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Absorption and Biotransformation of N-Ethyl FOSE and Tissue Distribution and Elimination of Carbon-14
after Administration of N-Ethyl FOSE-^^c in Feed
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0065
January 19, 1983
Conducted At:
During: Conducted By: Report By:
Riker Laboratories Inc. 3M Center St. Paul Minnesota 55144
October 1980 to February 1981
S. J. Gibson and J. D. Johnson
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S. J. Gibson
Date
Senior Laboratory Technician
Reviewed By:
J. D. Johnson MS Resea^ph Specialist
Date
M c . & w ifo/as
R. E. Ober PhD
Date
Manager, Drug Metabolism
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C03477
Table of Contents
Page
Summary ............................................. 1
Introduction ........................................ 1
Materials and Methods ............................... 1
Carbon-14 Labeled N-Ethyl FOSE .................... 1
Animals ........................................... 2
Dosing ............................................ 2
Preparation of Dose/Feed Mixture ................ 2
Administration of D o s e .......................... 2
Sample Collection ................................. 3
Radiometric Analyses .............................. 3
Sample Preparation for Radiometric Analyses ..... 3
Analyses of Samples ............................. 4
Isolation and Identification of Metabolites ....... 4
Extraction ...................................... 4
Column Chrcxnatography ........................... 4
Thin-Layer Chromatography of ColumnFractions .... 5
Results and Discussion .............................. 5
Extent and Route of Excretion and Tissue Distribution ....................................
Isolation and Identification of Metabolites .......
5 8
References .......................................... 10
List of Tables, Figures and Appendices.............. 11
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Page 1
Summary
After a single oral dose of N-ethyl FOSE-He (2-N-ethyl perfluorooctanesulfonamido ethanol) (mean dose, 10.13 mg/kg) 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 FOSE isolated from liver was identified by 19F-NMR analysis as the pgrfJUiprooctaneBulfonate 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 inter mediate in the production of other products. N-Ethyl FOSE is important in the study of FC-807 metabolism because the three esters of FC-807 are phosphate esters of N-ethyl FOSE and in addition FC-807 contains a trace fvO.8%) of N-ethyl FOSE. 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 biotransformation 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 FOSE
/ c 2h5 c 7f 1 5 *c f 2s o 2-n ^ CH2CH20h
* Denotes Position of Carbon-14 Label
N-Ethyl FOSE is 2-N-ethyl perfluorooctanesulfonamido ethanol.
The carbon-14 label is at the carbon a to the sulfur atom (see above structure). On the basis of the specific activity determination, chemical characterization, and radiochemical purity reported separately (1), the N-ethyl FOSE-14C was judged suitable for metabolism studies. The specific activity of the lot of N-ethyl FOSE-Hc used in these studies (Riker Isotope Inventory Number 468) is 0.483 jh 0.020 yCi/mg. The radiochemical purity determination was repeated on the N-ethyl FOSE-1^C dosing solution (see Appendix 1 - Table 1, and Appendix 1 Figures 1-5) and the N-ethyl FOSE-Hc was found to be at least 98% pure.
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Animals
Male Charles River-- CD 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 from 220 to 329 g, mean 277 g. Rats were dosed in groups of 3; individual rats were selected so that the body weights of rats within each group of 3 were within 21 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 FOSE-Hc solution was prepared by weighing N-ethyl FOSE-1^C into a 1 liter Class A volumetric flask, adjusting to volume with absolute ethanol, and mixing by inverting. The N-ethyl FOSE-1^C solution was transferred to a 4 liter beaker and Purina Ground Chow was added. The N-ethyl FOSE-I^C/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 FOSE-1^C/g. All of the rats in the groups sacrificed at 1, 2, and 4 days postdose consumed 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 fran -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 consumed. The mean dose was 10.13 mg/kg (see Appendix 3).
Charles River Breeding Laboratories, Wilmington, Massachusetts. -- Purina Lab Chow, Ralston Purina Company, St. Louis, Missouri.
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Sample Collection
Urine and feces were collected at 24 hour intervals for each rat sacrificed at 1, 2, 4, and 8 days postdose (groups of 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 ether; 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-- . 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 1 and 2 days postdose.
Radiometric 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 com bustion cones^-. 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 frcm biological samples was determined by combusting suitable blank homogenates (feces, liver, kidney, muscle, and spleen) spiked with N-ethyl FOSE-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 aliquots or 0.5 ml aliquots plus 0.5 ml water were pipetted directly into scintillation vials and 15 ml Aquasol* added. All samples were cold and dark adapted before counting.
-- Packard Instrument Company, Inc., 2200 Warrenville Road, Downers Grove, Illinois.
