Document jXLRaDrO3Nk4aa2RMJ5NmwRk

27,2000 One Generation Reproduction Study of PFOS Mevalonic Add/Cholesterol Challenge and NOEL Investigation in Rats Draft Study Outline fS -2- Study Objectives: This study will serve as a follow-up study to Combined Oral (gavagel Fertility. Developmental and Perinatal/Postnatal Reproduction Toxicity Study of PFOS in Rats (Argus Protocol 418-008,3M study number T-6295.9). The objectives of the study are as follows: 1) to determine whether or not co-administration of mevalonic acid or cholesterol can prevent or antagonize the adverse maternal and fetal effects (reduced maternal body weight gain, increased percent stillborn, decreased birth weight and decreased pup survival) observed in the previous study; and 2) to refine the no observed effect level (NOEL). GLP Status: A GLP study with appropriate QA audits; a signed QUA statement to be included in the final report. Animals: Female Harlan CD rats. Dose Groups: Thirteen: Vehicle Control; Mevalonic Acid Control; Cholesterol Control; 0.4 mg/kg PFOS; 0.8 mg/kg PFOS; 1 mg/kg PFOS; 1.2 mg/kg PFOS; 1.6 mg/kg PFOS; 1.6 mg/kg PFOS + mevalonic acid; 1.6 mg/kg PFOS + cholesterol; 2 mg/kg PFOS; 2 mg/kg PFOS + Mevalonic Acid; 2 mg/kg PFOS + cholesterol. Number Per Group: 22-28 in groups 1-3 & 8-13; 16-20 in groups 4-7. TOTAL: 246312 Test Article: Compound will be furnished by the Sponsor who is responsible for compound identity and purity. Reserve sample to be retained. Compound Administration: Females only, daily oral intubation, dose volume 5ml/kg. The vehicle (0.5 % Tween 80 in deionized water) will be prepared weekly. PFOS will be prepared daily as a suspension in deionized water with 0.5 % Tween 80. Mevalonic acid will be prepared fresh twice daily in deionized water. Cholesterol will be prepared fresh daily in olive oil. Compound administration will start 6 weeks prior to mating and continue through day 4 postnatal, (see table 1). 004594 Page 1 of 5 D.J. Luebker April 27, 2000 Table 1 - Dose Groups & Dosing Schedule Dose Groups Dosing schedule # Name 1st dosing 2nd dosing N Vz hr after 1st 1 Vehicle Control 30-40 Deionized H20 0.5 % Tween 80 2 Mevalonic Acid Control 22-28 500 mg/kg Mevalonic Acid 0.5 % Tween 80 3 Cholesterol Control 22-28 *500 mg/kg 0.5% Tween 80 Cholesterol 4 0.4 mg/kg PFOS 16-20 NA 0.4 mg/kg PFOS 5 0.8 mg/kg PFOS 16-20 NA 0.8 mg/kg PFOS 6 1 mg/kg PFOS 16-20 NA 1 mg/kg PFOS 7 1.2 mg/kg PFOS 16-20 NA 1.2 mg/kg PFOS 8 1.6 mg/kg PFOS 22-28 NA 1.6 mg/kg PFOS 9 1.6 mg/kg PFOS + 22-28 Mevalonic Acid 500 mg/kg Mevalonic A dd 1.6 mg/kg PFOS 10 1.6 mg/kg PFOS+ Cholesterol 22-28 *500 mg/kg Cholesterol 1.6 mg/kg PFOS 11 2 mg/kg PFOS 22-28 NA 2 mg/kg PFOS 12 2 mg/kg PFOS + Mevalonic Acid 22-28 500 mg/kg Mevalonic Add 2 mg/kg PFOS 13 2 mg/kg PFOS + Cholesterol 22-28 *500 mg/kg Cholesterol 2 mg/kg PFOS * Cholesterol dosing may occur before or after PFOS or Tween 80 dose. 3rddosing 5 hr after 2"d Deionized H20 500 mg/kg Mevalonic A dd NA NA NA NA NA NA 500 mg/kg Mevalonic A dd NA NA 500 mg/kg Mevalonic A dd NA Samples of dosing preparations will be collected once during the six weeks prior to mating, once during gestation and once during the postnatal period. A portion of each of these samples will be frozen and shipped to the Sponsor for possible analysis. A backup sample of each will be retained by the testing facility. Records will be maintained on how dosing preparations were made. Clinical Observations: Twice daily - approximately one hour after dosing and again 4-6 hours later. Body Weights: Weekly except when females are cohabiting with males during mating. Food Consumption: Weekly except when females are cohabiting with males during mating. Mating: After 6 weeks of compound administration, one male (Argus breeder colony) will be co-inhabited with each female. A female will be determined as mated upon the presence of sperm positive vaginal smear. The day of mating shall be considered day 0 of gestation. Pre-Birth Sacrifice and Examination: Just prior to delivery (morning of day 21 of gestation) 6-8 pregnant females in groups 1-3 and 8-13 will be sacrificed by CO2 asphyxiation and cesarean-section will be performed. A terminal body weight for each dam and a pooled terminal body weight for each litter will be obtained and recorded. Animals will be necropsied and any gross lesions will be described and recorded. 