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~ AR2G- 0956 geHg_ 06m. 75 KDT3 Summary Report 104-Week Dietary Chronic Toxicity and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium TSF Salt (PFOS: T-6295) in Rats . ! s [TIT & EPA-OTS 0008117937 PREPAREDFOR: COVANCE6S32T9U-1D8Y3 NUMBER: ~ E8i g3n = 52 zE ooo 002148 SUMMARY REPORT - WEEK 53 104 Week Dietary Toxicity and Carcinogenicity Study with Perfluorooetane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Rats Timeline Covance 6329-183: Tn-life start date: April 20, 1998 `Week 53 completed: April 25, 1999 Introduction `The purposeofthis study is to assess the chronic toxicity and carcinogenicityofthe test `material, T-6295 (Perfluorooctane sulfonic acid potassium salt, PFOS), when administered inthe diet to rats for at least 104 weeks. This summary report includes information through Week 53 and contains current findings it is not , however, the final report for the study. Covance Laboratories Inc. reserves the right of review of these data before issuing a final report, Methods Male and female CrLCD"(SD)IGS BR rats were assigned to groups according to the following design: Group 2T (0Con!tre) 43 Miidd-)H"igh) 5 (igh MNalue mofbAnFeiemmaarllse 70 70 0 7 0 7 Dietary Levels (ppm 0T-6295 02s0 25000 4 TA hecontrolanimalsreceived hebasaElrdetoanely.peers Bras b `FiWveeeakn4simaalnsdls1c4xfionr Gherpoautpocsel|ltuhlraroupgrhol5ifwerearteiosnacartifeicmeedasduurreinmgents, bacitoicvhietymiacnaallyasneasl,yasnesd (hpiasltompiattohyoll-oCgoyA(oWxeicdakti1o4no,nlmyi).tochondrial Tar6e2e0x5preissspeedralucpropomcoifanTe-6su2l9f5onic acid potassium (PROS);dose levels 4 Tsaecnriafinciemaanlismsaelsx.inTGhreosuepasni|maanlds wweereresadcriefisceid agfenarsaiantttleeeraisdmt 52 weeksofreaunent. : ! 002149 SMT95 Covance 6329-183 Observation of Animals Food was providedadlibitum, except when animals were fasted. Water was provided ad libitum. The animals were observed twice daily (a.m. and p.m.) for mortality and moribundity: findings were recorded as they were observed. At least once prior to treatment and weekly thereafter, each animal was removed from its cage and examined; `abnormal findingsoran indication of normal was recorded. Body weight data were recorded weekly for each animal for Weeks 1 through 17 and once every 4 weeks thereafter. Food consumption data were collected for each animal for Weeks 1 through 16 and once ever4y weeks thereafter. Clinical Pathology. Blood and urine samples were collected for hematology, clinical chemistry, urinalysis, and urine chemistry from 10 animals/sex/group in Groups 1 through 5 during Weeks 4, 14, 27, and 53. Animals were fasted overnight, and urine was collected chilled overnight (approximately 16 hours) before blood sampling. Blood was collected from a jugular vein. The anticoagulant was potassium EDTA for hematology tests. The samples for clinical chemistry and serum samples were collected without anticoagulant. When possible, a blood film was made for possible future examination from animals sacrificed at unscheduled intervals. A blood film was also made and held for `possible future examination from animals sacrificed during Week 53. Serum PFOS Analyses. During Weeks 4 and 14, blood samples were collected for `serum analyses from five animals/sex/group in Groups 1 through 5. During Week 53, blood samples were collected from five animals/sex/group in Groups 1 and 5. Animals `were fasted overnight. Blood (approximately 2 mL) was collected from a jugular vein, allowed to clot at room temperature, and centrifuged. Samples were collected without anticoagulant. Serum was harvested and stored in a freezer, set to maintain -60 to -80C, until sent to the Sponsor for analysesof PFOS levels. Termination. During Weeks 4 and 14, five animals/sex/group/intervianl Groups 1 through 5 were fasted overnight, bled for scrum chemistry, anesthetized with carbon dioxide, weighed, and exsanguinated. The abdominal cavity of each animal was opened, the liver was removed and weighed. Liver samples were collected for palmitoyl-CoA, cell proliferation, and PFOS concentration analyses and for mitochondrial activity (Week 4 sacrifice only) and histopathology (Week 14 sacrifice only). Animals sacrificed during Week 4 were discarded after liver collection. During Week 53, 10 animals/sex/group from ? 