Document JJ2p3D5EvGxRzvmaREkM00nYv

Attachments to Letter to C. Auer dated May 18,2000 Studies and Other Information on Certain Perfluorooctane Sulfonate-Related Compounds ARX&-032O 13. PFOS Additional Studies on Perfluorooctane Sulfonate Acute Toxicity 1) Acute Toxicity Tests for T-T-6684, Didecyldimethylammonium salt of perfluorooctanesulfonate: a) Acute Oral Toxicity Study of T-6684 in Rats (OECD Guidelines), Coming Hazleton, Inc., Project No. CHW 61101149, 3M Reference No. T-6684 January 31, 1997 b) Primary Dermal Irritation / Corrosion Study of T-6684 in Rabbits (OECD Guidelines), Coming Hazleton, Inc., Project No. CHW 61101150, 3M Reference No. T-6684 (didecyldimethylammonium salt of perfluorooctanesulfonate), January 10, 1997 c) Primary Eye Irritation / Corrosion Study of T-6684 in Rabbits (OECD Guidelines), Coming Hazleton, Inc., Project No. CHW 61101151, 3M Reference No. T-6684 (didecyldimethylammonium salt of perfluorooctanesulfonate), January 28, 1997 2) Acute Toxicity Tests for T-5898, lithium perfluorooctane sulfonate, 3M Ref. FC-94: a) Final Report, Acute Oral Toxicity Study of T-5898 in Rats, Hazelton Wisconsin, Study No. 40200468, April 22, 1994 b) Final Report, Primary Eye Irritation/Corrosion Study of T-5898 in Rabbits, Hazelton Wisconsin, Study No. 40200469, April 7, 1994 c) Final Report, Primary Dermal Irritation/Corrosion Study of T-5898 in Rabbits, Hazelton Wisconsin, Study No. 40200470, March 23, 1994 3) Acute Oral Toxicity - Rats, Biosearch, Inc., 3M Reference No. T-1388 (perfluorooctanesulfonic acid), March 4, 1976 Pharmacokinetic Studies 1) Draft Report, 5-Daily Dose Dermal Absorption / Toxicity Study of T-6684 in Rabbits, Covance Laboratories, Inc., Study No. 6329-200, 3M Reference No. T-6684 (didecyldimethylammonium salt of perfluorooctanesulfonate, slurry), July 11, 1997 -23 - ccso sa Attachments to Letter to C. Auer dated May 18,2000 Studies and Other Information on Certain Perfluorooctane Sulfonate-Related Compounds 2) Qualitative Investigation of the In Vitro Metabolism of T-6292 (n-ethyl FOSE), T6293 (n-ethyl FOSE phosphate diammonium salt(ester)), T-6294 (n-ethyl perfluorooctane sulfonamide) and T-6295 (perflurooctane sulfonate) by Rat and Human Hepatocytes Using Ion Spray LC/MS and LC/MS/MS, Advanced Bioanalytical Services, Inc., [Preliminary] Analytical Report, Report 96ADEM01.3M, November 12, 1996 Teratology 1) Memorandum from E. G. Lamprecht re Fetal Rat Lens Artifact - Summary of Developments to Date, Nov. 6, 1981 Analytical Ion Spray LC/MC Determination of Perfluoro Analytical Standards Provided by 3M Medical Department, Advanced Bioanalytical Services, Inc., No. 95MYHW01.3M, August 30, 1995 Studies in Progress 1) Protocol, Feces Method Development Metabolism Study for Perfluorooctanesulfonate Derivatives [N-EtFOSE, PFOS, and FOSA], 3M Strategic Toxicology Laboratory, Study Nos., T-636.17; T-6295.21; T-7132.3; ST-41, In-Life Start Date November 22, 1999, In-Life End Date November 24, 1999 2) Protocol, Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T-6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluorooctanesulfonamide (PFOSA 3M T-7091.1) in Rats, Pathology Associates International, Study No. 1132-100 - 24- C X SC 54 Attachments to Letter to C. Auer dated May 18,2000 Studies and Other Information on Certain Perfluorooctane Sulfonate-Related Compounds 13. PFOS Additional Studies on Perfluorooctane Sulfonate B ibliography Show ing Studies in 3M 's Possession Believed To Be In F IF R A Docket. REDACTED -25 - 0 5 6 55 Corning Hazleton Inc. P.O. Box 7545 Madison, WI 53707-7545 Deliveries: 3301 Kinsman Blvd., Madison, W I 53704 608.241.4471 608.241.7227 Fax C O R N I N G HazletOt Sponsor: 3M St. Paul, Minnesota FINAL REPORT Study Title: Acute Oral Toxicity Study of T-6684 in Rats (OECD Guidelines) Author: Steven M. Glaza Study Completion Date: January 31, 1997 Performing Laboratory: Corning Hazleton Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: CHW 61101149 Page 1 o f 29 iij\ I FEB-3B9T ! (t* m ------------------ fi _____ _________ _ C05S56 COMPLIANCE STATEMENT Acute Oral Toxicity Study of T-6684 in Rats (OECD Guidelines) CHW 61101149 This study was conducted in accordance with the Organisation for Economic Cooperation and Development and Principles o f Good Laboratory Practice, C(81)30(Final). Acute Studies Coming Hazleton Inc. _______ v~s wy~i Date 2 005657 