Document zzNvL23Q3Q8MOm259gR66XGpB

ARnc-om 1 08/13/99 T-6316.15; ST-30 N-EtFOSE Bile 3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY Protocol for Study No. T-6316.15; ST-30 N-EtFOSE Bile M ethod Development in Rats Study Objective: The objective o f this study is to collect urine and bile specimens from control and N EtFO SE treated rats for m ethod development. This study will support the N -EtFO SE ADME study in Cynom ologus monkeys currently underway at Covance M adison. Research Client: 3M Specialty Chemicals Division Biodegradable Surfactants Team 3M Center, Building 236 Saint Paul, M N 55133-3220 Sponsor: 3M Specialty Chemicals Division 3M Center, Building 236 Saint Paul, M N 55133-3220 Study Location: 3M Strategic Toxicology Laboratory 3M Center, Building 270-3S-06 room SB314 Saint Paul, M N 55133-3220 Study Director: Andrew M. Seacat, Ph.D. Sr. Research Toxicologist 3M M edical D ept. / C orporate Toxicology 3M Center, Building 220-2E-02 Saint Paul, M N 55133-3220 Ph.: 651-575-3161 FAX: 651-733-1773 Study Toxicologist Deanna Luebker, MS Advanced Research Toxicologist 3M M edical Dept. / C orporate Toxicology 3M Center, Building 220-2E-02 Saint Paul, M N 55133-3220 Ph: 651-737-1374 FAX: 651-733-1773 Proposed Study Tim eline: In-Life S tart Date: 8/12/1999 In-Life End Date: 8/20/1999 Regulatory Compliance: This study will be perform ed in the 3M Strategic Toxicology Laboratory under a defined protocol and classified as a "Class B Study" as explained in TOX SOP 0950, Strategic Toxicology Lab GLP Program Procedure. 004336 i 08/13/99 T-6316.15; ST-30 N-EtFOSE Bile Test M aterial: Dan H akes, Product Responsibility Liaison 3M Chemicals Division, will furnish highpurity N -EtFO SE. Id e n tific a tio n : N am e: N arrow Range N -Ethyl perfluorooctanesulfonam ido ethanol, FM 3923. M olecular Form ula: C8F17S02N(CH2CH3)CH2CH20H L o t N u m b er: L ots 30035, 30037, 30039 mixed and analyzed as one sample. P u rity : 9 8 .1 4 % S ta b ility : D ocum entation will be kept on file. Storage C onditions: U pon receipt, test material w ill be stored tightly sealed at room tem perature. C haracteristics: Inform ation on synthesis m ethods, com position o r other characteristics that define the test material will be kept on file. Anim als: S p e c ie s: Rat S tra in : Sprague Dawley Source: H arlan S pecifications: Bile Cannulated - Free End A ge a t in itia tio n o f treatm en t: 6-8 weeks W eight a t in itia tio n o f tre a tm e n t: approxim ately 150-250g N u m b er a n d sex: 20 males, 20 females T able 1 - Dose G roups G roup D ose 1 0 m g/kg 2 20 m g/kg 3 0 m g/kg 4 20 m g/kg N 5 5 5 5 Sex M ale M ale Fem ale Fem ale Id e n tific a tio n : AUA N um ber: ear tag w ith animal number or unique tail m ark. 2246 H usbandry: H ousing: U pon arrival at 3M all animals will be individually housed in standard cages. All rats from groups 2 and 4 and 1 each from groups 1 and 3 will be transferred to individual m etabolism cages following dosing and housed 004337 2 08/13/99 T-6316.15; ST-30 N-EtFOSE BUe there through day 4-post dose. All other rats will remain individually housed in standard cages throughout the study. D iet/W ater: H arlan Teklad LM-485 M ouse/R at Sterilizable Diet, supplied by H arlan Teklad, M adison, W I, and tap w ater will be provided to all rats ad libitum throughout the study. E nvironm ent: Environm ental controls for the animal room will be set to m aintain a tem perature o f 72 3F, hum idity o f 30-70% , a minimum o f 10 exchanges o f room air per hour and a 12 hour light/dark cycle. Dose and D osing Procedures: M ethod o f adm inistration/D ose p rep aratio n : A single 20m g/kg dose o f N -E t FO SE will be administered via oral gavage to ra ts in groups 2 and 4 on day zero o f the study. The dosing solution will be prepared as a 0.04% (4m g/m l) uniform solution in 2% Tw een 80 using a 15 ml tissue grinder. A volum e o f 5 ml suspension / kg body w eight will be adm inistered to each rat. R e-suspension o f solids will be perform ed w ith 5 strokes o f the tissue grinder pestel before each sample is draw n-up in the syringe for dosing. A single 5 ml / kg body weight dose o f 2% Tw een 80 w ill be administered via oral gavage to rats in groups 1 and 3 on day zero o f the study (see table 2). Observation o fA nim als: C linical O bservations: E ach animal will be observed daily for m ortality and m orbidity and notable findings will be recorded. A dditional findings will be recorded as they are observed. Body W eights: E ach animal will be weighed immediately prior to dosing and immediately prio r to euthanasia. Specim en Collection: F req u en cy (See table 2): B ile w ill be collected daily upon arrival at 3M. (All bile collected prior to dosing will serve as control bile.) U rine collections will be m ade on days 1 - 4 post dose. N ecropsies will be perform ed on day 4-post dose. 004338 3 Table 2 - Schedule Sun Mon Aug 15 D y-1 Bile coll Aug 16 DyO DOSE Bile coll. Tues Aug 17 Dy 1 postdose Bile colL Urine colL 08/13/99 T-6316.15; ST-30 N-EtFOSEBile Wed Aug 18 Dy 2 postdose Bile coll. Urine coll. Thurs Aug 12 Dy-4 Arrival @ 3M Bile colL Aug 19 Dy 3 postdose Bile colL Urine colL Fri Aug 13 D y-3 Bile coll. Aug 20 Dy 4 postdose Bile colL Urine coll. N ecropty Sat