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ST-295 Final Report ToxDocs 10-152 Final Report 28-Day Oral Study in Rats (ToxDocs Study Number 10-152, MTDID 8391) Study Director: Jill Hart, AAS, LATg 3M Toxicology Assessment & Compliance Assurance, 270-2A-08 Saint Paul, MN 55144 Phone: 651-733-8586 Principle Investigator: John Butenhoff, Ph.D., CIH, DABT 3M Medical Department, 220-6W-08 Saint Paul, MN 55144 Phone: 651-733-1962 "Testing Facility: 3M Strategic Toxicology Laboratory, 3M Center, 270-3S-06 St. Paul, MN 55144 i Experiment In-Life Start Date: Experiment In-Life End Date: 5-18-2010 7-1-2010 CONFIDENTIALITY STATEMENT `This report contains the unpublished results of research sponsored by 3M. These results may not be published, either wholly or in part, or reviewed or quoted in any other publication without the prior authorization of 3M. ie ST-295 Final Report ToxDoes 10-152 Study Summary "The objective of this study (ToxDocs Study Number 10-152) was to investigate the bilateral `pupillary reflex responses in male Sprague Dawley rats (n=18/dose group) during 28 daily oral doses with either vehicle control (DI water) or MTDID 8391 (ammonium perfluorobutyrate, NH"PFBA, 30 and 150 mg/kg/day). Interim serum samples were also collected weekly from a subsetofrats (n=6/dose group) during the study. All ats survived until scheduled necropsy. Twenty-four hours aftr the last treatment, ocular tissues (n=12/dose group) were removed and processed accordingly for histology and Western blot evaluation performed in the laboratory of Dr. Don Fox at University of Houston, School of Optometry. In addition, retina, serum, liver, and brain samples were harvested (n=6/dose group) and analyzed for PFBA concentrations by LC/MS-MS. `Twenty-four hours after the last oral treatment, mean (= SD) serum and liver PFBA concentrations were 14.25 + 10.08ug/mLand 4.46 + 4.20 ug/g, respectively, for the 30 `mg/kg/day dose group rats, andserumand liver PFBA concentrations were 25.08 + 11.02 ug/mL and 11.20 + 7.37 ug/g, respectively, for the 150 mg/kg/day dose group rats. MTDID 8391 concentrations were less than 1 ug/g in eachofthe retina and brain scollected (cerebellum, cortex, and restof the brain). Western blots and histology analysis for the ocular samples are pending a the time of preparationofthis report and will be appended to this report when they become available. Study Objective "The objective of this study was to investigate the bilateral pupillary reflex responses in male Sprague Dawley rats durinag 28-day oral dose study with either vehicle control (DI water) or MTDID 8391 (ammonium perfluorobutyrate). Rats received 28 consecutive daily dosesofeither vbielhaitcelrealcopnutpriolllaorry MreTflDeIx Dwe8r3e9e1v(aNluHaytePdFdBuAr,ing30thoertr1e5a0tmmegn/tkdp/edriaoyd).vTiaweonrtaly-gfaovuargeh.ouPresriafotdeircthe last treatment, ocular tissues from all ras wereremovedand processed accordingly for histology and biochemical analysis in accordance with methods provided by Dr. Don Fox, University of Houston, Schoolof Optometry. In addition, to characterize the toxicokinetic profile ofMTDID 8391 during the 28-day treatment period, interim blood samples were collected from a subgroup ofrats via tal vein bleeding on a weekly basis. Twenty-four hoursafter the last treatment, ocular and brain tissues were removed and stored frozen pending LC-MS/MS analyses. Experimental Procedures & Observation Test Material: [[lemiy [moms Other Identifiers Ammonium perfluorobutyrate, E-19895, NH,PFBA LotBatch Number | 3M NB# 140499-10/11 -2- ST-295 Final Report Molecular Weight Purity/Strength CFCF-CF-CONIL 