Document g2go5QJDMD3OEr5p7L8Qon9M9

loa) OECD 414-OPPTS 870.3700 Pilot Study 418-023A FINAL PILOT REPORT SANITI ZED DEC 0 9 2003 ORAL (GAVAGE) DOSAGE-RANGE DEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) 6lRATS SPONSOR'S STUDY NUMBER: T-7485.1 I Author Raymond G. York, Ph.D., DABT (Study Director) Study Completed On 15 October 2003 (Audited Final Pilot Report) PerfominP Laboratory Argus Research 905 Sheehy Drive, Building A Horsham, Pennsylvania 19044-1297 Laboratory Proiect ID Argus Research, Protocol Number: 418-023P 418-023P:PAGE 2 GOOD LABORATORY PRACTICE STATEMENT This study was conducted according to U.S. Food and Drug Administration (FDA) Good Laboratory Practice Regulations; Final Rule (21 CFR Part 58), the Japanese Ministry of Health and Welfare Good Laboratory Practice Standardfor Safety Studies on Drugs, and the European Economic Community Council decision on 28 July 1989 on the acceptance by the European Economic Community of an OECD decisiodrecomrnendationon compliance with principles of good laboratorypractice or the Organization for Economic Cooperation and Development (OECD) Good Laboratory Practice in the Testing of Chemicals [C(97)186/Final]. Any areas of noncompliance are documented in the study record. No deviations existed that affected the validity of the study. kajond G. York, Associate Director Argus Research Date Study Director 4 18-023P:PAGE 3 PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGE DEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) IN RATS SPONSOR'S STUDY NUMBER: T-7485.11 TABLE OF CONTENTS SUBJECT PAGE ABSTRACT 5 I. Purpose 7 11. Methods 7 111. Results 8 IV. Conclusion 10 Figure 1. Maternal Body Weights 11 Table 1. Clinical Observations and Necropsy - Summary 12 Table 2. Maternal Body Weights and Gravid Uterine Weights - Summary 13 Table 3. Maternal Body Weight Changes - Gestation - Summary 15 Table 4. Maternal Absolute Feed Consumption Values (g/day) - Summary 16 Table 5. Maternal Relative Feed Consumption Values (g/kg/day) - Summary 17 Table 6. Caesarean-Sectioning Observations - Summary 18 Table 7. Litter Observations (Caesarean-Delivered Fetuses) - Summary 19 Table 8. Clinical Observations - Individual Data 20 Table 9. Necropsy Observations - Individual Data 22 Table 10. Maternal Body Weights and Gravid Uterine Weights - Individual Data 24 Table 11. Maternal Feed Consumption Values - Individual Data 29 SUBJECT Table 12. Caesarean-Sectioning Observations - Individual Data 418-023P:PAGE 4 PAGE 31 Table 13. Litter Observations (Caesarean-Delivered Fetuses) - Individual Data 33 Table 14. Fetal'Sex, Vital Status and Body Weight - Individual Data 35 ATTACHMENT 1 - PROTOCOL A,ND AMENDMENT 40 ATTACHMENT 2 - ANALYTICAL REPORT 70 ATTACHMENT 3 - QUALITY ASSURANCE STATEMENT 81 TITLE: 418-023P:PAGE 5 ORAL (GAVAGE) DOSAGE-RANGE DEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) IN RATS ARGUS RESEARCH PROTOCOL NUMBER: 418-023P SPONSOR'S STUDY NUMBER: T-7485.11 ABSTRACT Forty presumed pregnant Crl:CDB(SD)IGS BR VAF/PlusB female rats were randomly assigned to five dosage groups (Groups I through V), eight rats per group. Suspensions of the test substance, Potassium Perfluorobutane Sulfonate (PFBS or T-7485), or the vehicle, aqueous 0.1YOcarboxymethylcellulose, were administered orally (gavage) once daily to these naturally-bred female rats on days 6 through 20 of presumed gestation (DGs 6 through 20) at dosages of 0 (Vehicle), 100,300, 1000 and 2000 mgikg/day. The dosage volume was 10 mL/kg, adjusted daily on the basis of the individual body weights recorded immediately before administration of the test substance or vehicle. * Checks for viability were made twice daily. Clinical observations were recorded daily before dosage, approximately 60 10 minutes after administration and on the day of sacrifice. Body weights were recorded daily during the dosage period and the day of sacrifice. Feed consumption values were recorded on DGs 0, 6, 9, 12, 15, 18 and 21. All rats were sacrificed on DG 2 1 and examined for the number and distribution of corpora lutea, implantation sites and uterine contents. The gravid uterus was excised and weighed. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Fetuses were weighed and examined for gross external alterations and sex. All rats survived to scheduled sacrifice. Clinical observations considered test substancerelated were limited to urine-stained abdominal fur in four rats in the 2000 mg/kg/day dosage group. Persistent adverse clinical observations were confirmed at necropsy. No additional gross lesions were identified. Rats in the 2000 mg/kg/day dosage group lost weight on DGs 6 to 9 and body weight gains were reduced at several intervals tabulated during the dosage period. Reflecting these effects of the test substance, body weight gain was reduced in the 2000 mg/kg/day dosage group for the entire treatment (DGs 6 to 21) and gestation (DGs 0 to 2 1) periods. Absolute and relative feed consumption values were reduced in the 2000 mg/kg/day dosage group on DGs 6 to 9 , 9 to 12, 12 to 15, 15 to 18 and 18 to 21, and for the entire treatment and gestation periods. 4 18-023P:PAGE 6 Fetal body weights were reduced in the 2000 mg/kg/day dosage group, as compared with the control group. No other Caesarean-sectioning or litter parameters were affected by dosages of the test substance as high as 2000 mg/kg/day. No fetal gross alterations occurred. I. Purpose 418-023P:PAGE 7 The purpose of this study was to provide information for the selection of dosages to be used in the developmental toxicity (embryo-fetal toxicity and teratogenic potential) study of Potassium Perfluorobutane Sulfonate (PFBS) administered orally via gavage to Crl:CDB(SD)IGS BR VAF/PlusB presumed pregnant female rats. 11. Methodsa The test substance, Potassium Perfluorobutane Sulfonate (PFBS or T-7485), a white powder, was received on 14 December 2000, and was stored at room temperature. The vehicle, 0.19'0 carboxymethylcellulose (CMC, medium viscosity), was prepared using reverse osmosis membrane processed deionized water (R.O. deionized water). The carboxymethylcellulose, an off-white powder, was received from Sigma Chemical Company, St. Louis, Missouri, on 10 October 1999, and was stored at room temperature. Test substance and vehicle formulations were prepared approximately every 10 days and stored refrigerated (2OC to SOC). Results of the concentration, homogeneity and stability analyses are available in ATTACHMENT 2. The 10 and 30 mg/mL samples were all found to be within f 10% of the target concentrations; the 100 mg/mL samples were 88.9% and 105% of target and the 200 mg/mL samples were 99.2% and 76.8% of target. Forty presumed pregnant Crl:CDB(SD)IGS BR VAF/PlusB female rats were randomly assigned to five dosage groups (Groups I through V), eight rats per group. Suspensions of the test substance or the vehicle were administered orally (gavage) once daily to these naturally-bred female rats on days 6 through 20 of presumed gestation (DGs 6 through 20) at dosages of 0 (Vehicle), 100,300, 1000 and 2000 mg/kg/day. The dosage volume was 10 mL/kg, adjusted daily on the basis of the individual body weights recorded immediately before administration of the test substance or vehicle. Checks for viability were made twice daily. Clinical observations were recorded daily before dosage, approximately 60 f 10 minutes after administration and on the day of sacrifice. Body weights were recorded daily during the dosage period and the day of sacrifice. Feed consumption values were recorded on DGs 0 , 6 , 9 , 12, 15, 18 and 21. All rats were sacrificed on DG 2 1 and examined for the number and distribution of corpora lutea, implantation sites and uterine contents. The gravid uterus was excised and weighed. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Fetuses were weighed and examined for gross external alterations and sex. a. Detailed descriptions of all procedures used in the conduct of this study are provided in the appropriate sections of this report and in the attached protocol and amendments. Deviations from the Protocol and the Standard Operating Procedures of the Testing Facility are available in the raw data. 111. Results 418-023P:PAGE 8 A. Mortality, Clinical and Necropsy Observations (Summary - Table 1; Individual Data - Tables 8 and 9) All rats survived to scheduled sacrifice. Clinical observations considered test substancerelated were limited to urine-stained abdominal fur in four rats in the 2000 mg/kg/day dosage group. Observations of red perinasal substance, red perioral substance, excess salivation and gasping in one rat in the 2000 mg/kg/day dosage group and localized alopecia on the limbs in one rat in the.300 mg/kg/day dosage group were not considered test substance related. Persistent adverse clinical observations were confirmed at necropsy. No additional gross lesions were identified. B. Maternal Body Weights, Body Weight Changes and Gravid Uterine Weights (Figure 1; Summaries - Tables 2 and 3; Individual Data - Table 10) Rats in the 2000 mg/kg/day dosage group lost body weight on DGs 6 to 9 and body weight gains were reduced at several intervals tabulated during the dosage period. Reflecting these effects of the test substance, body weight gain was reduced in the 2000 mg/kg/day dosage group for the entire treatment period (calculated as DGs 6 to 2 1) and the entire gestation period (calculated as DGs 0 to 21), compared to control group values. Gravid uterine weights were reduced (15.4%) for the rats in the 2000 mg/kg/day dosage group, compared to the control group. Reflecting this effect of the test substance, the corrected DG 2 1 body weight (DG 2 1 weight minus gravid uterine weight) was reduced (1 1.8%) in the 2000 mg/kg/day dosage group. Corrected body weight gain for the entire treatment period (calculated as DGs 6 to 2 1C) and the entire gestation period (calculated as DGs 0 to 21C) were also reduced in the 2000 mg/kg/day dosage group, compared to control group values. Body weights, body weight gains and gravid uterine weights were comparable in the 0 (Vehicle), 100,300 and 1000 rng/kg/day dosage groups. C. Maternal Absolute .(g/day) and Relative (g/kg/day) Feed Consumption Values (Summaries - Tables 4 and 5; Individual Data - Table 11) Absolute and relative feed consumption values were reduced in the 2000 mgikglday dosage group on DGs 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 2 1, compared to control group values. Reflecting this effect of the test substance, absolute feed consumption values for the entire treatment period (calculated as DGs 6 to 21) were reduced (19.2%) and the entire gestation period (calculated as DGs 0 to 21) were reduced (16.4%), compared to control group values. Relative feed consumption values for the entire treatment period were reduced (13.7%) and the entire gestation period were reduced (1 1.3%), compared to control group values. 