Document e7bmrz547ZKm1RXMBLk7Bjnk9

SRPT /^ Z - 3 4 Studv No. T-6295.30: ST69 Oxygen Saturation and pQ2 Levels in Rat Pups Borne to PFOS Exposed Dams 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., Advanced Research Toxicologist 3M Medical Dept. / Corporate Toxicology 3M Center, Building 220-2E-02 Saint Paul, MN 55144 Ph: 651-737-1374 FAX: 651-733-1773 Study Toxicologist: Andrew M. Seacat, Ph.D., DABT, Toxicology Specialist 3M Medical Dept. / Corporate Toxicology 3M Center, Building 220-2E-02 Saint Paul, MN 55144 Ph.: 651-575-3161 FAX: 651-733-1773 Sponsored by 3M Specialty Chemicals Division 3M Center Bldg 236 St. Paul, MN 55144 Page 1 o f 15 T-6295.30; ST69 Final Report Study Objective This study was the first of a series of exploratory pilot studies designed to investigate the mechanism of perinatal mortality in rats and mice borne to timed-pregnant dams dosed with PFOS during gestation. The hypothesis that PFOS interferes with normal respiration and/or gas exchange in the pups was investigated. In addition, amniotic fluid was analyzed for PFOS levels and specimens were gathered for possible future analysis (i.e., to investigate the ability of PFOS to interfere with the glucose metabolism). Method Summary This study was performed in the 3M Strategic Toxicology Laboratory under a defined protocol (1) and classified as a "Class B Study" as explained in TOX SOP 0950, Strategic Toxicology Lab GLP Program Procedure (2). All in-life procedures were approved by the 3M Laboratory Animal Review Committee (3M LARC) and are detailed in animal usage application 2001-0182 (3). Under the initial study protocol (1), dams were dosed on days 14-17 of gestation to reach of cumulative dose of 100 mg/kg. The intent was to allow these animals to litter, and to then sacrifice both dams and pups within 24 hours of birth. This dosing regime, however, resulted in very high rates of stillborn pups and early deliveries, greatly complicating the study and preventing the endpoints of interest (oxygen saturation and partial oxygen pressure in blood (p02)) from being examined. In response, the study protocol was amended and a second study as performed. As discussed in the protocol amendment (4), it is thought that an excessive dose of PFOS may have been delivered to the pups and/or that dosing period may have been too late in gestation. Under the amended study protocol, animals were treated via oral gavage with 25 mg PFOS per kg body weight per day for 3 consecutive days (days 12-14 of gestation) to reach a cumulative dose of 75 mg/kg. All animals, including dams, were euthanized via decapitation just prior to delivery. Pups were removed via caesarean-section (c-section), to standardize the time of data collection, and manually stimulated to breath prior to decapitation under a heat lamp set to maintain a temperature of ~ 95 to 98 degrees F. Blood and amniotic fluid (pooled from multiple amniotic sacs using a needle and syringe prior to removing each pup) were collected from each dam for PFOS analysis (see Appendix 1 for details on PFOS analytical method). Liver was also collected from each c-sectioned dam and flash frozen in liquid nitrogen for possible future analysis. Pups were decapitated within 5 minutes being removed from the placental sac. Immediately following decapitation of the pups, blood was drained onto a heparinized weigh boat and pipetted into an I-Stat cartridge for determination of p02 and other blood gases by the I-Stat method (described in the study protocol, 1). No oxygen saturation measurements using the pulse oximeter were obtained. One additional dam was received from the vendor. It was dosed with PFOS as described above and allowed to litter. This dam and her litter were then