Document XOvz8opLyZrjp7LdOQneNbOjd

Ad Sa 6 - N-Et FOSE alcohol Pharmacokinetic - / 0 0 0 3 oz Biotrreformation of '^C-N-Btbyl FOSE in Rats Aitar Administration in Feed for One Week .. 7 January 28, 1983 Conducted At: During: Conducted By: Report By: Reviewed By: Rlker Laboratories, Inc. 3M Center St. Paul, Minnesota 55144 October 1980 to June 1981 S. J. Gibson end J. D. Johnson ion, MS 'Specialist Date l-iS&xL /-Z0-S3 R. E. Ober, PhD Manager, Drug Metabolism Date 000303 Table of Contant Page Summary .............................................. 1 Introduction ................ 2 Material* and Method* ............................... 2 Carbon-14 Labeled H-Ethyl FOSE .... 2 A n i m a l * ..... ......................... 2 D o s i n g ....... 3 Preparation of Dose/Feed Mixture ................ 3 Administration of Dose ....................... 3 Sample Collection........ 3 Radiometric Analyses ............................... 4 Sample Preparation for Radiontrie Analyses ...... 4 Analyses of Samples ............................. 4 Metabolite Isolation and Identification .......... S Results and Discussion........ 8 References ........................... 9 List of F i g u r e s ..................... 10 List of Appendices .................................. 14 000304 Page 1 Summary A major metabolite of **C-N-ethyl COSE was isolated from rat feoea and ' identified as 2-N-ethyl perfluorooctanesulfonamido acetic acid (PFSAA). The metabolite accounta for at leaat 50* of the carbon-14 axcratad In feces between 24-48 houra after cessation of administration of a mixture of ^^C-N-ethyl FOSE/feed which rats were fed for 1 week. The mean total dose was 300 wq/kg/mmk. Although collection of feces for source material for metabolite isolation was the primary purpose of this study, the rats were fed normal chow for three weeks following the one week of ,4C-N-ethyl FOSE/feed administra tion and plasma and select tissues were collected at sacrifice and analysed for carbon-14 content. The mean carbon-14 contents expressed as ug of H-ethyl FOSE equlvalents/g of wet tissue in various tissues were: liver, 233; spleen, 20; and red blood cells, 30. Plasma contained 62 iig equivalents '*C-N-ethyl FOSE/ml of plasma. The tissue concentrations at 21 days following the last dose after 1 week of feeding '*C-N-etltfl FOSE are consistent with what would be expected after multiple dosing with a compound that is slowly eliminated. C0035 Page 2 Introduction N-Ethyl POSE la a product sold by 3M and is also a key intermediate in the synthesis of other products. In a previous study (1), it was shown that perfluorooctanesulfonate is a metabolite present in liver extracts from rats at 96 hours after administration in feed of a single dose of 1*C-N-ethyl POSE. Proa the same liver extracts, a second metabolite was isolated and tentatively identified as perfluorooctanesulfonaalde. Other unidentified metabolites were present in the liver extracts as was shown by thin-layer radioctaromatograms. In the present experiment, three rats were administered 1*C-N-ethyl POSE in feed for 1 week and feces were collected for two weeks after the dosing period. The feces collections from 24-48 hours after the last 14C-N-ethyl POSE dose were pooled and extracted. A metabolite was isolated and identified. The results are reported and discussed in the context of other fluorochemlcal metabolism studies. Materials and Methods Carbon-14 Labeled H-Bthyl POSE C 2h s C7P 15*CP2s o 2~n ~c h 2c h 2o h Denotes position of carbon-14 label N-Ethyl POSE is 2-ff-ethyl perfluorooctanesulfonaaido ethanol. The 1*C-N-ethyl POSE used in this study was identical to the '*C-N-ethyl POSE used in the single dose 14C-N-ethyl POSE study reported earlier) the details of the radiochemical purity determination repeated for these two studies is reported (1). The specific activity of the lot of '*C-N-ethyl POSE used in these studies (Riker Isotope Inventory Number 46B) is 0.483 ^ 0.020 vCi/mg. On the basis of the specific activity determination, chemical characterization, and radiochemical purity reported separately (2), the '*C-N-ethyl POSE was judged suitable for metabolism studies. Animal Prior to dosing, three male Charles River CD rats^ were conditioned overnight in Individual stainless steel metabolism cages. Body weights were: Rat No. 1, 269 g) Rat No. 2, 210 g; and Rat No. 3, 219 g. The rata had free access to food^ and water before and during the experiment. -r Charles River Breeding Laboratories, Wilmington, Massachusetts. -- Purina Lab Chow, Ralston Purina Company, St. Louis, Missouri. 