418-023P:PAGE 9 Absolute and relative feed consumption values were comparable in the 0 (Vehicle), 100, 300 and 1000 mg/kg/day dosage groups. D. Caesarean-Sectioning and Litter Observations (Summaries - Tables 6 and 7; Individual Data - Tables 12 through 14) Caesarean-sectioning observations were based on 8 (1OO%), 7 (87.5%),8 (loo%), 7 (87.5%)and 8 (100%) pregnant rats in the 0 (Vehicle), 100, 300, 1000 and 2000 mg/kg/day dosage groups, respectively. Male and female fetal body weights were reduced (12.2% and 13.0%, respectively), in the 2000 mg/kg/day dosage group, as compared with the control group. There were no other test substance-related effects on the following parameters evaluated at Caesarean-sectioning: litter averages for corpora lutea, implantations, litter sizes, live fetuses, early resorptions, percent resorbed conceptuses and percent live male fetuses. There were no dead fetuses or late resorptions, and no dams had whole litter resorptions. All placentae appeared normal. Totals of 121,96, 119, 101 and 118 live fetuses in the 0 (Vehcle), 100, 300, 1000 and 2000 mg/kg/day dosage groups, respectively, were examined externally for developmental alterations. No fetal gross alterations were identified. IV. Conclusion 418-023P:PAGE 10 Based on these data dosages of 0 (Vehicle), 100,300 and 1000mg/kg/day of PFBS are recommended for the developmental toxicity study in rats. The 100 mg/kg/day dosage is expected to be a no-observable-adverse-effect-level(NOAEL) for both maternal and embryo-fetal toxicity, and the 1000 mg/kg/day dosage is expected to produce minimal maternal toxicity and little or no developmental toxicity. an M. Hoberman, Ph.D., DABT Date J of Research Associate Director of k m & r c h and Study Director PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGE DEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANESULFONATE (PFBS) IN RATS (SPONSORS STUDY NUMBER: T-7485.11) 450 __---- -- MATERNAL BODY WEIGHTS Figure 1 U + 0 MGlKGlDAY 400 -I- 100 MGlKGlDAY 350 CI c3 v I1: c3 300 -0- 300 MGlKGlDAY --tI000 MGIKGIDAY -%E- 2000 MGlKGlDAY 250 P c Q A n 200 - T A T l b 1'1 112 15 114 15 DAY OF GESTATION 18 1'9 do $1 PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGEDEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBWANE SULFONATE (PFBS) IN RATS (SPONSOR'S STUDY NUMBER: T-7485.11) TABLE 1 (PAGE 1): CLINICAL OBSERVATIONS AND NECROPSY - SUMMARY P cN PROTOCOL 4 1 8 - 0 2 3 P : ORAL (GAVAGE)DOSAGE-RANGEDEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) IN RATS (SPONSOR'SSTUDY NUMBER: T - 7 4 8 5 . 1 1 ) TABLE 2 (PAGE 1 ) : MATERNAL BODY WEIGHTS AND GRAVID UTERINE WEIGHTS - SUMMARY MATERNAL BODY WEIGHT (0) DAY 0 MEANkS.D. 242.6 f 8.5 242.4 f 1.2 243.2 f 8.0 240.1 f 7.8 242.5 f 7.5 DAY 6 MEANfS.D . 279.9 f 7.8 281.3 f 5 . 1 287.1 f 8.9 278.3 f 7.6 280.0 f 13.8 DAY 7 MEANfS.D . 282.8 f 7.6 283.1 f 8.2 287.9 f 8.1 282.8 f 10.0 279.4 f 18.0 DAY 8 DAY 9 MEANfS.D. MEANfS .D. 288.8 f 7.2 293.2 f 8.9 289.0 f 4.7 295.0 f 6.8 293.9 f 7.4 297.2 f 9.5 286.6 f 9.5 291.8 f 9.4 279.4 t 283.4 f 19.7 7lb f 18.4 [ 7lb DAY 1 0 MEANfS.D. 302.2 f 6.9 303.7 f 8.4 305.8 f 8 . 1 296.1 f 11.2 292.1 f 18.2 DAY 11 MEANkS.D. 309.9 f 6.6 [ 61c 306.7 f 6.1 315.2 f 7.7 305.6 f 9.7 [ 7lb 297.6 f 13.9 t 71b DAY 1 2 MEANfS .D. 318.5 f 8.6 316.7 f 9.6 325.1 f 10.4 312.1 f 13.3 301.4 f 23.0 [ 7lb DAY 1 3 DAY 1 4 DAY 15 DAY 16 MEANfS .D. MEANfS .D. MEAN&. D. MEANfS.D. 323.1 f 9.5 327.9 i 9.6 337.1 * 11.3 347.8 f'10.9 318.6 f 7.8 326.8 f 10.7 335.6 110.3 346.0 f 10.5 330.1 f 9.3 334.0 f 9.4 345.2 f 9.4 354.1 f 11.8 319.8 f 15.6 325.0 f 13.2 332.3 f 15.1 338.7 f 19.4 306.8 [ 309.8 [ 318.3 [ 323.6 f 21.9 7lb f 21.3 7lb f 21.5 7lb f 23.7 - DAY 1 7 MEANfS .D. 360.9 f 11.7 356.0 f 10.0 367.5 f 11.0 353.7 f 19.8 [ 71b 334.1 f 22.9 [ 7lb DAY 1 8 MEANfS.D. 375.5 f 11.9 371.4 f 10.1 382.2 f 11.2 368.8 f 19.0 349.7 f 21.5 P DAY 1 9 MEANfS .D. 391.8 i 9.9 387.3 f 13.6 400.4 f 15.4 385.8 f 21.3 I 7lb 352.3 f 27.2 03 b [ 71b N .................................................................................................................................... cI> DAY = DAY OF GESTATION [ I = NUMBER OF VALUES AVERAGED a. Dosage occurred on days 6 through 20 of gestation. b . Excludes values for dam 13987 due to apparent injury. c. Excludes a value that appeared incorrectly recorded. w w PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGE DEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) IN RATS (SPONSOR'S STUDY NUMBER: T - 7 4 8 5 . 1 1 ) TRBLE 2 (PAGE 2) : MATERNAL BODY WEIGHTS AND GRAVID UTERINE WEIGHTS - SUMMARY GROUP I DOSAGE (MG/KGfDAY)a ..................................... -- 0 RATS TESTED N PREGNANT N 8 MATERNAL BODY WEIGHT (G) I DAY 20 DAY 21 MEANkS.D . . MEANkS D. 409.4 f 12.2 436.9 f 14.3 GRAVID UTERINE WEIGHTS DAY 21C c MEAN+S .D . MEANLS .D . 111.2 & 9.6 325.6 13.7 I1 100 7 400.8 k 15.2 427.6 f 18.5 100.9 i 7 . 3 326.6 i 17.5 I11 300 8 4 1 6 . 4 f 13.5 435.6 f 15.1 1 0 7 . 9 A 10.1 327.7 5 15.1 IV 1000 7 404.4 f 2 2 . 7 425.4 f 19.2 1 0 1 . 0 % 14.8 324.4 i 14.8 8 363.8 f 26.4 [ 71b 375.6 f 27.1 [ 7lb 94.1 2 10.7 2 8 7 . 3 26.1 P PROTOCOL 410-623P: ORAL (GAVAGE) DOSAGE-RANGEDEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBVPANE SULFONATE (PFBS) IN RATS (SPONSOR'S STUDY NUMBER: T-7405.11) TABLE 3 (PAGE 1) : MATERNAL BODY WEIGHT CHANGES - GESTATION - SUMMARY PREGNANT N 0 7 0 7 0 MATERNAL BODY WEIGHT CHANGE (GI DAYS 0 - 6 MEANAS.D . t37.2 f 5.2 t30.8 f 6.7 t43.9 2 0.0 t30.1 2 9.0 t37.5 2 9.7 DAYS DAYS 6- 9 9 - 12 DAYS 12 - 15 DAYS 15 - 10 DAYS 10 - 21 DAYS 6 - 21 DAYS DAYS 0 - 21 6 - 21C C! . MEANfS D. MEAN@. D. MEANfS.D . MEANfS . D. MEANfS .D. . MEANfS .D MEANfS.D . MEANLS .D . t13.4 f 2.6 t25.2 f 3.6 t10.6 f 3.0 t30.4 f 3.6 t61.4 f 6.9 t157,Of 10.8 t194.2f 14.2 t45.8 5 9.2 +13.7 f 3.9 t21.7 f 5.1 +10.0 f 5.1 t35.8 f 5.3 t56.1 f 9.8 t146.3f 15.9 t185.1f 17.8 t45.3 A 15.7 t10.1 f 10.1 t27.9 f 11.9 t20.1 f 5.0 t37.0 f 5 . 5 t53.4 f 13.2 t148.5f 14.6 t192.4f 18.1 t40.6 5 16.0 t13.6 f 7.1 t20.3 f 4.7 t20.1 f 7.4 t36.6 f 5 . 9 t56.6 f 3.2 t147.1* 14.5 tie5.3f 2 0 . 2 t46.1 2 13.2 t5.6 f 10.9 [ 71b +18.0 f 12.4 7lb t16.8 f 6.3 I 71b t31.4 f 5.7 [ 71b t25.8 f 25.6 [ 7lb t97.7 f 24.5 I 7lb t134.1f 23.1 [ 7lb t7.3 f 19.8 DAYS 0 - 21C C . MEANLS. D 403.0 2 13.6 t84.2 5 16.6 t84.5 5 17.6 t04.3 5 14.0 t44.8 2 21.2 .................................................................................................................................... DAYS = DAYS OF GESTATION a. Dosage occurred on days 6 through 20 of gestation. b. Excludee values for dam 13907 due to apparent injury. c. 21C = Corrected maternal body weight (day 21 of gestation body weight minus the gravid uterine weight). PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGEDEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) IN RATS (SPONSOR'S STUDY NUMBER: T-7485.11) TABLE 4 (PAGE 1) : MATERNAL ABSOLUTE FEED CONSUMPTION VALUES (G/DAY) - SUMMARY GROUP DOSAGE (MG/KG/DAY)a -____-________________ RATS TESTED 8 8 PREGNANT N 8 7 MATERNAL FEED CONSUMPTION (G/DAY) DAYS 0 - 6 MEAN@. D. 23.1 5 0.8 22.8 5 1.1 DAYS 6 - 9 MEANkS . D. 24.5 1: 1.0 23.6 f 2.2 DAYS 9 - 12 MEAN*S. D . 26.7 f 1.1 24.9 f 2.9 DAYS 12 - 15 MEANfS.D. 26.0 f 1.0 26.4 f 2.3 DAYS 15 - 18 MEAN@. D . 28.8 f 2.2 26.6 f 1.2 DAYS 18 - 21 MEAN@ .D. 26.4 f 1.8 25.7 f 2.5 DAYS 6 - 21 MEAN*S. D . 26.5 t 0.9 25.5 f 2.0 DAYS 0 - 21 MEANkS . D. 25.5 f 0.6 24.7 f 1.7 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - _ _ _ _ ~ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ------- - - DAYS = DAYS OF GESTATION [ 1 = NUMBER OF VALUES AVERAGED a. Dosage occurred on days 6 through 20 b. Excludes a value that was associated c. Excludes values for dam 13987 due to of gestation. with spillage. apparent injury. IV 1000 _. 8 8 7 V 2000 8 23.4 2 1.9 24.6 f 1.7 26.9 * 1.6 26.2 f 1.7 27.1 f 2.0 25.8 f 3.3 26.1 f 1.3 25.4 f 1.3 ----__----_- 22.5 5 1.0 23.6 f 2.7 25.0 f 2.1 24.9 f 1.4 26.2 f 2.3 25.9 f 2.9 25.1 f 1.7 24.4 f 1.2 20.9 2.9 I 7lb r22.2 f 6.0 71c r 23.0 f 2.6 71c 21.4 f 2.5 I 71c 23.1 i 2.5 I 71c 17.8 f 5.5 [ 21.5 *71c 2.1 [ 71c 21.3 f 2.0 _ _ _ _t_ _7_1_c _ _ _ _ _ _ - _ PREGNANT N 8 7 MATERNAL FEED CONSUMPTION (G/ KG/ DAY) DAYS 0 - 6 MEAN+S .D. 88.4 3.9 87.0 % 4.0 DAYS 6 - 9 MEANfS.D. 85.7 f 4.0 82.3 f 6.3 DAYS 9 - 1 2 DAYS 1 2 - 15 MEANiS.D. MEANkS ,D. 87.2 f 4.0 79.6 f 2.0 81.4 f 8 . 1 81.5 f 5.7 DAYS 15 - 1 8 DAYS 18 - 21 MEANkS .D. MEANiS .D. 81.2 f 6.7 65.5 f 3.4 75.6 f 2.6 64.7 f 5.0 DAYS DAYS 6 - 21 0 - 21 MEANkS .D. MEANfS .D. 78.7 f 2.5 77.1 f 2.2 76.2 f 4.6 75.2 f 4.1 ............................................................................. DAYS = DAYS OF GESTATION [ ] = NUMBER OF VALUES AVERAGED a . Dosage occurred on days 6 through 2 0 of g e s t a t i o n . b . ExCludes a value t h a t was a s s o c i a t e d with s p i l l a g e . c . Excludes values for dam 1 3 9 8 7 due t o apparent i n j u r y . 8 8 8 . 4 f. 6 . 7 84.4 f 6.9 86.5 f 4.8 78.4 f 5.0 74.8 f 5.1 63.3 f 8.2 76.3 f 4.2 75.4 f 4.2 7 8 86.7 2 2.9 82.7 f 8.6 82.8 f 4.7 7 7 . 4 f 1.6 75.0 f 3.8 6 5 . 3 2: 7 . 3 15.7 f 3.6 80.6 5 12.0 [ 71b 79.0 f 20.1 [ 7lc 78.2 f 6.9 [ 71c 6 9 . 2 f 5.3 I 71c 69.7 f 5.9 I 71c 4 9 . 2 f: 1 4 . 2 I 71c 68.1 f 4 . 4 [ 7lc 68.6 f 5.2 _ - _ - t- _ -7 _l c_ _ _ - _ _ - ..b h w , 'd +d PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGEDEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) IN RATS (SPONSOR'SSTUDY NUMBER: T-7485.11) TABLE 6 (PAGE 1): CAESAREAN-SECTIONING OBSERVATIONS - SUMMARY GROUP DOSAGE (MG/KG/DAY)a RATS TESTED N PREGNANT N(%) RATS PREGNANT AN0 CAESAREAN-SECTIONED ON DAY 21 OF GESTATION CORPORA LUTEA IMPLANTATIONS N MEAN2S.D . MEANkS.D. LITTER SIZES MEXi5S.D. LIVE FETUSES N MEANkS.D . DEAD FETUSES RESORPTIONS EARLY RESORPTIONS LATE RESORPTIONS N MEANkS .D , . N MEP.N+s. D N DAMS WITH ANY RESORPTIONS N(%) DAMS WITH ALL CONCEPTUSES RESORBED N(%) DAMS WITH VIABLE FETUSES N(%) PLACENTAE APPEARED NORMAL N (k 3 8 19.5 2 2.5 15.5 5 1.7 15.1 k 1.4 121 15.1 2 1.4 0 0.4 5 0 . 5 3 0.4 2 0.5 0 3 ( 37.5) O( O'.O) 8 (100.0) e(loo.0) 7 18.6 k 4.4 13.7 2 1.1 13.7 5 1.1 96 13.7 2 1.1 0 0.0 2 0.0 0 0.0 5 0.0 0 O( 0 . 0 ) O ( 0.0) 7 (100.0) 7 (100.0) 8 (100.0) 8 19.6 5 2.3 15.4 5 1.6 14.9 2 1.6 119 14.9 2 1.6 0 0.5 2 1.1 4 0.5 5 1.1 0 2( 25.0) O ( 0.0) 8 (100.0) 8 (100.0) 7( 87.5) e(loo.0) 7 19.1 2 4.4 14.6 2 2.6 14.4 2 2.5 101 14.4 5 2.5 0 0.1 A 0.4 1 0.1 2 0.4 0 1( 14.3) 8 17.6 2 1.5 14.9 5 0.8 14.8 2 0.9 118 14.8 2 0.9 0 0.1 2 0.4 1 0.1 2 0.4 0 1( 12.5) O ( 0.0) 7 (100.0) 7 (100.0) O( 0.0) 8 (100.0) _ _ _ _8 _(1_00_._0)_ _ _ - - _ _ _ _ _2_ 00 PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGE DEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) IN RATS (SPONSOR'S STUDY NUMBER: T-7485.11) TABLE 7 (PAGE 1) : LITTER OBSERVATIONS (CRESAREAN-DELIVERED FETUSES) - SUMMARY GROUP DOSAGE (MG/KG/DAY)a __-__^___________________ LITTERS WITH ONE: OR MORE LIVE FETUSES N ---- IMPLANTATIONS MEANkS .D . 8 15.5 2 1.7 LIVE FETUSES LIVE MALE FETUSES N MEANkS . D . N 121 15.1 1.4 47 S LIVE MALE FETUSES/LITTER MEANkS .D . 39.2 2 11.1 LIVE FETAL BODY WEIGHTS (GRAMS)/LIWER MALE FETUSES FEMALE FETUSES MEANkS .D . . MEAN2S.D MEANkS .D . 5.30 2 0.22 5.43 2 0.24 5.22 & 0.21 % RESORBED CONCEPTUSES/LIWER MEANkS .D . 2.2 5 3.1 I1 100 7 13.7 2 1.1 96 13.7 2 1.1 47 48.7 2 7.2 5.38 2 0.40 5.54 0.41 5.22 2 0.43 0.0 0.0 111 300 8 15.4 k 1.6 119 14.9 & 1.6 52 44.2 5 10.8 5.25 2 0.20 5.40 5 0 . 