Document GKz8daLLbMwG6n9b0RprKnkN4

Conducted At: Dur ing : Conducted By: Report By: Reviewed By: January 28, 1983 Riker Daboratorlea, Inc. 3M Center St. Paul, Kinneaota 55144 October 1980 to June 1981 S. J. Gibeon and J. D. Johnson v,L^ 'A?- r . E. Ober, PhD Manager, Drug Metabolism Date Tfhig of Contenta Sunaary ........*.... Introduction ................* Materials and Methods .................. Carbon-14 I-beled N-Ethyl FOSE ............. Animais ...................... Dosing ...................... Preparation of Dose/Feed Mixture ......... A d m in istra tio n of Dose ............ Sample Collection Radiometric Analyses ............ Sample Preparation for Radiometric AnalyseAnalyses of Samples ........... Metabolite Isolation and Identification ... Results and Discussion .................... References ................. List of Figures ....................... List of Appendices ...................... Peqe 1 2 2 2 2 3 3 3 3 4 4 4 5 8 9 10 14 Page 1 -- * :"S L 5 L 1 " ^ ^ - S i ' s u s j s r * - - - - 1-- * " " 1U .,, N U ,, U 1.=!.,^ J Alt cU.tto ! '" ^ " '." " * t" rit. ... < ... the pri-ary P" P" f ^.* of 14C-*-ethyl roSB/f.edad^" " analyzed for earbcn-1 content. Th ^ ln vmrioo tieeue were. ,, t! H-.thl S S q . n . i o . pl.m. cont.in. Ssss; iS " " "L r.irr.s ss ss rs s s rs - ssr-, j s s t s t s k s * - ** " Page 2 ss& z& zg^JB S :^ , 1 1.. >-- " " r PI" . r . ; r shown by 1c--ethyl EOSE in feed f 1 feceB collections cats were administereo ' the aoaing period. The rece were collected for two " elt "" `^ " .thyl FOSS dose were pooled ' r J S S ' i.'s . ' s s s s 3j r s s t - S J -- -- studies* Materia1 and Methods , ,,in n S -gthVl C7F , 5*CF2SO2-N-CH2CH20B .Denotes position of carbon-14 label H-Ethyl FOSE is ^ perfluorooctanesulfonamido ^ The 14c-s-ethyl EOSE used in t h U N - e X l ^ S ^ s t u S ^ e p o r t e d J U FOSE used in the single dose C u*" y determination repeatedfor the details of t h e r . d i ^ ^ c a l P u r i t y Bpeclflc activity of the lot of these two studies is reported ( > W - N - e t h y l *OSE used in these stu {Rilcer Isotope Inventory basis of the specific Number 468) is 0.482! "^ i c a /rtaracterisation, and radioch*ical activity determination, chemic 1 ^ . ,, . t h y l EOSE was judged purity reported separate Y Prior to dosing, three " le^ " t ' . t w l ' L ^ U TM ges!1 ody eights overnight in ^ " -- ** " 3' 2" 9` ~ -- - duri" the Page 3 pprreeppaarr^a*ti-o"n "-*--P-o-s-e/Peed M--i-x-t-u-re the preparation and the a***y orepared by wishing *50 *9 ,u. x 1C-H-ethyl r o ^ .ol^ion vaB pr lujietric flask, adjusting 1C-N-ethyl POSE into a 1 liter Claa h ^ inverting. The s f f f lijw s s iv r K r s s s a s r-- administration of P A feed cup a. attached with wi^ ^ ^ 0" 3 * ? 