000306 Page 3 Doting Preparation of Dose/Feed Mixture The '^C-N-ethyl FOSE/feed mixture, prepared juat prior to thla tody, waa also used for the single dose study (1). The details of the preparation and the assay for the carbon-14 content axe reported (1). X '*C-N-ethyl FOSE solution was prepared by weighing 250 eg 14C-N-ethyl FOSE into a 1 liter Class A volumetrie flask, adjusting to volume with absolute ethanol, and nixing by Inverting. The 1*C-N-ethyl FOSE solution was transferred to a 4 liter beaker and 500 g Purina Ground Chow was added. The t^C-H-ethyl FOSE/feed mixture was stirred for one hour then transferred to a glass tray and the ethanol was evaporated. The carbon-14 content of the dose/feed mixture waa determined by combustion (1). Administration of Dose A feed cup was attached with wire to the inside of each cage. A 130 g portion of Purina Ground Chow containing 0,53 mg **C-E-ethyl FOSE/g of chow was weighed into a separate wide-mouth, brown glass jar for each rat and portions of the mixture were frequently trans ferred to the corresponding feed cups. Care was taken not to place so much of the mixture in the cups that significant spilling by the rats could occur. The jars containing the 1*C-N-ethyl FOSE/feed stock mixture were kept tightly sealed at roam temperature. It took one week for the rats to eat 130 g of the 1*C-N-ethyl FOSE/feed mixture. The total dose of 1*C--ethyl FOSE administered was for Rat No. 1, 256 m g A g i Eat No. 2, 329 mg/kgi and Eat No. 3, 315 aig/kg. After the 1*C-N-ethyl FOSE/feed mixture was consumed (168 hours), the rats were given free access to normal chow. Sample Collection During the dosing period, urine and feces were collected and stored frozen (not analyzed at this time). Urine and feces were collected at 24 hour intervals for 1 week following cessation of administration of the 1*C-N-ethyl FOSE/feed mixture; thereafter, urine and feces were collected as pools for individual rats until sacrifice. At 21 days after the last ingestion of 14C-N-ethyl FOSE, the rats were anesthetized with diethyl ether; blood was drawn from the descending aorta and immediately transferred to a heparinized tube. Plasma was prepared promptly by centrifugation. Aliquots were taken for analysis and the samples were then frozen. The rats were sacrificed by exsanguination and liver, spleen, brain, kidneys, lungs, and testes were collected as whole organs. The femurs and tibias were collected for bone marrow sampling. Samples of subcutaneous and abdominal fat, skeletal muscle, and digestive tract (esophagus, stomach, and intestines), and the remaining carcass were collected for each rat. 0*00307 Page 4 Radio-- trie Analyses Sanpla Preparation races, lirai, and spleen were prepared foe carbon-14 analysis by homogenizing and allquoting a a m p l e of the hoaogenate into ocabustion oonea^-. Homogenizing was done in Waring blenders by adding nine parts of water to one part of biological -- terial. The hentogenates (1.0 g) were weighed into combustion cones in duplicate. Care was taken to alx the hoaogenate between asaplings. Samples of red blood cells were weighed into coabustion cones. All other aaaplas collected (see sample collection) were not analysed* these aaaples re-- in available for further analysis or for -- tabolite isolation/identlficatlon studies. Bo-- genates and aaaples weighed directly were conbuated with a Packard Model 306 oxldiser. Recovery of carbon-14 was daterainad by ccnbustlng suitable blank homogenates (feces and liver) spiked with '*C-N-ethyl POSE solution; these reference samples -- re oombusted at the beginning, middle, and end of the analytical sample set. Urine collections -- re saapled and counted directly; duplicate 1.0 ml aliquots of each sample -- re pipetted directly into scintillation vials and 15 ml Aquasol* was added. Plasma was saapled before freezing and counted directly* duplicate. 