Document xjDnpgvd29a25B27KkqoZLx9E

SRPT T-6295.8, T-6889.1, T-6316.4 DTI 5-A A Z226-oQ - 3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY Title: Inter-Species Comparison of Mechanisms Fluorochemical Toxicity following Intraperitoneal dosing of perfluorooctanesulfonate (PFOS, T-6295) or ammonium perfluorooctanoate (APFO, T-6889) in Rats and Guinea Pigs and oral dosing of N-ethyl perfluorooctanesulfonamido ethanol (N-EtFOSE, T-6316) in Rats. Final Report Date: May 25,2004 Study Numbers. T-6295.8, T-6889.1,6316.4 Strategic Toxicology Study Number: DT15-A Sponsor: 3M Specialty Chemicals Division 3M Center, Building 236 Saint Paul MN 55133-3220 Study Location: 3M Strategic Alternative Toxicology Laboratory 3M Center, Building 270-SB-181 Saint Paul, MN 55133-3220 Study Director: Andrew M. Seacat Ph.D., DABT Toxicology Specialist 3M Medical Dept. Corporate Toxicology and Regulatory Services 3M Center Building 220-2E-02 Study Toxicologist: Deanna Luebker M.S Senior Toxicologist 3M Medical Dept. Corporate Toxicology and Regulatory Services In-Life Start Dates Part A: In-Life End Dates Part A: In-Life Start Dates Part B: In-Life End Dates Part B: In-Life Start Date Part C: In-Life End Date Part C: 10/23/1997 and 11/17/1997 11/04/1997 and 12/1/1997 02/10/1998 and 03/11/1998 03/10/1998 and 03/13/1997 09/16/1998 09/18/1998 1 5 SRPT T-6295.8, T-6889.1, T-6316.4 DTI 5-A T able o f C ontents Introduction.......................................................................................................................................................................................3 M eth o d s..............................................................................................................................................................................................3 Test Materials........................ 4 Procedures................................................................................................................................................................................... 4 Part A: Procedures for a single intraperitoneal dosing o f PFOS (T-6295) and APFO (T-6889) in male rats and guinea pigs................................................................................................................................................................................ 4 Part B: Procedures for a Time-Course o f PFOS (T-6295) toxicity in male and female rats and guinea pigs following a single i.p. injection o f PFOS at two doses................................................................................................... 6 Part C Procedures for hepatic peroxisomal gene induction following oral dosing o f N-EtFOSE in rats...........6 Results and D iscussion................................................................................................................................................................... 7 Part A Results on the Toxicity o f PFOS and APFO in rats and guinea pigs..................................................................7 Results: Rats Part A ................................................................................................................................................................7 Results: Guinea pigs Part A .................................................................................................................................................. 8 Liver samples submitted for L-FABP analysis from rats and guinea pigs treated in Part A ...................................... 8 Conclusions from Part A :.......................................................................................................................................................... 8 Part B: Results o f Time-Course o f PFOS toxicity at two doses in male and female rats and guinea pigs...............8 Results: Rats Part B ................................................................................................................................................................8 Results: Guinea pigs Part B .................................................................................................................................................. 9 Part C: Results Oral dosing o f rats with 20 mg/Kg N-EtFOSE..................................................................................10 Conclusions.................................................................................................................................................................................... 10 Signatures........................................................................................................................................................................................ 11 Part A. Histopathology Report.................................................................................................................................................... 12 Rats...............................................................................................................................................................................................12 Guinea pigs................................................................................................................................................................................. 12 Tables:..............................................................................................................................................................................................13 Table 1. Part A. Sum mary o f Toxicity o f PFOS and APFO in m ale ra ts.............................................................. 13 Table 2. P art A. Sum mary o f Toxicity o f PFOS and APFO in m ale guinea p ig s................................................14 Table 3. Part B. Sum mary o f Body W eights in Rats, Means and Std D eviations............................................. 15 Table 4. Part B. Sum mary o f Body W eights in Guinea Pigs, M eans and Std D eviations...............................16 Table 5. Part B Liver to Body W eight ratios in rats..................................................................................................... 17 Figures............................................................................................................................................................................................. 18 Figure 1: Part B: Female Rat Body Weight Vs Time.........................................................................................................18 Figure 2: Part B: Male Rat Body Weight Vs Time.............................................................................................................19 Figure 3: Part B: Female Guinea Pig Body Weight Vs Time.......................................................................................... 20 Figure 4: Part B: Male Guinea Pig Body Weight Vs Time.............................................................................................. 20 Figure 4: Part B: Male Guinea Pig Body Weight Vs Time.............................................................................................. 21 Appendix 1. Deviations to the Protocol:.................................................................................................................................22 Appendix II. Part A: Toxicity o f PFOS and APFO Individual and Summary Data...................................................... 23 Part A: Rat d a ta ..................................................................................................................................................................... 23 Part A: Guinea Pig d ata....................................................................................................................................................... 25 Appendix III. Part B: Time course with PFOS. Individual Data.......................................................................................27 Part B. Individual Rat data Time course with PFOS..........................................................................................................27 Part B. Liver Weight to Body Weight ratio in male and female rats combined data. SAS analysis.................. 