002150 Covance 6329-183 _-- wTes Groups 1 and 5 were fasted overnight, bled for serum samples (five animals/sex/group), anesthetized with carbon dioxide, weighed, exsanguinated, and necropsied. The liver was weighed and liver sampleswere collected and frozen for analysis. Animals sacrificed during Weeks 14 and 53 had macroscopic observations recorded, selected organs `weighed, and selected tissues collected and preserved. Adrenals, brain, eyes, kidneys, liver, mesenteric lymph node, pancreas, spleen, testes, and ovaries were processed and examined microscopicfarlolmy animals sacrificed during Week 14 and on all tissues from the animals in Groups 1 and 5 that were necropsied during Week 53. Animals that died or were sacrificed at unscheduled intervals were also necropsied and microscopic examinations were done on selected tissues collected, but organ weights were not recorded. A blood film was taken for each animal sacrificed at unscheduled intervals and at the Week 3 interim sacrifice. Proliferation Cell Nuclear Antigen (PCNA) Evaluation. During the Week 4 and 14 tissue collections from animals in Groups | through 5 (five animals/sex/group), representative samples of the left lateral lobe and any macroscopic lesionsofthe liver were collected and preserved in zinc formalin. Liver samples were fixed and embedded, and blocks were sent to Pathology Associates International (PAI); PAIwillevaluate the samples (eft lateral lobe only) for PCNA. In addition, liver sections will be stained with hematoxylin and eosin and examined microscopically. Palmitoyl-CoA (PCoA) Oxidase Analyses. During the Week 4 and 14 tissue collections from animals in Groups 1 through S (five animal/sex/group). a sample (approximately 500 mg) of the right lateral lobeofthe liver was collected and flash-frozen in liquid nitrogen. The samples were stored in a freezer set to maintain -60 to -80C, until analyzed by Covance for PCoA oxidase activity. PFOS Analysis. During the Week 4 and 14 tissue collections for animals in Groups 1 through 5 (five animals/sex/group) and Week 53 for animals in Groups 1 and 5 (five animals/sex/group). a portion of the remaining liver was flash-frozen in liquid nitrogen and stored ina freezer set to maintain -60 to -80C, until sent to the Sponsor. "The Sponsor will analyze theliver samples for PFOS and metabolites. 3 002151 CovancIe M63T269-219853 Mitochondrial Analyses. During the Week 4 tissue collections for animals in Groups 1 through 5, liver samples (approximately 1.5 g) were collected and analyzed by the `Sponsor for mitochondrial activity. Bromodeoxyuridine Immunohistochemistry. Seven days before the Week 53 interim sacrifice, osmotic pumps (ALZET Model 2ML1) were surgically implanted from five animals/sex/group from Groups 1 and 5. The osmotic pumps were preloaded with approximately 2 mL of bromodeoxyuridine (BrdU) at a concentration of 20 mg/mL. The animals were anesthetized by administration of acepromazine-ketamine-xylazine, and one pump/animal was aseptically inserted subcutaneously (dorsal surface). The incision was closed with wound clips. and the animals were monitored during clinical observations until the timeofsacrifice to ensure that there are no clinical signs of infection. One Group 5 male implanted with osmotic pump was sacrificed before the Week 53 sacrifice. At the Week 53 sacrifice, BrdU immunohistochemistry was performedby PAI on the livers and duodenums from the five animals/sex/group from Groups 1 and S that received BrdU. In addition, sectionsofthe livers and duodenums from these animals were stained `with hematoxylin and eosin and examined microscopically. Results will be provided for inclusion in the final report. Results Clinical Observations and Survival. Adjusted survival data are summarized in Table 1. Clinical observation data are summarizedin Table 2. Survival after 53 weeks of treatment was 96.0%, 96.0%, 100%, 94.0%. 