0.5 ml aliquots plus 0.5 ml water -- re pipetted directly into scintillation vials and 15 ml Aquasol* added. All samples -- re cold and dark adapted before counting. Analyses of Samples All radio-- trie -- asura-- nts -- re done using a Packard Model 3380 Tri-Carb Liquid Scintillation Spectre-- ter. Por plasma and urine samples counted directly, the counting efficiency for each sample was determined by adding a known amount of internal standard to each sample and recounting. After each aample was corrected for back ground and for counting efficiency, the carbon-14 content was cal culated. Por ccmbusted amaples, counting efficiency for each sample was determined by use of the AES (Auto-- tic External Standardization) ratio method. To calibrate the external standard, a known amount of Internal standard was added to selected samples in the group (three with lew AES ratios and three with high rati-- ) and these samples -- re recounted. Por combusted samples, a correction was -- de for the recovery (man, 954) from the oxidizer. The percent recovery was based on reference samples of '*c-R-ethyl POSE combusted with each sample aet. -- Packard Instrument Company, Inc., 2200 Warrenvllle Road Doma r e Grove, Illinois. Page 5 Metabolite laoXatlon and Identification In this action, a lengthy procedure 1 described. X short descrip tion of this lengthy procedure follows: Feces honogenates were pooled and extracted with ether. The extract was chrosatographed on a silicic acid coluen and a fraction was rechromatographed on a seoond column, x major metabolite was located by thin-layer chroma tography (TLC) and isolated using preparative TIC. Comparison of Stf's in various systems with standard reference compounds suggested the structure to be CgFiySC^NCHjCHj. c b 2c o o h The data from gas chromatography/mass spectroscopy (QC/MS) confirm the identification. The fecal homogenate pool from feces collected 24-48 hours after the last adsdniatration of 1*C-N-ethyl FOSE was placed into twelve 45 ml centrifuge tubes (25 ml hcmogenate/tube). To each tube was added 15 ml diethyl ether and the mixtures were shaken for 15 minutes on a mechanical shaker. The tubes were centrifuged and the ether was removed. (Care was taken not to remove any of the interphase material). The extraction was repeated five tines. The ether was pooled end reduced with a rotating evaporator so that what remained was mostly the water that had been carried over with the ether. This water layer was extracted with chloroform (1:1, v:v). The layers were separated. Radicmetrically, the chloroform contained 5.9 mg of N-ethyl FOSE equivalents. The extraction removed *70* of the carbon-14 from the fecal homogenate. The chloroform layer was chromatographed by column chromatography (Onisiie^. was packed in chloroform into a 2.4 cm x 40 cm glass column). The chloroform extract of the aqueous residue from the ether extract was placed on the column. Five hundred ml of chloroform was passed through the colusrn and collected as one fraction. Following the 500 ml of chloroform, 500 ml of methanol was passed through the column, hadiometrleally, the chloroform fraction contained 4.6 mg equivalents of '*C-N-ethyl FOSE and the methanol contained 1.3 mg. Thus, the chloroform column fraction contained *55% of the material in the fecal homogenate. The procedure for thin-layer chromatography (TLC) analysis of carbon-14 labeled compounds has been described previously (1). The chloroform column fraction was analyzed by Tl and the radiochromatogram ia shewn in Figure 1. X component at Rf 0.43 comprised * 90% of the carbon-14 in this column fraction or *50% of the carbon-14 in the feces at 24-4B hours after the last 1*C-H-ethyl