28 Part B. Male rat LW/BW ratio summary.........................................................................................................................30 Part B. Female rat Liver weight summary..................................................................................................................... 32 Part B. Female rat LW/BW ratio Summary.................................................................................................................... 34 Part B. Kidney Weight to Body Weight ratio in male and female rats combined data. SAS analysis................ 36 Part B. Guinea Pig: Individual D ata....................................................................................................................................38 Part B. Guinea Pig Liver to Body Weight Ratio Summary Statistics:...................................................................... 39 Part B. Kidney Weight to Body Weight ratio in male and female guinea pigs. SAS analysis............................ 41 Appendix IV. Part C Oral dosing with N-EtFOSE in rats. Individual data......................................................................43 R e fe r e n c e s...................................................................................................................................................................................... 4 4 2 SRPT T-6295.8, T-6889.1, T-6316.4 DTI 5-A Introduction The research presented in this report was designed to elucidate the mechanism(s) that initiate peroxisomal proliferation in rats, but not necessarily in guinea pigs or primates, by some 3M fluorochemicals. The research objectives were to determine if the fluorochemicals disrupt the function of liver fatty acid binding proteins (L-FABPs) in both rats and guinea pigs and if so, to what extent. The intermediate objectives were to investigate the hypothesis that FC compounds alter L-FABP levels and/or functionality and that it is the disruption o f fatty acid processing leading to higher levels of unassociated long chain fatty acids in the cytoplasm, and that rats and guinea pigs will respond differently. Further objectives were to investigate and compare the molecular mechanisms o f peroxisome proliferation in rats and guinea pigs. Three fluorochemicals; perfluorooctane sulfonate (PFOS, T-6295), vV-ethyl perfluorooctanesulfonamido ethanol (iV-EtFOSE, T-6316) and perfluorooctanoate (APFO, T6889) were tested in this study that were either previously known (Sohlenius et al. 1994; Sohlenius et al. 1993) or suspected to cause peroxisome proliferation in the rat. The hypothesis tested was that these fluorochemicals would induce peroxisome proliferation in the rat, but not the guinea pig, and the data supported that hypothesis. The effect of these compounds on peroxisome proliferation in the rat, but not the guinea pig were presented in an abstract and poster presented at the Society o f Toxicology meeting in 2001 (Wallace etal. 2001). Other investigators using these fluorochemicals have also subsequently shown peroxisome proliferation with some of these compounds in vivo in rats or or in-vitro (Maloney and Waxman 1999; Yang et al. 2002; Berthiaume and Wallace 2002). L-FABPs isolated from certain rats and guinea pigs dosed in the current study were used to investigate the effect of those fluorochemicals on the binding o f a fluorescently labeled probe to L-LFABP. The detailed methods, results and conclusions o f the L-FABP analyses were published elsewhere (Nabbefeld 1998; Luebker et al. 2002), and are only briefly mentioned in the results and discussion section o f this report. Guinea pigs may serve as better surrogate models for human risk assessment than do other rodents because, like humans, they are resistant to peroxisome proliferation. However, the molecular and biochemical mechanisms that differentiate the response o f these species to peroxisome proliferators in general and for the perfluorosulfonamides in particular is unclear. Therefore, the specific aims o f this study were to: 1. Characterize the peroxisomal enzyme and fatty acid binding proteins response in the liver to these compounds 2. Perform toxicity tests such as serum clinical chemistry and to liver histology that may indicate an explanation for any the species differences in response to these compounds. 3. Correlate any observed alterations o f the above functions to liver and serum levels of perfluorosulfonamides and their metabolites. Methods This study, (DT-15A) was conducted in three segments, parts A, B and C under a broadly written protocol dated 9/18/1997 that had been approved by the 3M institutional animal use 3 7 SRPT T-6295.8, T-6889.1, T-6316.4 DT15-A committee (3MIACUC Animal use application # 2088). Part A consisted o f a study designed to compare the toxicity and peroxisomal proliferation in male rats and male guinea pigs dosed via intraperitoneal (i.p.) injection with PFOS or APFO. The results o f Part A led to further testing under part B, which consisted o f a dose-response and time-course for the observed effects of PFOS in both male and female rats and guinea pigs dosed via intraperitoneal (i.p.) injection. The results o f parts A and B led to the conclusion that i.p. dosing was not the best route of administration, therefore an exploratory oral dosing procedure in rats was performed with NEtFOSE in PartC Test Materials The T-numbers, chemical names, abbreviations used for these samples, and the chemical structures o f each o f the compounds that were tested in this study are given below. The currently accepted abbreviations and the ones generally used in this report for each compound are in bold: 1. Vehicle control (2% Tween 80). 2. T-6295: Perfluorooctane sulfonic acid, potassium salt (perfluorooctanesulfonate), PFOS, FC-95, Formula: C8F17S 0 3- K+, MW = 538.1 g/mole). 3. T-6889: Ammonium perfluorooctanoate (PFOA, APFO, FC-143, Formula: CyFisCOO-NH/, MW = 431.1 g/mole). 4. T-6316: N-ethylperfluorooctane sulfonamido ethanol (narrow Range N-Ethyl Perfluorooctanesulfonamido ethyl alcohol), N-EtFOSE, EtFOSE, Formula: C8F 17S 0 2N(C2H5)CH2CH2OH, MW = 571.06). 5. Wyeth-14643 (WY, MW = 323.79 g/mol) was obtained from Chemsyn Science Laboratories, Lexena, KS. Wyeth-14643 (WY), was added to DT15A as positive control dose group for hepatic peroxisome proliferation. The protocol stated that T-6868, N-ethyl perfluorooctane sulfonamide (FX-12, Sulfuramide TM), Formula CsFi7S 0 2NHC2H s) would be tested, however, T-6868 was not tested in DT15-A. Procedures Part A: Procedures for a single intraperitoneal dosing of PFOS fT-62951 and APFO fT-6889! in male rats and guinea pigs. For part A, a total o f 15 male rats and 13 male guinea pigs, 6-8 weeks in age and weighing between 150 and 250 grams were purchased from Charles River. Three to four male rats or guinea pigs per treatment group were used. Following an adaptation period o f not less than one week after arrival at 3M, animals were administered treatments by intraperitoneal (i.p.) injection, 4 8 SRPT T-6295.8, T-6889.1, T-6316.4 DT15-A at equimolar doses that were less than 40% o f the rat oral LD 50 for PFOS or the rat i.p. 24 hour LD 50 for APFO. There were solubility problems in com oil for the compounds. Therefore, doses o f each test material at 32 mM in 2% Tween-80 and using a dose volume o f 5 mL/Kg were used. The doses were calculated to be to the highest dose that could be delivered at less than 40% of the LD 50 for each test material. The selection o f these dose levels is discussed further in the protocol. Fresh solutions o f test substances were prepared for each dosing. The dose groups and final number o f animals in each dose group were as follows: 1. Control groups. The vehicle control group consisted of 2 male rats and three male guinea pigs. Each animal in the vehicle control group received a single i.p. injection 2% Tween-80. In addition, a no treatment control group consisted of two male rats that received no treatment were included in the study to check for any pronounced effects o f the vehicle control on the subtle endpoints of gene induction planned for this study. The biological and clinical chemistry data from the vehicle control group and the no treatment control rats were combined. 