QUALITY ASSURANCE STATEMENT CHW 61101149 This report has been reviewed by the Quality Assurance Unit o f Coming Hazleton Inc., in accordance with the Organisation for Ecomonic Cooperation and Development (OECD) Principles o f Good Laboratory Practice, C(81)30(Final). The following inspections were conducted and findings reported to the Study Director and management Inspection Dates From To 12/19/96 12/19/96 01/23/97 01/24/97 Phase Necropsy Data/Report Review Date Reported to Study Director 12/19/96 01/24/97 Date Reported to Management 12/19/96 01/24/97 Representative, Quality Assurance Unit ;-3 '-7 7 Date 005658 3 STUDY IDENTIFICATION Acute Oral Toxicity Study o f T-6684 in Rats (OECD Guidelines) CHW 61101149 Test Material Sponsor Sponsor's Representative Study Director Study Location Study Timetable Study Initiation Date Experimental (In-life) Start Date In-life End Date Experimental Termination Date Study Completion Date T-6684 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, M N 55133-3220 Roger G. Perkins, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, M N 55133-3220 (612) 733-3222 Steven M. Glaza Corning Hazleton Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Coming Hazleton Inc. 3301 Kinsman Boulevard Madison, W I 53704 November 27, 1996 December 5, 1996 December 19, 1996 January 31, 1997 January 31, 1997 005659 4 Acute Studies Steven M. Glaza Study Director Manager Steven R Sorenson Study Coordinator Jeffrey B. Hicks In-life Supervisor Rose M. Bridge Administrative Supervisor Toxicology Support Kathy Myers Manager Calvin L. H orton Supervisor KEY PERSONNEL Quality Assurance Sherry R W. Petsel Manager CHW 61101149 Laboratory Animal M edicine Cindy J. Cary, DVM Diplomate, ACLAM Supervisor Anatomical Pathology Thomas E. Palmer, PhD Anatomical Pathologist Deborah L. Pirkel/ Jack Serfort Supervisors Necropsy 5 & 05G8Q CONTENTS CHW 61101149 COMPLIANCE STATEM ENT.................................................................................................... 2 QUALITY ASSURANCE STATEM ENT..................................................................................3 STUDY IDENTIFICATION......................................................................... 4 K EY PERSONNEL....................................................... 5 O BJECTIV E.................................................................................................................................... 8 TEST MATERIAL............................................................................... 8 Identification.................... 8 Purity and Stability...................................................................................................................... 8 Storage and Retention.................................................................................................................8 Safety Precautions...................................................................................................................... 8 TEST SY STEM .............................................................................................................................. 9 Test Animal.................................................................................................................................. 9 H ousing................................................................................................. 9 Animal D ie t.................................................................................................................................. 9 Animal Selection and Grouping.............................. 9 Justification for Species Selection.......................................................................................... 10 P R O C E D U R E S .............. ..................................................................... 10 Preparation and Administration o f Test M aterial................................................................. 10 Reason for Route o f Administration.......................................................................................10 O b s e rv a tio n s ............................................................................................................................... 10 Pathology................................................................................................................................... 