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Absorption and Biotransformation of N-Ethyl FOSE and Tissue Distribution and Elimination of Carbon-14 after Administration of N-Ethyl POSE-14c in Feed January 19, 1983 Conducted At: During: Conducted By: Report By: Riker Laboratories, 3M Center St. Paul, Minnesota Inc. 55144 October 1980 to February 1981 S. J. Gibson and J. D. Johnson L/ S. J. Gibson Senior Laboratory Date Technician Reviewed By: J. D dohnsonl, 14S R esea-4h Specialist Date 18/2-7918.3 R. E. Ober, PhD Manager, Drug Metabolism Date Table of Contents Page summary 0000..* ...... .............. Introduction .............. materials and Methods Carbon-14 Labeled N-Ethyl FOSE ....o .....o.......... 1 Animals ooo.o.o ...... oo ... o-oo ....... 2 Dosing o.o ... oo...o ... o-oo.o.-o ... 2 Preparation of Dose/Feed Mixture 2 Administration of Dose ..o............ 2 Sample Collection ....... 3 Radicmetric Analyses ..o. .... o.o ..... o..ooo.oo ... o.o 3 Sample Preparation for Radicmetric Analyses 3 Analyses of Samples ...oo...o...oo..........o...o. 4 Isolation and Identification of Metabolites 4 Extraction 4 Column Chromatography 4 Thin-Layer Chromatography of Column Fractions 5 Results and Discussion o..o..........o..... 000.....0.0 5 Extent and Route of Excretion and Tissue Distribution.......o.oo..oo...o..oo..oo.o........ 5 Isolation and Identification of Metabolites 8 References ........o..............oo..Ooo..... 10 List of Tables, Figures and Appendices ....... 11 Page I Summary After a single oral dose of N-ethyl poSE-14C (2-N-ethyl perfluorooctaneoulfonamido ethanol) (mean dose, 10.13 mgag) in feed to fasted rats (groups of 3), at least 70% and probably more than 70% of the total carbon-14 is absorbed. Elimination of carbon-14 via urine and feces is very slow; from 0-32 days, only 63% of the dose is eliminated. Fecal elimination of carbon-14 is about 20-30 fold that of urine. The half-life of the disappearance from plasma of carbon-14 from day 1 to day 16 is 7.5 days. A metabolite of N-ethyl PIOSE isolated from liver was identified by 19F-Nt4R analysis as the egauorlooctanesulfonate anion. in addition, another metabolite from liver has been tentatively identified as perfluorooctanesulfonamide. Introduction in addition to being a product sold by 3M, N-ethyl FOSE is a key intermediate in the production of other products. N-Ethyl MSE is important in the study of FC-807 metabolism because the three esters of FC-807 are phosphate esters of N-ethyl MSE and in addition FC-807 contains a trace (-,O.St)of N-ethyl POSE. It would be expected that possible common metabolites occur from FC-807 and N-ethyl FOSE via different pathways or that after phosphate ester hydrolysis of FC-807 (systemic and/or gut) the biotransformation of N-ethyl FOSE and FC-807 follow similar pathways. Thus, the absorption, tissue distribution, and biatransformation of N-ethyl FOSE were investigated. The results are discussed in the context of other fluorochemical metabolism data from FC-807 and perfluorooctanesulfonate. Materials and Methods Carbon-14 Labeled N-Ethyl POSE ..-IC2H5 C7Fl5*CF2SO2-N,... CH2CE20H * Denotes Position of Carbon-14 Label N-Ethyl POSE is 2-N-ethyl perfluorooctanesulfonamido ethanol. The carbon-14label is at the carbon a to the sulfur atom (seeabove structure). On the basis of the specific activity determination, chemical characterization, and radiochemical purity reported separately (1), the N-ethyl poSE-14C was judged suitable for metabolism studies. The specific activity of the lot of N-ethyl POSE-14C used in these studies (Riker Isotope Inventory Number 468) is 0.483 + 0.020 pCi/mg. The radiochemical purity determination was repeated or;@e N-ethyl FOSE-14C dosing solution (see Appendix 1 - Table 1, and Appendix 1 Figures 1-5) and the N-ethyl FOSE-14C was found to be at least 98% pure. Page 2 Animals Male Charles River-SCD rats, eight weeks old, were conditioned to individual metal metabolism cages for 48 hours prior to dosing. The rats were fasted with free access to water for 24 hours prior to dosing. The body weights ranged frcm 220 to 329 g, mean 277 g. Rats were dosed in groups of 3; individual rate were selected so that the body weights of rats within each group of 3 were within gl grams of each other. The rats were allowed free access to Purina- Ground Chow and water after dosing. Dosing Preparation of Dose/Feed Mixture An N-ethyl POSE-14C'solution was prepared by weighing N-ethyl FOSE-14C into a 1 liter Class A volumetric flask, adjusting to volume with absolute ethanol, and mixing by inverting. The N-ethyl POSE-14C solution was transferred to a 4 liter beaker and Purina Ground Chow was added. The N-ethyl POSE-14C/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 was determined by combustion (see Appendix 2 and Appendix 2 - Table 1). Administration of Dose Each fasted rat was weighed immediately before being transferred to an individual metal metabolism cage. A feed cup was attached with wire to the inside of each cage to hold the single oral dose. The dose was a weighed amount of Purina Ground Chow containing 0.531 mg N-ethyl POSE-14C/g. All of the rats in the groups sacrificed at 1, 2, and 4 days postdose oonsumed the dose administered in a two hour period. For rats sacrificed at 8, 16, and 32 days postdose, a longer period of time was allowed to consume the dose (12 hours) although most of the fasted rats ate the feed/dose immediately. Any dose/feed mixture not consumed was weighed and the weight subtracted frcm the total dose/feed mixture given to each rat. By inspection, it appeared that very little of the feed was spilled by the rats. The dose administered each rat was calculated from the weight of feed consunked. The mean dose was 10.13 mg/kg (see Appendix 3). a Charles River Breeding Laboratories, Wilmington, Massachusetts. Purina Lab Chow, Ralston Purina Company, St. Louis, Missouri. Page 3 Sample Collection Urine and feces were collected at 24 hour intervals for each rat sacrificed at 1, 2, 4, and 8 days postdose (groupsof 3). Urine and feces were collected at intervals and respectively pooled for each rat sacrificed at 16 and 32 days postdose (groups of 3). At 1, 2, 4, 8, 16, and 32 days postdose, rats were anesthetized with diethyl etherl blood was drawn from the descending aorta and immediately transferred to a heparinized Vacutainer* tube. Plasma was prepared promptly by centrifugation. The rats were sacrificed by exsanguination and liver, spleen, kidneys, and lungs were collected as whole organs. Bone marrow was obtained from the femurs and tibias by splitting the bones and collecting the marrow on pieces of tared combustion pads-S. Samples of subcutaneous and abdominal fat, and muscle were collected. Digestive tract (esophagus, stomach, and intestines) and the remaining carcass were collected from rats sacrificed at I and 2 days postdose. Radionetric Analyses Sample Preparation for Radiometric Analyses Feces and major organs were prepared for carbon-14 analysis by homogenizing and aliquoting a sample of the homogenate into combustion cones4L. Homogenizing was done in Waring blenders by adding nine parts of water to one part of biological material. The homogenates were weighed into combustion cones in duplicate by taring the cone and adding 1.0 g of