2 2 5.14 5 0.22 3.1 5 6.1 IV 1000 7 14.6 5 2.6 101 14.4 & 2.5 58 58.0 15.6 5.21 2 0.36 5.30 0.38 5.12 5 0.38 0.9 5 2.3 V 2000 8 14.9 5 0.8 118 14.8 5 0.9 59 50.7 5 18.8 4.64 2 0.35 4.77 0.40 4.54 5 0.34 0.8 2.4 P .. cb PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGEDEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) IN RATS (SPONSOR'SSTUDY NUMBER: T-7485.11) TABLE 8 (PAGE 1) : CLINICAL OBSERVATIONS - INDIVIDUAL DATA E & N W .td. M N 0 PROTOCOL 419-023P: ORAL (GAVAGE) DOSAGE-RANGEDEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) IN RATS (SPONSOR'SSTUDY NUMBER: T-7495.11) TABLE 8 (PAGE 2 ) : CLINICAL OBSERVATIONS - INDIVIDUAL DATA PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGEDEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUORbBUTANE SULFONATE (PFBS) IN RATS (SPONSOR'S STUDY NUMBER: T-7485.11) TABLE 9 (PAGE 1): NECROPSY OBSERVATIONS - INDIVIDUAL DATA .................................................................................................................................... GROUP RAT DAY OF PREGNANCY DOSAGES DOSAGE (MG/KG/DAY) NUMBER NECROPSY STATUS ADMINISTERED OBSERVATIONS a .................................................................................................................................... I 0 13951 DG 21 P 15 ALL TISSUES APPEARED NORMAL. 13952 DG 2 1 P 15 ALL TISSUES APPEARED NORMAL. 13953 DG 21 P 15 ALL TISSUES APPEARED NORMAL. 13954 DG 21 P 15 ALL TISSUES APPEARED NORMAL. 13955 DG 21 P 15 ALL TISSUES APPEARED NORMAL. 13956 DG 21 P 15 ALL TISSUES APPEARED NORMAL. 13957 DG 2 1 P 15 ALL TISSUES APPEARED NORMAL. 13958 DG 21 P 15 ALL TISSUES APPEARED NORMAL. I1 100 13959 DG 21 P 15 ALL TISSUES APPEARED NORMAL. 13960 DG 21 P 15 ALL TISSUES APPEARED NORMAL. 13961 DG 2 1 NP 15 ALL TISSUES APPEARED NORMAL. 13962 DG 21 P 15 ALL TISSUES APPEARED NORMAL. 13963 DG 21 P 15 ALL TISSUES APPEARED NORMAL. 13964 DG 21 P 15 ALL TISSUES APPEARED NORMAL. 13965 DG 21 P 15 ALL TISSUES APPEARED NORMAL. 13966 DG 2 1 P 15 ALL TISSUES APPEARED NORMAL. 111 300 13967 DG 2 1 P 15 ALL TISSUES APPEARED NORMAL. 13968 DG 21 P 15 ALL TISSUES APPEARED NORMAL. 13969 DG 2 1 P 15 ALL TISSUES APPEARED NORMAL. 13970 DG 2 1 P 15 ALL TXSSUES APPEARED NORMAL. 13971 DG 2 1 P 15 ALL TISSUES APPEARED NORMAL. 13972 DG 21 P 15 ALL TISSUES APPEAFJD NORMAL. 13973 DG 21 P 15 ALL TISSUES APPEARED NORMAL. - 13974 DG 21 P __________________-_------------------ 15 ALL TISSUBS APPEARED NORMAL. DG = DAY OF PRESUMED GESTATION P = PREGNANT NP NOT PREGNANT a. Refer to the individual clinical observations table (Table 8) for external observations confirmed at necropsy. N N PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGEDEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE IPFBS) IN RATS (SPONSOR'SSTUDY NUMBER: T-7485.11) TABLE 9 (PAGE 2): NECROPSY OBSERVATIONS - INDIVIDUAL DATA --________________._____________________------------------------- GROUP RAT DAY OF PREGNANCY DOSAGES DOSAGE (MG/KG/DAY) NUMBER NECROPSY STATUS ADMINISTERED OBSERVATIONS a .................................................................................................................................... IV 1000 13975 13976 13977 13978 13979 13980 13 981 13982 DG 21 P DG 21 P DG 21 P DG 21 P DG 21 NP DG 21 P DG 21 P DG 21 P 15 ALL TISSUES APPEARED NORMAL. 15 ALL TISSUES APPEARED NORMAL. 15 ALL TISSUES APPEARED NORMAL. 15 ALL TISSUES APPEARED NORMAL. 15 ALL TISSUES APPEARED NORMAL. 15 ALL TISSUES APPEARED NORMAL. 15 ALL TISSUES APPEARIZD NORMAL. 15 ALL TISSUES APPEARED NORMAL. V 2000 13983 DG 21 P 15 ALL TISSUES APPEARED NORMAL. 13984 Dc 21 P 15 ALL TISSUES APPEARED NORMAL. 13985 DG 21 P 15 ALL TISSUES APPEARED NORMAL. 13986 M: 21 P 15 ALL TISSUES APPEARED NORMAL. 13987 DG 21 P 15 ALL TISSUES APPEARED NORMAL. 13988 DG 21 P 15 ALL TISSUES APPEARED NORMAL. 13989 . DG 21 P 15 ALL TISSUES APPEARED NORMAL. 13990 DG 21 P ......................................................... _ _ _ _ _1_5 _ - _ _ _ _AL_L_T_I_SSUES APPEARED NORMAL. -- DG = DAY OF PRESUMED GESTATION P = PREGNANT NP = NOT PREGNANT a. Refer to the individual clinical observation8 table (Table 8 ) for external observations confirmed at necropsy N w PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGE DmLOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE ( P F B S ) I N RATS (SPONSOR'S STUDY NUMBER: T-7485.11) TABLE 1 0 (PAGE 1 ) : MATERNAL BODY WEIGHTS AND GRAVID UTERINE WEIGHTS - INDIVIDUAL DATA DAY 18 19 20 21 GRAVID UTERINE WEIGHTS ____________________--------------------------------------------------*------------------------- 13951 P 13952 P 377. 359. 390. 377. 406. 388. 432. 423. 111.2 90.2 13953 P 394. 409. 427. 460. 115.3 13954 P 361. 385. 403. 426. 122.5 13955 P 388. 400. 422. 453. 115.8 13956 P 375. 396. 415. 434. 115.8 13957 P 376. 386. 410. 445. 111.4 13958 P 374. 391. 404. 422. 107.6 ................................................................................................ P = PREGNANT NP = NOT PREGNANT (VALUES EXCLUDED FROM AVERAGES) DAY = DAY OF PRESUMED GESTATION ALL WEIGHTS WERE RECORDED I N GRAMS ( G I . h) P PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGE DEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) I N RATS (SPONSOR'S STUDY NUMBER: T-7485.11) TABLE 1 0 (PAGE 2): MATERNAL BODY WEIGHTS AND GRAVID UTERINE WEIGHTS - INDIVIDUAL DATA _^-_____________________________________-------------------------------------------------------------------------------------------- RAT # GROUP I1 loo MG/KG/DAY .................................................................................................................................... PREGNANCY STATUS DAY 0 6 I 8 9 10 11 12 13 14 15 16 17 .................................................................................................................................... 13959 P 252. 291. 294. 295. 306. 316, 310. 332, 326. 345. 347. 358. 371. 13960 P 245. 280. 281. 286. 290. 296. 305. 311. 317. 326. 330. 341. 352. 13961 NP 13962 P 243. 237. 297. 285. 302. 289. 304. 291. 306. 296. 311. 307. 304. 308. 315. 316. 304. 323. 303. 324. 308. 334. 302. 344. 300. 364. 13963 P 243. 281. 288. 294. 302. 309. 314. 322. 328. 336. 350. 357. 360. 13964 P 13965 P 231. 240. 211. 218. 269. 283. 282. 289. 287. 292. 272.a 300. 291. 312. 304. 323. 306. 318. 313. 326. 327. 339. 337. 354. 347. 356. 13966 P 249. 277. 278. 286. 292. 294. 301. 309. 312. 318. 322. 331. 342. .................................................................................................................................... DAY 18 19 20 21 GRAVID UTERINE WEIGHTS .................................................................................................................................... 13959 P 390. 411. 420. 460. 103.1 13960 P 13961 NP 363. 308. 368. 311. 374. 309. 403. 307. - 9- -0-.-5 13962 P 370. 384. 405. 418. 112.7 13963 P 13964 P 13965 P 316. 366. 375. 392. 389. 391. 412. 395. 408. 438. 425. 434. 100.7 94.3 105.4 13966 P 360. 376. 392. 415. 99.9 .................................................................................................................................... P = PREGNANT N P = NOT PREGNANT (VALUES EXCLUDED FROM AVERAGES) DAY = DAY OF PRESUMED GESTATION ALL WEIGHTS WERE RECORDED I N GRAMS ( G I . a. V a l u e appeared incorrectly recorded and was excluded from group averages and statistical analyses. P N cn PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGEDEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) IN RATS (SPONSOR'S STUDY NUMBER: T-7485.11) TABLE 10 (PAGE 3 ) : MATERNAL BODY WEIGHTS AND GRAVID UTERINE WEIGHTS - INDIVIDUAL DATA DAY 18 19 20 21 GRAVID UTERINE WEIGHTS ........................................................................................ 13967 P 388. 412. 431. 449. 120.3 13968 P 369. 380. 401. 420. 113.0 13969 P 394. 414. 420. 447. 107.1 13970 P 374. 389. 406. 436. 97.5 13971 P 391. 412. 426. 459. 115.7 13972 P 387. 498. 422, 432. 108.6 13973 P 365. 378. 396. 426. 89.3 . .1 . . .1.3.9.7.4. .P. . . . . . . . .3.9.0... . . . . .4.1.0... . . . . .4.2.9... . . . . .4.1.6... . . . . . . . . . . . . . . 1. 1. 1. .. .8. . . . . . . . . . . . . . . . . . P = PREGNANT NP = NOT PREGNANT (VALUES EXCLUDED FROM AVERAGES) DAY = DAY OF PRESUMED GESTATION ALL WEIGHTS WERE RECORDED IN GRAMS (GI. PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGEDEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) IN RATS (SPONSOR'SSTUDY NUMBER: T-7485.11) TABLE 10 (PAGE 4): MATERNAL BODY WEIGHTS AND GRAVID UTERINE WEIGHTS - INDIVIDUAL DATA P Y PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGEDEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) IN RATS (SPONSOR'S STUDY NUMBER: T-7485.11) TABLE 10 (PAGE 5): MATERNAL BODY aIGHTS AND GRAVID UTERINE WEIGHTS - INDIVIDUAL DATA 00 b N w N 00 E: W 0 PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-QANGE DEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) IN RATS (SPONSOR'S STUbY NUMBER: T-7485.11) TABLE 12 (PAGE 1): CAESAREAN-SECTIONING OBSERVATIONS - INDIVIDUAL DATA w c PROTOCOL 4 1 8 - 0 2 3 P : ORAL (GAVAGE) DOSAGE-RANGEDEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBVPANE SULFONATE (PFBS) IN RATS (SPONSOR'SSTUDY NUMBER: T - 7 4 8 5 . 1 1 ) TABLE 1 2 (PAGE 2 ) : CAESAREAN-SECTIONINGOBSERVATIONS - INDIVIDUAL DATA 13975 9 7 9 7 16 0 0 0'0 0 0 0 0 0 9 7 16 12 11 2 3 1 3 9 7 6 11 5 10 6 16 0 0 0 0 0 0 0 0 0 10 6 16 17 10 27 13977 10 5 5 10 15 0 0 0 0 0 0 0 0 5 11 16 1 3 9 7 8 5 11 6 10 16 0 0 0 0 0 0 0 0 6 12 18 1 3 9 7 9 NOT PREGNANT 13980 6 8 9 5 14 0 0 0 0 0 0 0 0 13981 6 3 2 7 9 0 0 0 0 0 0 0 0 10 6 16 6 9 15 1 3 9 8 2 11 4 3 12 15 0 0 0 0 1 1 0 0 __________________-_____________________-----_------------------------------------- _5_ - - 1_4- _ _ 1 9 GROUP V 2000 MG/KG/DAY ................................................................................... ............................. ___--_________ 13983 9 5 7 7 14 0 0 0 0 0 0 0 0 0 7 7 14 8 9 17 13984 4 12 7 9 16 0 0 0 0 0 0 0 0 0 7 9 16 7 10 17 1 3 9 8 5 5 11 6 10 16 0 0 0 0 0 0 0 0 0 6 10 16 6 11 1 7 13986 8 6 6 8 14 0 0 0 0 0 0 0 0 0 6 8 14 6 10 16 13987 10 5 8 7 15 0 0 0 0 0 0 0 0 0 8 7 15 10 11 2 1 1 3 9 8 8 11 3 9 5 14 0 0 0 0 0 0 0 0 0 9 5 14 10 7 17 . 13989 6 9 7 a 15 0 0 0 0 0 0 0 0 0 7 8 15 9 9 18 13990 6 8 10 4 14 0 0 0 1 0 1 0 0 0 11 4 15 12 6 18 PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGEDEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) IN RATS (SPONSOR'S STUDY NUMBER: T-7485.11) TABLE 1 3 (PAGE 1): LITTER OBSERVATIONS (CAESARERN-DELIVERED FETUSES) - INDIVIDUAL DATA 15 5.52 5.29 5.35 15 0 0.0 12 5.36 5.06 5.22 12 0 0.0 16 5.29 5.07 5.11 16 0 0.0 17 5.27 5.10 5.18 18 15 5.86 5.66 5.75 16 1 5.6 1 6.2 16 5.47 5.04 5.20 16 0 0.0 15 5.60 5.37 5.48 15 0 0.0 15 5.08 5.19 5.14 16 1 6.2 .................................................................... I O M~G/KG/DAY .................................................................... 14 5.40 5.27 5.34 14 0 0.0 13 5.25 4.96 5.12 13 0 0.0 14 6.03 5.66 5.85 14 13 5.93 5.52 5.68 13 12 5.92 5.71 5.80 12 15 5.22 4.84 5.02 15 15 5.04 4.62 4.87 15 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGE DEVEELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBWANE SULFONATE ( P F B S ) I N RATS (SPONSOR'S STUDY NUMBER: T-7485.11) TABLE 1 3 (PAGE 2 ) : LITTER OBSERVATIONS ICAESAF@AN-DELIVERED FETUSES) - INDIVIDUAL DATA 13975 13976 13977 13978 13979 13980 13981 13982 _ _ -- _ _ - - - - - - _ - - - _ - - - - 4.99 4.76 4.89 4.81 4.57 4.74 5.41 5.32 5.38 5.59 5.20 5.32 5.48 5.82 4.97 5.30 5.70 4.97 5.38 5.78 4.97 __---_ 0.0 0.0 0.0 0.0 0.0 0.0 6.2 0 N W w P PROTOCOL 4 1 8 - 0 2 3 P : ORAL (GAVAGE) DOSAGE-RANGEDEVELOPMElTI'AL TOXICITY STUDY OF-POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) IN RATS (SPONSOR'S STUDY NUMBER: T - 7 4 8 5 . 