96.0%. and 100% for males in Groups 1, 2, 3,4, 5, and 6, respectively, and 100%, 94.0%, 94.0%, 96.0%, 98.0%, and 98.0% for females in Groups 1,2,3, 4.5, and 6, respectively. There were no apparent test material-related clinical observations noted through Week 53. Body Weights. Body weight and body weight change data are summarized in Tables 3 and 4. Males given 20.0 ppm had significantly lower mean body weights compared to thoseofanimals given the control material during Weeks 9 through 53, and females given 20.0 ppm had significantly lower body weights compared to thoseofanimals given the control material during Weeks 3 through 53. Mean body weights for males and females were similar in all the other treated groups compared to those of animals given the control 4 002152 CovancSe 63T29-s183 material. Males and females given 20.0 ppm gained less weight during the overall period from Weeks 1 through 53; the differences were statistically significant for the females. Animals at lower dose levels also had occasional statistically significant decreases in body weight gain; however, these occurrences were inconsistent over time between sexes and were not clearly dose related. Food Consumption. Food consumption data are summarized in Table 5. Although not always statistically significant, males given 20.0 ppm tended to consumeless food during `Weeks 1 through 24. Food consumption was similar for males given 20.0 ppm compared 10 those of animals given the control material during Weeks 28 through 52. Statistically significantly lower food consumption was noted for females given 20.0 ppm during `Weeks 2 through 44. Food consumption for males and females weresimilarin all the other treated groups compared to those of animals given the control material. `Test Material Consumption. Test material consumption data are summarized in Table 6. Animals were fed diets intended to provide 0, 0.5, 2.0, 5.0, 20.0 ppm. The amount of test material consumed by animals on a mg/kg of body weight/day basiswasas follows: (ppm) 05 20 5.0 20.0 20.0 (mg/kg body weight/day) Males Females 0.017-0.057 0.071-0.226 0.174-0.570 0.714-2.205 0.732-2.336 0.022-0.052 0.095-0.213 0.237-0.559 1.069-2.149 1.047-2.160 Clinical and Anatomic Pathology. Hematology. clinical chemistry, urinalysis, and urine chemistry data are summarized in Tables 7 through 22. Terminal body weights, absolute organ weights, organ-to-body weight percentages, and organ-to-brain weight ratios are summarized in Tables 23 and 24. Incidences of macroscopic and microscopic observations are summarizedin Tables 25 through 29. The Pathology Report contains a discussion of the data. ' 002153 - Covance 6329-183 OOOOOOOOOO Tews Dose Analyses. Results of the homogeneity. stability, and dose preparation analyses are in Tables 30 through 32. Several homogeneity analyses were conducted on each of several mixes in an attempt to resolve issues related to the inherent variability and lack of sufficient sensitivity in the analytical method for measuring (PFOS: T-6295). Mean valuesofthe homogeneity analyses for the mix done on March 18, 1998, ranged from 119-135%, and 107-118% of the theoretical concentrations for the diets containing 0.5 and 20 ppm (PFOS; T-6295), respectively. Mean values of the homogeneity analyses for the mix done on April 16, 1998, ranged from 96.2+122%, 98.5-107%. 97.6-101%, and 90.0-101%ofthe theoretical concentrations for the diet containing 0.5. 2, 5, and 20 ppm (PFOS; T-6295), respectively. Mean values of the homogeneity analyses for the mix done on April 22, 1998, ranged from 93.5-103% of the theoretical concentrations for the diet containing 20 ppm (PFOS; T-6295). Mean values of the homogeneity analyses for the mix done on June 18, 1998, ranged from 91.0-175%, 111-130%, 99.6-102a%nd, 90.5-97.0%of the theoretical concentrations for the diet containing 0.5, 2, 5, and 20 ppm (POS: T-6295). These results indicate that the mixing procedure produced a homogeneous distribution of the test material in the dose preparations: although variability `generally appeared slightly greater at the lower dietary concentrations. Stability analyses were conducted prestudy in an attempt to resolve issues related to the inherent variability and lackof sufficient sensitivity at levels in the analytical method for `measuring (PFOS; T-6295). Resultsofstability analyses for the mix conducted on March 18, 1998,ofsamples stored for 28 days at room temperature indicated that the. `mean concentrations were 78.8 %, 76.7%. 79.8% and 88.19% of the initial concentrations 0f0.5, 1, 2, and 20 ppm. respectively. Results of stability analyses for themix conducted on April 16. 1998,ofsamples stored for 33 days at room temperature indicated that the `mean concentrations were 112.3%, 119.6%, 113.9% and 126.7%ofthe initial concentrations of0.5. 