the homogenate. Care was taken to mix the homogenate between samplings. Samples of bone marrow, fat and red blood cells were weighed into combustion cones. Care was taken to weigh these samples promptly to avoid loss of weight by drying. Homogenates and samples weighed directly were combusted with a Packard Model 306 Oxidizer. Recovery of carbon-14 from biological samples was determined by combusting suitable blank hcmogenates (feces, liver, kidney, muscle, and spleen) spiked with N-ethyl POSE-14C solution; these reference samples were combusted at the beginning, middle, and end of the experimental sample set (see Appendix 4). Urine collections were sampled before freezing and were counted directly; duplicate 1.0 ml aliquots of each sample were pipetted directly into scintillation vials and 15 ml Aquasol* was added. Plasma was sampled before freezing and counted directly; duplicate 1.0 ml aliquoto or 0.5 ml aliquoto plus 0.5 ml water were pipetted directly into scintillation vials and 15 ml AquasolO added. All samples were cold and dark adapted before counting. a Packard Instrument Company, Inc., 2200 Warrenville Road, Downers Grove, Illinois. Page 4 Analyses of Samples All radimetric measurements were done using Packard Model 3380 and 3385 Tri-Carb Liquid Scintillation Spectrometers. For 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 sample was corrected for background with the appropriate blanks and for counting efficiency, the r-arbon-14 content of each sample was calculated. For combusted samples, counting efficiency for each sample was determined by use of the AES (Automatic External Standard) ratio method. To calibrate the external standard, a known amount of internal standard was added to selected samples in the group (three with low AES ratios and three with high ratios) and these samples were recounted. For oombusted samples, a correction was made for the recovery from the oxidizer. These recoveries were based on reference samples combusted with each sample set (see Appendix 4 - Tables 1-4). Isolation and Identification of Metabolites Extraction Aliquots of the 9:1 homogenates of liver for rats sacrificed at 2 days after dosing were pooled. The aliquots (by weight) contained a total of 1,052 ug equivalents of N-ethyl FOSE-14C. The homogenate was extracted three times with diethyl ether with back extractions of the ether with water. The final volume of ether was 1,500 ml and the final volume of water was 1,200 ml. The ether was evaporated and the residue was redissolved in methanol (Fraction 1). The water layer was acidified with 250 ml of 10% EC1 and stirred for an hour. The water layer was then extracted two times with diethyl ether. The ether was evaporated and the residue was redissolved in methanol (Fraction 2). The water was filtered and the filter cake was extracted two times with chlorofom-methanol 1:1 (v/v). The chloroform-mthanol was evaporated and the residue was redissolved in methanol (Fraction 3). Column Chromatography Three 2.0 cm in diameter columns were packed to 40 cm with silica gela in a chloroform slurry. The three fractions from the extraction of liver were chromatographed by placing the extract on the column and washing it into the bed with a small amount of chloroform. Each column war, eluted with 500 ml of CHC13 (Eluate A)f 500 ml of chlorofom-mthanol 1:1 (v/v) (Eluate B)p and 500 ml of methanol (Eluate C). The nine fractions were evaporated to dryness and redissolved in methanol and then analyzed for carbon-14 content. Unisil, activated silicic acid 100/120 mesh# Clarkson Chemical Company, Inc., Williamsport, Pennsylvania. Page 5 Thin-Layer Chromatography of C61umn Fractions Each of the six column fractions that contained carbon-14 (not methanol elutifon) were asmed by thin-layer chromatography. Small aliquots of the material were streaked on pre-adsorbent silica gel plates a and the plates were developed to 15 cm in the selected solvent system. The plates were then scraped in 0.5 cm segments into scintillation v@als containing methanol and 7.5 ml of a modified scintillant= was added. The contents of the scintillation vials were counted and the data were calculated and radioactivity per segment expressed as percent of total carbon-14 on the plate. Results and Discussion Extent and Route of Excretion and Tissue Distribution The results indicate that at least 70% of an oral dose of N-ethyl FOSE-14C administered in feed to previously fasted rats is absorbed. The data from analyses of feces for individual rats are shown in Table 1, the data for urine analyses are shown in Table 2, and the data for total excretion (feces plus urine) are shown in Table 3. These data are expressed as cumulative percent of dose excreted. (The data are listed as ug equivalents N-ethyl poSE-14C excreted per time period in Appendix 5 - Tables I and 2). As shown in Table 2, excretion of total carbon-14 via urine is not extensive; by 32 days postdose, <3.0% of the dose is eliminated via urine. The fecal elimination of carbon-14 is 20-30 fold more extensive than elimination via urine; however, the extent of carbon-14 elimination via feces is still only about 60% of the dose by 32 days postdose. The gastrointestinal transit time in fasted rate fed a nonabsorbed compound which does not affect the rate of excretion of feces is <30 hours (2). The fecal excretion rate of rats in this study is normal and does not seem to be affected by the N-ethyl poSE-14C (see Appendix 7 and Appendix 7 Table 1). The mean cumulative percent of dose excreted via feces by 48 hours postdose for the groups of 3 rats sacrificed at 2 days, 4 days, and 8 days postdose was 28.1, 14.4, and 15.2, respectively. Since carbon-14 levels found in feces at 48 hours or later after a single oral dose is most likely to represent material that has been absorbed and then excreted rather than unabsorbed compound, the cumulative percent of dose excreted via feces from time of dosing to 48 hours postdose (-.14-28%)is the upper limit of the estimate for percent of dose not absorbed. Thus, at least 70 percent of the dose of N-ethyl POSE-14C was absorbed. The digestive tracts (esophagus, stomache and intestines) were analyzed for carbon-14 content for individual rats in each of the groups of rats sacrificed at 24 and 48 hours postdose. The results (percent of dose) are shown in Table 4. (The data are expressed as vg N-ethyl POSE equivalents/g of tissue in Appendix 6.) Feces data from Table 1 for these 6 rats are repeated in Table 4. The mean total carbon-14 content of feces plus digestive tract with contents is 38.3% of the dose at 24 hours postdoze. However, the tissues and contents of the digestive tracts at 48 hours postdose contain a mean of 11.40 of the dose, and since the transit time of rats is faster than 48 a Analtech, 75 Blue Hen Drive, Newark, Delaware. Modified TSS: 25.2 g PPO, 1.01 g Dimethyl POPOP, and 3.8 1 toluene. Page 6 hours with normal rates of excretion (see Appendix 7 and Appendix 7 Table 1), it is probable that the 11.4% of the dose observed at 48 hours is not unabsorbed N-ethyl FOSE-14CI the 11.4% of the dose likely comprises carbon-14 labeled material associated with the tissues of the digestive tract and carbon-14 labeled material in the feces as a result of absorption of N-ethyl FOSE-14C with