorooctane sulfinate, potassium salt PFOSulfinate C8F17SO2  K+

Perfluorooctanoate, ammonium salt PFOA C8F15COO NH4 +

Perfluorooctane sulfonamide FOSA C8F17SO2NH2 RESULTS Degradation % after time: 0 Results: The analytical results demonstrate that when exposed to municipal wastewater treatment sludge for 35 days, the 2.582 mg/ L PFOS samples generated no quantifiable degradation products. PFOS was recovered at 2.553 ± 0.102 mg/ L in the pooled study samples and at 2.653 ± 0.083 mg/ L in the pooled abiotic samples. The measured concentration of PFOS was always 100 ± 7% of the expected concentration.

Breakdown products: No Remarks: None CONCLUSIONS The six sample point screening study established that PFOS is not biodegraded by the microbial populations of the municipal waste treatment inoculum used under the conditions tested.

Submitters' remarks: The submitters' assigned a data quality rating of 2 for this study since it was conducted as a non GLP study, but with the understanding that good data quality objectives be met.

No sample matrix spikes were included in this study. However, PFOS was recovered at expected concentrations, and previous duplicated results obtained during a related project showed excellent PFOS recoveries from sludge using the same extraction method. ENV/ JM/ RD( 2002) 17/ FINAL 249 A series of positive controls were not run with this study. However, the sludge used in this study was also used for the preparation of samples in other biodegradation studies, and in some of those studies, preliminary results demonstrated positive biological activity for degradation of test samples.

REFERENCE Lange, C. 2001b. The 35 day aerobic biodegradation study of PFOS. Pace Analytical Services, Inc. Minneapolis, MN. 3M Company. Minneapolis, MN. ENV/ JM/ RD( 2002) 17/ FINAL 250 PHOTODEGRADATION STUDY Title: Screening studies on the aqueous photolytic degradation of potassium perfluorooctane sulfonate ( PFOS)

TEST SUBSTANCE Identity: Perfluorooctanesulfonate potassium salt. May also be referred to as: PFOS, PFOS potassium salt, 1 perfluorooctanesulfonic potassium salt, or 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoropotassium salt.

Remarks: 3M production lot number 171. The test substance is a light colored powder at 25 º C. The purity was determined to be 86.4% by LC/ MS, 1H HMR, 19F NMR, and elemental techniques.

METHOD Method/ guideline followed: Based on OPPTS: 835.5270 and OECD Draft Document " Phototransformation of Chemicals in Water   Direct and Indirect Photolysis," August 2000.

Test Type: Direct and indirect photolysis GLP ( Y/ N): No Year study performed: 2001 Light Source: Suntest CPS+ or Suntest XLS+ lamp Light Spectrum ( nm): 290 800 nm Relative Intensity based on Intensity of Sunlight: 680 w/ m2 Spectrum of substance ( max lambda, max epsilon and epsilon 295): A UV/ Vis spectrum of a saturated aqueous solution of PFOS was recorded between 190 and 110 nm.

Test sample preparation: Aliquots of PFOS were added to 3 separate sets of VOA screw cap vials ( exposed, unexposed, and control vials) containing 5 mL of appropriate matrix. Test vials were placed in the photoreactor. Control vials were wrapped in aluminum foil, sealed in a plastic bag, and placed in the photoreactor.

Analytical Procedures: Samples were analyzed by quantitative LC/ MS and GC/ MS techniques.

Remarks: The duration of the study was 67 167 hours. The test media used were water, H2O2/ water ( 1: 1 molar equivalent), Fe2O3/ water ( Fe3+ at 24X molar excess), Fe2O3/ water with H2O2, and commercial ( Aldrich) humic material prepared as in OPPTS 835.5270. All tests included a series of unexposed controls ( kept in the dark) for the evaluation of any degradation reactions occurring without the presence of light. Solvent, matrix, and control blanks and spikes were tested under each condition.

RESULTS Concentration of Substance: Not stated ENV/ JM/ RD( 2002) 17/ FINAL 251 Temperature  C: 25 ± 3 Direct photolysis: No decomposition was observed.

Indirect photolysis: Data obtained from the Fe2O3 matrix samples ( with and without H2O2) were pooled to provide sufficient data to estimate the minimum half life. The mean standard deviation of these data indicate that the minimum environmental half life of PFOS due to indirect photolysis at 25 º C is greater than 3.7 years.

Breakdown products ( yes/ no): No Remarks: No evidence of direct or indirect photolysis of PFOS was observed under any of the conditions tested. Direct photolytic decomposition of PFOS was not observed based on loss of starting material, nor were any of the predicted degradation products detected above their limits of quantitation.

CONCLUSIONS No evidence of direct or indirect photolysis of PFOS was observed under any of the conditions tested. The mean and standard deviation of the observed PFOS concentrations in an aqueous Fe2O3/ H2O2 matrix indicate that the indirect photolytic half life of PFOS at 25 º C is greater than 3.7 years.

Submitters' Remarks: The authors assigned a Klimisch ranking of 2 for the data quality of this study.

Reviewer's Remarks: None REFERENCE Hatfield, T. 2001b. Screening studies on the aqueous photolytic degradation of potassium perfluorooctane sulfonate ( PFOS). 3M Environmental Laboratory. 3M Company, St. Paul, MN. Report number W2775. ENV/ JM/ RD( 2002) 17/ FINAL 252 BIOCONCENTRATION IN FISH Title: Perfluorooctanesulfonate, potassium salt ( PFOS): A flow through bioconcentration test with bluegill ( Lepomis macrochirus)

TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: Sample from 3M production lot number 217. The test substance was a white powder. The purity was determined to be 86.9% by LC/ MS, 1H NMR, 19F NMR, and elemental analysis techniques.

METHODS Method/ guideline followed: USEPA OPPTS 850.1730 and OECD 305 Test type: Flow through exposure with flow through depuration phase ( flow rate: approximately 6 volume additions per 24 hours)

GLP: Yes Year study performed: 2001 Species: Bluegill sunfish ( Lepomis macrochirus); mean length = 62 mm ( 56 66 mm); mean weight: 2.70 g ( 2.03 3.32 g); age ~ 7 months old Supplier: Osage Catfisheries, Inc. Osage Beach, Missouri Concentrations tested: 0, 0.086, and 0.87 mg/ L Uptake period: 62 days ( 0.086 mg/ L exposure); 35 days ( 0.087 mg/ L exposure   this exposure ended after 35 days due to fish mortality)

Depuration period: 56 days ( 0.086 mg/ L exposure); None ( 0.87 mg/ L exposure)

Analytical monitoring: Analyses of test solutions and fish tissues were performed at Wildlife International, Ltd. Water samples were diluted and analyzed by HPLC with a single quadruple mass spectrometric detection. Tissue samples were homogenized, extracted, diluted, and analyzed by HPLC with triple quadruple mass spectrometric detection. When determining the concentration of the test substance in the samples, the same and most prominent peak response for perfluorooctanesulfonate was used. No attempt was made to quantify on the basis of individual isomeric components. The LOQ was 0.05 mg/ L for water in this study. For tissue samples, the LOQ was calculated on an individual basis for each sample since each entire submitted sample, of differing weight, was extracted without an adjustment to constant weight.

Recovery was excellent in both water and fish tissues, ranging from 84.9 to 122% of fortification levels. Analytical results were not corrected for procedural recovery.

Collection of tissue samples: Fish were collected from test chambers by random selection at 12 time points during the 62 day uptake phase. They were euthanized, blotted dry, weighed, and measured. Fish ENV/ JM/ RD( 2002) 17/ FINAL 253 were then rinsed with dilution water, blotted dry again, and dissected into edible and nonedible fractions. The fractions were individually weighed. The head, fins, and viscera were considered to be nonedible tissue. The remaining tissue including skin was considered to be edible tissue.

Statistical methods: Whole fish concentrations were calculated based on the sum of the edible and nonedible parts. Steady state bioconcentration BCF values calculated from the tissue concentrations at apparent steady state divided by the mean water concentration. Tissue concentrations were considered to be at apparent steady state if 3 or more consecutive sets of tissue concentrations were not significantly different ( p> 0.05). Tissue concentrations were evaluated for normality and homogeneity of variance using the Shapiro Wilk's test and Bartlett's test, respectively. If the data did not meet the assumptions, data were transformed in an attempt to correct the data. Mean tissue concentrations were then compared using ANOVA and Dunnett's test.

The kinetic bioconcentration factor ( BCFK), uptake rate ( k1), and depuration rate ( k2) were calculated for the edible, nonedible, and whole fish exposed to 0.086 mg/ L PFOS using BIOFAC computer software. BIOFAC is a nonlinear parameter estimate routine, which estimates rate constants from a set of sequential time concentration data. These rate constants were then used to calculate a BCFK ( BCFK = k1/ k2).

Test conditions: Stainless steel aquaria ( 104 L) filled with approximately 80 L solution were used as exposure vessels. One vessel per concentration was used. The loading rate was 0.48 g fish/ L/ day ( 90 fish per vessel). The fish were fed flake food ( Ziegler Brothers, Inc., Gardners, PA) and given 16 hours of light and 8 hours of dark with a 30 minute transition period. The light intensity was 278 lux at the surface of the negative control vessel at test initiation.

Two stock solutions were prepared at 10 and 100 mg a. i./ L. Stock solutions were stirred with an electric top down mixer to aid in the solubilization of the test substance. After mixing, the stocks appeared clear and colorless. Stocks were prepared at approximately weekly intervals during the uptake phase. Stocks were injected into the diluter mixing chambers at a rate of 3.5 mL/ minute where they were mixed with dilution water at a rate of 350 mL/ minute to achieve the desired test concentrations. All final test solutions appeared clear and colorless. The dilution water was moderately hard well water with specific conductance of 313 umhos/ cm, hardness of 130 mg/ L, alkalinity of 178, and pH of 8.1. During the test, the dissolved oxygen levels ranged from 6.4 8.6 mg/ L, the temperature ranged from 21.7 to 22.0 º C, and the pH ranged from 7.9 to 8.2.

Remarks: None RESULTS Bioconcentration factors ( BCF):

0.086 mg/ L apparent steady state BCF:

Edible: 484 Nonedible: 1124 Whole fish: 856 0.87 mg/ L( study ended prior to achieving steady state) BCF:

Edible: 136 ENV/ JM/ RD( 2002) 17/ FINAL 254 Nonedible: 386 Whole fish: 278 BIOFAC Estimates ( using 0.086 mg/ L exposure):

Edible Nonedible Whole fish BCFK: 1866 4312 3614 Time to reach 50% clearance: 146 days 133 days 152 days PFOS Concentrations in Tissues of Bluegill Exposed to 0.086 mg/ L: ( Values are from 4 individual fish at each sample period.)