130 g portion of Purina Ground Ch wide-mouth, brown gl KSE/g of chow was veighed Into . ^ " "ture\-re frequently trans lar for each rat and portions of ^ ^ tQ place fffeeerrrrrreeedad tntooo t.h.e..c.o.rr--espo-nldnintgihhe*fe*ceuSdppscTutSphsie.t signi-f-i-c-a-n.t .s,,pi4l1l1i1 mng WW tthhee so such of the mixture in theJ " P* . ^ Uc-M-ethyl POSE/feed rats could occur. The jars containing tne___ t-BDeratur.. It too - 1 * * - <>* -- " "'-1 c.mnl. Collection *" were collected and stored During the dosing perif urine and feces were collected at froten (not analyzed at this tine)- aatlon of administration of 2<hour 'intervals for 1 e t ollcwi^ =" " r. urine and feces r e rhe 1*C-N-ethyl POSE/feed mixture, ther aacrifice. At 21 days f.n.5." - U - . " ar i r 4 : ; r TM " - . . ' . titer the last ingestion of the descending aorta and with diethyl ether; blood t ^ prepared Immediately transferred to a heparinl ^ ot analysis and the promptly by centrifugation. smaples r e then frozen. TM rata sacrificed by exsanguination teateB were collected as and liver, spleen, brain, collected for bone arrow whole organs. The ^ abdominal fat, skeletal muscle, sampling. Samples of stomach, and intestines), and the -~ Page 4 cUnmetric Analyses. C"T 1' Preparation Feces, U v .r . J > n ^ p l n r e homogenizing and alighting p in waring blander by hcoogenates 0- 9^ -- * homogenate between sampling*^ Saap = =iao5lation/identification tudie. - - " tabollte combusting suitable blank h ^ n" `nce ampit. were ebuted 1V-N-ethyl POSE solution) f y . analytical sple set. a ^ r ^ i n n i n g , middle. Erectly, duplicate 1.0 1 Drine collections were/^e^ipetted directly into aliquots of each sample were P P * plaama was sampled before^ vials and is 1 A!"!,i*ect`y f duplicate 0.5 -1 aliquot P *5 1 freezing and into scintillation vial, nd 15 ml -re cold - dark adapted befor counting- All radiometric measurements were Tri-Carb Liquid Scintillation samples counted directly,^ - n t i 9ng- Por"pit^a and urine ici.ncy for each sample was of internal standard to each ^ ~ tif s s r s ru f, ^ rr ;TM internal standard was,,fthreTw'ith high ratios) and these samples with low M S ratios and three w l t n g # correction was made for the were recounted. For cosbust!ed The ^ ,, n t recovery was recovery (mean, ) fron the POSE crusted with each based on reference samples or Packard Instrument Company. Downers Grove, I l l i n o i s . 2200 Warrenvill* Road, Page 5 .hollte Isolation_an Identifica,tlgn this ^ r .Ulclc acid column and a fracti located w thin-lay ehrow the structure to be C 8F 17S02NCH2CH3. CHjCOOH " n . 1.=*1 ^ L " " i S S j r r e s i ... p i " ln; ' " r s r s s 2 - " .rii r . s rjis ir s is - r ^ - r ir s * . - jr *r-- - s t s r r - r / , f f r.,:" TM js . 'is . r r . L ^ . r ^ ^ - ,, ,, r . n 1: 1 P = -rr :.f.;" ^ r by COIP. cblotoioc . * . * " ` r . ^ r . r : r r , _ s - s s r = - E s -s s - - n ,s rr" o tu .r. s ; ^ . a s s s . r0ou : r " - . 1 *-- ? " r F s 5 - S * " ,4c-i,- t^ n the chloroform fraction c"" ^ e* ' 9 Thus, the chloroform ool>n FOSE and the material In the fecal homogenate, fraction conta ne ; anaiysis of carbon-1* The procedure for O) chloroform labeled ooi>ounda haa been deacrib^ ^ ^ radlochromatogrn ia column fraction was analyied W 43 comptiaed 1.90 of the ahown in Figure 1. * " ^ " tion or k S0% of the carbon-14 in the carbon-14 in this column r" t 14c_H_ethyl FOSE ingestion. -- Onisilr activated silicic ac Company. Inc., Hilllamsport, id 100/120 pa. mesh, Clarkson Chemical Page 6 wasVcXed in benxene-chloroform with a rotating evaporator - y; - i s - placed on this cnd c"- - E " i S T S T . 