FOSE ingestion. -- Onisil, activated silicic acid 100/120 meah, Clarkson Chemical Company, Inc., Williamsport, FX. 000309 Page 6 The chloroform column fraction was evaporated with a rotating evaporator to a few ml and chromatographed by column chromatography, oniail* waa packed in benzene-chloroform (1t1, viv) in a 2.4 cm x 40 cm glass column and the chloroform column fraction was placed on this second column, fifty ml fractions were collected as 500 ml of benzenechloroform 0>1, v j v ) , 500 ml of benzene-chloroform (1i2, v j v ) , and then 500 ml of chloroform were passed through the column. Aliquots (100 ul) of the 50 ml fractions were analyzed radicmetrically, and most of the carbon-14 appeared to be in three 50 ml fractions collected from the 1i2 benzene-chloroform elutions (Fractions 17, 18, and 19). By inspection of the column during the chromatography and of the various fractions, it was apparent that much of the fecal pigments were separated from the carbon-14 by this column chromato graphy procedure. The pooled fractions 17, 18, and 19 contained 3.9 mg of '*C-H-ethyl FOSE equivalents. The pooled fractions 17, 18, and 19 from the second column were evaporated to a small volume and streaked on six 20 cm x 20 cm silica gel plates^. The plates were developed to 15 cm above the preadsorbent band in 100 ml of a mixture of 100 ml chloroform, 35 ml methanol, and 5 ml concentrated ammonita hydroxide. The carbon-14 was located by scraping 0.5 cm segments laterally from a center 5 cm x 20 cm section of the 20 cm x 20 cm plate. (Only a narrow strip `v.l cm was scraped.) The carbon-14 was located at fif 0.33. A 1.0 cm wide zone centered at Rj 0.33 was scraped from the six plates and the silica gel was eluted with methanol. The methanol from the elution was evaporated to a small volume and applied to a 20 cm x 20 cm silica gel plate that had been pre washed in methanol and air dried. The plate was developed to 15 cm in 100 ml of solvent prepared by mixing 100 ml chloroform, 100 ml methanol, and 2 ml concentrated acetic acid. The carbon-14 was located radicmetrically (a narrow band from the origin to the solvent front was scraped from the center of the plate laterally in 0.5 cm segments and counted). The Rj of the carbon-14 labeled material was 0.77. A 1.0 cm zone centered at Rj 0.77 was scraped and the silica gel was eluted with acetone and then chloroform-methanol (1:1, v:v). The eluates were combined, evaporated to a small volume, and streaked on a silica gel plate and developed to 15 cm in the same chloroform-methanol-acetic a d d solvent system as before. The carbon-14 was located by scraping a segment and counting. The ' carbon-14 was again located at Rj 0.77 . A 1.0 cm zone centered at R; 0.77 was scraped and the silica gel was eluted with acetone. The acetone eluate contained 1.0 mg of N-ethyl POSE equivalents. Figure 2 is a radiochromatogram of the carbon-14 labeled material eluted from the second plate with acetone) the TIC plate was developed in 100 ml of a mixture prepared from 100 ml chloroform, 100 ml methanol, and 2 ml concentrated acetic acid. -- Analtech, 75 Blue Ben Drive, Newark, Delaware. Page 7 Reference standards were prepared in ethanol and spotted along with aliquots of the purified (radlochealcally) etabollte on TIC plates. The structures of the reference standards werei 1) Cbf ,7802n h 2 2) C8r17S02*HC2H5 3) C 8F|7S03MCB2C02B C 28 5 4) C 8r 175O2tiaCB2C02B The plates were developed in 100 al of a mixture prepared from 100 ml chloroform, 35 methanol, and 5 ml concentrated ammonium hydroxide (System A) or in 100 ml of a mixture prepared from 100 al chloroform, 100 ml methanol, and 2 ml concentrated acetic acid (System B). The spots on the plate were located with acidified palladium chloride or brosphenol blue spray reagents. The Rj of the metabolite was confirmed radlometrically by scraping and counting. The Rj was 0.2B in System A and 0.77 in System B. The TLC of the metabolite and the reference standards showed that the Rf of standard 3 (C8F f7S02NCH2C02H ) was the same in