Uptake day Edible tissue, mg/ kg Nonedible tissue, mg/ kg Whole fish conc., mg/ kg 0 ( 4 hours) 0.167, 0.155, 0.144, 0.182 0.415, 0.519, 0.417, 0.497 0.293, 0.351, 0.286, 0.363 1 0.734, 0.726, 0.631, 0.806 1.68, 1.85, 1.72, 2.07 1.26, 1.34, 1.29, 1.53 3 1.73, 2.07, 2.03, 2.11 4.59, 5.50, 5.47, 5.97 3.21, 4.04, 4.18, 4.38 7 3.73, 4.25, 4.73, 6.25 10.2, 10.6, 11.9, 15.2 7.33, 7.66, 8.73, 11.4 14 11.4, 9.07, 13.7, 12.6 27.3, 23.2, 35.3, 32.6 20.2, 16.9, 26.0, 24.6 21 11.7, 12.0, 12.9, 10.6 33.3, 22.7 24.6, 24.4 23.3, 18.4, 19.8, 18.5 28 18.3, 13.7, 23.9, 23.1 49.4, 40.7, 65.3, 57.9 35.3, 29.2, 45.4, 44.1 35 22.6, 27.7, 23.8, 20.6 67.1, 73.3, 62.0, 59.1 46.3, 53.8, 46.6, 40.9 42 27.6, 25.3, 21.2, 27.6 64.0, 68.1, 54.4, 79.6 50.1, 49.4, 40.9, 56.3 49 33.3, 36.2, 39.0, 30.6 85.0, 95.1, 93.1, 77.7 62.8, 69.6, 70.8, 57.4 56 48.3, 38.9, 44.1, 38.3 122, 94.2, 73.2, 106 90.6, 71.6, 63.3, 74.8 62 42.4, 66.2, 42.2, 39.2 101, 112, 105, 96.4 77.0, 92.7, 79.6, 73.1 Depuration day 14 48.5, 31.8, 31.6, 42.0 124, 79.4, 81.8, 113 90.3, 60.4, 61.6, 85.3 28 26.0, 33.3, 38.7, 55.8 85.7, 95.1, 85.7, 94.8 58.2, 70.1, 68.1, 81.1 42 24.1, 31.2, 30.0, 33.0 71.7, 80.6, 78.3, 82.1 51.4, 61.4, 61.0, 62.2 56 21.1, 37.6, 32.9, 31.2 57.7, 80.3, 85.4, 84.4 41.6, 66.5, 65.8, 62.1 ENV/ JM/ RD( 2002) 17/ FINAL 255 PFOS Concentrations in Tissues of Bluegill Exposed to 0.87 mg/ L: ( Values are from 4 individual fish at each sample period.)

Uptake day Edible tissue, mg/ kg Nonedible tissue, mg/ kg Whole fish conc., mg/ kg 0 ( 4 hours) 1.46, 1.48, 1.19, 1.39 3.52, 4.37, 4.22, 4.06 2.71, 3.08, 2.84, 2.89 1 4.68, 6.59, 5.56, 5.64 11.1, 14.2, 13.3, 12.1 8.00, 10.9, 10.2, 9.47 3 17.3, 15.8, 19.0, 20.8 39.3, 42.0, 43.8, 51.8 30.5, 30.7, 34.5, 39.1 7 42.0, 44.0, 57.7, 46.8 100, 102, 102, 120 74.9, 77.0, 85.3, 89.8 14 87.1, 81.6, 90.7, 73.3 177, 207, 245, 214 141, 157, 180, 158 21 79.4, 117, 104, 102 201, 278, 246, 229 146, 210, 185, 172 281 102, 131, 107, 133 289, 372, 320, 361 205, 267, 232, 263 1Sampling of fish stopped after Uptake day 28 due to mortality.

Remarks: Test organism mortality was none in the negative control ( during both the uptake and depuration phases). At 0.086 mg/ L, one fish died after 49 days and one died after 59 days. None died during the depuration phase. At 0.87 mg/ L, mortality was first noted on day 9 and continued through day 35 of the uptake phase, at which time all fish either died or had been sampled.

Was control response satisfactory: Yes Statistical results: None CONCLUSIONS PFOS bioconcentrated in the tissues of bluegill sunfish during this study. Apparent steady state was attained on Day 49 for the fish exposed to 0.086 mg a. i./ L. Although Day 49, 56, and 62 tissue residues were not statistically significantly different, PFOS concentrations appeared to be still increasing during this time. Apparent steady state BCF values for edible, nonedible, and whole fish tissues were calculated to be 484, 1124, and 859, respectively.

PFOS depurated slowly. The BIOFAC estimates for the time to reach 50% clearance for edible, nonedible, and whole fish tissues were 146, 133, and 152 days, respectively.

Submitters' remarks: The authors assigned this study a Klimisch data reliability ranking of 1.

Reviewers' remarks: None REFERENCE Drottar, K., VanHoven, R., and H. Krueger. 2001. Perfluorooctanesulfonate, potassium salt ( PFOS): A flow through bioconcentration test with bluegill ( Lepomis macrochirus). Wildlife International, Limited. Project number 454A 134. 3M Company. St. Paul, MN. ENV/ JM/ RD( 2002) 17/ FINAL 256 Annex 6. Robust Summaries of Toxicology and Human Biomonitoring Studies PHARMACOKINETIC STUDIES Title: Absorption of FC 95 14C in Rats after a Single Oral Dose ( 1979)

TEST SUBSTANCE Identity: Potassium perfluorooctylsulfonate, CAS 2795 39 3 Remarks: FC 95 14C ( carbon 14 label alpha to sulfur atom, Riker Isotope Inventory Number 442). The specific activity is 0.459 + 0.008 uCi/ mg. Thin layer and column chromatography showed the FC 95 14C to be at least 99% radiochemically pure. The FC 95 14C was found to be suitable for metabolism studies. ( Synthesis described in Johnson and Behr, 1979).

METHOD Method/ guideline followed: NA Test type: in vivo Species/ strain/ cell type or line: rat, Charles River CD Sex: male Age and body weight range of animals used: 8 weeks, bw mean 285 g ( range 243 315)

Number of animals/ sex/ dose: 24 Route of administration: oral Vehicle: 0.9% NaCL solution containing 1.2 mg FC 95 14C/ 2.0 ml Doses: 4.2 mg/ kg average, single dose Excretion routes, body fluids, and tissues monitored and/ or sampled during study: red blood cells, plasma, urine, feces, spleen, digestive tract plus contents ( esophagus, stomach, small intestine, large intestine, and colon), and carcass Statistical methods used: mean, log mean concentration versus time least squares line Method remarks: Rats were conditioned to individual metal metabolism cages for 24 hours prior to dosing. Rats were allowed free access to Purina Ground Chow and water before and after dosing. Each non fasted rat was weighed immediately before being given a single oral dose of FC 95 14C. The dosing solution was prepared by adding ~ 200 mg of FC 95 14C to 0.9% NaCl, shaking for one half hour at moderate speed in a mechanical shaker, and centrifuging. The supernatant was removed and used for dosing solution. The carbon 14 content of the dosing solution was determined by direct counting. The dose was delivered with a 2.0 cc glass syringe ( Trylon) fitted with a stainless steel intubation tube. Recovery of total carbon 14 from series of FC 95 14C spiked, blank biological samples was used to adjust the recovery of the test samples to account for label loss during the experimental manipulations. ENV/ JM/ RD( 2002) 17/ FINAL 257 Groups of three rats were sacrificed by exsanguination at 1, 2, 6, 12, 24, 48, 96, and 144 hours post dose. Rats were anesthetized with diethyl ether and blood was drawn from the descending aorta of each rat and immediately transferred to a heparinized tube. Plasma was prepared promptly by centrifugation. In addition to plasma and red blood cells, total urine, total feces, spleen, digestive tract plus contents ( esophagus, stomach, small intestine, large intestine, and colon), and remainder of carcass were saved from each of the three rats in the 24 and 48 hours post dose groups for carbon 14 analysis.

RESULTS Detailed results: After a single oral dose of FC 95 14C ( mean dose, 4.2 mg/ kg) in solution to groups of three male rats, at least 95% of the total carbon 14 is systemically absorbed at 24 hours. The half life for elimination of total carbon 14 from plasma is 7.5 days.

The digestive tract and contents contained on the average, 3.45% of the dose. The mean fecal excretion is 1.55% of the dose at 24 hours and 3.24% at 48 hours. At 24 hours, the mean sum of total carbon 14 in feces and digestive tract plus contents is 5% of the dose. Some of this 5% likely represents systemically absorbed carbon 14 present either in the digestive tract tissues or in the digestive tract contents as a result of excretion. The data from the 48 hour post dose group of rats are consistent with the 24 hour post dose data. Thus, at least 95% of the FC 95 14C dose was absorbed from solution after administration to nonfasted rats. The major portion of the radioactivity recovered was found in the carcass. The carcass data are not as reliable as the other tissue data since large volume homogenates were necessary and homogeneity of sample aliquots was difficult to assure. There is some excretion of total carbon 14 in urine ( 1 2%/ day). The spleens from the 24 hour and 48 hour post dose rats were analyzed for total carbon 14 content, and the percent of the dose in the whole organ was ~ 0.2%. The concentrations of total carbon 14 in red blood cells and plasma were compared. The mean ratio of red blood cell to plasma concentration at 24 and 48 hours is 0.25 and 0.39, respectively. Thus, at 24 and 48 hours after a single oral dose of FC 95 14C, there is no selective retention of carbon 14 in red blood cells.

The half life of elimination from plasma was determined by analysis of plasma samples from groups of three rats at 1, 2, 6, 12, 24, 48, 96, and 144 hours after a single oral dose of FC 95 14C. The log of mean concentration versus time for these data was plotted. The least squares line through the individual points from 24 to 144 hours for these data fits the equation: Cp = 15.65e^( 0.00387t) where Cp is plasma concentration. The half life of elimination from plasma is 179 hours ( 7.5 days). Thus, elimination from plasma of total carbon 14 after a single oral dose of FC 95 14C is slow.

Metabolites measured: none CONCLUSIONS agree REFERENCE Absorption of FC 95 14C in Rats after a Single Oral Dose. Riker Laboratories, Inc., Subsidiary of 3M, St. Paul, MN. Project No. 890310200. Johnson, JD, Gibson, SJ, and Ober, RF, October 26, 1979. ENV/ JM/ RD( 2002) 17/ FINAL 258 Title: Extent and Route of Excretion and Tissue Distribution of Total Carbon 14 in Rats after a Single Intravenous Dose of FC 95 14C ( 1979)

TEST SUBSTANCE Identity: Potassium perfluorooctylsulfonate, CAS 2795 39 3 Remarks: FC 95 14C ( carbon 14 label alpha to sulfur atom, Riker Isotope Inventory Number 442). The specific activity is 0.459 + 0.008 uCi/ mg. Thin layer and column chromatography showed the FC 95 14C to be at least 99% radiochemically pure. The FC 95 14C was found to be suitable for metabolism studies. ( Synthesis described in Johnson and Behr, 1979).