1.2, and 20 ppm, respectively. `The mean concentrations of the dose preparation analysesforall levels ranged from 44.4%-276% of the theoretical concentrations (including the reassay and retention sample. analyses). Inherent variability and lackofsufficient sensitiviatty low levels in the. analytical method for measuring (PFOS:T-6295) resulted in homogeneity, stability, and. routine analysis data that were in many cases outside of the standard limitsof+/- 15%. 6 002154 Covance 6329-183 3M T6295 `These results will be reevaluated in conjunction with the analytical results from the blood and liver level determinations when they are provided by the Sponsor. 7 002155 PATHOLOGY REPORT `Week 14 and 53 Interim Sacrifices SM T205 Covance 6329-183 SUMMARY `The purposeofthis study is to assess the chronic toxicity and carcinogenicityofthe test `material, Perfluorooctane Sulfuric Acid Potassium Salt (PFOS) when administered in the diet to rats for at least 104 weeks. The test material is being administered at dose levels of 0.5,2.0, 5.0, and 20.0 ppm. This interim report discusses findings through the first 53 weeksofstudy. Dietary administration of PFOS for approximately 53 weeks was associated with mildly `higher urea nitrogen for males and females fed 5.0 or 20.0 ppm; mildly lower glucose for `males and females fed 20.0 ppm; mildly to moderately lower cholesterol for males and females fed 20.0 ppm; and mildly higher alanine aminotransferase for males fed 20.0 ppm. None of these effects were considered adverse. There was no effect on hepatic `palmitoyl-CoA oxidase activity. `Terminal body weights in males and females at Week 14 were comparable between control and treated groups. At the Week 14 interim sacrifice, absolute and relative liver weights `were significantly increased in the males given 20.0 ppm. In females given 20.0 ppm, only the liver-to-body weight percentage was significantly increased. Test material-related `histomorphologic changes were limited to the liver in the males given 5.0 or 20.0 ppm and in the females given 20.0 ppm. The changes consisted of hypertrophy of hepatocytes in vcaecnutorlialtoibounl.ar aTrheeasiinncimdaelnecse aanndd fseemvaelreist,y oafndthmeicdhzaonngaelstotecnednetdritloobbuelagrrheaetpeartoicnytthiec males. At the Week 53 interim sacrifice, terminal body weights were significantly decreased in the females given 20 ppm. In the males, absolute and relative liver weights were increased in the group receiving 20 ppm. In addition, absolute and relative spleen weights were decreased in males given 20 ppm. There were no clearor consistent gross observations at the Week 53 sacrifice that could be attributed to the administrationofthe test material. At the Week 53 sacrifice, centrilobular hepatocytic hypertrophy and vacuolation was 8 002156 Covance 6329-183 _-- Tew increased in incidence and severity in the males given 20 ppm. In the females given 20 ppm, generally, only centrilobular hypertrophy was seen, and the change was less severe than that noted in the males. In addition, minimal to slight centrilobular hepatocytic pigment was found in the females given 20 ppm. There were no other histomorphologic changes that could be associated with the administration ofthe test material. Findings in liversinseveral unscheduled deaths given 20 ppm resembled those seen in animals sacrificed at Week 53. METHODS Six groupsofCrl:CD'(SD)IGS BR rats were studied using the following study design. Group 1 (Control) 2 (Low) 3 (Mid) 4 (Mid-High) 5 (High 6 (High Recovery) NumofbAneimarls Male Female 70 70 60 60 60 60 60 60 70 70 40 40 Dietary Levels (ppm T-6295) 0 0.5 20 5.0 20.0 20.0 During Weeks 4, 14, 27, and 53, blood and urine were collected for hematology, clinical chemistry, urinalysis, and urine chemistry tests from 10 animals/sex in Groups 1 through 5. Five animals/sex in Groups 1 through 5 were sacrificed during Week 4; livers were. collected and weighed. A portion of the liver was shipped to the Sponsor for PFOS analysis and mitochondrial activity, a portion was shipped to Pathology Associates International for hepatocellular proliferation rate measurements by proliferation cell nuclear antigen (PCNA), anda third portion was used for determination of palmitoyl-CoA oxidase activity. During Weeks 14 