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Analyses of Samples
All radiometric 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 carbon-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 combusted 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 yg equivalents of N-ethyl FOSE-14C. The homo
genate was extracted three times with diethyl ether with back ex
tractions 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% HC1 and stirred
for an hour. The water layer was then extracted two times with
diethyl ether. The ether was evaporated and the residue was re
dissolved in methanol (Fraction 2). The water was filtered and
the filter cake was extracted two times with chloroform-methanol
1:1 (v/v). The chloroform-methanol was evaporated and the residue
was redissolved in methanol (Fraction 3).
p
Column Chromatography
Three 2.0 cm in diameter columns were packed to 40 cm with silica gel-- 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 was eluted with 500 ml of CHCI3 (Eluate A), 500 ml of chloroform-methanol 1:1 (v/v) (Eluate B), 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.
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Thin-Layer Chromatography of Column Fractions
Each of the six column fractions that contained carbon-14 (not methanol elution) were assayed by thin-layer chromatography. Small aliquots of the material were streaked on pre-adsorbent silica gel plates^- 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 vials containing methanol and 7.5 ml of a modified scintillant-- was added. The contents of the scintilla tion vials were counted and the data were calculated and radio activity 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-I^C 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 yg equivalents N-ethyl F0SE-14C excreted per time period in Appendix 5 - Tables 1 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 rats 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 FOSE-1*C (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 (<v14-28%) is the upper limit of the estimate for percent of dose not absorbed. Thus, at least 70 percent of the dose of N-ethyl FOSE-14C was absorbed.
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The digestive tracts (esophagus, stomach, 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 yg N-ethyl FOSE 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 postdose. However, the tissues and contents of the digestive tracts at 48 hours postdose contain a mean of 11.4%
of the dose, and since the transit time of rats is faster than 48
lui
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-14C; 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-^C with subsequent elimination via bile. It follows that some of the carbon-14 in feces before 48 hours is likely absorbed N-ethyl F0SE-1*C and then excreted N-ethyl FOSE derived material. Thus, the absorption of N-ethyl FOSE-- '^C is probably greater than 70%.
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, kidneys, 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 yg N-ethyl FOSE-14C equivalents/g of tissue. [The same data (not normalized) are listed in Appendix 8 ].
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 FOSE-14c and/or its metabolites were lost during preparation of the samples (N-ethyl FOSE-^^C 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 1 for groups of rats (3 rats/group). The ratio (liver/plasma) 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-^C, the mean liver/plasma, spleen/plasma, 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 FC-807-14C, the mean liver/plasma, spleen/plasma, and bone marrow/plasma carbon-14 level ratios were 22.1, 202.8, and 48.1, respectively. Thus, the pattern of distribution of carbon-14 after FC-807-^C administration is so different from the pattern after N-ethyl FOSE-1*C administra-
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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 biotransforma tion is more complex than a simple conversion to N-ethyl FOSE by hydrolysis of the phosphorus-oxygen bond with subsequent biotrans formation 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-1*C 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 FOSE (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 frcm plasma of carbon-14 from day 1 to day 16 (Figure 1) is 7.5 days after N-ethyl FOSE-14C administration. Hcwever, 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 yg equivalents on day 16, 2 . 1 yg equivalents on day 32). The plasma half-life value of 7.5 days for carbon-14 after N-ethyl FOSE-'^C oral administration is the same as that reported for carbon-14 after oral administration of potassium perfluorooctanesulfonate-14C to male rats (estimated from day 1 to day 6 ) (4). As with N-ethyl F0SE-1 *C, 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 F0SE-1*C and potassium perfluorooctanesulfonate-1^ . In addition, for both compounds^, 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 frcm 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 frcm plasma are inaccurate and should not be attempted.
-- The change in rate of elimination of total carbon-14 from plasma is not unique to N-ethyl FOSE-14C and perfluorooctanesulfonate-14C; it is quite common for other compounds.
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Isolation and Identification of Metabolites
The liver homogenate pool (9:1, water/tissue) from rats sacrificed at 48 hours after a b ingle oral dose of N-ethyl F0SE-1*c was ex tracted 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 from the liver homogenate for each fraction was: Fraction 1, 30.1%; 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 (0%). Thus, of the 1052 yg equivalents of N-ethyl F0SE-1*C present in the feces pool, 705 yg 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-methanol (Eluate B); 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 (Eluates A and B) were chromatographed by thin-layer chromatography. Radiochromatograms from thin-layer chroma tography of these six fractions are shown in Figures 3-12. When chromatographed in the same solvent system (100 chloroform, 35 methanol, 5 ammonium 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 Eluate B indicate that the radiochemical purity of the metabolite is *95%.
The extraction Fraction 2 (acid-ether) Eluate B (chloroform-methanol) was labeled metabolite Fraction I and submitted to S. Pathre of the Central Analytical Laboratory for 19F-NMR analysis on the Varian XL-100 and XL-200 Spectrometers. The results are reported in Appendix 9; the metabolite was identified as the perfluorooctanesulfonate anion.