monitored to assess viability after birth using this study design. Page 2 of 15 T-6295.30; ST69 Final Report Results Initial Study In-life observations from the initial study can be found in Table 1. Four control animals and six PFOS treated animals were included in this study. One control and one treated animal were not pregnant. Pregnant control animals gained an average of 33.0 7.8 grams from day 14-17 of gestation while treated pregnant animals gained only an average of 8.6 12.2 grams. All control animals littered on day 21 of gestation and all treated animals littered on either day 20 or 21 of gestation. Control litters appeared normal while treated litters were either borne dead or died shortly after birth (within approximately 10 minutes). Liver and sera were gathered from some dams and pups and flash frozen for possible future analysis. An attempt was made to measure oxygen saturation using a pulse oximeter, and p02 levels with an I-Stat blood gas analyzer on those pups borne alive. No useful data was gathered for either parameter. It was decided that the pulse oximeter method was not suitable for use on such small pups and that future blood gas analysis would be limited to the I-Stat method. ' Amended Study In-life observations from the amended study can be found in Table 2. Three control animals and 4 PFOS treated animals were included in this study. All animals were pregnant. Control animals gained an average of 99.5 0.7 grams from day 12-21 of gestation while treated animals gained only an average of 74.8 11.4 grams. Dams were palpitated on gestation day (GD) 19 by Dr. DeWayne Walker (3M Lab Animal Veterinarian) and pups were determined to be too immature to survive if delivered. One control animal was decapitated and c-sectioned on GD20 and pups (n=12) were unable to breath and survive. C-sectioning of the remaining dams was thus postponed on the recommendation of Dr. Walker until early morning (5:30 - 6:30am) on GD21. The remaining 2 control animals were decapitated and c-sectioned on GD21, and litters of 10 and 11 pups were delivered, all appearing normal. Three of the treated dams were also decapitated and c-sectioned and litters of 10-13 pups were delivered. Blood was evident in the uterine horns of each of the PFOS treated dams. Treated pups had difficulty breathing and some died within minutes after birth. It was determined that these treated pups were not as mature as the control pups. The remaining PFOS dam was allowed to litter. She delivered 13 pups (10 on GD22 am, 3 on GD23 am), all borne dead or dying within minutes of birth. I Stat data from the pups can be found in Table 3. The only parameter that significantly different between control and PFOS treated groups (p<0.05) was pH, with controls averaging 7.14 0.08 (N=2) and PFOS treated averaging 7.07 0.03 (N=8). No significant difference in p02 levels (mmHg) or 02 saturation (%) was found between treated and control pups. Control p02 levels averaged 30 4 mmHg while levels in PFOS treated pups averaged 35 6 mmHg. Oxygen saturation averaged 39 1 % in controls and 44 13 % in PFOS treated pups. Systemic p02 represents the 02 levels at the exact time the blood is collected and is readily altered by a few breaths (5). Literature values for p02 from control pups delivered from c-sectioned decapitated dams are ~ 60 mmHg when taken within 30 seconds of birth and after at least one-breath (5). This is equivalent to vaginally bom pup P02 levels (5). The p02 levels found in the current study are substantially lower than those cited in the literature. This may be a result of the Page 3 o f 15 T-6295.30; ST69 Final Report increased time the pups were allowed to breath on their own prior to decapitation (5 minutes as opposed