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CXR Biosciences Ltd. James Lindsay Place DUNDEE DD15JJ q1.26-33Z2 C,ntarn r.~.o C REPORT 13-WEEK STUDY TO INVESTIGATE THE REVERSIBILITY OF PERFLUOROOCTANE SULFONATE (PFOS)-INDUCED EFFECTS IN MALE SPRAGUE DAWLEY RATS. CXR STUDY NUMBER : Sponsor: Sponsors Representative: Sponsor's study identifier: Sponsor's material identifier: CXR0486s 3M Dr John Butenhoff 3M Medical Department 3M Center Building 220-06-W-08, St. Paul, Minnesota 55144-1000 06-301 MTDID-208 Study Director: Test facility "In Life" Start Date : "In Life" Finish Date : CIRCULATION B M Elcombe, CXR Biosciences CXR Biosciences, James Lindsay Place, Dundee Technopote, Dundee, DD1 5JJ . and Biological Sciences Resource Unit (BSRU), Dundee University, Dundee, DDl 5EH. 29' November 2006 28"' February 2007 Signed Original : Copies to : Study File/Archives Sponsor Study Director Sponsor > Author_ Barbara T1co1 e, Study rector Approved (Director, CXR Biosciences CXR0486s Final Report Page 1 of 128 CONTENTS CONTENTS ... . ... . .. . . .. . . ... ... . . .. . . .. . ... . ... . .. . ... . .. . . .. . ... . .. . . .. . ... . .. . . .. . ... . .. . . .. . ... . ... . ... . .. . . .. . ... . ... . . . . .. 2 SUMMARY ... . ... . .. . . .. . .... .... . .. . . .. . ... . ... . .. . . .. . .. . . .. . ... . .. . . .. . . .. . ... . .. . ... . .. . . .. . . .. . ... . .. . . .. . . .. . ... . .. . . .. . .. 4 l . INTRODUCTION . . .... ... . . .. . . .. . ... . ... ... . . .. . ... . .. . ... . .. . . .. . . .. . ... . .. . ... . .. . . ... . .. . ... . .. . . .. . . .. . . .. . .. . . .. . .. 7 2. ANIMALS (Scientific Procedures) ACT 1986 COMPLIANCE . . ... . ... . .. . . .. . . .. . . .. . .. . . . . . .. 7 2.1 Licensee Responsibilities . . .. . ... . ... ... . . .. . ... . .. . ... . .. . . .. . .. . . ... . .. . ... . .. . . .. . ... . ... . .. . . ... . .. . . .. . ... . . . . . . 8 3 . MATERIALS AND METHODS . . . .. . . .. . ... . .. . ... . .. . . .. . ... . ... ... . ... . .. . . .. . ... . ... . .. . . .. . . .. . ... . .. . . .. . .. 8 3 .1 Test Substances . .. . .... .... . .. . . .. . ... . ... . .. . ... . .. . . .. . ... . .. . . .. . ... . .. . . .. . ... . .. . . .. . . .. . ... . ... . ... . .. . ... . ... . . .. . . 8 3 .2 Safety Precautions . ... .... . .. . . .. . ... . ... . .. . . .. . .. . . ... ... . .. . . .. . . .. . ... . .. . . .. . .. . . .. . . .. . ... . .. . . .. . . .. . . .. . .. . . .. . .. 8 3 .3 Diet . .. . . .. . ... . ... . ... . . . . . ... .... . .. . . .. . ... . ... . .. . . .. . .. . . ... ... . .. . . .. . . .. . ... . .. . ... . .. . . ... . .. . . .. . .. . . .. . . .. . . .. . .. . . . . . .. 8 3 .4 Animals . ... . ... . ... . . . . .... .... . .. . . .. . ... . ... . .. . ... . .. . . .. . ... . ... . .. . ... . ... . .. . . .. . ... . .. . . .. . . .. . ... . .. . . .. . . .. . ... . . . . .. 8 3 .5 Animal Accommodation and Husbandry ... . .. . ... . .. . . .. . ... . .. . . .. . ... . ... . .. . . .. . ... . ... . .. . . ... ... . . .. . . 9 4. EXPERIMENTAL DESIGN . . . .. . ... . ... . .. . . .. . ... . .. . ... . .. . . .. . ... . .. . . .. . ... . .. . . .. . . .. . ... . ... . .. . . ... ... . . .. . . 9 5 . EXPERIMENTAL PROCEDURES . . .. . . .. . ... . .. . . .. . .. . . .. . ... . ... . .. . . .. . .... .. . . .. . . . .. .... . . . . . .. . .. . . .. 10 5 .1 Bodyweight .. . ... . . .. . . .. .... . .. . . .. . . .. . ... . ... . .. . . .. . ... . .. . . .. . ... . .. . . .. . ... . ... . .. . .... ... . . . . . ... .... . .. . . .. . ... . .. 10 5 .2 Clinical Observations ... . ... . ... . .. . ... . ... . .. . . .. . .... .. . . .. . . .. . ... . .. . ... . .. . . . . . . ... ... . . . . . ... .... . .. . . .. . . .. . .. 10 5 .3 Food Consumption . .. . . .. . ... . ... . ... . .. . ... . .. . . . . . .... .. . . .. . . ... ... . .. . ... . ... . . .. . ... ... . . . . . . .. . ... . .. . . .. . . .. . .. 10 5 .4 Intercurrent Deaths . .. . ... . ... . ... . ... . .. . ... . .. . . .. . . .. . ... . .. . ... . .. . . .. . . .. . .. . . . . . .... . .. . . . . . . .. .... . .. . . .. . . .. . .. 10 5.5 Terminal Procedures . . .. . ... . . .. . ... . .. . . ... .. . . . .. . ... ... . . . . . . .. ... . . . . . ... . .. . . .. . ... . .. . . .. . . .. . . .. . ... . .. . . .. . .. 11 6. BIOCHEMICAL MEASUREMENTS . . . . . . ... ... . . . . . .. . ... . . .. . ... ... . . .. . . .. . ... . .. . . .. . ... . . .. . .. . . .. . .. 11 6.1 Clinical Chemistry. . ... . .. . . .. . . .. . ... . . . . . ... ... . . . . . ... ... . . . . . ... ... . .. . . . .. ... . . .. . ... . .. . . .. . . .. . ... . .. . . .. . . .. . . . 11 6.2 Peroxisome Proliferation .. . . .. . ... . . . . . ... ... . ... .... ... . . . . . ... ... . .. . . .. . . ... . .. . ... ... . . .. . . .. . ... . ... . .. . . .. . . . 12 6.3 DNA Content of Liver... . .. . . ... ... . . .. . ... .... . .. .... . .. . . . . . ... .... .. . . ... . ... . .. . ... .... . .. . . .. . . .. . ... . .. . . .. . . . 12 6.4 Total Cytochrome P450 . .. . . ... .... . .. . . .. . ... . .. . ... . ... . .. . ... . ... . .. . . .. . ... . .. . . .. . ... . ... . ... . .. . ... . ... . .. . . . 12 6.5 Lauric Acid 12-Hydroxylation (LAH) . . .. . . .. . ... . .. . . .. . ... . .. . . .. . ... . ... . .. . . .. . ... . ... . .. . ... . ... . ... . . 12 6.6 Pentoxyresorufrn-O-depentylation (PROD) . ... . .. . . .. .... . . . . . .. . ... . ... . .. . . .. . . .. . ... . .. . . . . . . ... ... . . 12 6.7 Testosterone 6P-Hydroxylation . . ... . .. . ... . ... . .. . . .. . ... . .. . . .. . ... . .. . . .. . ... . .. . . .. . . .. . ... . .. . . .. . ... . .. . . . 12 6.8 Protein Determination .... . .. . . .. . ... . ... . .. . . .. . ... . .. . . .. . ... . .. . . .. . ... . .. . . .. . ... . ... . .. . . .. . ... . .. . . . . . . ... .... . 12 6.9 Statistical Analysis . . ... . ... . .. . . .. . ... . ... . .. . . .. . ... . .. . . .. . .. . . ... ... . ... . .. . . .. . ... . .. . . .. . ... . .... . .. . . .. . . .. .... . 13 6.10 Additional Analyses . . ... . .. . . .. . ... . ... . .. . . .. . . .. . ... . .. . ... . ... . .. . . .. . ... . .. . . .. . ... . .. . . . . . . ... ... . . .. . . .. . ... . 13 7. HISTOPATHOLOGY .. . . .. . ... . .. . ... . . .. . ... . .. . ... . .. . . .. . . .. . ... . .. . ... . .. . . .. . ... . ... . .. . ... . . ... ... . . . . . ... .... . 13 8. SPECIMEN SHIPMENT . . ... . ... . .. . . .. . ... . ... ... . ... . ... . .. . ... . .. . . .. . . ... ... . .. . . .. . . .. . . .. . ... ... . . . . . . .. . ... . 13 9. PROTOCOL AMENDMENTS . .. . . .. . . ... ... . .. . ... . ... . . . . ... . .. . . .. . ... . .. . . . . . . ... ... . . .. . ... . .. . . .. . . .. . . .. . 13 10. RESULTS . . .. . . .. . ... . ... . .. . . .. . . .. . .... .. . . .. . . ... ... . .. . . ... .... . .. .... ... . . . . . ... .... . . . . ... . ... . ... . .. . . .. . .. . . .. . . .. . 13 10.1 Bodyweights, Food Consumption and Liver Weights . . . ... .... . .. . . .. . .. . . ... . .. . . .. . ... . .. . ... . 13 10.1 .1 Bodyweights . .. . ... . ... . ... . . .. . ... ... . . . . . . .. ... . . .. . ... .... . . . . ... . ... . . . . ... . ... . .. . . .. . ... . ... . .. . . .. . ... . ... . .. . 13 10.1 .2 Food consumption . . . ... . . .. . ... .... . .. . . .. . ... . .. . . .. .... . .. . ... . ... . .. . . .. .... . .. . . .. . ... . ... . .. . . .. . . .. . ... . .. . 15 10.1 .3 Liver weights . .. ... . .... ... . . .. . ... . ... . .. . . .. .... . .. . . .. . ... . .. . ... . .. . . .. . . .. . ... . .. . . .. . . .. . . .. . ... . .. . . .. . . ... . .. 15 10.2 Clinical Chemistry. . . ... . .. . . ... . .. . ... . .. . . .. . . .. . ... . .. . . .. . ... . .. . . .. . ... . .. . . .. . ... . .. . . .. . ... . ... . ... . . . . . ... ... 18 10.3 Liver Biochemistry . . ... . ... . ... . .. . . .. . ... . .. . . .. . ... . .. . . .. . ... . .. . . .. . ... . ... . .. . . .. . ... . ... . .. . . .. . ... . . . . . ... . .. 23 10.3 .1 DNA content of liver ... . ... . .. . . .. . ... . ... . .. . ... . ... . .. . ... . .. . . .. . ... . ... . .. . ... . ... . ... . .. . . ... ... . . .. . ... . .. 23 10.3 .2 Peroxisome proliferation . . ... . .. . . .. . ... . .. . . . . . . .. . ... . .. . . .. . ... . . .. .... ... . . .. . . .. .... . .. . . .. . .. . . ... . .. . . . 25 10.3 .3 Total cytochrome P450 content of liver . . ... ... . .. . . ... ... . . .. . ... ... . . . . . . .. . ... . .. . . .. . ... . ... . .. . . . 26 10.3 .4 Lauric acid hydroxylation . ... ... . . .. . ... ... . . .. . . .. .... .. . . . .. . ... . .. . ... . .. . . .. . . .. . ... . .. . . .. . . .. . ... . .. . . . 27 10.3 .5 Pentoxyresorufin-O-depentylation .. . ... . . . . ... . ... . .. . ... ... . . .. . ... . ... . .. . . .. . . .. . ... . .. . . .. . ... . . .. . . 28 10.3 .6 Testosterone hydroxylation .. . ... . ... . .. . ... . ... . .. . ... . ... . .. . . .. . ... . .. . . .. . . .. . ... . .. . . .. . . .. . ... . . . . . ... . 29 10.4 Histopathology .. ... . . .. . . .. .... . .. . . .. . . .. . ... . .. . . .. . ... . .. . . .. . ... . .. . . .. . ... . ... . .. . ... . ... . .. . . .. . ... . ... . .. . ... . . 30 10.4.1 Liver H&E . . .... ... . . .. . . .. . ... . ... . .. . . .. . ... . .. . . .. . . .. . ... . .. . . .. . ... . .. . ... . .. . . .. . . .. . .... ... . . . . . . .. . ... . .. . . .. . 30 10.4.2 Hepatic Apoptosis . . . ... . .. . . .. . ... . .. . . .. . .... .. . . . .. .... ... . . . . . .. . ... . . . . . ... ... . . . . . . .. . ... . .. . . .. . ... . ... . .. . 30 CXR0486s Final Report Page 2 of 128 10.4.3 Cell proliferation in liver. . . ... . ... . ... . ... . .. . . . .. . ... .... . ... . .. . ... . . .. . .... .... . ... . .. . . .. . ... . . ... ... . . ... 31 10.4.4 Thyroid H&E . . .. . . .. . .... .... . .. . . .. . . .. . ... . .... .. . . .. . .... . ... . ... . ... . .. . . .. . .... .... . .. . . ... . ... ... . .... ... . . .. . 32 10.4.5 Thyroid in situ cell death ... . .. . . .. . ... . .... ... . . .. . ... . ... . ... . ... . ... . .. . . ... . ... . ... . ... . ... . .. . . ... . ... . ... 32 10.4 .6 Cell proliferation in thyroid ... . .. . . .. . . ... .... . ... ... . ... . ... . ... . ... . . .. . ... .... . ... . ... . ... . . .. . ... .... . ... 32 11 . DISCUSSION . .... .... . .. . . ... ... . ... . ... . ... . . .. . ... . ... . ... . .. . . .. . . .. . .... .... . .. . . .. . ... . . .. . .... .... . ... . .. . ... . ... 34 12. DATA STORAGE AND ARCHIVING . . .. . ... . ... . .. . . .. . . ... . ... . ... . ... . .. . . . .. . ... .... . ... . .. . . .. . . .. 34 13. REPORT.. . .. . . .. . .... . ... . .. . . ... . ... . .. . . ... .... . .. . . .. . . .. . ... . .... .. . . .. . . ... . ... . ... . .... ... . . . . . ... . ... . ... . ... . ... ... 34 14. QUALITY ASSURANCE .. . . .. . ... .... . ... . .. . . .. . ... . .... ... . . .. . ... . ... . ... . ... . ... . . .. . ... .... . ... . ... . ... . .. 34 15. STUDY DIRECTORS STATEMENT . .. . . ... . .. . .... .... . .. . ... . . .. . ... . .... .... . .. . . .. . ... . . .. . ... . .... .. 34 APPENDIX 1 . PATHOLOGY REPORT . . ... . ... . ... . ... .... . ... . .. . . .. . . .. . .... .... . ... . ... ... . . .. . . .. . .... . . 35 SUMMARY " The mean ingested doses of Test Items calculated for the 7 days of administration were: 1.93 0.01 (PFOS 20ppm) and 9.65 0.08 (PFOS 100ppm) mg Test Item / kg bwt / day. " No adverse clinical observations were made throughout the study . " Administration of dietary PFOS (20ppm or 100ppm) to rats for 7 days did not affect body weights . " Absolute liver weights were increased following exposure to PFOS l00ppm for 7 days, reaching 115% of control values . After 28 days on control diet, following exposure to PFOS 20ppm for 7 days, absolute liver weights were decreased to 87% of control values . Relative liver weights were increased following exposure to both PFOS 20ppm and PFOS 100ppm for 7 days to approximately 112% and 120% of control values, respectively. After 28 and 56 days receiving control diet relative livers weights had returned to control values . However, after 84 days on control diet, following exposure to both dietary concentrations of PFOS, relative liver weights were again increased, to approximately 113% of control values . The biological significance, if any, of this is unclear . " A lack of overt hepatotoxicity was indicated for all compounds by the absence of toxicologically significant increases in plasma ALT and AST . " Mean plasma cholesterol concentrations were decreased, in a dose dependant manner, by PFOS 20ppm and PFOS 100ppm after 7 days of treatment . Following a return to control diet for 28 days, plasma cholesterol concentrations were still decreased in a dose dependant manner . After 56 days reversal, cholesterol concentrations had recovered to near control levels in animals administered both concentrations of PFOS ; however plasma cholesterol concentrations were still decreased after 84 days reversal in animals treated with PFOS 100ppm. " Administration of PFOS 100ppm to rats for 7 days led to a small increase (127% of control) in hepatic peroxisomal (3-oxidation, as determined by measurement of CNinsensitive palmitoyl CoA oxidation (PCO) . This increase was maintained after 28 and 56 days return to control diet. Eighty-four days after withdrawal of PFOS 100ppm, PCO activity was returning to control levels . Administration of PFOS 20ppm did not increase PCO activity. " Administration of PFOS 20ppm and PFOS 100ppm to rats for 7 days resulted in CXR0486s Final Report Page 4 of 128 marked increases in hepatic total P450, in a dose dependent manner, to 124% and 190% of control values, respectively . Reversal to control diet for up to 84 days had no effect on the increased hepatic total P450 content . " Lauric acid 12-hydroxylation (12-OH LAH) is used as a marker for cytochrome P450 4A activity . PFOS administration at 20ppm had no effect on 12-OH LAH after 7 days on diet, whilst administration at 100ppm increased the 12-OH LAH by approximately 2-fold after 7 days on diet, and 3-fold after 28 and 56 days on control diet (Table 15) . After 84 days on control diet, however, 12-OH LAH activity had returned to control values. " Pentoxyresorufin-O-depentylation (PROD) is used as a marker for cytochrome P450 2B activity. PFOS administration at 20ppm initially decreased the PROD activity by approximately half the control value after 7 days on diet. Reversal to control diet for up to 84 days had no effect, and the decrease in activity was maintained throughout. PFOS administration at 100ppm increased the PROD activity by approximately 3.5fold after 7 days on diet. Reversal to control diet resulted in increased activity of approximately 1 .5-fold relative to control values after 28 days; a return to control values after 56 days ; and a decrease of approximately half the control value after 84 days . The decreases in PROD are believed to be incidental and not of biological significance . Testosterone 6R-hydroxylation (Test-6p-OH) is used as a marker for cytochrome P450 3A activity. PFOS administration at 20ppm had no effect on the Test-6(3-OH activity until, after 84 days on control diet, a slight increase was seen. This is not considered to be biologically significant. PFOS administration at 100ppm initially increased the Test-6(3-OH activity by 2.3-fold after 7 days on diet. Reversal to control diet resulted in a return to control values after 28 days followed by an increase in activity of approximately 2.2- and 1 .9-fold after 56 days and 84 days, respectively . " In the liver, histopathology revealed slightly decreased glycogen vacuolation in animals treated with 20ppm and 100ppm PFOS at each sacrifice period . A minimal to moderate, centrilobular hepatocellular hypertrophy was noted, dose related in severity, in animals treated with 20ppm PFOS and 100ppm PFOS for 7 days . This finding was still present after a 4-, 8- or 12-week treatment-free recovery period . An unequivocal