and 53, necropsies were performed on five and 10 animalssex, respectivelyinGroups | trough 5. At necropsy, macroscopic observations were recorded, organ weights were obtained, and tissues were placed in fixative as specified by the protocol. In addition, liver samples were collected for PFOS analysis (Weeks 14 and 53), hepatocellular proliferation rate measurement [by PCNA at `Week 14 and by bromodeoxyuridine (BrdU) immunohistochemistry at Week 53] and palmitoyl-CoA oxidase determination (Week 14). A section of duodenum was also B 002157 CovanceIM63T26-210853 collected at Week 53 for BrdU immunohistochemistry. At unscheduled necropsies for animals that died or were sacrificed because of poor health, macroscopic observations were recorded, and tissues were placed in fixative. but organ weights were not obtained. Microscopic examinations were done on selected tissues (adrenals, brain, eyes, kidney, liver, mesenteric lymph node, pancreas. spleen, testes. and ovaries) from the animals necropsied during Week 14 and on all tissues from the animals in Groups 1, and that were necropsied during Week 53. In addition, microscopic examinations were done on the tissues from animals that died or were sacrificed due to poor health. Statistically significant differences cited in the Results and Discussion section are based on comparisons between the control and treated groups. RESULTS AND DISCUSSION Clinical Pathology `Weeks 4, 14, 27, and 53, There were relatively few statistically significant or otherwise notable differences for clinical pathology results between the control and treated groups. `Those differences that were the most consistent over time were considered to be. associated with administration of the test material. These included mildly higher urea nitrogen for males and females fed 5.0 or 20.0 ppm; mildly lower glucose for males and females fed 20.0 ppm; mildly to moderately lower cholesterol for males and females fed 20.0 ppm; and mildly higher alanine aminotransferase for males fed 20.0 ppm. There were no correlative microscopic renal findings for the minor change in urea nitrogen. The findings for alanine aminotransferase, and possibly for glucose and cholesterol, were likely associated with the histopathological findings of hepatocellular hypertrophy and vacuolation. None of the clinical pathology effects were considered adverse. Of uncertain relationship to the test material were statistically higherurea nitrogen for `males fed 2.0 ppmand statistically lower glucose for females fed 2.0 or 5.0 ppm at Week 53. These findings were not as consistent over time as those considered to be effects of the test material, and the magnitudes of these differences were very small. : 10 002158 - Covance 6329-183 Tews All other statistically significant differences for clinical pathology results between the control and treated groups were considered incidental. Most of these differences did not affect animals fed the highest dose level, and none of these differences were consistent over time. Anatomical Pathology Week 14, Terminal Body Weight and Organ Weights. Terminal body weights were comparable between control and treated groups. Absolute and relative liver weights were: significantly increased in the males given 20.0 ppm. In females given 20.0 ppm, only the liver-to-body weight percentage was significantly increased. The absolute spleen weight was significantly decreased in the females given 20.0 ppm. as was the absolute lung weight in females given 2.0, 5.0, or 20.0 ppm. Spurious, significant increases in left thyroid/parathyroid-to-body weight percentages were seen in females given 5.0 or 20.0 ppm. Macroscopic Observations. There were no macroscopic observations that could be: attributed to the administration of the test material Microscopic Observations. Test material-related histomorphologic changes were limited to the liver in the males given 5.0 or 20.0 ppm and in the females given 20.0 ppm. The. changes consisted of hypertrophy of hepatocytes in centrilobular areas and midzonal to centrilobular hepatocytic vacuolation. The incidence and severityofthe changes tended to be greater in the males. `There were no potentially test material-related lesions in the remaining tissues examined. Week 53 Interim Sacrifice, Body and Organ Weights. In the females, terminal body weights