to 30 seconds). Dam sera and amniotic fluid PFOS levels can be found in Table 4. Control sera and amniotic fluid PFOS levels averaged 0.017 0.003 ug/ml and 0.011 0.003 ug/ml respectively. The ratio of sera to amniotic fluid PFOS levels for controls averaged 1.7:1. PFOS treated sera and amniotic fluid PFOS levels averaged 68.3 15.0 ug/ml and 16.4 1.2 ug/ml respectively. The ratio of sera to amniotic fluid PFOS levels for the PFOS treated dams averaged 4.2:1. Conclusions The low numbers of animals used in this exploratory pilot study prevents definitive conclusions from being drawn. The results of the I Stat analysis suggest, however, that PFOS does not interfere with normal respiration and/or gas exchange in neonatal rat pups borne to PFOS exposed dams. Results of the dam sera and amniotic fluid PFOS analysis suggest that PFOS distributes more readily into the sera than into the amniotic fluid (approximately 4:1 in PFOS treated animals). PFOS was, however, detectable in the amniotic fluid of all animals tested. This shows that neonatal rat pups borne to PFOS treated dams are exposed to PFOS in utero via the amniotic fluid. The dose used in the amended study, 25 mg/kg/day on days 12-14 of gestation (cumulative dose of 75 mg/kg/day) produced death in live borne pups within minutes of birth. A dose producing death within the first 6 - 2 4 hours of live birth is desired to allow time for data collection and observation. It is also thought that a single dose may produce the same effect as multiple daily doses. Thus, a dose response study will be performed to determine if the dosing regime can be limited to a single administration and to find a dose level that will extend life to 6-24 hours following birth. Page 4 o f 15 List of Tables Table 1: Initial Study In-Life Data Table 2: Amended Study In-Life Data ' Table 3: Amended Study I-StatData (Pups) Table 4: Amended Study Maternal Sera and Amniotic Fluid PFOS Levels T-6295.30; ST69 Final Report Page 5 of 15 T-6295.30; ST69 Final Report Table 1: Initial Study In-Life Data Dose (GDs 14-17) Body Weight (g) Change Animal # c1R05311 c1R05312 c1R05316 c105317 (mg/kg/day) Cumulative 0 0 0 0 GD14 GD15 GD16 GD17 GD18 GD 14-17 Litter day 266 272 281 295 NA 29 GD21 322 331 343 364 NA 42 GD21 280 287 295 308 324 261 255 258 263 266 28 GD21 2 NA 1R05313 25 100 296 304 307 306 NA 10 GD20 1R05314 25 100 315 322 330 339 NA 1R05315 1R05318 1R05319 1R05320 25 100 318 320 318 310 NA 25 10C 314 314 325 316 307 25 100 257 249 235 222 213 25 100 27 288 293 294 278 Gestation Day (GD) Not Applicable or Not Available (NA) 24 GD20 -8 GD20 2 GD21 -35 NA 15 GD21 Comments (am) no count, all normal (am) no count, all normal am) 13 pups all normal Not pregnant (pm) no count, pups borne alive but dying within 10 minutes (pm) started to litter, performed C-Sect., all pups dead 9 pups in am, 1 in pm, all borne dead (am) 2 pups, borne dead Not pregnant (am) 1 pup, borne dead Page 6 of 15 T-6295.30; ST69 Final Report Table 2: Amended Study In-Life Data Dose (GDs 12-14) Body We ght(g C- Number Cumulative Change Litter section pups/ Animal # (mg/kg/day) (mg/kg) GD12 GD13 GD14 GD19 GD20 GD21 GD 12-21 day day litter comments 1R06028 1R06029 1ft06030 pups not mature enough to 0 0 241 248 259 320 329 NA NA NA GD20 12 breath & survive 0 0 280 290 293 350 363 379 99 NA GD21 10 normal 0 c 287 294 296 352 371 387 100 NA GD21 11 normal not as mature as controls, difficulty breathing, some dying before we could decipatate 1R06031 them (within minutes); blood in 25 75 255 269 256 311 330 345 90 NA GD21 13 uterine horn not as mature as controls, difficulty breathing, some dying before we could decipatate 1R06032 them (within minutes); blood in 25 75 255 262 254 302 319 332 77 NA GD21 11 