subsequent elimination via bile. It follows that some of the carbon-14 in feces before 48 hours is likely absorbed N-ethyl FOSE-14C and then excreted N-ethyl FOSE derived material. Thus, the absorption of N-ethyl POSE-14c is probably greater than 701. The results of analyses of liver, spleen, kidneys, lungs, and red blood cells for carbon-14 content by combustion and for carbon-14 in plasma by direct counting are shown in Table 5 for individual rats in all groups. (The data are expressed as percent of dose). Also included in Table 5 are results of analyses for carbon-14 content of digestive tract and'carcass of rats sacrificed at 24 and 48 hours postdose. The data from analyses of liver, spleen, kidneysp lungs, red blood cells, plasma, bone marrow, digestive tract, carcass, subcutaneous fat, abdominal fat, and muscle are normalized to a 10 mg/kg dose and are shown in Table 6 as ug N-ethyl POSE-14C equivalents/g of tissue. [The same data (not normalized) are listed in Appendix 81. Mean total recovery of radioactivity from rats sacrificed at 24 hours and 48 hours (sum of amount in tissue (Table 5) and amount excreted (Table 3)) was 86%. Since a correction for recovery from the combustion analyses was made, these results seem low. It is probable that some of the N-ethyl POSE-14C and/or its metabolites were lost during preparation of the samples (N-ethyl FOSE-14C is slightly volatile at room temperature). From Table 5, it is apparent that as with FC-807 (3), potassium perfluorooctanesulfonate (4), and ammonium perfluorooctanoate (5) a significant portion (mean, 9.5%) of the dose of carbon-14 is retained in the liver at 32 days after a single oral dose of the labeled compound. The mean log carbon-14 levels (normalized to a 10 mg/kg dose) in liver and plasma are plotted versus time in Figure I for groups of rats (3 rats/group). The ratio (liver/plama) of carbon-14 levels are plotted versus time in Figure 2. From Figure 2, it is apparent that the liver/plasma ratio is not constant and the equilibrium is not established until 16 days postdose. Selective retention of a biotransformation product in the liver with more rapid clearance of N-ethyl FOSE-14C and/or other metabolites would be a possible explanation for this pattern. At 32 days after a single oral dose of N-ethyl FOSE-14Cg the mean liver/plasmaj,spleen/plasmas and bone marrow/plasma carbon-14 level ratios were 11.8, 0.4, and 0.4, respectively. At 124 days after a single iv dose of PC-807-14C, the mean liver/plasma, spleen/plawAa, and bone marrow/plama carbon-14 level ratios were 22.1, 202.8, and 48.lr respectively. Thus, the pattern of distribution of carbon-14 after FC-807-14C administration is so different from the pattern after N-ethyl POSE-14C administra- page 7 tion that despite the two different routes of administration (oral versus iv) and the difference in duration of the experiments (time from dosing to sacrifice), it is probable that FC-807 biotransformation is more complex than a simple conversion to N-ethyl POSE by hydrolysis of the phosphorus-oxygen bond with subsequent biotransformation of the released alcohol as N-ethyl FOSE. The mean liver/plasma, spleen/plasma, and bone marrow/plasma carbon-14 level ratios calculated from data reported for potassium perfluorooctanesulfonate (6) are 9.3, 0.2, and 0.2, respectively, at 89 days after a single intravenous dose. Thus, in contrast to the tissue carbon-14 ratio data from FC-807, the perfluorooctanesulfonate-14C tissue/plasma ratio data are similar to-the N-ethyl FOSE-14C tissue/plasma ratio data. Although perfluorooctanesulfonate is shown to be a metabolite of N-ethyl POSE (see below, this report), these tissue/plasma ratio similarities should'not be interpreted as evidence that the carbon-14 labeled material at later times (32 days) in liver is due only to perfluorooctanesulfonate. The half-life of the disappearance from plasma of carbon-14 from day 1 to day 16 (Figure 1) is 7.5 days after N-ethyl FOSE-14C administration. However, after day 16 the disappearance is much slower since the mean carbon-14 levels on day 32 are about the same as the mean levels on day 16 (2.2 jig equivalents on day 16, 2.1 ug equivalents on day 32). The_@lasma half-life value of 7.5 days for carbon-14 after N-ethyl FOSE 4c oral administration is the same as that reported for carbon-14 after oral administration of potassium perfluorooctanesulfonate-14C to male rats (estimated fr(xnday 1 to day 6) (4). As with N-ethyl POSE-14C, it is apparent that sometime after a few days (>6 days) the rate of disappearance of carbon-14 from plasma after administration of perfluorooctanesulfonate-14C decreases to a much lower rate. Overall, the pattern of carbon-14 distribution in tissue and the half-life values for the first few days post oral dosage are similar for N-ethyl POSE-14C and potassium perfluorooctanesulfonate-14C. In addition, for both compounds a , the rates of disappearance of carbon-14 seem to decrease so that later plasma values for carbon-14 levels are somewhat higher than would be predicted from the earlier levels and the half-life values. Thus for these compounds in rats (and likely other species), predictions of plasma levels at later times based on half-life estimates of elimination from plasma are inaccurate and should not be attempted. a The change in rate of elimination of total carbon-14 frcm plasma is not unique to N-ethyl POSE-14C and perfluorooctanesulfonate-14C; it is quite common for other compounds. Page 8 Isolation and Identification of Metabolites The liver homogenate pool (9:1, water/tissue) from rats sacrificed at 48 hours after a single oral dose of N-ethyl FOSE-14C was extracted with ether (Fraction 1) then with acid-ether (Fraction 2); after filtration, the filter cake was extracted with chloroformmethanol 1:1 (Fraction 3). The percent of carbon-14 extracted frm the liver homogenate for each fraction was: Fraction 1,,30.10; Fraction 2, 25.1%; and Fraction 3, 11.8%. The water layer separated by filtration before the 1:1 chloroform-methanol extraction of the filter cake contained no detectable carbon-14 (00). Thus, of the 1052 ug equivalents of W-ethyl POSE-14C present in the feces pool, 705 ug equivalents (67%) was extracted. The 3 fractions were chromatographed on separate silica gel columns and the columns were eluted successively with chloroform (Eluate A); 1:1 chloroform-iwthanol (Eluate B)l and methanol (Eluate C). There was very little carbon-14 removed from any of the columns by methanol (Eluate C). For each of the liver homogenate extractions that were chromatographed on a silica gel column, there are two column eluates (A and B) containing carbon-14. The relative carbon-14 content of Eluates A and B for each fraction are shown in Table 7. The six column fractions (Bluates A and B) were chromatographed by thin-layer chromatography. Radiochromatograms from thin-layer chromatography of these six fractions are shown in Figures 3-12. When chromatographed in the same solvent system (100 chloroform, 35 methanol, 5 mmonium hydroxide) (Figures 3-5), the three column eluates (B's) from the three extractions (ether, acid-ether, 1:1 chloroform-methanol) each have one major peak, and comparison of the radiochromatograms suggests that each contain the same metabolite. The extraction