10 Statistical A nalyses................................................................................................................... 11 Location o f Raw Data, Records, and Final Report...............................................................11 R ESU LTS.......................................................................................................................................11 M ortality.....................................................................................................................................11 Body W eights.............................................................................................................................11 Clinical Signs.......................................... 11 P a t h o l o g y ............................................... 12 D ISC U SSIO N ................................................................................................................................ 12 6 c <jij 3L CHW 61101149 SIGNATURE................................................................................................................................. 12 REFERENCE................................................................................................................................. 12 PATHOLOGY REPORT..............................................................................................................13 TABLE 1 Mortality Summary.....................................................................................................14 2 Individual and M ean Body Weights/ Body Weight Gains (g ).......................................15 3 Individual Clinical Signs......................................................................................................16 4 Individual Pathology Comments........................................................................................17 A P P E N D IX .....................................................................................................................................18 Protocol T P 2069.......................................................................................................................19 Protocol Amendment No. 1 ............................................................................. ......................28 Protocol Amendment No. 2 .................................................................................................... 29 7 005662 OBJECTIVE CHW 61101149 The objective o f this study was to assess the acute oral toxicity produced when the test material is administered by the oral route (gavage) to rats.1 All procedural times presented in this report fall within the acceptable ranges as specified in the Wisconsin facility o f Coming Hazleton (CHW) Inc. Standard Operating Procedure (SOP). TEST MATERIAL Identification The test material was identified as T-6684 and described as an off-white liquid. Purity and Stability The Sponsor assumes responsibility for purity and stability determinations (including under test conditions). Storage and Retention The test material was stored at room temperature. Any unused test material will be returned to the Sponsor after issuance o f the final report according to CHW SOP. Safety Precautions The test material handling procedures were according to CHW SOPs and policies. 00S6G 3 8 TEST SYSTEM CHW 61101149 Test Animal Young adult albino rats o f the Crl:CD (SD)BR strain were procured from Charles River Laboratories, Inc. on November 4, 1996 (Portage, Michigan facility) and N ovember 12, 1996 (Raleigh, N orth Carolina facility). Housing After receipt, the animals were acclimated for a period o f at least 7 days. During acclimation and throughout the study, the animals were separated by sex and group housed in screen-bottom stainless steel cages. Environmental controls for the animal room were set to maintain a temperature o f 19 to 25C, a relative humidity o f 50% 20%, and a 12-hour light/12-hour dark lighting cycle. In cases where variations from these conditions existed, they were documented and considered to have had no adverse effect on the study outcome. Animal Diet The animals were provided continuous access to Laboratory Rodent Diet #5001, PM I Feeds, Inc., and water except for approximately 17 to 20 hours before test material administration when food, but not water, was withheld. The feed is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Samples o f the w ater are periodically analyzed. There were no known contaminants in the feed or water at levels that could be expected to interfere with or affect the results o f the study. Animal Selection and Grouping Five male and five female healthy, acclimated rats, weighing from 255 to 299 g and approximately 10 to 13 weeks o f age, were used for a single dose level o f 5,000 mg/kg o f body weight. The animals were identified by animal number and corresponding ear tag throughout the study. 