3 5 - - p5oo - - - and column. Fifty . |>n*ane-chloroform 0 -14m^ ts chloroform <l1. vv>. 500 l ot " " through the column. Aliquots then 500 ml of chloroform wer P analysed radiometrically Too " ? Of the 50 rnl ^ " ^ I r ^ 'o ^ ' thr.e 50 .1 f.tion- .nA most of the carbon-14 appear .lutionB (Tractions 17, 18, Sleeted fra. the 1.2 1 " i n g the c h r o m s t o g r . p ^ and 19). By inspection of th' t that much of the !*=*ato_ Of the various fractiona.it ... .PP ,4 w thi. column cht" " pigment. fraction. 17. 18, -- contained !!fmg ^ " Mi-ethyl FOSE equivalents. The pooled fractions 17. and H f cm " 20 '* ,1UC` bzx s v - ' i s i a ~ s r" was located by scraping 0.5 " " 9J( 2Q ,, plate. (Only a narrow IVT.V5 cm x 20 cm ,ectlon ^ The catbon-14 was located at Rf 0.33. trip -1 -- was - p a d fra. the six and applied to a 20 cm * 20 " * } *he plat. was develop to 15 cm washed in methanol and "ir sv'aixing 100 ml chloroform, 100 ml in 100 ml of solvent prepared by , ?he carbon-14 was Z Z Z o l , and 2 1 concentrated acetic * = igln the advent located radiaaetrically (a "a toe plate laterally in 0.5 cm front was raped fra. the cent carbon-14 labeled material aegments and counted). was 0.77. A 1.0 cm zone ^ at R 0.77 was scraped and the 1f chiorofor-methanol 0-1- silica gel was eluted g r a t e d to a email volume, and v:v). The eluates were combine , e^ loped to 15 cm in the same r U e d on a . H i ^ i Z Z l Z Z l Z Z Z ^ as before. The chloroform-methanol-acetlc gment and counting. The - carbon-14 -as located by craping a - ^ ^ ^ ,, ,, centered carbon-14 was again located at Rf waS emted with acetone. S . mS ^ t l aS 5 ^ . 0 mg of R-ethyl ROSE equivalents. Figure 2 is a plate was 75 Blue Ben Drive, Hewart, D e l w a r e -- Analtech, Page 7 1) C 8P 17S02HH2 2) C BP 17S02NHC2H5 3) C8P17SO2NCB2CO2H C2H5 4) C 8F 17S02NaCH2C02H f . ; ^ r , K S `-- - , 1. .1 of - i n - p f P " " " 'M hydrcocide (System A) or in 100 ,, ,, ,, ,, t r a t e d acetic acid chloroform. 100 . 1 - t h ` n ' * ^ at e" t " Seated with acidified (Svatem B). The spote on the Plate reagente. The R f of S-- - --- ri" ? n " ^ . r r s C2H5 S ta ^ ^ - ^ ^ tr s ^ " r QC/MS analyaia (aee Appendix 1) . ^ L ? : 14^ > OC/HS d ata in d ic a te id e n tic a l r e t e n t i spectrum w ith o f .S n d .rd 3. In a d d itio n , i t th e s tr u c tu r e o f s ta n d a rd 3. - " * with C e l l , the 1 0 - 1 * ' " " t 't ' r hy. r i i c 2h5 .. . . < . - . 