Document v1pwEEe4p6xDaEKo70GgVzV9w

aulison, WI. 53707-7545 tliveries: 3301 Kinsman Blvd., Madison, WI 53704 608.241.4471 608.241.7227 Fax A IU X -0 M 2 0 C O R N IN G Hazleton Sponsor: 3M St. Paul, Minnesota FINAL REPORT Study Title: Acute Oral Toxicity Study o f T-6669 in Rats (OECD Guidelines) A uthor: Steven M. Glaza Study Completion Date: January 10, 1997 Performing Laboratory: Corning Hazleton Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: CHW 61001760 Page 1 o f 35 id JAN I 3 B97 i 1 000051 COMPLIANCE STATEMENT Acute Oral Toxicity Study o f T-6669 in Rats (OECD Guidelines) CHW 61001760 This study was conducted in accordance with the Organisation for Economic Cooperation and Development and Principles of Good Laboratory Practice, C(81)30(Final) with the exception that analysis o f the test material mixtures for concentration, homogeneity/solubility and stability was not conducted. Acute Studies Coming Hazleton Inc. Date 2 QUALITY ASSURANCE STATEMENT CHW 61001760 This report has been reviewed by the Quality Assurance Unit o f Coming Hazleton Inc., in accordance with the Organisation for Economic 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 Phase 10/24/96 10/24/96 Necropsy 12/29/96 12/30/96 Data/Report Review Date Reported to Study Date to Director Management 10/24/96 12/30/96 10/24/96 12/30/96 _____ / - 10-71 Representative, Quality Assurance Unit Date CQ0053 3 STUDY IDENTIFICATION Acute Oral Toxicity Study o f T-6669 in Rats (OECD Guidelines) CHW 61001760 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-6669 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 Roger G. Perkins, PhD, DABT 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612) 733-3222 Steven M. Glaza Coming Hazleton Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Corning Hazleton Inc. 3301 Kinsman Boulevard Madison, WI 53704 October 9, 1996 October 10, 1996 November 14, 1996 January 10, 1997 January 10, 1997 4 CC0C54 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. Horton Supervisor KEY PERSONNEL Quality Assurance Sherry R. W. Petsel Manager CHW 61001760 Laboratory Animal Medicine Cindy J. Cary, DVM Diplomate, ACLAM Supervisor Anatomical Pathology Thomas E. Palmer, PhD Anatomical Pathologist Deborah L. Pirkel/ Jack Serfort Supervisors Necropsy Anne Mosher Supervisor Pathology Data 00005 5 CONTENTS CHW 61001760 COMPLIANCE STATEMENT...................................................................................................2 QUALITY ASSURANCE STATEM ENT.................................................................................3 STUDY IDENTIFICATION....................................................................................................... 4 KEY PERSONNEL....................................................................................................................... 5 O B JEC TIV E.................................................................................................................................. 8 TEST M ATERIAL........................................................................................................................ 8 Identification............................................................................................................................... 8 Purity and Stability..................................................................................................................... 8 Storage and Retention............................................................................................................... 8 Safety Precautions.....................................................................................................................8 TEST SY STEM .............................................................................................................................9 Test A nim al................................................................................................................................ 9 H ousing....................................................................................................................................... 9 Animal D ie t................................................................................................................................ 9 Animal Selection and Grouping............................................................................................... 9 Justification for Species Selection......................................................................................... 