Document jJyxDK8kbzpjmw2BXbYx51v9

Study No. T-6295.31: ST77 AN INVESTIGATION OF THE MECHANISM OF TOXICITY IN ADULT RATS EXPOSED TO HIGH LEVELS OF PERFLUOROOCTANESULFONATE (PFOS). Study Location: 3M Strategic Toxicology Laboratory Corporate Toxicology 3M Medical Department 3M Center, Building 270-SB-314 St. Paul, MN 55144 Study Director: Deanna J. Luebker, M.S., Senior Toxicologist 3M Medical Dept. / Corporate Toxicology & Regulatory Services 3M Center, Building 220-2E-02 Saint Paul, MN 55144 Ph: 651-737-1374 FAX: 651-733-1773 Laboratory Management: John L. Butenhoff, Ph.D., CIH, DABT, Staff Scientist 3M Medical Dept. / Corporate Toxicology & Regulatory Services 3M Center Building 220-2E-02 - Saint Paul, MN 55144 Ph.: 651-733-1962, FAX: 651-733-1773 Sponsored by 3M Specialty Chemicals Division 3M Center Bldg 236 St. Paul, MN 55144 Page 1 of 14 T-6295.31; ST77 Final Report Study Objective The objective of this study was to investigate the hypothesis that Perfluorooctanesulfonate (PFOS) causes damage to muscle mitochondria, possibly leading to rhabdomyolysis and lactic acidosis at toxic exposure levels. This may explain the relatively rapid onset of toxicity and precipitous decline seen in prior rodent and primate toxicity studies as well as provide insight in to the mechanism by which PFOS decreases viability in the neonatal rat and mouse. Methods Summary This study was performed in the 3M Strategic Toxicology Laboratory under a defined protocol (1, 2) and classified as a "Class B Study" as explained in TOX SOP 0950, Strategic Toxicology Lab GLP Program Procedure (3). All in-life procedures were approved by the 3M Laboratory Animal Review Committee (3M LARC) and are detailed in animal usage application 2001-0182 (4). Details of the study methods can be found in the study protocol and protocol amendment (1, 2). Briefly, adult male rats (12 per dose group) were administered either 400 mg/kg PFOS or vehicle (2% tween 80) via oral gavage on day zero of the study. The dose level of 400 mg/kg was chosen as it has been demonstrated to cause toxicity in previous rodent studies and, because this study was designed to investigate the mechanism of toxicity, a toxic dose was required. In the original study design, 6 rats per dose group were to be euthanized 24-hours post-dose and the remaining 6 rats per dose group were to be euthanized 48-hours post dose. However, a decision was made to humanely euthanize all animals at 24-hours post-dose due to signs of pain and distress (hunching and extreme lethargy) and severe diarrhea in treated animals. All animals were euthanized via C02 asphyxiation. Plasma, liver, and kidneys were collected from each animal. In addition, urine samples from 6 rats per dose group were collected approximated 24-hours after dosing. Plasma lactate/pyruvate ratios were examined as a marker of mitochondrial dysfunction. Urinary myoglobin, plasma myoglobin , and plasma creatine kinase (CK) were examined as markers of muscle damage. Due to limited sample size, a decision was made to eliminate the clinical chemistry (cholesterol, blood urea nitrogen (BUN), and potassium) and PFOS analysis from the original study design. No analysis of liver or kidneys was performed. All results were compared between treated and control animals and analyzed to determine statistical differences using the students T-test. Results As expected, weight loss was observed in all animals, regardless of treatment, following dosing. Treated animals, however, lost significantly more weight than control animals (P < 0.01) and exhibited signs of diarrhea (Table 1 and Figure 1). Liver weight was significantly increased in treated rats (P < 0.01) (Table 1 and Figure 2). All values for plasma and urinary myoglobin were below the detection limit of lug/L and 1 mg/L, respectively (Table 1). Plasma lactate (measured as lactic acid) was significantly lower in treated animals than in control animals (P < 0.01) Page 2 of 14 T-6295.31 ; ST77 Final Report (Table 1 and Figure 3), while no significant difference was observed in plasma pyruvate levels (Table 1 and Figure 4). Due to the