uterine horn not as mature as controls, difficulty breathing, some dying 1R06033 before we could decipatate them (within minutes); blood in 25 75 216 227 219 251 266 281 65 NA GD21 10 uterine horn 10'on GD22 (am),"3 6n GD23 1R06034 25 Gestation Day (GD) 75 232 232 220 268 291 299 GD22 67 & 23 NA (am), all borne dead or died shortly after birth (within 13 minutes) Not Applicable or Not Available (NA) Page 7 of 15 T-6295.30; ST69 Final Report Table 3: Amended Study 1Stat Data (Puds) cumulative maternal Animal # Dose dose (dam) Group (mg/kg) p C02 p 02 BE HC03(m TC02 02 sat Na K ICa++ Hct Hgb pup#* pH (mmHg) (mmHg) (mmol/L) mol/L) (mmol/L) (%) (mmol/L) (mmol/L) (mmol/L) (%PCV) (<j/dL) 1R06030 Control 1R06030 Control 0 1-5 7.08 80.0 0 6-10 7.20 46.1 32 27 -8 23 -9 18 26 38 19 39 131 130 9 0.74 39 13 >9 0.33 29 10 Control Averaqe Control St Dev 7.14 63.1 0.08 24.0 30 4 -9 21 14 23 39 51 131 1 9 0.54 34 12 0 0.29 72 1-5 7.07 76.5 33 -8 22 25 41 128 >9 0.75 35 12 6-8 6.99 76.1 24 -13 18 21 20 128 8 0.32 34 12 1R06031 PFOS 75 9-11 7.05 63.7 38 -13 17 19 49 129 >9 0.74 37 13 1-5 7.10 70.7 34 -7 22 24 44 133 7 0.30 28 10 1R06032 PFOS 75 6-10 7.09 51.3 36 -13 16 17 49 125 8 0.37 29 10 1-5 7.08 67.7 29 -9 20 22 33 121 9 0.31 20 7 6-8 7.09 49.3 41 -14 15 17 58 124 >9 0.39 35 12 1R06033 PFOS 75 9-11 7.07 40.5 42 -16 12 13 58 122 8 NA 18 6 PFOS Average 7.07 62.0 35 -12 18 20 44 126 8 0.45 30 10 PFOS St Dev 0.03 13.4 6 33 4 13 4 1 0.20 73 T-Test control/treated 0.04 0.47 0.15 0.11 0.17 0.21 0.29 0.09 0.12 0.33 0.23 0.27 * Blood samples pooled from multiple pups per litter for each reading. ; Not available: NA ' Page 8 of 15 Table 4: Amended Stud) Maternal Sera and Amniotic Fluid PFOS Levels serum Cumulative PFOS Animal # Dose Group Dose (mg/kg) (ug/ml) Amniotic Fluid sera:amniotic PFOS (ug/ml) fluid PFOS ratio 1R06028 control 1R06029 control R06030 control 0 NA 0 0.019 0 0.015 NA 0.009 0.013 NA 2.1:1 1.2:1 1R06031 1R06032 1R06033 1R06034 Control Average Control Std Dev PFOS PFOS PFOS PFOS 0.017 0.003 75 56.0 75 64.0 75 85.0 75 NA 0.011 1.65:1 0.003 0.64 16.0 3.5:1 17.7 3.6:1 15.5 5.5:1 NA ---------------------- m PFOS Average PFOS Std Dev Not Availa Die (NA) 68.3 16.4 15.0 1.2 4.2:1 1.13 T-6295.30; ST69 Final Report Page 9 of 15 Signatures: Prepared By: T-6295.30; ST69 Final Report i/\ Deanna Luebker, MS Advanced Research Toxicologist Study Director fotQCLL Date 7W Andrew Seacat, Ph.D. Toxicology Specialist Study Toxicologist ________/ / 2 . /o'U Date Reviewed By: John Butenhoff, Ph.D. Corporate Toxicology Management // s Date Page 10 o f1 5 T-6295.30; ST69 Final Report References 1. 3M Medical Department, Corporate Toxicology Protocol for Study No. T-6295.30 DT-69 Oxygen Saturation and p02 Levels in Rat Pups Borne to PFOS Exposed Dams, 2001. 2. 3M Medical Department, Corporate Toxicology Strategic Toxicology Laboratory Standard Operating Procedure (TOX SOP) No. 0950 - GLP Program Procedure, 1999. 3. 3M Lab Animal Review Committee Animal Usage Application No. 2001-0182. MECHANISTIC RESEARCH IN TIMED PREGNANT RATS AND MICE AMENDMENT TO AUA # 2001-0182, 2001. 4. 3M Medical Department, Corporate Toxicology Amendment #1 for Study No. T-6295.30 DT-69 Oxygen Saturation and p02 Levels in Rat Pups Borne to PFOS Exposed Dams, 2001. 