2. PFOS. A single i.p. injection o f 32 mM PFOS in 2% Tween-80 (final concentration =17.4 mg PFOS/mL) was given at a volume of 5 mL/Kg (final dose ~ 87.15 mg/Kg) to a total three male rats and four male guinea pigs. 3. APFO. A single i.p. injection of 32 mM APFO in 2% Tween-80 (final concentration =13.8 mg APFO/mL) was given at a volume of 5 mL/Kg (final dose ~ 70 mg/Kg) to a total four male rats and three male guinea pigs. 4. Wyeth-14643 (WY). A single i.p. injection o f WY in corn oil (final concentration = 52.45 mg/mL W Y ) was given at a volume o f 1 mL/Kg (final dose ~ 52.3 mg/Kg) to a total four male rats and three male guinea pigs. Example dose calculation for 32 mM PFOS i.p. at 5 ml/Kg, assuming an average weight o f 350 g for the guinea pig this came to: (0.35 kg)(0.005 L/kg)(538.1 g/mol)(0.032 mol/L) = 30.1 mg/guinea pig, or approximately 86 mg/Kg PFOS. All animals were observed for mortality and signs o f toxicity during the first four hours after dosing, at 24 hours and daily thereafter for the duration o f the study. Body weights were recorded immediately prior to dosing, daily thereafter, and at necropsy. Following the first exposure and up to four times throughout the in-life phase o f the study, the animals were housed individually in metabolic cages overnight, and urine and feces were collected. Animals were sacrificed by CO2 and gross necropsy performed on either day 12 or day 14 post-dose. Organ tissues (liver, kidney, testes and pancreas) and urine and feces were stored frozen at -80C for biochemical analysis. Sections of liver kidney, testes and pancreas were placed in 10% buffered formaldehyde and submitted for histological analyses by light microscopy. Some livers were perfused with gluteraldehyde and submitted for histological analysis by electron microscopy. Blood samples were collected at necropsy by heart puncture to analyze for test compound, metabolite formation and to monitor for changes in blood chemistry. 5 7 SRPT T-6295.8, T-6889.1, T-6316.4 DT15-A Part B: Procedures for a Time-Course o f PFOS (T-6295) toxicity in male and female rats and guinea pigs following a single i.p. injection o f PFOS at two doses. Based on the results of Part A (discussed below) the doses, dose volumes, and the time points chosen for sample analyses were modified. A time course o f 3, 6,12 and 28 days was conducted to measure peroxisome proliferation induction in the liver o f male and female rats and guinea pigs treated with either 16 mg/Kg or 100 mg/Kg PFOS. A total of 22 rats and 20 guinea pigs, 12 male and 10 female rats, and 10 male and 10 female guinea pigs, 6-8 weeks in age and weighing between 150 and 250 grams were purchased from Charles River. Following an adaptation period o f not less than one week after arrival at 3M, animals were administered treatments by intraperitoneal (i.p.) injection. Eight rats and eight guinea pigs (4/sex) were treated in each fluorochemical dose group. The dose groups were 16 mg/Kg PFOS and 100 mg/Kg PFOS. To limit the volume o f the i.p. injection, a 100 mg/mL suspension of PFOS was made by dissolving 200 mg PFOS in 0.5 mL com oil then adding 1.5 ml 2% Tween 80 and creating an emulsion in a glass tissue grinder. A single i.p. injection o f the 100 mg/mL PFOS suspension was given at a volume o f 1 mL/Kg to the 100 mg/Kg dose group animals, and at a volume o f 0.16 mL/Kg to the animals in the 16 mg/Kg dose group. One animal per sex per species per fluorochemical dose group were sacrificed on days 3, 6,12 and 28 post-dose. Two animals per sex per species received the vehicle control (25% com oil in 2% Tween 80), one vehicle control animal each at a volume o f volume o f 1 mL/Kg, corresponding to the 100 mg/Kg dose group animals, and at 0.16 mL/Kg, corresponding to the 16 mg/Kg dose group. One animal per sex per species per dose group were sacrificed on days 3, 6,12 and 28 post-dose. Two male rats received WY as a positive control group. One of the positive control rats received a single i.p. injection o f a 100 mg/mL suspension o f WY at a volume o f 1 mL/Kg to for a total dose or 100 mg/Kg WY. The other positive control rat received a single i.p. injection o f a 100 mg/mL suspension o f WY at a volume o f 0.16 mL/Kg for a total dose o f 16 mg/Kg dose group WY. The positive control group rats were sacrificed on day 3 post-dose. Part C Procedures for hepatic peroxisomal gene induction following oral dosing o f N-EtFOSE in rats. The results o f parts A and B led to the conclusion that i.p. dosing was not the best route of administration, therefore an exploratory oral dosing procedure in rats was performed with NEtFOSE in Part C. The purpose was to generate samples for peroxisomal gene induction assays in liver, and for N-EtFOSE metabolite analysis in liver and serum. A dose of 20 mg/Kg/day NEtFOSE for two days was chosen as an initial screening dose. This dose was based on a dietary study with 300 ppm N-EtFOSE that caused liver effects and induced palmitoyl Co A oxidase activity in liver over a 4-week period in which the average N-EtFOSE consumption in the dietary study was calculated to be approximately 23 mg/Kg/day for the second week o f the study (Ref. 3M Medical Dept. T-6316.1). Three male rats were dosed with 20 mg/Kg/day N-EtFOSE for two days. A 4 mg/mL suspension of N-EtFOSE in 2% Tween 80 was prepared in a glass 6 /O SRPT T-6295.8, T-6889.1, T-6316.4 DTI 5-A tissue grinder and delivered to the rats by oral gavage at a dose volume of 5 mL/Kg for two consecutive days. Two rats were dosed with the vehicle control, 2% Tween 80at a dose volume of 5 mL/Kg for two consecutive days. The animals were euthanized and necropsied on day 3 post-dose. Livers were weighed and chunks o f liver were frozen in liquid nitrogen then put in polypropylene vials, placed on dry ice and then stored at -80. Serum was obtained by centrifugation from blood collected by heart puncture at necropsy and stored at -80 The ~ 2g liver samples that were flash frozen in liquid nitrogen were sent on dry ice for analysis of gene induction to: Kendall B. Wallace, Ph.D. D.A.B.T. Professor, Dept of Biochemistry and Molecular Biology School of Medicine 10 University Drive Duluth, MN 55812-2496 Results and Discussion Part A Results on the Toxicity o f PFOS and APFO in rats and guinea pies. Results: Rats Part A In rats, there were no statistically significant changes in body weight. Liver weight and liver-tobody weight ratios were increased following treatment with all three compounds, particularly APFO; however, these changes were not statistically significant (Table 1; Appendix 1). The serum clinical chemistry analysis revealed that cholesterol was lowered to below the limit of detection o f 45 mg/dL in rats treated with PFOS. Glucose was significantly lowered in rats treated with PFOS and WY. No statistically significant changes occurred in the clinical chemistry parameters o f rats treated with APFO. The histological results for 3 out o f four rats treated i.p. with 52 mg/Kg WY (Animal numbers 7R4845 7R4847 and 7R4837) showed macroscopic masses o f white spots on the liver surface covered in thin scar tissue and evidence o f mononuclear infiltration. Multifocal microscopic subchronic granulomas were noted. The histological results for rats treated i.p. with APFO (animal numbers 7R04851-53) showed enlarged livers and kidneys. Macroscopic lesions o f mottled black spots on one liver were noted (7R04851). Microscopically, this animal had a pad o f scar tissue with an imbedded granuloma on the liver surface. The histological results for one rat treated with PFOS (7R04838) showed signs o f liver damage repair. All other rats, including the control group rats, had no significant change present in liver, kidney, or pancreas. 