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From the percent of total carbon-14 extracted from the liver homo genate pool (67%) and the total carbon-14 recovered from the columns in 1 : 1 chloroform-methanol and the percent composition 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-1^C 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 perfluorooctanesulfonate 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 8-10). As with the chloroform-methanol eluates, comparison of the three column eluates suggests that most of the carbon-14 is the same metabolite. The extraction Fraction 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) (Figure 12).
The extraction Fraction 1 (ether) Eluate A (chloroform) was labeled metabolite Fraction II and submitted to S. Pathre of the Central Analytical Laboratory for 1 4F-NMR 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 perfluorooctanesulfonate, 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-I^c is metabolite Fraction II.
Further refinement of these extractions and separations would likely result in a higher estimate of the percentage of perfluorooctanesul fonate and the metabolite tentatively identified as perfluorooctane sulfonamide 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 8-10) 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.
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Page 10 References
1. Johnson, JD and Behr, FE: Synthesis and Characterization of N-Ethyl FOSE-Hc (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.
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List of Tables, Figures, and Appendices
Table 1:
Cumulative Excretion of Total Carbon-14 in Feces After an Oral Dose of N-Ethyl FOSE-^C in Feed to Rats (Mean Dose, 10.13 mgAg)
Table 2:
Cumulative Excretion of Total Carbon-14 in Urine After an Oral Dose of N-Ethyl F0SE-14C in Feed to Rats (Mean Dose, 10.13 mg/kg)
Table 3:
Cumulative Excretion of Total Carbon-14 in Urine and Feces After an Oral Dose of N-Ethyl FOSE-^C in Feed to Rats (Mean Dose, 10.13 mg/kg)
Table 4:
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 mgAg) at 24 and 48 Hours Postdose
Table 5: Carbon-14 Content in Tissues After an Oral Dose of N-Ethyl F0SE-14c in Feed to Rats (Mean Dose, 10.13 mgAg)
Table 6 : Carbon-14 Content in Tissues After an Oral Dose of N-Ethyl FOSE-)^C in Feed to Rats (Mean Dose, 10.13 mgAg)
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 mg/kg 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 FOSE-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 FOSE-Hc in Feed NB-53102-43-44, NB-56531-5
Figure 3: Thin-Layer Radiochranatogram of Extraction Fraction 1 (ether) Eluate B (1:1 chloroform-methanol) NB-51579-36
Figure 4: Thin-Layer Radiochranatogram of Extraction Fraction 2 (acid-ether) Eluate B (1:1 chloroform-methanol) NB-51579-36
Figure 5: Thin-Layer Radiochranatogram of Extraction Fraction 3 (1:1 chloroform-methanol) Eluate B (1:1 chloroform-methanol) NB-51579-36
Figure : Thin-Layer Radiochranatogram of Extraction Fraction 2 (acid-ether) Eluate B (1:1 chloroform-methanol) NB-51579-35
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List of Tables, Figures, and Appendices (Con't)
Figure 7s Thin-Layer Radiochrcmatogram of Extraction Fraction 2 (acid-ether) Eluate B (1:1 chloroform-methanol) NB-51579-35
Figure 8 : Thin-Layer Radiochrcmatogram of Extraction Fraction 1 (ether) Eluate A (chloroform) NB-51579-36
Figure 9: Thin-Layer Radiochrcmatogram of Extraction Fraction 2 (acid-ether) Eluate A (chloroform) NB-51579-36
Figure 10: Thin-Layer Radiochromatogram of Extraction Fraction 3 (1:1 chloroform-methanol) Eluate A (chloroform) NB-51579-36
Figure 11: Thin-Layer Radiochromatogram of Extraction Fraction 1 (ether) Eluate 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 Systems for N-Ethyl FOSE-1 NB-51806-41
Appendix 1 Figure 1: Thin-Layer Radiochromatogram of N-Ethyl FOSE-1 4c Dosing Solution, Plate No. 1 NB-51806-42
Appendix 1 Figure 2: Thin-Layer Radiochromatogram of N-Ethyl FOSE-1 4c Dosing Solution, Plate No. 2 NB-51806-42
Appendix 1 Figure 3: Thin-Layer Radiochromatogram of N-Ethyl FOSE-1 4c Dosing Solution, Plate No. 3 NB-51806-42
Appendix 1 Figure 4: Thin-Layer Radiochromatogram of N-Ethyl FOSE-1 4c Dosing Solution, Plate No. 4 NB-51806-42