METHOD Method/ guideline followed: NA Test type: in vivo Species/ strain/ cell type or line: rat, Charles River CD Sex: male Age and body weight range of animals used: 8 weeks, bw mean 288 g ( range 262 303)

Number of animals/ sex/ dose: 6 Route of administration: iv, via tail vein Vehicle: 0.9% NaCL solution containing 1.2 mg FC 95 14C/ 2.0 ml Doses: 4.2 mg/ kg average, single dose Excretion routes, body fluids, and tissues monitored and/ or sampled during study: urine, feces, liver, plasma, kidney, lung, spleen, bone marrow, adrenals, skin, testes, muscle, fat, eye, brain Statistical methods used: mean, standard deviation Method remarks: Rats were conditioned to individual metal metabolism cages for 24 hours prior to dosing. The rats were allowed free access to Purina Ground Chow and water before and after dosing. Each rat was weighed, anesthetized with diethyl ether, then given a single iv dose using a 3.0 cc disposable plastic syringe fitted with a 26 gauge 1/ 2" needle. Urine and feces were collected at intervals for each of the six rats for 89 days. At 89 days post dose, the rats were anesthetized with diethyl ether; blood was drawn from the descending aorta, animals were sacrificed by exsangination, and tissue samples were collected.

RESULTS Detailed results: By 89 days post dose, mean urinary excretion was 30.2+ 1.5% of total C 14 administered. Mean cumulative fecal excretion was 12.6+ 1.2%. The authors note that radioactive content in feces was too low to measure after 64 days. At day 89, mean tissue C 14 concentrations above one ug FC 95 14C ENV/ JM/ RD( 2002) 17/ FINAL 259 equivalents/ g were as follows: liver, 20.6; plasma, 2.2; kidney, 1.1; and lung, 1.1. Other tissues such as muscle, skin, bone marrow, and spleen had concentrations ranging from 0.2 to 0.6 ug/ g. There was a difference in C 14 content of subcutaneous fat ( 0.2 ug/ g) and abdominal fat (<= 0.08 ug/ g). Very little C 14 was found in whole eye ( 0.16 ug/ g) and no detectable C 14 was found in brain. Only liver and plasma contained a substantial percentage of dose at 89 days post dose, 25.21% and 2.81%, respectively. The low levels of radioactivity found for kidney, lung, testes, and spleen are due in part to blood still contained in these organs when homogenized.

Mean Excretion of Total Carbon 14 in Urine Over Time Collection Period Percent Dose ( Days) During Period 0 0.5 0.91 0.5 1 0.77 1 2 1.21 2 3 1.03 3 4 0.93 4 5 0.83 5 6 0.71 6 7 0.76 7 8 0.75 8 9 0.68 9 10 0.68 10 11 0.59 11 12 0.58 12 13 0.59 13 14 0.55 14 15 0.54 15 16 0.51 16 17 0.48 17 18 0.43 18 19 0.39 19 21 0.84 21 23 0.78 23 25 0.66 25 27 0.68 27 29 0.68 29 32 0.86 32 36 1.05 36 40 0.99 40 43 0.75 43 47 0.92 47 50 0.68 50 54 0.78 54 57 0.61 57 61 0.79 61 69 1.50 69 78 1.64 78 89 2.08 Total 30.2 Mean Excretion of Total Carbon 14 in Feces Over Time ENV/ JM/ RD( 2002) 17/ FINAL 260 Collection Period Percent Dose ( Days) During Period 0 0.5 0.049 0.5 1 0.842 1 2 0.795 2 3 0.649 3 4 0.656 4 5 0.577 5 6 0.510 6 7 0.588 7 8 0.482 8 9 0.421 9 10 0.387 10 11 0.370 11 12 0.296 12 13 0.310 13 14 0.281 14 15 0.276 15 16 0.272 16 17 0.187 17 18 0.163 18 19 0.129 19 21 0.311 21 23 0.302 23 25 0.262 25 27 0.208 27 29 0.202 29 32 0.223 32 36 0.526 36 50 1.530 50 64* 0.833 Total 12.6 * The radioactive content of the feces was too low to measure after 64 days.

Metabolites measured: none.

CONCLUSIONS agree REFERENCE Extent and Route of Excretion and Tissue Distribution of Total Carbon 14 in Rats after a Single Intravenous Dose of FC 95 14 C. Riker Laboratories, Inc., Subsidiary of 3M, St. Paul, MN. Johnson, JD, Gibson, SJ, and Ober, RE , December 28, 1979. ENV/ JM/ RD( 2002) 17/ FINAL 261 Title: Cholestyramine Enhanced Fecal Elimination of Carbon 14 in Rats after Administration of Ammonium [ 14C] Perfluorooctanoate or Potassium [ 14C] Perfluorooctanesulfonate ( 1984)

TEST SUBSTANCE Identity: Potassium perfluorooctanesulfonate ( 14C PFOS) Ammonium perfluorooctanoate ( 14C PFO)

Remarks: 14C PFOS: sp act 0.46 uCI/ mg, radiochemical purity > 99%, 14C label in PFOS is adjacent to sulfur 14C PFO: sp act 0.51 uCI/ mg, radiochemical purity > 98%

METHOD Method/ guideline followed: NA Test type: in vivo Species/ strain/ cell type or line: rat, Charles River CD Sex: male Age and body weight range of animals used: 12 weeks, 300 342 g Number of animals/ sex/ dose: 5 Route of administration: iv Vehicle: 0.9% NaCl, 2 ml/ rat Doses: Potassium [ 14C] Perfluorooctanesulfonate ( PFOS): 3.4 mg/ kg mean, single dose, 0.56 mg/ ml PFOS control animals: 3.5 mg/ kg mean Ammonium [ 14C] Perfluorooctanoate ( PFO): 13.3 mg/ kg mean, single dose, 2.1 mg/ ml PFO control animals: 13.5 mg/ kg mean Excretion routes, body fluids, and tissues monitored and/ or sampled during study: Urine, plasma, red blood cells, liver Statistical methods used: mean, standard deviation, Student's t test Method remarks: Rats were housed in individual stainless steel metabolism cages and fasted with free access to water for 24 hrs prior to receiving the fluorochemicals. The radiolabeled compounds were administered as single intravenous doses ( lateral tail vein). Two ml of dosing solution was administered to each rat. Ten rats were dosed with each compound. Five rats from each group were fed cholestyramine ( dried and ground resin Z 620), 4% in feed ( Purina Lab Chow), for 14 days after administration of PFO and for 21 days after administration of PFOS. Control rats were administered radiolabeled fluorochemical but were not treated with cholestyramine. In order to allow comparison of the radiometric results on an absolute basis, the ENV/ JM/ RD( 2002) 17/ FINAL 262 radiolabel doses were not adjusted for individual body weights. Urine and feces samples were collected at intervals for individual rats in each group until 14 days after 14C PFO administration and 21 days after 14C PFOS administration. At these times, rats were anesthetized with diethyl ether and exsanguinated by drawing blood from the descending aorta. Plasma and red blood cells were prepared promptly by centrifugation. Liver was collected as the whole organ and stored frozen until analysis.

RESULTS Detailed results: After 21 days of cholestyramine treatment, the mean percentage of 14C PFOS dose eliminated via feces ( 75.8 + 5.0) was 9.5 fold the mean percentage of dose eliminated via feces by control rats ( 8.0 + 0.8). After adjustment for the amount of carbon 14 excreted in urine ( 18% for controls and 5% for cholestyramine treated), the amounts of carbon 14 remaining to be excreted are 19% for cholestyraminetreated rats and 74% for control rats. After 14C PFOS administration, the mean liver carbon 14 content at 21 days represents 11% and 40% of the dose for cholestyramine treated and control rats, respectively. Mean plasma and red blood cell carbon 14 concentrations are significantly lower after 21 days of cholestyramine treatment.

After 14 days of cholestyramine treatment, the mean percentage of 14C PFO dose eliminated via feces ( 43.2 + 5.5) was 9.8 fold the mean percentage of dose eliminated via feces by control rats ( 4.4 + 1.0). After adjustment for the amount of carbon 14 excreted in urine ( 67% for controls and 41% for cholestyramine treated), the amounts of carbon 14 remaining to be excreted are 16% for cholestyraminetreated rats and 28% for control rats. After 14C PFO administration, the mean liver carbon 14 content at 14 days represents 4% and 8% of the dose for cholestyramine treated and control rats, respectively. Mean plasma and red blood cell carbon 14 concentrations are significantly lower after 14 days of cholestyramine treatment.

Carbon 14 Concentration ( expressed as ug eq/ g tissue or ml fluid) Treatment Group Liver Plasma Red Blood Cells 14C PFOS Cholestyramine 9.4+ 1.6* 0.9+ 0.1* 0.3+ 0.1* Control 35.6+ 5.6 6.9+ 0.6 1.8+ 0.4 14C PFO Cholestyramine 12.1+ 2.1* 5.1+ 1.7* 1.8+ 0.7* Control 22.3+ 6.2 14.7+ 6.8 4.2+ 2.4 * Significantly different from control values ( p< 0.05)

The authors conclude that the high concentration of 14C PFOS or 14C PFO in liver at 2 to 3 weeks after dosing and the fact that cholestyramine treatment enhances fecal elimination of carbon 14 by nearly 10 fold suggest that there is a considerable enterohepatic circulation of 14C PFOS and 14C PFO.

Metabolites measured: none CONCLUSIONS agree ENV/ JM/ RD( 2002) 17/ FINAL 263 REFERENCE Johnson, J. D., Gibson, SJ, and Ober, RE ( 1984). Cholestyramine Enhanced Fecal Elimination of Carbon 14 in Rats after Administration of Ammonium [ 14C] Perfluorooctanoate or Potassium [ 14C] Perfluorooctanesulfonate. Fundamental and Applied Toxicology 4, pages 972 976.

See also Johnson, J. D., Gibson, SJ, and Ober RE ( 1984). Enhanced elimination of FC 95 14C and FC 143 14C in rats with cholestyramine treatment. Project No. 8900310200, Riker Laboratories, Inc. St. Paul, MN. ENV/ JM/ RD( 2002) 17/ FINAL 264 Title: Oral ( Gavage) Pharmacokinetic Study of PFOS in Rats, Analytical Laboratory Report, Determination of the Presence and Concentration of Perfluorooctanesulfonate ( PFOS) in Serum, Liver, Urine, and Feces Samples TEST SUBSTANCE Identity: Perfluorooctylsulfonate, potassium salt, CAS 2795 39 3 Remarks: Purity 86.9%, Lot # 217 METHOD Method/ guideline followed: This study was conducted in compliance with United States Food and Drug Administration ( FDA) Good Laboratory Practice ( GLP) Regulations 21 CFR Part 58, with the exceptions noted on page 3 of report.