7 n SRPT T-6295.8, T-6889.1, T-6316.4 DTI 5-A Results: Guinea pies Part A One guinea pig (Animal number 7G0253) that was treated with 100 mg/Kg APFO died overnight following treatment. No tissues were collected from that animal and the initial body weight (358 g) o f that animal is not included in the tables or statistical analyses. The cause of death is unknown, but may have been compound related. The guinea pigs treated with PFOS had significantly lower weight gains compared to control. The guinea pigs treated with APFO had significantly greater liver to body weight ratios compared to control. One o f four guinea pigs treated with 17.4 mg/Kg i.p. PFOS (7G02254) showed signs of liver damage repair. No significant changes were present in the liver, kidney, testes, or pancreas of any of the other guinea pigs examined microscopically (7G2004 through 7G2013). Tissues from two out o f three control guinea pigs (7G2255 & 7G2256) were never analyzed and were disposed of. Liver samples submitted for L-FABP analysis from rats and guinea pies treated in Part A. Certain liver samples from control and PFOS treated rats and guinea pigs were used for the isolation o f liver fatty acid binding protein at the University of San Francisco (Animals 7G02004-control guinea pig, 7G02005-PFOS guinea pig, 7R04844-control rat and 7R04848PFOS rat). The goal of the LFABP study was to assess the effect of FCs on L-FABP function as evaluated by the ability of the fluorescent fatty acid analogue 11- (5dimethylaminonapthalenesulphonyl) - undecanoic acid (DAUDA) to bind to L-FABP isolated from rats and guinea pigs treated and not treated with PFOS in vivo. Results showed a decreased maximum binding capacity of L-FABP from PFOS treated rats without an increase in Kd. The complete methods and the results o f those analyses are reported elsewhere (Luebker et al. 2002; Nabbefeld 1998). Conclusions from Part A: The histological results of liver damage with evidence of scar tissue on the surface o f the livers of some animals 12 or 14 days after treatment with WY, APFO, and/or PFOS led us to consider that the doses, dose volumes, route o f administration and the time points chosen for part A were not necessarily the best model for the objectives of the study. Further analyses o f tissues for total organic fluorine, electron microscopy, or peroxisome proliferation from part A were aborted and Part B was planned. Part B: Results of Time-Course of PFOS toxicity at two doses in male and female rats and guinea pigs. Results: Rats Part B All rats treated with either 16 mg/Kg or 100 mg/Kg PFOS gained weight following dosing and maintained body weights that were slightly less than control values (Table 3, Figures 1 and 2). Statistical analyses o f body weight suggested that there were no significant differences between treated and control, but had very little power because o f the low N (statistics not shown). Liver weights were increased in both male and female rats treated with 100 mg/Kg dose groups (Appendix 3). The liver to body weight ratios in rats were significantly increased in combined 8 SRPT T-6295.8, T-6889.1, T-6316.4 DT15-A male and female liver to body weight ratio values from the 100 mg/Kg dose group versus control values (Table 5, Appendix 3). The female rats had more o f an increase in liver to body weight ratios than did the male rats. Hypertrophy and white pigmentation were noted by gross observation at necropsy in some o f the high dose group rats (Appendix 3). Kidney weights and kidney to BW ratios were not significantly affected in rats (Appendix 3). Results: Guinea pigs Part B PFOS was more toxic to guinea pigs than rats, and apparently more toxic to female guinea pigs than male guinea pigs. Three o f the four female guinea pigs, and one male guinea pig treated with 100 mg/Kg PFOS i.p. died on day 2 o f the study. The remaining female guinea pig lost 12% of its initial body weight by day 6 and was humanely euthanized on day 6. All other guinea pigs gained weight and maintained body weights that were slightly lower than control values throughout the time course (Table 4, Figure 4 ).The guinea pig kidney to body weight ratios from all animals, males and females combined, were significantly increased by treatment with 100 mg/Kg PFOS i.p. (Appendix III). Female guinea pigs in particular were found to be very sensitive to PFOS toxicity, evidenced by the fact that three out o f four female guinea pigs died within the first 24 hours after dosing with a 100 mg/Kg dose o f PFOS in 25% com oil and 75% Tween 80 and the remaining female guinea pig lost weight up to day 6 post-dose. In contrast, one out o f four male guinea pig died after a i.p. dose of 100 mg/Kg dose of PFOS in 25% com oil and 75% Tween 80 at that dose, and the remaining 3 male guinea pigs survived and gained weight following treatment. The administration o f the 100 mg/Kg PFOS dose mixed with 25% com oil may have contributed to the observed toxicity o f PFOS to guinea pigs dosed in part B. None o f the male guinea pigs died following i.p. administration of a 100 mg/Kg PFOS dose in 2% Tween 80 at a volume o f 5 mL/Kg, whereas one male and three female guinea pigs died when the same dose was delivered in 25% com oil at a volume o f lmL/ Kg. There is a precedent for delivery o f PFOS in com oil by oral gavage to be more toxic than when delivered in an aqueous solution. The use o f com oil in the dose preparation has previously led to a clear difference in the oral LD 50 of PFOS in rats. The rat 24 hour oral LD-50 for PFOS in a 20:80 acetone: com oil suspension was determined to be 251 mg/kg (Dean et al. 1978). In contrast, the rat oral LD-50 for PFOS in an aqueous suspension was determined to be 1.25 - 2.5 g/kg (Gabriel 1976). No deaths or gross lesions were observed 48 hours after dosing male and female rats with a single oral gavage dose o f 250 mg/kg aqueous suspensions o f PFOS (Goldenthal et a l 1979). Thus, the use o f a mixture of 25% com oil in an aqueous suspension of 2% Tween 80 as the vehicle to deliver PFOS may have contributed to the observed increased toxicity of PFOS to guinea pigs when compared to an 2% Tween 80 vehicle alone. Conclusions for Part B: Intraperitoneal administration of PFOS led to the formation o f scar tissue on the liver. The findings in this study led to the decision that i.p. dosing was not the appropriate route of administration, and that subsequent studies should be conducted following oral administration of these compounds. 9 5 SRPT T-6295.8, T-6889.1, T-6316.4 DTI 5-A Part C: Results Oral dosing o f rats with 20 mg/Kg N-EtFOSE There was no apparent effect of treatment on body weight or liver weight (Appendix IV). No signs of toxicity or liver effects were apparent by gross observations at necropsy. The rats used were o f different ages and initial body weights, therefore, statistics were not performed on these data. The liver specimens were analyzed for induction o f mRNA for peroxisome proliferation specific genes. There was no apparent indication o f gene induction by N-EtFOSE by Northern Blot analysis, however the total mRNA was partially degraded in all samples, therefore, a quantitative analysis o f the results was not performed. Further analysis o f these samples was aborted, and the tissues were disposed of. Conclusions Standard toxicity tests o f serum clinical chemistry and microscopic examination o f the liver obtained in Parts A o f this study indicated that male guinea pigs were more sensitive to the toxic effects o f PFOS and APFO than were male rats. In Part B, three out o f four female guinea pigs and one out of four male guinea pigs died overnight, and the remaining female guinea pig was sacrificed on day 6 due to excessive weight loss, following i.p treatment with 100 mg/Kg PFOS. In contrast, none o f the male or female rats died following alOO mg/Kg i.p. dose o f PFOS. These data showed that the guinea pigs, females in particular, were very sensitive to PFOS toxicity relative to rats. The findings in this study led to the conclusion that intraperitoneal dosing was not the appropriate route of administration, and that subsequent studies should be conducted following oral administration of these compounds to test the stated hypotheses. An initial oral dose study with 20 mg/Kg N-EtFOSE in male rats showed no apparent effects by gross observations therefore the doses were considered to be low as there were no apparent liver effects, and the gene induction studies were aborted. Therefore, Oral dosing and higher doses of N-EtFOSE were deemed necessary for future experiments. 