Appendix 1 Figure 5: Thin-Layer Radiochromatogram of N-Ethyl FOSE-1 4c Dosing Solution, Plate No. 5 NB-51806-42
Appendix 2
Determination of Carbon-14 Content of N-Ethyl FOSE-1 4c Dose/Feed Mixture
Appendix 2'Table 1: Carbon-14 Content of N-Ethyl FOSE-1 4c Dose/Feed Mixture
Appendix 3
Rat Weights and Amount of N-Ethyl FOSE-1 4c Dose/Feed Mixture Administered to Each Rat
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LiBt of Tables, Figures, and Appendices (Con't)
Appendix 4:
Determination of Recovery of Total Carbon-14 From Blank Biological Samples Spiked With N-Ethyl FOSE-14C
Appendix 4-Table 1:
Recovery of Total Carbon-14 From Blank Biological Samples Spiked With N-Ethyl F0SE-14C, Combustion Set No. 1
Appendix 4-Table 2:
Recovery of Total Carbon-14 Fran Blank Biological
Samples Spiked With N-Ethyl F0SE-14c, Combustion Set No. 2
Appendix 4-Table 3:
Recovery of Total Carbon-14 From Blank Biological Samples Spiked With N-Ethyl FOSE-14c, Combustion Set No. 3
Appendix 4-Table 4:
Recovery of Total Carbon-14 From Blank Biological Samples Spiked With N-Ethyl F0SE-1 *C, Combustion Set No. 4
Appendix 5-Table 1:
Total Carbon-14 in Feces After an Oral Dose of N-Ethyl FOSE-1^C in Feed to Rats (Mean Dose, 10.13 mg/kg)
Appendix 5-Table 2: Appendix 6 :
Total Carbon-14 in Urine After an Oral Dose of N-Ethyl FOSE-- 1^C in Feed to Rats (Mean Dose, 10.13 mg/kg)
Carbon-14 Content in Digestive Tract (plus contents) and Feces After an Oral Dose of N-Ethyl FOSE-1*C in Feed to Rats (10.13 mg/kg)
Appendix 7:
Comparative Data Showing Normal Fecal Excretion for Rats
Appendix 7-Table 1: Comparative Data Showing Normal Fecal Excretion for Rats
Appendix 8 :
Carbon-14 Content in Tissues After an Oral Dose of N-Ethyl FOSE-1*C in Feed to Rats (Mean Dose, 10.13 mg/kg)
Appendix 9:
Report of Central Analytical Laboratory Analysis of Metabolite Fractions I and II
t 0^491
Page 14
' Table 1
Cumulative Excretion of Total Carbon-14 in Feces After an Oral Dose of N-Ethyl FOSE-14C
in Feed to Rats (Mean Dose, 10.13 mg/kg)
Collection Period (Days)
Rat Identification
AB
C
Mean 4 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
1 Day Group
29.982.
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
16 Day Group
66.27
60.39
63.88 66.73
32 Day Group 47.62 56.93
22.55 21.65 + 8.82
11.80 15.46 4 3.60 20.59 28.10 4 7.11
9.53 16.89 21.74 26.08
7.51 4 14.42 4 19.85 4 24.04 4
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 4 2.71 15.16 4 10.98 28.10 4 9.17 35.99 4 9.21 40.44 4 10.18 44.07 4 10.36 47.11 4 10 .0 0 49.49 +_ 9.41
62.79 63.15 4_ 2.96
56.72 56.07 4 8.15 59.36 61.01 4- 5.10
-- Data are expressed as percent of dose excreted during collection period.
Notebook Reference: NB-55673-37 and 38
0*3492
Page 15
Table 2
Cumulative Excretion of Total Carbon-14 in Urine After an Oral Dose of N-Ethyl FOSE-1*C
in Feed to Rats (Mean Dose, 10.13 mg/kg)
Collection Period (Days)
Rat Identification
AB
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
1 Day Group
0.13^-
0.12
0.14 0.23
2 Day Group 0.09 0.17
0.11 0.25 0.36 0.50
4 Day Group 0.10 0.24
0.34
0.44
0.10 0.17
0.30 0.62 0.88 0.97 1.06 1.12
8 Day Group 0.06 0.18 0.28 0.36 0.61 0.67 0.72 0.77
16 Day Group
2.45
1.19
32 Day Group
1.85
2.69
0.11 0.12 + 0.01
0.11 0.20
0 . 1 1 + 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 + 0.02 0.19 + 0. 02 0.31 + 0.03
0.47 + 0.13
0.67 + 0.18
0.75 + 0.19 0.83 + 0.20 0.90 + 0.19
1 . 1 0 1.58 + 0.75
1.62 2.05 + 0.56
-- Data are expressed as percent of dose excreted during collection period.
Notebook Reference: NB-55673-38
e o a 493
Page 16
Table 3
Cumulative Excretion of Total Carbon-14 in Urine and Feces After an Oral Dose of N-Ethyl FOSE-^c
in Feed to Rats (Mean Dose, 10.13 mgA9)
Collection Period pays)
Rat Identification
AB
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
30.11--
1 Day Group 12.53
19.13 34.96
2 Day Group 15.69 29.15
7.85 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 Group 7.77
27.34 38.49 44.77 49.82 53.22 55.58 57.12
16 Day Group
68.72
61.58
32 Day Group
68.58
59.62
22.6 6 21.77 + 8.82
11.91 15.58 + 3.61 20.79 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
60.98 63.06 + 4.83
-- Data are expressed as percent of dose excreted during collection period.