difference in the severity and incidence of this finding between the various recove,ry periods was not observed, indicating an incomplete reversibility during the various recovery periods used. " Administration of PFOS 20ppm and PFOS 100ppm for 7 days decreased the hepatic apoptotic index to approximately 70% and 40% of control values, respectively. Reverting to control diet did not reverse this decrease inFhepatic apoptotic index, and CXR0486s Final Report Page 5 of 128 the decrease continued, reaching a minimum of 30% of control values after 84 days for PFOS 20ppm treated animals, and 20% of control values after 56 days for PFOS 100ppm treated animals . " PFOS 20ppm and PFOS 100ppm administration for 7 days both increased the hepatocellular labelling index (S-phase) by 2.5- and 5.9-fold, respectively. Reversal to control diet resulted in a return to control values after 28 days. " In the thyroid gland, no microscopic findings considered to be related to the treatment with the test items were recorded . The number of apoptotic cells found in all treatment groups, at all time points, was minimal . Regardless of treatment, it was usual to find either no cells or just one cell per thyroid staining in the TUNEL assay as apoptotic . " 20ppm PFOS and 100ppm PFOS treatment had no effect on thyroid S-phase activity at any time point studied . CXR0486s Final Report Page 6 of 128 1. INTRODUCTION A variety of non-genotoxic chemicals elicit hepatocellular adenomas and carcinomas when administered to rats in long-term studies. The appearance of tumours is preceded by the rapid and early stimulation of hepatomegaly . The liver enlargement seen following the administration of such "liver growth carcinogens" to rats is a result of both hypertrophy and hyperplasia. Depending on the particular chemical, the hypertrophy in characterised by proliferation of peroxisomes and/or smooth endoplasmic reticulum, induction of peroxisomal enzymes and induction of cytochrome P450 isoforms. The hyperplasia is characterised by stimulation of hepatocellular proliferation (S-phase, labelling index) and inhibition of apoptosis . PFOS has previously been shown to elicit marked liver growth in rats, and this study aims to characterise the reversibility of this hepatomegaly in these terms. The reversibility of thyroid effects, following the administration of PFOS to rats, will also be investigated . This study used liver, serum, thyroid and duodenal (for assessing BrdU labelling) samples generated over a time course including exposure for 7 days followed by withdrawal of treatment for 4, 8 and 12 weeks . The following parameters were studied : " Bodyweights " Liver weights (relative and absolute) " Clinical chemistry (5 parameters) . " Haematoxylin and eosin (H&E) histopathology (liver and thyroid) " Apoptotic indices analysed using the TUNEL assay (liver and thyroid) " BrdU incorporation analysed as a measure of cell proliferation (liver and thyroid) " The DNA content of liver was measured using the diphenylamine reaction " Acyl CoA oxidase activity was determined in liver heavy pellets as CN-insensitive palmitoyl CoA oxidation " Total P450 content was determined in liver microsomes " Lauric acid hydroxylation (LAH, CYP4A) was determined in liver microsomes " Pentoxyresorufin-O-depentylation (PROD, CYP2B) was determined in liver microsomes " Testosterone hydroxylation (CYP 3A) was determined in liver microsomes 2. ANIMALS (Scientific Procedures) ACT 1986 COMPLIANCE The in-life procedures undertaken during the course of this Study were subject to the provisions of the United Kingdom Animals (Scientific Procedures) Act 1986. The Act, administered by the UK Home Office, regulates all scientific procedures in living animals which may cause pain, suffering, distress or lasting harm and provides for the designation of CXR0486s Final Report Page 7 of 128 establishments where procedures may be undertaken, the licensing of trained individuals who perform the practical techniques, and the issue of project licences for specified programmes of work. This Study complied with all applicable sections of the Act and the associated Codes of Practice for the Housing and Care of Animals used in Scientific Procedures and the Humane Killing of Animals under Schedule 1 to the Act, issued under section 21 of the Act. 2.1 Licensee Responsibilities The procedure performed was PD1 as detailed in the PD project licence (60/3005) . The severity limit for this procedure is MODERATE. Mr F Simeons returned the animals at the start of the study under the PD project licence . 3. MATERIALS AND METHODS 3.1 Test Substances Perfluorooctane sulfonate potassium salt (PFOS K+, Lot no. 217, purity 87.6%), supplied by 3M Company, St. Paul Minnesota, USA, was a white powder . It was handled, and its identity verified, according to CXR Biosciences SOP 0010. This SOP describes routine procedures for the receipt, identification, labelling, handling, sampling and storage of Test and Reference Items . These procedures are implemented to ensure that: " Test and Reference Items used are those Items specified in Study Protocols . " Handling and storage of Test and Reference Items preserves their identity and integrity during use . 3.2 Safety Precautions The normal safety precautions as detailed in the relevant Sop's and COSHH assessments were applied, no additional precautions were considered necessary . 3.3 Diet RM1 powdered diet (Special Diet Services Ltd., Stepfield, Witham, Essex, UK) was used; Biological Sciences Resource Unit (BSRU) hold the specification of the diet. The Sponsor requested the dietary route of administration . The test diets, containing 20ppm PFOS or 100ppm PFOS, were prepared by CXR Biosciences (Laboratory Method Sheet [LMS] TSC-002) and samples were retained for analysis by the Sponsor . Rats were fed RM1 powdered diet or test diets ad libitum throughout the study . 3.4 Animals A sufficient number of male 6-7 week old Charles River Sprague Dawley (CD) rats were obtained from Charles River, UK. On arrival in the BSRU the rats were housed 2 per cage on sawdust in solid -bottom, polypropylene cages. The rats were acclimatised for a period of at CXR0486s Final Report Page 8 of 128 least 5 days before use . No animals were excluded from the study . Environmental enhancing additions were not used in the cages prior to the start of the Study. The Sprague Dawley (CD) rat was the test system of choice for this study because previous work on PFOS has been undertaken using this strain . In addition, CXR Biosciences has extensive experience in the use of this strain of rat, and the strain is well accepted by the regulatory authorities. 3.5 Animal Accommodation and Husbandry The environment was controlled in the animal room to provide conditions suitable for the Sprague Dawley (CD) strain of rat. The temperature was maintained within a range of 1923C and relative humidity within a range of 40-70l0 . There were a nominal 14-15 air changes per hour. Twelve-hour periods of light were cycled with twelve-hour periods of darkness. Drinking water was taken from the local supply and provided in bottles. Drinking water was provided ad libitum prior to and throughout the study . Prior to the start of the study the rats were allowed powdered RM1 diet ad libitum . 4. EXPEREVIENTAL DESIGN The animals were uniquely numbered by ear-punch and randomly allocated to groups up to one week after arrival . An experimental card was placed on each cage and showed the project licence code, treatment group, study number, sex and individual numbers of the rats within, and identified the Home Office Licensee. In addition, these cards were colour coded to correlate with the coding for the treatment group on the diet boxes and jars. The study consisted of one control and two test groups, each containing 40 male animals . Control animals received powdered RM1 diet ad libitum for the duration of the study . The test groups of animals were administered either 20ppm or 100ppm PFOS in the diet ad libitum for 7 days. On Day 8 the test diets were withdrawn and all animals were administered powdered RM1 diet ad libitum for the remainder of the study . The dietary concentrations of PFOS were prepared with purity correction . The corresponding PFOS concentrations in the powdered RM1 diet will be analyzed by the Sponsor. Ten animals from each group were sacrificed on days 8, 36, 64 and 92. Animals were implanted with osmotic pumps (Alzet 2ML1) containing bromodeoxyuridine (BrdU, 15mg/mL in phosphate buffered saline [PBS], pH7.4) 5 days before termination, whilst still receiving the .treatmqnt .re$imen (Table 1). CXR0486s Final Report Page 9 of 128 TABLE 1 EXPERIMENTAL DESIGN Group 1 2 3 Colour code Blue Green Yellow Animal No 1-10 11-20 21-30 31-40 41-50 51-60 61-70 71-80 81-90 91-100 101-110 111-120 Treatment Dates Control PFOS 20ppm PFOS l00ppm Arrival 21/11/06 Start 29/11/06 21/11/06 29/11/06 21/11/06 29/11/06 ~ ~ Implant Kill O1/12/06 06/12/06 29/12/06 03/01/07 26/01/07 31/01/07 23/02/07 28/02/07 01/12/06 06/12/06 29/12/06 03/01/07 26/01/07 31/01/07 23/02/07 28/02/07 O1/12/06 06/12/06 29/12/06 03/01/07 26/01/07 31/01/07 23/02/07 ~ 28/02/07 ~ 5. EXPEREVIENTAL PROCEDURES 5.1 Bodyweight The bodyweight of each rat was recorded at the start of the study . The animals were weighed weekly on the same day of the week. All animals were weighed prior to termination . Bodyweights were recorded electronically and records of the weights were stored in the Study File . 5.2 Clinical Observations Prior to the start of the study, all rats were observed to ensure that they were physically normal and that they exhibited normal activity. Each rat was observed at least once daily during the study. Clinical abnormalities of individual animals were recorded in the Study Diary . This was kept in the BSRU until completion of the in life phase of the study and then transferred to the Study File for retention and archiving . 5.3 Food Consumption Food consumption was measured every time the diet jars were changed / refilled, and when the animals were sacrificed, for the first week of the study so that food consumption could be recorded. Following reversion to control diet on Day 8, food consumption was not recorded. 5.4 Intercurrent Deaths There were no rats requiring euthanasia during the study. nor were there any intercurrent deaths. CXR0486s Final Report Page 10 of 128 5.5 Terminal Procedures On the day of termination the rats were weighed and transferred to the post mortem room. The rats were killed by exposure to a rising concentration of CO2. Approximately 6 to 10 mL of venous blood was taken by cardiac puncture and dispensed at equal volume into uncoated tubes (to obtain serum) and lithium/heparin-coated tubes (to obtain plasma) . The tubes were mixed on a roller for 10 min then cooled on ice. Serum and plasma were prepared by centrifugation (2,000 rpm for 10 min at 8 - 10C) then stored at approximately -70C pending analysis. The pellet was discarded . " The liver was removed from each animal and weighed. " 2 samples of liver, approximately 2mm strips, were taken, one from the Left lobe and one from the Median lobe. These were placed in 10% neutral buffered formaldehyde (NBF), for approximately 48hours, for BrdU immunohistochemistry and analysis of the apoptotic index . " 2 samples of liver, approximately 2mm strips, were taken, one from the Left lobe and one from the Median lobe. These were placed in NBF, for at least 1 week, for H&E histopathology . " 1 sample of liver, of approximately 0.25g, was weighed and then flash frozen in liquid nitrogen then stored at approximately -70C for DNA determination . " 2 samples of liver, of approximately lg each, were flash frozen in liquid nitrogen then stored at approximately -70C prior to dispatch, on dry ice, to the Sponsor for further analytical determination under a separate protocol " The remainder of the liver was weighed and scissor-minced in ice-cold 1 .15% (w/v) KCl prior to subcellular fractionation according to Laboratory Method Sheet (LMS) Cent-001 . Samples of homogenate, heavy pellet and microsomes were stored at approximately -70C prior to analysis . Samples of cytosol were stored at approximately -70C prior to dispatch, on dry ice, to the Sponsor for further analytical determination under a separate protocol . " 1 sample of duodenal small intestine, of approximately lcm, was taken and placed in NBF, for approximately 48 hours, as a positive control for BrdU immunohistochemistry. " The thyroid was removed from each animal, including the parathyroid gland together with trachea and oesophagus. This tissue was placed in NBF, for approximately 48 hours, for BrdU immunohistochemistry, analysis of the apoptotic index and H&E histology . 6. BIOCHEMICAL MEASUREMENTS 6.1 Clinical Chemistry Plasma samples, prepared as described, above; were assayed for ALT ; AST, cholesterol, CXR0486s Final Report Page 11 of 128 glucose and triglycerides using a Roche Integra 400 automated analyser according to the manufacturer's instructions. System control samples and calibration standards were those supplied by the manufacturer . Results were maintained in the Study File. 6.2 Peroxisome Proliferation CN- -insensitive acyl CoA oxidation was determined spectrophotometrically in liver heavy pellet, using palmitoyl CoA as a substrate, according to LMS Spec003 . Results were expressed as nmol NAD+ reduced/min/mg protein . Results were maintained in the Study File. 6.3 DNA Content of Liver The DNA content of liver was measured spectrophotometrically in whole tissue using the diphenylamine reaction, according to LMS Spec005 . Results were expressed as pg DNA/g liver and mg DNA/whole liver . Results were maintained in the Study File. 6.4 Total Cytochrome P450 The total cytochrome P450 content of liver microsomes was determined by measuring the carbon monoxide difference spectrum of ferrocytochrome P450 according to LMS Spec002. Results were expressed as nmol total P450/mg protein . Results were maintained in the Study File. 6.5 Lauric Acid 12-Hydroxylation (LAH) The activity of CYP4A in liver microsomes was determined using lauric acid and LC-MSMS. Results were expressed as nmols 12-OH lauric acid formed/ lOminutes/mg protein . Results were maintained in the Study File. 6.6 Pentoxyresorufin-O-depentylation (PROD) The activity of CYP2B in liver microsomes was determined spectrofluorometrically by the formation of resorufin from pentoxyresorufin, according to LMS Fluor-002 . Results were expressed as pmols resorufin formed/minute/mg protein . Results were maintained in the Study File. 6.7 Testosterone 6(3-Hydroxylation The activity of CYP3A in liver microsomes was determined by HPLC followed by UV detection, according to LMS HPLC-006 . Results were expressed as nmols hydroxytestosterone formed/minute/mg protein . Results were maintained in the Study File . 6.8 Protein Determination The protein concentration of tissue heavy pellets and microsomes was determined in aqueous solutions using a modification of the method of Lowry et al., (1951) and bovine serum albumin standards, according to LMS Spec001 . Results were maintained in the Study File. CXR0486s Final Report Page 12 of 128 6.9 Statistical Analysis Statistical comparisons between treated and control groups have been undertaken for all numerical data sets. The statistical analyses performed are documented with the results . 6 .10 Additional Analyses The Sponsor has requested no additional analyses. 7. HISTOPATHOLOGY Following fixation, all fixed liver and thyroid samples were processed, embedded and 5 1tM sections cut according to LMS Pat-005, Pat-006 and Pat-007 . Sections were: " Stained using H&E, according to LMS Pat-010, Pat-O11 and Pat-012 . " Analysed for in situ cell death. The method used was an indirect TUNEL labelling assay, according to LMS Pat-014 . " Analysed for BrdU incorporation as a measure of cell proliferation . The method used was an indirect BrdU labelling assay, according to LMS Pat-008. H&E sections were evaluated by Dr. med. vet. Ortwin Vogel, Goethestraf3e 26, 24116 Kiel, Schleswig-Holstein, Deutschland . The histopathology report was maintained in the Study File and as an appendix to the Study Report . 