005664 9 ____________________________________________________________ CHW 61101149 Justification for Species Selection Historically, rats have been used as a representative of a rodent species and are preferred by various regulatory agencies. PROCEDURES Preparation and Administration of Test Material An individual dose o f the test material was calculated for each animal based on its fasted body weight and administered as a single dose by gavage. The test material was administered at a volume o f 5.05 mL/kg o f body weight based on an average bulk density of0.99g/m L. Reason for Route of Administration Historically, the oral route has been the route o f choice for administering a known amount o f test material. Observations Clinical observations were conducted at 1, 2.5, and 4 hours after test material administration and daily thereafter for 14 days. Mortality checks were conducted twice a day (morning and afternoon) for 13 days after test material administration and again the morning o f Day 14. Body weights were determined before test material administration (Day 0), at Day 7, and at termination o f the in-life phase (Day 14). Pathology A t termination o f the in-life phase, all surviving animals were euthanized. All animals, whether found dead during the study or euthanized, were subjected to an abbreviated gross necropsy examination and any abnormalities were recorded. After necropsy, the animals were discarded and no tissues were saved. 10 005665 Statistical Analyses No statistical analyses were required by the protocol. ____________ CHW 61101149 Location of Raw Data, Records, and Final Report The raw data, records, and an original signed copy o f the final report will be retained in the archives of CHW in accordance with CHW SOP. RESULTS M ortality A summary o f the survival rate is in Table 1. One female was found dead on D ay 7. N o other mortality was observed. The estimated oral LD50values for male and female rats w ere determined to be greater than 5,000 mg/kg o f body weight. Body Weights Individual and mean body weights and body weight gains are in Table 2. All surviving animals exhibited body weight gain with the exception o f 3 females which exhibited body weight losses o f 1 to 22 g during the first week o f the study and one male which exhibited a weight loss o f 66 g during the second week. Clinical Signs Individual clinical signs are in Table 3. All animals appeared normal with the exception o f the one female which died during the study which exhibited red-stained face, wet/yellow-stained urogenital area, hunched posture, tremors, hypersensitivity to touch, and tonic convulsions. In addition, one male was noted to have a missing front tooth on Day 11. This animal subsequently appeared thin on Days 12 through 14. This animal's weight loss noted above is probably due to the missing tooth and is not considered to be test material related. C05G66 11 ______________________________________________________________________________ CHW 61101149 Pathology Individual gross necropsy findings are in Table 4. A summary report by the study pathologist is on Page 13. There were no test material related lesions in any o f the animals. DISCUSSION The acute oral toxicity o f T-6684 was evaluated in male and female rats at a dose level o f 5,000 mg/kg. The estimated LDso value for male and female rats was determined to be greater than 5,000 mg/kg. The only mortality was the death o f one female on Day 7. The only test material related clinical signs o f toxicity were in one male which included a missing upper right front tooth and thin appearance and the female which died during the study and included red-stained face, wet/yellow-stained urogenital area, hunched posture, tremors, hypersensitivity to touch, and tonic convulsions. All surviving animals exhibited body weight gain with the exception o f three females which exhibited body weight losses o f 1 to 22 g during the first week o f the study and one male which exhibited a weight loss o f 66 g during the second week. The weight loss in the male animal was due to a non-test material related condition. There were no test material related lesions observed at necropsy. Steven M. Glaza Study Director Acute Studies SIGNATURE W - _______ u V ^V T l Date REFERENCE 1. "Acute Oral Toxicity," Organisationfo r Economic Cooperation and Development Guidelinesfo r Testing o f Chemicals, Section 4, Health Effects, Number 401, Paris Cedex (February 24, 1987). 