Notebook Reference: NB-55673-39 and 40
c 0,2434
Page 17
Table 4
Carbon-14 Content in Digestive Tract (plus contents) and Feces After an Oral Dose of N-Ethyl FOSE-^c in Feed to Rats (Mean Dose, 10.13 mg/kg) at 24 and 48 Hours Postdose
Rat Identification
Time PostDose (Hours)
Digestive Tract (plus contents)
Feces
1A IB 1C Mean jf S.D.
24
12.842-
29.98
24
22.40
12.41
24
14.60
22.55
16.61 + 5.09 21.65 8.82
2A 2B 2C Mean +_ S.D.
48
11.10
34.73
48
13.03
28.98
48
10.10
20.59
11.41 + 1.49 28.10 +7.11
-- Data are expressed as percent f dose. Notebook Reference: NB-51806-49-51
00.3495
Page IS
Table 5
Carbon-14 Content In Tissues After an Oral Dose of N-Ethyl F O S E - K c in Feed to Rats (Mean Dose, 10.13 mg/kg)
Rat Identification
Liver--
m
Spleen^- Kidneye^- Lungs--
Red Blood Celled
Digestive
Plasma^-
Tract--
Carcass^-
1A 1B 1C
Mean
16.85 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.B2
7.00
2.55 3.10 2.80
2.82
12.84 22.40 14.60
16.61
18.76 23.48 17.40
19.88
2A 2B 2C
Kean
4A 4B 4C
He an
17.72 18.23 24.17
20.04
0.14 0.10 0.16
0.13
21.59 18.47 17.69
19.25
0.14 0.12 0.13
0.13
0.66 0.77 1.00
0.81
0.77 0.98 0.75
0.83
0.35 0.40 0.59
0.45
0.43 0.43 0.38
0.41
5.89 5.77 3.62
5.09
5.44 5.85 5.74
5.68
2.06 2.17 3.32
2.52
2.67 2.96 2.72
2.78 .
11.10 13.03 10.10
11.41
__ d -- --
12.30 15.62 19.29
15.74
__ -- --
8A SB 8C
Mean
15.44 12.B3 18.30
15.52
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.B4
3.00
1.15 1.19 2.05
1.46
-- -- --
----
-- --
16A 16B 16C
Mean
.32A 32B 32C
Mean
9.52 12.10 10.32
10.65
0.02 0.02 0.03
0.02
7.84 12.71
8.04
9.53
0.02 0.03 0.01
0.02
0.18 0.16 0.27
0.20
0.18 0.31 0.16
0.22
0.09 0.09 0.14
0.11
0.07 0.13 0.07
0.09
0.77 0.99 1.08
Q.95
0.28 0.87 0.30
0.48
0.72 0.85 1.16
0.91
0.78 1.13 0.64
0.85
--
--
,,
-- --
--
--
--
__
-- --
--
-- Data are expressed as percent of dose in tissue. -- Data are estimates of percent of dose present in red blood cells. Red blood cell volume
26.3 ml/kg body weight (7). -- Data are estimates of percent of dose present in plasma. Plasma volume 31.3 m l A 9 body d weight (7). -- Sample was not taken.
Notebook References: NB-56531-8 and 9, NB-51806-49, and NB-53102-49
00,3490
Page 19
Table 6
Carbon-14 Content in Tissues After an Oral Dose of
H-Ethyl r0SE-14C in Feed to Rats (Mean Dose, 10.13 mgAg)
Rat
Red Blood
Bone Digestive
Subcut. Abdcnt.