both systeais k H5 and different than the Rj of standards 1, 2, and 4. The ,4Cmetabolite was submitted to 3M Central Research (J. R. Schroepfer) for QC/MS analysis (see Appendix 1). GC/MS data Indicate Identical retention tines and an exact matdi of El spectrum with standard 3, and the Cl spectrum of the metabolite matches exactly with the Cl spectrum of standard 3. In addition, it is possible to assign the major fragments observed consistent with the structure of standard 3. Overall, the identical thin-layer chromatography Rf's, identical gc retention times, identical El and Cl spectrum, and the interpretation of structural data from mass spectra show that the metabolite is C eF 17SO2NCB2C02H c 2b 5 The methanol fraction from the first silica gel column was evaporated and spotted on a silica gel plate and developed to 15 cm in 100 ml of a mixture of 100 ml chloroform, 100 ml methanol, and 2 ml acetic acid. The radiochromatogram is shown in Figure 3. Page 8 Result and Dlacuaaion A major metabolite of '*C-N-ethyl p o s e was isolated from feoes sad identified as 2-N-ethyl perfluorooctanesulfonasiido acetic acid (PPSAA). The astabollte accounts for at least SOt of the carbon-14 excreted in a feces pool at 24-48 hours after the last dose (see Appendix 2) in a regimen in which rats were fed '^C-H-ethyl POSE for 1 week. Since the extraction is from feces collected (24-48 hours after the last dose) following a week of dosing, the contents reflect what would be expected at various times after a aingle dose. As reported previously (1) by one week following a single dose of -ethyl POSE, *504 of the carbon-14 is eliminated via feces. These data suggest that a large portion of the carbon-14 eliminated via feces is PPSAA. In addition, three other metabolites were extracted iron feces. Together, these other unidentified metabolites account for *15% of the carbon-14 in this pool. In a previous experiment, perfluorooetanesulfonate was identified in liver extracts from rats at 48 hours after a single oral dose of 1*C-N-etbyl FOSE (1). Perfluorooctanesufonate accounted for at least 22% of the carbon-14 in the liver pool (4.4% of dose). Pram the same extracts, a second metabolite which accounted for at least 32% of the carbon-14 in the liver pool was tentatively identified as perfluorooctanesulfonamide (6.4% of dose) (1). Identification of perfluorooetanesulfonate in liver as a metabolite at 4.4% of the dose after a single dose and identification of PPSAA as a metabolite comprising 50% of the carbon-14 in feces during the second twenty-four hour period after the last dose following a week of feeding 1*C-N-ethyl POSE establishes that both compounds are major metabolites of N-ethyl FOSE. However, these data do not lead to conclusions as to the relative proportion of the biotransformation to either compound. Since it can be assumed that the time course of elimination of PPSAA and perfluorooetanesulfonate is vastly different [based on the amount of PPSAA in feces and on previous metabolism studies on perfluorooctaneaulfonate which is eliminated slowly via feces (<5% of dose/week) (3)) and since the possibility that PPSAA is further biotransformed to perfluorooctaneaulfonate exists, it would require a kinetic study to ascer tain the rate and extent of biotransfonaation of N-ethyl POSE to these compounds. Por the same reasons given for PPSAA, the relative extent of the biotransformation of N-ethyl POSE to the three metabolites tentatively Identified as perfluorooctanesulfonamide (1) and the metabolites in the methanol column fraction (Figure 3) (which, since they have not been identified, may include perfluorooetanesulfonate and perfluorooctaneaulfonaaide) is not established with these data. The mean carbon-14 content expressed as ug of N-ethyl POSE equivalents/g of wet tissue in various tissues were: liver, 233; spleen, 20; and red blood cells, 30. Plasma contained 62 wg equivalent! '*C-N-ethyl FOSE/nl of plasma. (Individual tissue and plasma concentrations are listed in Appendix 3.) The tissue concentrations at 21 days following the last dose after 1 week of feeding ,4C-N~ethyl POSE are consistent with net would be expected from multiple dosing with a slowly eliminated compound. 