5. Vaillancourt C., Berger N., and Boksa P. (1999) Effects of vaginal birth versus Caesarean section birth with general anesthesia on blood gasses and brain energy metabolism in neonatal rats. Experimental Neurology 160:142-150. Page 11 of 15 Appendix 1 3M CorporateToxicology Analytical Laboratory Bldg 236 T-6295.30; ST69 Final Report To: From: Compound: Date Deanna Luebker Andrew Seacat David J. Ehresman Oxygen Saturation and p 02 Levels in Rat Pups Borne to PFOS Exposed Dams 3M Medical Department Study Number T-6295.30 3M Strategic Toxicology Lab Folder Number DT-69 December 10,2001 Pregnant female rats were exposed to PFOS to evaluate the pup fetal lung maturity in an attempt to determine rat pup oxygen levels at birth. Part of this study involved determining the concentration of PFOS in the female rat serum and in the amniotic fluid post exposure to correlate with the Oxygen Saturation levels of the exposed and control pups. Samples of the rat serum and amniotic fluid from both exposed and control animals were deliver to the Strategic Toxicology Laboratory for analysis by LC-MS. The amniotic fluid samples were collected at the time of Caesarean section delivery for both the control and dosed animals. Control animals were pregnant females rats with the same approximate gestational status. Control amniotic fluid and serum were collected from them for analysis. Analytical Method: PFOS levels were determined from both the rat serum and amniotic fluid using a single extraction in ethyl acetate. The pH of the tested solution (100 uL; serum or amniotic fluid) was adjusted to an approximate pH of 3.0 with the addition of 400 uL of a 0.2 N formic acid buffer solution (pH = 3.0). Internal standard (PFHS) was added prior to the extraction and the mixture was shaken on a mechanical shaker for 1 hour. The tubes were then centrifuged at 3000 rpm's for 5 minutes and the ethyl acetate transferred to a clean polypropylene tube. The ethyl acetate was dried to dryness using a gentle stream of nitrogen gas on the N-Evap drier. The resultant residue was re-dissolved in 250 uL of 25% acetonitrile and 75% 10 mm ammonium acetate buffer. This mixture was then filtered through a 0.2 micron syringe filter and placed in a polypropylene injection vial. 25 uL was injected on to the LC-MS system. Page 12 of 15 Appendix 1 T-6295.30; ST69 Final Report The liquid chromatograph used was the Finnigan MAT Spectra system P4000. The column used was a Keystone Fluorophase (150 x 2.1 mm) speciality column featuring a selective, fluorinated, branched-chain alkyl packing. The flow rate was maintained at a constant 0.4 ml/min. A gradient flow of acetonitrile was used to elute the compounds of interest starting with 15% acetonitrile and ending with a concentration of 70 % acetonitrile over a six and one half minute gradient program. There was a constant methanol concentration of 10% methanol with the balance of the mobile phase being 10 mm ammonium acetate buffer. The initial buffer concentrations were returned to starting 6.5 minutes into the run and reached at 8.0 minutes. Column equilibration period was then 2.5 additional minutes before the next injection for a cycle time of 10.5 minutes per injection. All compounds of interest eluted within the first 5 minutes of the gradient run. The Finnigan TSQ system was operated in the MS/MS mode using the Electro Spray source in the negative ionization mode with a constant source potential of 3.0 kV applied. The reactant gas in the Q2 region of the triple quad system was argon. The method involved the transition of the following mass ions: Compound Base Peak (01 mass) MS/MS Ion 02 Offset Voltage Int. Std. (PFHS) 399.0 -- 80 60 volts PFOS 499.0 80 60 volts The capillary heater was held at a constant 300 degrees C. All quantitative calculations were based on the MS/MS ion ratios between the compound of interest (PFOS) and the Internal Standard (PFHS). Study Results: Animal Number Status 6029 Control 6030 Control 6031 6032 6033 Dosed Animal Dosed Animal Dosed Animal Serum PFOS Level 19 ng/mL 15 ng/mL 56.0 ug/mL 64.0 ug/mL 85.0 ug/mL Amniotic Fluid Level 9 ng/mL 13 ng/mL 16.0 ug/mL 17.7 ug/mL 15.5 ug/mL Due to the high concentration of PFOS in the serum a 