10 PROCEDURES............................................................................................................................ 10 Preparation and Administration o f Test M aterial................................................................ 10 Reason for Route o f Administration......................................................................................10 Observations............................................................................................................................. 10 P a th o lo g y .................................................................................................................................. 10 Statistical Analyses..................................................................................................................11 Location o f Raw Data, Records, and Final Report.............................................................. 11 RESU LTS..................................................................................................................................... 11 M ortality................................................................................................................................... 11 Body W eights........................................................................................................................... 11 Clinical Signs............................................................................................................................ 11 Pathology.................................................................................................................................. 12 DISCU SSIO N ...............................................................................................................................12 6 CO0056 CHW 61001760 SIGNATURE............................................................................................................................... 12 REFERENCE............................................................................................................................... 13 PATHOLOGY REPORT............................................................................................................ 14 TABLE 1 Mortality Summary............................................................................................................ 15 2 Individual and Mean Body Weights/ Body Weight Gains (g )...................................... 16 3 Individual Clinical Signs.................................................................................................... 18 4 Individual Pathology Comments...................................................................................... 21 A P P E N D IX ...................................................................................................................................23 Protocol T P 2069.....................................................................................................................24 Protocol Amendment No. 1 ...................................................................................................33 Protocol Amendment No. 2 ...................................................................................................34 Protocol Amendment No. 3 ...................................................................................................35 00005? 7 OBJECTIVE CHW 61001760 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 of Coming Hazleton (CHW) Inc. Standard Operating Procedure (SOP). TEST MATERIAL Identification The test material was identified as T-6669 and described as a white powder. Purity and Stability The Sponsor assumes responsibility for purity and stability determinations (including under test conditions). Analysis o f the test material mixture for concentration, homogeneity/solubility and stability was not conducted Storage and Retention The test material was stored at room temperature. Any unused test material will be returned to the sponsor after issuance of the final report according to CHW SOP. Safety Precautions The test material handling procedures were according to CHW SOPs and policies. 8 000058 TEST SYSTEM CHW 61001760 Test Animal Young adult albino rats o f the Crl.CD (SD)BR strain were procured from Charles River Laboratories, Inc. on September 23, October 7, October 21, 1996 (Portage, Michigan facility) and on October 1 and October 15, 1996 (Kingston, New York 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, PMI 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 of the water are periodically analyzed by CHW. 