1 1 ) TABLE 14 (PAGE 1): FETAL SEX, VITAL STATUS AND BODY WEIGHT - INDIVIDUAL DATA .................................................................................................................................... GROUP I o MG/KG/DAY _--__-___-______________________________-------------------------------------------------------------------------------------------- FETUS# 1 2 3 4 5 6 7 8 9 1 0 11 1 2 1 3 1 4 1 5 1 6 17 1 8 1 9 20 2 1 22 2 3 .................................................................................................................................... RAT # CLs 1 3 9 5 1 1 0 / 8 FA FA MA MA FA FA FA / MA FA FA FA FA FA MA FA 13952 7/10 5.37 5.19 5.69 5.63 5.31 5.46 5.43 5.24 5.38 4.91 5.42 4.75 5.60 5.51 5.34 MA FA MA FA MA MA / FA MA MA FA FA FA 5.12 4 . 6 6 5.42 5.11 5.25 5.66 5.04 5.24 5.50 5.38 5.05 5.15 1 3 9 5 3 6/16 FA FA FA FA FA / FA MA FA FA MA FA MA FA FA FA FA 4 . 9 9 4.86 4.97 5.46 4.96 5.22 5.09 5.07 5.03 5.33 4.96 5.44 4.89 5.01 5.35 5.14 1 3 9 5 4 6 / 1 5 FA MA FA MA MA FA / E FA MA MA FA FA FA FA MA MA MA FA 13955 8/10 4.15 4.06 4.02 5.39 5.54 5.37 MA FA MA FA FA FA 5 . 2 0 5.63 5.43 5 . 0 8 5.40 5 . 1 4 4.88 5 . 3 2 4 . 9 8 5.04 5 . 2 5 MA FA / MA FA MA E FA MA FA MA 13956 10/12 5 . 8 1 5.65 5.93 5.55 5.15 5.66 6.19 5.46 5.67 5.65 5.80 FA FA FA FA FA MA / MA MA FA FA FA 5.82 5.13 5.72 5.86 FA MA FA MA MA 5 . 0 7 5 . 1 4 4 . 9 8 5 . 0 1 5 . 0 9 5 . 4 8 5 . 2 0 5 . 4 4 3.03 5 . 2 0 5 . 3 2 4 . 5 8 5 . 2 7 5 . 0 3 5 . 4 7 5 . 9 7 1 3 9 5 7 8 / 8 FA MA MA MA MA MA FA FA / MA FA MA FA FA FA FA 5.57 5.64 5.60 5.50 5.66 5.45 5.05 5.50 5.58 5 . 2 1 5.75 5 . 6 9 5.40 5.28 5.23 1 3 9 5 8 1 0 / 1 2 MA FA FA MA MA MA / FA FA E FA MA FA FA FA MA FA 4 . 4 4 5.38 5.49 5.16 5.32 5.36 4.16 5.04 5.50 5.15 5.04 4.97 5.11 5.02 5.40 ________________________________________-------------------------------------------------------------------------------------------- M r MALE F = FEMALE A = ALIVE E = EARLY RESORPTION " / " DENOTES POSITION OF CERVIX CLS = CORPORA LUTEA/OVARY FETAL BODY WEIGHTS WERE RECORDED IN GRAMS ( G ) . ? w cn PROTOCOL 4 1 8 - 0 2 3 P : ORAL (GAVAGE) DOSAGE-FlANGE DEVELOPMENTAL TOXICITY STUDY OF POTASSItJM PERFLUOROBUTANE SULFONATE (PFBS) I N RATS (SPONSOR'S STUDY NUMBER: T - 7 4 8 5 . 1 1 ) TABLE 1 4 (PAGE 2 ) : FETAL SEX, VITAL STATUS ANC BODY WEIGHT - INDIVIDUAL DATA 1 3 9 6 2 1 0 / 8 FA FA MA FA FA MA FA MA / MA MA MA MA FA FA 13963 6/12 5.41 5 . 6 1 6.32 5.89 5.79 6.02 5.53 6.09 6.16 6.08 5.69 5.88 5.58 5 . 8 4 FA MA FA MA / MA FA FA FA FA MA MA FA FA 13964 4/ 8 5 . 4 7 5 . 9 0 5 . 6 2 6.18 6 . 0 3 5 . 1 7 5.34 5 . 4 6 5 . 5 8 6 . 0 8 5 . 4 7 5 . 7 5 5 . 8 1 FA FA MA FA / MA FA FA MA MA MA FA FA 5 . 7 6 5 . 6 5 6 . 1 3 5 . 7 5 5 . 6 5 5 . 6 2 5 . 1 8 6.03 6.06 5 . 7 2 5 . 5 9 5 . 8 3 1 3 9 6 5 6 / 1 2 MA FA FA FA MA MA / FA FA FA MA FA MA FA MA MA 5.15 5.11 5.08 4.79 5.06 5.39 4.41 4.59 4.84 5.13 4.82 5.20 5.12 5.18 5.46 1 3 9 6 6 9/13 MA FA MA FA MA MA / MA FA FA MA MA FA MA F A MA _ _ _ _ _ _ _ _ _ _ _ _5_._2 8_ _4_. 6_2_5_._0 7_ _4_. 7_3_5_._1 5_ _5_.1_1_4_.9_5_ 4_._6 2_ _4-.3- 4- - -5-. -2-7- -5-.-0-3- -4-.-9-5- -4-.-4-9- -4-.-4-6- -5-.-0-5- - - - - - - - - - - - - - - - - - - M = CLS MALE F = FEMALE A = CORPORA LUTEA/OVARY = RLIVE FETAL E = BODY EARLY RESORPTION 'I/" DENOTES WEIGHTS WERE RECORDED I N GRAMS . POSITION (G) OF CERVIX P c M 537b N W 'd PROTOCOL 418-023P: ORAL (GAVAGE) DOSAGE-RANGEDEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) IN RATS (SPONSOR'S STUDY NUMBER: T-7485.11) TABLE 14 (PAGE 3): FETAL SEX, VITAL STATUS AND BODY WEIGHT - INDIVIDUAL DATA RAT # CLs 13967 7/15 13968 9/11 13969 9/12 13970 10/12 13971 8/ 9 13972 7/10 13973 5/12 13974 6/15 MA 5.27 FA 5.34 MA 5.16 MA 5.63 FA 5.13 FA 5.16 MA 5.27 MA 5.33 FA FA FA 5.06 4.89 4.94 FA FA MA 5.49 5.65 5.71 MA FA MA 5.01 5.30 5.12 E MA FA 5.72 5.48 MA FA FA 5.43 5.29 5.45 MA MA FA 5.39 5.61 5.19 FA FA / FA 5.37 5.14 5.18 FA MA FA 4.86 5.42 5.15 FA FA FA / MA FA 5.07 4.86 5.09 5.15 4.77 FA FA FA / MA FA 5.02 5.28 5.52 5.79 5.14 FA FA MA FA / FA 5.33 4.92 5.24 5.02 4.86 FA / FA FA FA MA 5.39 5.33 5.50 5.15 5.56 MA E MA MA / FA 5.85 6.03 5.86 4.74 MA FA / FA FA FA 4.90 5.15 4.76 5.27 5.18 FA MA FA M A M A 4.77 5.12 5.37 5.15 5.50 FA / FA MA FA MA 4.91 4.33 5.04 4.49 5.17 FA 4.89 FA 5.46 FA 4.83 E MA 5.64 FA 5.19 MA 5.83 FA 4.75 MA 5.34 MA 5.60 MA 4.88 FA 5.38 FA 5.81 FA 4.79 MA 5.36 FA 4.97 FA FA 4.86 4.81 FA MA 5.30 5.75 MAMA 4.96 5.27 MAE 5.82 FA FA 5.30 5.13 MAMA 5.29 5.18 MA 5.15 MA FA 5.33 4.73 FA 4.96 MA 5.46 MA 5.51 MA 5.20 MA 5.70 FA 5.25 MA 5.22 MA 5.38 MA 5.38 MA 5.26 FA 4.89 FA 5.40 MA 5.19 MA 5.39 MA 4.84 FA 5.58 FA 5.72 MA 5.03 FA 5.08 FA 5.61 PROTOCOL 4 1 8 - 0 2 3 P : ORAL (GAVAGE) DOSAGE-RANGE DEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBmANE SULFONATE (PFBS) IN RATS (SPONSOR'S STUDY NUMBER: T - 7 4 8 5 . 1 1 ) TABLE 1 4 (PAGE 4 ) : FETAL SEX. VITAL STATUS AND BODY WEIGHT - INDIVIDUAL DATA 13980 10/ 6 13981 6/ 9 13982 5/14 FA 5.06 MA 5.97 MA 5.50 FA MA MA 5.16 5.40 5.46 MA / FA MA 5.87 5.56 5.99 FA FA / MA 5.39 5.17 4.13 FA 5.29 FA 5.80 FA 4.63 MA 5.52 MA 5.8'7 MA 4.95 MA 5.31 FA 5.74 FA 4.70 FA 5.26 MA 5.53 MA 5.08 MA / FA 5.56 5.02 MA 5.66 MA E 4.82 FA 5.65 MA 4.97 MA 5.66 MA 5.09 FA 5.24 MA 4.89 FA 5.69 MA 5.12 MA 4.85 MA 5.24 M = MALE F = FEMALE A = ALIVE E = EARLY RESORPTION " / " DENOTES POSITION OF CERVIX CLS = CORPORA LUTEA/OVARY FETAL BODY WEIGHTS WERE RISCORDED I N GRAMS ( G ) . w oa PROTOCOL 418-023P: ORAL (GAVAGE)DOSAGE-RANGE DEVELOPMENTAL TOXICITY STUDY OF POTASSIUM PERFLUOROBUTANE SULFONATE (PFBS) IN RATS (SPONSOR'S STUDY NUMBER: "-7485.11) TABLE 14 (PAGE 5): FETAL SEX, VITAL STATUS AND BODY WEIGHT - INDIVIDUAL DATA .................................................................................................................................... GROUP V 2000 MG/KG/DAY .................................................................................................................................... FETUS# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 ______________________________________r_-------------------------------------------------------------------------------------------- RAT # C L s 13983 E/ 9 MA MA MA MA FA FA MA / MA MA FA MA FA FA MA 4.10 4.48 4.52 4.71 4.13 3.95 4.29 4.17 4.30 4.35 4.43 4.22 4.29 4.34 13984 7/10 FA FA FA FA FA MA MA / FA FA FA MA FA FA MA FA FA 4.28 4.87 4.32 4.97 4.63 4.91 5.07 4.65 4.55 4.53 5.16 4.40 4.66 4.64 4.86 4.82 13905 6/11 Mh Mh FA FA FA FA / MA FA FA FA MA MA FA FA FA FA 5.92 5.62 5.42 4.99 5.41 4.98 5.51 5.04 5.04 5.13 5.02 5.23 5.35 5.15 5.31 5.35 13986 6/10 MA MA MA MA FA MA / FA MA FA FA FA FA MA MA 5.39 5.35 5.44 5.11 4.71 5.10 4.87 5.03 4.97 4.63 4.42 4.80 4.96 4.98 13987 10111 MA FA MA MA FA MA FA FA / MA MA MA MA MA FA MA 5.01 4.70 4.58 4.15 4.62 4.18 4.33 4.12 4.59 4..46 4.17 4.38 4.25 4.39 4.84 1 13988 101 7 MA MA MA MA FA FA FA MA MA / MA MA MA MA MA 4.51 4.19 4.53 4.36 4.43 4.36 4.04 4.06 4.25 4.69 4.13 4.44 4.35 4.65 13989 9/ 9 FA MA FA FA FA FA FA 1 MA MA FA MA FA MA FA MA 4.53 4.91 4.50 4.56 4.44 4.41 2.66 4.82 4.40 4.54 4.93 4.17 3.26 3.95 4.86 13990 12/ 6 E FA FA FA FA MA MA MA MA FA FA / MA MA FA FA 4.82 4.74 4.41 4.49 4.87 4.75 4.74 4.56 4.38 4.93 5.24 4.86 4.94 4.40 .................................................................................................................................... M = MALE F = FEMALE A = ALIVE E = EARLY RESORPTION "ID"ENOTES POSITION OF CERVIX CLe = CORPORA LUTEA/OVARY FETAL BODY WIGHTS WERE RECORDED IN GRAMS ( 0 ) . .. m 4 18-023P:PAGE 40 ATTACHMENT 1 PROTOCOL AND AMENDMENT ~PRIMEDICA 418-023P:PAGE 4 1 Argus Research Lahoratories. Inc. 905 Sheehp Drive, Building A Horsham, PA I9044 Telephone: (2 15) 4443-87 IO Telefax: (215) 443-8587 PROTOCOL 418-023P SPONSOR'SSTUDY NUMBER: T-7485.11 STUDY TITLE: PURPOSE: TESTING FACILITY: STUDY DIRECTOR: SPONSOR: STUDY MONITOR: Oral (Gavage) Dosage-Range Developmental Toxicity Study of Potassium PerfluorobutaneSulfonate (PBSF) in Rats The purpose of this study is to provide information for the selection of dosages to be used in the developmental toxicity (embryo-fetal toxicity and teratogenic potential) study of Potassium Perfluorobutane Sulfonate (PBSF) administered orally via gavage to Crl:CD@(SD)IGS BR VAF/PIusB presumed pregnant female rats. Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, Pennsylvania 19044-1297 .Telephone: (215 ) 443-8710 Telefax: (215) 443-8587 Raymond G. York, Ph.D., DABT Associate Director of Research raymond.york@ primedica.com 3M Corporate Toxicology 3M Center, Building 220-2E-02 St. Paul, Minnesota 55144-1000 Ph.D., DABT 3M Corporate Toxicology 3M Medical Department Telephone: Telefax: REGULATORY ClTATlONS: 4 18-023P:PAGE 42 Protocol 418-023P Page 2 U.S. Environmental Protection Agency (1998). Health Effects Test Guidelines; Prenatal Developmental Toxicity Study. Office of Prevention, Pesticides and Toxic Substances (OPPTS) 870.3700, August, 1998. U.S. Environmental Protection Agency (1997). Toxic Substances Control Act (TSCA) Test Guidelines; Final Rule. Prenatal DevelopmentalToxicity, 799.9370 (crossreferencedto OPPTS 870.3700). Federal Register, August 15, 1997. Organization for Economic Cooperationand Development (1981). OECD Guidelines for Testing of Chemicals. Section 4, No. 414: Teratogenicity, adopted 12 May 1981. Japanese Ministy of Agriculture, Forestry and Fisheries (1985). Guidance on Toxicology Study Data for Application of Agricultural Chemical Registration. 59 NohSan No. 4200. U.S. EnvironmentalProtection Agency. Toxic Substances Control Act (TSCA); Good Laboratory Practice Standards; Final Rule. 40 CFR Part 792. U.S. Environmental Protection Agency. Federal Insecticide, Fungicide and Rodenticide Act (FIFRA); Good Laboratory Practice Standards; Final Rule. 40 CFR Part 160. Organization for Economic Cooperationand Development (1998). The Revised OECD Principles of Good Laboratory Practices [C(97)186/Final]. Japanese Ministry of Agriculture, Forestry and Fisheries (1984). Good Laboratory Practice Standards. 