1:500 dilution was required to bring the concentration down to be within the quantitation limits of the established standard curve for the dosed animals. The dosed animal amniotic fluid was diluted 1:100 before the concentration was within the acceptable curve limitations. The routine standard curves were established using 6 points between 10 ng/mL and 500 ng/mL. Page 13 of 15 Appendix 1 T-6295.30; ST69 Final Report The R squared on the curve used for the calculated results was 0.9926 for the straight serum and amniotic fluid results run on the control animals and the 1:100 dilution of the dosed animal amniotic fluid extracts. The 1:500 dilution of the dosed rat serum curve had an R squared value of 0.9997. Straight serum, diluted serum, and control dams amniotic fluid standard curve graph from original Finnigan Xcalibur program. Page 14 of 15 Appendix 1 T-6295.30; ST69 Final Report jIM1 '! I lh id u! - i v il i S ' . w J p - j M atjnryCft Lune M eturM36 L in g MatLituO? . i L r - A L*>g MeturtjCB Luno MaturivOS 2 : r'fTi S:c . 50 ng/m L Std tO O n ^ m L Std 250n Q /m L S tC 500 n o /n i. Std ^ 3*3.*. f^fas^-v.---- . J i n k _j : 0420S 149808 229373 4B7404 748888 STB f 'S p ^ S ^ k ^ m K' s i Z ; r. *44477 *46174 482161 *69421 ::c c c 50 000 100.000 250 000 500 000 2 4 .3 :1 . 51.323 , 96.98* 24S335 500 632 _> r h* ; 385 3 1.14 4 -0.27 5 01 4 ResoonteHxi 6 4 r\-iT .I'K lI `it-.ii'.| ` f ` * * * * i, i M k KT u *M 'Ij-'T , n r r i. r /rrL ng/rrv. no/m l 4 6` 465 465 458 452 ^ e t 3 ^CtT 3Jy,itS".X-r;'v'2 -\v '';Vt,i"^- '" ^ ^ ^^ ^ ^ ^ (3 ,> ' ' - r - j " . - - i - w w * . - "t c i j w - -V-, r . r-F v-->v - 7.Vf\ *7 *"~ ^ jz~ " - v" ,-*- ;'. ' * 3 ^ ^ * -v -m -i - V-.. * ;i2/2lMiin-SJsoS-UT This graph represents the standard curve used with the 1:500 diluted specimens. Note: This curve was based on 5 points starting at 25 ng/mL through 500 ng/mL. The 10 standard was dropped on this run due to low detector response. The raw data and printed reports are contained in labeled 3 ring binders in the Bldg. 236 Strategic Toxicology LC-MS Laboratory. All original methods and raw data are stored as files that are backed up to Zip discs from the Finnigan Xcalibur system. These files would need to be re-loaded to a compatible Finnigan Xcalibur system to function properly. Page 15 of 15 PCTL f r ? 3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY Protocol for Study No. T-6295JO DT-69 Oxygen Saturation and pQ 2 Levels in Rat P ups Borne to PFOS Exposed Dams Study Objective: This study is the first of a series designed to investigate the mechanism of perinatal mortality in rats and mice borne to timed-pregnant dams dosed with PFOS during gestation. The hypothesis that will be tested in this study is that PFOS interferes with normal respiration and/or gas exchange in the pups. In addition, specimens will be gathered to investigate the ability of PFOS to interfere with the glucose metabolism. Research Client: 3M Specialty Chemicals Division 3M Center, Building 236 Saint Paul MN 55133-3220 Sponsor: 3M Specialty Chemicals Division 3M Center, Building 236 Saint Paul MN 55133-3220 Study Location: 3M Strategic Toxicology Laboratory 3M Center, Building 270-SB314 Saint Paul, MN 55144-1000 Study Director: - Deanna J. Luebker, M.S. Advanced Research Toxicologist 3M Medical Dept. / Corporate Toxicology 3M Center Building 220-2E-02 Saint Paul, MN 55144-1000 Ph.: 651-737-1374, FAX: 651-733-1773 Study Toxicologist: Andrew M. Seacat Ph.D. Toxicology Specialist 3M Medical Dept. / Corporate Toxicology 3M Center Building 220-2E-02 Saint Paul, MN 55144-1000 Ph.: 651-575-3161, FAX: 651-733-1773 Proposed Study Timeline: In-Life S tart Date: September 24,2001 In-Life End Date: October 2, 2001 Regulatory Compliance: This study will be performed in the 3M Strategic Toxicology Laboratory in the "spirit of GLP". A defined protocol will be in place and the study will be classified as "Class B" as explained in TOX SOP 0905, Strategic Toxicology Lab GLP Program Procedure. All in- Page 1 o f 5 T-6295.30; ST69 dosing and 5 ml dosing solution / kg body weight will be administered to each rat in the PFOS treatment group. Control rats will receive 5ml/kg 2% Tween 80 on days 16-19 of gestation. TABLE 1: Dosing Regime Dose Groups N Dosing days A. Control (2% Tween 80) B. PFOS treatment (25 mg/kg/day PFOS) 4 A.1: 9 /2 4 -9/27/01 A.2: 9 /2 5 -9 /2 8 /0 1 4 B .l: 9/24 - 9/27/01 B.2: 9 /2 5 -9 /2 8 /0 1 Cumulative Dose (mg/kg) 0 lOOmg/kg Sacrifice dates A l: N = 2 on Monday 10/1/01 A 2: N = 2 on Tuesday 10/2/01 B .l: N = 2 on Monday 10/1/01 B.2: N = 2 on Tuesday 10/2/01 Observation o fAnimals: . Clinical Observations: Each animal will be observed daily (excluding weekends) for mortality and morbidity and notable findings will be recorded. Body Weights: Dams will be weighed immediately prior to each dosing. Terminal body weights will be recorded on dams and litters. Sample Collection: Number & Frequency Animals will be sacrificed within 24 hours of delivery. Dams will be euthanized via C 02 asphyxiation and pups via decapitation. Animals have been ordered such that four will litter Sunday night, September 30th. The remaining four will litter"'* Monday night, October 1st. The animals that litter/are borne on Sunday night will be sacrificed on Monday and the animals that litter/are borne Monday night will be sacrificed on Tuesday, within 24 hour of their birth. It is estimated that 10 to 14 pups will be bom per litter. It is presumed that the dams will cannibalize pups that are stillborn or that die during the night. Eight of the remaining pups (if available) per litter will be used in the study. Three of the eight pups from each litter will be identified (marked) as time-course pups. The oxygen saturation in these pups will be monitored at several recorded time points throughout the day using the oximeter method (described below) to determine changes in oxygen saturation over time. The other pups on the study will be monitored for oxygen saturation by the oximeter method, then immediately sacrificed to obtain blood for partial oxygen pressure in blood (p02) determination by the I-Stat method (described below). Near the end of the study, the time-course pups will be sacrificed to obtain blood for p02 determination by the I-Stat method. Any remaining pups may be sacrificed to obtain serum and tissues for possible future analysis. Method o f CoUection/Measurement Oxygen saturation will be measured with a pulse oximeter (Ohmeda), and p02 will be measured with an I-stat blood gas analyzer (Abbot Labs). Oxygen saturation and pulse rate will be measured on each pup using an Ohmeda Biox 3740 Pulse Page 3 o f 5 Signatures: /Jha Deanna J. Luebker, M.S. Advanced Research Toxicologist Study Director Andrew M. Seacat, Ph.D. Toxicology Specialist Study Toxicologist T-6295.30; ST69 jlIqQJ.-- Date Date Page 5 of 5 " ' PCTL 10/29/01 T-6295.30; DT-69 Amendment #1 3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY Amendment #1 for Study No. T-6295.30 DT-69 Oxygen Saturation and pQ2 Levels in Rat Pups Borne to PFOS Exposed Dams Sponsor: Study Location: Study Director: 3M Specialty Chemicals Division 3M Strategic Alternative Toxicology Laboratory Deanna J. Luebker, M.S. Background: The initial study performed under this protocol resulted in very high rates o f stillborn pups and early deliveries. This greatly complicated the study, preventing the endpoints o f interest from being examined. It is believe that these complications