were significantly decreased in the group given 20 ppm. Terminal body weights in the males were comparable between control and treated groups. In the males, absolute and relative liver weights were increased in the group receiving 20 ppm. In addition, absolute and relative spleen weights were decreased in males given 20 ppm. Significantly decreased left thyroid/parathyroid weights were considered to be spurious due to the absence of a contralateral effect. . n 002159 Covanc3eM62T256-219853 In view of the significant decreased in body weight in females given 20ppm, significant increases in organ-to-body weight percentages for brain, kidney, liver, and spleen may be ofno toxicological importance. Decreased absolute weights in the left adrenal gland and bilateral adrenal-to-brain weight ratios may also represent changes secondary to the body weight loss. Macroscopic Observations. There were no clear or consistent gross observations at the Week 53 sacrifice that could be attributed to the administration of the test material. Microscopic Observations. Centrilobular hepatocytic hypertrophy and vacuolation was increased in incidence and severity in the males given 20 ppm. In the females given 20 ppm, generally, only centrilobular hypertrophy was seen, and the change was less severe than that noted in the males. In addition, minimal to slight Centrilobular hepatocytic pigment was found in the females given 20 ppm. There were no other `histomorphologic changes that could be associated with the administration of the test `material. Unscheduled Deaths Through Week 53 Large, mottled, or diffusely dark livers were noted in 2/3 males and 1/1 females given 20ppm. There were no other gross observations that could be attributed to the `administration of the test material. Microscopically, several animals given 20 ppm exhibited liver changes that were similar or related to those seen at the Week 53 Interim Sacrifice. Robert L. Hall, DVM, PhD Diplomate, ACVP (Clinical Pathology) Richard D. 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E wommEE NDED 1 3 10 10 | roe omngan 23&8wn 9 g 88 . nates n QS cmeegpan SOgS 2 roe comma O88S Q3 QS ie 30 Covance 6329-183 3MT-6295 Table 30 Results of Homogeneity Analyses (ppm) Mixed 3/18/98 104-WEEK DIETARYCH`SRUOLNFIOCNITCOXAICCIIDTPYOTAANSDSICUAMRCSIANLOTGE(PNRIOCSI:TTY-ST29U5)DYINWRIATTHS PERFLUOROOCTANE. _--_-- T-6295 (ppm) --Sam--pleH s Locmatiopn _e ReRpepls liiccaal ttee 0o0.55c~a ~ t2i 200 on Top 1 0.605 23 2 0.588 214 Mean 0597119) 214 (107) Middle 1 0.657 2 0.690 Men 0674(135) 26 25 236118) Bottom 1 0.601 26 2 0.631 24 Mean 0616(123) 22.5113) a Each value in parenthesis is the percentof theoretical -- 002350 Covance 6329-183 3M T-6295 Table 30 (Continued) Results of Homogeneity Analyses (ppm) Mixed 4/16/98 104-WEEK DIETARYCHSRUOLFNOINCITCOXAICCIIDTPYOATNASDSCIUAMSRACLTI(NPROOSG; ET-S6N2T5IU5)DCIYNIWRITATTYHS PERFLUOROOCTANE -_-- O00 p Samplo e Locan tion _RReepplliceatee 0--T 0_.55 2 E 2 T-62095S s(5ppm) 0 ) 20 Top 1 om 2 os 222 5.36 203 5.48 181 174 3 Mean 0406 212 0.557(11)" 212(106) 4.04 4.96 (99.2) 18.4 18.0(90.0) Middle 1 048 2 04m 228 201 490 490 30486 Mean 0.481(96.2) 2.09 213(107) 5.28 5.03 (101) 197 19.5 213 202 (101) Bottom 1 0955 2 0s19 2.04 470 203 5.00 17.6 187 3 Mean 0359 0611122) 184 197(98.5) 494 488(97.6) 184 182910) a Each value in parenthesis is the percent oftheoretical. : 002351 Covance 6329-183 3MT-6295 `Table 30 (Continued) Resultsof Homogeneity Analyses (ppm) Mixed 6/18/98 104-WEEKDIETARY CHSRULOFNOINCICTOAXCIICDITPYOTAANSDSICUARMCSIANLOTG(EPNFIOCSI:TTY62S5T5U)DIYN WRAITTSH PERFLUOROOCTANE -_ 000000 Sapmploe Loocactihon _ReRepplliiccataes00.55 T-6295 (ppm) 2 2 0s5 0 2 Top 1 0566 2 110 238 271 487 5.18 203 187 3 Mean 095s 0874(175) 223 244(122) 490 498(%9.6) 186 19.2(96.0) Top 1 2 00.448830 - - - - - - 3 0401 - Mean 0.455910) - -- -- Middle 10617 2 06x 3 0635 Mean 0.630(126) 250 211 205 222(111) Bottom 1 0640 3.08 2 069% 229 3 0532 241 Mean 0.622(124) 259(130) a b Each value Reassay. in parenthesis is the percent of theoretical 5.15 5.43 470 509(102) 5.08 512 5.15 5.12(102) 183 178 182 18.1(905) 194 205 182 19.4 (97.0) 002352 Covance 6329-183 3MT-6295 `Table 30 (Continued) Results of Homogeneity Analyses (ppm) Mixed 04/22/98 104-WEEK DIETARYC`HSRUOLNFOINCITCOAXCIICDITPYOTAANSDSICUARMCSIANLOTG(EPNRIOCSI:TTY-6S2T95U)DIYN