005667 12 PATHOLOGY REPORT CHW 61101149 There were 10 rats (five males, five females) necropsied. One female died on Test Day 7 and the remaining animals were euthanized and necropsied at the termination o f the study. The dose level, day o f death, and gross observations recorded for each animal are in the Individual Pathology Comments that follow this report. A t necropsy, the only lesions observed were in the female that died on test. The ventral haircoat was stained and the glandular portion o f the stomach had multiple, dark red, pinpoint foci. These changes were considered incidental findings and unrelated to the test material. There were no visible lesions in any o f the animals that survived to study termination.. Thomas E. Palmer, PhD Pathologist (61101149.fin) 010797 Date 005668 13 Table 1 Mortality Summary CHW 61101149 Dose Level (mg/kg) Mortality Results Sex No. Died/ No. Dosed* 5,000 5,000 M F 0/5 1/5, Day 71 Superscript number indicates number of animals found dead on the indicated day. 005669 14 Table 2 Individual and M ean Body W eights/ Body W eight Gains (g) CHW 61101149 Animal Number Day 0 Weight _______ Day 7_______ Weight Gain* M ales (5,000 mg/kg) C15368 C15363 C15374 C15367 C15337 264 270 299 298 298 310 46 305 35 352 53 324 26 346 48 Mean 286 327 42 Day 14 Weight Gain* 334 70 339 69 382 83 372 74 280 -18 341 56 Females (5,000 mg/kg) C12896 C12898 C l2905 C l2903 C l2908 284 270 273 286 255 213 -71 270 0 251 -22 285 -1 247 -8 (209)7 303 294 301 271 - 33 21 15 16 Mean 274 253 - 20 292 21 * Gain from Day 0 body weight. ( ) Value in parantheses is a dead body weight. Superscript number indicates the day the animal was found dead. 005670 15 Table 3 Individual Clinical Signs CHW 61101149 Animal Number Observation C15368 Appeared normal C l 5363 Appeared normal C l 5374 Appeared normal C15367 Appeared normal C15337 Appeared normal Missing upper right front tooth Thin appearance Hour 1.0 2.5 4.0 / / / / - - Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Males (5,000 mg/kg) S /// / / S / S - - - - Females (5,000 mg/kg) C12896 Appeared normal Yellow-stained urogenital area Red-stained face Wet urogenital area Tremors Hypersensitive to touch Tonic convulsions Hunched posture Found dead (after clinical observation) C12898 Appeared normal / C12905 Appeared normal C12903 Appeared normal C12908 Appeared normal -- _ --- / - . . / _ - - -/ / - - - - - / S S / // / Condition existed. Condition not evident 005671 16 Table 4 Individual Pathology Comments Dose Level: 5,000 mg/kg of Body Weight CHW 61101149 Animal Number C15368 C15363 C15374 C15367 C15337 C12896 . Sex M M M M M F C12898 F C 12905 F C l2903 F C l2908 F - N ot applicable. Test Day Died Sacrificed 14 14 14 14 14 7 - 14 14 14 14 Necropsy Observation No visible lesions. No visible lesions. No visible lesions. No visible lesions. No visible lesions. The haircoat o f the entire ventral surface is stained w ith dark red and dry material. The mucosal surface o f the glandular portion o f the stomach has multiple, dark red, pinpoint foci. No visible lesions. No visible lesions. No visible lesions. No visible lesions. 005672 17 APPENDIX Protocol TP2069 Protocol Amendment No. 1 Protocol Amendment No. 2 CHW 61101149 18 5 ou CHW 61101149 Sample Submittal Form This form is to be used when submitting samples fo r routine acute testing. Special testing needs can be easily arranged by contacting the Acute Studies Departm ental (60S) 241-7292. CH W S tud y N o. C . / / c a ^ / * f a r Enclose with samples and send to: Com ing H azleton Inc. 3301 Kinsman Boulevard M adison, W isconsin 53704 Subm itted bvf f \ Q>1 f , A*T D ale Sam ple Sant: i / 'i i f p !A. C om pany:_________________________________________________________ N um ber of Reports Required: 3 Full GLP Com pliance: Yes _____F D A (21C FR S 8) --------No --------- EPAfTSCA-- 40 CFR 792) ___ _ EPA (FIFRA-- 40 CFR 160) - X OECD _____MAFF -------- MOHW Sam ple Nam e: 7^~ (o (g * T - (a C R C~ ^ ~7~-- < P 1& ___________________________ Physical Description: ____________ !