59 NohSan No. 3850. REGULATORY COMPLIANCE: This study will be conducted in compliance with the Good Laboratory Practice (GLP) regulations cited above. All changes or revisions of this protocol shall be documented, signed by the Study Director and the Sponsor, dated and maintained with the protocol. The Testing Facility's Quality Assurance Unit (QAU) will audit the protocol, the raw data and the report, and will inspect critical phases of those portions of the study conducted at the Testing Facility in accordance with the Standard Operating Procedures of Argus Research Laboratories, Inc. Should any portion of the study be conducted by a subcontractor or by the Sponsor, the QAU for that facility will conduct critical phase inspections and audit respective results and reports for that study portion according to the SOPSof that facility. Such critical phase inspection reports and report audits will be submitted by that facility to the Study 418-023P:PAGE 43 Protocol 418-023P Page 3 Director. The dates of the inspections and report submissions will be incorporated into a QAU Statement generated by that facility and provided to the Testing Facility for inclusion in the final report. The final report will include a compliance statement signed by the Study Director that the report accurately reflects the raw data obtained during the performance of the study and that all applicable GLP regulations were followed in the conduct of the study. Should significant deviations from GLP regulations occur, each will be described in detail, together with how the deviation might affect the quality or integrity of the study. SCHEMATIC OF STUDY DESIGN AND STUDY SCHEDULE: See AlTACHMENT 1 to the protocol. TEST SUBSTANCE AND VEHICLE: Identification: Test Substance: Potassium PerfluorobutaneSulfonate (PBSF o T-7485). Lot numb r will be documented in the raw data. The Sponsor will provide to the Testing Facility documentationor certification of the identity, composition, method of synthesis, strength and purity of the test substance. Vehicle: Aqueous 0.1% carboxymethylcellulose(CMC) (medium viscosity) prepared using reverse osmosis membrane processed deionized water (R.O. deionized water). Lot number will be documented in the raw data. Neither the Sponsor nor the Study Director is aware of any potential contaminants likely to be present in the vehicle that would interfere with the results of this study. Therefore, no analyses other than those mentioned in this protocol will be conducted. Safetv Precautions: Gloves, dust-mist/HEPA-filtered mask, appropriate eye protection and uniform/lab coat to be worn during formulation preparation and dosage. In addition, Tyvek sleeves and a half-face respirator will be worn and preparation will be conducted in a chemical fume hood when using the bulk test substance. The Material Safety Data Sheet (MSDS) is attachedto the protocol (ATTACHMENT2). Storaqe: 4 18-023P:PAGE 44 Protocol 418-023P Page 4 Bulk Test Substance: Bulk Vehicle Components: Prepared Test Article and Vehicle Formulations: Room temperature. Room temperature 2C to 8C All test substance shipments should be addressedto the attention of Julian Gulbinski, Manager of Formulations, at the previously cited Testing Facility address and telephone number. Shipments should include information concerning storage conditions and shipping cartons should be labeled appropriately. The recipient should be notified in advance of shipment. FORMULATlON: Frequency of Preparation: Formulations (suspensions) will be prepared (approximately every 10 days) at the Testing Facility. Detailed preparation procedures will be attachedto this protocol (ATTACHMENT 3). Adiustment for Puritv: The test substance will be considered 100% pure for the purpose of dosage calculations. Testinq Facilitv Reserve Samples: The Testing Facility will reserve a sample (1 g) of each lot of bulk test substance and bulk vehicle component used during the course of the study. Samples will be stored under the previously cited conditions. ANALYSES: Results of required analyses will be provided to the Testing Facility for inclusion in the study report. Samples additional to those described below may be taken if deemed necessary during the course of the study. Bulk Test Substance Sampling: 418-023P:PAGE 45 Protocol 418-023P Page 5 A sample (lg)of the test substance will be taken on the last day of treatment and sent (ambient conditions) to the Sponsor for analysis. This sample will be sent to: ~ Ph.D. 3M EnvironmentalTechnology & Safety Services 935 Bush Avenue Building 2-3E-09 St. Paul, Minnesota 55133-3331 Telephone: Telefax: The recipient will be notified in advance of sample shipment. Analvses of Prepared Formulations: Homoqeneitv: Homogeneity analyses of the prepared formulations will not be conducted. Information is on file with the Sponsor to document the homogeneity of the test substance in the prepared vehicle over the range of concentrations to be used in this study. This information will be provided to the Study Director for inclusion in the final report. Concentration: Concentration of the prepared formulations will be verified during the course of this study. Duplicate samples (2 mL each) will be taken from the first and last preparation on the day prepared. One sample of each set will be shipped for analysis; the remaining samples will be retained at the Testing Facility as backup samples. Backup samples will be stored under the previously cited conditions and discarded at the Testing Facility upon the request of the Sponsor. Stabilitv: Stability data for prepared formulations bracketing the range of concentrations in this study are on file with the Sponsor and will not be determined during the conduct of this study, This information will be provided to the Study Director for inclusion in the final report. Shippinq Instructions: a - 0 Protoco4l 1481-023P. ~A8GE 46 Page 6 Samples to be analyzed will be shipped (on cold packs) to previously cited address. Ph.D., at the The recipient will be notified in advance of sample shipment. DISPOSlTlON: Preparedformulations will be discarded at the Testing Facility. All remaining bulk test substance will be returned to: 3M SMMG EHS&R 3M Center Building 236-16-10 St. Paul, MN 55144-1000 TEST SYSTEM: SpeciedStrain and Reason for Selection: The Crl:CD@(SD)IGS BR VAF/PIu@ rat w a s selected as the Test System because: 1) it is one mammalian species accepted and widely used throughout industry for nonclinical studies of developmentaltoxicity (embryo-fetal toxicity/teratogenicity); 2) this strain has been demonstrated to be sensitive to developmentaltoxins; and 3) historical data and experience exist at the Testing Number: Initial population acclimated: Population selected for study: 60 virgin female rats. 40 mated female rats (8 per dosage group). Bodv Weiqht and Aqe: Female rats will be ordered to have body weights of 200 g to 225 g each at receipt, at which time they will be expected to be at least 60 days of age. Actual body weights recordedthe day after receipt will be documented in the raw data. - Sex: Female rats will be given the test substance. Male rats of the same source and strain will be used only as breeders and are not considered part of the Test System. Source: 4 18-023P:PAGE47 Protocol 418-023P Page 7 Charles River Laboratories, Inc. The rats will be shipped in filtered cartons by air freight and/or truck from Charles River Laboratories, Inc., to the Testing Facility. Identification: Rats are permanently identified using MoneIGD self-piercing ear tags (Gey Band and Tag Co., Inc., No. MSPT 20101). Male rats are given unique permanent identification numbers upon assignment to the Testing Facility's breeder male rat population. Female rats are assigned temporary numbers at receipt and given unique permanent identification numbers when assigned to the study on the basis of day 0 of presumed gestation body weights. ANIMAL HUSBANDRY: All cage sizes and housing conditions are in compliance with the Guide for the Care and Use of Laboratory Housinq: The rats will be individually housed in stainless steel wire-bottomed cages except during the cohabitation period. During cohabitation, each pair of rats will be housed in the male rat's cage. No nesting materials will be supplied because the female rats will be sacrificed before parturition is expected. Room Air, Temperature and Humidity: The animal room is independently supplied with at least ten changes per hour of 100% fresh air that has been passed through 99.97% HEPA filters. Room temperature will be maintained at 64F to 79F (18C to 26C)and monitored constantly. Room humidity will also be monitored constantly and maintained at 30% to 70%. Liqht: An automatically-controlled 12-hour light: 12-hour dark fluorescent light cycle will be maintained. Each dark period will begin at 1900 hours EST. - Diet: Rats will be given Certified Rodent Dieto #5002 (PMI Nutrition International) available ad libitum from individual feeders. Water: 4 18-023P:PAGE 48 Protocol 418-023P Page 8 Water will be available ad libitum from individual bottles attached to the cages or from an automatic watering access system. All water will be from a local source and passed through a reverse osmosis membrane before use. Chlorine will be added to the processedwater as a bacteriostat; processedwater is expected to contain no more than 1.2 ppm chlorine at the time of analysis. Water is analyzed monthly for possible bacterial contaminationand twice annually for possible chemical contamination. Contaminants: Neither the Sponsor nor the Study Director is aware of any potential contaminants likely to be present in the certified diet or in the drinking water at levels that would interfere with the results of this study. Therefore, no analyses other than those routinely performed by the feed supplier or those mentioned in this protocol will be conducted. RANDOMlZATlON AND COHABITATION: Upon arrival, male and female rats will be assigned to individual housing on the basis of computer-generatedrandom units. After acclimation, virgin female rats will be cohabited with breeder male rats, one male rat per female rat. The cohabitation period will consist of a maximum of five days. Female rats with spermatozoa observed in a smear of the vaginal contents andlor a copulatory plug observed in situ will be considered to be at day 0 of presumed gestation and assigned to individual housing. Healthy mated female rats will be assigned to dosage groups based on computergenerated (weight-ordered) randomization procedures. ADMINISTRATION: Route and Reason for Choice: The oral (gavage) route was selected for use because: 1) in comparison with the dietary route, the exact dosage can be accurately administered; and 2) it is one possible route of human exposure. Method and Frequency: Female rats will be given the test substance once daily on days 6 through 20 of presumed gestation. Dosages will be adjusted daily for body weight changes and given at approximately the same time each day. Rationale for Dosaqe Selection: 4 18-023P:PAGE 49 Protocol 418-023P Page 9 Dosages were selected on the basis of a 28-day (gavage) study of T-7485 in rats (Redfield Laboratories Study Number 132-007)with an approximate 14-day recovery period which resulted in no mortality or abnomal clinical observations as well as no changes in body weights, body weight changes, feed consumption, peripheral neuropathy, motor activity, audio/visual response, hematology,clinical chemistry, gross pathology and histopathology. The administration of 900 mg/kg/day T-7485 resulted in significant increases in male liver and female kidney weights when compared to control animals and establishes the no-observable-adverse-effect-level(NOAEL) at 300 mg/kg/day T-7485. The highest dosage level selected should induce some overt developmental and/or maternal mortality. The intermediate dosage levels should produce minimal observed toxic effects. The lowest dosage level should not produce any evidence of either maternal or developmental toxicity. Dosaqe Levels, Concentrations and Volumes: Group I II 111 IV V Number of Rats 8 8 8 8 8 Dosage (mgkglday) 0 100 300 1000 2000 Concentration (mglmL) 0 10 30 1 00 200 Volume (mVkg) 10 10 10 10 10 Argus Batch Number 