Test type: in vivo Species/ strain/ cell type or line: rat/ Sprague Dawley/ pregnant Crl: CD( R) BR VAF/ Plus( R)

Sex: F0: female, F1: both Age and body weight range of animals used: 60 days, 200 225 g Number of animals/ sex/ dose: F0: 16, F1: 5 male and 5 female pups/ litter Route of administration: oral Vehicle: 0.5% Tween( R) 80 in R. O. deionized water, dosage volume 5 ml/ kg Doses: 0 ( vehicle), 0.1, 0.4, 1.6, and 3.2 mg/ kg/ day in volume of 5 ml/ kg, once daily beginning 42 days prior to cohabitation, and continued through day 14 or day 20 of presumed gestation. Only the F0 females were dosed.

Excretion routes, body fluids, and tissues monitored and/ or sampled during study: F0 urine, feces, serum, liver. F1 liver and serum.

Statistical methods used: mean and standard deviation.

Method remarks: Serum, urine, and feces specimens were collected from adult female rats ( F0 dams) before mating and at gestation day ( GD) 7, GD 15 and GD 21. Liver specimens were collected from F0 dams at termination of the study ( GD 21). A total of 54 pooled serum and liver specimens were collected from fetuses on GD 21. Specimens were sent to the 3M Environmental Laboratory and the contract labs to be analyzed for PFOS. ENV/ JM/ RD( 2002) 17/ FINAL 265 RESULTS Detailed results: Average Results for the Analysis of Serum Samples ( ug/ ml) 0 mg/ kg 0.1 mg/ kg 0.4 mg/ kg 1.6 mg/ kg 3.2 mg/ kg Day 0 dam 0.0723 8.89 40.7 160 318 Day 7 dam 0.126 7.82 40.9 154 105 Day 15 dam 0.0926 8.80 41.4 156 275 Day 21 dam 0.0714 4.24 26.2 136 155 Day 21 Fetal 0.125 9.07 34.3 101 165 Average Results for the Analysis of Liver Samples ( GD 21) Dose Group PFOS Conc. ( ug/ g) PFOS Conc. ( ug/ g) ( mg/ kg/ day) Female Adult ( F0) Fetal Liver ( F1) 0 0.288 0.169 0.1 29.2 7.93 0.4 107 30.6 1.6 347 86.7 3.2 610 230 Average Results for the Analysis of Urine Samples from F0 Dams 0 mg/ kg 0.1 mg/ kg 0.4 mg/ kg 1.6 mg/ kg 3.2 mg/ kg Day 0 < LOQ 0.0497 0.302 0.959 1.53 Day 7 < LOQ 0.0620 0.308 1.10 1.60 Day 15 0.00905 0.0685 0.526 0.622 0.563 Day 21 0.0194 0.0574 0.555 2.71 1.61 Average Results for the Analysis of Feces Samples from F0 Dams 0 mg/ kg 0.1 mg/ kg 0.4 mg/ kg 1.6 mg/ kg 3.2 mg/ kg Day 0 0.0380 0.499 2.42 10.3 23.9 Day 7 0.0155 0.490 2.16 9.19 33.0 Day 15 0.0322 0.662 2.93 11.1 29.5 Day 21 0.0342 0.416 2.39 9.94 20.1 In general, there was a dose related increase in the levels of PFOS in the liver and serum of the dams and the fetuses. PFOS was also observed in the control dams, as well as the control fetuses. On sacrifice on GD21, the levels of PFOS were much higher in the liver than in the serum for the dams. The levels of PFOS remained fairly steady in the serum of the dams from GD0   GD 15, but the levels dropped at GD21. In the GD21 fetuses, the level of PFOS in the serum was generally comparable to the level observed in the dams, whereas the level of PFOS in the fetal livers was well below that seen in the dams.

Results Remarks: As stated in the report. " 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, indicate that the data are quantitative to 50% or greater."

Metabolites measured: none ENV/ JM/ RD( 2002) 17/ FINAL 266 CONCLUSIONS Conclusions: Under the conditions of the present studies, PFOS was observed in the livers, urine, feces and sera of all female rats dosed with PFOS during the in life phase of the study. Additionally, PFOS was observed in fetal liver and serum taken during gestation from the same group of female rats.

Agree REFERENCE Study Title: Oral ( Gavage) Pharmacokinetic Study of PFOS in Rats Analytical Laboratory Report Title: Determination of the Presence and Concentration of Perfluorooctanesulfonate ( PFOS) in Serum, Liver, Urine, and Feces Samples 3M Medical Department Study: T 6295.12, Argus In Life Study: # 418 013, FACT TOX 110, 3M Laboratory Request No. U2849, 3M Environmental Laboratory, May 4, 2001. ENV/ JM/ RD( 2002) 17/ FINAL 267 Title: Oral ( Gavage) Pharmacokinetic Recovery Study of PFOS in Rats, Analytical Laboratory Report, Determination of the Concentration of Perfluorooctanesulfonate ( PFOS) in the Serum, Liver, Urine, and Feces of Crl: CDBR VAF/ Plus ® Rats Exposed to PFOS via Gavage TEST SUBSTANCE Identity: Perfluorooctylsulfonate, potassium salt ( FC 95), CAS 2795 39 3 Remarks: Purity 86.9%, Lot # 217 METHOD Method/ guideline followed: This study was conducted in compliance with United States Food and Drug Administration ( FDA) Good Laboratory Practice ( GLP) Regulations 21 CFR Part 58, with the exceptions noted on page 3 of report. It does not appear these exceptions would significantly impact the results or conclusions. The analytical phase completed at the 3M Environmental Laboratory was performed in accordance with 3M Environmental Technology and Safety Services Standard Operating Procedures.

Test type: in vivo Species/ strain/ cell type or line: rat/ Sprague Dawley/ pregnant Crl: CD( R) BR VAF/ Plus ® Sex: F0: female, F1: both Age and body weight range of animals used: 65 days, 192 231 g Number of animals/ sex/ dose: F0: 8, F1: 5 male and 5 female pups/ litter Route of administration: oral Vehicle: 0.5% Tween( R) 80 in R. O. deionized water, dosage volume 5 ml/ kg Doses: 0 ( vehicle), 0.1 and 1.6 mg/ kg/ day in volume of 5 ml/ kg ( 0.00, 0.02, and 0.32 mg/ mL), once daily beginning 43 days prior to cohabitation until confirmed evidence of mating. Only the F0 females were dosed.

Excretion routes, body fluids, and tissues monitored and/ or sampled during study: F0 urine, feces, serum, liver. F1 liver and serum.

Statistical methods used: means, standard deviations, and percentages Method remarks: Rat dams were exposed to PFOS via gavage prior to and during mating. Exposure to PFOS was halted on the first day of presumed gestation. Pups were not directly exposed to PFOS, but may have been exposed in utero and during lactation. Male rats of the same source and strain were used only as breeders and were not administered the test article or considered part of the test system.

Urine and fecal samples were collected from F0 female rats for the following intervals: one day prior to initiation of cohabitation to the following morning, days 6 to 7, 14 to 15, and 20 to 21 of presumed ENV/ JM/ RD( 2002) 17/ FINAL 268 gestation ( DG 6 to 7, 14 to 15, and 20 to 21), and days of lactation ( DL) 21 to 22. Blood samples were collected from each of the maternal rats on the day cohabitation was initiated ( prior to cohabitation), DG 7, 15 and 21, and DL 14 and 22. Day 1 of lactation was defined as the day of birth. On DL 4, litters were culled to five male pups and five female pups per litter, where possible. Sera specimens were collected from pooled litter samples on DL 21. On DL 22, all surviving Generation F0 and Generation F1 animals assigned to the study were sacrificed, and a liver specimen was collected from each animal. The liver from each pup was collected and pooled per litter. Blood samples were collected and pooled per liter. Urine, fecal, serum and liver samples were shipped to the Sponsor for analysis. On days 1 to 4 of the 43 day premating period, F0 female rats received 25% greater dose due to an incorrect calculated amount of test substance in vehicle.

RESULTS Detailed results:

Average Results for the Analysis of Sera Samples ( PFOS Conc. ug/ ml) DG0 DG7 DG15 DG21 DL14 DL21 DL22 0.0 mg/ kg F0 0.100 0.0796 0.0742 < LLQ 0.0542 NS 0.0492 0.0 mg/ kg F1 NS NS NS NS NS 0.0531 NS 0.1 mg/ kg F0 9.21 7.24 5.68 2.58 1.63 NS 0.979 0.1 mg/ kg F1 NS NS NS NS NS 1.80 NS 1.6 mg/ kg F0 161 129 90.6 39.5 20.6 NS 14.1 1.6 mg/ kg F1 NS NS NS NS NS 27.1 NS Average Results for the Analysis of Liver Samples ( PFOS Conc. ug/ g) DL22 0.0 mg/ kg F0 0.243 0.0 mg/ kg F1 0.174 0.1 mg/ kg F0 6.15 0.1 mg/ kg F1 5.00 1.6 mg/ kg F0 59.7 1.6 mg/ kg F1 56.2 Average Results for the Analysis of Urine Samples ( PFOS Conc. ug/ ml) DG0 DG6/ 7 DG14/ 15 DG20/ 21 DL21/ 22 0.0 mg/ kg F0 0.00819 0.0100 0.00685 0.00614 < LOQ 0.1 mg/ kg F0 0.0905 0.0307 0.0327 0.0231 0.00555 1.6 mg/ kg F0 2.11 0.888 0.613 0.340 0.0334 Average Results for the Analysis of Feces Samples ( PFOS Conc. ug/ g) DG0 DG6/ 7 DG14/ 15 DG20/ 21 DL21/ 22 0.0 mg/ kg F0 ND ND ND ND ND 0.1 mg/ kg F0 0.601 0.399 0.294 0.119 0.0522 1.6 mg/ kg F0 10.9 8.39 4.83 2.06 0.387 In general, there was a dose related increase in the levels of PFOS in the liver and serum of the dams and the levels in the serum decreased with time. The levels of PFOS were much higher in the liver than in the serum of the dams and the pups. The levels of PFOS were similar in the liver of the dams and pups, while the levels in the serum were slightly higher in the pups than in the dams. ENV/ JM/ RD( 2002) 17/ FINAL 269 Results Remarks: As stated in the report. " 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, indicate that the data are quantitative to 50% or greater."

Metabolites measured: none CONCLUSIONS Statement of Conclusion Under the conditions of the present study, perfluorooctanesulfonate was observed in all sample types of all Generation 0 test system animals dosed with the test substance during the in life phase of the study, and in all sample types of their offspring ( Generation F1).