____________________ ____________________________________________ S pecial Handling Precautiot(s: /ft 9 f)S s 4 /V V ^ < fy O * 7 - 7 ~ 7 . 4) Test m aterial purity and stability inform ation (including under test conditions) on file with S po nso r Y es ____No Test m ixture analysis for concenlration/hom ogeneity/stability to be conducted: ____ Y es* _____by Sponsor _____ by CHW Sam ple Disposal: ^ Return to Sponsor at following address: _No ..J L -k > q r^ --------------3 ix 9 c . D _________ 6la _______ SC s s W - / qq . Dispose of according to CHW SOPs Sam ple Storage Requirem ents: y X k Room temperature ____ Refrigerated ____ O ther______________________________ * A t additional cost to'S ponsor (CHW w ill contact Sponsor as to these additional charges). Tests A cu te O ral T o x ic ity in R ats ------ TP8084 Up and down LDS0 procedure ------ TP3206 FHSA screen; 5M-5F at 5.0 g/kfl ------ Conduct defined study Hdeath occurs at 5.0 g/kg ___ TP3013 EPA screen; 5M-5F at 5.0 g/kg . . ---- Conduct defined study Hdeath occurs at 5.0 g/kg _X_TP2069 OECD screen; 5M-5F at 5.0 g/kg ------ Conduct defined study If death occurs at 5.0 g/kg Special instructions: A cu te D erm a l T o x ic ity in R abbits ------TP3207 FHSA screen; 5M-5F at 2.0 g/kg ___ TP3016 EPA screen; 5M-5F at 2.0 g/kg ------ Conduct defined study If death occurs at 2.0 g/kg ------TP2070 OECD screen; 5M-5F at 2.0 g/kg ------ Conduct defined study if death occurs at 2.0 g/kg Special instructions:. -h JD PtCChAt- / r g r o R g & A ^ i. - J - - 4 4 W Fo r CHW U se O nly ---------- Protocol Issue D ate: Study Director: t\-- P rim ary S kin Irrita tio n ___ TP3208 FHSA; 6 rabbits-1 abraded, 1 1ntact slte/rabblt __ TP3014 EPA; 6 rabbits-1 intact slte/rabblt -X T P 2 0 7 1 OECD; 3 rabbfls-1 Intact sha/rabbH ------ TP4206 DOT corrosivity; 6 rabbits-1 Intact sfta/rabbit ------ TP7145 Phototoxicity; 6 rabbKs-2 Intact sltes/rabbit (one site w ith UVA exposure) Special Instructions:_________________________________ P rim ary E ye Irrita tio n ___ TP6360 Low-volume procedure; 6 rabbits unwashed ___ TP3209 FHSA; 6 rabbits unwashed ------ TP2012 1978 EPA; 6 rabbits unwashed, 3 washed ------ TP3015 1982 EPA; 6 rabbits unwashed -X J P 2 0 7 2 OECD; 3 rabbits unwashed ___ 3 rabbits washed at 4 seconds ------ 3 rabbits washed at 30 seconds Special in stru ctio n s:_______________________________ G u in ea P ig S e n s itiza tio n ___ TP2017 EPA Magnusson-Kligman m axim ization TP6164.EC OECD/EC Magnussun-Kligman m axim ization ------TP2008 Buehler sensitization ___ TP6289 Photoallergenic contact derm atitis (Arm strong) Special In stru ctio n s:____________________________________ W hite copy-- C H W Yellow copy-- Subm itter 005673 19 CHW 61101149 POST O f f i c i BOX 7 6 4 S MADISON. WISCONSIN S7707-7B4S COAMING la b o ra to ry Services Company Sponsor: 3H St. Paul, Minnesota PROTOCOL TP2069 Study. Title: Acute Oral Toxicity Study in Rats (0EC0 Guidelines) Date: June 1, 1993 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Pro.iect Identification: HWI 005674 20 CHW 61101149 TP2069 Page 2 STUDY IDENTIFICATION Acute Oral Toxicity Study in Rats (OECD Guidelines) HWI No. Test Material Sponsor Sponsor's Representative Study Director Study Location Proposed Study Timetable Experimental Start Date Experimental Termination Date Final Report Date 6>f/to H I * ) (See sample submittal form) 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 John L. Butenhoff, PhD 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 Week of Week of I 7 - ^ 7 Week of / - 2 7 - ^ 7 2] 005G7S CHW 61101149 TP2069 Page 3 1. Study Acute Oral Toxicity Study In Rats (OECD Guidelines) 2. Purpose To assess the acute oral toxicity produced when the test material 1s administered by the oral route (gavage) to rats 3. Regulatory Compliance This study will be conducted in accordance with the following Good Laboratory Practice Regulatlons/Standards/Guldellnes: [ ] Conduct as a Nonregulated Study ( ] 21 CFR 58 (FDA) [ ] 40 CFR 160 (EPA-FIFRA) [ ] 40 CFR 792 (EPA-TSCA) E><C(8I)30 (Final) (OECD) ( ) Notification No. 3850, August 10, 1984 (Japanese MAFF) [ ] Notification No. 313, March 31, 1982, and as amended by Notification No. 870, October 5, 1988 (Japanese MOHW) All procedures In this protocol are In.compliance with the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study does not unnecessarily duplicate any previous work. 4. Quality Assurance For regulated studies, the protocol, study conduct, and the final report will be audited by the Quality Assurance Unit in accordance with Hazleton Wisconsin (HW1) Standard Operating Procedures (SOPs) and policies. 