WRAITTSH. PERFLUOROOCTANE -_-- T-6295 (ppm) Sampplee Looccaatoionn _ReRpelplliecaattee __ 2200 Top 1 189 2 18.4 Mean 187 93.5) Middle 1 201 2 19.6 Mean 19.9.99.5) Bottom 1 211 2 201 Mean 20.6 (103) a Each value in parenthesis is the percentof theoretical 002353 `Table 31 Covance 6329-183 3M T-6295 ResultsofStability Analyses (ppm) Mixed 3/18/98 104-WEEKDIETARY CHSRULOFNOINCICTOAXCIICDIPTOYTAANSDSICUAMRSCAILNOTG(EPNRIOCSI;TTY-6S29T5U)DIYN WRAITTSH PERFLUOROOCTANE S_tora--ge C--onditions Initial Replicate 1 2 Mean 0.5 0657 0690 0.674(135 T6295 (ppm) 10 v2c0 7 20 115 1.51 229 267 26 25 133(133) 248(124) 23.6(118) 28 days, room temperature 1 0507 2 0554 111 1.94 0937 201 207 208 Mean 0.531(106) 102(102) 1.98 (99.0) 20.8 (104) a R`emsidudlltesoffotrhtehedo0s.5e-parnedpa2r0a-tiloenvselfsorwehroemodgeernieveidtyfranoamlytshies.sample collected from the b Each valuein the parenthesis is the percentoftheoretical -- 002354 Table 31 (Continued) Covance 6329-183 3MT-6295 Results ofMSitaxbeidlit4y/1A6n/a9l8yses (ppm) 104WEEKDIETARYCHSRUOLNFOINCICTOAXCICDITPYOTAANSDSICUAMRCSIANLOTG(EPNRIOCSI:TTY-6S29T5U)DIYN WRIATTHS PERFLUOROOCTANE --St_ora--ge_Con--dimtions Replicate 0.5 T6295 (ppm) 0 20 7 20 Initial 1 0478 2 0478 228 490 201 490 19.7 19.5 3 Mean 0486 0481(962) 209 2.13(107) 5.28 5.03(101) 213 202(101) 33 days, room temperature. 1 os 2 0553 257 5.64 254 583 284 21 Mean 0.540 (108) 256(128) 5.74(115) 25.6(128) a Rperseuplatrsatwieornsefdoerrihvoemdogfernoemitthyeasnaamlypsliescollected from the middle of the dose b Each value in the parenthesisi the percent of theoretical. 002355 Covance 6329-183 3MT-6295 Table 32 Results of Dose Preparation Analysis (ppm) 104-WEEKDIETARYCHSRULOFNOINCITCOXAICICDITPYOTAANSDSICUAMRCSIANLOTG(EPNRIOCSI;TTY-S62T9U5)DIYNWRIATTSH. PERFLUOROOCTANE. T-6295 (ppm) _t M--ixDate Replicate e0e5 M 10 20 0% 20 3/18/98 1 06s7 2 06% 115 229 236 151 2.67 25 Mean 0.674135 133(133) 248 (124) 23.6 (118) a Resultsfor the collected from t0.h5e-maindddl2eo0-ftlehveeldsowseerperdeeprairvaetdiofnrsofmorthheomsoagmepnleeity analysis. b Eachvaluein the parenthesis the percentoftheoretical. " 002356 Covance 6329-183 3MT-6295 `Table 32 (Continued) Resultsof Dose Preparation Analysis (ppm) 104-WEEK DIETARY C'HSRUOLNFOINCITCOAXCIICDITPYOTAANSDSICUARMCSIANLOTG(EPNRIOCS:ITTY-6S29T5U)DIYN WRAITTSH PERFLUOROOCTANE T-6295 (ppm) MixDate Replice 0 05 20 50 20 anes 4123/98 1b 04m 2b 04718 30-0486 Mean - 0481(962F 1b 2b Mean - 0s 03m 0377(154) 228 201 209 2130107) 207 176 1920960) 490 490 5.28 S503(101) 654 492 S573(115) 197 195 213 202(101) 199 213 20.6 (103) 4123/98 423198 4130198 1 - 0674 2-083 Mean - 06050121) 1. 2Mean - 0s 0406 0462924) 1b 2b Mean - oss 0478 0515103) - 212 211 212(106) 40.77880 278(556) 455 612 53400) 5.55 6.03 S79(116) -: 26 21 22.4(112) 430098" 1 -- Me2 m . - - - 5.24 - - 524 - - 524105 - a Resuls for the 0.5-, 2.0-, 5.0- and 20-ppm levels were derived from the sample collected from the middleofthe dose preparations for homogeneity analysis. b Below the limit of quantitation (<0.4 ppm). Each valueinthe parenthesis is the percent of theoretical. d Reassay. e Retention. 002357 `Table 32 (continued) Covance 6329-183 3MT-6295 Results of Dose Preparation Analysis (ppm) 104-WEEK DIETARY CSHURLOFNOINCICTOAXCIICDITPYOTAANSDSICUAMRCSIANLOTGE(PNRIOCSI;TTY-6S29T5U)DIYN WRIATTSH PERFLUOROOCTANE. MixDate Replicate 0 0.5 T6295 (ppm) 20 50 20 517198 1 0962 0406 173 437 182 2 0942 039% 174 431 182 Mean 0.952 0398(19.6f 174(87.0) 434(868) 182 (910) s/7198 1 LI 0202 - - - 2 LIZ 0557 - - - Mean 112 0380(760) - - - 517198 1b 0476 - - - 2b 0347 - - - Mean - 0412(824) - - - S498 spi 1b 2b Mean 1 <075 2 <075 Mean - 04m 0402 0438(87.6) 0157 0349 0253(506) 252 5.48 2.54 5.06 253(127) 527(105) 1.60 4.98 1.86 627 173(865) 563(113) 198 21s 207 (104) 196 209 203(102) sr2198* 1 b 0.248 - - - 2b 0326 - - - Mean - 0287(57.4) - - . 5128/98 1b 0a 195 619 2b 0453 229 649 Mean - 0448(89.6) 212(106) 634(127) 'b Below the limitof quantitation (<0.4 ppm). Each value in the parenthesis is the percent of theoretical. d Reassay. e Retention. The low standard (0.4 ppm) was not used for the calibration curve. 