10 SRPT T-6295.8, T-6889.1, T-63 !6.4 DTI 5-A Signatures Prepared by: ____ JY). r/is -/x c d________________________________ Andrew M. Seacat, Ph.D., DABT. Toxicology Specialist 3c Study Director Date /5 SRPT T-6295.8, T-6889.1, T-6316.4 DT15-A P a rt A. Histopathology Report. Elden Lamprecht 3M Lab Animal Research Services: Rats 7R4845 Macroscopic mass imbedded in liver surface covered in thin scar tissue. Its substance has been dissolved leaving a foamy matrix o f protein and mononuclear cells. The interface of the mass and liver consists of foamy mononuclear cells. 7R4847 Macrospopic mass protruding from liver surface covered by thin layer of scar tissue. Its substance has been dissolved leaving a foamy matrix o f protein and mononuclear cells. The interface of the mass and liver consists of foamy mononuclear cells. 7R4851 A pad of scar tissue with an imbedded granuloma on the liver surface. 7R4837 Multifocal microscopic subchronic granulomas are present in liver. 7R4838 Parietal pleura of liver is markedly thickened with repair tissue. A generalized mild periportal vacuolation of hepatocytes is present. Some degredation o f hepatocytes may be present. All other rats had no significant change present in liver, kidney or pancreas. Guinea pigs 7G02254 -A macroscopic pad of granulomatous inflammation present on the surface. It has a foamy appearance with pore contents being dissolved leaving. The matrix which remains is composed o f mononuclear cells, some o f which are engorged or vacuolated. The pad is covered by an increasingly thick layer of scar tissue. Scar tissue interfaces with the liver parenchyma. No change in liver parenchyma. A second smaller pad is present on another location o f the liver. 7G2255 & 7G2256 - never analyzed and disposed of. 7G2004 - 7G2013 - No significant changes were present in the liver, kidney, testes or pancreas. 12 t(o SRPT T-6295.8, T-6889.1, T-6316.4 DT15-A Tables: Table 1. Part A. Summary of Toxicity of PFOS and APFO in male rats Table 1. Rats Parameter Control PFOS APFO Avg SD avg SD %control avg SD %control avg WY S D |% control Body w t (g) day1 Body wt (g) day12/14 weight gain 332 122 369 79 72 4 258 12.5 341 9.8 83 11.5 77.7 92.4 115.3 311 125.9 413 95.8 102 41.7 93.6 111.9 142.0 300 75.7 398 56.0 99 20.3 90.2 107.9 137.2 Liver w t(g ) liver/body wt 17 0.033 5.47 0.023 18.90 0.04 1.41 0.033 111.2 112.7 26.63 0.060 6.43 0.010 156.6 181.8 20.58 0.05 3.4 0.0 121.1 151.5 Clinical Chemistry Choi mg/dL Ca mg/dL Phos mg/dL TBIL mg/dL Alb mg/dL TP mg/dL BUN mg/dL GLU mg/dL ALKP U/L AST U/L ALT U/L LDH U/L Na+ mmol/L K+ mmol/L Cl- mmol/L CREA mg/dL GGT U/L TRIG mg/dL 54 11.6 12.6 0.25 3.50 6.23 15.3 263 335 255 79.7 391 142 8.53 101 0.57 8.0 171 8.19 <45 ' 0.10 11.0 0.85 11.8 0.30 0.10 0.20 3.43 0.15 6.13 3.3 15 29.5 149f 81.0 337 310 91 10.8 80 144 335 1.15 144 0.83 6.6 1.5 99 0.12 0.40 0.00 8.00 130 77 0.00 NA12 0.20 94.8 0.90 93.6 0.00 40.0 0.21 98.1 0.21 98.4 1.5 100.5 26 56.8 87 100.7 8.2 35.7 10 100.7 55.2 85.5 3.1 101.6 1.7 77.0 1.0 97.7 0.0 70.6 0.00 100.0 34 45.4 60 11.65 12.55 0.13 3.73 6.48 14.5 254 374 88 87 334 145 8.50 101 0.43 8.0 141 12.8 0.44 1.62 0.05 0.39 0.48 1.29 160 62.8 9.9 6.58 90.1 2.1 1.00 0.82 0.05 0.0 53 111.1 100.4 99.9 50.0 106.4 103.9 95.1 96.7 111.9 34.5 109.1 85.2 102 99.6 99.7 75.0 100.0 82.7 60 11.43 11.78 0.10 3.43 6.28 14.75 175 316 138 67 717 151 7.13 98.5 0.48 8.0 175 5.3 0.80 0.32 0.00 0.10 0.24 2.63 3 9 .9t 67 173 12.5 993 9.56 0.42 3.00 0.10 0.0 32 111.6 98.5 93.7 40.0 97.9 100.7 96.7 66.5 94.3 54.3 84.0 183.2 106.1 83.5 97.2 83.8 100.0 102.5 1. The limit o f Detection for Cholesterol (CHOL) was 45 mg/dL. 2. NA N ot applicable f Significantly different from control values by Student's T-test 13 /7 SRPT T-6295.8, T-6889.1, T-6316.4 DT15-A Table 2. Part A. Summary of Toxicity o f PFOS and APFO in male guinea pigs Table 2 Guinea Pigs Control Parameter Avg SD Avg PFOS SD %control avg APFO SD %control Avg WYETH SD %control Body w t (g) d1 Body w t (g) d12/14 weight gain 333.00 422.00 89.00 17.09 335.50 17.62 385.00 2.89 4 9 .50T 20.9 20.3 17.9 100.8 91.2 5 5 .6T 316.67 18.72 377.67 44.56 61.00 25.98 95.1 89.5 68.5 322.00 401.67 79.67 11.36 10.02 1.53 96.7 95.2 89.5 Liver (g) liver/body w t d12/14 17.00 0.04 0.50 0.00 17.45 3.43 0.05 0.010 102.6 125.0 17.57 1.16 1- 0 .0 5 1 0.00 103.3 116.7 18.10 0.05 1.77 0.00 106.5 112.6 Clinical Chemistry Choi mg/dL 55.00 10.00 51.25 7.46 93.2 47.33 4.04 Ca mg/dL 11.70 0.56 10.83 0.36 92.5 11.07 0.57 Phos mg/dL 11.70 2.25 8.53 1.49 72.9 8.87 0.45 TBIL mg/dL 0.47 0.64 0.10 0.00 21.4 0.10 0.00 Alb mg/dL 2.47 0.31 2.25 0.17 91.2 2.37 0.21 TP mg/dL 5.00 0.46 4.73 0.26 94.5 4.83 0.25 BUN mg/dL 18.33 3.79 16.25 2.87 88.6 15.33 0.58 GLU mg/dL 266.67 178.75 148.50 49.29 55.7 130.33 8.62 ALKP U/L 349.00 107.48 283.50 39.14 81.2 326.67 38.63 AST U/L 151.00 132.18 53.50 6.45 35.4 65.67 20.11 ALT U/L 63.33 7.51 52.75 5.19 83.3 51.00 4.00 LDH U/L 891.33 1050.23 192.50 50.40 21.6 264.00 68.61 Na+ mmol/L 139.67 3.51 141.00 1.83 101.0 142.00 2.83 K+ mmol/L 9.90 2.55 5.30 1.50 53.5 7.70 0.57 Cl- mmol/L 103.67 0.58 103.00 3.56 99.4 102.00 0.00 CREA mg/dL 0.40 0.10 0.38 0.05 93.8 0.33 0.06 GGT U/L 10.45 13.51 12.50 1.29 119.6 12.67 2.08 TRIG mg/dL 1. 2. 268.33 108.15 117.75 54.71 43.9 140.33 37.81 The limit o f Detection for Cholesterol (CHOL) was 45 mg/dL. NA Not applicable t Significantly different from control values by Student's t-test 86.1 47.67 4.62 86.7 94.6 11.27 0.45 96.3 75.8 8.43 0.55 72.1 21.4 0.10 0.00 21.4 95.9 2.37 0.06 95.9 96.7 4.67 0.15 93.3 83.6 18.00 2.65 98.2 48.9 151.67 52.25 56.9 93.6 328.00 75.11 94.0 43.5 70.67 20.40 46.8 80.5 38.00 15.59 60.0 29.6 238.00 22.54 26.7 101.7 # D IV /0 ! # D IV/0 ! # D IV/0 ! 77.8 # D IV/0 ! # D IV /0 ! # D IV/0 ! 98.4 # D IV/0 ! # D IV/0 ! # DIV/0 ! 83.3 0.33 0.06 83.3 121.2 11.33 0.58 108.5 52.3 128.67 13.58 48.0 14 SRPT T-6295.8, T-6889.1, T-6316.4 DT15-A Table 3. Part B. Summary of Body Weights in Rats, Means and Std Deviations Gender Dose PFOS Day Mean Body Weight Std Dev N (mg/Kg) Post-Dose F Cont F Cont F Cont F Cont F Cont F 16 F 16 F 16 F 16 F 16 F 100 F 100 F 100 F 100 F 100 M Cont M Cont M Cont M Cont M Cont M 16 M 16 M 16 M 16 M 16 M 100 M 100 M 100 M 100 M 100 M Cont M Cont M Cont M Cont M Cont 1 3 6 12 28 1 3 6 12 28 1 3 6 12 28 1 3 6 12 28 1 3 6 12 28 1 3 6 12 28 1 3 6 12 28 194 NR 211 228 250 183 192 207 215 223 192 185 195 221 239 244 NR 290 337 413 241 278 292 327 419 251 247 279 334 386 244 NR 290 337 413 10 2 NA 0 72 NA 1 NA 1 64 NA 1 73 72 NA 1 44 NA 1 63 22 NA 1 92 NA 0 11 2 NA 1 NA 1 11 4 NA 1 17 3 21 2 NA 1 94 NA 1 12 3 21 2 NA 1 92 NA 0 11 2 NA 1 NA 1 SRPT T-6295.8, T-6889.1, T-6316.4 DT15-A Table 4. Part B. Summary of Body Weights in Guinea Pigs, Means and Std Deviations Gender Dose PFOS Day Post-Dose Mean BW (g) Std Dev N (mg/Kg) F Cont F Cont F Cont F Cont F Cont F 16 F 16 F 16 F 16 F 16 F 100 F 100 F 100 F 100 F 100 M Cont M Cont M Cont M Cont M Cont M 16 M 16 M 16 M 16 M 16 M 100 M 100 M 100 M 100 M 100 1 3 6 12 28 1 3 6 12 28 1 3 6 12 28 1 3 6 12 28 1 3 6 12 28 1 3 6 12 28 365 49 2 NR NA 0 398 70 2 500 NA 1 573 NA 1 359 23 4 349 NA 1 393 17 3 442 26 2 464 NA 1 355 26 4 NR NA 0 317 NA 1 NA NA 0 NA NA 0 370 7 2 NR NA 0 429 10 2 511 NA 1 626 NA 1 377 18 4 371 NA 1 415 25 3 478 24 2 610 ? 