5. Test Material A. Identification (See sample submittal form) B. Physical Description (See sample submittal form) C. Purity and Stability The Sponsor assumes responsibility for purity and stability determinations (including under test conditions). Samples of test material/vehicle mixture(s) (if applicable) for concentration, solubility, homogeneity, and stability analyses will be taken before administration if requested by the Sponsor. These samples (if taken) will be sent to the Sponsor after experimental termination for possible analysis. 005676 22 CHW 61101149 TP2069 Page 4 0. Storage (See sample submittal form) E. Reserye_ganip1fi,; Studies of less than 4 weeks 1n experimental duration will not have reserve samples retained. Reserve sample(s) of each batch/lot of test material will be taken if this study 1s more than 4 weeks 1n experimental duration. The test material reserve sample will be stored at HWI In a freezer set to maintain a temperature of below O'C for 10 years per HWI SOP. The Sponsor will be contacted after 10 years for disposition in accordance with the appropriate regulatory Good Laboratory Practices. F. Retention Any unused test material will be discarded after Issuance of the final report, unless directed otherwise by the Sponsor. G. Safety Precautions As required by HWI SOPs and policies 6. Experimental Design A. Animals (1) Species Rat (2) Strain/Source Crl :CD BR/Charles River Laboratories, Inc. [ ] Hsd:Sprague Dawley SD*/Harlan Sprague Dawley, Inc. (3) Age at Initiation Young adult (4) Weight at Initiation 200 to 300 g (5) Number and Sex 5 males and 5 females for the initial dose level 5 males and/or 5 females for any additional dose levels (if required) (6) Identification Individual numbered ear tag 005677 23 CHW 61101149 TP2069 Page 5 (7) Husbandry (a) Mousing Separated by sex and group housed In screen-bottom stainless steel cages (heavy gauge) (b) fiod Rodent Chow* 15001 (Purina Hills, Inc.) id libitum except for overnight before test material administration. The food 1s routinely analyzed by the manufacturer for nutritional components and environmental contaminants. (c) Water Ad libitum from an automatic system. Samples of the water are analyzed by HWI for total dissolved solids, hardness, and specified microbiological content and for selected elements, heavy metals, organophosphates, and chlorinated hydrocarbons. (d) Contaminants There are no known contaminants 1n the food or water that would Interfere with this study. (e) Environment Environmental controls for the animal room will be set to maintain a temperature of 19 to 25*C, a relative humidity of 50% +20%, and a 12-hour light/12-hour dark cycle. (f) Acclimation At least 7 days (8) Selection of Test Animals Based on health and body weight according to HWI SOPs. An adequate number of extra animals will be purchased so that no animal in obviously poor health is placed on test. (9) Justification for Species Selection Historically, rats have been used as representative of a rodent species and are preferred by various regulatory agencies. B. Dose Administration (1) Dose Level A single dose of 5,000 mg/kg of body weight will be administered to five males and five females. If no test material-related mortality is produced at this level, no 005678 24 CHW 61101149 TP2069 Page 6 further testing will be required. If any mortality occurs at the 5,000 mg/kg dose level, additional dose levels may be added at the direction of the study director In order to meet the objectives of the study. (2) Dose Preparation and Administration All animals will receive the same concentration of test mixture per dose level. If a solid, the test material will be suspended 1n an appropriate vehicle. If a liquid, the test material will be dosed undiluted, using the bulk density to determine the dose volume. If the material 1s an