8. SPECIIVIEN SHIPMENT The powdered RM1 diets, cytosol, serum, and the liver samples designated for further analytical determination by the Sponsor were shipped on dry ice and sent to: Dave Ehresman 3M Company 3M Center Building 236-C 148 St. Paul, MN 55144 9. PROTOCOL AMENDMENTS There. were no changes to the protocol, and no Protocol Amendments have been signed by the Study Director. 10. RESULTS 10.1 Bodyweights,, Food Consumption and Liver Weights. 10.1.1 BodyWeights . , Administration of dietary PFOS (20ppm) to rats for 7 days followed by 28 days on control CXR0486s Final Report Page 13 of 128 diet lead to decreased body weights (Table 2). This effect was not deemed toxicologically significant . No adverse clinical observations accompanied the decreased body weights . Table 2. Terminal Bodyweights Bodyweight (g) Days on diet/reversal Oppm PFOS 20ppm PFOS l00ppm 7/0 296.4 30.1a 286.5 34.1 282.0 32.5 (100 10.2) (96 .7 11 .5) (95 .1 11.0) 7/28 423.3 46.0 382.2 30.8* 378.2 59.9 (100 10.9) (90 .3 7.3) (89 .4 14.1) 7/56 504.3 61.4 456 .1 57.8 476.6 32.8 (100 12.2) (90 .5 11 .5) (94 .5 6.5) 7/84 557.8 78.7 524.3 89.5 509.8 54.1 (100 14.1) (94 .0 16.1) (91 .49.7) a Values are Mean SD. Values in parenthesis are mean % control SD. n = 10 per group. A Student's t-test (2-sided) was performed on the results; *statistically different from control p<0 .05 ; **p<0.01 ; *** p<0 .001 . 700 .- soo , - - - soo r .oT 400 ~ v 0 a p 300 ; c G d H 200 100 - 7 Days on test diet Terminal Bodyweights 28 Days reversal 56 Jays mersal Days on diet 84 Days reeersal CXR0486s Final Report Page 14 of 128 10.1.2 Food consumption . The food consumption of the PFOS 20ppm and PFOS 100ppm-treated rats were essentially the same as control values throughout the experimental period (Table 3.). When diet consumption was calculated per unit body weight, the PFOS 20ppm and PFOS 100ppm-treated rats were essentially the same as control values throughout the experimental period. Table 3. Food Consumption Days on diet 0 m PFOS 20 m PFOS 100 m Diet consumed (g) / Rat / Week 7 194.112 .7 191 .715 .1 188 .823.1 (100 6.5) (98 .7 7.8) (97 .3 11 .9) Diet consumed (g) / K Bwt / Day 7 94.23.6 96.74.4 96.58 .0 (1003.78) (102.64.7) (102.58 .5) Test Item Consumption : m Test Item / K Bwt / Day 7 00 1 - -- 1 .930.01 1 9.650.08 a Values are Mean SD. Values in parenthesis are mean % control SD. n = 10 per group. A Student's t-test (2-sided) was performed on the results ; *statistically different from control p<0 .05 ; ** p<0 .01 ; *** p<0 .001 . 10.1.3 Liver weights. Absolute liver weights were increased following exposure to PFOS 100ppm for 7 days, reaching 115% of control values (Table 4). After 28 days on control diet, following exposure to PFOS 20ppm for 7 days, absolute liver weights were decreased to 87% of control values . Table 4. Absolute Liver Weights Liver weight (g) Days on Oppm PFOS PFOS ' diet/reversal 20ppm 100ppm I 7/0 13 .40 1 .38" 14.45 1 .71 15.42 1.85* 'I (100 10.31) (107.86 12.78) (115.08 13.80) ~I 7/28 18.04 2.88 15.76 1 .24* 17.46 3.46 ' (100 15.95) (87 .36 6.86) (96 .80 19.18) i 7/56 20.19 2.61 18.23 3.48 19.19 1 .76 ' (100 12.92) (90 .30 17.23) (95 .07 8.73) 7/84 20.23 3.40 21 .56 5.20 21 .03 2.74 (10016.79) (106 .5825 .72) (103.9513.55) ', a Values are Mean SD. Values in parenthesis are mean % control SD. n = 10 per group. A Student's t-test (2-sided) was performed on the results; *statistically different from control p<0 .05 ; **p<0.01 ; *** p<0 .001 . CXR0486s Final Report Page 15 of 128 25 - 20 rn I t I " I ~ 15 id 0 < 10 Absolute Liver Weights Control " PROS 20ppm o PFOS 100ppm 0 7 Days on test diet 28 Days reeersal 56 Days reRrsal Days on diet 84 Days re%ersal After normalisation with respect to bodyweight, relative liver weights were increased following exposure to both PFOS 20ppm and PFOS 100ppm for 7 days to approximately 112% and 120% of control values, respectively (Table 5). After 84 days on control diet, following exposure to both dietary concentrations of PFOS, relative liver weights were again increased, to approximately 113% of control values . Table 5. Liver/bod wei ht ratio (%) Liver/bodyweight ratio (%) Days on Oppm PFOS PFOS diet/reversal 20ppm l00ppm 7/0 4.530 .29a 5.060.38** 5.480.36*** (1006 .46) (111 .738.40) (120.917.86) 7/28 4.25 0.34 4.07 0.34 4.61 0.44 (100 8.12) (95 .71 8.02) (108.50 10.27) 7/56 4.02 0.36 3 .98 0.38 4.03 0.27 (100 9.05) (99 .03 9.58) (100.27 6.68) 7/84 3.63 0.39 4.09 0.51 * 4.12 025** (100 10.62) I (112.78 14.03) (113.587.01) a Values are Mean SD. Values in parenthesis are mean % control SD. n = 10 per group . A Student's t-test (2-sided) was performed on the results; *statistically different from control p<0 .05 ; **p<0 .01 ; *** p<0 .001. CXR0486s Final Report Page 16 of 128 Relative Liver Weights Control " PFOS 20ppm 0 PFQS 100ppm 6 7 Days on test diet 28 Days reversal 56 Days reoersal Days on diet ti4 Days reversal CXR0486s Final Report Page 17 of 128 10.2 Clinical Chemistry The lack of overt hepatotoxicity was indicated by the absence of toxicologically significant increases in plasma ALT and AST (Tables 6 and 7). Table 6. Plasma Alanine Aminotransferase Activity Days on diet/reversal Oppm ALT (U/L) PFOS 20ppm PFOS 100ppm ' 7/0 110 .60 18.64a 93 .97 10.47* 90.04 16.66* (100 16.86) (84 .96 9.47) (81 .41 15 .06) 7/28 110.32 17.16 117 .56 54.52 102 .78 22.45 (100 15.56) (106.56 49.42) (93 .17 20.35) 7/56 90.54 15.55 89.07 22.47 103 .85 29.44 (100 17.18) (98 .38 24.82) (114.70 32.52) 7/84 84.15 16.68 84.82 18.03 99.36 36.44 (100 19.83) (100.8021 .42) (118.0743.31) a Values are Mean SD . Values in parenthesis are mean Io control SD. n = 10 per group. A Student's t-test (2-sided) was performed on the results; *statistically different from control p<0 .05; **p<0.01 ; *** p<0 .001 . Plasma ALT 180 o Control PFOS 20ppm a PFOS 100ppm 7 Days on test diet 28 Days reversal 56 Days re\ersal Days on diet 84 Days reversal CXR0486s Final Report Page 19 of 128 p. 19 Table 7. Plasma As artate Transaminase Activity AST (U/L) Days on diet/reversal Oppm PFOS 20ppm PFOS 100ppm 7/0 102 .25 12.09a 103 .94 10.47 109 .97 12.75 (100 11.83) (101 .65 10.24) (107.55 12.47) 7/28 168 .98 38.11 151 .51 58.79 136 .02 28.85* (100 22.55) (89.66 34.79) (80 .49 17.07) 7/56 124 .75 24.75 128 .30 26.87 120 .31 22.49 (100 19.84) (102.85 21 .54) (96 .44 18.03) 7/84 184 .34 44.30 186 .13 40.44 177 .97 66.32 (100 24.03) (100.97 21 .94) (96 .54 35.98) a Values are Mean SD . Values in parenthesis are mean % control SD. n = 10 per group. A Student's t-test (2-sided) was performed on the results ; *statistically different from control p<0 .05; **p<0.01 ; *** p<0 .001 . I 300 250 Plasma AST o Control ', * PFOS 20ppm ~ 0 PFOS t00ppm ~ 7 Days on test diet 28 Days re%ersal 56 Days re%ersal Days on diet 84 Days reversal CXR0486s Final Report Page 19 of 128 Mean plasma cholesterol concentrations were decreased, in a dose dependant manner, by PFOS 20ppm and PFOS 100ppm after 7 days of treatment (Table 8). Following a return to control diet for 28 days, plasma cholesterol concentrations were still decreased in a dose dependant manner. After 56 days reversal, cholesterol concentrations were recovered to near control levels in animals administered both concentrations of PFOS; however this recovery was not present after 84 days reversal in animals treated with PFOS 100ppm . Table 8. Plasma Total Cholesterol Concentration Days on Total Cholesterol (mmoUL) ' Oppm PFOS PFOS diet/reversal 20ppm 100ppm 7/0 2.730.44a 2.170.37** 1 .770.39*** (100 16.16) (79 .63 13.49) (64.84 14.15) 7/28 2.290 .24 1 .610.33*** 1 .430.44*** (100 10.49) (70 .19 14.30) (62 .59 19.26) 7/56 2.19 0.35 1 .96 0.26 2.00 0.37 (100 15.93) (89 .44 11 .77) (91 .36 16.87) 7/84 2.17 0.36 1 .98 0.40 1 .75 0.38* (100 16.68) (91 .37 18.49) (80 .70 17.63) a Values are Mean SD . Values in parenthesis are mean % control SD. n = 10 per group. A Student's t-test (2-sided) was performed on the results; *statistically different from control p<0 .05 ; **p<0.01 ; *** p<0 .001 . 35r-_______- Plasma Cholesterol E3 Control o PROS 20plorn O PROS 100ppm 7 Days on test diet 28 Days reeersal 56 Days reeersal Days on diet 84 Days re\ersal CXR0486s Final Report Page 20 of 128 p. 21 Mean plasma glucose concentrations showed only one significantly different value after 56 days reversal following 7 days of PFOS at 100ppm (Table 9). This is not considered to be biologically significant . Table 9. Plasma Glucose Concentration Glucose (mmol/L) Days on Oppm PFOS PFOS diet/reversal 20ppm 100ppm 7/0 22.51 3.47a 22.92 3.58 22.26 2.93 (100 15.44) (101 .82 15.90) (98 .92 13.02) 7/28 19.98 3 .75 19.43 4.98 20.40 2.81 (100 18.76) (97 .28 24.94) (102.12 14.06) 7/56 17.87 2.67 19.34 2.82 23.18 3.29*** (100 14.96) (108.24 15.77) (129.71 18.44) 7/84 18.43 2.24 17.70 2.20 19.86 2.21 (100 12.17) (96.02 11 .92) (107.76 11 .97) a Values are Mean SD. Values in parenthesis are mean % control SD. n = 10 per group. A Student's t-test (2-sided) was performed on the results; *statistically different from control p<0 .05 ; **p<0.01 ; *** p<0 .001 . 30 25 20 O E E 15 0 u t7 10 5 Plasma Glucose Control " PFOS 20ppm OPFOS 100ppm 7 Days on test diet 28 Days reversal 56 Days reversal Days on diet 84 Days re%ersal CXR0486s Final Report Page 21 of 128 After 7 days on diet there was a marked reduction in mean plasma triglyceride levels following administration of l0ftppm PPFOS. The mean plasma triglyceride concentrations recovered to near control levels upon reversal to control diet (Table 10). Table 10. Plasma Triglyceride Concentration Triglycerides (mmoUL) Days on diet/reversal Oppm PFOS 20ppm PFOS l00ppm 7/0 1 .510 .44a 1 .580.53 1 .020.41* (100 28.92) (104.65 35.48) (67 .44 27.17) 7/28 1 .72 0.74 1 .32 0.32 1 .26 0.24 (100 43 .17) (76 .92 18.60) (73 .31 13.84) 7/56 1 .70 0.63 2.02 0.70 1 .26 0.34 (100 36.74) (118.81 41.38) (74 .25 19.80) 7/84 1 .66 0.50 2.35 0.99 1 .58 0.65 (100 30.09) (141.35 59.47) (95 .02 39.18) a Values are Mean SD. Values in parenthesis are mean % control SD. n = 10 per group. A Student's t-test (2-sided) was performed on the results ; *statistically different from control p<0 .05 ; **p<0 .01 ; *** p<0 .001 . Plasma Triglycerides 3.5 - 3,0 i 7 Days on test diet 28 Days rewsal 58 Days re~ersal Days on diet 84 Days re%ersal CXR0486s Final Report Page 22 of 128 p. 23 10.3 Liver Biochemistry 10.3.1 DNA content of liver The mean hepatic concentration of DNA was decreased by PFOS 20ppm and PFOS 100ppm treatments after 7 days on diet. Upon reversal to control diet, PFOS 20ppm treated animals recovered to near control levels after 28 days, whilst PFOS 100ppm treated animals did not recover to this level until 84 days (Table 11). Table 11 . Liver DNA Concentration (m DNA/ liver) Liver DNA (mg/g liver) I Days on Oppm PFOS PFOS diet/reversal 20ppm l00ppm 7/0 2.253 0.163" 2.043 0.165* 1 .669 0.406*** (100 7.23) (90 .65 7.34) (74.07 18.04) 7/28 2.179 0.132 2.109 0.339 1 .562 0.355*** (100 6.04) (96 .77 15.56) (71 .69 16.28) 7/56 2.162 0.504 1 .844 0.259 1 .655 0.325* (100 23.30) (85 .28 11 .96) (76 .52 15.03) 7/84 1 .915 0.226 1 .918 0.408 1 .740 0.153 (100 11 .78) (100.12 21.28) (90 .87 7.97) a Values are Mean SD. Values in parenthesis are mean % control SD. n = 10 per group. A Student's t-test (2-sided) was performed on the results; *statistically different from control p<0 .05 ; **p<0.01 ; *** p<0 .001 . 3-OT---2.5 --- -------- -- ------------ --Liver DNA concentration (mg DNA/g liver) o Control * PFOS 20ppm I * PFOS 100ppm j -------------- 7 Days on test diet CXR0486s Final Report 28 Days reversal Days on diet 56 Days reeersal Page 23 of 128 84 Days rmrsal Although neither PFOS administration at 20ppm nor 100ppm decreased the total liver content of DNA after 7 days on diet, upon reversal to control diet they both decreased the total liver content of DNA after 28 and 56 days of reversal . A return to control levels was seen after 84 days on control diet for both dose levels (Table 12). Table 12. Total Liver DNA Content (mg DNA/whole liver) Liver DNA (mg/whole liver) Days on diet/reversal Oppm PFOS 20ppm PFOS 100ppm 7/0 30.21 3.95a 29.43 3 .33 25.59 6.37 (100 13.08) (97 .43 11.02) (84.72 21 .09) 7/28 39.10 5 .30 31.71 3 .74** 26.83 6.27*** (100 13.56) (81 .09 9.57) (68 .61 16.03) 7/56 43 .45 10.91 33.14 5.09* 32.10 8 .08* (100 25.10) (76 .26 11.72) (73 .87 18.59) 7/84 38.80 7 .55 40.44 9.99 36.91 4.37 (100 19.45) (104.22 25.74) (95 .12 11 .25) a Values are Mean SD. Values in parenthesis are mean % control SD. n = 10 per group. A Student's t-test (2-sided) was performed on the results; *statistically different from control p<0 .05; **p<0.01 ; *** p<0 .001 . 60 I 50 40 .ma 0 ;aZo 30 E 20 10 --- - Total liver DNA content (mg DNA/whole liver) 7 Days on test diet 28 Days reversal 56 Days reeersal Days on diet Control PFOS 20ppm ', o PFOS t00ppm , 84 Days reversal CXR0486s Final Report Page 24 of 128 p. 25 10.3.2 Peroxisome proliferation Administration of PFOS 100ppm to rats for 7 days followed by up to 84 days reversal on control diet resulted in marked increases in hepatic peroxisomal (3-oxidation, as determined by measurement of CN-insensitive palmitoyl CoA (PCO) oxidation, at all time points studied . Administration of PFOS 20ppm, however, had no effect or a slight decrease in PCO (Table 13). Table 13. Hepatic Cyanide-insensitive Palmito 1 CoA Oxidation (PCO) PCO (nmol NAD+ reduced/minute/mg protein) Days on diet/reversal Oppm PFOS 20ppm PFOS l00ppm 7/0 11 .51 1 .104 9.64 2.09* 14.66 2.37** (100 9.59) (83 .73 18.15) (127 .31 20.57) 7/28 15.31 1 .57 14.672.71 21 .632.01*** (100 10.25) (95 .77 17.67) (141 .24 13.16) 7/56 16.49 1 .87 16.23 1 .40 21 .40 1 .53*** (100 11 .34) (98 .44 8 .48) (129.80 9.28) 7/84 15.17 1 .51 13 .20 2.18* 16.47 1 .21 * (100 9.97) (86 .99 14.36) (108.59 7.99) a Values are Mean SD. Values in parenthesis are mean % control SD . n = 10 per group. A Student's t-test (2-sided) was performed on the results ; *statistically different from control p<0.05 ; **p<0.01 ; *** p<0 .001 . 