1 376 7 4 359 NA 1 373 35 2 456 NA 1 NR NA 0 SRPT T-6295.8, T-6889.1, T-6316.4 DT15-A Table 5. Part B Liver to Body Weight ratios in rats Dose Group Cont 16 mg/Kg PFOS 100 mg/Kg PFOS Species Rat Rat Rat Gender M 8t F M&F M&F N Mean 4 0.05 8 0.05 8 0.06T Conttrol 16 mg/Kg 100 mg/Kg GP GP GP 4 0.04 8 0.04 4 0.04 'Significantly different from control values by Dunnett's t-test Std Dev 0.01 0.01 0.01 0.003 0.002 0.001 SRPT T-6295.8, T-6889.1, T-6316.4 DT15-A Figures Figure 1: Part B: Female Rat Body Weight Vs Time. Time-Course o f PFOS toxicity at two doses, 16 mg/Kg and 100 mg/Kg. Female Rat BW Vs Time ---- Cont - -1 6 r 100 Days Post-dose SRPT T-6295.8, T-6889.1, T-6316.4 DT15-A Figure 2: Part B: Male Rat Body Weight Vs Time. Time-Course of PFOS toxicity at two doses, 16 mg/Kg and 100 mg/Kg. Male Rat BW Vs Time SRPT T-6295.8, T-6889.1, T-6316.4 DT15-A Figure 3: Part B: Female Guinea Pig Body Weight Vs Time. Time-Course o f PFOS toxicity at two doses, 16 mg/Kg and 100 mg/Kg. SRPT T-6295.8, T-6889.1, T-6316.4 DTI 5-A Figure 4: Part B: Male Guinea Pig Body Weight Vs Time. Time-Course o f PFOS toxicity at two doses, 16 mg/Kg and 100 mg/Kg. 3-S SRPT T-6295.8, T-6889.1, T-6316.4 DTI 5-A Appendix 1. Deviations to the Protocol: 1. The protocol states that there would be Five male and five female rats and guinea pigs per treatment group, with 5 treatment groups for a total o f 50 rats and 50 guinea pigs. In Part A, only male animals were used. Three to four animals per treatment group were used and a total of 15 male rats and 13 male guinea pigs were used in Part A. In Part B, a total o f 20 rats and 20 guinea pigs, 10 males and 10 females per species were used. Thus, in both parts A and B, a total o f 35 rats and 33 guinea pigs were used. 2. The protocol stated that N-ethyl perfluorooctane sulfonamide (FX-12) and Nethylperfluorooctane sulfonamido ethanol (FC-10, N-EtFOSE, T-6316) would be dosed. These two dose groups were removed from the protocol, and a positive control, Wyeth14643 (WY), a model persoxisome proliferators, was added as a dose group. 3. The protocol stated that all treatments would be administered in corn oil. PFOS (T-6295) and APFO (T-6889) were administered in 2% Tween 80. Wyeth-14643 (WY) was administered in 25% com oil. 4. The protocol stated that Dunnett's t-test would be performed on all data. Student's t-test were performed instead where noted. 5. The protocol stated that all treatments would be given by i.p. dosing. Dosing by oral gavage was performed in Part C. 22 SRPT T-6295.8, T-6889.1, T-6316.4 DTI 5-A Appendix II. Part A: Toxicity of PFOS and APFO Individual and Summary Data Part A: CONTROL PFOS Rat data animal # 7r)-xxxx Nvh. *1 C1 Nvh. *2 C2 Vh *3 4844 Vh A*4 Avg 4836 SD *1 4848 *2 4849 *3 7850 Avg Parameter Body wt 245 418 332 122 272 254 248 258 (9)cl1 Body wt 328 320 487 369 79.4 344 349 330 341.0 (9) d12 wtgain wt gain % #DIV/0 #DIV/0! ! 74.7 69 72 4.0 30% 17% 23% 0.1 72 26% 95 82 83.0 37% 33% 0.3 SD 12.5 9.8 11.5 0.1 Liver (g) liver/body wt Kidney (g) Testis (g) 12,5 0.04 3.2 3.3 15.9 0.05 2.9 3.4 23.2 0.00 0.05 3.2 4.9 2.9 3.9 17 0.03 3.6 3.4 5.5 0.02 0.9 0.4 0.00 3.3 3.3 17.9 0.05 2.9 3.3 19.9 0.06 3.2 3.1 18.9 0.04 3.1 3.2 1.4 0.03 0.2 0.1 Clinical Chemistry Choi mg/dL 52 63 Ca mg/dL 11.5 11.7 Phos mg/dL 13.4 11.7 TBIL mg/dL 0.1 0.1 Alb mg/dL 3.5 3.7 TP mg/dL 6.2 6.4 BUN mg/dL 13 15 GLU mg/dL 244 248 ALKP U/L 271 326 AST U/L 98 89 ALT U/L 67 77 LDH U/L 332 286 Na+ mmol/L 141 143 K+ mmol/L 9.2 8.8 Cl- mmol/L 101 100 CREA mg/dL 0.5 GGT U/L 88 TRIG mg/dL 70 131 47 54.0 8.2 <45 <45 <45 NA 11.6 11.6 0.1 10.8 11.2 11 11.0 12.6 12.6 0.9 10.9 12.7 11.7 11.8 0.1 0 7 0.3 0.3 0.1 0.1 0.1 0.1 3.3 3.5 0.2 3.5 3.6 3.2 3.4 6.1 6.2 0.2 6.3 6.2 5.9 6.1 13 20 15.3 3.3 14 17 15 15.3 297 263 29.5 119 166 163 149.3 451 290 334 80.9 276 436 299 337.0 114 719 255 309 84 100 89 91.0 82 93 79 10 70 90 81 80 556 391 144 277 387 340 334 143 142 1.2 148 142 144 144 7.6 8.5 0.8 5.3 8.5 5.9 6.6 103 101 1.5 98 99 100 99.0 0.5 0.6 0.1 0.4 0.4 0.4 0.4 8 8.0 0.0 8 8 8 8.0 120 361 170 129 57 117 58 77.3 NA 0.2 0.9 0.0 0.2 0.2 1.5 26.3 86.5 8.2 10 55.2 3.1 1.7 1.0 0.0 0.0 34.4 23 <27 SRPT T-6295.8, T-6889.1, T-6316.4 DT15-A Part A: Rat data (cont) animal # 7R0-xxxx Parameter Body wt (q)d1 Body wt (g) d12 wtqain wt gain % Liver (q) liver/body wt Kidney (g) Testis (q) APF 0 *1 *2 *3 A*4 av9 485 485 483 4851 2 3 8 227 253 265 498 310 385 349 363 555 413 158 70% 28.9 96 38% 19.6 98 37% 23.6 57.0 102 11% 39% 34.4 26.6 0.08 0.06 0.07 0.06 0.06 3.9 3.5 3.4 4.8 3.9 3.6 3.0 3.4 3.6 3.4 WY SD *1 2 *3 A * 4 Ayg_. SD 484 484 484 5 6 7 4837 125 264 258 263 413.0 299.5 75.7 95 367 368 376 41. 7 103 110 113 0.2 39% 43% 43% 6.4 19.6 18.6 18.5 482 398.3 56.0 69 17% 25.6 98.8 20.3 35% 0.1 20.5 3.4 0.0 0.05 0.05 0.05 0.6 3.7 3.2 3.4 0.3 3.1 3.4 3.2 0.05 4.3 3.7 0.05 3.7 3.4 0.0 0.5 0.3 Clinical Chemistry , Choi mq/dL 53.0 56.0 52.0 79.0 60.0 12 65.0 63.0 60.0 53.0 Ca mq/dL 11.5 11.4 11.4 12.3 11.7 0.4 12.6 11.1 11.2 10.8 Phos mq/dL 11.2 12.0 12.1 14.9 12.6 1.6 12.1 12.0 11.5 11.5 TBIL mq/dL 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.1 Alb mq/dL 3.4 3.4 3.9 4.2 3.7 0.4 3.4 3.5 3.3 3.5 TP mq/dL 6.1 6.1 6.6 7.1 6.5 0.5 6.1 6.3 6.1 6.6 BUN mq/dL 14.0 13.0 16.0 15.0 14.5 1.3 17.0 12.0 13.0 17.0 GLU mq/dL 206 175 145 491 254 160 210 142 139 209 ALKP U/L 419 284 379 415 374 63 332 401 284 245 AST U/L 94 84.0 98.0 76.0 88.0 9.9 76.0 8.0 76.0 394 ALT U/L 88 95 86 79 87 6 52 74 62 80 LDH U/L 340 234 309 451 334 90 262 233 167 2205 Na+ mmol/L K+ mmol/L Cl- mmol/L CREA mg/dL GGT U/L 145 142 145 147 145 2 165 146 144 7.0 8.9 9.1 9.0 8.5 1.0 7.3 7.4 6.5 101 100 101 102 101 1 100 96 96 0.4 0.4 0.5 0.4 0.0 0.5 0.4 0.4 8.0 8.0 8.0 8.0 8.0 0.0 8.0 8.0 8.0 149 7.3 102 0.6 8.0 TRIG mq/dL 107 118 119 220 141 53 196 130 175 198 * = Histoloov missing data A = sacrificed on dav 14 rather than 12 Limit of detection for Cholesterol = 45 mg/dl 60.3 11.4 11.8 0.1 3.4 6.3 14.8 175 316 139 67 717 5.3 0.8 0.3 0.0 0.1 0.2 2.6 40 67 173 12 993 151 10 7.1 0.4 99 3 0.5 0.1 8.0 0.0 175 32 SRPT T-6295.8, T-6889.1, T-6316.4 DT15-A Part A: Guinea Pig data animal# 7G 0-XXXX Parameter Body wt (g) d1 Body wt (g) d12 wt gain Liver (g) liver/body wt Kidney (g) Testis (g) *1 2004 317 408 91 17.2 0.04 3.9 1.2 Vehicle A*2 A*3 Avg 2256 2255 331 417 86 16.7 0.04 3.4 1.6 351 333 442 422 91 17.7 0.04 4.1 1.5 89 17.2 0.04 3.8 1.4 SD 17 18 3 0.5 0.00 0.4 0.2 *1 2005 313 387 74 13.1 0.03 3.5 1.0 *2 2006 329 371 42 16.4 0.04 3.9 0.8 PFOS *3 2007 a*4 2254 337 369 32 20.7 0.06 4.4 0.9 363 413 50 19.6 0.05 4.1 1.6 Avg 336 385 50 17.5 0.05 4.0 1.1 SD 21 20 18 3.4 0.01 0.4 0.4 Clinical Chemistry Choi mg/dL 55 Ca mg/dL 12 Phos mg/dL 10 TBIL mg/dL 0.1 Alb mg/dL 2.2 TP mg/dL 4.6 BUN mg/dL 14 GLU mg/dL 118 ALKPU/L AST U/L 87 alt U/L 56 LDH U/L 277 Na+ mmol/L 143 K+ mmol/L 8.1 Cl- mmol/L 104 CREA mg/dL 0.3 GGT U/L TRIG mg/dL 157 65 12 14 1.2 2.8 5.5 21 465 425 303 71 2104 136 103 0.5 20 373 45 11 11 0.1 2.4 4.9 20 217 273 63 63 293 140 11.7 104 0.4 1 275 55 10 12 1 12 2 0.5 0.6 2.5 0.3 5.0 0.5 18 4 267 179 349 107 151 132 63 8 891 1050 140 4 9.9 2.5 104 1 0 0.10 10 14 268 108 50 45 48 62 11 11 11 11 8 7 8 11 0.1 0.1 0.1 0.1 2.1 2.2 2.2 2.5 4.5 4.6 4.7 5.1 14 14 20 17 117 130 125 222 278 340 264 252 58 47 49 60 49 59 55 48 183 149 173 265 142 139 140 143 5.0 4.3 4.4 7.5 103 101 100 108 0.3 0.4 0.4 0.4 12 14 13 11 98 94 80 199 51 11 9 0.1 2.3 4.7 16 149 284 54 53 193 141 5.3 103 0.38 13 118 7 0 1 0.0 0.2 0.3 3 49 39 6 5 50 2 1.5 4 0.05 1 55 25 SRPT T-6295.8, T-6889.1, T-6316.4 DT15-A Part A: Guinea Pig data (cont) APFO WYETH (in com oil) animai # 7GO-XXXX Parameter Body wt (g) d1 Body wt (g) d12 wt gain Liver (g) liver/body wt Kidney (g) Testis (g) *1 2008 *2 2009 *3 2010 309 355 46 16.8 0.05 4.2 1.1 338 429 91 18.9 0.04 3.9 0.9 303 349 46 17.0 0.05 3.8 1.0 avg SD *1 2011 *2 2012 *3 Avg 2013 SD 317 19 378 45 61 17.6 0.05 4.0 1.0 26 1.2 0.00 0.2 0.1 317 398 81 16.2 0.04 4.1 0.9 335 413 78 19.7 0.05 4.8 1.8 314 394 80 18.4 0.05 4.6 0.8 322 402 80 18.1 0.05 4.5 1.2 11 10 2 1.8 0.00 0.4 0.6 Clinical Chemistry Choi mg/dL <45 <45 52 52 NA 53 45 45 48 5 Ca mg/dL 11 11 12 11 1 11 11 12 11 0 Phos mg/dL 9 8 9 90 9 88 8 1 TBIL mg/dL 0.1 0.1 0.1 0.1 0.0 0.1 0.1 0.1 0.1 0.0 Alb mg/dL TP mg/dL 2.3 2.2 2.6 2.4 0.2 2.4 2.3 2.4 2.4 0.1 4.8 4.6 5.1 4.8 0.3 4.7 4.5 4.8 4.7 0.2 BUN mg/dL 15 16 15 15 1 19 20 15 18 3 GLU mg/dL 132 121 138 130 9 121 122 212 152 52 ALKP U/L 323 290 367 327 39 408 259 317 328 75 AST U/L 88 49 60 66 20 66 93 53 71 20 alt U/L 51 47 55 51 4 47 20 47 38 16 LDH U/L 342 213 237 264 69 250 252 212 238 23 Na+ mmol/L K+ mmol/L 144 140 7.3 8.1 142 3 7.7 0.6 NA NA NA NA Cl- mmol/L 102 102 102 0 NA NA CREA mg/dL 0.3 0.3 0 4 0.33 0.06 0.4 0.3 0.3 0.33 0.06 GGT U/L 11 15 12 13 2 11 11 12 11 1 TRIG mg/dL 127 111 183 140 38 127 143 116 129 14 * = Histology nissing data A= sacrificed on day 14 rather than 12 26 3D SRPT T-6295.8, T-6889.1, T-6316.4 DT15-A Appendix in . Part B: Time course with PFOS. Individual Data Part B. Individual Rat data Time course with PFOS. Animal # Gender Dose Species BW BW BW BW BW LW LW/BW KW KW/BW 8R00344 8R00345 8R00346 8R00347 8R00340 8R00341 8R00342 F F F F F F F PFOS (mg/Kg) 100 100 100 100 16 16 16 Rat Rat Rat Rat Rat Rat Rat (9) (9) (g) (g) (g) (9) d1 d3 d6 d12 d28 188 185 ? ? ? 