Identif icat ion Divert SpleerA KidneysS- LungeS- CellsS- Plasma^- MarrowS Tract-- Carcass^- Fat- FatA MuscleS
1A IB 1C
Mean
32.4 46.7 30.4
37.2
6.72 6.88 6.06
6.55
8.87 14.39
8.54
10.60
8.07 11.68
7.78
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 9.51 6.15
7.90
0.40 0.42 0.34
0.39
2A 2B 2C
Mean
4A 4B 4C
Mean
30.4 41.8 41.4
37.9
43.1 43.8 39.2
42.0
5.76 4.68 7.26
5.90
6.55 7.41 6.65
6.87
6.70 8.00 10.44
8.38
8.56 10.98
9.54
9.69
6.21 6.57 8.76
7.18
18.83 20.10 12.25
17.06
5.53 6.36 9.46
7.12
8.06 8.83 7.59
8.16
18.28 20.86 19.34
19.49
7.53 8.84 7.69
8.02
10.42 10.59 11.35
10.79
8.27 12.03
8.08
9.46
7.97 9. B0 8.67
8.81
__ b -- --
w--
1.43 1.93 2.39
1.92
3.96 4.16 3.95
4.03
3.26 3.99 4.28
3.84
__ -- --
_
2.15 2.99 1.82
2.32
2.08 1.75 1.74
1.86
0.22 0.37 0.52
0.37
1.58 1.36 1.32
1.42
8A 8B BC
Mean
26.1 24.0 33.6
27.9
2.09 1.75 3.87
2.57
3.25 3.35 6.16
4.25
2.79 2.55 6.34
3.89
8.19 7.46 12.27
9.31
2.85 3.15 5.50
3.83
3.27 2.83 5.48
3.86
--
---
--
--
0.53 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
32A 32B 32C
Mean
20.4 27.9 26.3
24.9
0.81 0.84 1.08
0.91
19.7 32.8 20.4
24.3
0.74 1.33 0.62
0.90
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
1.81 3.47 1.75
2.34
1.61 2.3B 1.32
1.77
0.B1 2.58 0.86
1.42
1.89 2.79 1.54
2.07
0.96 0.98 1.48
1.14
-- --
0.67 1.46 0.61
0.91
--*
--
--
0.03 0.04
0.16
--
0.13 0.01
0.16
0.35 0.03
0.20
--
0.17 0.03
0.17
----
0.08 0.00
0.13
--
0.28
0.00
0.28
--
0.06 0.00
0.09
--
0.14
0.00
0.17
Data is normalized to a 10 m g A g dose and expressed as tig N-ethyl FOSE-1*C equivalents/g. -- Sample was not taken.
Notebook References: NB-56531-5 and 6, NB-53102-43 and 44
C03497
Page 20
Table 7
Relative Carbon-14 Content of Eluates A and B for Extraction Fractions 1, 2 and 3
Fraction 1 (ether)
Eluate A (chloroform)
93%
Fraction 2 (acid-ether)
Fraction 3 (1 : 1 chloroform-methanol)
23% 61%
Eluate B (1 : 1 chloroform-
methanol)
19%
61%
22%
Total % recovered
112%
84%
Notebook Reference: NB-51579-34
83%
003498
yg N-Ethyl FOSE- C equivalents/gram
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..and 32 Days Post Oral Dose of N-Ethyl FOSE- c in Feed
DAYS POSTDOSE
C 0^493
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, jgd 32 Days Post Oral Dose of N-Ethyl FOSE- in Feed
C'OcioOO
Page 23
Figure 3
Thin-Layer Radiochromatogram of Extraction Fraction 1 (ether) Equate B (1:1 chloroform-methanol)
Pre-adsorbent SGF Uniplate: 100 chloroform 35 methanol 5 ammonium hydroxide
DISTRNCE FROM ORIGIN (CM)
C OviSOl
Page 24
Figure 4
Thin-Layer Radiochromatogram of Extraction Fraction 2 (acid ether) Eluate B (1:1 chloroform-methanol)
Pre-adsorbent SGF Uniplate:
100 chloroform 35 methanol 5 ammonium hydroxide
Total CPM on Plate 4,363
DISTANCE FROM ORIGIN (Oil
C0502
Page 25
Figure 5
Thin-Layer Radiochromatogram of Extraction Fraction 3 (1:1 chloroform-methanol) Eluate B (1:1 chloroform-methanol)
Pre-adsorbent SGF Uniplate:
100 chloroform 35 methanol 5 ammonium hydroxide
DISTANCE PROM ORIGIN (CM)
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
DISTANCE FROM ORIGIN (CHI
(03504
* i. 0
Page 27
Figure 7 :
Thin-Layer Radiochromatogram of Extraction Fraction 2 (acid-ether) Eluate B (1:1 chloroform-methanol)
Pre-adsorbent SGF Uniplate:
100 chloroform
100 methanol 2 acetic acid
DISTANCE FROM 0RI6IN (CM) 00305
Page 28
Figure 8
Thin-Layer Radiochromatogram 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
DISTANCE FROH ORIGIN (CM)
003S0G
Page 29
Figure 9
Thin-Layer Radiochromatogram 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
DISTANCE FROM ORIGIN (CM)
COas07
Page 30
Figure 10
Thin-Layer Radiochromatogram of Extraction Fraction 3 (1:1 chloroform-methanol) Eluate A (chloroform)
Pre-adsorbent SGF Uniplate: 100 chloroform 35 methanol 5 ammonium hydroxide
Total CPM on Plate = 1,216
DISTANCE FROM ORIGIN (CM)
003508
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
DISTANCE FROM ORIGIN (CM)
003509
Page 32
Figure 12
Thin-Layer Radiochromatogram of Extraction Fraction 1 (ether) Eluate A (chloroform)
Pre-adsorBent SGF Uniplate:
100 chloroform 100 methanol
2 acetic acid
Total CPM on Plate = 2,882
DISTANCE FROM ORIGIN (CM)
003510
Appendix 1 - Table 1 Thin-Layer Chromatography Systems for N-Ethyl POSE-1Aq
Plate No.