7 Days on test diet 28 Days reversal 56 Days reeersal 64 Days reversal CXR0486s Final Report Page 25 of 128 10.3.3 Total cytochrome P450 content of liver Administration of PFOS 20ppm and PFOS 100ppm to rats resulted in marked increases in hepatic total P450. Administration of PFOS increased total P450 in a dose dependent manner . Reversal to control diet for up to 84 days had no effect on hepatic total P450 content (Table 14). Table 14. Hepatic Total P450 Content Days on diet/reversal Oppm PFOS 20ppm PFOS ' 100ppm 7/0 0.860 .12a 1 .070.24* 1 .630.17*** (100 13.90) (123.53 27.82) (189.47 20.09) 7/28 0.650.05 0.870.17*** 1 .330.18*** (100 7.70) (134.16 26.38) (205 .06 28.35) 7/56 0.680.14 0.950.16*** 1 .120.18*** (100 20.27) (139.08 22.86) (164.68 26.70) 7/84 0.600.15 0.800.12** 1 .01 0.11*** (100 25.40) (132.12 19.85) (167 .27 18.81) a Values are Mean SD. Values in parenthesis are mean Io control SD. n = 10 per group. A Student's t-test (2-sided) was performed on the results; *statistically different from control p<0 .05 ; **p<0.01 ; *** p<0.001 . Microsomal Total P450 Control PROS 20ppm p PFOS 100ppm 7 Days on test diet 28 Days reversal 56 Days reversal Days on diet 84 Days reversal CXR0486s Final Report Page 26 of 128 p. 27 10.3.4 Laurie acid hydroxylation Lauric acid 12-hydroxylation (12-OH LAH) is used as a marker for cytochrome P450 4A activity . PFOS administration at 20ppm had no effect on 12-OH LAH after 7 days on diet, whilst administration at 100ppm increased the 12-OH LAH by approximately 2-fold after 7 days on diet, and 3-fold after 28 and 56 days on control diet (Table 15) . After 84 days on control diet, however, 12-OH LAH activity had returned to control values . Table 15. Laurie Acid 12-Hydroxylation Lauric Acid 12 Hydroxylation (nmoUlOminutes/mg protein) Days on diet/reversal Oppm PFOS 20ppm PFOS l00ppm 7/0 5.952 .24a 6.622 .12 12.281.73*** (100 37.62) (111 .34 35.65) (206.35 29.09) 7/28 2.40 1 .09 2.75 0.48 6.91 2.40*** (100 45.65) (114.96 19.92) (288.53 100 .13) 7/56 2.01 0.76 1 .89 0.53 5 .72 1 .92*** (100 38.10) (94 .18 26.23) (285.20 95 .70) 7/84 2.56 1 .16 2.48 0.79 3 .41 1 .73 (100 45 .436) (97 .05 31 .04) (133.23 67.75) a Values are Mean SD. Values in parenthesis are mean Io control SD . n = 10 per group. A Student's t-test (2-sided) was performed on the results ; *statistically different from control p<0 .05; **p<0.01 ; *** p<0 .001 . Microsomal Lauric Acid 12 Hydroxylation 16 . o Control - - - w Pros 2opprn o PFos i ooppm 7 Days on test diet 28 Days re%ersal 56 Days reversal 84 Days reversal ' CXR0486s Final Report Page 27 of 128 10.3.5 Pentoxyresorufin-O-depentylation Pentoxyresorufin-O-depentylation (PROD) is used as a marker for cytochrome P450 2B activity . PFOS administration at 20ppm initially decreased the PROD activity by approximately half the control value after 7 days on diet. Reversal to control diet for up to 84 days had no effect, and the decrease in activity was maintained throughout (Table 16) . PFOS administration at 100ppm increased the PROD activity by approximately 3.5-fold after 7 days on diet. Reversal to control diet resulted in an increase in activity of approximately 1 .5fold after 28 days; a return to control values after 56 days ; and a decrease of approximately half the control value after 84 days . Table 16. Pentoxyresorufin-O-depentylation Days on diet/reversal PROD (pmols resorufm formed/minute/mg protein) Oppm PFOS PFOS 20ppm 100ppm 7/0 3.970.84a 1 .950.61*** 14.363 .51*** (100 21.22) (49 .19 15.48) (361 .52 88.35) 7/28 3.12 1 .12 1 .57 0.58** 5 .06 1 .95* (100 35.97) (50 .16 18.44) (161 .99 62.37) 7/56 2.66 0.54 1 .17 0.49*** 2.56 0.86 (100 20.32) (43 .99 18.55) (95 .91 32.30) 7/84 2.710 .97 1 .200.37*** 1 .180.38*** (100 35.86) (44 .37 13.77) (43 .73 14.05) A Values are Mean SD . Values in parenthesis are mean Io control SD. n = 10 per group. A Student's t-test (2-sided) was performed on the results; *statistically different from control p<0 .05 ; **p<0.01 ; *** p<0 .001 . 20 - - 1 18 j- c 18 - .v. i a 14 tI- E 12 ~- w i I E 1o t o j c 8I t m 12 s 0 a 4 2 Pentoxyresorufin-O depentylation I - - -T - - ., I I _ s_ Control m PFOS 20ppm t7 PFOS 100ppm ', I 7 Days on test diet 28 Days reversal S6 Days reversal 84 Days reversal CXR0486s Final Report Page 28 of 128 p. 29 10.3.6 Testosterone hydroxylation Testosterone 6(3-hydroxylation (Test-6(3-OH) is used as a marker for cytochrome P450 3A activity . PFOS administration at 20ppm had no effect on the Test-6(3-OH activity until after 84 days on control diet. This is not considered to be biologically significant (Table 17). PFOS administration at 100ppm initially increased the Test-6(3-OH activity by 2.3-fold after 7 days on diet. Reversal to control diet resulted in a return to control values after 28 days followed by an increase in activity of approximately 2.2- and 1 .9-fold after 56 days and 84 days, respectively . Table 17. Testosterone 6/3 hydroxylation Days on diet/reversal Test-6(3-OH (nmols 6(3-OH formed/minute/mg protein Oppm PFOS PFOS 20ppm l00ppm 7/0 3 .66 0.93a 4.24 1 .27 8.48 2.34*** (100 25.45) (115 .88 34.87) (231 .87 64.06) 7/28 2.92 0.63 3.43 1 .20 3.39 0.63 (10021 .63) (117.51 41 .17) (116.1021 .48) 7/56 2.54 0.54 1 .63 1 .35 5.60 1 .93*** ! (100 21 .29) (64 .34 53 .22) (220.67 75.84) 7/84 2.120.74 2.970.89* 4.06 1 .31*** ' (100 34.66) (139.72 41 .84) (191 .09 61 .70) a Values are Mean SD. Values in parenthesis are mean % control SD. n = 10 per group. A Student's t-test (2-sided) was performed on the results; *statistically different from control p<0 .05 ; **p<0.01 ; *** p<0 .001 . 'i I 12 T----------- Microsomal Testosterone 6beta Hydroxylation ----------- --------- 10 Control * PFOS 20ppm O PFOS 100ppm 0 a a c loE I o iE 4 II - I c 2 I 7 Days on test diet 28 Days re%ersal 56 Days reversal 84 Days re%ersal CXR0486s Final Report Page 29 of 128 10.4 Histopathology 10.4.1 Liver H&E Microscopically, treatment related findings were recorded in the liver. When compared with the control animals, a slight decrease in the usual glycogen induced hepatocellular vacuolation was noted in the animals of the 20ppm and 100ppm PFOS groups at each sacrifice period . Additionally, a minimal to moderate, centrilobular hepatocellular hypertrophy was noted, dose related in severity, in the animals of the 20ppm PFOS and 100ppm PFOS after 7 days of treatment. This finding was still present after a 4-, 8- or 12-week treatment-free recovery period. An unequivocal difference in the severity and incidence of this finding between the various recovery periods was not observed, indicating an incomplete reversibility during the various recovery periods used. 10.4.2 Hepatic Apoptosis Administration of PFOS 20ppm and PFOS 100ppm for 7 days decreased the hepatic apoptotic index to approximately 70% and 40% of control values, respectively . Reverting to control diet did not reverse this decrease in hepatic apoptotic index, and the decrease continued, reaching a minimum of 30% of control values after 84 days for PFOS 20ppm treated animals, and 20% of control values after 56 days for PFOS 100ppm treated animals (Table 18) . Table 18. Hepatic A o totic Indices Apoptotic Index (%) Days on Oppm PFOS PFOS diet/reversal 20ppm l00ppm 7/0 0.22 0.07a 0.15 0.07* 0.09 0.03*** (100 32.73) (69 .92 30.44) (41 .76 13.93) 7/28 0.230 .13 0.100.04** 0.080.03** (100 53.68) (41 .79 17.86) (32 .68 14.03) 7/56 0.21 0.08 0.100.06** 0.040.03*** (100 36.82) (47 .26 27.49) (19 .17 13.62) 7/84 0.230 .07 0.070.05*** 0.070.02*** (100 29.79) (30 .70 t 22.55) (29 .24 +-9 .73) a Values are Mean SD. Values in parenthesis are mean % control SD. n = 10 per group. A Student's t-test (2-sided) was performed on the results; *statistically different from control p<0 .05; **p<0.01 ; *** p<0 .001 . CXR0486s Final Report Page 30 of 128 Hepatic Apoptotic Indices 0 .4 p. 31 7 Days on test diet 28 Days reversal 56 Days reversal Days on diet 84 Days reversal 10.4.3 Cell proliferation in liver PFOS 20ppm and PFOS 100ppm administration for 7 days both increased the hepatocellular labelling index (S-phase) by 2.5- and 5 .9-fold, respectively (Table 19). Reversal to control diet resulted in a return to control values after 28 days . Table 19. Hepatic S-phase Labelling Indices Labelling Index (%) Days on Oppm PFOS PFOS diet/reversal 20ppm l00ppm 7/0 1 .220:80a 2.830.85*** 7.143.93*** (100 65.77) (231 .97 69.58) (585.99 322.37) 7/28 1 .41 0.68 2.44 2.09 1.77 1 .13 (100 48.23) (173.23 148 .40) (126.01 80.52) 7/56 0.50 0.22 1 .00 1 .00 0.68 0.54 (100 43.81) (200.05 200.63) (136.91 107 .27) 7/84 1 .30 0.44 0.67 0.46** 1 .08 0.60 (100 34.16) (51 .43 35 .53) (83 .07 46.08) a Values are Mean SD. Values in parenthesis are mean % control SD. n = 10 per group. A Student's t-test (2-sided) was performed on the results ; *statistically different from control p<0 .05 ; **p<0 .01 ; *** p<0 .001 . CXR0486s Final Report Page 31 of 128 12---- 10 -- 8 ,x m -o . c . c a m J 4- Hepatic S-Phase t7 Control " PFOS 20ppm , * PFOS 100pprn ', 7 Days on test diet 28 Days reversal Days on diet 56 Days reversal 84 Days re%ersal 10.4.4 Thyroid ME In the thyroid gland, no microscopic findings considered to be related to the treatment with the test items were recorded. 10.4.5 Thyroid in situ cell death The level of apoptosis found in both treatment groups, at all time points, was minimal . It was usual to find either no cells, or just one cell per thyroid, staining in the TUNEL assay as apoptotic, regardless of treatment. 10.4.6 Cell proliferation in thyroid PFOS 20ppm and PFOS 100ppm treatment had no effect on S-phase at any time point studied (Table 20) . CXR0486s Final Report Page 32 of 128 p. 33 Table 20. Th roid S-phase Labelling Indices Labelling Index (%) I Days on diet/reversal 7/0 Oppm 2.62 0.21 a PFOS' 20ppm 2.70 0.56 PFOS' ' l00ppm 3 .28 1 .10 (100 8.14) (103.16 21 .38) (125.34 42.08) 7/28 1 .50 0.33 1 .70 0.27 1 .82 0.38 (10021 .81) (113.49 18 .00) (121 .5625.44) 7/56 1 .12 0.33 1.30 0.26 1 .32 0.25 (100 29.70) (116.27 23 .60) (118.41 22.55) 7/84 1 .02 0.31 1 .21 0.24 1 .18 0.07 (10030.58) (118.6123.52) (115 .036.57) a Values are Mean SD. Values in parenthesis are mean % control SD. n = 10 per group; 1 n = 5 per group. A Student's t-test (2-sided) was performed on the results ; *statistically different from control p<0.05 ; **p<0 .01 ; *** p<0.001 . 5 4.5 - 4 3.5 0 3 K d D C '- 2 5 C 1 d a a J 15~ . 0 5 II- _ 0 7 Days on test diet Thyroid S-Phase 28 Days reoersal Days on diet 56 Days reeersal 84 Days reversal CXR0486s Final Report Page 33 of 128 11. DISCUSSION In this study, PFOS appeared to exhibit the combined hepatic effects of a peroxisome proliferator and "phenobarbital-like" enzyme inducer. Thus, PFOS administration for 7 days generally reflected the results obtained in a previous study, CXR0481 . As in the earlier study no effects of PFOS on the thyroid were observed . Both histopathologically and biochemically, reversibility was not complete after 7 days of PFOS treatment followed by up to an 84 day treatment-free recovery period . Hepatic apoptosis, particularly, was still severely inhibited after this reversal time. Likewise the induced microsomal cytochrome P450 content and representative monoxygenase activities were maintained throughout the treatment-free recovery period . This latter observation was reflected by the maintenance of hepatocellular hypertrophy . 12. DATA STORAGE AND ARCHIVING The data has been held in a Study File according to CXR Biosciences SOPs 0003 and 0005. On completion of the study, the following items will be retained in the CXR Archive : " Study protocol and possible amendments " Study Report and possible amendments " Study File (including Study Diary) " Relevant correspondence All items will be kept for two years from the date of signing the Study Report unless, in the opinion of CXR Biosciences, the quality of the specimens no longer allows evaluation. At the end of the two-year period, the Study Sponsor will be contacted and the items either sent to the Study Sponsor or retained by CXR Biosciences under a separate agreement . 13. REPORT The Study Report has been prepared as described in CXR Biosciences SOP 0004. 14. QUALITY ASSURANCE Study activities at the Test Facility (CXR Biosciences Ltd and BSRU) have been conducted according to relevant SOPs. There were no deviations from this protocol. No claim of GLP compliance has been made for this study . 15. STUDY DIRECTORS STATEMENT Final Report signature by the Study Director indicates acceptance of responsibility for the validity and integrity of the Study data . CXR0486s Final Report Page 34 of 128 APPENDIX 1. PATHOLOGY REPORT CXR0486s Final Report Page 35 of 128 PATHOLOGY REPORT TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . CXR 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2dl PREPARED BY : Dr . med . vet . Ortwin Vogel Veterinary Pathologist CXR0486s Final Report Page 36 of 128 PATHOLOGY REPORT TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TABLE OF CONTENTS AUTHENTICATION PAGE CXR I 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 PAGE : 1 PRINCIPAL SECTION SUMMARY METHODS RESULTS CONCLUSIONS REFERENCES 2 - 3 4 - 5 6 - 7 8 - 9 10 EXPLANATION OF CODES AND SYMBOLS SUMMARY TABLES SUMMARY INCIDENCE OF GRADINGS BY ORGAN/GROUP/SEX NECROPSY STATUS : TERMINAL SACRIFICE GROUP (KO) SUMMARY INCIDENCE OF GRADINGS BY ORGAN/GROUP/SEX NECROPSY STATUS : RECOVERY / POST-TREATMENT GROUP (R1) SUMMARY INCIDENCE OF GRADINGS BY ORGAN/GROUP/SEX NECROPSY STATUS : RECOVERY / POST-TREATMENT GROUP (R2) SUMMARY INCIDENCE OF GRADINGS BY ORGAN/GROUP/SEX NECROPSY STATUS : RECOVERY / POST-TREATMENT GROUP (R3) INDIVIDUAL ANIMAL DATA TABLE OF INDIVIDUAL MICROSCOPIC FINDINGS (AOFT) ANIMAL HEADING DATA DOSE GROUP 01 TEXT OF MICROSCOPIC FINDINGS DOSE GROUP 01 ANIMAL HEADING DATA DOSE GROUP 02 11 12 13 14 15 16 - 27 28 29 - 48 49 CXR0486s Final Report Page 87 of 128 PATHOLOGY REPORT TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TABLE OF CONTENTS TEXT OF MICROSCOPIC FINDINGS DOSE GROUP 02 ANIMAL HEADING DATA DOSE GROUP 03 TEXT OF MICROSCOPIC FINDINGS DOSE GROUP 03 PAGE CXR II 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2dl PAGE : 50 - 69 70 71 - 90 CXR0486s Final Report Page-38 of 128 PATHOLOGY REPORT PAGE CXR 1/ 90 0486s TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 AUTHENTICATION The undersigned hereby declares that the histopathology data in this report were compiled by him, and that they reflect accurately the raw data records . Dr . med . vet . Ortwin Vogel Veterinary Pathologist TPC Toxicologic Pathology Goethestr . 26 D-24116 Kiel Consultancy CXR0486s Final Report Page 39 of 128 PATHOLOGY REPORT PRINCIPAL SECTION TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . SUMMARY PAGE CXR 2/ 90 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 To investigate the reversibility of Perfluorooctane Sulfonate (PFOS)-induced effects, pathomorphological examinations were performed on 120 male Sprague Dawley rats . The rats were allocated to 3 dose groups each consisting of 40 males and received the test item via the feed at dose levels of 20 ppm "PFOS" or, 100 ppm "PFOS" (groups 02 and 03, respectively) for a treatment period of 7 days . The rats of group 01 received the same diet without any test item and served as control animals . Ten rats of each group were assigned for the sacrifice at the end of the treatment period (test day 8) and ten animals each were assigned to the sacrifices after a 4-week (R1), 8-week (R2) and 12-week (R3) recovery period . At the end of the treatment/recovery periods, the rats were sacrificed and de- tailed necropsies performed . Histopathological examination was performed on the liver and thyroid from all animals of all groups . Microscopically, treatment related findings were recorded in the liver . When compared with the control animals, a slight decrease in the usual glycogen induced hepatocellular vacuolation was noted in the animals of groups 02 (20 ppm PFOS) and 03 (100 ppm PFOS) on each sacrifice period . In addition, a minimal to moderate, centrilobular hepatocellular hypertrophy was noted, dose related in severity, in the animals of groups 02 (20 ppm PFOS) and 03 (100 ppm PFOS) after 7 days of treatment . This finding was still present in animals of groups 02 and 03 sacrificed after a 4-, 8- or 12-week treatment-free recovery period . An unequivocal difference in the severity and incidence of this finding between the various recovery periods was not observed indicating an incomplete reversibility during the various recovery periods used in this study . In the thyroid gland, no microscopic finding was recorded, that is considered to be related to the treatment with the test items . All other microscopic findings recorded did not distinguish significantly treated rats from vehicle control rats or the differences noted were regarded as random events . All these findings are considered to be spontaneous in nature and within the normal background pathology commonly seen in rats of these strains and age . CXR0486s Final Report Page 40 of 128 PATHOLOGY REPORT PRINCIPAL SECTION TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . SUMMARY PAGE CXR 3/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 Under the conditions of this study, the repeated administration of 20 or 100 ppm "PFOS" via the feed to rats of the Sprague Dawley strain for a treatment period of 7 days produced a centrilobular hepatocellular hypertrophy, that was not completely reversible during a 4-, 8- or 12-week treatment-free recovery period . CXR0486s Final Report Page 41 of 128 PATHOLOGY REPORT PRINCIPAL SECTION TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . METHODS PAGE CXR 4/ 90 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2dl Group Design for Pathological Evaluation Dose Pathdata Test item+ Number of Rats* Animal numbers Group Group Males Males . ....... .... .... .... . ... . .... ....... ....... . ... .... .... .... ... .... . .. . . .. 