10.06 0.054 188 ? 188 ? ? 12.7 0.068 195 ? 198 222 ? 13.8 0.062 196 ? 199 219 239 13.4 0.056 189 192 ? ? ? 9.31 0.048 187 ? 215 ? ? 12.7 0.059 180 ? 202 220 ? 10.6 0.048 (9) 0.88 0.9 0.88 0.97 0.98 0.87 0.87 0.005 0.005 0.004 0.004 0.005 0.004 0.004 8R00343 8R00339 8R00348 8R00334 8R00335 8R00336 8R00337 8R00330 8R00331 8R00332 8R00333 8R00329 8R00338 F F F M M M M M M M M M M 16 V Conti1 V Cont2': 100 100 100 100 16 16 16 16 V Conti1 V Cont2,! Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat 177 ? 203 210 223 10.8 201 ? 216 ? ? 11.1 187 ? 206 228 250 10.4 244 247 ? ? ? 14.8 247 ? 279 ? ? 18 265 ? 291 348 ? 21.8 248 ? 267 319 386 19.4 253 278 ? ? ? 13.5 240 ? 288 ? ? 15.5 227 ? 277 312 ? 15 245 ? 310 341 419 10.8 250 ? 297 ? ? 12.7 237 ? 282 337 413 22.1 0.048 0.051 0.042 0.06 0.065 0.063 0.05 0.049 0.054 0.048 0.026 0.043 0.054 1.06 1 1.01 1,18 1.08 1.58 1.5 1.3 1.3 1.32 1.06 1.17 1.57 0.005 0.005 0.004 0.005 0.004 0.004 0.004 0.005 0.005 0.004 0.003 0.004 0.004 8R0004 M Wyeth 1J Rat 520 513 8R0003 M Wyeth 2" Rat 488 485 Notes: 1. V cont 1 = 0.16 mL/Kg dose volume of the vehicle, 25% corn oil in 80% Tween 80. 2. V cont 2 = 1.0 mL/Kg dose volume of the vehicle, 25% corn oil in 80% Tween 80. 3. Wyeth 1 = Positive control high dose 100 mg/Kg Wyeth 14643. Wyeth animal data was excluded from further analysis. 4. Wyeth 2 = Positive control low dose 16 mg/Kg Wyeth 14643. Wyeth animal data was excluded from further analysis. Gross observations: Animal number 8R0336 Male rat 100 mg/Kg dosegroup, necropsied on day 6 post-dose, had white edges on liver. Animal number 8R0336 Male rat 100 mg/Kg dosegroup, necropsied on day 12 post-dose, had a hypertrophic liver with white edges. Animal number 8R0337 Male rat 100 mg/Kg dosegroup, necropsiedon day 28 post-dose, had an abnormal, thickened liver with white edges. Sac day 3 6 13 28 3 6 13 28 6 28 3 6 13 28 3 6 13 28 6 28 3 3 27 Part B. Liver Weight to Body Weight ratio in male and female rats combined data. SAS analysis _________________________ LW/BW By DosePFOS(mg/Kg)__________________________ D osePFOS(m g/Kg) Student's t 0.05 D un n ett's 0.05 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.419071 0.350727 0.007664 0.0524 20 Source Model Error C Total Analysis of Variance DF Sum of Squares 2 0.00072030 17 0.00099850 19 0.00171880 Mean Square 0.000360 0.000059 0.000090 F Ratio 6.1317 Prob>F 0.0099 Means for Oneway Anova Level Number Mean 100 8 0.059750 16 8 0.047500 Cont 4 0.047500 Std Error 0.00271 0.00271 0.00383 Level 100 16 Cont Std Error uses a pooled estimate of error variance Means and Std Deviations Number Mean Std Dev Std Err Mean 8 0.059750 0.006018 0.00213 8 0.047500 0.009562 0.00338 4 0.047500 0.005916 0.00296 Means Comparisons Dif=Mean[i]-Mean[j] 100 100 0.000000 16 -0.01225 Cont -0.01225 16 0.012250 0.000000 0.000000 Cont 0.012250 0.000000 0.000000 Alpha= 0.05 Comparisons for each pair using Student's t t 2.10980 Abs(Dif)-LSD 100 16 Cont 3Z, DT15 11/16/98 study 100 16 Cont -0.00808 0.004165 0.002348 0.004165 -0.00808 -0.0099 0.002348 -0.0099 -0.01143 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.42255 Abs(Dif)-LSD 100 100 -0.00928 16 0.002967 Cont 0.000881 Positive values show pairs of means that are significantly different. 33 29 Part B. Male rat LW/BW ratio summary LW/BW By DosePFOS(mg/Kg) DT 15 11/16/98 study Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.423397 0.258653 0.009697 0.0512 10 Source Model Error C Total Analysis of Variance DF Sum of Squares 2 0.00048335 7 0.00065825 9 0.00114160 Mean Square 0.000242 0.000094 0.000127 F Ratio 2.5700 Prob>F 0.1456 Means for Oneway Anova Level Number Mean 100 4 0.059500 16 4 0.044250 Cont 2 0.048500 Std Error 0.00485 0.00485 0.00686 Level 100 16 Cont Std Error uses a pooled estimate of error variance Means and Std Deviations Number Mean Std Dev Std Err Mean 4 0.059500 0.006658 0.00333 4 0.044250 0.012447 0.00622 2 0.048500 0.007778 0.00550 Means Comparisons Dif=Mean[i]-Mean[j] 100 100 0.000000 Cont -0.011 16 -0.01525 Cont 0.011000 0.000000 -0.00425 16 0.015250 0.004250 0.000000 Alpha= 0.05 Comparisons for each pair using Student's t t 2.36464 Abs(Dif)-LSD 100 Cont 100 -0.01621 -0.00886 16 -0.00096 3 </ 30 DT15 11/16/98 study Cont 16 -0.00886 -0.00096 -0.02293 -0.01561 -0.01561 -0.01621 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.70602 Abs(Dif)-LSD Cont 100 -0.01173 Cont -0.02624 16 -0.01848 Positive values show pairs of means that are significantly different. 31 35 Part B. Female rat Liver weight summary DT15 11/16/98 study Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.315261 0.119621 1.444065 11.487 10 Source Model Error C Total Analysis of Variance DF Sum of Squares 2 6.720735 7 14.597275 9 21.318010 Mean Square 3.36037 2.08532 2.36867 F Ratio 1.6114 Prob>F 0.2657 Means for Oneway Anova Level Number Mean 100 4 12.4900 16 4 10.8525 Cont 2 10.7500 Std Error 0.7220 0.7220 1.0211 Level 100 16 Cont Std Error uses a pooled estimate of error variance Means and Std Deviations Number Mean Std Dev Std Err Mean 4 12.4900 1.68258 0.84129 4 10.8525 1.39751 0.69875 2 10.7500 0.49497 0.35000 Means Comparisons Dif=Mean[i]-Mean[j] 100 100 0.00000 16 -1.63750 Cont -1.74000 16 1.63750 0.00000 -0.10250 Cont 1.74000 0.10250 0.00000 Alpha= 0.05 Comparisons for each pair using Student's t t 2.36464 Abs(Dif)-LSD 100 16 100 -2.41456 -0.77706 Cont -1.21722 32 3 (a DTI 5 11/16/98 study 16 Cont -0.77706 -1.21722 -2.41456 -2.85472 -2.85472 -3.41470 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.70602 Abs(Dif)-LSD 100 16 Cont Cont -1.64415 -3.28165 -3.90768 Positive values show pairs of means that are significantly different. 33 37 Part B. Female rat LW/BW ratio Summary DT15 11/16/98 study RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.541849 0.410949 0.005991 0.0536 10 Source Model Error C Total Analysis of Variance DF Sum of Squares 2 0.00029715 7 0.00025125 9 0.00054840 Mean Square 0.000149 0.000036 0.000061 F Ratio 4.1394 Prob>F 0.0651 Means for Oneway Anova Level Number Mean 100 4 0.060000 16 4 0.050750 Cont 2 0.046500 Std Error 0.00300 0.00300 0.00424 Level 100 16 Cont Std Error uses a pooled estimate of error variance Means and Std Deviations Number Mean Std Dev Std Err Mean 4 0.060000 0.006325 0.00316 4 0.050750 0.005500 0.00275 2 0.046500 0.006364 0.00450 Means Comparisons Dif=Mean[i]-MeanO] 100 100 0.000000 16 -0.00925 Cont -0.0135 16 0.009250 0.000000 -0.00425 Cont 0.013500 0.004250 0.000000 Alpha= 0.05 Comparisons for each pair using Student's t t 2.36464 Abs(Dif)-LSD 100 16 100 -0.01002 -0.00077 Cont 0.001231 34 DT15 11/16/98 study 16 Cont -0.00077 0.001231 -0.01002 -0.00802 -0.00802 -0.01417 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.70602 Abs(Dif)-LSD Cont 100 -0.00054 16 -0.00979 Cont -0.01621 Positive values show pairs of means that are significantly different. 