8-41 8-023P-A(Day.Month.Year) 6-41 8-023P-B(Day.Month.Year) 5-41 8-023P-CIDav.Month.Yearl 5-418-023P-D(Day.Month.Year) - 6-418-023P-E(Day.Month.Year) TESTS, ANALYSES AND MEASUREMENTS: Viability: All Periods: At least twice daily. Clinical Observations and/or General Appearance: Acclimation Period: Weekly. Predosage Period: Day 0 of presumed gestation. I Dosage Period: Daily before dosage. Postdosage observations will be recorded approximately 60+10 minutes after administration. Day of Sacrifice: Once. 418-023P:PAGE 50 Protocol 418-023P Page 10 Clinical observations may be recorded more frequently than cited above, if deemed appropriate by the Study Director and/or Study Monitor. Bodv Weiqhts: Acclimation Period: Weekly. Predosage Period: Day 0 of presumed gestation. Dosage Period: Daily. Day of Sacrifice: Prior to sacrifice. Feed Consumption Values (recorded and tabulated): Predosage Period: Dosage Period: Day 0 of presumed gestation. Days 6, 9 12, 15, 18 and 21 of presumed gestation. Day of Sacrifice: Feed left recorded. Feed consumption values may be recorded more frequently if it is necessary to replenish the feed. These intervals will not be tabulated. Matinq Performance: Mating will be evaluated daily during the cohabitation period and confirmed by observation of spermatozoa in a smear of the vaginal contents and/or a copulatory plug observed in situ. Caesarean-Sectioninq Observations: Rats will be Caesarean-sectionedon day 21 of presumed gestation. To minimize bias, Caesarean-sectioning and subsequent fetal observationswill be conducted without knowledge of dosage group. The gravid uterus will be excised and weighed. The fetuses will be removed from the uterus and placed in individual containers. The rats will be examined for number and distribution of: Corpora Lutea. Implantation Sites. 4 18-023P:PAGE 5 1 Protocol 418-023P Page 11 Live and Dead Fetuses. (A live fetus is defined as one that responds to stimuli; a dead fetus is defined as a term fetus that does not respond to stimuli and that is not markedly autolyzed; dead fetuses demonstrating marked to extreme autolysis are considered to be late resorptions.) Early and Late Resorptions. (A conceptus is defined as a late resorption if it is grossly evident that organogenesis has occurred; if this is not the case, the conceptus is defined as an early resorption.) Fetal Observations: To minimize bias, fetal observations will be conducted without knowledge of dosage group. Fetuses will be examined for sex and for gross external alterations. Late resorptions and dead fetuses will be examined for gross external alterations to the extent possible. The body weight of each fetus will be recorded. Only body weights of live fetuses will be used to determine litter fetal body weight averages. Fetuses with gross external alterations will be fixed in Bouin's solution; all other fetuses will be discarded. Representative photographs of fetal gross external alterations will be taken. METHOD OF SACRIFICE: Rats will be sacrificed by carbon dioxide asphyxiation. Live fetuses will be sacrificed by an intraperitoneal injection of BeuthanasiaB-D Special, manufactured by ScheringPlough Animal Health. NECROPSY: Gross lesions will be retained in neutral buffered 10% formalin for possible future evaluation. Unless specifically cited below, all other tissues will be discarded. Scheduled Sacrifice: On day 21 of presumed gestation, female rats will be Caesarean-sectioned, and a gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. The gravid uterus will be excised and weighed. Uteri of apparently nonpregnant rats will be examined while being pressed between glass plates to confirm the absence of implantation sites. Rats Found Dead or Moribund: 418-023P:PAGE 52 Protocol 418-023P Page 12 Rats that die or are sacrificed because of moribund condition, abortion or premature delivery will be examined for the cause of death or moribund condition on the day the observation is made. The rats will be examined for gross lesions. Pregnancy status and uterine contents of female rats will be recorded. Gravid uterine weights will not be recorded if precluded by autolysis. Aborted fetuses, delivered pups and/or concepti in uteri will be examined to the extent possible, using the methods described above. Uteri of apparently nonpregnant rats will be examined while being pressed between glass plates to confirm the absence of implantation sites. STATISTICAL EVALUATION: Averages and percentages will be calculated. Litter values will be used where appropriate. Additional procedures and/or analyses may be performed if deemed appropriate. DATA ACQUISITION, VERIFICATION AND STORAGE: Data generated during the course of this study will be recorded either by hand or using the Primedica Argus Automated Data Collection and Management System and the Vivarium Temperatureand Relative Humidity Monitoring System. All data will be tabulated, summarized and/or statistically analyzed using the Primedica Argus Automated Data Collection and Management System, the Vivarium Temperatureand Relative Humidity Monitoring System, Microsoft Excel [part of Microsoft Off ice 97 (version SR-2)]and/or The SAS System (version 6.12). Records will be reviewed by the Study Director and/or appropriate management personnel within 21 days after generation. All original records will be stored in the archives of the Testing Facility. All original data will be bound and indexed. A copy of all raw data will be supplied to the Sponsor upon request. Preserved tissues will be stored at the Testing Facility at no charge for one year after mailing of the draft final report, after which time the Sponsor will be contacted to determine the disposition of these materials. RECORDS TO BE MAINTAINED: Protocol and Amendments. Test Substance, Vehicle andlor Reagent Receipt, Preparation and Use. Animal Acquisition. Randomization Schedules. Mating History. Treatment (if prescribed by Staff Veterinarian). General Comments. Clinical Observations and/or General Appearance. Blood Sample Collection, Processing and Shipment (if required). Body Weights. Feed Consumption Values. Caesarean-Sectioning and Fetal Observations. Gross Necropsy Observations. Organ Weights (if required). Photographs (if required). Study Maintenance (room and environmental records). Feed and Water Analyses. Packing and/or Shipment Lists. 418-023P:PAGE 53 Protocol 418-023P Page 13 KEY PERSONNEL: Executive Director of Research: Mildred S. Christian, Ph.D., Fellow, ATS Director of Research: Alan M. Hoberman, Ph.D., DABT Associate Director of Research and Study Director: Raymond G. York, Ph.D., DABT Director of Laboratory Operations: John F. Barnett, B.S. Manager of Animal Operations and Chairperson, Institutional Animal Care and Use Committee: Dena C. Lebo, V.M.D. Director of Operations and Compliance: Barbara J. Patterson, B.A. Director of Study Management: Valerie A. Sharper, M.S. Consultant, Veterinary Pathology: W. Ray Brown, D.V.M., Ph.D., ACVP The Study Director may provide periodic updates of study progress to the Sponsor. Draft summary tables of unaudited computer-recordeddata may accompany these updates. Statistical analyses will not be performed on these interim data. An unaudited letter report for the purpose of dosage selection for the full study will be prepared immediately following completion of the in-life phase. An audited report will be prepared including: abstract, summaries of the methods, results and conclusion; table of contents; Study Director's GLP compliance statement; copy of the protocol; amendments; QAU statement; summary and individual tables; and reports of supporting data (if appropriate). The report (will be included) (will not be included) as an appendix to the full study report. The Sponsor will receive one copy of the draft report and two copies of the final report. 418-023P:PAGE 54 Protocol 418-023P Page 14 INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE STATEMENT: The procedures described in this protocol have been reviewed by the Testing Facility's Institutional Animal Care and Use Committee. All procedures described in this protocol that involve study animals will be conducted in a manner to avoid or minimize discomfort, distress or pain to the animals. The Sponsor's signature below documents the fact that information concerning the necessity for conducting this study and the fact that this is not an unnecessarily duplicative study may be obtained from the Sponsor. No alternative (in vitro) procedures were available for meeting the stated purposes of the study. REFERENCES: 1. Christian, M.S. and Voytek, P.E. (1982). ln Vivo Reproductive and Mutagenicity Tests. EnvironmentalProtection Agency, Washington, D.C. National Technical Information Service, U.S. Department of Commerce, Springfield, VA 22161. 2. Christian, M.S. (1984). Reproductive toxicity and teratology evaluations of naltrexone (Proceedingsof Naltrexone Symposium, New York Academy of Sciences, November 7, 1983), J. Clin. Psychiat. 45(9):7-10. 3. Lang, P.L.(1988). Embryo and Fetal Developmental Toxicity (Teratology) Control Data in the Charles River Cr/:CDBBR Rat. Charles River Laboratories, Inc., Wilmington, MA 01887-0630. (Data base provided by Argus Research Laboratories, Inc.) 4. Institute of LaboratoryAnimal Resources (1996). Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, D.C. PROTOCOL APPROVAL: FOR THE TESTING FACILITY Alan M. Hoberman, Ph.D., DABT Director of Research Study Director FOR THE SPONSOR 418-023P:PAGE 55 Protocol 418-023P Page t 5 418-023P:PAGE 56 AllACHMENT 1 SCHEMATIC OF STUDY DESIGN AND STUDY SCHEDULE & ATTACHMENT 1 418-023P:PAGE 57 Protocol 418-023P Page 1 of 2 STUDY SCHEMATIC DOSAGE-RANGE DEVELOPMENTAL TOXICITY STUDYa Start of Dosage Cohabitation I Rats Day 6 Of Presumed Gestation End of Dosage CaesareanSectionin$ Day 20 of Presumed Gestation Day 21 of Presumed Gestation -Dosage Period. a. For additional details, see "Tests, Analyses and Measurements" section of the protocol. b. Fetal evaluations (all fetuses - external examinations). ATTACHMENT 1 4 18-023P:PAGE 58 Protocol 418-023P Page 2 of 2 SCHEDULEa 19 DEC 00 24 DEC 00 PM - 29 DEC 00 AM 25 DEC 00 29 DEC 00 31 DEC 00 - 18 JAN 01 15 JAN 01 -19 JAN 01 26 JAN 01 29 MAR 01 Animals Arrive - Acclimation Begins. Cohabitation Period. First Possible Day 0 of Presumed Gestation. Last Possible Day 0 of Presumed Gestation. Dosage Period (Days 6 through 20 of presumed gestation). Caesarean-Sectioning Period (Day 21 of presumed gestation). Unaudited Letter Report. Audited Summary Report. __--_-------r---------- a. The study initiation date is the date the Study Director signs the protocol. 