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 Ten male and 10 female healthy, acclimated rats, weighing from 208 to 269 g and approximately 8 to 12 weeks o f age, were assigned to two treatment groups o f 250 and 500 mg/kg o f body weight. Each dose level consisted o f 5 male and 5 female rats. The animals were identified by animal number and corresponding ear tag throughout the study. 9 ___________________________________________________________________ CHW 61001760 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 The test material was mixed with distilled water to a concentration o f 0.025 g/mL for the 250 mg/kg dose level and 0.050 g/mL for the 500 mg/kg dose level. The prepared test material mixtures appeared to be solutions. An individual dose o f the respective test mixture was calculated for each animal based on its fasted body weight and administered by gavage at a volume o f 10 mL/kg o f body weight. The test material mixtures were stored at room temperature until administered. 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 and mortality checks 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 of Day 14. Body weights were determined before test material administration (Day 0). Additional body weights were determined at Day 7, at termination o f the in-life phase (Day 14), or at death when survival exceeded 1 day. Pathology At termination o f the respective in-life phase for each dose level, all surviving animals were euthanized. All animals, whether found dead during the study or euthanized, were 10 coooso CHW 61001760 subjected to an abbreviated gross necropsy examination and any abnormalities were recorded. After necropsy, the animals were discarded and no tissues were saved. Statistical Analyses No statistical analyses were required by the protocol. 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 o f CHW in accordance with CHW SOP. RESULTS Mortality A summary o f the observed mortality is in Table 1. No mortality was observed at the 250 mg/kg dose level. Two males and all five females treated at 500 mg/kg were found dead within 4 days o f test material administration. No other mortality was observed. Based on the observed mortality, the estimated oral LD50 values were determined to be greater than 500 mg/kg for males and between 250 and 500 mg/kg for females. Body Weights Individual and mean body weights and body weight gains are in Table 2. All surviving animals exhibited body weight gain throughout the study. Clinical Signs Individual clinical signs are in Table 3. All animals treated at 250 mg/kg appeared normal during the study with the exception o f two females which exhibited red-stained face and/or wet urogenital area within 24 hours o f test material administration. Clinical signs o f toxicity observed in the animals treated at 500 mg/kg included red-stained face, yellowstained or wet urogenital area, hypoactivity, hunched posture, staggered gait, excessive cooosi CHW 61001760 salivation, and death. The surviving animals treated at 500 mg/kg returned to a normal appearance by Day 7. Pathology Individual gross necropsy pathology findings are in Table 4. A summary report by the study pathologist is on Page 14. There were no test material related lesions observed at necropsy. DISCUSSION The acute oral toxicity of T-6669 was evaluated in male and female rats when administered as a single gavage dose at levels o f 250 and 500 mg/kg o f body weight. Based on the observed mortality, the estimated oral LD50 values for male rats was determined to be greater than 500 mg/kg and between 250 and 500 mg/kg for females. All mortality occurred at the 500 mg/kg dose level within 4 days o f test material administration. All animals treated at 250 mg/kg appeared normal during the study with the exception o f two females which exhibited red-stained face and/or wet urogenital area within 24 hours o f treatment. Clinical signs of toxicity observed in the animals treated at 500 mg/kg included red-stained face, yellow-stained or wet urogenital area, hypoactivity, hunched posture, staggered gait, and excessive salivation. Animals surviving to the end of the observation period exhibited body weight gain during the study. The gross necropsy examinations did not reveal any test material related lesions. SIGNATURE Stever Study Director Acute Studies Date 12 C00062 CHW 61001760 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). 