01 Ol 0 (Control) 10** 1 - 10 10*** 11 - 20 10**** 21 - 30 10***** 31 - 40 02 02 20 ppm PFOS 10** 10*** 10**** 10***** 41 - 50 51 - 60 61 - 70 71 - 80 03 03 100 ppm PFOS 10** 10*** 10**** 10***** 81 - 90 91 - 100 101 - 110 111 - 120 + * ** *** **** ***** Perfluorooctane sulfonate potassium salt (PFOS K+), 3M Company, St . Paul, Minnesota, USA . strain : Sprague Dawley (CD), (Breeder : Charles River, UK) ; These animals are listed in the tables under necropsy status K0 . These animals are listed in the tables under necropsy status R1 . These animals are listed in the tables under necropsy status R2 . These animals are listed in the tables under necropsy status R3 . Administration of the Test Item The in-life part of the study was performed at CXR Biosciences Ltd ., James Lindsay Place, Dundee DDl 5JJ, UK . The animals were administered 20 ppm PFOS (group 02) or 100 ppm PFOS (group 03) in the diet ad libitum for 7 days . The animals of group 01 received the same diet without the test item and served as control animals . Necropsy and Histopathology At the end of the assigned study periods (test days 8, (sacrifice group KO), 36 (sacrifice group R1), 64 (sacrifice group R2) and 92 (sacrifice CXR0486s Final Report Page 42 of 128 PATHOLOGY REPORT PRINCIPAL SECTION TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . METHODS PAGE CXR 5/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 group R3), the rats were killed by exposure to a rising concentration of C02 . Two samples of the liver (one from the left lobe, one from the median lobe) and the thyroid including parathyroid gland together with trachea and oesophagus were taken and preserved in 10s neutral buffered formaldehyde (NBF) . Histotechnique was performed at Progenix Research UK Ltd ., Suite 1 Forth House, Burnside Business Court, Inverkeithing, Fife, KY11 1NZ, Scotland . The preserved liver and thyroid samples of all rats of all groups were trimmed, processed, embedded in paraffin wax and sectioned at a nominal thickness of about 5 ~im . All sections were stained with haematoxylin and eosin . One section of each organ sample was examined by the undersigned pathologist by light microscope . Data compilation The animal heading data recorded by CXR at postmortem examination were taken from the post-mortem records and entered manually into the PathData Computer system, current version, under Pathology No . 07059 (PathData is a registered trademark of Pathology Data Systems, Inc .) . Microscopic findings are recorded by the undersigned pathologist during histopathological examination using on-line data input into the pathology computer system . All microscopic observations are presented for each rat in the "Table of Individual Microscopic Findings" as well as in full descriptive terms in the "Individual Animal Data - Text of Gross and Microscopic Findings" . The incidence of microscopic findings is also presented in tabular form . Incidence tables were created by the Pathdata software . Histologic alterations were described, wherever possible, according to their distribution, severity and morphologic character . Severity scores were assigned as given under "Explanation of Codes and Symbols" . The slides were evaluated on December 19/20, 2007 . Archive For archiving purposes, the raw data including a CD-ROM containing the electronic study data and the microscopic slides will be returned to CXR Biosciences Ltd ., James Lindsay Place, Dundee DD1 5JJ, UK . CXR0486s Final Report Page 43 of 128 PATHOLOGY REPORT PRINCIPAL SECTION TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . RESULTS PAGE CXR 6/ 90 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 Microscopic findings At histopathologic examination, few microscopic findings were recorded . These findings are described in detail in the "Individual Animal Data Text of Gross and Microscopic Findings" . THYROID : There were no microscopic related to the treatment with findings any test recorded items in that are considered to be either sacrifice group . LIVER : Sacrifice at the end of the treatment period (sacrifice KO) : .... ... .... .... .... .... . ... .... .... ....... . ... .... ....... ... A minimal to moderate, periportal hepatocellular vacuolation that is considered to be due to an intracytoplasmic glycogen accumulation was prominent in the control animals of group 01 . Hepatocellular vacuolation was also present, similar in incidence and severity, in the animals of group 02 (20 ppm PFOS) and, with a slight decreased mean severity (mean 2 .0) when compared with the control animals (mean 2 .5), of group 03 (100 ppm PFOS) Centrilobular hepatocellular hypertrophy was noted, minimal to slight in degree, in treated rats only . This finding occurred in 8 animals of group 02 (20 ppm PFOS, mean severity 1 .0) and all animals of group 03 (100 ppm PFOS, mean severity 1 .8) . Sacrifice after a 4-week treatment-free recovery period (sacrifice Rl) : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .----- . . . . . . . . . . . . . Hepatocellular vacuolation similar to the finding noted in the animals of the KO-sacrifice was recorded also in all control animals of this sacrifice group . A slight decrease in incidence and severity of this finding was noted in groups 02 (20 ppm PFOS, 7 animals affected, mean severity 1 .9) and 03 (100 ppm PFOS, 6 animals affected, mean severity 1 .8) when compared with the control group (all ten animals affected, mean severity 2 .0) . A minimal to moderate animals only occurring ty 1 .2) and all ten rats hepatocellular hypertrophy was noted in in 5 rats of group 02 (20 ppm PFOS, mean of group 03 (PFOS 100 ppm, mean severity treated severi2 .1) . CXR0486s Final Report Page 44 of 128 PATHOLOGY REPORT PRINCIPAL SECTION TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . RESULTS PAGE CXR 7/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 Sacrifice after a 8-week treatment-free recovery period (sacrifice R2) : . .... . .... ..... ......... .... . .... ........ ......... .... . ... . ..... ....... A similar hepatocellular vacuolation as recorded in the liver of the animals sacrificed at the end of the treatment period (KO-sacrifice) was also noted, minimal to moderate in degree, in all control rats of groups Ol and almost all treated animals of groups 02 and 03 . A slight decrease in incidence and severity of this finding was noted in groups 02 (20 ppm PFOS, 9 animals affected, mean severity 2 .1) and 03 (100 ppm PFOS, 9 animals affected, mean severity 1 .7) when compared with the control group (mean severity 2 .4) . Hepatocellular hypertrophy occurred only in treated was noted, minimal in degree, in 4 animals of group severity 1,0) and, slight to moderate in degree, group 03 (100 PFOS, mean severity 2 .1) . animals . This finding 02 (20 ppm PFOS, mean in all ten animals of Sacrifice after a 12-week treatment-free recovery period (sacrifice R3) : .... . ..... .... ..... .... .... . .... ........ ..... ........ . .... . .... .... .... . Hepatocellular vacuolation similar to the finding noted in the animals of the KO-sacrifice was recorded also in all control animals . A slight decrease in the incidence and/or severity of this finding was noted in groups 02 (20 ppm PFOS, 10 animals affected, mean severity 2 .0) and 03 (100 ppm PFOS, 7 animals affected, mean severity 1 .7) when compared with the control group (all 10 animals affected, mean severity 2 .6) . Hepatocellular hypertrophy occurred in treated animals only . This finding was recorded, minimal in degree, in 5 males of group 02 (20 ppm PFOS, mean severity 1 .0) and in all 10 males of group 03 (100 pph PFOS, mean severity 1 .7) . The type, incidence and severity of all other microscopic alterations recorded did not indicate a relationship to the treatment with the test item or the differences noted were regarded as random events . These findings were regarded to be spontaneous in nature and within the normal background pathology commonly seen in rats of these strains and age . CXR0486s Final Report Page 45 of 128 PATHOLOGY REPORT PRINCIPAL SECTION TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . CONCLUSIONS PAGE CXR 8/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2dl In this 13-week toxicity study of the test item "PFOS" administered with the feed to Sprague Dawley rats at dose levels of 0 ppm (control), 20 ppm PFOS or 100 ppm PFOS, a hepatocellular vacuolation occurred in rats of all groups and sacrifice periods . The severity of this finding appeared to be decreased in treated groups when compared with the control group in all sacrifice periods . This kind of vacuolation is considered to be due to the intracytoplasmic presence of glycogen and is quite commonly observed in well-fed, untreated rodents . The difference between control and treated animals noted in this study at different sacrifice periods is considered to be related to the treatment with the test items . A minimal to slight centrilobular hepatocellular hypertrophy was noted, dose related in severity, in the animals of groups 02 and 03 sacrificed at the end of the 7-day treatment period (KO) . In the absence of any hepatocellular hypertrophy in the control animals, this alteration is considered to be related to the treatment with the test item . A large number of medicinal agents with different chemical structures and therapeutic activities produce liver enlargement when given in high doses to species used in toxicity studies (Greaves, 1990) . This enlargement that may be characterized morphologically by hepatocyte hypertrophy, hepatocyte hyperplasia or both may be accompanied by an increase in activity of the hepatic microsomal drug metabolizing enzymes in the absence of any morphogical evidence of hepatocellular damage . The changes are typically reversible on cessation of treatment . In this study, however, hepatocellular hypertrophy was also present, similar in incidence and severity, in both treated groups sacrificed after a 4-, 8- or 12-week treatment-free observation period . An unequivocal difference in the severity and incidence of this finding between the various recovery periods was not observed . This indicates, that a complete recovery did not occur during the recovery periods used in this study . In the thyroid gland, are considered to be either sacrifice group . there were no microscopic findings recorded that related to the treatment with any test items in All other microscopic findings recorded did not distinguish significantly treated rats from vehicle control rats or the differences noted were regarded as random effects . All these findings are considered to be spontaneous in nature and within the normal background pathology commonly seen in rats of these strains and age . CXR0486s Final Report Page 46 of 128 PATHOLOGY REPORT PRINCIPAL SECTION TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . CONCLUSIONS PAGE CXR 9/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 Under the conditions of this study, the repeated administration of 20 or 100 ppm "PFOS" via the feed to rats of the Sprague Dawley strain for a treatment period of 7 days produced a centrilobular hepatocellular hypertrophy, that was not completely reversible during a 4-, 8- or 12-week treatment-free recovery period . CXR0486s Final Report Rage 47 of 128 PATHOLOGY REPORT PRINCIPAL SECTION TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . REFERENCES PAGE CXR 10/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 Greaves P . (1990) : Histopathology of Preclinical Toxicity Studies : Interpretation and Relevance in Drug Safety Evaluation . Digestive System I, 278-392 . Elsevier, Amsterdam New York Oxford CXR0486s Final Report Page 48 of 129 PATHOLOGY REPORT TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PE'1DS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . EXPLANATION OF CODES AND SYMBOLS PAGE CXR . 11/ 90 . 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 CODES AND SYMBOLS USED AT ANIMAL LEVEL : M = Male animal KO = Terminal sacrifice group R1 . . .R9 = Recovery / post-treatment group 1 . . .9 CODES AND SYMBOLS USED AT ORGAN LEVEL : 0 = Tissue not present for histologic examination = Histologic examination not required + = Organ examined, findings present = Organ examined, no pathologic findings noted (AOFT only) ( = Only one of paired organs examined/present CODES AND SYMBOLS USED AT FINDING LEVEL : GRADE 1 GRADE 2 GRADE 3 P ( = Minimal / very few / very small = Slight / few / small = Moderate / moderate number / moderate = Finding present, severity not scored = Finding unilateral in paired organs size CXR0486s Final Report Page 49 .of .128 PATHOLOGY REPORT SUMMARY TABLES TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . PAGE CXR 12/ 90 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 SUMMARY INCIDENCE OF GRADINGS BY ORGAN/GROUP/SEX Necropsy Status : TERMINAL SACRIFICE GROUP (KO) Sex Dose Group No . Animals per Dose Group Males 01 02 03 10 10 10 Females 01 02 03 - - - LIVER No .Examined 10 10 10 - - - - mononuclear cell GRADE 1 5 3 3 - - - infiltrate(s) TOTAL AFFECTED 5 3 3 - - - MEAN GRADE/TISS .AFFECTED 1 .0 1 .0 1 .0 - - - - (mixed) inflammatory GRADE 1 7 8 6 - - - cell infiltration TOTAL AFFECTED 7 8 6 - - - MEAN GRADE/TISS .AFFECTED 1 .0 1 .0 1 .0 - - - - vacuolation GRADE 1 1 - 1 - - - GRADE 2 3 2 8 - - - GRADE 3 6 8 1 - - - TOTAL AFFECTED 10 10 10 - - - MEAN GRADE/TISS .AFFECTED 2 .5 2 .8 2 .0 - - - - hepatocellular hypertrophy GRADE 1 - 8 2 - - - GRADE 2 - - 8 - - - TOTAL AFFECTED - 8 10 - - - MEAN GRADE/TISS .AFFECTED - 1 .0 1 .8 - - - - mitotic figures GRADE 1 7 7 8 - - - TOTAL AFFECTED 7 7 8 - - - MEAN GRADE/TISS .AFFECTED 1 .0 1 .0 1 .0 - - - THYROID GLAND No .Examined 9 9 4 - - - - branchiogenic cyst GRADE 1 - 1 - - - - TOTAL AFFECTED - 1 - - - - MEAN GRADE/TISS .AFFECTED - 1 .0 - - - - CXR0486s Final Report Page 50 of 128 PATHOLOGY REPORT SUMMARY TABLES TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . PAGE CXR 13/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 SUMMARY INCIDENCE OF GRADINGS BY ORGAN/GROUP/S$:)C Necropsy Status : RECOVERY / POST-TREATMENT GROUP (R1) Sex Males Females Dose Group No . Animals per Dose Group 01 02 03 01 02 03 10 10 10 - - - LIVER - congestion No .Examined 10 10 10 - - - GRADE 1 - 1 - - - - GRADE 2 - 1 - - - - TOTAL AFFECTED - 2 - - - - MEAN GRADE/TISS .AFFECTED - 1 .5 - - - - - mononuclear cell GRADE 1 6 5 3 - - - infiltrate(s) TOTAL AFFECTED 6 5 3 - - - MEAN GRADE/TISS .AFFECTED 1 .0 1 .0 1 .0 - - - - (mixed) inflammatory GRADE 1 9 6 9 - - - cell infiltration TOTAL AFFECTED 9 6 9 - - - MEAN GRADE/TISS .AFFECTED 1 .0 1 .0 1 .0 - - - - vacuolation GRADE 1 1 1 1 - - - GRADE 2 8 6 5 - - - GRADE 3 1 - - - - - TOTAL AFFECTED 10 7 6 - - - MEAN GRADE/TISS .AFFECTED 2 .0 1 .9 1 .8 - - - - hepatocellular hypertrophy GRADE 1 - 4 - - - - GRADE 2 - 1 9 - - - GRADE 3 - - 1 - - - TOTAL AFFECTED - 5 10 - - - MEAN GRADE/TISS .AFFECTED - 1 .2 2 .1 - - - - mitotic figures GRADE 1 4 8 6 - - - TOTAL AFFECTED 4 8 6 - - - MEAN GRADE/TISS .AFFECTED 1 .0 1 .0 1 .0 - - - THYROID GLAND No .Examined 8 10 10 - - - - branchiogenic cyst GRADE 1 - - 2 - - - TOTAL AFFECTED - - 2 - - - MEAN GRADE/TISS .AFFECTED - - 1 .0 - - - CXR0486s Final Report Page 51 of 128 PATHOLOGY REPORT SUMMARY TABLES TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . PAGE CXR 14/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2dl SUMMARY INCIDENCE OF GRADINGS BY ORGAN/GROUP/SEX Necropsy Status : RECOVERY / POST-TREATMENT GROUP (R2) Sex Males Dose Group No . Animals per Dose Group 01 02 03 10 10 10 LIVER - congestion No .Examined 10 10 10 GRADE 1 - 1 - Females 01 02 03 - - - - - - - - - TOTAL AFFECTED - 1 - - - - MEAN GRADE/TISS .AFFECTED - 1 .0 - - - - - mononuclear cell GRADE 1 5 - 3 - - - infiltrate(s) TOTAL AFFECTED 5 - 3 - - - MEAN GRADE/TISS .AFFECTED 1 .0 - 1 .0 - - - - (mixed) inflammatory GRADE 1 8 9 10 - - - cell infiltration TOTAL AFFECTED 8 9 10 - - - MEAN GRADE/TISS .AFFECTED 1 .0 1 .0 1 .0 - - - - vacuolation GRADE 1 1 2 3 - - - GRADE 2 4 4 6 - - - GRADE 3 5 3 - - - - TOTAL AFFECTED 10 9 9 - - - MEAN GRADE/TISS .AFFECTED 2 .4 2 .1 1 .7 - - - - hepatocellular hypertrophy GRADE 1 - 4 - - - - GRADE 2 - - 9 - - - GRADE 3 - - 1 - - - TOTAL AFFECTED - 4 10 - - - MEAN GRADE/TISS .AFFECTED - 1 .0 2 .1 - - - - mitotic figures GRADE 1 1 3 2 - - - TOTAL AFFECTED 1 3 2 - - - MEAN GRADE/TISS .AFFECTED 1 .0 1 .0 1 .0 - - - CXR0486s Final Report Page 52 of 128 PATHOLOGY REPORT SUMMARY TABLES TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . PAGE CXR 15/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 SUMMARY INCIDENCE OF GRADINGS BY ORGAN/GR0UP/SZ}C Necropsy Status : RECOVERY / POST-TREATMENT GROUP (R3) Sex Males Dose Group No . Animals per Dose Group 01 02 03 10 10 10 LIVER No .Examined 10 10 10 - mononuclear cell GRADE 1 6 7 4 infiltrate(s) Females 01 02 03 - - - - - - - - - TOTAL AFFECTED 6 7 4 - - - MEAN GRADE/TISS .AFFECTED 1 .0 1 .0 1 .0 - - - - (mixed) inflammatory GRADE 1 10 6 8 - - - cell infiltration TOTAL AFFECTED 10 6 8 - - - MEAN GRADE/TISS .AFFECPED 1 .0 1 .0 1 .0 - - - - vacuolation GRADE 1 - 3 1 - - - GRADE 2 4 4 6 - - - GRADE 3 6 3 - - - - TOTAL AFFECTED 10 10 7 - - - MEAN GRADE/TISS .AFFECTED 2 .6 2 .0 1 .9 - - - - hepatocellular hypertrophy GRADE 1 - 5 3 - - - GRADE 2 - - 7 - - - TOTAL AFFECTED - 5 10 - - - MEAN GRADE/TISS .AFFECTED - 1 .0 1 .7 - - - - mitotic figures GRADE 1 - 1 2 - - - TOTAL AFFECTED - 1 2 - - - MEAN GRADE/TISS .AFFECTED - 1 .0 1 .0 - - - CXR0486s Final Report Page 53 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA PAGE CXR 16/ 90 0486s TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 TABLE OF INDIVIDUAL MICROSCOPIC FINDINGS (AOFT) DOSE GROUP : 01, Control ANIMAL NUMBER : 1 2 3 4 5 6 7 8 9 10 MKO MKO MKO MKO MKO MKO MKO MKO MKO MKO LIVER + + + + + + + + + + - mononuclear infilt . . 