004595 Page 2 of 5 D.J. Luebker April 27, 2000 Serum/plasma and liver samples will be taken from each dam and her pups (pup samples pooled by litter) and milk will be collected from each dam. Liver and serum wiE be analyzed for LDL, HDL, total cholesterol, and triglycerides. Plasma mevalonic acid levels will be measured. Milk wEl be analyzed for total cholesterol Additional Ever and sera samples wEl be frozen (-20C) and shipped on dry ice to Kris J. Hansen, Ph.D., 3M Environmental Technology and Safety Services, 935 Bush Avenue, BuEding 2-3E-09, St. Paul MN 55133-3331 for PFOS analysis. A portion of each Ever sample wiE be flash frozen in Equid nitrogen and shipped on dry ice to the Sponsor for possible future analysis (i.e., Ever adenine nucleotides & HMG-CoA reductase inhibition). Additional Ever tissue wEl be coEected and fixed in gluteraldehyde for possible future analysis by electron microscopy. Female Parturition: Remaining rats wEl be aEowed to deEver their Etters. Pup Examinations: Pups wEl be counted, sexed, weighed and observed for moribundity/mortaEty on postnatal days 0, 1,2, 3 and 4. Rats Found Dead or Moribund: Rats that die or are sacrificed because of moribund condition or abortion wEl be examined for cause of death or moribund condition on the day the observation is made. Aborted fetuses and/or in-utero pups wiE be examined to the extent possible. When possible, the foEowing specimens wEl be coEected and analysis performed: Rats wEl be necropsied and any gross lesions wEl be described and recorded. Serum/plasma and Ever samples will be taken (individually for each dam and her pups pup samples pooled by Etter). Liver and serum wiE be analyzed for LDL, HDL, total cholesterol and triglycerides. Plasma mevalonic acid levels will be measured. Milk wiE be coEected from each dam and analyzed for total cholesterol Additional Ever and sera samples wEl be frozen (-20C) and shipped on dry ice to Kris J. Hansen, Ph.D., 3M Environmental Technology and Safety Services, 935 Bush Avenue, BuEding 2-3E-09, St. Paul MN 55133-3331 for PFOS analysis. A portion of each Ever sample wiE be flash frozen in Equid nitrogen and shipped on dry ice to the Sponsor for possible future analysis (i.e., Ever adenine nucleotides & HMG-CoA reductase inhibition). Additional Ever tissue wiE be coEected and fixed in gluteraldehyde for possible future analysis by electron microscopy. Myocardial tissue wEl be coEected for possible histology and electron microscopy. Day 4 Postnatal Sacrifice and Examination: Sixteen to 20 dams in each dose group and aE of their pups wiE be sacrificed by CO2 asphyxiation on day four postnatal A terminal body weight for each dam and a pooled terminal body weight for each Etter wiE be obtained and recorded. Animals wEl be necropsied and any gross lesions wEl be described and recorded. Serum/plasma and Ever samples wEl be taken from each dam and her pups (pup samples pooled by sex for each Etter) and miEc samples wEl be coEected from each dam. Liver and serum wEl be analyzed for LDL, HDL, total cholesterol and triglycerides. Plasma mevalonic acid levels wiE be measured. Milk wiE be coEected from each dam and analyzed for total cholesterol. Additional Ever and serum samples wiE be frozen (-20C) and shipped on dry ice to Kris J. Hansen, Ph.D,, 004596 Page 3 of 5 D.J. Luebker April 27, 2000 3M Environmental Technology and Safety Services, 935 Bush Avenue, Building 2-3E09, St. Paul, MN 55133-3331 for PFOS analysis. A portion of each liver sample will be flash frozen in liquid nitrogen and shipped on dry ice to the Sponsor for possible future analysis (Le., liver adenine nucleotides & HMG-CoA reductase inhibition). Additional liver tissue will be collected and fixed in gluteraldehyde for possible future analysis by electron microscopy. Myocardial tissue will be collected from each dam and four of her pups (2 males and 2 females) for possible histology and electron microscopy. G rp# Group N am e Table 2 - Scheduled Sacrifices (N dams/group/Sacrifice) 1 2 3 4 5 6 7 8 9 10 11 12 13 0) Pre-Birth s (dy-1) uocs Ifl Dy 4 Postnatal TOTAL N/grp 6-8 16-20 22-28 6-8 16-20 22-28 6-8 16-20 22-28 16-20 16-20 16-20 16-20 16-20 16-20 16-20 16-20 6-8 16-20 22-28 6-8 16-20 22-28 6-8 16-20 22-28 6-8 16-20 22-28 6-8 16-20 22-28 6-8 16-20 22-28 Vehicle control Mevalonic acid control Cholesterol Control 0.4 mg/kg PFOS 0.8 mg/kg PFOS 1 mg/kg PFOS 1.2 mg/kg PFOS 1.6 mg/kg PFOS 1.6 mg/kg PFOS + mevalonic acid 1.6 mg/kg PFOS + ___ cholesterol___ 2 mg/kg PFOS 2 mg/kg PFOS + mevalonic ______ acid 2 mg/kg PFOS + cholesterol 004597 Page 4 of 5 D.J. Luebker Study Director: Deanna J. Luebker, M.S. 3M Corporate Toxicology 3M Medical Department 3M Center Bldg. 220-2E-02 Saint Paul, MN 55144-1000 Telephone 651/737.1374 FAX 651/733.1773 Alternative Study Director: John Butenhoff, Ph.D., DABT, CIH 3M Corporate Toxicology 3M Medical Department 3M Center Bldg. 220-2E-02 Saint Paul, MN 55144-1000 Telephone 651/733.1962 FAX 651/733.1773 In-Life Phase Investigator: Argus Analytical Phase Investigator: 1) Argus 2) Kris J. Hansen, Ph.D., 3M Environmental Technology and Safety Services, Bldg. 02-3E-09 935 Bush Avenue, Building 2-3E-09, St. Paul, MN 55133-3331 Telephone 651/778.6018 FAX 651/778.6176 April 27, 2000 004598 Page 5 of 5 D.J. Luebker