Fraction 2 (acid-ether) Eluate B was chromatographed in a second solvent system (100 butanol, 10 acetic acid, 10 water) (Figure 6) and in a third solvent system (100 chloroform, 100 methanol, and 2 acetic acid) (Figure 7). The three radiochromatograms of Fraction 2 Bluate B indicate that the radiochemical purity of the metabolite is -.95%. The extraction Fraction 2 (acid-ether) Eluate B (chlorofom-methanol) was labeled metabolite Fraction I and submitted to S. Pathre of the Central Analytical Laboratory for 19F-NKR analysis on the Varian XL-100 and XL-200 Spectrometers. The results are reported in Appendix 91 the metabolitewas identifiedas the perfluorooctane- sulfonate anion. Page 9 From the percent of total carbon-14 extracted from the liver homogenate pool (679) and the total carbon-14 recovered from the columns in 1:1 chloroform-methanol and the percent couposition of these eluates by thin-layer chromatography (95%) it can be estimated that at least 22% of the carbon-14 in liver at 48 hours after a single dose of N-ethyl FOSE-14C is due to perfluorooctanesulfonate-14C. It is quite likely that due to loss during extraction and to loss during chromatography that the actual amount of perfluorooctaneoulfonate is somewhat greater than 22%. The chloroform column eluates from the three extraction fractions were chromatographed using thin-layer chromatography in the same solvent system (100 chloroform, 35 methanol, 5 ammonium hydroxide) (Figures B-10). As with the chloroform-mthanol eluates, comparison of the three oolumn eluates suggests that most of the carbon-14 is the same metabolite. The extraction Fract' ion 1 (ether) Eluate A was chromatographed in a second solvent system (100 butanol, 10 acetic acid, 10 water) (Figure 11) and in a third solvent system (100 chloroform, 100 methanol, and 2 acetic acid) (Figure12). The extraction Fraction I (ether) Eluate A (chloroform)was labeled metabolite Fraction II and submitted to S. Pathre of the Central Analytical Laboratory for 14F-NKR analysis on the Varian XL-100 and XL-200 Spectrometers. The results are reported in Appendix 9. The metabolite was tentatively identified as perfluorooctanesulfonamide. Similar to the calculation for perfluorooetanesulfonate, it can be estimated that at least 32% of the carbon-14 present in liver at 48 hours after a single oral dose of N-ethyl FOSE-14C is metabolite Fraction II. Further refinement of these extractions and separations would likely result in a higher estimate of the percentage of perfluorooctanesulfonate and the metabolite tentatively identified as perfluorooctanesulfonamide with respect to the total.carbon-14 present. However, since these estimates establish that the two metabolites are present in substantial quantities, further refinement of the extraction and separation is not planned. The presence of other peaks in radiochromatograms (Figures B-10) and the 33% of the carbon-14 in the liver homogenate that was not extractable by these conditions indicate that other unidentified metabolites are present. 0 4D Page 10 References 1. Johnson, JD and Behr, FE: Synthesis and Characterization of N-Ethyl POSE-14C (Report), December 11, 1980. 2. Thompson, RC and Hollis, OL: Irradiation of the Gastrointestinal Tract of the Rat by Ingested Ruthenium-106. Am J Physiol 194, 308-312, 1958. 3. Johnson, JD: Absorption of FC-807-14C in Rats After a Single Oral Dose (Report), July 10, 1979. 4. Johnson, JD: Absorption of FC-95-14C in Rats After a Single Oral Dose (Report), October 26, 1979. 5. Johnson, JD: Absorption of FC-143-14C in Rats After a Single Oral Dose (Report), December 28, 1979. 6. Johnson, JD: Extent and Route of Excretion and Tissue Distribution of Total Carbon-14 in Rats After a Single Intravenous Dose of FC-95-14C (Report), December 28, 1979. 7. Altman, PL and Dittmer, DS: Blood and Other Body Fluids. Bethesda, Maryland, Federation of American Societies for Experimental Biology, 1971, p.5. Page 11 4ist of Tables, Figures, and Appendices Table 1: Cumulative Excretion Oral Dose of N-Ethyl 10.13 mg/kg) of Total POSE-14C Carbon-14 in Feces After an in Feed to Rats (Mean Dose, Table 2: Cumulative Excretion Oral Dose of N-Ethyl 10.13 mg/kg) of Total POSE-14C Carbon-14 in Urine After an in Feed to Rats (Mean Dose, Table 3: Table 4: Cumulative Excretion of Total Carbon-14 in Urine and Feces After an Oral Dose of N-Ethyl POSE-14C in Feed to Rats (Mean Dose, 10.13 mgag) Carbon-14 Content in Digestive Tract (plus contents) and Feces After an Oral Dose of N-Ethyl FOSE-14C in Feed to Rats (Mean Dose, 10.13 mg/kg) at 24 and 48 Hours Postdose Table 5: Carbon-14 Content in Tissues After an Oral Dose of N-Eihyl POSE-14C in Feed to Rats (Mean Dose, 10.13 mgag) Table 6: Carbon-14 Content in Tissues After an Oral Dose of N-Ethyl p,OSE-14C in Feed to Rats (Mean Dose, 10.13 mg/kg) Table 7: Relative Carbon-14 Content of Eluates A and B for Extraction Fractions 1, 2, and 3 Figure 1: Mean Log Carbon-14 Levels (Normalized to a 10 mgag Dose) in Liver and Plasma of Rats (Groups of 3) at 1, 2, 4, 8, 16, and 32 Days Post Oral Dose of N-Ethyl poSE-14C in Feed NB-53102-43-44, NB-56531-5 Figure 2: Ratio of Carbon-14 Level in Liver/Carbon-14 Level in Plasma of Rats (Groups of 3) at 1, 2, 4, 8, 16, and 32 Days Post Oral Dose of N-Ethyl POSE-14C in Feed NS-53102-43-44, NB-56531-5 Figure 3: Thin-Layer Radiochrcmatogram of Extraction Fraction 1 (ether) Eluate B (1:1 chloroform-mthanol) NB-51579-36 Figure 4: Thin-Layer Radiochrcmatogram of Extraction Fraction 2 (acid-ether) EluatBe (1:1 chloroform-methanol) NB-51579-36 Figure 5: Thin-Layer Radiochrcmatogram of Extraction Fraction 3 (1:1 ch3.oroform-methanol) Eluate B (ltl chloroform-methanol) NB-51579-36 Figure 6: Thin-Layer Radiochromatogram of Extraction Fraction 2 (acid-ether) Eluate B (1:1 chlorofom-methanol) NB-51579-35 Page 12 List of Tables, Figures, and Appendices (Con't) Figure 7: Thin-Layer Radiochrmatogram of Extraction Fraction 2 (acid-ether) Eluate B (1:1 chlorofom-methanol) NB-51579-35 Figure 8: Thin-Layer Radiochrcmatogram of Extraction Fraction 1 (ether) Eluate A (chloroform) NB-51579-36 Figure 9: Thin-Layer Radiochromatogram of Extraction Fraction 2 (acid-ether) Eluate A (chloroform) NB-51579-36 Figure 10: Thin-Layer Radiochrcmatogram of Extractiori Fraction 3 (1:1 chloroform-methanol) Eluate A (chloroform) NB-51579-36 Figure 11: Thin-Layer Radiochromatogram of Extraction Fraction 1 (ether) Bluate A (chloroform) NB-51579-35 Figure 12: Thin-Layer Radiochromatogram of Extraction Fraction 1 (ether) Eluate A (chloroform) NB-51579-35 Appendix 1-Table 1: Thin-Layer Chromatography FOSE-14C NB-51806-41 Systems for N-Ethyl Appendix 1-Figure 1: Thin-Layer Radiochromatogram of N-Ethyl FOSE-14C Dosing Solution, Plate No. 1 NB-51806-42 Appendix 1-Figure 2: Thin-Layer Radiochromatogram of N-Ethyl FOSE-14C Dosing Solution, Plate No. 2 NB-51806-42 Appendix 1-Figure 3: Thin-Layer Radiochromatogram of N-Ethyl FOSE-14C Dosing Solution, Plate No. 3 NB-51806-42 Appendix 1-Figure 4: Thin-Layer Radiochromatogrm of N-Ethyl FOSE-14C Dosing Solution, Plate No. 4 NB-51806-42 Appendix 1-Figure 5: Thin-Layer Radiochrmatogram of N-Ethyl FOSE-14C Dosing Solution, Plate No. 5 NB-51806-42 Appendix 2: Determination of Carbon-14 Content of N-Ethyl POSE-14C D6se/Feed Mixture Appendix 2-Table 1: Carbon-14 Mixture Content of N-Ethyl FOSE-14C Dose/Feed Appendix 3: Rat Weights and Amount of N-Ethyl IPOSE-14C Dose/Feed Mixture Administered to Each Rat Page 13 List of Tablest Figures, and Appendices (Con't) Appendix 4: Appendix 4-Table 1: Appendix 4-Table 2: Appendix 4-Table 3: Appendix 4-Table 4: Appendix 5-Table 1: Appendix 5-Table 2: Appendix 6: Appendix 7: Appendix 7-Table 1: Appendix 8: Appendix 9: Determination of Recovery of Total Carbon-14 From Blank Biological Samples Spiked With R-Ethyl POSE-14C Recovery of Total Carbon-14 Frm Blank Biological Samples Spiked With N-Ethyl FOSE-14C, Combustion Set No. 1 Recovery of Total Carbon-14 Samples Spiked With N-Ethyl Set No. 2 From Blank Biological poSE-14C, Combustion Recovery of Total Carbon-14 Samples Spiked With N-Ethyl Set No. 3 From Blank Biological POSE-14C, Combustion Recovery of Total Carbon-14 Samples Spiked With N-Ethyl Set No. 4 From Blank Biological POSE-14C, Combustion Total Carbon-14 in Feces After an Oral Dose of N-Ethyl POSE-14C in Feed to Rats (Mean Dose, 10.13 mg/kg) Total Carbon-14 in Urine After an Oral Dose of N-Ethyl POSE-14C in Feed to Rats (Mean Doser 10.13 mg/kg) Carbon-14 Content in Digestive Tract (plus contents) and Feces After an Oral Dose of N-Ethyl POSE-14C in Feed to Rats (10.13 mg/kg) Comparative Data Showing Normal Fecal Excretion for Rats Comparative Data Showing Normal Fecal Excretion for Rats Carbon-14 Content in Tissues After an Oral Dose of N-Ethyl FOSE-14C in Feed to Rate (Mean Doser 10.13 mg/kg) Report of Central Analytical Laboratory Analysis of Metabolite Fractions I and II Page 14 Table 1 Cumulative Excretion of Total Carbon-14 in Feces After an Oral Dose of N-Ethyl POSE_ 14C in Feed to Rats (?4eanDose, 10.13 mg/kg) Collection Period (Days) Rat Identification A B c Mean + S.D. 0-1 0-1 1-2 0-1 1-2 2-3 3-4 0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 0-16 0-16 16-32 29.98t I Day Group 12.41 18.99 34.73 2 Day Group 15.60 28.98 7.74 14.04 20.03 23.56 4 Day Group 5.27 12.32 17.78 22.48 2.36 5.61 25.77 37.41 42.83 47.14 50.65 53.36 8 Day Group 7.71 27.16 38.21 44.41 49.21 52.55 54.86 56.35 66.27 16 Day Group 60.39 63.88 66.73 32 Day Group 47.62 56.93 22.55 21.65 + 8.82 11.80 20.59 15.46 + 3.60 28.10 -; 7.11 9.53 16.89 21.74 26.08 7.51 + 14.42 19.85 24.04 2.14 2.31 1.99 1.85 4.31 12.70 20.32 26.15 29.27 32.52 35.83 38.77 4.79 + 15.16 28.10 35.99 40.44 44.07 47.11 49.49 2.71 10.98 9.17 9.21 10.18 10.36 10.00 9.41 62.79 63.15 + 2.96 56.72 59.36 56.07 + 8.15 61.01 5.10 Data are expressed as percent of dose excreted during collection period. Notebook Reference: NB-55673-37 and 38 Page 15 Table 2 cumulative Excretion of Total Carbon-14 in Urine After an Oral Dose of N-Ethyl FOSE-14C in Feed to Rats (Mean Dose, 10.13 mg/kg) Collection Period (Days) Rat Identification A B c Mean + S.D. 0-1 0-1 1-2 0-1 1-2 2-3 3-4 0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 0-16 0-32 0.13@0- 0.14 0.23 0.11 0.25 0.36 0.50 0.10 0.17 0.30 O@62 0.88 0.97 1.06 1.12 2.45 1.85 1-Day Group 0.12 2 Day Group 0.09 0.17 4 Day Group 0.10 0.24 0.34 0.44 8 Day Gr 0.06 0.18 0.28 0.36 0.61 0.67 0.72 0.77 16 Day Group 1.19 32 Day Group 2.69 0.11 0.12 + 0.01 0.11 0.20 0.11 + 0.03 0.20 + 0.03 0.11 0.22 0.33 0.43 0.11 + 0.01 0.24 + 0.02 0.34 0.02 0.46 0.04 0.09 0.21 0.34 0.44 0.53 0.61 0.71 0.81 0.08 + 0.19 + 0.31 0.47 0.67 0.75 0.83 0.90 0.02 0.02 0.03 0.13 0.18 0.19 0.20 0.19 1.10 1.58 + 0.75 1.62 2.05 + 0.56 Data are expressed as percent of dose excreted during collection period. Notebook Referenceo. VB-55673-38 Page 16 Table 3 Cumulative Excretion of Total Carbon-14 in Urine and Feces After an Oral Dose of N-Ethyl poSE-14C in Feed to Rats (Mean Dose, 10.13 mgag) Collection Period (Days) Rat Identification A B c 14ean + S.D. 0-1 0-1 1-2 0-1 1-2 2-3 3-4 0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 0-16 0-32 30.11-M I Day Group 12.53 19.13 34.96 2 Day Group 15.69 29.15 7.65 14.29 20.39 24.06 4 Day Group 5.37 12.56 18.12 22.92 2.46 5.78 26.07 38.03 43.71 48.11 51.71 54.48 8 Day Grou_p 7.77 27.34 38.49 44.77 49.82 53.22 55.58 57.12 68.72 16 Day Group 61.58 68.58 32 Day Group 59.62 22.66 21.77 + 8.82 11.91 20.79 15.58 + 3.61 28.30'; 7.12 9.64 17.11 22.07 26.51 7.62 + 14.65 20.19 24.50 2.14 2.30 1.98 1.83 4.40 12.91 20.66 26.59 29.80 33.13 36.54 39.58 4.88 + 2.69 15.34 10.98 28.41 9.14 36.46 9.19 41.11 10.26 44.82 10.44 47.94 10.06 50.39 9.46 63.89 64.73 + 3.64 60i.98 63.06 + 4.63 Data are expressed as percent of dose excreted during collection period. Notebook Reference: NB-55673-39 and 40 Page 17 Table 4 Carbon-14 Content in Digestive Tract (plus contents) and Feces After an Oral Dose of IN-Ethyl POSE-14C in Feed to Rats (Mean Dose, 10.13 mg/kg) at 24 and 48 Hours Postdose Rat Identification 1A 1B ic Mean + S.D. 2A 2B 2C Mean + S.D. Time PostDose (Hours) 24 24 24 48 48 48 Digestive Tract (plus contents) Feces 12.84t 22.40 14.60 16.61 + 5.09 29.98 12.41 22.55 21.65 + 8.82 11.10 13.03 10.10 11.41 + 1.49 34.73 28.98 20.59 28.10 + 7.11 -S Data are expressed as percent of dose. Notebook Reference: NB-51806-49-51 Page IS Table 5 Carbon-14 Content in Tissues After an Oral Done of N-Zthyl FOSZ-14C in Poed to Rats plean Dos*, 10.13 ogag) Rat Identification L Kidneys@L Lungs, Red Sir Caller- plasmac- Digestive Tract-E Carcasm4L IA IB ic Mean 16.BS 17.98 16.95 17.26 0.16 0.13 0.11 0.13 0.71 1.12 0.80 0.88 0.43 0.60 0.39 0.47 6.39 6.80 7.82 7.00 2.55 3.10 2.60 2.82 12.84 22.40 14.60 16.61 18.76 23.46 17.40 19.98 2A 2D 2C Kean 17.72 16.23 24.17 20.04 0.14 0.10 0.16 0.13 0.66 0.77 1.00 0.81 0.35 0.40 o.s9 0.4s S.89 5.77 3.62 5.09 2.06 2.17 3.32 2.52 11.10 13.03 10.10 11.41 12.30 15.62 19.29 IS.74 4B 4C Mean 21.59 0.14 0.77 0.43 S.44 2.67 - 16.47 0.12 0.98 0.43 5.85 2.96 17.69 0.13 0.7S 0.38 5.74 2.72 19.25 0.13 0.83 0.41 S.68 2.78 SA as oc Kean 15.44 12.83 16.30 IS.S2 0.04 0.04 0.09 0.'06 0.32 0.27 0.54 0.38 0.16 0.14 0.36 0.22 2.78 2.37 3.84 3.00 1.15 1.19 2.OS 1.46 16A 16B 16C Mean 9.52 12.10 10.32 10.65 0.02 0.02 0.03 0.02 0.18 0.16 0.27 0.20 0.09 0.09 0.14 0.11 0.77 0.99 1.08 0.95 0.72 0.85 1.16 0.91 .32A 32B 32C Mean 7.84 12.71 8.04 9.S3 0.02 0.03 0.01 0.02 0.18 0.31 0.16 0.22 0.07 0.13 0.07 0.09 0.28 0.87 0.30 0.48 0.78 1.13 0.64 O.BS a Data are express*d as percent of doe* in tissue. Data art estimates of percent of done present in red blood calls. Red blood cell volume 26.3 al/kg body weight (7). Data are estimates of percent of dose present in plasma. Plasma volume a 31.3 al/kg body weight (7). Smple was not taken. Not*book Referencesi MD-56S31-8 wd 9, M-SlSC6-49, and MD-53102-49 Page 19 Table 6 Carbon-14 Content in Tissues After an Oral Dose of N-Ethyl poSS-14C in IP**d to Rats (Kean Dose, 10.13 mg/kg) Rat Identification Liv*r-S Spl*erv-a-Rid..Y.E Rod Blood Lun 9*S Cell&S PlammA Done Digestive a a Narrow Tract-;- CarcasA Subeut. Abdom. PatA ]PatE Hscia IA is lc Mean 32.4 48.7 30.4 37.2 6.72 6.88 6.06 6.55 0.87 14.39 8.54 10.60 8.07 11.68 7.76 9.18 21.80 27.24 25.69 24.91 7.31 10.42 7.74 8.49 12.31 14.73 11.02 12.69 11.77 31.41 10.78 17.99 2.27 3.17 2.03 2.49 6.40 7.79 4.92 6.37 8.04 9.Sl 6.15 7.90 0.40 0.42 0.34 0.39 2h 2B 2C Mean 4B 4C Mean 30.4 41.8 41.4 37.9 43.1 43.8 39.2 42.0 S.76 4.68 7.26 S.90 6.55 7.41 6.65 6.87 6.70 8.00 10.44 8.36 8.s6 10.98 9.s4 9.69 6.21 G.S7 8.76 7.10 18.83 20.10 12.2S 17.06 S.S3 