0G0o l 2 Page 9 References 1. Gibson SJ, Johnson JDs Absorption and Blotranaformation of N-Ethyl POSE and Tissue Distribution and Elimination of Carbon-14 After Administration of N-Etbyl POSE-1*C In Peed to Rats (Report) January 19, 1983. 2. Johnson, JD and Behr, FEr Synthesis and Characterization of tt-Sthyl POSE-1*C (Report) December 11, 1980. 3. Johnson JDs Extent and Route of Excretion and Tissue Distribution of Total Carbon-14 in Rats After a Single Intravenous Dose of FC-95-,4C (Report) December 28, 1979. 000313 Page to Lit of Figure Figure 1i Thin-Layer Radlochrcaatograa of Chlorofora Coluan Fraction HB-56531-19 Figure 3> Thin-Layer Fadlochromatograa of Coluan Fractions 17, 18, and 19 NB-56531-23 .Figure 3: Thin-Layer Radlochrcaatograa of Methanol Coluan Fraction MB-36531-28 000314  Page 11 Figure 1 Thin-Layer Radiochromatogram of Chloroform Column Fraction Pre-adsorbent SGF Uniplate: 100 chloroform 35 methanol 5 ammonium hydroxide Total CPM on Plate 19,221 000G1S Page 12 Figure 2 Thin-Layer Radiochromatogram of Column Fractions 17, IS, and 19 Pre-adsorbent SGF Uniplate: 100 chloroform 100 methanol 2 acetic acid Total CPU on Plate " 18,428 00 >316 Page 13 Figure 3 Thin-Layer Radiochromatogram of Methanol Column Fraction Fre-adsorbent SGF Uniplate: 100 chloroform 100 methanol 2 acetic acid Total CPM on Plate - 1,742 000317 Page 14 Lit of Appendice Appendix It Technical Report Sunary, Haas Spectral Analysis of 14C-M-Ethyl FOSE HB-563l-28u-28x Appendix 2t Excretion of Total Carbon-14 in Feces After an Oral Dose of 'c-H-Bthyl FOSE Fed to Rats for 1 Week NB-5S531-1 3-14 Appendix 3i Carbon-14 Content at 21 Days in Tissues After an Oral Dose of 'C-W-Ethyl FOSE Fed to Rats for 1 Weak KB-56531-14-16 000318 WH RWm 7*7-1VA A ppnalx i TECHNICAL REPORT SUMMARY March 13, 1981 TO: TECHNICAL COMMUNICATIONS CENTER - 201-2CN n p e tu n t - If r tpo rt It p rin act an both tU a te fp tp a r. m nd m o co p irt to TCC.) fHlaa MPL Nnmlwr CENTRAL RESEARCH LABORATORIES. Analytical and Properties Research Laboratory 0502 rieteei PrUtNumtor Service to Rlker - Isolation of Trace Fluorochemical* R^rtT' Haas Spectral Analysis of N-Ethyl AR No. 7454 - P0SE-,sC Labeled - March 13, 1981 TT A000007 W-- on NiimW 454 J. 0. Johnon - 218-2-02 A u aw rl -- -------------------------------------------------- Tssssjaniwssaa------- J. N, Schroepfer 076647 n m ibiI Hihwwi ' Na. Ifr^tMMIIlCtKaMlw M C U R IT Y ^ tCom .ny Coalman lia fipa riN A u tta rim iaal KIVWOMOS: (Salaci ta rira (ram 3M niaaaunic. Su fSM t thar CUORINT O M C T IV fl Request No. C58236 TM C H IM tC A L ^ n ta trrn r ~ O v B No CR1AF Analytical Report Project Mo. 91505026 Requestor - J. C. Johnson Chemical Analysis v J/MS . S^/MS R C PO ftT A S S T R A C T : (200-7SO wordal T N i abanaat in to n ealia h OtamtarlaO by tka T aarn ical C am m m iiranon C a n u r to atari S f a n ta Cnme m y R a o . I l la Comany aairf iOam lal m atariaL CI/MS Negative MS Based on the agreement of the results from the two samples using GC, El MS, and Cl MS, the structure of the major component In the sample metabolite is probably Identical to the structure of the major component In the standard-N-ethyl FOSE. Negative Cl MS of the methylated standard can help detect this compound but cannot confirm the structure by Itself. tfrtrmoiUm m a; Cai J /u /t/ 000319 . CENTRAL ANALYTICAL LABORATORY R e p o rt N o .____________________ D a t e ____ ___ rgh J 91 __ ___ 'W Subject: Kese Spectre! Analysis of N-Ethyl FOS E - ^ C Labeled R e q u e s to r: __v.JL.JehnAPA.. D ept. N a m e . __S i'iV _______ P r o j. N o .