Agree REFERENCE Study Title: Oral ( Gavage) Pharmacokinetic Recovery Study of PFOS in Rats Analytical Laboratory Report Title: Determination of the Concentration of Perfluorooctanesulfonate ( PFOS) in the Serum, Liver, Urine, and Feces of Crl: CDBR VAF/ Plus ® Rats Exposed to PFOS via Gavage, 3M Environmental Laboratory Report No. FACT TOX 111, Laboratory Request No. U2994, 3M Ref. No. T 6295.14, Argus In Life Study 418 015, May 4, 2001. ENV/ JM/ RD( 2002) 17/ FINAL 270 Title: Half Life Study of PFOS in Serum, 2000 TEST SUBSTANCE Identity: PFOS METHOD Study design: PFOS half life study on retired workers from the Decatur, Alabama plant Manufacturing/ Processing/ Use: N/ A Hypothesis tested: To determine the half life of PFOS in retired perfluorochemical production workers.

Study period: Nov. 1998 to 2003 Setting: N/ A Total population: 27 retirees from the Decatur, Alabama and Cottage Grove, Minnesota plants Subject selection criteria: The participants volunteered for this study Total # of subjects in study: 18 males Comparison population: N/ A Participation rate: Unknown Subject description: Retirees ranged in age from 55 74 years, worked in the plant for an average of 28 years, average time from retirement to start of study was 30 months ( range: 5 130 months).

Health effects studied: N/ A Data collection methods: Blood sera samples collected every 6 months Details on data collection: No information was provided as to how the blood was drawn, stored, etc.

Exposure period: Unknown.

Description/ delineation of exposure groups/ categories: N/ A Measured or estimated exposure: N/ A Exposure levels: N/ A Statistical methods: Medians and ranges calculated.

Other methodological information: Half lives were calculated assuming a one compartment model. A log linear relationship was used to estimate the serum fluorochemical elimination half life in participating ENV/ JM/ RD( 2002) 17/ FINAL 271 retirees. In this log linear relationship, the slope of the line is related to the elimination constant via the equation slope =  kel( 2.303). Once the elimination constant is calculated, the half life is determined using the relationship t1/ 2 = 0.693/ kel.

If 3 data points were not available for any of the subjects and if there was a lack of fit to the model, that retiree was not included in the analyses. Eighteen participants met these requirements.

RESULTS Describe results: The median serum half life of PFOS was 270 days, with a range of 139 to 640 days. It should be noted that the difference in serum PFOS levels between retirees was quite large ( 0.2  2.0 ppm).

Study strengths and weaknesses: For most of the participants not included in the analysis, the second measurement was higher than the first. Therefore, the data did not fit the model and they were excluded. Although this may justify not including those participants in the analysis, it is an indication of the many limitations of the data. It is stated in the report that neither age nor number of months retired was associated with the serum PFOS half life calculations; however, this statement is not supported with any data in the report. In addition, no individual data were provided in the report and the relationship between number of years exposed in the workplace and PFOS levels and half life were excluded. Retirees were excluded from these calculations if their second measurement was higher than the first. It is unknown why this occurred, but the exclusion of those retirees introduces bias to the results. Also, elimination of PFOS occurs via urine and feces; however, these measurements were not taken. Therefore, it cannot be determined that the half life suggested by the preliminary results reported here represents a true elimination half life from the body. Finally, the effect of continued non occupational, low level exposure on the halflife is unknown.

Research sponsors: 3M Environmental Lab Consistency of results: These results are not consistent with the first study in which 3 retirees were followed over a 5 year period. In that study, the estimated half life was 3 to 4 years. There is no explanation in this current report as to why there is such a large difference in the results or why none of the retirees in the current study didn't fall within the range of the first study.

CONCLUSIONS N/ A REFERENCE Determination of Serum Half Lives of Several Fluorochemicals, June 8, 2000, 3M Company. FYI 0700 1378, 8( e) Supplemental Submission, 8EHQ 0373/ 0374. ENV/ JM/ RD( 2002) 17/ FINAL 272 ACUTE TOXICITY STUDIES Title: An Acute Inhalation Toxicity Study of T 2306 CoC in the Rat. 1979 TEST SUBSTANCE Identity: Potassium perfluorooctylsulfonate, CAS No.: 2795 39 3 Remarks: Dust, PFOS ( T 2306 CoC). Purity not specified.

METHOD Method/ guideline followed: Similar to OECD 403 GLP: N, no QA/ QC indicated Year study performed: 1979 Species/ Strain: Rat/ Sprague Dawley Sex ( Males/ females/ both): Both No. of animals/ sex/ dose: 5/ sex/ group Route of Administration: Inhalation Remarks: Concentrations of 1.89, 2.86, 4.88, 6.49, 7.05, 13.9, 24.09, 45.97 mg/ l PFOS were administered to eight test groups. A Wright dust feed mechanism with dry air at a flow rate of 12 to 16 liters per minute was used to administer the PFOS dust. Rats were exposed for 1 hour. The test group rats weighed 201 299 g at study initiation. The control group rats weighed 203 263 g at study initiation. The control rats were exposed to dry air at a flow rate of 12 liters per minute. All other protocols were the same as the test group rats. The rats were observed for abnormal signs prior to exposure, at 15 minute intervals during the 1 hour exposure, at removal from the exposure chamber, hourly for four hours after exposure, and daily thereafter for 14 days. Individual bodyweights were recorded on Day 0 ( prior to exposure), Day 1, Day 2, Day 4, Day 7, and Day 14. It is reported that all animals dying spontaneously were necropsied as soon as possible after death. Blood samples were collected on Day 14 from all surviving animals, but analyses were not provided.

RESULTS LC50 = 5.2 ( 4.4   6.4) mg/ l, ( 95% confidence limits); referenced method of Litchfield and Wilcoxon Number of deaths at each dose level ( by sex):

0.0 mg/ l: 0/ 10; 1.89 mg/ l: 0/ 10; 2.06 mg/ l: 1/ 10; 4.88 mg/ l: 2/ 10; 6.49 mg/ l: 8/ 10; 7.05 mg/ l: 8/ 10; 24.09 mg/ l: 10/ 10 ( authors did not provide summary by sex)

Remarks: The highest dose group, 45.97 mg/ l, was not used in the LC50 calculations and terminated on Day 2. At that point, only 5 animals survived and blood samples were taken at termination. The 13.9 mg/ l group was also terminated early ( Day 1) because of a mechanical problem during exposure. These animals ENV/ JM/ RD( 2002) 17/ FINAL 273 were also not used in the LC50 determination.

In the 24.09 mg/ l exposure group, all animals died by Day 6. At 7.05 and 6.49 mg/ l there was 80% mortality with last deaths at Day 10. At 4.88, 2.86, and 1.89 mg/ l there was 20%, 10%, and 0% mortality, respectively. At 2.86 mg/ l, deaths occurred on Day 7 and 10. At 1.89 mg/ l, one death occurred on Day 12. The rats in all these groups showed signs of toxicity including emaciation, red material around the nose or other nasal discharge, yellow material around the anogenital region, dry rales or other breathing disturbances, and general poor condition. Abnormal in life observations were reported to be less frequent in the lower exposure groups.

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

CONCLUSIONS LC50 = 5.2 ( 4.4   6.4) mg/ l, ( 95% confidence limits). Only conclusion provided; seems reasonable with available data REFERENCE Rusch, G. M., W. E. Rinehart and C. A. Bozak. 1979. An Acute Inhalation Toxicity Study of T 2306 CoC in the Rat. Project No. 78 7185, Bio/ dynamics Inc. ENV/ JM/ RD( 2002) 17/ FINAL 274 Title: Fluorad Fluorochemical Surfactant FC 95 Acute Oral Toxicity ( LD50) Study in Rats. 1978.

TEST SUBSTANCE Identity: Potassium perfluorooctylsulfonate, CAS No.: 2795 39 3 Remarks: FC 95. Purity not specified.

METHOD Method/ guideline followed: Similar to OECD 401 GLP ( Y/ N): N, no QA/ QC indicated Year study performed: 1978 Species/ Strain: Rat/ Charles River CD Sex ( Males/ females/ both): both Number of animals/ sex/ dose: 5/ sex/ dose Vehicle: 20% acetone/ 80% corn oil Route of Administration: gavage Remarks: Levels of 100, 215, 464, and 1000 mg/ kg PFOS were tested. All dose levels were administered as volumes of 10ml/ kg body weight. The rats weighed 172 212 g at the beginning of the study immediately prior to dosing and weights were recorded at Day 7 and Day 14. The rats were observed for abnormal signs during the four hours after exposure, and daily thereafter for 14 days. It is reported that all animals dying spontaneously were grossly necropsied, as well as all rats that survived to the end of the 14 day study.

RESULTS LD50: 251 ( 199 318) mg/ kg, ( 95% confidence limits); 3 references for statistical tables are given.

Number of deaths at each dose level ( by sex): 100 mg/ kg: 0/ 5 males, 0/ 5 females; 215 mg/ kg: 2/ 5 males, 1/ 5 females; 464 mg/ kg: 5/ 5 males, 5/ 5 females; 1000 mg/ kg: 5/ 5 males, 5/ 5 females Remarks: All rats in the 464 and 1000 mg/ kg dose groups died before the end of the study. Three animals in the 215 mg/ kg group died prematurely. It appears signs of toxicity most frequently observed included: hypoactivity, decreased limb tone, and ataxia. At necropsy observations included: yellowstained urogenital region, stomach distention and signs of irritation of the glandular mucosa, and lung congestion. No differences between sexes were noted.

LD50 male rats: 233 ( 160 339) mg/ kg ( 95% confidence limits) LD50 female rats: 271 ( 200 369) mg/ kg ( 95% confidence limits) ENV/ JM/ RD( 2002) 17/ FINAL 275 CONCLUSIONS None specified beyond LD50 REFERENCE Dean, W. P., D. C. Jessup, G. Thompson, G. Romig, and D. Powell. 1978. Fluorad Fluorochemical Surfactant FC 95 Acute Oral Toxicity ( LD50) Study in Rats. Study No. 137 083, International Research and Development Corporation. ( Includes Acute Oral Toxicity Study in Rats with T 2297 CoC. Project No. 78 1433A, Biosearch, Inc.) . ENV/ JM/ RD( 2002) 17/ FINAL 276 SKIN IRRITATION Title: Eye and Skin Irritation Report on Sample T 1117. 1974 TEST SUBSTANCE Identity: Potassium perfluorooctylsulfonate, CAS No.: 2795 39 3 Remarks: FC 95, Sample T 1117. Purity not specified.

METHOD Note pH of test material: Not specified Method/ Guideline followed: Not specified Test Type:        Species/ strain/ cell type: Rabbits/ albino Sex ( males/ females/ both): Not specified Number of animals/ sex/ dose: 6 total Total dose: appears to be 1.0 gram, 0.5g placed on each of 2 prepared test sites ( intact wet, abradedwet total dose not specified Vehicle: None? Not specified Length of time test material is in contact with animal/ cell: 72 hr Grading scale: Separate scores for erythema formation and edema formation are summed. Reference source not provided.

Remarks: Six albino rabbits had their hair clipped from their backs and flanks, and five tenths of one gram ( 0.5 g) of test material was placed on abraded wet or intact wet prepared test sites, then covered with gauze patches. After 24 hours and 72 hours the coverings were removed and the degree of erythema and edema was recorded according to a standardized scale.