aerosol, it will be discharged Into a beaker and administered as a liquid. Individual doses will be based upon the animal's body weight taken just before test material administration, and administered by gavage. The animals will have food withheld for 17 to 20 hours before test material administration. The prepared test mixtures will be stored at room temperature until administration. After administration, any remaining test mixtures will be discarded. (3) Reason for Route of Administration Historically, the oral route has been the route of choice for administering a known amount of test material. C. Observation of Animals (1) Clinical Observations At approximately 1, 2.5, and 4 hours after test material administration and daily thereafter for at least 14 days for clinical signs and twice daily (a.m. and p.m.) for mortality. Observations may be extended when directed by the study director. (2) Body Weights Before experimental initiation, at 7 and 14 days after test material administration, and at death (when survival exceeds 1 day) 0. Pathology At termination of the experimental phase, surviving animals will be euthanized. All animals, whether dying during the study or euthanized, will be subjected to an abbreviated gross necropsy examination and all abnormalities will be recorded. After necropsy, the animals will be discarded and no tissues will be saved. 005679 25 CHW 61101149 TP2069 Page 7 E- Statistical Analyses Other than LDS0 calculations (when applicable) no statistical analyses are required. 7. Report A final report Including those Items listed below will be submitted. Description of the test material Description of the test system Procedures Dates of experimental initiation and termination Tabulation of mortality data by sex and dose level Description of any toxic effects Tabulation of mean body weights by sex and dose level LD.0 calculations for each sex with 95% confidence intervals (when applicable) Gross pathology flndings/gross pathology report (when applicable) 8. location of Raw Data. Records, and Final Report Original data, or copies thereof, will be available at HWI to facilitate auditing the study during Its progress and before acceptance of the final report. When the final report Is completed, all original paper data, including those items listed below will be retained in the archives of HWI according to HWI SOP. Protocol and protocol amendments Dose preparation records In-life records Body weights Dose administration Observations Anatomical pathology records Study correspondence Final report (original signed copy) The following supporting records will be retained at HWI but will not be archived with the study data. Animal receipt/acclimation records Water analysis records Animal room temperature and humidity records Refrigerator and freezer temperature records Instrument calibration and maintenance records 005680 26 CHW 61101149 PROTOCOL APPROVAL TP2069 Page 8 Oohrt L. Butenhoff, PhD ' Sponsor's Representative 3H Steven H. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. _---- TUiuM/i S . Representative Quality Assurance Unit Hazleton Wisconsin, Inc. (TP2069.3M) Date _______ Q ///9* Date 005681 27 CHW 61101149 CHW No. C * t r o i t ` -ti PROTOCOL AMENDMENTS Amendment No. I Effective AT, Portion of Protocol Being Modified: Applicable sections of the protocol. Reason for Modification: To identify the location where the study will be conducted and to reflect a company name change from Hazleton Wisconsin, Inc. (HWI) to Corning Hazleton Inc. fCHWl. replace wherever applicable the following changes Modification: ________ Corning Hazleton Inc. fCHWl_______________________________ 3301 Kinsman Boulevard. ____________________________ Madison. WI 53704________________________________________ (G21/01-07-91) Study Director Approval: y. G5GS2 28 CHW 61101149 CHW No. PROTOCOL AMENDMENTS > 0 /1 ^ Amendment No. Q Effective___f^cHen/Tee. ~is /yc. Portion of Protocol Beinq Modified: Paoe 4. 6. Exoerimental Desion: A. Animals m Strain/Source Reason for Modification: To correctlv identify the nomenclature used for animals recelvec from Charles River Laboratories. Inc. Modificaticin: Reolace this section with the followina chanae: ___ It ) Str'ain/Source K Crl:CD(SD)BR/Charles River Laboratories, Inc. Hsd:Soraoue Dawlev SD/Harlan Soraoue Dawlev. Inc. (G21/01-07-91) Study Director Approval: 'Wiy-- 005G83