98.8p%ure,28.9% in water ToxDocs 10-152 Combi T7 se ] Species Selection: `The Animal Usage Application protocol (AUA 2008-0459) requires the use ofa small rodent speciesto optimize useoftest material. Sprague Dawley rats were selected as they are commonly used in oral toxicity testing to generate data for human health risk assessment. Animal Husbandry: Housin~g Rats from the main study group were individually housed in standard solidbottom cages except during dosing which requiring their removal from cages. Diet/Wate~r commercially available certified rodent chow and tap water were provided to all aniamdaliblitsum throughout the study. Environment Environmental controls for the animal room weresetto maintain a temperatureof 72 3F, humidityof 30-70%, a minimumof 10 exchanges of room air per hour, and a 12-hour light/dark cycle. The 12-hour light/dark cycle was altered on the dayofpupillary reflex examination, in which the room was kept dark in the moming until the pupillary reflex examination was completed. Exposure: Dose Grow umolkg | Volume : Suiulel Control istiled water) 30 mykg/day |S [| oo| 0 |orem [pe 150mykg/day EID EE E al Thos refers toth sal form o he main component ofthe ext article. 24 hourdsospeost last. 24 hours post ast 24 hourspost ast. MTDID 8391 was prepared in vehicle (distilled water) as 6 and 30 mg/mL. Five ml. of the dosing solution was orallygivento rats per kg body weights for 28 consecutive days. -3- ST-295 Final Report ToxDocs 10-152 Observation: Each animal was observed daily when practical for morbidity/mortality. Body Weights: All ats were weighed weekly. The body weight information was used to calculate the daily dosing volumesforthe subsequent 7 days until next scheduled weighing. All rats were also weighed prior to necropsy. Pupillary Reflex Evaluation: Pre-dosing and interim pupillary reflex evaluations were performed on allrats throughout the study based on methods provided by Dr. Don Fox. Interim Specimen Collection: Interim (weekly) blood samples were collected from a subsetofrats (n=6/dose group) via tail vein bleeding. The blood samples were allowed to clot foar period of 15 10 30 minutes at roomtemperatureand spun down in a centrifuge at 2000 x g for 15 minutes to obtain serum. The serum samples were carefully transferred to labeled 0.6-mL polypropylene microcentrifuge tubes. Serum, urine, and fecal samples were stored frozen at 70C pending LC-MS/MS analysis. Necropsy and Specimen Collection: All the rats were euthanized by decapitation 24 hours after last dose. Immediately after decapitation, ocular tissues were removed and processed accordingly for either Westen bolts, histology, or LC-MS/MS evaluation.Liverand brain tissues (sectioned and labeled as: cerebellum, cortex, and the restofthe brain) were also removed from a subset of rats pending LC-MS/MS analyses. Results & Discussion Al rats survived until scheduled necropsy. Data for body weights and liver weights are `summarized in Table 1. All MTDID 8391-dosed rats gain weight similar o control rats. Bilateral pupillary reflex evaluation results are presented in Table 2. The corresponding ocular Western blots and histology data are pending, and they will be appended to this report when they become available. LC-MS/MS data are presented in Table 3. Twenty-four hours after the last oral treatment, serum `and liver PFBA concentrations were 14.25 10.08 ug/mL. and 4.46 4.20 ug/g, respectively, for 4. ST-295 Final Report ToxDocs 10-152 the 30 mg/kg/day dose group rats, and serum and liver PFBAconcentrationswere 25.08 + 11.02 ug/L and 11.20 7.37 ug/g, respectively, for the 