1 column was evaporated The m ethanol fr a c tio n f r th e ' i r ^ d' ^ l o ^ d to 15 cm in 100 m l o f ra -c iEd . TThe4- ra"di-ochSrom a*togrSam ie* shrown- in gV - 1-1 ~ t U Page p.aults and Discussion A major metabolite of ' identified a. 2-N-thyl The metabolite accounts tor m TeUc^S 50% of the carbon-1 4 .xcret.din a do#e (see Appendix 2) in feces pool at 24-48 bouts a 14c-*-thyl FOSE for 1 week. si"e e regimen in which rate "*r* ** (24_4B hour* after the laet do) " J S S - * " < s ; r i " ; ~.traction is ftTM tece* collected { ,,fleet what would be expected ,t various times after a single ^* x r0SE( ,,50% of the carbon-1* ek foUowing a single dost of . l.rge portion of the is eliminated via carbcn-14 eliminated i^pFSAA. In addition, three other Together, these other unidentified -rrr.: sr-~i. - - -- perfluorooctanesulfonate was identified in In a previous experiment, liver extracts from rats a.fee h,,o-u-ra,nea`sffuttfeeorrna,ate,inagclceo'uonrtaeld m for a**tleastt ...........-- octanesulfonamide (6.4% of dose) ( ) ln Uver as a metabolite at Identification of f tflU ^ i " " Be i!d identification of PFSAA as a 1 .4% of the dose after a ingl ,. in {ecee during the second metabolite comprising 50% of t e following a week of feeding twenty-four hcx,r P * * o S . are major metabolites 1*c--ethyl FOSE establishes that bo h o m p o ^ ^ as to ^j**telatlve^proport^on*of ^"biotransfor^tion to either co^und. Since it can be assumed t h a t ,, ^ y T i f fent "b a s ^ o n ^ amount of Ih1iS.iebiUF -t SAA - further * ? rsc'er- "r r compounds. of roSE toN ' e t h y l For the same reasons given for PFSAA, the '`^ ^ ^ ' " t e n t a t i v e l y transformation of N-ethyl FOSE to the t metabolites in the identified as perfluorooctjnesulfon^ide t 1 they have ,t been include"?!^luorooctanesulfonate and perfluorooctane- sulfonamide) is not established with these data. of wet tissue in various vg ^uW.lent. W-X-ethyl blood cells, 30. Plasma.contained 62 y g q_ ^ ^ a com.entratlon. are FOSE/ml of plasma. (Individual ,,,,--trations at 21 days following listed in Appendix 3.) **)* lic-x-ethyl FOSE are consistent expound. 2'^-1 -- "lth * -l w l y -liB page 9 References Min i s t r a t i o n of "-Ethyl ru" JJaannuuaarryy 1199.. 11998833.. chBr.cteri.tion of -Ethyl ^ .,,a Behr. BE. Synthesis and Characteri 2. 3. JoofhTno.talJCa'rbon-1* Jm T*^*T S . " s" " TC-9S-U C (Report) Deceaber 28, 1979. Tage '0 r.it of Figur Figure is Figure 2s .Figure 3: ssttx rsx sa '""*"* PERCENT OF TOTRL CPH ON PERTE Page 11 Figure 1 Thin-Layer Radiochromatogram of %zzzsrChloroform Column Fraction . 5 ammonium hydroxide Page 12 Figure 2 Thin-Layer Radiochromatogram of Column Fractions 17, IB, and 19 Pre-adsorbent SGF Uniplate: 100 chloroform 100 methanol 2 acetic acid Total CFM on Plate - 18,428 PERCENT OF TOTRL CPM ON PLRTE Page 13 Figure 3 Thin-Layer Radlochromatogram of Methanol Column Fraction Pre-adsorbent SGF Uniplate: 100 chloroform 100 methanol 2 acetic acid Total CPM on Plate 1,742 Page 14 r.iat of Appendice Appendix 1 POSE NB-56531-2BU-2BX ^ " " SF" " " of Hc-H-Ethyl Appendix 2: Appendix 3 Excrtion of Total -rbon-14 ^ I*c-H-Ethvl FOSE Fed to Rat* tt ^-^531^3^' a ipiichs Aftet n Oral Doae C.rbon-1* Contentt 21 W ^ l n Tl. ^ ^.565^-14-16 14c-W-Ethyl FOSE Fed to Rata r lit- TECHNICALREPQRT s u m m a r y t o :TECHN.CAL COMMUNICATIONS CENTER - 1 4 