were a result o f a dose being delivered to the pups that was in excess of what was necessary to achieve the desired decrease in pup viability. As was stated in the original study protocol, "studies conducted by the EPA NHEERL have demonstrated that a cumulative dose of roughly 100 mg/kg will reduce pup viability by approximately 50% within the first 5 days of lactation". In the EPA studies, however, Tween 20 was used for a vehicle rather than Tween 80, as has been used in the majority o f PFOS oral dosing studies. A greater amount of PFOS is presumed to be absorbed when using Tween 80 as compared to Tween 20. Thus, we believe a cumulative dose of 100 mg/kg with Tween 80 is in excess o f what is necessary in these studies. In addition, the EPA has demonstrated that the later in gestation dosing occurs, the greater the rate of pup mortality. The period of dosing used the under the original study protocol, days 14-17 of gestation, may have been too late to administer such a high dose of PFOS. Objective: The purpose of this amendment is to adjust the dosing regime used under the original protocol to prevent in utero death and early deliveries. In addition, amniotic fluid will be collected and analyzed for PFOS and caesarean-sections (c-sections) will be performed rather than allowing the dams to deliver. The purpose o f the c-sections is to standardize the time o f data collection. A follow-up study will thus be performed under the original study protocol with the following modifications: 1. Page 1. Study Objective: In addition to the originally stated objective, amniotic fluid will be collected from c-sectioned rats to measure PFOS levels. 2. Page 1. Proposed Study Timeline: The in-life start date for the follow-up study will be November 7,2001 and the in life end date will be November 14, 2001. 3. Page 2. Animal Number: Six animals will be used in the follow-up study. 4. Page 2-3. Dose and Dosing Procedures: Page 1 o f 3 10/29/01 T-6295.30; DT-69 Amendment #1 All six animals will arrive at 3M on day 11 of gestation. Three rats will be treated via oral gavage with 25 mg/kg PFOS per kg body weight per day for 3 consecutive days (days 12-14 of gestation) to reach a cumulative dose of 75 mg/kg. PFOS will be prepared as described in the original protocol. The remaining three rats will serve as controls and receive 5ml/kg body weight per day of 2% Tween 80 on days 12-14 of gestation. Any additional rats received from the vendor will be dosed with PFOS as described above and allowed to litter. Pups and dams will be monitored through day 5 of lactation to assess the dosing regime or sacrificed to collect tissues for additional analyses. 5. Page 3. Sample Collections, Number and Frequency All animals, including dams, will be euthanized via decapitation on gestation day 19. Pups will be removed via c-sectioning. In addition to that described in the original protocol, amniotic fluid will be collected from each dam following decapitation. Liver and sera may be collected from some, not all, of the dams and pups. 6. Page 4. Sample Handling Amniotic fluid will temporally be stored at -70C in the Strategic Toxicology Lab, building 270. For PFOS analysis, the amniotic fluid will be shipped on dry ice to Dave Ehersman, building 236. Liver and sera will be handled as described in the original protocol. 7. Page 4. Analysis In addition to the analysis outlined in the original protocol, PFOS level will be compared between PFOS treated and control dose groups and analyzed for statistical differences. Page 2 of 3 Signatures: 10/29/01 T-6295.30; DT-69 Amendment #1 Study Director Andrew M. Seacat, Ph.D. Toxicology Specialist Study Toxicologist ...... Date ` Dan Hakes Sponsor Representative . Date Page 3 o f 3