218 23 22.1(1L 002358 Table 32 (continued) Covance 6329-183 3M T-6295 Results of Dose Preparation Analysis (ppm) 104-WEEK DIETARYCH`RSOULNFIOCNITCOXAICCIDITPYOTAANSDSICUAM SRACLTI(NPROOS;GTE-S6N2T9UI5)DCIYNIWRIATTTSYH. PERFLUOROOCTANE MixDae Replicste 0 05 T-6295 (ppm) 20 50 20 614198 1b 2b Mean - oes 195 491 182 0610 1.66 539 203 0.614(123) 181(905) 5.15(103) 19.3(%.5) 61198 61898 1b 2b Mean - 0446 05% 0.493(986) 1b 0617 2b 063 3-063 Mean - 06300126) 225 191 208(104) 2.50 211 205 222(111) 522 481 502(100) 5.15 5.43 4.70 509(102) 190 169 180 (90.0) 183 17.8 182 18.1(90.5) 76198 1b ose 1.60 390 179 2b 0519 1.90 413 174 Mean - 0542(108) 175(87.5) 402(804) 17.7(885) 6/98 813198 z1 -.Mean - 1b 2b Mean = - 04m 0569 0524(105) - 207 204 206(103) 476 5.41 509002) 5.60 495 528(106) - 189 199 19.4(97.0) a Results for the 0.5-, 2.0-, 5.0- and 20-ppm levels were derived from the sample collected from the middleof the dose preparations for homogeneity analysis b Below the limit ofquantitation (<0.4 ppm). Eachvaluein the parenthesis is the percent of theoretical. d Reassay. & Reassayed due to unacceptable calibration curve for original assays. 002359 `Table 32 (continued) Covance 6329-183 3MT-6295 Results of Dose Preparation Analysis (ppm) 104-WEEKDIETARY CSHURLOFNOINCICTOAXCIICDITPYOTAANSDSICUAMRCSIANLOTG(EPNRIOCSI:TTY-S62T9U5)DIYNWRIATTHS PERFLUOROOCTANE. T-6295 (ppm) MixDate Replicate 0 05 20 50 20 9/1098 1b 2b Mean - 0am 223 458 17.1 2: 227 4.64 168 138Q76 225(113) 4610922) 17.0(850) 90/98* 1 - 0213 2. ox; - - - - - - Mean - 022044) - - - 9/1098" o-w - - - 2-12 - - . Mean - LISI) - - - onoss* 1 - ob 2 - hb - - - - . 9/1098" 1 04s - - 2-038 . - Mean - 0431862) - - 10/8/98 1b 2b Mean 0836 179 5.68 04 183 490 0659132) 181(90.5) 529(106) 'b Below the limit of quantitation (<0.4 ppm). Each value in the parenthesis is the percent of theoretical. d Reassay. f RReeatesnstaiyoonf.the retention. 179 172 17.6(85.0) 002360 Covance 6329-183 3M T-6295 `Table 32 (continued) Results of Dose Preparation Analysis (ppm) 104:WEEK DIETARYCSHURLOFNOINCITCOAXCIICDITPYOATNASDSCIAURMCSIANLOTGE(PNFIOCSI:TTY6S2T55U)DYIWNRIATTHS PERFLUOROOCTANE -- ee-- r ------ T-629-- 5T(eppgm) -- -------- -- MixDateeeReplicate 0nS0.5 ri 20ne 50 D 20 B. 10/8/98 Io. - 2 0s (30F - -. .- 10/8/98 1 4m - - 2-1; - - Mean - 0747049) - - 10/8/98" 11/5/98 1 - 0474 2-047 Men - 0486972) 1b 2b Mean - 0546 0556 0551(110) - 184 205 195(97.5) - 433 445 439 (87.8) 12/3/98 Ib ob 2 bb Mean - - 1.65 386 151 360 158(79.0) 378(75.6) 12/3198" 1- 2 Mean - 0607 198 - 0s 1.69 - 0573(115) 184(920) - b Belowthelimitofquantitation (<0.4 ppm). d Reassay. Each value in the parenthesis s the percent of theoretical e Retention. f Reassayofthe retention & Group will reassayed due to unacceptable chromatography. - - 163 154 15.9(79.5) 146 166 15.6 (78.0) - - 002361 Table 32 (continued) Covance 6329-183 3MT-6295 ResultsofDose Preparation Analysis (ppm) 104-WEEK DIETARY CHSRULOFNOINCICTOAXCIICDIPTOYTAANSDSICUAMRCSIANLOTG(EPNFIOCSI:T-Y6S25T5U)DIYN WRAITTSH PERFLUOROOCTANE _-- T-6295 (ppm) R MixR DateMReeplim cate S0 OS05 W20O0 50 w 20 o 12/31/98 1b 2b Mean - 033% 0246 189 1.69 442 190 an 19.4 0293 (586 17989.5) 4.57 (914) 19.2(96.0) 12/31/98 1- 2. 0.442 0422 - - - - - - Mean - 0432864) - . . 1128199 1b oe 2 be Mean - - 167 an 17.6 205 492 17.1 186(93.0) 482 (96.4) 17.4(87.0) 128/99 21 - bb -- -- -- 1728/99 r- b 2 b Mean - - - - - - - - 225/99 1 1498" 0907 206 5.67 2b 082 679 7.48 Mean - 0880(176) 443222) 6.58(132) 2025/99 1b 2b Men b 0.450 056 1.64 491 206 4.56 0488976) 185025) 474948) b Below the limit of quantitation (<0.4 ppm). d Each valueinthe Reassay. parenthesis i the percentof theoretical. This level was outsideofthe modified standard curve and will be retested. 20.1 20.1 20.1(101) - - 002362 Table 32 (continued) Covance 6329-183 3MT-6295 ResultsofDose Preparation Analysis (ppm) 104:WEEK DIETARY CHSRULOFNOINCITCOAXCIICDITPYOTAANSDSICUAMRCSIANLOTG(EPNFIOCITTY-S62T9U5)DIYN RWAITTSH PERFLUOROOCTANE MixDate Replicate 0 05 1 20 50 20 3125/99 1b 0414828 191 544 2 bb 297 5.59 185 199 Mean - - 244122) 5.52(110) 192 (96.0) 3/25/99* 1 2 - 1.63 0702 218 191 - - - - Mean - 117234) 205103) - - 422199 Ib ob 2 bb Mean - - 197 473 17.7 204 572 188 201(10) 523(105) 183 (91.5) 4122/99" 5120099 1- 2Mean - Ib 2b Mean - 0.431 0.405 0418836) 058 0s 0557(111) -176 1.68 172(86.0) 54.9006 498(996) 465 4.67 4.66(932) -. 178 20.1 19.0(95.0) 'b Below the limit of quantitation (<0.4 ppm). Each value in the parenthesis is the percentoftheoretical. d Reassay. 002363