RESULTS Results: In all cases it is reported the primary skin irritation scores were 0; which indicates no reddening or swelling detected. Primary irritation score: zero Remarks: No indication of reliability. No QA/ QC. No effects reported.

CONCLUSIONS No irritation. Inadequate information is presented in report to evaluate quality of study and validity of conclusion.

REFERENCE J. A. Biesemeier and D. L. Harris. 1974. Eye and Skin Iritation Report on Sample T 1117. Project No. 4102871, WARF Institute Inc. ENV/ JM/ RD( 2002) 17/ FINAL 277 EYE IRRITATION Title: Eye and Skin Irritation Report on Sample T 1117. 1974 TEST SUBSTANCE Identity: Potassium perfluorooctylsulfonate, CAS No.: 2795 39 3 Remarks: T 1117. Purity not specified.

METHOD Note pH of test material: Not specified Method/ Guideline followed: Not specified Test Type: in vivo Species/ strain/ cell type or line: Rabbit/ New Zealand White Sex ( males/ females/ both): Not specified Number of animals/ sex/ dose: 6/ single dose Total dose: it appears 0.1 gram, ( protocol states either 0.1 ml or 0.1g and T 1117 is reported to be a solid { FC 95} by 3M), total dose not specified Length of time test material is in contact with animal/ cell: could be 72 hours, But at 1 hr observation and at each scoring after " any accumulated discharge or residue of test material was flushed from the eye."

Observation period: 1hr, 24 hr, 48 hr, 72 hr Scoring method used: Not specified or referenced  " The reaction to the test material was read according to the scale of scoring for damage to the cornea, iris, and the bulbar and palpebral conjunctivae "

Remarks: Rabbits were placed in collars so they could not rub their eyes. One tenth of a gram ( 0.1 g) of the test substance was instilled in one eye, the other eye was left untreated as a control. It is reported that the reaction to the test material was read against a scale of damage to the cornea, iris, and the bulbar and palpebral conjunctivae at 1, 24, 48, and 72 hours after treatment. The scale criteria are not presented or referenced. Each time the eyes were scored, any accumulated discharge or residue of test material was flushed from the eye. It appears that scores were maximal at 1 hour and 24 hours after treatment then decreased over the rest of the study.

RESULTS Corrosive: no Irritation score: Only total scores provided. 1 hr: 8.00; 24 hr: 9.33; 48 hr: 3.33; 72 hr: zero Tool used to assess score: Not specified Description of lesions: none Remarks: Inadequate description and discussion in report. Scores appear reduced in all rabbits over time. Decreases were noted at 48hrs and at 72hrs values were zero. ENV/ JM/ RD( 2002) 17/ FINAL 278 CONCLUSIONS Only conclusion provided in study is that test substance is irritating to eyes. Inadequate information is presented in report to evaluate quality of study and validity of conclusion.

REFERENCE J. A. Biesemeier and D. L. Harris. 1974. Eye and Skin Iritation Report on Sample T 1117. Project No. 4102871, WARF Institute Inc. ENV/ JM/ RD( 2002) 17/ FINAL 279 GENETIC TOXICITY STUDIES Title: MUTAGENICITY EVALUATION OF T 2O14 CoC IN THE AMES SALMONELLA/ MICROSOME PLATE TEST, 1978 TEST SUBSTANCE Identity: Potassium perfluorooctylsulfonate, CAS # 2795 39 3 Remarks: T 2014 CoC, FC 95, Purity not specified.

METHOD Method/ Guideline followed: Ames, 1975 Test type: Reverse mutation Test system: Bacteria; Yeast GLP: N Year study performed: 1977 Species/ Strain/ cell type/ cell line: Salmonella typhimurium TA100, TA1535, TA1537, TA1538, TA09, Saccharomyces cerevisiae D4 Metabolic activation: 0.1 ± .05 ml S9 homogenate of Aroclor 1254 induced Sprague Dawley rat liver Concentrations tested: 0.01 µ g/ plate, 1.0 µ g/ plate, 10.0 µ g/ plate, 100 µ g/ plate, 500 µ g/ plate nonactivated; 0.1 µ g/ plate, 1.0 µ g/ plate, 10.0 µ g/ plate, 100 µ g/ plate, 500 µ g/ plate activated Statistical methods used: None Remarks: There were no significant protocol variations. ( 1) For the time when the test was done ( 1977) a single plate per concentration was routine; ( 2) the negative control was the solvent DMSO; the positive controls were chosen according to strain being tested and activation condition and included ethyl methanesulfonate, quinoline hydroxide, nitroflourene , 2 anthramine; and 2 dimethylnitrosamine ( 3) a limited repeat study was done with strain TA100 both with and without activation because the testing laboratory believed that there was some evidence of mutagenicity with this strain. The doses tested without activation were 100 µ g/ plate, 500 µ g/ plate, and 1000 µ g/ plate and 500 µ g/ plate, 1000 µ g/ plate, and 2000 µ g/ plate with activation. However, a review of the data shows that was originally thought to be mutagenicity was within the normal variation of the assay. The repeat was inadequate because the doses tested were too high and too toxic to shed any light on possible mutagenic activity; there were no signs of mutagenicity in any of the other strains tested. The test with Saccharomyces was also negative. ( 4) criteria to evaluate results were as follows: dose response over 3 concentrations with lowest increase equal to 3X the solvent control for TA1535, TA1537 and TA1538. Dose response over 3 concentrations with lowest increase equal to 3X background for TA100 and 2x 3X background for TA98 and D4. ENV/ JM/ RD( 2002) 17/ FINAL 280 RESULTS Overall results: positive, negative, ambiguous: Negative Genotoxic effects ( unconfirmed, dose response, equivocal   with/ without activation): Negative with and without activation Cytotoxic concentration: 1000 µ g/ ml both with and without activation.

Statistical results: No statistics performed.

Remarks: None CONCLUSIONS Author's conclusions are accurate but for the wrong reasons. The test chemical is negative not because it was negative on repeat testing but because what was taken as mutagenicity in the first test was within normal variation of the assay.

REFERENCE Litton Bionetics, Inc. Kensington, Maryland 20795 1978. Mutagenicity Evaluation of T 2014 CoC in the Ames Salmonella/ Microsome Plate Test. Final Report. Submitted to: 3M Company, Saint Paul, Minnesota 55101 ENV/ JM/ RD( 2002) 17/ FINAL 281 Title: SALMONELLA   ESCHERICHIA COLI/ MAMMALIAN MICROSOME REVERSE MUTATION ASSAY WITH PFOS, 1999 TEST SUBSTANCE Identity: Potassium perfluorooctylsulfonate, CAS # 2795 39 3 Remarks: FC 95, T 6295, purity not specified.

METHOD Method/ Guideline followed: Ames et al., 1975; Green and Muriel, 1976; Maron and Ames, 1983 Test type: Reverse mutation Test system: Bacterial GLP: Y Year study performed: 1999 Species/ Strain/ cell type/ cell line: Salmonella typhimurium TA1535, TA100, TA98, TA1537 Escherichia coli WP2uvrA Metabolic activation: 0.1 ml S9 liver homogenate from Aroclor 1254 induced Sprague Dawley rats Concentrations tested: S. typhimurium: 33.3 µ g/ plate, 100 µ g/ plate, 333 µ g/ plate, 1,000 µ g/ plate, 3,330 µ g/ plate, and 5,000 µ g/ plate µ g/ plate with activation and 0.333 µ g/ plate, 1.00 µ g/ plate, 3.33 µ g/ plate, 10.0 µ g/ plate, 33.3 µ g/ plate plate, 100 µ g/ plate, 3333 µ g/ plate, 1,000 µ g/ plate and 5,000 µ g/ plate without activation. E. coli: 33.3 µ g/ plate, 100 µ g/ plate, 3333 µ g/ plate, 1,000 µ g/ plate, 3,330 µ g/ plate, and 5,000 µ g/ plate both with and without activation.

Statistical methods used: None Remarks: There were no significant protocol deviations. ( 1) There were 3 plates per test concentration and control; the positive controls were strain and activation condition specific and included benzo[ a] pyrene, 2 nitrofluorene, 2 aminoanthracene, sodium azide, ICR 191 and 4 nitroquinoline N oxide. The vehicle control was DMSO; ( 2) the solvent was DMSO; ( 3) the assay was not repeated. ( 4) For the test article to be considered positive in strains TA98, TA100 and WP2uvrA, there had to be at least a 2 fold increase in the mean revertants per plate over that of the appropriate vehicle control. The increase had to be accompanied by a dose response to increasing concentrations of the test article. For strains TA1535 and TA1537 there had be at least a 3 fold increase in the mean revertants per plate over that of the appropriate vehicle control. The increase had to be accompanied by a dose response to increasing concentrations of the test article.

RESULTS Overall results: positive, negative, ambiguous: Negative Genotoxic effects ( unconfirmed, dose response, equivocal   with/ without activation): PFOS was not ENV/ JM/ RD( 2002) 17/ FINAL 282 genotoxic when tested either with or without metabolic activation.

Cytotoxic concentration: Cytotoxicity was noted at 5000 µ g/ plate without metabolic activation. This cytotoxicity was evidenced by a slight reduction in the bacterial lawn.

Statistical results: Results were not evaluated statistically.

Remarks: There were no test specific confounding factors. Mutation frequencies were within the range of the vehicle controls.

CONCLUSIONS Author's conclusions are that PFOS is negative in this assay. This is accurate.

REFERENCE Mecchi, M. S. 1999. Salmonella   Escherichia Coli/ Mammalian Microsome Reverse Mutation Assay with PFOS. Covance Laboratories Inc. ( Covance) Vienna, Virginia 22182 Final Report Covance Study No.: 20784 0 409. Submitted to: 3M Corporate Toxicology St. Paul, Minnesota 55144 1000 ENV/ JM/ RD( 2002) 17/ FINAL 283 Title: CHROMOSOMAL ABERRATIONS IN HUMAN WHOLE BLOOD LYMPHOCYTES WITH PFOS, 1999 TEST SUBSTANCE Identity: Potassium perfluorooctylsulfonate, CAS # 2795 39 3 Remarks: FC 95, purity not specified.

METHOD Method/ Guideline followed: Galloway, 1994 Test type: In vitro cytogenetics Test system: Human cells in culture GLP: Y Year study performed: 1999 Species/ Strain/ cell type/ cell line: Human lymphocytes Metabolic activation: Aroclor 1254 induced rat liver S9 homogenate, 15.0 µ L/ ml, plus NADP and isocitric acid.

Concentrations tested: 12.5 µ g/ ml, 24.9 µ g/ ml, 49.7 µ g/ ml, 99.3 µ g/ ml, 149 µ g/ ml, 199 µ g/ ml, 249 µ g/ ml, 299 µ g/ ml, 349 µ g/ ml, 449 µ g/ ml, 599 µ g/ ml without activation.