decreased plasma lactate levels observed in treated animals, the plasma lactate/pyruvate ratio was significantly lower (P < 0.01) in treated animals (Table 1 and Figure 5). Plasma CK levels were significantly increased in treated animals as compared to control animals (P < 0.01) (Table 1 and Figure 6). Discussion The diagnosis of rhabdomyolysis is made primarily from measurement of circulating CK. A rise in CK is usually observed 2-12 hours after muscle injury with peak concentrations occurring within 1-3 days and a decline after 3-5 days of injury. Serum myoglobin usually increases before a rise in CK and drops more rapidly than does the concentration of CK. Myoglobinuria is also expected with rhabdomyolysis. The role of myoglobin is to store and carry oxygen and thus maintain the ability of the muscles to contract. If the concentration of myoglobin in the urine is high, the urine may take on a red-brown or cola colored appearance (6). In the current study, significant elevations in plasma CK were observed in treated animals. This effect has also been observed in previously performed PFOS and ammonium perfluorooctanoate (APFO) primate studies. Despite this evidence of muscle damage, elevations of urinary or serum myoglobin could not be observed because all measured values in treated and control animals were below the limit of detection. Possible explanations are that, due to limited sample size, it was not possible to accurately measure myoglobin levels or that a rat-specific assay is necessary. The assay used in the current study was not specific for the rat, but developed for use. with human samples. In addition, the sample collection may have occurred after an initial rise and fall in circulating serum myoglobin levels. However, the urine of treated rats in the current study did not take on a red-brown color as would be expected with increased levels of urinary myoglobin. Blood lactate/pyruvate ratios are widely used as a noninvasive test for the detection of mitochondrial toxicity (7). Despite previous observations (see study background in the protocol) suggesting that PFOS may cause mitochondrial toxicity, the lactate/pyruvate ratios measured in this study do not support this hypothesis. In addition, the decreased lactate levels observed in this study do not support the hypothesis that PFOS treatment leads to lactic acidosis. Despite this, it is obvious that irritation/stress occurs in PFOS treated animals that cause elevations in circulating levels of CK. It is possible that the animals in this study had not yet progressed to frank rhabdomyolysis, and that if sampled at a later time point, would have shown increased lactate and lactate/pyruvate ratios as expected. Conclusions The results of this study show that PFOS causes an increase in plasma CK levels prior to overt moribundity. This supports the hypothesis that muscle damage occurs in PFOS treated rats. Because neither urinary nor plasma myoglobin could be detected in control or treated samples, no conclusions can be drawn and further studies are required to investigate these endpoints as well as to explain the increased CK levels observed in this and other PFOS and APFO studies. Future studies should, if possible, utilize a rat-specific assay for urinary and serum myoglobin. Although expected increases in lactate and lactate/pyruvate ratios were not observed, further Page 3 of 14 T-6295.31; ST77 Final Report studies are required to investigate these endpoints and to determine if PFOS causes damage to muscle mitochondria, possibly leading to rhabdomyolysis and lactic acidosis at toxic exposure levels. It is possible that the animals in this study had not yet progressed to frank rhabdomyolysis. It may be more appropriate to investigate these endpoints by dosing at lower levels over a period of days to reach the same cumulative dose; thus allowing for the progression to a state of overt rhabdomyolysis. Page 4 of 14 List of Tables and Figures: Table 1: Raw data Figure 1: Body Wight Change (day 0- day 1) Figure 2: Liver Weight Figure 3: Plasma Lactate Figure 4: Plasma Pyruvate Figure 5: Plasma Lactate/Pyruvate Figure 6: Plasma Creatine Kinase T-6295.31; ST77 Final Report Page 5 of 14 T-6295.31; ST77 Final Report Table 1: Raw Data Animal number 13XX 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 dose group (mg/kg PFOS) 0 0 0 0 0 0 0 0 0 0 0 0 400 400 400 400 400 400 400 400 400 400 400 400 Body Weight day 0 (dosing) 248 240 220 227 233 222 230 253 241 236 236 219 249 242 240 239 242 232 250 220 242 213 246 230 Body Weight day 1 (24 Hr Post Dose) 232 231 203 221 216 207 207 229 218 211 212 199 223 218 213 214 217 210 222 192 215 190 217 206 Body Weight .Change -16 -9 -17 -6 -17 -15 -23 -24 -23 -25 -24 -20 -26 -24 -27 -25 -25 -22 -28 -28 -27 -23 -29 -24 Liver weight (g) 9.5 9.3 8.0 9.3 8.6 8.0 9.4 7.8 9.0 9.0 9.0 8.2 9.7 10.1 9.5 9.4 8.7 10.0 9.2 8.7 9.5 8.7 10.2 8.6 Urinary Myoglobin (mg/L) NA NA NA NA NA NA <1 <1 <1 <1 <1 <1 NA NA NA NA NA NA <1 <1 <1 <1 <1 <1 plasma lactate (mmol/L) 6.1 6.7 6.5 8.1 5.8 5.3 4.3 6.0 7.4 7.7 6.3 NA 5.9 3.7 4.4 3.5 4.3 5.7 1.4 5.3 7.0 6.2 5.5 NA plasma pyruvate (mmol/L) 0.02 0.02 0.02 0.03 0.02 0.01 0.02 0.04 0.02 0.02 0.04 NA 0.02 0.02 0.02 0.04 0.02 0.02 0.02 0.03 0.03 0.02 0.03 NA lactate / pyruvate 305 335 325 270 290 530 215 150 370 385 158 NA 295 185 220 88 215 285 70 177 233 310 183 NA plasma creatine kinase (U/L) 129 110 88 132 100 127 84 102 103 110 86 77 109 NA 154 329 77 391 264 NA 1112 NA NA NA plasma myoglobin (ug/L) <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 NA <1 NA <1 <1 <1 NA <1 NA NA NA Page 6 of 14 0.00 -5.00 - 10.00 -15.00 - 20.00 Figure 1: Body Weight Change (Day 0 - Day 1) Control " N = 12 ` v ' i ' i .. * < V Jh'"' <';y>! '1.' t > >t1t : 4 , > -, V ' :' ; f ' . , ' f, ,S ' > ` <<O < * Treated N = 12 -25.00 -30.00 = statistically significant difference from control p < 0.01 Dose Group T-6295.31; ST77 Final Report grams Page 7 o f 14 Figure 2: Liver Weight T-6295.31; ST77 Final Report grams Page 8 o f 14 Figure 3: Plasma Lactate T-6295.31; ST77 Final Report mmol/L Page 9 of 14 Figure 4: Plasma Pyruvate T-6295.31; ST77 Final Report mmol/L Page 10 o f 14 (mmol/L)/(mmol/L) 450.00 400.00 Figure 5: Plasma Lactate/Pyruvate 350.00 300.00 250.00 5 31 $ I * ** 200.00 150.00 100.00 50.00 4- '~ , < N = 11 0.00 Control = statistically significant difference from control p < 0.01 Dose Group N = 11 Treated T-6295.31; ST77 Final Report Page 11 o f 14 800.00 700.00 Figure 6: Plasma Creatine Kinase ** 600.00 500.00 400.00 300.00 * 200.00 100.00 0.00 - 100.00 -- -- , 1T -, ** - ' f * fr Control N = 12 = statistically significant difference from control p < 0.01 Dose Group N= 7 Trettted T-6295.31; ST77 Final Report Page 12 o f 14 References T-6295.30; ST69 Final Report 1. 3M Medical Department, Corporate Toxicology Protocol for Study No. T-6295.31 ST77. 2. An investigation of the mechanism of toxicity in adult rats exposed to high levels of Perfluorooctanesulfonate (PFOS), 2001. 3. 3M Medical Department, Corporate Toxicology Amendment #1 for Study No. T6295.31 ST-77. An investigation of the mechanism of toxicity in adult rats exposed to high levels of Perfluorooctanesulfonate (PFOS), 2001. 4. 3M Medical Department, Corporate Toxicology Strategic Toxicology Laboratory Standard Operating Procedure (TOX SOP) No. 0950 - GLP Program Procedure, 1999. 5. 3M Lab Animal Review Committee Animal Usage Application No. 2001-0182, MECHANISTIC RESEARCH IN TIMED PREGNANT PREGNANT RATS AND MICE - AMENDMENT TO AUA # 2001-0182, 2001. 6. Omar M.A., Wilson J.P., and Cox T.S. (2001) Rhabdomyolysis and HMG-CoA reducatse inhibitors. Annals of Pharmacotherapy 35, 1096-1107. 7. De Pinieux G., Chariot P., Ammi-Said M., Louam F., Lejonc J.L., Astier A., Jacotot B., and Gherardi R. (1996). Lipid-lowering drugs and mitochondrial function: Effects of HMG-CoA reductase inhibitors on serum ubiquinone and blood lactate/pyruvate ratio. Br J Clin Pharmacol 42: 333-337. Page 13 of 14 Signatures: Prepared By: Deanna Luebker, M6 Study Director Reviewed By: John Butenhoff, Ph.D. DABT, CIH Laboratory Management Dan Hakes Sponsor Representative T-6295.30; ST69 Final Report ( j'-i Date / Date *fA 3/0 7 )ate Page 14 of 14