6.36 9.46 7.12 8.06 8.83 7.S9 8.16 18.28 20.86 19.34 19.49 7.53 8.84 7.69 8.02 10.42 lo.s9 11.3S 10.79 7.97 9.80 8.67 8.81 8.27 12.03 8.06 9.46 -b - - 1.43 1.93 2.39 1.92 -- b - - 3.96 4.18 3.95 4.03 2.15 2.99 1.82 2.32 3.26 3.99 4.28 3.84 2.08 1.75 1.74 1.86 0.22 0.37 0.52 0.37 1.58 1.36 1.32 1.42 BA es ac Mean 26.1 24.0 33.6 27.9 2.09 1.75 3.87 2.57 3.2S 3.3S 6.16 4.25 2.79 2.SS 6.34 3.89 6.19 7.46 12.27 9.31 2.BS 3.15 5.50 3.83 3.27 - 2.83 - 5.48 - 3.86 - - O.S3 0.37 0.33 - 0.98 0.22 0.24 - 0.69 0.47 0.95 - 0.73 0.35 0.51 16A 16B 16C Mean 20.4 27.9 26.3 24.9 0.81 0.84 1.08 0.91 1.88 1.99 2.98 2.28 1.43 1.76 2.29 1.83 2.18 2.95 3.16 2.76 1.70 2.13 2.84 2.22 0.96 0.98 1.48 1.14 - 0.03 0.04 0.16 0.13 0.01 0.16 0.35 0.03 0.20 0.17 0.03 0.17 32h 19.7 0.74 1.81 1.61 0.81 1.89 0.67 32B 32.8 1.33 3.47 2.38 2.58 2.79 1.46 - 32C 20.4 0.62 1.75 1.32 0.86 1.54 0.61 - Moan 24.3 0.90 2.34 1.77 1.42 2.07 0.91 - a Data is normalized to a 10 mg/kg dome &M k Smple was not taken. expressed as pg O-ethyl POSE-14C equivalents/g. Notebook Referencest NB-56531-5 and 6, NB-53102-43 and 44 0.08 0.28 0.06 0.14 0.00 0.00 0.00 0.00 0.13 0.28 0.09 0.17 Page 20 Table 7 Relative Carbon-14 Content of Eluateg A and B for Extraction Fractions 1, 2 and 3 Fraction I (ether) Eluate A 93% (chloroform) Fraction 2 (acid-ether) Fraction 3 (1:1 chlorofom-methanol) 23% 61% Eluate B 19% 61% 22% (1:1 chloroform- methanol) . Total % 112% 84% 83% recovered Notebook Reference: NB-51579-34 Page 21 Figure 1 Mean Log Carbon-14 Levels (Normalized to a 10 mg/kg Dose) in Liver and Plasma of Rats (Groups of 3) at 1, 2, 4, 8, 16,tnd 32 Days Post Oral Dose of N-Ethyl POSE- C in Feed loo- Liver u >4 .a z Plasma T-- I I I I I I l@ 0 2 4 5 8 10 12 14 16 18 20 22 24 25 20 3D 32 DAYS POSTOOSE Page 22 Figure 2 Ratio of Carbon-14 Level in Liver/Carbon-14 Level in Plasma of Rats (Groups of 3) at 1, 2, 4, 8, 16, lid 32 Days Post Oral Dose of N-Ethyl FOSE- C in Feed U7 2 z z -3 t&3 z 0 o .2 1 6 8 10 la 14 15 10 20 n 24 26 38 30 32 ORYS POSTOOSE page 23 Figure 3 Thin-Layer Radjochromatogram of Extraction Fraction 1 (ether) Eluate B (1:1 chloroform-methanol) Pre-adsorbent SGF Uniplate: 100 chloroform 35 methanol 5 ammonium hydroxide Total CPM on Plate 1,125 cc 5! 40 30- 20 10- 0 -1 0 '1W %-F2w - N3V %w 4I 5 9 7 0 9 101112131415 DISTRNCE rrom ORIGIN (CM) page 24 so- Figure 4 Thin@-Layer Radiochromatogram of Extraction Fraction 2 (acid ether) Eluate B (1:1 chloroform-methanol) Pre-adsor@ent SGF Uniplate: 100 chloroform 35 methanol 5 ammonium hydroxide Total CPM on Plate 4,363 SD- 40- -3 c 30- 20- io-@ 0 -1 0 -3 5 7 0 9 10 11 12 13 1-115 DISTRNCE FROM ORIGIN (CM) page 25 00- Figure 5 Thin-Layer Radiochromatogram of Extraction Fraction 3 (1:1 chloroform-methanol) Eluate B (1:1 chloioform-methanol) Pre-adsorbent SGF Uniplate: 100 chloroform 35 methanol 5 ammonium hydroxide Total CPM on Plate 664 40- -3 30- LL3 20to- 0 3 It 5 6 7 5 9 to 11 12 13 14 15 I)ISTRNCE FROM ORIGIN (CH) Page 26 Figure 6 Thin-Layer Radiochromatogram of Extraction Fraction 2 (acid-ether) Eluate B (1:1 chloroform-methanol) Pre-adsorbent SGF Uniplate: 100 butanol 10 water 10 acetic acid Total CPM on Plate 1,650 eL cc 3D- 20 10 2 3 4 5 5 7 0 9 10 11 12 13 14 15 DISTRNCE RROM ORIGIN (Cli) Page 27 Figure 7 Thin-Layer Radiochromatogram of Extraction Fraction 2 (acid-ether) Eluate B (1:1 chloroform-methanol) Pre-adsorbent SGF Uniplate: Total CPM on Plate 1,474 100 chloroform 100 methanol 2 acetic acid 70- 0 4030-m 20- 2 3 4 5 5 7 a 9 10 11 12 13 14 15 DISTRNCE FROM ORIGIN (CM) Page 28 Figure 8 Thin-Layer Radi0chromatc>gram of Extraction Fraction 1 (ether) Eluate A (chloroform) Pre-adsorbent SGF Uniplate: 100 chloroform 35 methanol 5 ammonium hydroxide Total CPM on Plate 4,983 soE%J 30- bi 20to- -1 0 1 2 3 4 5 5 7 0 9 10 it 12 13 14 15 DISTRNCE RROM ORIGIN (CM) Page 29 Figure 9 Thin-Layer Radi0chromatogram of Extraction Fraction 2 (acid-ether) Eluate A (chloroform) Pre-adsorbent SGF Uniplate: 100 chloroform 35 methanol 5 ammonium hydroxide Total CPM on Plate 1,020 so- -3 30- 20- to- -1 0 1 2 3 4 5 5 7 a 9 io ii-ii i@ 14 is DISTFINCEFROM ORIGIN (Cti) Page 30 Figure 10 Thin@-Layer Radiochromatogram of Extraction Fraction 3 (1:1 chloroform-methanol) Eluate A (chloroform) Pre-adsorbient SGF Uniplate: 100 chloroform 35 methanol 5 atmnonium hydroxide Total CPM on Plate 1,216 so- 40. -3 m 3020to- 01 1 5 0, 7 0 9 10 11 12 13 14 15 DISTRNCE FROM ORIGIN (CM) page 31 Figure 11 Thin-Layer Radiochromatogram of Extraction Fraction 1 (ether) Eluate A (chloroform) Pre-adsorbent SGF Uniplate: 100 butanol 10 water 10 acetic acid Total CPM on Plate 2,879 30- 30- to- 9 0 -1 0 1 2 3 4 5 0 7 a 9 10 11 12 13 14 15 DISTFINCE FROM ORIrol-N(CM) Page 32 Figure 12 Thin-Layer Radiochromatogram of Extraction Fraction I (ether) Eluate A (chloroform) Pre-adsor6ent SGF Uniplate: 100 chloroform 100 methanol 2 acetic acid Total CPM on Plate 2,882 soCL -3 30ti 20- to- o+-oeGieee"eeoe". 1 0 1 2 3 4 5 6 7-S 9 10 11 12 l@3-i4-f5 oisTRNcERROM ORIGIN(CM) Appendix 1 - Table 1 Thin-Layer Chromatography Systems for N-Ethyl POSE-14C Plate No. 1 2 3 4 5 Solvent System a Rf b of N-Ethyl poSE-14C 100 chloroform 100 acetone 100 chloroform 100 methanol 2 acetic acide iso chloroform 50 methanol 5 ammonium hydroxidec- 100 chloroform 35 methanol c 5 ammonium hydroxide 100 butanol 10 water c 10 acetic so d@-- 0.70 0.90 0.90 1.00 0.77 Solvents were prepared volume:volumey a 100 ml aliquot of solvent mixture was added to the chromatography tank. Rf is of major (> 98%) peak on the thin-layer chromatography plate. Acetic acid and ammonium hydroxide were concentrated. Notebook Reference: NB-51806-41-42 Appendix 1 - Figure I Thin-iiyer Radiochromatogram of N-Ethyl FOSE- C Dosing Solution, Plate No. 1 Pre-adsorbent SGF Uniplate: 100 chloroform 100 acetone Total CPM on Plate 5,393 CL so- 4030- 2010- 0 12 3 4 a 9 10 11 12 13 14 15 DISTRNCE FROM ORIGIN (CM) Appendix 1 - Figure 2 Thin-@lyer Radiochromatogram of N-Ethyl FosE- c isosing Solution, Plate No. 2 Pre-adsorbent SGF Uniplate: 100 chloroform 100 methanol 2 acetic acid Total CPM on Plate 5,310 go- 70CL 00. so- 40- 0 1; Li 30- C3 20- to- -1 0 1 2 3 4 5 5 7 0 9 10 11 12 13 1 is DISTRNCE FROM ORIGIN (CM) Appendix 1 - Figure 3 Thin-iiyer Radiochromatogram of N-Ethyl POSE- c i:6sing Solution, Plate No. 3 Pre-adsorbent SGF Uniplatd: Total CPM on Plate 5,356 150 chloroform 50 methanol 5 ammonium hydroxide 70- 3 CL 00- SD403020- -1 0 1 2 3 4 5 5 7 a 9 10 11 12 13 14 15 DISTHNGE FRM ORIGIN (CM) Appendix 1 - Figure 4 Thin-ityer Radiochromatogram of N-Ethyl FOSE- C posing Solution, Plate No. 4 Pre-adsorbent SGF Uniplate: 100 chloroform 35 methanol 5 ammonium hydroxide Total CPM on Plate 5,154 70 OD -3 40- t; 30- 0 7 0 9 10 11 12 13 14 is DISTRNCE RROM ORIGIN (CM) Appendix 1 - Figure 5 Thin-ityer Radiochromatogram of N-Ethyl FOSE- C Dosing Solution, Plate No. 5 Pre-adsorbent SGF Uniplate: 100 butanol 10 water 10 acetic acid Total CPM on Plate 5,258 70IL 403D2010- 0-1 2 3 4 5 5 7 6 9 10 11 12 13 11 is DISTRNCE FRM ORIGIN (CM) Appendix 2 Determination of Carbon-14 Content of N-Ethyl POSE-14C Dose/Feed Kixture Five aliquots of the dose/feed mixture were weighed into tared combustion cones and pads,a:o-n a five-place analytical balance. The carbon-14 content of the dose/feed aliquots was determined by combustion