nporan,-ltnp o n > > P ' ^ ^ bo0' ~'<`' ~ a"~ " oTCa> >" --------March 13, 1981 KtVWOMOS: {ltdw n tramaw TaShpMliucanbaW-uSnura".)ottw CRLAP Analytical Report Chemical Analysis t -/MS . \_-/MS Cl/MS Negative MS BttcrtOr1O-*MAduthorit*"*) cuaaiKT oajacrivi: Request No. C5B236 'Project No. 91505026 Requeator - J. 0. Johnaon - M ^ ! S s a s = *___________ ________ - , rih *w -- . a MvTM** Ta'"tolCm""""" ""1C^"' . -f ehe reaulta from the two aamples using Rased on the greemen trueture of the major component in cc, El MS, and Cl MS, the struct the structure cj ms u. rS compound but cannot confirm the structure y " ' " thl* Inform*tonL m w iMlUia: aJC/1 , w **>-' '**. ' CENTRAL analytical l a b o r a t o r Report No. D a t e . ----------M-a--r-c--h-- 12l-- -- ^ t- Mas. Spectral Analy.l. of H-Ethyl FOSE--C W -- Subject. Mae P Riher_____ _ . Dept* R eq u esto r: P March R e q u e st NNoo. - -C-5-8-236 -- D *t8d R e p o rt: P roJ. No.A IA ?---- T0 /CHjCH3 C.FiiSOiN ___ 'CHjCOOH t,4--3. the requestor aeperated v-i<-e wee an extract fro* rat feces " viai, *ch containing Th* * T le ^ s y t c i m s and divided equally iato thr on various TLC sjst lOOpg. 100,000 cpm Of O, or y^erimental. -hnllte and atandard; each metabolite *"d ."nd`" bie j ll.t. the M - p l e * ^ " d ' " bl. II li.t. the c"a'` S , S S i X - * - - " **" ~ " niecueelon Tor this dicusion^w^ " ^ ^ ^ j i '-.i,,,iite and the standard, specif identical ^ " T. r r ^ ; retention time (8.6 - i r s s . - = - ^ 3 "-- t0 the El -- *pef ^ 6W*--p" b-blv sample ^ " y each contain the .' J ^ * t "high-a8S fragment is the sane in each -- pi r r / o ^ e -. primarily^three . J, Cl aasa spectrum ot m e at 600 / z - a P " ` rl ^ 1.I. The Cl r. " ' J H '' ^ r s : - r " for the parent ion AR Ho. 7454 March 13, 1961 Tag* 1 ronclualon ,,-nl.a ualng GC. El MS. ethyl TOSE. Sample Sample metabolite of N-Ethyl FOSE l-C methylated Standard of H-Ethyl TOSE ethylated Standard of N-Ethyl TOSE TABLE I Method of GC CC GC GC D? DP Mode of Operation El CI El CI -CI -CI Masa Spectrum Mumber AA5624 AA5625 AA5622 AA5623 AA5639 AA563B TABLE II u.ei.n 2740 r- Chromatograph ' eter, glees. 5ZOV101 on 80/100 Column: 2 100*C to 330*C at 10'C/min. Oven Program: temperature: 270'C ~ -----or temperature : 260*C (V.ri.n T O ) - * . - 1C .le/mln. esh Chrom G-KP ..,,Pont 21-49TM Spectrometer Source: Chemical Ionization Ci gas: Iaobutane M a Range: 15-617 m lz Separator: Single tage jet t o 4 200v for Negative CI) Multiplier: Bendi* Ch.nneltroA&> (et j. N. Sehroepfer JHS/ra I Appendix 2 Excretion of Totel Carbon-14 in Feces r an Oral Dose of U C H M t h j l FOSE Fed to Rata for 1 Week- Tine Period of Collection (Bra) 0-24 24-48 48-72 72-96 96-120 120-144 144-168 168-504 \ 5.74^ 2.10 1.52 1.14 0.66 0.49 0.48 2.35 Rat Ruaber 23 4.68 3.12 1.92 1.12 0.65 0.52 0.43 2.23 5.28 3.23 1.87 1.33 0.66 0.65 0.44 2.39 X + 5D 5.23 + 0.53 2.82 + 0.63 1.77 + 0.22 1.19 + 0.12 0.66 + 0.00 0.55 + 0.09 0.45 + 0.03 2.32 + 0.09 Total dotes fed rats were: Rat no. Appendix 3 C.rbon-U content >n Orel Doee of 'C-*-thyl