4 18-023P:PAGE 59 ATTACHMENT 2 MATERIAL SAFETY DATA SHEET MATERIAL SAFETY DATA SHEET (Experimental) 3M 3M C e n t e r St. Paul, Minnesota 55144- 1000 1-800-364-3577 o r (651) 737-6501 (24 hours) C o p y r i g h t , 2000, Minnesota M i n i n g and Manufacturing Company, A l l r i g h t s reserved. Copying andlor downloading o f t h i s i n f o r m a t i o n f o r t h e purpose of p r o p e r l y u t i l i z i n g 3M p r o d u c t s i s allowed provided that: 1 ) t h e i n f o r m a t i o n i s c o p i e d i n full w i t h no changes u n l e s s p r i o r agreement i s o b t a i n e d f r o m 3M, and 2) n e i t h e r the copy nor the o r i g i n a l i s resold o r otherwise d i s t r i b u t e d w i t h t h e i n t e n t i o n of e a r n i n g a p r o f i t thereon D I V I S I O N : 3M SPECIALTY MATERIALS MATERIAL : L-7038 DEVELOPMENTAL MATERIAL ISSUED: December 08, 2000 SUPERSEDES: October 11, 2000 DOCUMENT: 04-4734-2 418-023P:PAGE 60 POTASSIUM . . . . . NONAFLUOROBUTANESULFONATE 2 9 4 2 0 - 4 9 - 3 - 98 POTASSIUM GAMMAHYDRO OCTAFLUOROBUTANE SULFONATE ............................. Unknown -2 The components o f t h i s p r o d u c t a r e i n compliance w i t h t h e chemical n o t i f i c a t i o n r e q u i r e m e n t s of TSCA. A l l a p p l i c a b l e c h e m i c a l i n g r e d i e n t s i n t h i s m a t e r i a l a r e l i s t e d on t h e European I n v e n t o r y o f E x i s t i n g Chemical Substances (EINECS), o r a r e exempt polymers whose monomers a r e l i s t e d on EINECS. BOILING POINT: ................. NIA VAPOR PRESSURE: . . . . . . . . . . . . . . . . N / A VAPOR DENSITY: . . . . . . . . . . . . . . . . . NIA EVAPORATION RATE:. . . . . . . . . . . . . . NIA SOLUBILITY I N WATER: . . . . . . . . . . . 5 % SPECIFIC GRAVIPI:.... .......... ca. 0.54 Water=l Bulk Density 7-8 PERCENT VOLATILE: . . . . . . . . . . . . . . N/A pH: ............................ 5.5 . 7.5 (5% Aqueous) VISCOSITY: ..................... N/A MELTING POINT:... . . . . . . . . . . . . . . 265 C lb/gal APPEARANCE AND ODOR: Solid, white, odorless, granular ---____._---____-_-_---------------.-.-------------------A b b r e v i a t i o n s : N I D - Not Determined N/A - Not A p p l i c a b l e CA - A p p r o x i m a t e l y 418-023P:PAGE 61 MSDS: L-7038 DEVELOPMENTAL MATERIAL December 0 8 , 2000 PAGE 2 __.______________--_----------------.--------------- _3_. F_IRE_AN_D E_XPL_OS_ION_HA_ZAR_D D_ATA_ _ _ _ - - - - - - - - - - - . - - - - - - - - - - - - - - - - - - - - - - FLASH POINT:... ................ Non-Flammable FLAMMABLE LIMITS - LEL: ........ N/A AFLUATMOMIGANBLITEIOLNIMTITEMSPE- RUAETUL:RE.:.. ........... N/A N/A EXTINGUISHING MEDIA: . Water , Carbon d i o x i d e , Dry chemical , Foam SPECIAL FIRE FIGHTING PROCEDURES: Wear f u l l p r o t e c t i v e c l o t h i n g , i n c l u d i n g helmet, s e l f - c o n t a i n e d , p o s i t i v e p r e s s u r e o r p r e s s u r e demand b r e a t h i n g a p p a r a t u s , bunker and pants, bands around arms, waist and l e g s , f a c e mask, and protective covering f o r exposed areas of t h e head. coat UNUSUAL FIRE AND EXPLOSION HAZARDS: See Hazardous Decomposition section f o r p r o d u c t s of combustion ___________________-___________________ . . 4 . . . . R. .E.A.C.T.I.V.I T. Y. . .D.A.T.A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . STABILITY: S t a b l e INCOMPATIBILITY - MATERIALS/CONDITIONS TO AVOID: None known. HAZARDOUS POLYMERIZATION: Hazardous polymerization W i l l not occur. HAZARDOUS DECOMPOSITION PRODUCTS: Carbon Monoxide and Carbon Dioxide, Oxides of S u l f u r , Hydrogen Fluoride, Toxic Vapors, Gases o r Particulates. SPILL RESPONSE: Observe precautions from other sections. Ventilate area. Collect s p i l l e d m a t e r i a l . Use wet sweeping compound o r water t o avoid dusting. Clean up residue. Place i n a closed container. RECOMMENDED DISPOSAL: Incinerate i n an i n d u s t r i a l o r commercial f a c i l i t y i n the presence of a combustible m a t e r i a l . Combustion p r o d u c t s w i l l i n c l u d e HF. Disposal a l t e r n a t i v e : Dispose of waste product i n a f a c i l i t y permitted t o accept chemical waste. ............................................................................. A b b r e v i a t i o n s : N/D - Not Determined N/A - Not Applicable CA - Approximately MSDS: L-7038 DEVELOPMENTAL MATERIAL December 08, 2000 4 18-023P:PAGE 62 PAGE 3 ENVIRONMENTAL DATA: Not determined. REGULATORY INFORMATION: V o l a t i l e O r g a n i c Compounds: N/D. VOC Less H20 & Exempt S o l v e n t s : NID. Since regulations vary, consult applicable regulations or authorities b e f o r e d i s p o s a l . U.S. EPA Hazardous Waste Number = None (Not U.S. EPA Hazardous). EPCRA HAZARD CLASS: FIRE HAZARD: No PRESSURE: No REACTIVITY: No ACUTE: Yes CHRONIC: Yes EYE CONTACT: Immediately f l u s h eyes w i t h l a r g e amounts o f water. Get immediate medical attention. SKIN CONTACT: Wash a f f e c t e d a r e a w i t h soap and w a t e r . INHALATION: I f signslsymptoms occur, remove person t o f r e s h a i r . I f signstsymptoms continue, c a l l a physician. I F SMLLOWED: If swallowed, c a l l a p h y s i c i a n i m m e d i a t e l y . Only induce v o m i t i n g a t ' the i n s t r u c t i o n o f a p h y s i c i a n . Never g i v e a n y t h i n g by mouth t o an unconscious person. EYE PROTECTION: A v o i d eye c o n t a c t . The f o l l o w i n g s h o u l d be worn alone o r i n c o m b i n a t i o n , as a p p r o p r i a t e , t o p r e v e n t eye c o n t a c t : Wear v e n t e d g o g g l e s . Wear s a f e t y g l a s s e s w i t h s i d e s h i e l d s . SKIN PROTECTION: A v o i d s k i n c o n t a c t . Wear a p p r o p r i a t e g l o v e s when h a n d l i n g t h i s m a t e r i a l . A p a i r o f g l o v e s made f r o m t h e f o l l o w i n g m a t e r i a l ( s ) a r e recommended: neoprene, n i t r i l e r u b b e r . Use one o r more o f t h e f o l l o w i n g personal p r o t e c t i o n i t e m s as necessary t o prevent s k i n contact: head covering, coveralls. Protective garments (other than - - -gl.ov-e-s)-s-ho-u_ld _be _ma-de-o-f -e _i t h_e r _o f _t h e- _f o l_l o wIi_n g _m a_t e r_i a l_s : _ _ _ _ _ _ _ _ _ _ _ _ - - - - - - - . - A b b r e v i a t i o n s : N/D : Not Determined N/A - Not A p p l i c a b l e CA A p p r o x i m a t e l y 4 18-023P:PAGE 63 MSDS: L-7038 DEVELOPMENTAL MATERIAL December 08, 2000 PAGE 4 ________-_______----------.------------------------ _7_. P_REC_AU_TIO_NAR_Y I_NFO.R_MA_TIO.N. _ .(c_on-tin-u-ed-)- - - - . . - - . - . - - - - - - . - . - - - - - - - - - - - - - - - - - P l a i n Tyvek o r coated Tyvek o r s i m i l a r disposable c o v e r a l l . RECOMMENDED VENTILATION: P r o v i d e a p p r o p r i a t e l o c a l exhaust when p r o d u c t i s heated. P r o v i d e appropriate l o c a l exhaust ventilation a t transfer points. Provide s u f f i c i e n t v e n t i l a t i o n t o m a i n t a i n emissions below recommended exposure l i m i t s . I f exhaust v e n t i l a t i o n i s not adequate, use appropriate respiratory protection. RESPIRATORY PROTECTION: Avoid breathing o f airborne material. Avoid breathing of dust. S e l e c t one of t h e f o l l o w i n g NIOSH approved r e s p i r a t o r s based on a i r b o r n e c o n c e n t r a t i o n o f c o n t a m i n a n t s and i n accordance w i t h OSHA r e g u l a t i o n s : half-mask dust and m i s t r e s p i r a t o r , f u l l - f a c e dust and mist respirator. PREVENTION OF ACCIDENTAL INGESTION: 00 n o t e a t , d r i n k o r smoke when u s i n g t h i s p r o d u c t . Wash exposed areas t h o r o u g h l y w i t h soap and w a t e r . Wash hands a f t e r h a n d l i n g and b e f o r e e a t i n g . Do n o t i n g e s t . RECOMMENDED STORAGE: S t o r e away from heat. when n o t i n use. Keep c o n t a i n e r d r y . Keep c o n t a i n e r c l o s e d FIRE AND EXPLOSION AVOIDANCE: Not applicable. Nonflammable. OTHER PRECAUTIONARY INFORMATION: No smoking: Smoking w h i l e u s i n g t h i s p r o d u c t can r e s u l t i n c o n t a m i n a t i o n o f t h e t o b a c c o a n d / o r smoke and l e a d t o t h e f o r m a t i o n o f the hazardous decomposition products mentioned i n the Reactivity Data s e c t i o n o f t h i s MSDS. S t o r e work c l o t h e s s e p a r a t e l y f r o m o t h e r c l o t h i n g , food and tobacco products. EXPOSURE LIMITS INGREDIENT VALUE UNIT TYPE AUTH SKIN* POTASSIUM NONAFLUOROBUTANESULFONATE .......... . . . . . . . . . POTASSIUM GAMMAHYDRO OCTAFLUOROBUTANE SULFONATE 0.1 NONE MGIMS NONE TNA 3M Y NONE NONE * SKIN NOTATION: L i s t e d substances i n d i c a t e d w i t h ' Y ' under SKIN r e f e r t o the p o t e n t i a l contribution t o the o v e r a l l exposure by the cutaneous route i n c l u d i n g mucous membrane and eye, e i t h e r by a i r b o r n e o r , more p a r t i c u l a r l y , by d i r e c t contact w i t h t h e substance. Vehicles can a l t e r s k i n absorption. ---_______i______-_-___________________ A b b r e v i a t i o n s : NID - Not Determined NIA - Not A p p l i c a b l e CA - Approximately 4 18-023P:PAGE 64 MSDS: L-7038 DEVELOPMENTAL MATERIAL December 08, 2000 EXPOSURE LIMITS (continued) PAGE 5 I_N_G_R_ED_ I_E_N_T_ _ - _ . _ _ . - - - - - - . - - - - - - - - - - - - - - - - -V-A- L-U- E- - - -U-N- -IT- - - - - . - * T- Y- -P-E- - - -A-U-T-H- - - -S-K-IN- ' -SOURCE OF EXPOSURE L I M I T DATA: 3M: 3M Recommended Exposure G u i d e l i n e s - NONE: None E s t a b l i s h e d ________________-_--------------------------.----_8_. H-EA_LTH- H-A-ZA-RD-D-AT_A - . - - - - - - - - - - - - - - - . - - - - - - - - . - - - - - - - - * - - - - - - - - - - - EYE CONTACT: Moderate Eye I r r i t a t i o n : signslsymptoms can i n c l u d e redness, pain, tearing, and hazy vision. swelling, SKIN CONTACT: Contact with the s k i n during product use i s not expected t o r e s u l t i n significant .irritation. INHALATION: S i n g l e overexposure, above recommended g u i d e l i n e s , may cause: I r r i t a t i o n (upper respiratory): signslsymptoms can include soreness of t h e nose and t h r o a t , coughing and sneezing. I F SWALLOWED: May be h a r m f u l i f swallowed. PHYSICAL DATA SECTION CHANGED SINCE O c t o b e r 11, 2 0 0 0 FIRE AND EXPL. SECTION CHANGED SINCE O c t o b e r 11, 2000 ENVIRONMENTAL INFO. SECTION CHANGED SINCE October 11, 2 0 0 0 PRECAUTIONARY INFO. SECTION CHANGED SINCE O c t o b e r 11, 2 0 0 0 ISSUE ISSUE ISSUE ISSUE .-.______-_._.--_---------------------.--------.------A b b r e v i a t i o n s : NID - N o t Determined NIA - N o t A p p l i c a b l e CA - A p p r o x i m a t e l y 418-023P:PAGE 65 MSDS: L-7038 DEVELOPMENTAL MATERIAL December 08, 2 0 0 0 PAGE 6 ............................................................................. The i n f o r m a t i o n i n t h i s M a t e r i a l S a f e t y D a t a Sheet (MSDS) i s b e l i e v e d t o be c o r r e c t as o f t h e d a t e i s s u e d . 3M MAKES NO WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING, BUT NOT LIMITED TO, ANY IMPLIED M R R A N T Y OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE OR COURSE OF PERFORMANCE OR USAGE OF TRADE. User i s r e s p o n s i b l e f o r d e t e r m i n i n g whether t h e 3M p r o d u c t i s f i t f o r a p a r t i c u l a r purpose and s u i t a b l e f o r u s e r ' s method o f use o r a p p l i c a t i o n . Given t h e v a r i e t y o f f a c t o r s t h a t can a f f e c t t h e use and a p p l i c a t i o n of a 3M p r o d u c t , some o f which a r e uniquely w i t h i n t h e u s e r ' s knowledge and c o n t r o l , i t i s e s s e n t i a l t h a t t h e u s e r e v a l u a t e t h e 3M p r o d u c t t o d e t e r m i n e whether i t i s f i t f o r a p a r t i c u l a r purpose and s u i t a b l e f o r u s e r ' s method of use o r a p p l i c a t i o n . 