with a Packard L4odel 306 Oxidizer. Recovery of carbon-14 was determined to be 84.10 (see Appendix 4 and Appendix 4 - Table 1). This recovery was uniformly low throughout the combustion sample set, thus the data were corrected using this recovery factor. Packard Instrument Company, Inc., 2200 Warrenville Road, Downers Grove, Illinois. Appendix 2 - Table 1 Carbon-1'4 Content of N-Ethyl POSE-14C Dose/Feed 14ixture ug N-Ethyl POSE-14C equivalents/g overall 523.69 535.61 517.61 517.37 559.24 530.7 + 17.58 Notebook Reference: W-51806-45 Appendix 3 Rat Weights and Amount of N-Ethyl FOSE- 14C Dose/Feed Mixture Administered to Each Rat Rat Weight (g) Amount (g) of Dose/ Identification at Time of Dose Feed Mixture Consumed Dose in mg/kg IA 236 IB 221 ic 242 2A 231 2B 220 2C 237 4A 315 4B 329 4C 327 8A 270 SB 289 sc 288 16A 280 16B 289 16C 276 32A 317 32B 309 32C 301 4.48 4.24 3.61 4.48 4.26 4.49 6.11 6.37 6.34 5.23 5.61 5.58 5.42 5.61 5.35 6.15 5.99 5.84 SE + S.D. 10.07 10.18 7.92 10.29 10.28 10.05 10.30 10.28 10.29 10.28 10.30 10.28 10.27 10.30 10.29 10.30 10.29 10.30 10.13 + 0.56 Notebook Reference: NB-56531-16b-s and NB-51806-46 Appendix 4 Determination of Recovery of Total Carbon-14 Frco Blank Biological Samples Spiked With N-Ethyl FOSE-14C For each of the four sets of samples ccmbusted, five replicates of 10 ul, 50 ul, and 100 ul of diluted N-ethyl FOSE-14C dosing solution were aliquoted with calibrated micropipettors directly into scintillation vials. At the same time using the same solution and pipets, either five or six replicates of 10 ul, 50 ul, and 100 ul were aliquoted directly into combustion cones containing I g blank biological material (fecal, liver, spleen, or muscle homogenates). The combustion oones were dried and then pelletized with 5 cm ashleas filter paper. Blank filter paper pellets were combusted and the solvents collected in the vials to which the FC-95-14C had been'added directly. One of each of the 10 ul, 50 ul, and 100 ul N-ethyl POSE-14C spiked pellets were routinely combusted at the beginning, middle, and end of each set of samples. After correction for background and counting efficiency, percent recovery was calculated by comparing mean results from direct addition and combustion. The recovery data for four sets of samples that were analyzed on different days for total carbon-14 (N-ethylPOSE-14C) are shown in Appendix 4 - Tables 1-4. The mean recoveries for the four sample sets are 84.1%, 92.7%, 93.3%, and 95.0%. The recoveries were uniformly low throughout the combustion sample setst so the data were corrected using the appropriate recovery factors. Appendix 4 - Table I Recovery of Total Carbon-14 From Blank Biological Samples Spiked With N-Ethyl POSE_ 14c, Combustion Set No. 1 Combusted Spiked Biological Samples -i of Liver Feces Feces Feces Liver Liver Liver and Feces 10 UlA 50 ul 100 ul b 1563;- 1648 1682 8328 8329 7768 16381 14905 -15581 1750 1748 1713 8219 7605 7475 15724 15693 14498 10 ul 50 ul 100 ul 1917 9547 18943 Direct Addition Samples 1907 1912 1946 1962 9558 9609 9605 9819 18415 18654 19154 18741 1684 7954 15464 I; 1929 9628 18781 10 ul 1684 x 100 - 87.3% 1929 50 ul 7954 x 100 - 82.6% 9629 100 ul 15464 x 100 - 82.3% 18761 Overall sunples + S.D. recovery used for correction of oxidized i4.1 + 2.8%. a Amount of ti-ethylFOSE-14C spiking solution added. k Data are expressed as dpm. Notebook Reference: NB-51806-44 Appendix-4 - Table 2 Recovery of Total Carbon-14 From Blank Biological Samples Spiked With N-Ethyl POSE-14c, Combustion Set No. 2 Combusted Spiked Biological Samples of of Spleen Spleen Muscle Muscle Liver Spleen Muscle x of Spleen, Muscle, and Liver 10 ill-! b 1650;-- 1892 50 til 9392 7837 100 ul 18645 18613 1701 9397 16306 1622 1985 9387 8775 -- S 19135 1771 1661 8615 9392 18629 16306 1806 8927 18023 10 ill 50 ul 100 Ill 1933 9877 19743 Direct Addition Samples 1938 1713 1925 1919 9829 19735 9883 19546 9861 9931 19523 19338 1886 9876 19577 10 tLl 1806 x 100 n 95.76% 1866 50 ul 8927 x 100 - 90.36% 9876 100 til 18023 x 100 19577 92.06% Overall samples + S.D. recovery used for correction of oxidized i2.7 + 2.8%. a Amount of N-ethyl FOSE-14C spiking solution added. Data are expressed as dpm. Spiking error; sample was not used. Notebook Reference: NB-53102-46 Appendix 4 - Table 3 ,Recovery of Total Carbon-14 From Blank Biological Samples Spiked With N-Ethyl roSE-14c, Combustion Set No. 3 Combusted Spiked Biological Samples 10 ul-S 5'0 ul Spleen b 1978z- Spleen 2071 9530 -- c 100 ul 18332 1807D Feces 2084 10347 18944 Feces Feces i of Spleen 2052 1979 2025 10061 10049 9530 19641 19778 18201 of Feces 2038 10152 19454 ;of Spleen and Feces 2032 9841 18828 10 ul 50 til 100 ul 2050 10624 21668 Direct Addition Samples 2055 10664 2085 10609 2087 2105 10525 10466 20617 21263 20810 21253 2076 10578 21122 10 ul 2032 x 100 - 97.9% 2076 50 ul 9841 x 100 - 93.00 10578 100 til 18828 x 100 21122 89.1% Overall samples + S.D. recovery used for correction of oxidized i3.3 + 4.4%. a Amount of N-ethyl FOSE-14C spiking solution added. k Data are expressed as dpm. c Spiking error; sample was not used. Notebook Reference: NB-53102-53 Appendix 4 - Table 4 I Recov ery of Total Carbon-14 From Blank Biological Samples Spiked With N-Ethyl FOSE-14C, Combustion Set No. 4 Combusted Spiked Biological Samples 'E of Kidney Kidney Kidney Kidney Feces Feces and Feces 10 ul-S 2420@l 50 ul 11892 100 ul 22569 2386 11876 22444 2210 2341 2310 11844 11584 10782 226 22 22867 21969 2333 11596 22494 10 vl 2412 50 Ul 11299 100 ill 24816 Direct Addition Samples 2394 2384 2413 2425 12222 12232 12214 12127 24405 24289 24873 24753 i 2406 12019 24627 10 ul 2333 x 100 - 96.970 2406 50 ul 100 -ILl 11596 x 100 - 96.48% 22494 x 100 - 91.34% 12019 24627 Overall x- + S.D. recovery used for correction of oxidized samples a 95.0 + 3.1%. a Amount of N-ethyl FOSE-14C spiking solution added. k Data are expressed as dpm. Notebook Reference: NB-56531-10 Appendix 5 Table 1 Total'Carbon-14 in Feces After an Oral Dose of N-Ethyl FOSE-14C in Feed to Rats (Mean Dose, 10.13 mgag) collection Period (Days) Rat Identification A B c Mean + S.D. 0-1 0-1 1-2 0-1 1-2 2-3 3-4 0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 0-16 0-16 16-32 712.8@a 451.5 374.4 251.0 204.2 194.4 114.4 65.6 90.3 323.1 150.4 119.6 97.5 75.3 1906.0 2085.0 93.0 1 Day Group 279.3 2 Day Group 352.8 302.6 4 Day Group 178.0 238.5 184.7 159.0 8 Day Group 229.6 579.0 329.0 184.6 142.8 99.4 68.7 44.2 16 Day Group 1797.8 32 Day Group 1513.8 .295.8 432.1 474.7 + 219.9 281.2 209.5 361.8 + 85.5 295.5 82.7 320.6 247.8 163.2 146.0 249.9 + 230.2 + iao.8 139.8 71.3 23.0 16.0 22.9 127.7 248.3 225.7 172.7 92.3 96.2 98.0 86.9 141.0 + 82.8 305.9 + 249.4 371.4 170.9 226.8 83.6 128.5 + 31.6 105.1 + 12.7 88.1 16.8 68.8 22.1 1782.6 1828.8 + 67.3 1757.6 1785.5 + 286.6 81.7 156.8 + 120.5 a Data are expressed as jigN-ethyl FOSE-14C equivalents/sample collection period. Notebook References: NB-51806-50, NB-55673-30-33 Appendix 5 Table 2 Total darbon-14 in Urine After an Ora3.Dose of N-Ethyl poSE-14C in Feed to Rats (Mean Dose, 10.13 MgAg) Co3.lection Period (Days) Rat Identification A B c Mean + S.D. 0-1 0-1 1-2 0-1 1-2 2-3 3-4 0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 0-16 0-32 3.15@@-' 1 Day Group 2.73 3.30 2..16 2 Day Group 2.11 1.71 3.72 4.67 3.72 4.71 4 Day Group 3.31 4.62 3.31 3.25 2.86 2.34 4.20 10.42 8.46 2.45 2.63 1.78 8 Day Group 2.05 3.95 3.31 2.60 8.34 1.85 1.51 1.37 70.34 16 Day Group 35.46 60.36 32 Day Group - 85.44 2.11 2.66 + 0.52 2.65 2.18 2.69 + 0.60 2.02 0.27 3.76 3.62 3.76 3.22 3.60 + 4.30 + 3.60 3.73 0.25 0.59 0.25 0.85 3.12 4.08 4.86 3.53 3.12 2.47 2.92 2.81 2.68 + 3.46 + 4.12 5.52 6.64 2.26 2.35 1.99 0.56 0.97 0.78 4.27 3.05 0.35 0.74 0.74 31.27 45.69 + 21.45 50.07 65.29 + 18.19 Data are expressed as ug R-ethyl POSE-14C equivalents/sample collection period. Notebook Reference: NB-51806-52-55 Appendix 6 Carbon-14 Content in Digestive Tract (plus contents) and Feces After an Oral Dose of N-Ethyl POSE-14C in Feed to Rats (Mean Dose, 10.13 mgag) Rat identification Time PostDose (Hours) Digestive Tract (plus contents) Feces IA 1B ic Mean + S.D. 24 11.85!