Solvent System^-
Rf^ of N-Ethyl FOSE-14C
1 100 chloroform 100 acetone
0.70
2 100 chloroform 100 methanol 2 acetic acid^-
0.90
3 150 chloroform
0.90
50 methanol
5 ammonium hydroxide^-
4 100 chloroform
1.00
35 methanol
5 ammonium hydroxide--
5 100 butanol 10 water 10 acetic acid^-
0.77
-- Solvents were prepared volume:volume; a 100 ml aliquot of k solvent mixture was added to the chromatography tank. -- Rf is of major (> 98%) peak on the thin-layer chromato
graphy plate. -- Acetic acid and ammonium hydroxide were concentrated.
Notebook Reference: NB-51806-41-42
coasil
Q.
Appendix 1 - Figure 1 Thin-|yer 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
DISTANCE FROM ORIGIN (CM)
co^sis
Appendix 1 - Figure 2
Thin-l^gyer Radiochromatogram of N-Ethyl FOSE-X c loosing Solution, Plate No. 2
Pre-adsorbent SGF Uniplate:
100 chloroform 100 methanol
2 acetic acid
DISTRNCE FROM ORIGIN (CM) 003513
Appendix 1 - Figure 3
Thin-]jigyer Radiochromatogram of N-Ethyl FOSE- C Dosing Solution, Plate No. 3
Pre-adsorbent SGF Uniplat:
150 chloroform 50 methanol 5 ammonium hydroxide
DISTANCE FROM ORIGIN (CM)
('0JS14
Appendix 1 - Figure 4
Thin-]ji|yer Radiochromatogram of N-Ethyl FOSE- C Dosing Solution, Plate No. 4
Pre-adsorbent SGF Uniplate:
100 chloroform 35 methanol 5 ammonium hydroxide
Total CPM on Plate = 5,154
DISTANCE PROM ORIGIN (CM)
CO violS
Appendix 1 - Figure 5 Thin-J|yer Radiochromatogram of N-Ethyl FOSE- C Dosing Solution, Plate No. 5 Pre-adsorbent SGF Uniplate: 100 butanol
10 water 10 acetic acid
DISTANCE FROM ORIGIN (CM)
CO uC)
Appendix 2
Determination of Carbon-14 Content of N-Ethyl FOSE-t4C Dose/Feed Mixture
Five aliquots of the dose/feed mixture were weighed into tared combustion cones and pads-- on a five-place analytical balance. The carbon-14 content of the dose/feed aliquots was determined by combustion with a Packard Model 306 Oxidizer. Recovery of carbon-14 was determined to be 84.1% (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.
a Packard Instrument Company, Inc., 2200 Warrenville Road, Downers Grove, Illinois.
Appendix 2 - Table 1
Carbon-14 Content of N-Ethyl FOSE-^C Dose/Feed Mixture
pg N-Ethyl FOSE-14C equivalents/g
Overall x *
523.69 535.61 517.61 517.37 559.24 530.7 _+ 17.58
Notebook Reference: NB-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/ Dose in
Identif ication at Time of Dose Feed Mixture Consumed m g A g
1A 236 IB 221 1C 242
2A 231 2B 220 2C 237
4A 315 4B 329 4C 327
8A 270 8B 289 8C 288
16A 280 16B 289 16C 276
32A 317 32B 309 32C 301
4.48 4.24 3.61
10.07 10.18
7.92
4.48 4.26 4.49
10.29 10.28 10.05
6.11 6.37 6.34
10.30 10.28 10.29
5.23 5.61 5.58
10.28 10.30 10.28
5.42 5.61 5.35
10.27 10.30 10.29
6.15 5.99 5.84
10.30 10.29 10.30
x + S.D. - 10. 13 + 0.56
Notebook Reference: NB-56531-16b-s and NB-51806-46
C03519
Appendix 4 Determination of Recovery of Total Carbon-14 From Blank Biological Samples Spiked With N-Ethyl FOSE-14c
For each of the four sets of samples combusted, five replicates of 10 ul, 50 ul, and 100 ul of diluted N-ethyl FOSE-^C 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 1 g blank biological material (fecal, liver, spleen, or muscle homogenates). The combustion cones were dried and then pelletized with 5 cm ashless 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 FOSE-^c 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 com bustion. The recovery data for four sets of samples that were analyzed on different days for total carbon-14 (N-ethyl FOSE-14C) are shown in Appendix 4 - Tables 1-4. The mean recoveries for the four sample sets are 64.1%, 92.7%, 93.3%, and 95.0%. The recoveries were uniformly low throughout the combustion sample sets, so the data were corrected using the appropriate recovery factors.