12.5 µ g/ ml, 24.9 µ g/ ml, 49.7 µ g/ ml, 99.3 µ g/ ml, 149 µ g/ ml, 199 µ g/ ml, 249 µ g/ ml, 349 µ g/ ml, 449 µ g/ ml with activation Statistical methods used: Cochran Armitage test for linear trend; Fisher's Exact Test Remarks: There were no significant protocol deviations. ( 1) Each concentration was tested in replicate; each replicate was considered an independent unit. The negative control for the nonactivation assay was DMSO at 10 µ l/ ml, which was the highest concentration used in the test cultures; in the activation assay it was DMSO plus the S9 mix; the positive control was mitomycin C for the nonactivation assay and cyclophosphamide for the activation assay. Three concentrations of each positive control were tested. Cultures were exposed to chemical for 3 hours and harvested 22 hours later. One hundred metaphases from each replicate of the useable treatment cultures and the solvent and one dose of the positive control were used; mitotic index was evaluated by analysing the number of mitotic cells in at least 1000 cells per culture; ( 2) the solvent for the chemical was DMSO; ( 3) there was no follow up study done although in a study such as this where there are negative results after 3 hours incubation with a 22 hour harvest time a second study with a continuous exposure of 22 hours for the nonactivated portion of the assay is recommended. ( 4) The test article would have been considered positive if there had been a significant increase ( p< 0.01) in the number of cells with chromosomal aberrations at one or more concentrations. The test article was considered negative because there was no significant increase observed in the number of cells with chromosomal aberrations at any concentration tested. ENV/ JM/ RD( 2002) 17/ FINAL 284 RESULTS Overall results: positive, negative, ambiguous: Negative Genotoxic effects ( unconfirmed, dose response, equivocal   with/ without activation): Negative both with and without activation.

Cytotoxic concentration: 299 µ g/ ml without metabolic activation and 199 µ g/ ml with activation were the first cytotoxic concentrations tested as evidenced by a reduction in mitotic index.

Statistical results: Negative Remarks: Mitotic index was reduced 38%, 8% 15%, 15%, 12%, 19%, 24%, 69% and 92% in cultures treated with 12.5 µ g/ ml, 24.9 µ g/ ml, 49.7 µ g/ ml, 99.3 µ g/ ml, 149 µ g/ ml, 249 µ g/ ml, 299 µ g/ ml, 149 µ g/ ml and 449 µ g/ ml without activation. Aberrations were analysed from cultures treated 199 µ g/ ml, 249 µ g/ ml, 299 µ g/ ml, and 349 µ g/ ml.

With metabolic activation, mitotic index was reduced by 12%, 41%, 71%, and 53% in cultures treated with 49.7 µ g/ ml, 199 µ g/ ml, 249 µ g/ ml, and 299 µ g/ ml. Aberrations were analysed from cultures treated with 99.3 µ g/ ml, 149 µ g/ ml, 199 µ g/ ml, and 299 µ g/ ml. Only 27 and 4 metaphases were available for analysis from cultures treated with 299 µ g/ ml.

CONCLUSIONS Author's conclusions are that PFOS does not cause mutation in human lymphocytes; this is correct as stated.

REFERENCE Murli, H. 1999. Chromosomal Aberrations in Human Whole Blood Lymphocytes with PFOS. Covance Laboratories Inc. ( Covance) Final Report. Covance Study No.: 20784 0 449. Submitted to: 3M Corporate Toxicology, St. Paul Minnesota 55144 1000. ENV/ JM/ RD( 2002) 17/ FINAL 285 Title: Unscheduled DNA Synthesis in Rat Liver Primary Cell Cultures with PFOS, 1999 TEST SUBSTANCE Identity: Potassium perfluorooctylsulfonate, CAS # 2795 39 3 Remarks: T 6295, purity not specified.

METHOD Method/ Guideline followed: Williams, 1977; Williams, 1980; Butterworth et al., 1987 Test type: Unscheduled DNA Synthesis in Mammalian Cells in Culture Test system: Primary cells in culture GLP: Y Year study performed: 1999 Species/ Strain/ cell type/ cell line: Primary hepatocytes from a Fischer 344 rat male rat.

Metabolic activation: None Concentrations tested: 15 concentrations between 0.025 µ g/ ml and 4000 µ g/ ml. Six, 0.5 µ g/ ml, 1.0 µ g/ ml, 2.5 µ g/ ml, 5 µ g/ ml, 10.0 µ g/ ml and 25.0 µ g/ ml, chosen for evaluation based upon cytotoxicity.

Statistical methods used: None Remarks: There were no significant protocol deviations. ( 1) Triplicate cultures on coverslips were incubated for 19.6 20.0 hours, then the assay was terminated and 3H thymidine added to the cultures for 30 minutes after which the cells were fixed, dried over night, coverslips were mounted on slides, dipped in emulsion and stored for 6 days at 2 8o C after which the emulsions were developed, fixed and stained. 150 cells per dose were read ( 50 from each coverslip) and the mean net nuclear grain count determined. ( 2) The solvent for the assay was DMSO; ( 3) there was no follow up repeat study; ( 4) the positive control was 2 AAF; ( 5) for a treatment to be considered positive, there must be an increase in the mean net nuclear grain count to at least 5 grains per nucleus above the concurrent vehicle control value, and/ or an increase in the number of nuclei with five or more net grains such that the percentage of these nuclei in test cultures is 10% above the percentage seen in the vehicle control cultures. The positive control satisfied both of these criteria.

RESULTS Overall results: positive, negative, ambiguous: Negative Genotoxic effects ( unconfirmed, dose response, equivocal   with/ without activation): Negative Cytotoxic concentration: Excessive cytotoxicity at and at 50.0 µ g/ ml; weak cytotoxicity at 25.0 µ g/ ml. Cell morphology was suitable for analysis at and below 25.0 µ g/ ml. ENV/ JM/ RD( 2002) 17/ FINAL 286 Statistical results: The results were not evaluated statistically.

Remarks: There were no test specific confounding factors.

CONCLUSIONS The author concludes that PFOS is negative in this assay. This is accurate.

REFERENCE Cifone M. A. 1999. Unscheduled DNA Synthesis in Rat Liver Primary Cell Cultures with PFOS. Covance Laboratories Inc. Vienna, VA 22182 Final Report. Covance Study No.: 20780 0 447. Submitted to 3M Corporate Toxicology St. Paul, MN 55144 1000 ENV/ JM/ RD( 2002) 17/ FINAL 287 Title: MUTAGENICITY TEST ON T 6295 IN AN IN VIVO MOUSE MICRONUCLEUS ASSAY, 1996 TEST SUBSTANCE Identity: Potassium perfluorooctylsulfonate, CAS # 2795 39 3 Remarks: T 6295, FC 95, purity not specified.

METHOD Method/ Guideline followed: Heddle, 1983 Test type: Micronucleus GLP: Y Year study performed: 1996 Species/ Strain: Mouse; Crl: CD 1 ® ( ICR) BR Sex: Males & Females No. animals/ sex/ dose: 5/ sex/ dose Vehicle ( if used): Deionized water Route of administration: Oral Doses: 237.5 mg/ kg, 450 mg/ kg, 950 mg/ kg Frequency of treatment: Single dose Statistical methods used: Analysis of variance; Dunnet's t test Remarks: There were no significant protocol deviations. ( 1) Animals were 9 weeks and 1 day old at start of dosing males; weight range for the males was 29.9   37.0 g; for females it was 23.1 29.2 g; ( 2) the vehicle was deionized water; ( 3) the test lasted 72 hours; ( 4) the test material was administered as a single oral dose; ( 5) all treatment groups were sampled at 24, 48 and 72 hours; ( 6) the vehicle control was H2O; the positive control was 80 mg/ kg cyclophosphamide dissolved in water and administered by gavage. Controls were sampled at 24 hours only. Control groups consisted of 5 males and 5 females each. ( 7) No clinical examinations were made. ( 8) No necropsies or other gross examinations were made on these animals. ( 9) Micronuclei were evaluated in the bone marrow of treated animals. Frequency of PCEs vs. NCEs was determined by scoring the number of PCEs and NCEs in the optic fields while scoring the first 1000 erythrocytes. A positive was judged by an increase in micronucleated polychromatic erythrocytes over levels observed in the vehicle controls in either sex or at any harvest time. Bone marrow toxicity was judged by a significant reduction in PCE/ NCE ratios in either sex at any harvest time. ( 10) The M. T. D. was chosen on the basis of 2 preliminary dose selection assays both of which showed significant toxicity at the highest dose tested. ENV/ JM/ RD( 2002) 17/ FINAL 288 RESULTS Effect on mitotic index or PCE/ NCE ratio by dose level and sex: PCE: NCE Ratio 237.5 mg/ kg 24 hours: males 0.57 ± 0.11; females 0.52 ± 0.10 48 hours: males 0.48 ± 0.04; females 0.80 ± 0.10 72 hours: males 0.39 ± 0.11; females 0.42 ± 0.14 450 mg/ kg 24 hours: males 0.75 ± 0.11; females 0.59 ± 0.08 48 hours: males 0.71 ± 0.05; females 0.37 ± 0.07 72 hours: males 0.29 ± 0.06; females 0.40 ± 0.12 950 mg/ kg 24 hours: males 0.56 ± 0.13; females 0.59 ± 0.08 48 hours: males 0.54 ± 0.08; females 0.44 ± 0.11 72 hours: males 0.17 ± 0.05; females 0.17 ± 0.05 Genotoxic effects ( unconfirmed, dose response, equivocal): Negative Statistical results: The PCE: NCE ratio was reduced in 237.5 mg/ kg males at 48 and 72 hours; in 450 mg/ kg males at 72 hours and in 450 mg/ kg females at 48 hours and in 950 mg/ kg males at 48 and 72 hours and in 950 mg/ kg females at 72 hours. There was no statistically significant increase in the number of micronucleated PCEs over the controls in any treatment group. The positive control induced a significant increase in the number of mPCE in both males and females and reduced the PCE: NCE ratio in females only at 24 hours.

Remarks: ( 1) Animals were examined approximately 1 2 hours before sampling for signs of toxicity and mortality. Animals in the 237.5 mg/ kg group remained healthy throughout the treatment period. ( 2) Both males and females in the 950 mg/ kg dose group began dying about 22 hours after treatment. Also at 22 hours 2 males in the 950 mg/ kg dose group went into convulsions when their cage was opened but recovered in a few minutes. At about 46 hours after treatment 1 female from the 450 mg/ kg dose group and more males and females from the 950 mg/ kg dose group were found dead and at about 71 hours after treatment, one male from the 950 mg/ kg dose group was found dead. All surviving animals appeared normal at that point. ( 3) No other clinical signs were noted or reported. ( 4) Body weight changes were not reported. ( 5) Food and water consumption were not reported. ( 6) There was no increase in the percent of micronucleated PCEs at any dose level tested or at any time period sampled.