@ 64.04 24 31.98 105.79 24 8.54 57.38 17.46 + 12.69 75.74 + 25.24 2A 2B 2C Mean + S.D. 48 8.51 91.79 48 12.37 115.74 48 8.12 44.15 9.67 + 2.35 83.89 + 36.44 a Data are expressed as ug N-ethyl POSE-14C equivalents/g. Notebook Reference: NB-51806-49-50 Appendix 7 Comparative Data Showing Normal Fecal Excretion for Rats Appendix 7 - Table 1 shows comparative data showing normal fecal excretion for rats. Grams of feces excreted by rats at 24 hour intervals for a 7 day postdose period are given for rats in this study and for rats used as control groups in previous studies (PC-Experiments 8 and 9). These data show that the fecal excretion rates of rats in this study are comparable to the excretion rates of rats used as control groups in 2 other studies. Appendix 7 - Table I Comparativi Data Showing Normal Fecal Excretion for Rate + S.D. of Co3lection 3 ra7ts from the Period (Days) present study + S.D. of 5 con@rol rats from FC-Bxp. 8 + S.D. of 5 cont@rol rats from PC-Exp. 9 0-1 8.0 + 2.6BL'-b 10.29 + 3.68 1-2 9.47 + 2.94 7.79 + 1.58 2-3 10.79 + 1.14S 10.27 + 2.04 3-4 11.61 + 0.91S 10.33 + 2.22 4-5 8.92 + 2.47S 8.51 + 2.63 5-6 9.78 + 2.17S 9.11 + 1.79 6-7 10.69 + 1.27S 6.65 + 1.93 7.04 + 4.38 9.00 + 2.21 8.30 + 1.20 9.74 + 1.25 9.15 + 0.86 9.57 + 1.54 8.62 + 1.07 a Data are expressed as grams of feces excreted per collection period. This data is from the 2 Day Group of this study. This data is from the 8 Day Group of this study. Notebook References: NB-56531-16u-16v and 16dd-16hh, and M-53102-29t-29bb and 42q Appendix B Carbon-14 Content in Tissues After an Oral Dose of N-Ethyl POSE-14C in Ye*d to Rats IK*an Dose, 10. 13 AgAg) Rat Identification Liver-E Spl*eni KidneymA Red Blood Done Digemtive a a Subaut. Abdon. a Lungs! Cella! Plasm*a- Narrow- Tract-;- Careas . Pat-S PatA Muscle@l 1A ID ic Moan 32.55 49.58 24.05 35.39 6.77 7.00 4.80 6.19 6.93 14.65 6.76 10.11 8.13 11.69 6.16 8.73 21.gS 27.73 20.3S 23.34 7.36 10.61 6.13 8.03 12.40 is.00 8.73 12.04 11.85 31.98 8.54 17.46 2.29 3.23 1.61 2.36 6.44 7.93 3.90 6.09 8.10 9.68 4.87 7.55 1.79 2.21 0.97 1.66 2A 2B 2C Kean 4A 4D 4c Kenn 31.25 42.95 41.58 38.59 5.93 4.81 7.30 6.01 44.42 44.95 40.34 43.24 6.75 7.62 6.84 7.07 6.89 8.22 10.49 8.53 8.82 11.29 9.82 9.98 6.39 6.75 8.80 7.31. 19.38 20.66 12.31 17.45 6.30 9.08 7.81 8.40 18.63 21.44 19.90 20.06 5.69 6.54 9.51 7.25 7.76 9.10 7.92 8.26 10.72 10.69 11.41 11.01 8.21 10.07 8.92 9.07 8.51 12.37 8.12 9.67 b 1.47 1.98 2.40 1.93 b 4.07 4.30 3.97 4.11 2.21 3.07 1.87 2.38 3.35 4.10 4.30 3.92 2.14 1.80 1.79 1.91 1.13 1.65 1.66 1.55 1.63 1.40 1.36 1.46 SA es ac Mean 26.81 24.65 34.52 28.66 2.15 1.80 3.98 2.64 3.34 3.45 6.33 4.37 2.87 2.63 6.52 4.01 8.42 7.68 12.61 9.57 2.94 3.24 5.66 3.95 3.36 2.91 5.63 3.97 - 0.54 0.38 0.34 - 1.01 C.23 0.25 0.71 0.48 0.98 0.75 0.36 0.52 16A 16B 16C Mean 20.97 28.67 27.09 25.58 0.83 0.87 1.11 0.94 1.93 2.05 3.07 2.35 1.47 1.81 2.36 1.88 2.24 3.04 3.2S 2.64 1.75 2.19 2.92 2.29 0.99 1.01 1.52 1.17 0.03 0.13 0.36 0.04 0.01 0.03 0.16 0.16 0.21 - 0.17 0.03 0.18 32A 32B 32C Mean 20.25 33.78 20.95 24.99 0.76 1.37 0.64 0.92 1.86 3.S7 1.90 2.41 1.66 2.45 1.36 1.82 0.83 2.6S 0.89 1.46 1.95 2.67 1.59 2.14 0.69 I.SO 0.63 0.94 - 0.08 0.00 0.13 - 0.28 0.00 0.29 - 0.06 0.00 0.09 - 0.14 0.00 0.17 Data are expressed as ug N-othyl POSZ-14C equivalents/g. b Smple was not tak*n. Notebook Reforencess 99-51806-47-49, VS-53102-43-44, 47-52, and 54, MD-5633i-11-12 am PWM&747-11-A Appendix 9 TECHNICALREPORTSUMMARY Oein June 9,* 1981 -fo:YFCHNICAL COMMUNICATIONS CENTER - 201- Worgwt - ifrppwtisprintedan boO *Wn of~, tendtm copan toTCC-1 CENTitAL RESEARCH LABORATORIES. Analytical-and Properties Research Service to Riker - loolation of Trace Fluorochemicals -Rwo-rt-Tiog . Perfluorooetane Sulfanic Acid - A Rat-Livar AR No. 7474 - Metabolite of FM-3422 - June 9-. 1981 -To S. ii Gibson, J. D. Johnson - 218-2-02 AWNWIV S. V. Pathre MCLOITY 10 ow icompwiv ConfkbntWI civew I$Pdel Audwrintion) MA CHEIWIICAL REGISTRY KEVWO@tos: (Selecwtwo frorn3M Thsmrw. Swggm other MOW.WAIO WML) CURRENT OBACTIVE: Request No. C57427 CRLAP Analytical Report Project No. 91505026 Requestor - S. J. Gibson, J. D. Johnson Chemical Analysis Laboratory DGPL Numbw 2 I Nwmbw A000007 I%wcwNtumbet 474 233150 -N& ofPea" includiCng nttmt 3 Nm Chomi=isReported [3 yo E) No 004MT AWTITACT:(2M2$0 warddThisabnrm infomotioinsdistribubtVetdheT@MnicaClomnwnicatioCnesntetro oWt 3M'wotoODWO&W R&D. ItisCGMPWW conkfMntiWOWUWIBI. A rat-liver setabolite acid by Isy-)=. is Identified as perfluorooctane sulfonic irjwmmwLnwiwn lmlime: gm CONFIDENTL4kL CENTRAL ANALYTICAL LABORATORY Report No. WWW --- 7-4-7-4---- IIlk Subject: Perfluorooctane Sulfoule Acid - A Rat-Liver Metabolite of YM-3422 S. J. Gibson itequestor:--.J.PD-a -,Ighwv- Dept. Name ----VJW ------ Proj. N0.91505026- Request No. ..0.7.42-Z------- Dated..AqRng?.-t:rL,(MZf.g.---- R"rt: Two metabolites Isolated from the liver of a rat administered 14C-labeled YM-3422 were submitted for spectroscopic analysis. These two metabolites were labeled as I-CH$Cl-MaDH aluted and II-CHS aluted. T4cHs CF3CF2-CF2(CF2)3-CFZCFIN-CRI-CH20H FM-3422 Experimental Both samples were reconstituted in CD30D. The 19F-NMR spectra on these samples were obtainod on the Varian XL-100 and XL-200 NMR spectrometers. Metabolite 'I 197 *M Metabolite 11 '97 WM 13875N 30190X Results The chemical shifts (ppo upfield from CFCls) of the major peaks In the fluorine spectra are given below. The peak frequencies are normalized to 6(CF3) 81.0 ppm. 1 81.0 114.3 120.4 121.5 122.4 126.0 11 81.0 112.9 120.2 121.8 122.8 126.2 Discussion Both spectra were typical of perfluorooctaneoulfonyl derivatives. The metabolite I was Identical to perfluorooctane sulfonic acid as determined by comparing the reference 19Y NMR (11252X) of the latter with that of 1. CFS - CF2 - CF2 81.0 126.0 122.4 (CF2)3 121.5 CF2 - CF2 120.4 114.3 S03H The spectrum of the metabolite 11 was very similar to that of I except the chemicalshift of the fluoramethylonealpha to the sulfonyl group. It is observed at 112.9 ppa in 11, 1.4 ppm upfiold from that in 1. The 112.9 ppm peak, I AR No. 7474 op June 9, 1981 Page 2 although not unambiguously,can be assigned to the alpha fluoromethylene of the sulfonamide (-SO2NH2)group: CF3 - CFI - CFS - (CF2)3 - CF2 - CF2 - 112.9 SOgNH2 Conclusion The liver metabolite labeled I-,CHC13-N*OHis Identifiedan perfluorooctane oulfonicacid and that labeled ll-(MCISis suggestedan perfluorooetane oulfanamlide. S. V. Pathre SVP/ra