000062 13 PATHOLOGY REPORT CHW 61001760 There were 10 rats (five male, five female) each from dose levels o f 250 and 500 mg/kg necropsied. All animals given 500 mg/kg, except for three males, died on test. The surviving 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. At necropsy, there were few findings and all o f these were considered incidental and unrelated to the test material. In the animals that died on test, one male was partially cannibalized, one female had multiple, dark brown areas o f variable size in the glandular mucosa o f the stomach, and both horns o f the uterus in another female were filled with clear fluid. The pelvis o f both kidneys in one female given 250 mg/kg was observed to be large. There were no visible lesions in the remaining animals. Pathologist (61001760.fin) 121696 Date 14 0 0 0 0 6 4 Table 1 Mortality Summary CHW 61001760 Dose Level Mortality Results (mg/kg) _ Sex No. Died/ No. Dosed* 250 M 0/5 250 F 0/5 500 M 2/5, Days 3 1, 4 1 500 F 5/5, Days 0 1, l \ 3*,42 Superscript number indicates number o f animals found dead on the indicated day. 000065 15 Table 2 Individual and Mean Body Weights/ Body Weight Gains (g) CHW 61001760 Animal Number Day 0 Weight C12551 C12550 C12611 C12612 C12613 269 269 248 259 253 Mean 260 Day 7 Weight Gain* Males (250 mg/kg) 330 61 325 56 307 59 296 37 296 43 311 51 Day 14 Weight Gain* 383 114 339 70 375 127 354 95 375 122 365 106 Females (250 mg/kg) C12371 C12367 C12368 C12369 C12370 261 248 235 259 252 294 33 285 37 280 45 303 44 287 35 Mean 251 290 39 * Gain from Day 0 body weight. 315 54 294 46 285 50 309 50 288 36 298 47 16 C 0 0 0 S 6 Table 2 (Continued) Individual and Mean Body Weights/ Body Weight Gains (g) CHW 61001760 Animal Number Day 0 Weight C12231 C 12227 C l2228 C 12229 C 12230 213 221 208 217 215 Mean 215 Day 7 Weight Gain* Males (500 mg/kg) 249 (167)4 (168)3 271 274 36 - - 54 59 265 50 Day 14 Weight Gain* 302 89 --- 320 103 334 119 319 104 Females (500 mg/kg) C l2276 C l2277 C l2278 C l2279 Cl 2280 248 223 224 246 236 ( 2 3 1)3 (175)4 t (234)' (193)4 - - ---- -- Mean 235 -- -- Gain from Day 0 body weight. t Animal was found dead on the day o f dosing. No body weight required. ( ) Value in parentheses is a dead body weight and was not considered in calculating the mean. Superscript number indicates the day the animal was found dead. Not applicable. 000067 17 Table 3 Individual Clinical Signs CHW 61001760 Animal Number Observation C12551 Appeared normal C12550 Appeared normal C12611 Appeared normal Cl 2612 Appeared normal C12613 Appeared normal Hour______________________________________ Day 1.0 2.5 4.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Males (250 mg/kg) / y / / y / y / y / y C12371 Appeared normal C l 2367 Appeared normal Red-stained face C l 2368 Appeared normal CI2369 Appeared normal Red-stained face Wet urogenital area C l 2370 Appeared normal Condition existed. Condition not evident. / . - - / / -- - - - Females (250 mg/kg) y / y- / y / y / / y y 18 C0 0 0S8 Animal Number Observation C12231 Appeared normal Red-stained face Yellow-stained urogenital area C l 2227 Appeared normal Red-stained face Hypoactivity Hunched posture Staggered gait Found dead C12228 Appeared normal Red-stained face Hypoactivity Yellow-stained urogenital area Found dead C l 2229 Appeared normal Red-stained face C12230 Appeared normal / Condition existed. Condition not evident Table 3 (Continued) Individual Clinical Signs CHW 61001760 Hour 1.0 2.5 4.0 ----- Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M ales (500 mg/kg) -- - - - / - - -- / ----------- / ------- --- / --- -- - / - - - - - -- -- / - - - - - - / 19 0 00 0 69 Table 3 (Continued) Individual Clinical Signs CHW 61001760 Animal Number Observation Hour 1.0 2.5 4.0 Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 C 12276 Appeared normal Red-stained face Wet urogenital area Hypoactivity Hunched posture Found dead --------- --- C 12277 Appeared normal Red-stained face Wet urogenital area Hypoactivity Hunched posture Found dead - ---------- C l2278 Appeared normal Excessive salivation Found dead -- C 12279 Appeared normal / Red-stained face --- Wet urogenital area - - - Hypoactivity --- Staggered gait --- Found dead (after --- clinical observation) C 12280 Appeared normal Red-stained face --- Wet urogenital area - - - Hypoactivity --- Hunched posture --- Staggered gait --- Found dead - ** Condition existed. Condition not evident. Females (500 mg/kg) -- - - - - --- - - -- - ** - -/ - -/ - - * G00070 20 Table 4 Individual Pathology Comments Dose Level: 250 mg/kg of Body Weight CHW 61001760 Animal Number C12551 C12550 C12611 C12612 C12613 C12371 Sex M M M M M F C12367 F C12368 F C l2369 F C12370 F - Not applicable. Test Day Died Sacrificed 14 14 14 14 14 14 14 14 14 14 Necropsy Observation No visible lesions. No visible lesions. No visible lesions. No visible lesions. No visible lesions. Both o f the kidneys have a large pelvis. No visible lesions. No visible lesions. No visible lesions. No visible lesions. 000071 21 Table 4 (Continued) Individual Pathology Comments Dose Level: 500 mg/kg of Body Weight CHW 61001760 Animal Number C l2231 C l2227 C l2228 C l2229 C12230 C l2276 Sex M M M M M F Cl 2277 C l2278 C l2279 F F F C l2280 F - Not applicable. Test Day Died Sacrificed - 14 43- 14 - 14 3- 401- 4- Necropsy Observation No visible lesions. The right flank was cannibalized. No visible lesions. No visible lesions. No visible lesions. The glandular mucosa o f the stomach has multiple dark brown areas, up to 2 x 1 mm. No visible lesions. No visible lesions. The lumen in the bilateral horns of the uterus is filled with clear fluid. No visible lesions. 000072 22 APPENDIX Protocol TP2069 Protocol Amendment No. 1 Protocol Amendment No. 2 Protocol Amendment No. 3 CHW 61001760 000073 23 CHW 61001760 rEDICflL 2E 02 Sample iSubmittal Form This form Is to be used when submitting temples 1er routine acute testing. Special tasting needs can bo easily arranged By contacting the Acute Studies Department at BOB)241-72S2. chw study no. <<./.<=? 7 0 ca_______ E nelw Y ^sem pb* and send to: , _. w S o S l n K I 704 Submitted by: QE R f f c e f c f ^ S DataSampleSent: Com pany: ? t V \ ___________________________________________________ N um ber o l Reports Required: 3 ________ Full O L P Com pliance: * L Yee _____FDA (21 CFR SB) -------- No ----------EPA (TSC A -- 40C FR 7B 2) _____ EPA (FIFRA-- 40 CFR 160) -X -O E C O _____ MAFF --------- M O H W Sample Nam e: T~C t*(eQ_____________________________________________________ ________________________________ Physical Description: U i U l T g S^< O --------------------------------------------------------------------------- Special Handling Precautions: S E A r t S P S -2 -____________________________________ _ Test m aterial purity and stability f o r m a tio n (Including under test conditions) on (lie with Sponsor. J L . Y e s ____ No _2LTest m ixture analysis (or concentratlon/homogenelty/stablllly to be conducted: ____ Yes* by Sponsor ---------by CHW Sam ple Disposal: Return to Sponsor st following address: L <J. 7,ciceT r ?friSc_g>_________ U 53-3S-Q7- CLPTEJI . s f j w , jut* tN -ic Dispose ol according to CHW SOP* --i_ No Sample Storage Requirements: Room temperature _____Refrigerated ____ Other__________________________ * At additional oosl to Sponsor (C HW will contact Sponsor ss to these additional chargee). Tests A cute O rel Toxicity in Rate ____TP8084 Up end down LOSO procedure ____TP3206 FHSA screen: 6M-SF et 5.0 g/kg ____Conduct defined study Hdeath occurs at 5.0 g/kg ____TP3013 EPA screen; SM-SF at S.O g/kg ------ Conduct defined study It death occurs at S.0 g/kg _ 1 L t P206 O E C p screen; M -S F at 6.0 g/kg ____Conduct defined study Kdeath occurs at 6.0 ghtg Special Instructions; ---------------------------------------------------------------- Acute Dermal Toxicity In Rabbits ____TP3207 FHSA screen: 5M-5F at 2.0 g/kg ___ TP3018 EPA screen; SM-SF at 2 0 g/kg ____ Conduct defined study It death occurs at 2.0 g/kg ___ TP2070 OECD screen; SM-SF at 2.0 g/kg ____ Conduct defined study II deeth occure et 2.0 g/kg Special Instructions:______________________________________ For CHW Use Only Protocol Issue Date: Study Director: _ ______ nK. Primary Skin Irritation ____TP320B FHSA; 6 rebbks-1 abraded, 1 1ntact elts/rsbbk ____TP3014 EPA: 6 rabbits-1 Intact afte/ribbtt ____TP2071 OECD; 3 rabbns-1 Intact elta/rabbll ____TP4206 DOT corrosivity; 6 rabblta-l Intact alla/rabblt ____TP7145 Phototoxicity; 6 rabbks-2 Intad sltes/rabblt (one eke with UVA exposure) Special Instructions; - P rim ary E ye Irritation ,___ TP630 Low-volume proceduto; 6 rebbke unwashed ___ TP320S FHSA; B rabbits unwashed ____TP2012 1978 EPA; 6 rabbits unwashed. 