CONCLUSIONS The author concludes that PFOS is negative in the mouse bone marrow micronucleus assay. This is an accurate assessment.

REFERENCE Murli, H. 1996. Mutagenicity Test on T 6295 in an In Vivo Mouse Micronucleus Assay. Corning Hazelton Inc. ( CHV), Vienna, Virginia 22182. Final Report. CHV Study No.: 17403 0 455. Submitted to 3M St. Paul, Minnesota 55144 1000. ENV/ JM/ RD( 2002) 17/ FINAL 289 Title: IN VITRO MICROBIOLOGICAL MUTAGENICITY ASSAYS OF 3M COMPANY COMPOUNDS T 2247 CoC AND T 2248 CoC, 1978 TEST SUBSTANCE Identity: T 2247 CoC; L 4299, a 50% by weight solution of the diethanolammonium salt of perfluorooctanesulfonate in water T 2248 CoC; 22.5% of a reaction product of ethyl and methyl methacrylates and 22.5% of the pyridinium chloride salt of an: N methylperfluorooctanesulfonamidoethanol based glutaryl amide.

Remarks: T 2247 CoC, T 2248 CoC, purity not specified METHOD Method/ Guideline followed: Ames et al., 1975; Zimmermann and Schwaier, 1967; Brusick and Mayer, 1973 Test type: Reverse Mutation; Recombination Test system: Salmonella typhimurium; Saccharomyces cerevisiae GLP: N Year study performed: 1978 Species/ Strain/ cell type/ cell line: Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100; Saccharomyces cerevisiae D3 Metabolic activation: 0.5 ml of 10% S9 liver homogenate from Aroclor 1254 induced rats.

Concentrations tested: Plate incorporation assay: 10 µ g/ plate, 50 µ g/ plate, 100 µ g/ plate, 500 µ g/ plate, 1000 µ g/ plate, 5000 µ g/ plate Dessicator method: 0.1 ml/ dessicator, 0.5 ml/ dessicator, 1.0 ml/ dessicator, 5.0 ml/ dessicator Yeast recombination: 0.1%, 0.5%, 1.0%, 5.0% Yeast repeat assay at 1.0%, 2.0%, 4.0%, 5.0%

Statistical methods used: None Remarks: There were no significant protocol deviations. ( 1). The plate incorporation assay and the S. cerevisiae assay were performed with both chemicals and with one plate per test concentration; the dessicator assay was performed with T 2247 CoC using two plates per concentration but used only strains TA 98 and TA100 for the test. However, given the complexity of the dessicator assay and the limitations involved in setting it up, this is acceptable; ( 2) the positive controls were chosen according to the strain and activation conditions and included sodium azide, 9 aminoacridine, 2 nitrofluorene and 2 anthramine for the plate incorporation assay; 1,1 dichloroethylene for the desiccator assay with T 2247 CoC and 1,2,3,4 diepoxybutane for the S. cerevisiae assay. The negative control group for all assays was water. ( 3) The plate incorporation assay with both agents and the yeast assay with T 2248 were repeated; the desiccator assay was run only once. ( 4) For the desiccator assay, plates were prepared as for the standard assay but no test chemical was added to the agar. The strains tested were S. typhimurium TA98 and TA 100. The test was performed both with and without metabolic activation. Plates without lids were placed side by side in ENV/ JM/ RD( 2002) 17/ FINAL 290 a perforated shelf in a 9 liter desiccator. A known volume of T 2247 was added to a glass Petri dish that was placed in the center of and attached to the bottom of the shelf. In decreasing order, 5.0 ml, 1.0 ml, 0.5 ml and 0.1 ml of test chemical were added to the desiccator. The negative control chemical was water; the positive control chemical was 1,1 dichloroethylene. Both were treated in the same manner as T 2247. The desiccator was sealed and placed on a magnetic stirrer plate in a room maintained at 37o C. A magnetic stirrer with vanes was placed in the base of each desiccator to ensure adequate dispersion of the chemical. Plates were incubated for 8 hours, removed from the desiccators, their lids replaced and they were incubated at 37o C for an additional 42 hours before revertants were counted.

RESULTS Overall results: positive, negative, ambiguous: All tests were negative.

Genotoxic effects ( unconfirmed, dose response, equivocal   with/ without activation): Negative both with and without activation.

Cytotoxic concentration: T 2247 was not cytotoxic. In the plate incorporation assay, T 2248 was toxic to strain TA1538 at 1000 µ g/ plate and to all other strains at 5000 µ g/ plate when tested without activation. It was toxic at 1000 µ g/ plate to strain TA1537 and at 5000 µ g/ plate for all other strains when tested with metabolic activation. T 2248 was slightly toxic to S. cerevisiae D3 at 5% concentration without metabolic activation.

Statistical results: No statistical results were determined.

Remarks: In the first assay with T 2248 and S. cerevisiae D3 without metabolic activation there seemed to be some slight indication of mutagenicity at the highest concentration tested, 5%. The assay was repeated at 1%, 2%, 4%, and 5% concentrations with and without activation. There was no indication of a mutagenic dose response and the testing laboratory concluded that T 2248 did not cause recombination in S. cerevisiae D3. There were no test specific confounding factors in any aspect of the test.

CONCLUSIONS The testing laboratory concluded that T 2247 and T 2248 were nonmutagenic for S. typhimurium TA1535, TA100, TA1537, TA1538, and TA98 when tested in a plate incorporation assay with and without metabolic activation; that T2247 did not induce mutation in S. typhimurium TA98 and TA100 when tested in a dessicator assay for volatile chemical and that neither chemical induced recombination in S. cerevisiae D3. These conclusions are accurate.

REFERENCE Simmon, V. F. 1978. IN VITRO MICROBIOLOGICAL MUTAGENICITY ASSAYS OF 3M COMPANY COMPOUNDS T 2247 CoC AND T 2248 CoC. SRI International, Final Report. Prepared for 3M Company, St. Paul, Minnesota 55101. ENV/ JM/ RD( 2002) 17/ FINAL 291 REPEAT DOSE STUDIES Title: First ninety day rhesus monkey toxicity study, 1979 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: Rhesus monkey Sex: Males and females Number of animals per dose group: 2/ sex/ group Route of administration: Gavage Doses tested and frequency: 0, 10, 30, 100, 300 mg/ kg/ day Post observation period: None Statistical methods used: None Remarks: Distilled water was used for the vehicle control. The males weighed 3.05 3.80 kg at study initiation and the females weighed 2.75 4.10 kg. The monkeys were observed daily for general clinical signs and body weights were recorded weekly. Hematological and clinical chemistry analyses and urinalysis were conducted at the beginning of the study. The study was terminated after 20 days due to the death of the monkeys. At necropsy the heart, liver, adrenals, spleen, pituitary, kidneys, testes/ ovaries and brain were weighed. The thyroid/ parathyroid were weighed after fixation. Tissues were preserved in buffered neutral 10% formalin; the eyes were preserved in Russell's fixative. The following organs from control and all treated groups were examined microscopically: adrenals, aorta, brain, esophagus, eyes, gallbladder, heart ( with coronary vessels), duodenum, ileum, jejunum, cecum, colon, rectum, kidneys, liver, lung, skin, mesenteric lymph node, retropharyngeal lymph node, mammary gland, nerve ( with muscle), spleen, pancreas, prostate/ uterus, bone/ bone marrow ( rib junction), salivary gland, lumbar spinal cord, pituitary, stomach, testes/ ovaries, thyroid, parathyroid, thymus, trachea, tonsil, tongue, urinary bladder and vagina. ENV/ JM/ RD( 2002) 17/ FINAL 292 RESULTS NOAEL ( dose and effect): None LOAEL ( dose and effect): None Toxic response/ effects by dose level: All of the monkeys in the treated groups died.

Statistical results: None Remarks: The monkeys in the 300 mg/ kg/ day group died between days 2 4, the monkeys in the 100 mg/ kg/ day group died between days 3 5, the monkeys in the 30 mg/ kg/ day group died between days 7 10, and the monkeys in the 10 mg/ kg/ day group died between days 11 20 of treatment. The monkeys from all the groups showed similar signs of toxicity including decreased activity, emesis with some diarrhea, body stiffening, general body trembling, twitching, weakness, convulsions and prostration. At necropsy, several of the monkeys in the 100 and 300 mg/ kg/ day groups had a yellowish brown discoloration of the liver; histologic examination showed no microscopic lesions. Congestion, hemorrhage and lipid depletion of the adrenal cortex was noted in all treated groups. No other lesions were noted.

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

REFERENCE Goldenthal, E. I., D. C. Jessup, R. G. Geil and J. S. Mehring. 1979. Ninety day subacute rhesus monkey toxicity study. Study No. 137 087, International Research and Development Corporation, Mattawan, MI. ENV/ JM/ RD( 2002) 17/ FINAL 293 Title: Second ninety day rhesus monkey toxicity study, 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: Rhesus monkey Sex: Males and females Number of animals per dose group: 2/ sex/ group Route of administration: Gavage Doses tested and frequency: 0, 0.5, 1.5, 4.5 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: Distilled water was used for the vehicle control. The males weighed 2.55 3.55 kg at study initiation and the females weighed 2.7 3.75 kg. The monkeys were observed daily for general clinical signs and body weights were recorded weekly. Hematological and clinical chemistry analyses and urinalysis were conducted at the beginning of the study and after 30 and 90 days of treatment. 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.

RESULTS NOAEL ( dose and effect): None ENV/ JM/ RD( 2002) 17/ FINAL 294 LOAEL ( dose and effect): 0.5 mg/ kg/ day: Soft stools, diarrhea, anorexia, emesis, occasional decreases in activity; slight reduction in serum alkaline phosphatase.

Toxic response/ effects by dose level: 4.5 mg/ kg/ day  4/ 4 monkeys died between weeks 5 7, clinical signs ( anorexia, emesis, black stool, dehydration), significant reduction in serum cholesterol, marked diffuse lipid depletion in the adrenals, moderate diffuse atrophy of pancreatic acinar cells, moderate diffuse atrophy of serous alveolar cells. 1.5 mg/ gk/ day   clinical signs ( soft stools, diarrhea), reduced body weight, reduced serum alkaline phosphatase activity and serum potassium ( females), reduced serum cholesterol ( 1/ 2 females), reduced inorganic phosphate ( 1/ 2 females). 0.5 mg/ kg/ day   clinical signs, ( soft stools, diarrhea), soft stools, diarrhea, anorexia, emesis, occasional decreases in activity; slight reduction in serum alkaline phosphatase.

Statistical results: The statistical results are presented, but should be viewed with caution due to the small number of animals. 4.5 mg/ kg/ day  significant reduction in serum cholesterol 1.5 mg/ kg/ day   significant reduction in serum alkaline phosphatase activity and serum potassium ( females)