35 39 DTI5 11/16/98 study Part B. Kidney Weight to Body Weight ratio in male and female rats combined data. SAS analysis. KW/BW By DosePFOS(mg/Kg) DosePFOS(mg/Kg) DTI 5 11/16/98 study Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.007634 -0.10912 0.000618 0.00435 20 Source Model Error C Total Analysis of Variance DF Sum of Squares 2 0.00000005 17 0.0000065 19 0.00000655 Mean Square 2.5e-8 3.824e-7 3.447e-7 F Ratio 0.0654 Prob>F 0.9369 Means for Oneway Anova Level Number Mean 100 8 0.004375 16 8 0.004375 Cont 4 0.004250 Std Error 0.00022 0.00022 0.00031 Level 100 16 Cont Std Error uses a pooled estimate of error variance Means and Std Deviations Number Mean Std Dev Std Err Mean 8 0.004375 0.000518 0.00018 8 0.004375 0.000744 0.00026 4 0.004250 0.000500 0.00025 Means Comparisons Dif=Mean[i]-Mean[j] 100 100 0.000000 16 0.000000 Cont -0.00013 16 0.000000 0.000000 -0.00013 Cont 0.000125 0.000125 0.000000 Alpha= 0.05 Comparisons for each pair using Student's t t 2.10980 Abs(Dif)-LSD 100 16 100 -0.00065 -0.00065 16 -0.00065 -0.00065 Cont -0.00067 -0.00067 Cont -0.00067 -0.00067 -0.00092 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method Idl 2.37772 Abs(Dif)-LSD Cont 100 -0.00078 16 -0.00078 Cont -0.00104 Positive values show pairs of means that are significantly different. Part B. Guinea Pig: Individual Data Animal Gen Dose Species # der PFOS (mg/Kg) 8G00283 F 100 GP Guinea Pig data 2-10-98 BW BW BW BW BW LW LW/ (g) (g) (g) (g) (g) (g) d1 d3 d6 d12 d28 BW ratio 360 ? 317 ? ? 12.9 0.041 KW (g) 1.46 8G00281 8G00282 8G00284 8G00276 8G00277 8G00278 8G00279 8G00275 8G00280 8G00271 8G00272 8G00273 8G00274 8G00266 8G00267 8G00268 8G00269 8G00265 8G00270 F F F F F F F F F M M M M M M M M M M 100 100 100 16 16 16 16 Cont Cont 100 100 100 100 16 16 16 16 Cont Cont GP GP GP GP GP GP GP GP GP GP GP GP GP GP GP GP GP GP GP 383 ? ? ? ? ? ? ? 355 ? ? ? ? ? ? ? 321 ? ? ? ? ? ? ? 332 349 ? ? ? 356 ? 387 ? 360 ? 392 ? ? 410 460 ? 377 423 464 399 ? 447 500 573 330 ? 348 ? ? 365 359 ? ? ? 381 ? 348 ? ? 380 ? 377 ? 397 456 ? ? ?? 14 15.8 20.4 19.9 23.5 12.7 15.9 14.7 19.1 ? 0.04 0.04 0.044 0.043 0.041 0.036 0.044 0.042 0.042 ? ? 1.67 1.85 1.75 2.01 1.56 1.98 1.83 2.13 ? 375 371 ? ?' ? 16.8 0.045 1.84 364 ? 394 ? ? 16.1 0.041 1.74 366 ? 409 461 ? 17.3 0.038 1.84 402 ? 442 495 610 27 0.044 2.7 365 ? 436 511 626 27 0.043 2.65 375 ? 422 ? ? 18.0 0.043 1.98 KW/ BW ratio 0.005 0.004 0.004 0.004 0.004 0.004 0.006 0.005 0.005 0.005 0.004 0.004 0.004 0.004 0.005 Sac Notes day 6 -12% BWi day 6 Dead day 2 Dead day 2 Dead day 2 3 6 13 28 28 6 3 6 13 Dead day 2 3 6 13 28 28 6 Part B. Guinea Pig Liver to Body Weight Ratio Summary Statistics: LW/BWBy DosePFOS(mg/Kg) *3 DT15 11/16/98 study Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.059254 -0.08548 0.002487 0.041687 16 Source Model Error C Total Analysis of Variance DF Sum of Squares 2 0.00000506 13 0.00008038 15 0.00008544 Mean Square 0.000003 0.000006 0.000006 F Ratio 0.4094 Prob>F 0.6723 Means for Oneway Anova Level Number Mean 100 4 0.042250 16 8 0.041875 Cont 4 0.040750 Std Error 0.00124 0.00088 0.00124 Level 100 16 Cont Std Error uses a pooled estimate of error variance Means and Std Deviations Number Mean Std Dev Std Err Mean 4 0.042250 0.001258 0.00063 8 0.041875 0.002475 0.00088 4 0.040750 0.003304 0.00165 Means Comparisons Dif=Mean[i]-Mean] 100 100 0.000000 16 -0.00038 Cont -0.0015 16 0.000375 0.000000 -0.00112 Cont 0.001500 0.001125 0.000000 Alpha= 0.05 Comparisons for each pair using Student's t t 2.16037 Abs(Dif)-LSD 100 16 100 -0.0038 -0.00291 16 -0.00291 -0.00269 Cont -0.0023 -0.00216 Cont -0.0023 -0.00216 -0.0038 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method W 2.46358 Abs(Dif)-LSD 100 100 -0.00433 16 -0.00338 Cont -0.00283 Positive values show pairs of means that are significantly different. 40 DT15 11/16/98 study Part B. Kidney Weight to Body Weight ratio in male and female guinea pigs. SAS analysis KW/BW By DosePFOS(mg/Kg) 41 *5 DT15 11/16/98 study Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.58887 0.520349 0.000443 0.004467 15 Source Model Error C Total Analysis of Variance DF Sum of Squares 2 0.00000338 12 0.00000236 14 0.00000573 Mean Square 0.000002 1.964e-7 4.095e-7 F Ratio 8.5939 Prob>F 0.0048 Means for Oneway Anova Level Number Mean 100 4 0.005250 16 7 0.004143 Cont 4 0.004250 Std Error 0.00022 0.00017 0.00022 Level 100 16 Cont Std Error uses a pooled estimate of error variance Means and Std Deviations Number Mean Std Dev Std Err Mean 4 0.005250 0.000500 0.00025 7 0.004143 0.000378 0.00014 4 0.004250 0.000500 0.00025 Means Comparisons Dif=Mean(i]-Mean(j] 100 100 0.000000 Cont -0.001 16 -0.00111 Cont 0.001000 0.000000 -0.00011 16 0.001107 0.000107 0.000000 Alpha= 0.05 Comparisons for each pair using Student's t t 2.17882 Abs(Dif)-LSD 100 Cont 100 -0.00068 0.000317 Cont 0.000317 -0.00068 16 0.000502 -0.0005 16 0.000502 -0.0005 -0.00052 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.49055 Abs(Dif)-LSO 100 Cont 16 100 -0.00078 0.000219 0.000415 Positive values show pairs of means that are significantly different. DT15 11/16/98 study Appendix IV. Part C Oral dosing with N-EtFOSE in rats. Individual data. Animal# Gende Dose N-EtFOSE Specie BW BW LW( LW/B r (mg/Kg/day) for two s (g) (g) g) W days. d1 d3 Sacrifice day post dose Necropsy Date 8R0217 9 8R0246 6 8R0246 7 M M M Control Control Control Rat 384.6 388.7 17.3 0.045 Rat 416.3 420.4 20.1 0.048 Rat 453.7 461.2 21.9 0.047 3 9/18/98 3 9/18/98 3 9/18/98 8R0217 6 8R0217 7 8R0217 8 M M M 20 Rat 20 Rat 20 Rat 523.7 539 30.2 0.056 485.6 488.4 23.8 0.049 497.3 503 23.8 0.047 3 9/18/98 3 9/18/98 3 9/18/98 Note, 20 mg/Kg is approximately 300 ppm in the diet. Rats were dosed on two consecutive days, 9/16/1998, and 9/17/1998 with 20 mg/Kg/day NEtFOSE, then sacrificed on 9/18/1998. 43 V7 DTI 5 11/16/98 study References Berthiaume, J., and Wallace, K. B. (2002). Perfluorooctanoate, Perfluorooctane Sulfonate, and N-ethyl-perfluorooctanesulfonamido ethanol; Peroxisome Proliferation and Mitochondrial Biogenenesis. Toxicology Letters 129, 23 - 32. Dean, W. P., Jessup, D. C., Thompson, G., Romig, G., and D, P. (1978). Fluorad Fluorochemical Surfactant FC-95 acute oral toxicity (LD50) study in rats. International Research and Development Corporation, Mattawan, MI. Gabriel, K. L. (1976). Acute oral toxicity in rats o f T-13891. Biosearch, Inc., Philadelphia, PA. Goldenthal, E. I., Jessup, D. C., Geil, R. G., and Jefferson, N. D. (1979). Metabolism study with FC-95 in rats. International Research and Development Corporation, Mattawan, MI. Luebker, D. J., Hansen, K. J., Bass, N. M., Butenhoff, J. L., and Seacat, A. M. (2002). Interactions of fluorochemicals with rat liver fatty acid-binding protein. Toxicology 176, 175-85. Maloney, E. K., and Waxman, D. J. (1999). trans-Activation o f PPARalpha and PPARgamma by structurally diverse environmental chemicals. Toxicol Appl Pharmacol 161,209-18. Nabbefeld, D. (1998). Investigation o f the Effects of Fluorocarbons on Liver Fatty AcidBinding Protein. In Environmental Health, p. 50. University o f Minnesota, Minneapolis. Sohlenius, A. K., Andersson, K., Bergstrand, A., Spydevold, O., and De Pierre, J. W. (1994). Effects of perfluorooctanoic acid--a potent peroxisome proliferator in rat-on Morris hepatoma 7800C1 cells, a rat cell line. Biochim BiophysActa 1213, 63-74. Sohlenius, A. K., Eriksson, A. M., Hogstrom, C., Kimland, M., and DePierre, J. W. (1993). Perfluorooctane sulfonic acid is a potent inducer o f peroxisomal fatty acid betaoxidation and other activities known to be affected by peroxisome proliferators in mouse liver. Pharmacol Toxicol 72, 90-3. Wallace, K. B., Luebker, D. J., Butenhoff, J. L., and Seacat, A. M. (2001). Perfluorooctane sulfonate and 2-(N-ethylperfluorooctanesulfonamido)-ethyl alcohol are peroxisome proliferators in rats, but not guinea pigs. Toxicologist 60, 348 Abstract ID: 1657. Yang, Q., Xie, Y., Alexson, S. E., Nelson, B. D., and DePierre, J. W. (2002). Involvement o f the peroxisome proliferator-activated receptor alpha in the immunomodulation caused by peroxisome proliferators in mice. Biochem Pharmacol 63, 1893-900. 44