&\?1>2QZ Request No. ...... D ated ___ R ep o rt: The requestor submitted, for RC/MS analysis, a saaple aetabollee of N-Ethyl FOSE-1*C labeled and a standard of H-Bthyl FOSE having the following structure: ^CHj CHj C , F JTS02K C H j COOH The saaple aecabolite was an extract froo rat feces which the requestor separated on various TLC systems and divided equally into three vials each containing 100,000 cpa of 1'C, or about lOOpg. Experimental Diazomethane (CHjN j ) reacted with the saaple metabolite and standard; each saaple had an air atreaa blown over It to evaporate excess CBjN j . Electron Iapact (El) and Chemical Ionization (Cl) Baas spectra resulted from the sample aetabollee and standard Introduced Into the sues spectrometer (MS) via a gas chromatograph (GC). Table I lists the saaple analyzed, the method of Introduction, the node of MS operation and the aass spectrum number. Table II lists the conditions used. Negative Cl aaaa spectra resulted from the standard methylated and unaathylated Introduced directly Into the MS. Discussion For this discussion, when I aentlon the saaple metabolite and the standard, I will refer to their methylated ferns unless specified otherwise. The major component in the CC trace of the sample metabolite and standard showed Identical retention time (9.6 minutes). The El mass spectrum of this component In the sample metabolite compared exactly to the El mass spectrum of the major component in the standard. They each contain the same base peak-- 116 n/z-- probably due to (-N(CHjCK,)CHjCOOCHj) . The largest abundant high-mass fragment Is the same In each saaple -540 m/z which la due to C,FitSOj N(CHi CH,)-CH}-. Both aass spectra show a very small fragment at 600 a/z-- s parent Ion of 599 m/z + 1, which la probably due to a self Cl phenomenon observed in othgr samples. The Cl mass spectra of the two samples compare exactly. Both sample spectra show primarily three aass spectral fragments: 116, 540, and 600 a/z. The negative Cl mass spectrum of the unmethylated standard shows a 596 for the parent ion + 1. The negative Cl maas spectrum of the methylated standard shows a barely detectable fragment at 600 m/z, a fragment at 527 a/z (loss of 72 m/z from 599 m/z), a fragment ac 493 m/z (probably due to CtFirSO?), and the base peak at 191 m/z (probably due to loss of 419 m/zCiFi?). 000320 ^ ^-- ' AX No. 7454 March 13, 1981 Tage 2 Conclualoo Baaad on the agreement of Che results from the two samples using GC, 1 MS, and Cl MS, the structure of the major component In the sample metabolite la probably Identical to the structure of the major component In the standaxd-Nethyl FOSE. Sample Sample metabolite of N-Ethyl FOSE `"C methylated Standard of N-Ethyl FOSE methylated Standard of N-Ethyl FOSE TABLE I Method of Introduction CC GC CC CC DP DP Mode of Operation El Cl El Cl -Cl -Cl Mass Spectrum Nwabar AA5624 AA5625 AA5622 AA5623 AA5639 AA5638 TABLE XI Varan 2740 Cea Chromatograph Column; 2 meter, glass, 51 0V101 on 80/100 meah Chron G-HP Oven Progran: 100*C to 330*C at lO'C/nin. Inlector temperature: 270*C Detector temperature: 260*C (Vartan FID) Carrier gaa: helium at 25 mla/mln. duPont 21-491B Mesa Spectrometer Source; Chemical Ionization Cl gaa: Isobutane Mass Range; 15-617 m/z Separator; Single stage jet Multiplier; Bendlx Channeltroi (set to 4200v for Negative Cl) " 3. N. Schroepfer JMS/rs I 0003Z 1 Appendix 2 Excretion of Total Carbon-14 In Feces After an Oral Dose of 1*C-N-Ethvl FOSE Fed to Rats for 1 Week- Time Period of Collection (Hrs) 1 Rat Humber 23 x + SD 0-24 24-4B 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 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 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 doses fed rats werei Rat Ho. 1, 256 mg/kg; Rat Ho. 2, 329 mg/kg; and Rat Ho. 3, 315 mg/kg. -- Data are expressed as mg of H-ethyl FOSE equivalents excreted during time period. 000322 Appendix 3 Carbon-14 Content at 21 Days in Tlaaues After an Oral Dose of ,4C-*-Bthyl POSE Fed to Hats for 1 Week-- Rat Nuaber Liver Spleen Plasaa Red Blood Cells 1 220.8^ 21.6 61.9 2 211.3 17.5 55.5 3 266.6 21.8 68.9 28.3 24.8 37.4 Mean 4 SD 232.9 + 29.6 20.3 + 2.4 62.1 6.70 30.2 + 6.5 -- Total doses fed rata were: Rat No. 1, 256 ag/kgi Rat No. 2. . 329 ag/kg> and Rat No. 3, 315 ng/kg. -- Data are expressed as ug of N-ethyl FOSE equlvalcnts/g of wet tissue or al of fluid. 000323