Aug 14 Dy-2 Bile coll. M ethod o f Specimen Collection: B ile will b e collected from each rat daily upon arrival at 3M . The exteriorized portion o f the catheter will be rem oved from th e pouch o f the protective jacket and the w ire plug in the Free End catheter w ill be rem oved. A newly opened syringe will be used to w ithdraw n bile from the catheter o f each rat. The bile from each rat will be equally split and placed into 2 labeled tubes. The wire plug will be inserted back into the catheter follow ing bile collection and the exteriorized portion o f the catheter place in th e pouch o f the protective jacket to prevent catheter chewing. Each tu b e o f bile will be frozen (-70 C ) until analysis. U rine w ill be collected from each metabolism cage on days 1-4 post dose. For each urine specim en, the initial volume will be recorded and the sides o f the urine collection apparatus will be washed w ith 10-20 ml deionized w ater. The urine and w ash from each cage will be collected in a 50 ml tube and the final volum e will be brought to 45 ml w ith additional deionized w ater. E ach urine specimen will be frozen (-70 C) until analysis. O n day 4 post dose animals will be euthanized by CO2 and gross necropsy perform ed. D uring necropsy, blood ( 6 m l) will be collected via the abdom inal aorta and transferred to blood collection tubes w ithout anticoagulant. B lood sam ples will be allowed to clot for a period o f 15 to 30 m inutes at room tem perature, and the clot will be spun dow n in a centrifuge a t 1100 x g for 5 m inutes. The serum will be transferred to labeled 1.5 ml m icrofuge tubes and centrifuged again at 2000 x g to rem ove any remaining red blood cells. E ach sera sample will then be transferred to a separate labeled polypropylene microfuge tube and (-70 C ) until analysis. E ach liver will be rem oved, rinsed in saline, w eighed, placed into a labeled sterile sample bag and frozen (-70 C) until analysis. Specimen H andling and Analysis: Specim ens will tem porarily be stored in a freezer set to m aintain -60 to 80C in the Strategic Toxicology Laboratory. <04339 4 08/13/99 T-6316.15; ST-30 N-EtFOSEBe For m etabolite analysis, urine, liver and sera will be packed in dry ice and shipped to: K ris Hansen, Ph.D. 3M Environm ental Technology and Safety Services 935 Bush Avenue St. Paul, M N 55133-3331 Ph: 612-778-6081, FAX: 612-778-6176. F o r analytical m ethod developm ent purposes, bile wll be packed in dry iced and shipped to: K arsten Levsen Fraunhofer Institut filer Toxikologie und A erosolforschung N ikolai Fuchsstr 1. D-30625 H annover GERMANY The Strategic Toxicology laboratory will retain one h alf o f all th e bile specim ens collected for in-house m ethod developm ent, verification analysis and/or as a retain for possible future analysis. All results will be provided for inclusion in the final report. The num ber, type and date o f collection o f specimens to be generated for analysis are as follows: Table 3 - Specimens S pecim en day -4 Bile for 20 Fraunhofer Bile for in- 20 house analysis U rine - Serum - Liver - day day day day 0 day 1 day 2 day 3 day 4 Total -3 -2 -1 PD PD PD PD 20 20 20 20 20 20 20 20 180 20 20 20 20 20 20 20 20 180 - - - - 12 12 12 12 48 - - - - - - - 20 20 - - - - - - - 20 20 Data Analysis: D ata collected on N -EtFO SE m etabolite concentrations in serum, liver and urine using validated m ethods will be analyzed for toxicokinetic param eters and for statistically significant differences betw een groups using Students T -test and/or ANOVA. D ata collected during th e m ethod developm ent and validation phases o f bile and urine m etabolite analysis w ill be analyzed for precision and accuracy as necessary to develop a validated m ethod. Provided that enough o f the retained bile and urine sam ples are 004340 5 08/13/99 T-6316.15; ST-30 N-EtFOSEBile available follow ing the developm ent o f validated m ethod they may then be analyzed for N EtFO SE m etabolite levels under the validated m ethods to generate data for use in a definitive report. R esp o n sib ilities: Deanna L uebker and Andrew Seacat will be responsible for dosing the animals, collecting in-life specimens, perform ing the necropsies, collecting and sending specimens fo r analysis and may perform additional experim ents on th e collected tissues for m ethod developm ent o r verification. K ris H ansen, 3M Environm ental, will be responsible for analysis o f serum and liver tissues, and th e urine samples following developm ent o f a validated m ethod for urinary m etabolites o fN -E t FO SE if possible. K arsten Levsen, Fraunhofer Institut filer Toxikologie und A erosolforschung w ill be responsible fo r the developm ent o f a validated m ethod fo r biliary m etabolites and the subsequent analysis o f the retained bile samples, provided there is a sufficient supply. A ndrew Seacat and D eanna Luebker will draft a final report and ensure th e report receives appropriate 3M review before a final report is issued. 004341 6 Signatures: ___ Q mJ aJcJ Dr. Andrew Seacat Senior Research Toxicologist Study D irector /& Deanna Luebker, MSr Advanced Toxicologist Study Toxicologist 08/13/99 T-6316.15; ST-30 N-EtFOSE Bile ___ #/! D ate ?<:/&?<?<?________________ _____________ D ate 004342