3M p r o v i d e s i n f o r m a t i o n i n e l e c t r o n i c f o r m as a s e r v i c e t o i t s customers. Due t o t h e remote p o s s i b i l i t y t h a t e l e c t r o n i c t r a n s f e r may have r e s u l t e d i n e r r o r s , o m i s s i o n s o r a l t e r a t i o n s i n t h i s i n f o r m a t i o n , 3M makes no representations as t o i t s completeness o r accuracy. I n a d d i t i o n , i n f o r m a t i o n o b t a i n e d f r o m a database may n o t be as c u r r e n t as the i n f o r m a t i o n i n t h e MSDS a v a i l a b l e d i r e c t l y f r o m 3M. 4 18-023P:PAGE 66 AlTACHMENT 3 TEST SUBSTANCE PREPARATION PROCEDURE 418-023P:PAGE 67 AITACHMENT 3 Protocol 418-023P Version: 418-023P(18DEC 00) TEST SUBSTANCE PREPARATION PROCEDURE Page 1 of 2 Test Substance: PBSF Vehicle: Aqueous 0.1YOCMC (medium viscosity) A. Purpose: The purpose of this procedure is to provide a method for the preparation of dosage suspensions of PBSF for oral (gavage) administration to rats on Primedica Argus Research Laboratories, Inc., Study 418-023P. B. General Information: 1. All suspension containers will be labeled and color-coded. Each label will specify the protocol number, test substance identification, Argus batch number, concentration, dosage level, preparation date, expiration date and storage conditions. 2. Suspensionswill be prepared: - - Daily Weekly - - X Approximatelyevery ten days For- days of use By Sponsor 3. Suspensions will be prepared at a final dosage volume of mUkg. 4. Safety: - X Nitrile or neoprene gloves, uniform/lab coat, goggles or safety - glasses with side shields - X Dust-Mist Respirator if u s e d in a chemical fume hood X Half-Face Respirator if not used in a chemical fume hood - Full-Face Respirator/Positive Pressure Hood - X Tyvek Suit or tyvek apron and sleeves 5. Dosage suspensions adjusted for YoActivity/Purity or Correction Factor: - Yes - Oh Activity - X No (Calculations based on 100%) - % Purity - Correction Factor , 6. Sampling requirements: Cited in protocol 7. Storage: Cited in protocol 418-023P:PAGE 68 AlTACHMENT 3 Protocol 418-023P Version: 418-023Pl18 DEC 00) Page 2 of 2 TEST SUBSTANCE PREPARATION PROCEDURE NOTE: Prior to test substance preparation accurately measure the required amount of the appropriate vehicle (R.O.deionized water should be used for calibration purposes) in a graduated cylinder, pour the required amount of vehicle into a beaker. Carefully mark each beaker at the meniscus. This mark will be used during the preparation to bring the test substance slurry up to volume. C. Dosage Suspension Preparation: 1. Weigh the required amount of test substance on a piece of weigh paper or into an appropriately sized mortar (see PREPARATION CALCULATIONS). 2. If weigh paper is used, transfer the test substance to an appropriately sized mortar. Ifnecessary, grind the test substance into a fine powder. Slowly add a small amount of vehicle and grind. Continue to add vehicle slowly and grind the vehicle and the test substance together to form a fine slurry. Transfer the vehicle/test substance slurry to a marked beaker. 3. Rinse the mortar and pestle with additional vehicle to remove any remaining test substance. Transfer rinse to beaker. 4. Add additional vehicle to the beaker to bring volume up to the mark. Place on magnetic stir plate and agitate prior to and during administration and/or sampling. 5. Repeat steps (1) through (4) for each concentration. ate: 2 - 2 - M - ~ d Clarification: J NO -Yes [see attached clarification form] 4 18-023P:PAGE 69 ~ ~ ~ e s e u c h -. k 905 Sbeetr). ;tkivc, Haphmr,PA 19044 TclepbDnc: (215) 443-8710 Tclefix: (U5)443-8587 PROTOCOL 418-023P ORAL (QAVAGE) DOSAGEZbWGE D W E L O P m U XHIClTY STUDY OF POTASSIUM PERFLUOROBVTANE -ONATE (PFBS) XN RATS SPONSORSSTUDY NUMB= T-7485.11 Ammrtment 1 - 26 Fcbroary 2001 Any revisions made to'this finalized amendment must be made by subsequent amendment. 418-023P:PAGE 70 ATTACHMENT 2 ANALYTICAL REPORT 418-023P:PAGE 71 ANALYSIS OF DOSING SOLUTIONS USED AT ARGUS RESEARCH LABORATORY FOR SPONSOR'S STUDY NUMBERS T-7485.11, T-7485.12, AND T-7485.13 (ARGUS RESEARCH LABORATORY PROTOCOL NUMBERS: 418-023P, 418-023 AND 418-021) STUDY ID: A343.1 SPONSOR STUDY NOS. T-7485.11 T-7485.12 T-7485.13 Southern Research Institute 2000 Ninth Avenue South P.O. Box 55305 Birmingham, AL 35255-5305 418-023P:PAGE 72 SUMMARY A total of 30 formulated dose samples including blanks and vehicles ranging in concentration from 3 to 200 m g / d were analyzed by analytical method BACG 3533 to determine the concentration of perfluorobutanesulfonate (PFBS) in the formulated mixture. All dose formulations were found to be within _+ 10% of the reported concentration except for four samples, 100 m g / d dated 1/11/01, 100 m g / d dated 2/23/01, 100m g / d date 9/4/01 and 200 mg/mL dated 1/11/01. These three samples were found to have measured concentrations of 88.9 %, 86.3 %, 76.6% and 76.8 % of target values, respectively. KEY PERSONNEL Raymond G. York, Ph.D., D.A.B.T Study Director Argus Research Laboratories James D. Johnson, M.S., MBA Manager Bioanalytical Chemistry Group Gregory S . Gorman, Ph.D. Staff Chemist Bioanalytical Chemistry Group Lara Cook, M.S. Research Associate I1 Bioanalytical Chemistry Group 4 18-023P:PAGE 73 418-023P:PAGE 74 1. OBJECTIVE The objective of this study was to determine the dose concentration of PFBS in the supplied dosing solutions received from the sponsor. 2. SAFETY All necessary procedures to ensure safety of the analysts were based on information contained in the Material Safety and Data Sheets (MSDS), provided by the producer of the test article and the study director. 3. Compliance The study described in this final report was conducted in accordance with the EPA Good LaboratoryPractice Regulations (40 CFR Part 160 and 792), OECD Principles of Good Laboratory Practice [C(97)186/FinalJ. For studies 418-023 and 418-023P Good Laboratory Practice Standards (1984) for W F 59 Nohsan No. 3850 were met. The final report accurately reflects the raw data obtained during the performance of the study. There were no adverse circumstancesthat affected the quality or integrity of the study. 4. EXPERIMENTAL 4.1 Analytical Procedures The sample preparation and analysis procedures as described in the analytical method BACG 3533 were employed for all analyses. Each sample was allowed to warm to room temperature and was then vortexed well before being sampled. An aliquot was taken from each and diluted as described in the method. (Note: the 66.7 rng/rnL and higher concentrated samples often were suspensions and the entire sample volume in some cases were diluted to the target range). Multiple calibration curves were prepared over a concentration range of 500 to 10,000 ng/mL and analyzed along with the samples as described in the method. The correlation coefficient for each curve was greater than 0.9982. 4.2 Results The results of the analysis are presented in the Tables I-III corresponding to the sponsors study number at the end of the report. 418-023P:PAGE 75 5.0 Conclusion A total of 30 dose formulation samples ranging in concentration fiom 3 to 200 mg/mL were analyzed by BACG 3533. All samples except for four, 100 mg/mL dated 1/11/01, 100 mg/mL dated 2/23/01, 100 mg/mL dated 9/4/01 and 200 mg/mL dated 1111 were found to be within k 10 % of the reported concentration. Table I Dose Formulation Analysis of PFBS in 0.1% CMC Sponsor Study Number T-7485.13 4 18-023P:PAGE 76 * SrnalI amount of material detected in the undiluted sample is considered insignificant when compared to amount found in diluted samples and is beiieved to be the result of carry-over from a previously run standard injection. 2Averageof two results. Table II Dose Formulation Analysis of PFBS in 0.1% CMC Sponsor Study Number T-7485.12 418423P:PAGE 77 Dose (mg/mL) 0 (2123) 0 (318) 10 (2/23) 10 (318) 30 (2123) 30 (318) 100 (2/23) 100 (3/8) - Dilution Conc. (ng/mL) Conc.(mg/mL) Yo no dilution not detected no dilution not detected Theoretical ---- ---_ --____ I---- 3200 331 1 10.3 103 3200 31 92 31 92 3207 31 12 31 54 10.0 29.2 29.6 100 97.5 98.8 3200 2760 86.3 86.3 3200 3383 106 106 Table 111 Dose Formulation Analysis of PFBS in 0.1% CMC Sponsor Study Number T-7485.11 418-023P:PAGE 78 6.0 Approvals Lara Cook, M.S. Date Research Associate I1 Bioanalytical Chemistry Group G;eg&y S . Goman, Ph.D. Date Staff Chemist Bioanalytical Chemistry Group Tina Rogers, Ph.D. Date Director Safety Assessment Department u. k.;kiO:ork,Phx, Date Argus esearch Laboraton 418-023P:PAGE 79 ' , 41.8-023P:PAGE 80 Quality Assurance Statement Final Report On Analysis of Dosing Solutions Used at Argus Research Laboratory for Sponsor's Study Numbers T-7485.11, T-7485.12, and T-7485.13 (Argus Research Laboratory Protocol Numbers: 418-023P, 418-023, and 418-021) A343.1 Listed below are the phases andor procedures performed by Southern Research Institute that were inspected and audited by the Quality Assurance Unit during the study described in the report. Findings were reported to the study director and management periodically. Phaseflrocedures Protocol Review t I Dose Formulation Concentration Analysis Inspection/ Audit Date 5/14/01 511 410 1 Date Management Notified 5/14/01 I 611410 I Date Study Director -1 Notfzed' I I 1/18/02 1118/02 Data Audit and Draft Report Review 12/3/01-1~16/01; 1/14/021-117/02 1/18/02 1/18/02 Final Report 1/30/02 113 0102 1/30/02 ' The study director is off-site. Assurance/Quality Control z Date 4 18-023P:PAGE 8 1 ATTACHMENT 3 QUALITY ASSURANCE STATEMENT 418-023P:PAGE 82 QUALITY ASSURANCE STATEMENT Argus Protocol: 418-023P Sponsor's Study Number: T-7485.11 Study Director: Raymond G. York, Ph.D., DABT The protocol, critical phases, raw data and pilot report were inspected by the Quality Assurance Unit (QAU), to assure conformance with: U.S. Environmental Protection Agency. Toxic Substances Control Act (TSCA); Good Laboratory Practice Standards; Final Rule. 40 CFR Part 792. U.S. Environmental Protection Agency. Federal Insecticide, Fungicide and Rodenticide Act (FJFRA); Good Laboratory Practice Standards; Final Rule. 40 CFR Part 160. Organization for Economic Cooperation and Development (1998). The Revised OECD Principles of Good Laboratory Practices [C(97)186FinalI. Japanese Ministry of Agriculture, Forestry and Fisheries (1984). Good Laboratory Practice Standards. 59 NohSan No. 3850. The undersigned indicate that the report is an accurate representation of the raw data. Data provided by the Sponsor or a subcontractor were not audited by the Primedica Argus Quality Assurance Unit. 418-023P:PAGE 83 The QAU inspection and report audit dates are listed below: Date@)Findings Submitted to Study Inspection Phase Inspection Date(s) Director Protocol 21 DEC 00 21 DEC 00 Test Substance Administration 05 JAN 01 17 JAN 01 Test Substance Formulation 11 JAN 01 11 JAN 01 Caesarean-Sectioning 16 JAN 01 17 JAN 01 In-Life Raw Data 29-31 JAN01 31 JAN01 Formulation Data 21 FEB 01 21 FEB 01 Report Tables 21,22 MAR 01 22 MAR 01 Report Text 27MAR01 27MAR01 Date(s) Findings Submitted to Management 21 DEC 00 17 JAN 01 11 JAN01 17 JAN01 31 JAN 01 21 FEB 01 22 MAR 01 27 MAR 01 A atthew J. Vaneman, B.S. Date Senior Manager, Regulatory Compliance