150 mg/kg/day dose group rats. PFBA concentrations were less than 1 ug/g in eachofthe retina and brain regions collected (cerebellum, cortex; and restofthe brain). Se ST-295 Final Report Signatures Report Prepared By: LAJie Host. Jill Hart Study Director Reviewed By: Pb 2 Lelio John Butenhoff, Ph.D., DABT, CIH Principle Investigator "ToxDocs 10-152 KITA 01 Date a Gm zon Date "6 ST-295 Final Report ToxDocs 10-152 Table 1: Data for body weights and liver weights _-- pro-Dose] wee2[ waka [wees[(TProeT rDops|| DGoossee Taman| wBeon0vt|| wBeoadyn|| WBeiogdhyt|| wGeiegdht|| (F5ol% || eiWegl n | 0 |e |e |o [oCre7s I0 Toa | ra | sor| saa| sa=sJ| Wa | 1 [IomCeos TW 23s |0 70 0|2 oo| wiz| ss | 3 Wa| [oI rgor[vi | 2a 0| ges 0| sri|aJ 8[sA r| 3 wa| [ToorRaessseTVviTT226011112z5o6rTom00T To3 rTo6 se5 || WW6 aa]] [IoS Creo TXT2s 0 280 6 Tr0T= a0 Toi0| Wa] [[CFoorrsseeT TnXTo2as ||22677TTso0r4sTTo3r3[6500 T| WWaa]] I[FCorEesTT 226 |0 2660Top0T5 s[50 |13A7] [[oCRoRssooreT TW T 224201 | 228666TToovrerTTossT[soere ||t1a6s0 [oI msoe|v 2o0 r | go0r| sor| 516 |= so | 11630| [TooRmsattto[vviir1T5201|0r2sT|5s8r6rT|o3easT|omsaos || Waa|| [[OoRmSerTTXXT Tor a 3Toa stn T[o3rmer T[woeerTe Twwaa]| [[oCoomamt[[xxu[2ps0||oens || sgos||saszt|| sseos1|aa]| [[oCmoomette[ TT1T1a2s 1|227o66T|o5or|Tosew T[osrerr || WWaa|| [[CCoommeettssTWW1T2a3r1122070Te5o0T TooeTToosooT| WWa || [Io0 mCszo [vo|2r6 | sop|5 [ so0 r| Wa| T[oomRsazzsz[[vvaw[|2s2e0o||s2e66o|| g3a0s0||3so0r || ssiosa || 16653|| I[S CComes TXT2410 12800 S00 6Too0 r0TZ soW |165 [[ooRmssssrsTTXXiTT2aa322806Too0s6[T90o4TToosa T| 115605 T[oCmoRssssesT TXvT2255001228e6 || o5tta5T|oooT[ooesse ||1t3a0s|| . CCCC 00 0 0 0 20 1 [["CooRRssssaz[xxvviiI [6a6||22oe8r|| 521o70 ||osrais|| ssaess || aazi|] [CCoCmsEss[wv1225 |260 0 |7o6 | wJ oo|soa |wa|| [CComs0se[xv1200 |20 s| 3000| srJ o | so0 s | ma]| [[CCoomrssseo[[wxwvwi[5p1es ||270767[|350106 || s5o3r|| soieea || wwaa|] TComCaso[xi [261|200 6 | 3170 | 5309| so20| ma]| CCCC 0 0 0 70 02 0 || [[oCRoeRassee[|xxvvi|2656112s6o6r || 2t8a8 ||3506[|ssai1|| WWAa]] 7. ST-295 Final Report ToxDocs 10-152 Table 2: Bilateral pupillary reflex data Sue| o>me fe) aa eTvrr|ar coiraenrteoon]s cSoeratneir]ne feeeems] =E E=te]] vee E=3=21 = 1 E] ffEoeesvTrv]]] E == t= = = = E = = e ffEeorw e aw]irr]] = E= r] ] SRE EH ee E = oT ] d ffpreeeertr]]] E= r]= = E = r = E = t f ffrext] = = a = E = H em = E E] H t fffe rsvr t]] = E = r] = E = r = = f C fos em] v = = == = ] f= = rie] = e =] w = pCfoers satmexr] FE = H t= = = = H = = =] c feeoseim] E= = T = ==. | = = BE Effoeesisvtvi]] F E= t e=t] = a = Et f ffoemeaTtw]i]] = = a E= -- r] Ei EB t] e foCnr sam] FE EHT r] =1 ==] = --E--T-- ffeesetor]] B E E T= = = ch] f= Eetr e] ffeeesssrst]]] F E BE H = == E= --F --=-- ffseovr]} --E ----t-- -- = = EH ] ee ==nt efesisTo]a] --E----1------= = == ] ] = l E] = r eTsoie]} --B--E--] == = =E=a] aT ffeooWrsaaisrsetR zTTTexawrvrA]]]| ------A E 1 C =t] F = =E] =] wee = = EE=]| 5. ST-295 Final Report ToxDocs 10-152 Table 3: Data for Serum, liver, and brain PFBA Concentrations Liver Restofthe | Retnal Serum PBA | (PFBAa] (PF[aAo]oart 24|]|[CPoFrBsAb]oaltu2m4| 5oi |r DailyDose(mg)| A2800uS| 24FOS ourspost.|hourspost (on 2024| 24hours mr" | Poagsag || os"t9do9ne|| l"os9ao9ne||TIgy | | POE agg Lo [Mem|subn SUE oUDo|wlDe |SUSE tion [so [7 wa "na | Na | NA | NA | NA| Le fem me am Lem ama en [so[7008 | 420 | 027 | 029 | 025 |009| [Mean| 2508 | 1120| 083 | 085s | 082 |032 | ["so [102 |757 | 085 | 08 | 067 |020| LreLspOecQtifvoerlys.erum, liver, brain, and retina were ng/mL, 25 ng/g, 25 ng/g, and S ng/g. 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