3 washed ____TP3016 1BB2 EPA; 6 rebbtle unwashed ____TP2072 OECD; 3 rabbits unwashed ____3 rabbits washed at 4 seconde ------ 3 rabbits washed et 30 seconde Special Instructions:_______________________________ _ Guinea Pig Sensitization ___ TP2017 EPA Megnusson-Kllgman maximization ___ TP6I64.E C OECD/EC Magnussun-KIIgman maximization ___ TP2008 Bushier sensitization ___ TP626B PhotoallergenIc contact dermatitis (Armstrong) Special Instructions: ______________________________________ W hile copy-- C H W Yellow copy-- Submitter 000074 24 CHW 61001760 * O S T OF F I CE BOX 7 S4 S M A D IS O N . W IS C O N S IN 5 3 7 0 7-7545 , C O M K M O HOOrHW S w w * C o a w w PPH;or: 3M St. Paul, Minnesota PROTOCOL TP2069 Study Title: Acute Oral Toxicity Study in Rats (OECO Guidelines) Date: June 1, 1993 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: HWI C, ;<ao iTC-o 000075 25 CHW 61001760 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 C,io o i 7 c . o (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 -7 Week of /a-*7-^ Week of r a - ^ - ^ 000076 26 CHW 61001760 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 is administered by the oral route (gavage) to rats 3. Regulatory Compliance This study will be conducted in accordance with the following Good Laboratory Practice Regulations/Standards/Guidelines: [ J Conduct as a Nonregulated Study [ ] 21 CFR 58 (FDA) [ ] 40 CFR 160 (EPA-FIFRA) [ ] 40 CFR 792 (EPA-TSCA) M C (81 )30 (Final) (OECD) [ j 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. Oualitv 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 (HWI) Standard Operating Procedures (SOPs) and policies. 5. Test Material A. Identification (See sample submittal form) 8. 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. 000077 27 CHW 61001760 TP2069 Page 4 D. Storage (See sample submittal form) E. Reserve Samples Studies of less than 4 weeks in experimental duration will not have reserve samples retained. Reserve sample(s) of each batch/lot of test material will be taken if this study is more than 4 weeks in experimental duration. The test material reserve sample will be stored at HWI in a freezer set to maintain a temperature of below 0*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 txj" 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 000078 28 CHW 61001760 TP2069 Page 5 (7) Husbandry (a) Housing Separated by sex and group housed In screen-bottom stainless steel cages (heavy gauge) (b) Food Rodent Chow* #5001 (Purina Hills, Inc.) ad 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 in 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 000079 29 CHW 61001760 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 in 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 is 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. coooso 30 CHW 61001760 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 LDc0 calculations for each sex with 95* confidence Intervals (when applicable) Gross pathology findings/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 cooosi 31 CHW 61001760 PROTOCOL APPROVAL TP2069 Page 8 -- .7,-- John L. Butenhoff, PhD '' Sponsor's Representative 3M OatT 7 fJ __ ___________________________________________ ^VA~!> Steven H. G l a z a T ) Date Study Director Acute Toxicology Hazleton Wisconsin, Inc. ------ S Representative Quality Assurance Unit Hazleton Wisconsin, Inc. (TP2069.3M) ________ k /l/fa Date 0000S2 32 CHW 61001760 CHW No. PROTOCOL AMENDMENTS C . / Q G /7C.Cs Amendment No. I Effective Q c r c > g g < 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. (CHW1. replace wherever applicable the following changes Modification: _____________ Corning Hazleton Inc. fCHWl_______________________________ 3301 Kinsman Boulevard. Madison. WI 53704________________________________________ (G21/01-07-91) Study Director Approval: C000S3 33 CHW 61001760 CHW No. .'<=><a/7C.O PROTOCOL AMENDMENTS Amendment No. Effective Portion of Protocol Being Modified: Page 4. 6. Experimental Pesian: A. Animals (2) Strain/Source_______________ Reason for Modification: To correctly identify the nomenclature used for animals received from Charles River Laboratories. Inc.____________________________________ Modification: Replace this section with the following change: 21 Strain/Source P d Crl:CD(SD)BR/Charles River Laboratories, Inc. f Hsd:Spraoue Dawlev SD/Harlan Sprague Dawlev. Inc. (G21/01-07-91) Study Director Approval: 34 C-00034 CHW 61001760 CHW No. PROTOCOL AMENDMENTS 61001760 35 OOOOS5