1. 1. 1. 1. . . . 1. . - inflammatory cell 1. . 1. . . 1. 1. 1. 1. 1. - vacuolation, hepat . 3. 3 . 2. 2. 3. 2. 3. 1. 3. 3. - mitotic figures . 1. . 1. 1. 1. 1. 1. . 1. ........ ........ .... . ... . .................. .... .... ................... ... THYROID GLAND ( - - - (- 0 - - - - - CXR0486s Final Report Page 54 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA PAGE CXR 17/ 90 0486s TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFbS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . PATHOL . NO . : 07059 V00 DATE : 19-MAR-OS PathDataSystem V6 .2d1 TABLE OF INDIVIDUAL MICROSCOPIC FINDINGS (AOFT) DOSE GROUP : 01, Control ANIMAL NUMBER : 11 12 13 14 15 16 17 18 19 20 MR1 MR1 MR1 MR1 MR1 MR1 MR1 MR1 MR1 MR1 LIVER + + + + + + + + + + - mononuclear infilt . 1. . . . 1. 1. 1. 1. 1. . - inflammatory cell 1. 1. 1. . 1. 1. 1. 1. 1. 1. - vacuolation, hepat . 2. 2. 1. 2. 2. 2. 3. 2. 2. 2. - mitotic figures . . . 1. . 1. . 1. . 1. . . .... ..... .................. .... ................. ..... .... ............. . THYROID GLAND - - - - - - - 0 0 (- CXR0486s Final Report R~ge~. 55,of128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA PAGE CXR 18/ 90 0486s TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 TABLE OF INDIVIDUAL MICROSCOPIC FINDINGS (AOFT) DOSE GROUP : 01, Control ANIMAL NUMBER : 21 22 23 24 25 26 27 28 29 30 MR2 MR2 MR2 MR2 MR2 MR2 MR2 MR2 MR2 MR2 LIVER + + + + + + + + + + - mononuclear infilt . . . 1. 1. . 1. 1. . . 1. - inflammatory cell . 1. 1. 1. 1. 1. . 1. 1. 1. - vacuolation, hepat . 3. 3. 3. 2. 3. 2. 1. 2. 3. 2. - mitotic figures . . . . . . . 1. . . . ........ ............ .... ............ ........ ... .... ................ ... . . THYROID GLAND - - - (- ( - - - - - - CXR0486s Final Report Page 56 of-128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA PAGE CXR 19/ 90 0486s TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 TABLE OF INDIVIDUAL MICROSCOPIC FINDINGS (AOFT) DOSE GROUP : 01, Control ANIMAL NUMBER : 31 32 33 34 35 36 37 38 39 40 MR3 MR3 MR3 MR3 MR3 MR3 MR3 MR3 MR3 MR3 LIVER - mononuclear infilt . + + + + + + + + + + 1. . 1. . . . 1. 1. 1. 1. - inflammatory cell 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. - vacuolation, hepat . 3. 2. 3. 3. 3. 2. 3. 3. 2. 2. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . THYROID GLAND - - - - - - - - - (- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . CXR0486s Final Report Rage 57 -of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA PAGE CXR 20/ 90 0486s TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2dl TABLE OF INDIVIDUAL MICROSCOPIC FINDINGS (AOFT) DOSE GROUP : 02, PFOS 20 ppm ANIMAL NUMBER : 41 42 43 44 45 46 47 48 49 50 MKO MKO MKO MKO MKO MKO MKO MKO MKO MKO LIVER + + + + + + + + + + - mononuclear infilt . . . 1. . . 1. . 1. . . - inflammatory cell 1. 1. 1. 1. 1. 1. . . 1. 1. - vacuolation, hepat . 3. 3. 3. 3. 3. 2. 2. 3. 3. 3. - hypertrophy, hepat . 1. 1. 1. 1. 1. . 1. . 1. 1. - mitotic figures 1. 1. 1. . . 1. 1. . 1. 1. .............. ..... .... ........ ........... ............... ........ .... .... THYROID GLAND ( - (- ( - 0 - + - (- (- + - ectopic thymus . . . . . ( P. . . . . - branchiogenic cyst . . . . . . . . . ( 1. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CXR0486s Final Report Page 58 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA PAGE CXR 21/ 90 0486s TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 TABLE OF INDIVIDUAL MICROSCOPIC FINDINGS (AOFT) DOSE GROUP : 02, PFOS 20 ppm ANIMAL NUMBER : 51 52 53 54 55 56 57 58 59 60 MR1 MR1 MR1 MR1 MR1 MR1 MR1 MR1 MR1 MR1 LIVER + + + + + + + + + + - congestion . . . . 2. 1. . . . . - mononuclear infilt . . 1. . . 1. 1. 1. . 1. . - inflammatory cell 1. 1. . 1. . . 1. 1. . 1. - vacuolation, hepat . 2. . 2. . . 2. 1. 2. 2. 2. - hypertrophy, hepat . 1. . . 2. . . . 1. 1. 1. - mitotic figures 1. 1. 1. . . 1. 1. 1. 1. 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . THYROID GLAND - - - - - - - - - - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . . . CXR0486s Final Report Page 59 of 128 . PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA PAGE CXR 22/ 90 0486s TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 TABLE OF INDIVIDUAL MICROSCOPIC FINDINGS (AOFT) DOSE GROUP : 02, PFOS 20 ppm ANIMAL NUMBER : 61 62 63 64 65 66 67 68 69 70 MR2 MR2 MR2 MR2 MR2 MR2 MR2 MR2 MR2 MR2 LIVER + + + + + + + + + + - congestion . . . . . . . 1. . . - inflammatory cell 1. 1. 1. 1. 1. 1. 1. 1. . 1. - vacuolation, hepat . 3. 3. 1. 2. 2. 1. 2. . 2. 3. - hypertrophy, hepat . 1. 1. . . . . 1. . . 1. - mitotic figures . 1. . . . . 1. . . 1. .................. .... ........ ........... .... ........... ........ .... ..... THYROID GLAND ( - - - (- 0 - - - - - .. ................ .... ............ ....... .... .... ....... .... .... .... ..... CXR0486s Final Report PageW~oU128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA PAGE CXR 23/ 90 0486s TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PE`OS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2dl TABLE OF INDIVIDUAL MICROSCOPIC FINDINGS (AOFT) DOSE GROUP : 02, PFOS 20 ppm ANIMAL NUMBER : 71 72 73 74 75 76 77 78 79 80 MR3 MR3 MR3 MR3 MR3 MR3 MR3 MR3 MR3 MR3 LIVER + + + + + + + + + + - mononuclear infilt . - inflammatory cell - vacuolation, hepat . - hypertrophy, hepat . - mitotic figures 1. . 1. 1. 1. 1. 1. . 1. . 1. 1. . . . 1. 1. 1. . 1. 3. 2. 1. 1. 2. 1. 2. 3. 2. 3. . . 1. 1. 1. . 1. . . 1. . . . . 1. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . THYROID GLAND - - 0 (- - - - - - - . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . CXR0486s Final Report Page.61-of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA PAGE CXR 24/ 90 0486s TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 TABLE OF INDIVIDUAL MICROSCOPIC FINDINGS (AOFT) DOSE GROUP : 03, PFOS 100 ppm ANIMAL NUMBER : 81 82 83 84 85 86 87 88 89 90 MKO MKO MKO MKO MKO MKO MKO MKO MKO MKO LIVER + + + + + + + + + + - mononuclear infilt . 1. 1. 1. . . . . . . . - inflammatory cell 1. . . 1. 1. 1. . 1. . 1. - vacuolation, hepat . 2. 1. 2. 3. 2. 2. 2. 2. 2. 2. - hypertrophy, hepat . 2. 1. 1. 2. 2. 2. 2. 2. 2. 2. - mitotic figures . 1. 1. 1. 1. . 1. 1. 1. 1. ........ ............ .... . .................. .... ............... ........ ... THYROID GLAND 0 0 0 0 0 - - - 0 (- CXR0486s Final Report Page 62 of 128 ' PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA PAGE CXR 25/ 90 0486s TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 TABLE OF INDIVIDUAL MICROSCOPIC FINDINGS (AOFT) DOSE GROUP : 03, PFOS 100 ppm ANIMAL NUMBER : 91 92 93 94 95 96 97 98 99 100 MR1 MR1 MR1 MR1 MR1 MR1 MR1 MR1 MR1 MR1 LIVER - mononuclear infilt . - inflammatory cell - vacuolation, hepat . + + + + + + + + + + . . . . . 1. . . 1. 1. 1. 1. . 1. 1. 1. 1. 1. 1. 1. 2. 2. 2. 2. . 1. 2. . . . - hypertrophy, hepat . 3. 2. 2. 2. 2. 2. 2. 2. 2. 2. - mitotic figures 1. 1. 1. . . 1. 1. . . 1. .. .... ........................ .... ..................... .................. THYROID GLAND - - - - - - - + - + - branchiogenic cyst . . . . . . . ( 1. . ( 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . CXR0486s Final Report Page 63 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA PAGE CXR 26/ 90 0486s TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 TABLE OF INDIVIDUAL MICROSCOPIC FINDINGS (AOFT) DOSE GROUP : 03, PFOS 100 ppm ANIMAL NUMBER : 101 102 103 104 105 106 107 108 109 110 MR2 MR2 MR2 MR2 MR2 MR2 MR2 MR2 MR2 MR2 LIVER + + + + + + + + + + - mononuclear infilt . . . 1. . . . 1. . 1. . - inflammatory cell 1. 1. 1. 1. 1. 1. 1. 1. 1. 1. - vacuolation, hepat . 2. 1. 2. 2. 2. 2. 2. . 1. 1. - hypertrophy, hepat . 3. 2. 2. 2. 2. 2. 2. 2. 2. 2. - mitotic figures 1. . 1. . . . . . . . . .................... .... ................... ... . .... ........... .... .... .. THYROID GLAND - - (- - (- - - - - - CXR0486s Final Report Page 64 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA PAGE CXR 27/ 90 0486s TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFbS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 TABLE OF INDIVIDUAL MICROSCOPIC FINDINGS (AOFT) DOSE GROUP : 03, PFOS 100 ppm ANIMAL NUMBER : 111 112 113 114 1,15 116 117 118 119 120 MR3 MR3 MR3 MR3 MR3 MR3 MR3 MR3 MR3 MR3 LIVER + + + + + + + + + + - mononuclear infilt . . 1. . 1. 1. . . . 1. . - inflammatory cell 1. 1. 1. 1. . 1. 1. 1. 1. . - vacuolation, hepat . 2. 2. 1. . 2. 2. . 2. 2. . - hypertrophy, hepat . 1. 2. 2. 2. 1. 2. 2. 1. 2. 2. - mitotic figures . . . . . . 1. . . 1. .............. .............. .... .......................... ............... THYROID GLAND - (- (- - - - - - - - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CXR0486s Final Report Page.fi,5 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . ANIMAL HEADING DATA DOSE GROUP : 01, Control PAGE CXR . 28/ 90 . 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2dl ANIMAL SEX DEFINED AND FINAL TEST FIRST AND LAST DATE OF NUMBER M/F STATE OF NECROPSY DAYS DAY UNDER TEST NECROPSY . ........... ..... ... . ........ .. ...... ............... ........ :.. . .... 1 M 2 M KO KO KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 8 29-NOV-06 06-DEC-06 06-DEC-06 3 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 4 M 5 M 6 M KO KO KO KO KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 8 29-NOV-06 06-DEC-06 06-DEC-06 8 29-NOV-06 06-DEC-06 06-DEC-06 7 M 8 M 9 M KO KO KO KO KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 8 29-NOV-06 06-DEC-06 06-DEC-06 8 29-NOV-06 06-DEC-06 06-DEC-06 10 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 11 M Rl Rl 36 29-NOV-06 03-JAN-07 03-JAN-07 12 M Rl Rl 36 29-NOV-06 03-JAN-07 03-JAN-07 13 M R1 R1 36 29-NOV-06 03-JAN-07 03-JAN-07 14 M R1 Rl 36 29-NOV-06 03-JAN-07 03-JAN-07 15 M R1 R1 36 29-NOV-06 03-JAN-07 03-JAN-07 16 M R1 R1 36 29-NOV-06 03-JAN-07 03-JAN-07 17 M R1 R1 36 29-NOV-06 03-JAN-07 03-JAN-07 18 M R1 R1 36 29-NOV-06 03-JAN-07 03-JAN-07 19 M Rl R1 36 29-NOV-06 03-JAN-07 03-JAN-07 20 M R1 R1 36 29-NOV-06 03-JAN-07 03-JAN-07 21 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 22 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 23 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 24 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 25 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 26 M 27 M R2 R2 R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 64 29-NOV-06 31-JAN-07 31-JAN-07 28 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 29 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 30 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 31 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 32 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 33 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 34 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 35 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 36 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 37 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 38 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 39 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 40 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CXR0486s Final Report Page 66 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PF!OS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 29/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present organ examined, no pathologic findings noted * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 2 ... . ..... .................. .... . ..................... .... ................ * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate .(s), periportal-/biliar, -vacuolation, hepatocellular, periportal, grade 3 -mitotic figures, grade l THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted grade 1 CXR0486s Final Report Rege 67 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 30/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-OS PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 3 * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 4 . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present Organ examined, no pathologic findings noted CXR0486s Final Report Page 68 of 1.^8 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 31/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL NO . 5 * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, -vacuolation, hepatocellular, periportal, grade 3 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Tissue not present for histologic examination grade 1 * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 6 . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Rage,69pf .128, PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 32/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 7 * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL NO . : 8 .............. ..... ... . .... .... ........... ............... .... .... . ... .... * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 70 of T28 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR,Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 33/ 90 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2dl MALE * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL NO . : 9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 10 ... . .... ..... ......... ......... .... ...................... .... ......... ... * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 7'I of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 34/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : Rl DAYS ON TEST . 36 * ANIMAL N0 . : 11 * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, diffuse, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : Rl DAYS ON TEST . 36 * ANIMAL N0 . : 12 .... ................ .... .... ............... .... ................... .... ... * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 72 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 35/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 13 -- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, -vacuolation, hepatocellular, diffuse, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted grade 1 * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 14 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -vacuolation, hepatocellular, periportal, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 73 o# .128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 36/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 15 . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, diffuse, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 16 . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 74 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 37/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 17 * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 18 . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Tissue not present for histologic examination CXR0486s Final Report PagB 75 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 38/ 90 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, diffuse, grade 2 THYROID GLAND (BOTH LOBES) : Tissue not present for histologic examination * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL NO . : 20 * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present Organ examined, no pathologic findings noted CXR0486s Final Report Page 76 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 39/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -vacuolation, hepatocellular, periportal, grade 3 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 22 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, -vacuolation, hepatocellular, diffuse, grade 3 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted grade 1 * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL NO . : 23 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .--- . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periporta,l-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 CXR0486s Final Report Page 77 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 40/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE CONT ./FF . ANIMAL N0 . : 23 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL NO . : 24 .. ................ .... ................... .... .... ............... . ... ..... * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present Organ examined, no pathologic findings noted * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 25 . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present organ examined, no pathologic findings noted CXR0486s Final Report Page 78 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 41/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 26 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL NO . : 27 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, -vacuolation, hepatocellular, periportal, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted grade 1 CXR0486s Final Report Page 79 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 42/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 28 * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, diffuse, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 29 * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page"80 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 43/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 30 . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periporta;l-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL NO . : 31 . ..... ..... .... .............. .... ................. .... . .... ..... ........ . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page Z;I,Qf 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 44/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 32 . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL NO . : 33 .. ................ .... ............ ....... . ... ................... . ... ..... * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, diffuse, grade 3 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 62 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFpS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 45/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 34 * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, -vacuolation, hepatocellular, diffuse, grade 3 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted grade 1 * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 35 * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, -vacuolation, hepatocellular, diffuse, grade 3 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted grade 1 CXR0486s Final Report Page 83 of.,128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 46/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 36 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, diffuse, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 37 * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, diffuse, grade 3 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 94 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 47/ 90 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 38 * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periporta.l-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 39 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, diffuse, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 85 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 01, Control PAGE CXR 48/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2dl MALE * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL NO . : 40 * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present Organ examined, no pathologic findings noted CXR0486s Final Report Rage 86 of l28 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . ANIMAL HEADING DATA DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 49/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 ANIMAL SEX DEFINED AND FINAL TEST FIRST AND LAST DATE OF NUMBER M/F STATE OF NECROPSY DAYS DAY UNDER TEST NECROPSY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 42 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 43 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 44 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 45 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 46 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 47 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 48 M KO KO 8 29-'NOV-06 06-DEC-06 06-DEC-06 49 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 50 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 51 M R1 R1 36 29-NOV-06 03-JAN-07 03-JAN-07 52 M 53 M 54 M 55 M 56 M 57 M R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 36 29-NOV-06 03-JAN-07 03-JAN-07 36 29-NOV-06 03-JAN-07 03-JAN-07 36 29-,NOV-06 03-JAN-07 03-JAN-07 36 29-'NOV-06 03-JAN-07 03-JAN-07 36 29-NOV-06 03-JAN-07 03-JAN-07 36 29-NOV-06 03-JAN-07 03-JAN-07 58 M 59 M 60 M R1 R1 R1 R1 R1 R1 36 29-NOV-06 03-JAN-07 03-JAN-07 36 29-NOV-06 03-JAN-07 03-JAN-07 36 29-NOV-06 03-JAN-07 03-JAN-07 61 M 62 M 63 M R2 R2 R2 R2 R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 64 29-NOV-06 31-JAN-07 31-JAN-07 64 29-NOV-06 31-JAN-07 31-JAN-07 64 M 65 M 66 M 67 M R2 R2 R2 R2 R2 R2 R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 64 29-NOV-06 31-JAN-07 31-JAN-07 64 29-NOV-06 31-JAN-07 31-JAN-07 64 29-NOV-06 31-JAN-07 31-JAN-07 68 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 69 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 70 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 71 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 72 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 73 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 74 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 75 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 76 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 77 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 78 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 79 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 80 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 . ........................ ... . ..................... .... . ................. . CXR0486s Final Report Page 87. of. 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 50/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 41 * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 -hepatocellular hypertrophy, centrilobular, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present organ examined, no pathologic findings noted * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 42 .................... .... ................... .... .... ............... .... ... * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 -hepatocellular hypertrophy, centrilobular, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present organ examined, no pathologic findings noted CXR0486s Final Report 'Page 68 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 51/ 90 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL NO . : 43 * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 -hepatocellular hypertrophy, centrilobular, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present organ examined, no pathologic findings noted * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 44 * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 -hepatocellular hypertrophy, centrilobular, grade 1 THYROID GLAND (BOTH LOBES) : Tissue not present for histologic examination CXR0486s Final Report .Pagq 8,99f. 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 52/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 45 . ........ ........ .... .... ............ ........ ... .... ................... . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 -hepatocellular hypertrophy, hepatocellular, centrilobular, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 46 * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : -ectopic thymus, unilateral CXR0486s Final Report Page 00 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 53/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL NO . : 47 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL NO . : 48 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, -vacuolation, hepatocellular, diffuse, grade 3 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present Organ examined, no pathologic findings noted grade 1 CXR0486s Final Report Page 91 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, . 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 54/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 49 ...... ............ .... ............ ........... .... ............... . ... ..... * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 -hepatocellular hypertrophy, centrilobular, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present Organ examined, no pathologic findings noted * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL NO . : 50 . ........ ........ .... .... ............ ....... .... .... ........... ........ .. * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 -hepatocellular hypertrophy, centrilobular, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : -branchiogenic cyst, unilateral, grade 1 CXR0486s Final Report Page 02 of128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 55/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : Rl DAYS ON TEST . 36 * ANIMAL NO . : 51 ..... ..... ....................... .... ...................... .... .......... * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 52 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .--- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 9,3 .of.128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 56/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL NO . : 53 .............. ..... ... . .... .... ........... ............... ........ .... .... * MICROSCOPIC FINDINGS LIVER : -vacuolation, hepatocellular, periportal, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL NO . : 54 ........ ............ .... ................... .... ....................... ... * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 94 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 57/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 55 ..... ..... ....................... .... ...................... .... .......... * MICROSCOPIC FINDINGS LIVER : -congestion, grade 2 -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 56 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -congestion, grade 1 -mononuclear cell infiltrate(s), periportal-/biliar, -vacuolation, hepatocellular, periportal, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted grade 1 CXR0486s Final Report Rage 95 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 58/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2dl MALE * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL NO . : 57 * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : Rl DAYS ON TEST . 36 * ANIMAL N0 . : 58 .. ........... ........ . .... .... ........... .... ... ........ ............ ... . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 96'of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 59/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 59 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 60 . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : . Organ examined, no pathologic findings noted CXR0486s Final Report Page 97 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 60/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL NO . : 61 * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 -hepatocellular hypertrophy, centrilobular, grade 1 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present organ examined, no pathologic findings noted * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 62 . . ........... ........ . ....................... ... ........ ............ ... . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, pe-riportal, grade 3 -hepatocellular hypertrophy, centrilobular, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page-'438 of A28 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 61/ 90 . 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 63 * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, -vacuolation, hepatocellular, diffuse, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted grade 1 * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 64 * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present organ examined, no pathologic findings noted CXR0486s Final Report Page,99 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 62/ 90 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 MAL E * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL NO . : 65 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, -vacuolation, hepatocellular, diffuse, grade 2 THYROID GLAND (BOTH LOBES) : Tissue not present for histologic examination grade 1 * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 66 ... .... .... ............ . ... ............... .... .... ................... .... * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, diffuse, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 100 of 'F28 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (P5OS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 63/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 67 . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, diffuse, grade,2 -hepatocellular hypertrophy, centrilobular, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL NO . : 68 . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -congestion, grade 1 -(mixed) inflammatory cell infiltration, focal, THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted grade 1 CXR0486s Final Report ,Pagq 'F09 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 64/ 90 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 69 . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -vacuolation, hepatocellular, periportal, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 70 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 -hepatocellular hypertrophy, centrilobular, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 102 of 428 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR . 65/ 90 . 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL NO . : 71 .. .... ....................... . .... ..................... .... ......... ..... * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL NO . : 72 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 103 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 66/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 73 ...... .... ........ .... ................ .... ... .................... ... . .... * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -vacuolation, hepatocellular, diffuse, grade 1 -hepatocellular hypertrophy, centrilobular, grade 1 THYROID GLAND (BOTH LOBES) : Tissue not present for histologic examination * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 74 .................... .... ................... .... .... ................... ... * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -vacuolation, hepatocellular, periportal, grade 1 -hepatocellular hypertrophy, centrilobular, grade 1 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present organ examined, no pathologic findings noted CXR0486s Final Report Page 104 of 1~8 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 67/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2dl MALE * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 75 . . . . . . . . . . . . . . . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade l -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 76 . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page . 105 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 68/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 77 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 78 .................... .... ................... ....................... .... ... * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 106~of~A28 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 02, PFOS 20 ppm PAGE CXR 69/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 79 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ., . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, -vacuolation, hepatocellular, periportal, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted grade 1 * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL NO . : 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 -hepatocellular hypertrophy, centrilobular, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Pogo 1A7cof'1?8 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . ANIMAL HEADING DATA DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 70/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 ANIMAL NUMBER SEX M/F DEFINED AND FINAL STATE OF NECROPSY TEST DAYS FIRST AND LAST DAY UNDER TEST DATE OF NECROPSY ~ .~~8~1~ " M ~~~~~~~K~O~~ "" ~~ KO . . .~~~~ """ 8 ~29~ -NO~ V-0~ 6 06-DEC-06 . 06-DEC-06 82 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 83 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 84 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 85 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 86 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 87 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 88 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 89 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 90 M KO KO 8 29-NOV-06 06-DEC-06 06-DEC-06 91 M R1 Rl 36 29-NOV-06 03-JAN-07 03-JAN-07 92 M R1 R1 36 29-NOV-06 03-JAN-07 03-JAN-07 93 M R1 Rl 36 29-NOV-06 03-JAN-07 03-JAN-07 94 M R1 R1 36 29-NOV-06 03-JAN-07 03-JAN-07 95 M R1 R1 36 29-NOV-06 03-JAN-07 03-JAN-07 96 M 97 M 98 M R1 R1 R1 Rl R1 R1 36 29-NOV-06 03-JAN-07 03-JAN-07 36 29-NOV-06 03-JAN-07 03-JAN-07 36 29-NOV-06 03-JAN-07 03-JAN-07 99 M R1 R1 36 29-NOV-06 03-JAN-07 03-JAN-07 100 M R1 R1 36 29-NOV-06 03-JAN-07 03-JAN-07 101 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 102 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 103 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 104 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 105 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 106 M R2 R2 64 29-NOV'-06 31-JAN-07 31-JAN-07 107 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 108 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 109 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 110 M R2 R2 64 29-NOV-06 31-JAN-07 31-JAN-07 111 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 112 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 113 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 114 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 115 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 116 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 117 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 118 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 119 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 120 M R3 R3 92 29-NOV-06 28-FEB-07 28-FEB-07 .... .... ........ .... .... . ... ................... .... .... .... ........... . .. CXR0486s Final Report Rage 106'of128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFpS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 71/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 81 ... . ....................... ..... .... ...................... ............... * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Tissue not present for histologic examination * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 82 . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, -vacuolation, hepatocellular, periportal, grade 1 -hepatocellular hypertrophy, centrilobular, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Tissue not present for histologic examination grade 1 CXR0486s Final Report Page _409,of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 72/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL NO . : 83 . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . :. . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 1 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Tissue not present for histologic examination * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL NO . : 84 . . . . . . . . . . . .. . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 3 -hepatocellular hypertrophy, centrilobular, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Tissue not present for histologic examination CXR0486s Final Report Page 110 of A28 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 73/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL NO . : 85 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ., . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Tissue not present for histologic examination * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 86 .... .......... ............. . .... ........ .................. ............... * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 111 ,of128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS . DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 74/ 90 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 87 . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 88 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 112 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 75/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 89 . . .... ..... .................. .... ................. ..... .... .............. * MICROSCOPIC FINDINGS LIVER : -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Tissue not present for histologic examination * STATE AT NECROPSY : KO DAYS ON TEST . 8 * ANIMAL N0 . : 90 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Only one of paired organs, examined/present Organ examined, no pathoogic findings noted CXR0486s Final Report Page, 11-3 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 76/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 91 ....... ........ . .... .... ............... .... ........... .... .... .... .... ... * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 3 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : Rl DAYS ON TEST . 36 * ANIMAL N0 . : 92 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 114 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PPOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 77/ 90 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 93 . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL NO . : 94 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 11;5 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 78/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL NO . : 95 .. ........ ........ .... ............ .... .... ... .... ............... . ... ..... * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 96 ....... ................ ............ ........ ... ............... .... . ... .... * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 1 -hepatocellular hypertrophy, centrilobular, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 116 of'128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 79/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 97 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 98 . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : -branchiogenic cyst, unilateral, grade 1 CXR0486s Final Report Regal 1.7 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 80/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2dl MALE * STATE AT NECROPSY : R1 DAYS ON TEST . 36 * ANIMAL N0 . : 99 * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : Rl DAYS ON TEST . 36 * ANIMAL N0 . : 100 .............. ........ . ................... ....................... .... .... * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -hepatocellular hypertrophy, centrilobular, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : -branchiogenic cyst, unilateral, grade 1 CXR0486s Final Report Page'118 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 81/ 90 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 101 * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 3 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL NO . : 102 . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 1 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Raga 139 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 82/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 103 . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present organ examined, no pathologic findings noted * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 104 * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 120 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 83/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL NO . : 105 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present organ examined, no pathologic findings noted * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL NO . : 106 . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES .) : Organ examined, no pathologic findings noted CXR0486s Final Report P;ige 129 .of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 84/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 107 . . . . . . . . . . . . . . . . . . . : . . . . . . . . . . . . . . . .. . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 108 * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 122 of T28 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 85/ 90 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL NO . : 109 . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periport 1-/biliar, grade 1 -(mixed) inflammatory cell infiltration, ;ocal, grade 1 -vacuolation, hepatocellular, periportal, grade 1 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R2 DAYS ON TEST . 64 * ANIMAL N0 . : 110 . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 . -vacuolation, hepatocellular, periportal, grade 1 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 12.3,~of 1,28 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 86/ 90 0486s PATHOL . NO . : 07059 VOO DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 111 . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 112 .............. ......... .... .... ........... ............... ........ .... .... * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present Organ examined, no pathologic findings noted CXR0486s Final Report Page 124 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 87/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 113 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 1 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Only one of paired organs examined/present Organ examined, no pathologic findings noted * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 114 . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 125,of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 88/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 115 ................ .... ........ .... ........ ... .... ........... .... . ... ....... * MICROSCOPIC FINDINGS LIVER : -mononuclear cell infiltrate(s), periportal-/biliar, -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted grade 1 * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL NO . : 116 .......... ........ .... ............ .... .... ... .................... ... . .... * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 126 of 128 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 89/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 , MALE * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL NO . : 117 . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -hepatocellular hypertrophy, centrilobular, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 118 . . . . . . . . . . . . . . . . . ., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 127 of 1;28 PATHOLOGY REPORT INDIVIDUAL ANIMAL DATA TEST ITEM TEST SYSTEM SPONSOR : Perfluorooctane Sulfonate (PFOS) : RAT, 13 weeks, feeding : CXR Biosciences Ltd . TEXT OF MICROSCOPIC FINDINGS DOSE GROUP : 03, PFOS 100 ppm PAGE CXR 90/ 90 0486s PATHOL . NO . : 07059 V00 DATE : 19-MAR-08 PathDataSystem V6 .2d1 MALE * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 119 * MICROSCOPIC FINDINGS LIVER : -mononuclear cell'infiltrate(s), periportal-/biliar, grade 1 -(mixed) inflammatory cell infiltration, focal, grade 1 -vacuolation, hepatocellular, periportal, grade 2 -hepatocellular hypertrophy, centrilobular, grade 2 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted * STATE AT NECROPSY : R3 DAYS ON TEST . 92 * ANIMAL N0 . : 120 . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * MICROSCOPIC FINDINGS LIVER : -hepatocellular hypertrophy, centrilobular, grade 2 -mitotic figures, grade 1 THYROID GLAND (BOTH LOBES) : Organ examined, no pathologic findings noted CXR0486s Final Report Page 128 of 128 00 0 0 N 0\0 ig 00 E u oNivi a o -v > ~jQ -a8u ~~t3 e0"'~+ p uQ 3c_O ~C ~ tC~ C M r4~`~C f,, ~ ~ ~w m C p V ;, c a lA .' u 01 i u O p `1 (U ~ a E ~ o'7s C iO ~~ LG7 ~O rl N a) ~ R ~cocp ;j Y o'itnn ~VW -~''`--'~ a~c, e.c o= a O ~ L-~ O ~ ~ ~ - - .x CO 'o ~ ~~10+ ~ O ~ ~1 ~ - 0 p c ~75 '3: .Ti,-~~ 3 ai i a ~~a~oac -d Z 3 =v .~- W u Q ,`z.... c U C H C 8 o 3 +or y a~ ~ E ~ c U o EM CLA.M $ .oo. .`n a V2 ~d~ ~ y c Z " G O Y c ~ ao w O N .q > O w V W E ~3m O ca+ Bc a~ tc5~~J2G3 a0- ~ ~ ,~ > '~ o 3 ~ = O U w c c E c o aU (U E $ a~~~g8 _ it ~I o a Y 1v $'o c :03 ~ a C ~~Q,cu a~a~_n.a "~ a $ f' Y ~Lu uf c H N rs N -r~ :~ ~ ~ frl H 1~'0 ~ 10 41 O Q- s o O ~ ~ 7 rob u 012 ~ 3'r > ~ p ~o~+h-vN 3 a a~ o a~ '(ff V +r cd bA cd & c o j C) 0 O oid cis 0 E ,L_ C W 7 W C3 0 W a O U f a fl W W m 0 b 0 IrD O Y 0 0 e' 1'n flt > D~ 3 h m ~ -c m 3 3~ 0 ~I 0.3 ~ ~ 3 ~z n-o oaQ~ .. 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