coaseo
Appendix 4 - Table 1
Recovery of Total Carbon-14 Fran Blank Biological Samples Spiked With N-Ethyl F0SE-1*C,
Combustion Set No. 1
Combusted Spiked Biological Samples
x of Liver Feces Feces Feces Liver Liver Liver and Feces
10 yl- 1563^ 1648 1682 1750 1748 1713 50 yl 8328 8329 7768 8219 7605 7475 100 1 16381 14905 15581 15724 15693 14498
1684 7954 15464
10 yl 50 yl 100 yl
Direct Addition Samples 1917 1907 1912 1946 1962 9547 9558 9609 9605 9819 18943 18415 18654 19154 18741
X 1929 9628 18781
10 yl
1684 v 100 - 87. 3% 1929
50 yl
7954 x 100 82.6% 9629
100 yl
15464 x 100 - 82.3 18781
Overall X + S.D. recovery used1 for correction of oxidized samples - 84.1 +. 2.8.
Amount of N-ethyl FOSE-14C spiking solution added. -- 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 FOSE-14c,
Combustion Set No. 2
Combusted Spiked Biological Samples
x of x of Spleen Spleen Muscle Muscle Liver Spleen Muscle
x of Spleen, Muscle, and
Liver
10 M l 50 Ml 100 Ml
165( 1892 9392 7837 18645 18613
1701 9397 16306
1622 1985
9387 8775 _ 19135
1771
1661
8615 9392
18629 16306
1806 8927 18023
10 vl 50 Ml 100 Ml
1933 9877 19743
Direct Addition Samples________________________ x
1938 1713 1925 1919
1886
9829 9883 9861 9931
9876
19735 19546 19523 19338
19577
10 Ml
1808 x 100 - 95.76% 1886
50 Ml
100 Ml
8927 x 100 - 90.36% 18023 x 100 92.06%
9876
19577
Overall x jh S.D. recovery used for correction of oxidized samples 92.7 + 2.8%.
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
C03522
- }
Appendix 4 - Table 3
'Recovery of Total Carbon-14 From Blank Biological Samples Spiked With N-Ethyl FOSE-14C,
Combustion Set No. 3
Combusted Spiked Biological Samples
x of "x of Spleen Spleen Feces Feces Feces Spleen Feces
10 Hi50 Hi
1978^9530
2071 _
2084 10347
2052 1979 10061 10049
2025 9530
2038 10152
100 Hi
18332 18070 18944 19641 19778 18201 19454
x of Spleen and Feces
2032
9841
18828
10 Hi 50 Hi 100 Hi
2050 10624 21668
Direct Addition Samples 2055 2085 2067 2105 10664 10609 10525 10466 20617 21263 20810 21253
X
2076 10578 21122
10 Hi
2032 x 100 * 97.9% 2076
50 Hi
100 Hi
9841 x 100 - 93.0% 18828 x 100 - 89.1%
10578
21122
Overall x _+ S.D. recovery used for correction of oxidized samples 93.3 + 4.4%.
Amount of N-ethyl FOSE-14C spiking solution added. -- Data are expressed as dpm. -- Spiking error; sample was not used.
Notebook Reference: NB-53102-53
cSZ3
Appendix 4 - Table 4
Recovery of Total Carbon-14 From Blank Biological Samples Spiked With N-Ethyl FOSE-^C,
Combustion Set No. 4
Combusted Spiked Biological Samples
jc of Kidney Kidney Kidney Kidney' Feces Feces and Feces
10 u l - 2420^ 2386
2210 2341 2310
2333
50 u l 11892 11876 11844 11584 10782
11596
100 u l 22569 22444 22622 22867 21969
22494
10 u l 50 u l 100 u l
2412 11299 24816
Direct Addition Samples
2394
2384 2413 2425
12222 12232 12214 12127
24405 24289 24873 24753
X 2406 12019 24627
10 u l
2 3 3 3 x 100 ,, 9 6 . 97% 2406
50 u l
100 u l
1 1 5 9 6 ,, 10 0 - 96i.48% .2 2 4 9 4 x 10 0 9 1 . 3 4 %
12019
24627
Overall x + S.D. recovery used for correction of oxidized samples 9 5 . 0 + 3 . 1 % .
g- Amount of N-ethyl FOSE-^C spiking solution added. -- Data are expressed as dpm.
Notebook Reference: NB-56531-10
C0viS24
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 mg/kg)
Collection Period (Days)
Rat Identification
AB
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^-
1 Day Group 279.3
432.1 474.7 + 219.9
451.5 374.4
2 Day Group 352.8 302.6
281.2 361.8 + 85.5 209.5 295.5 + 82.7
251.0 204.2 194.4 114.4
4 Day Group 178.0 238.5 184.7 159.0
320.6 247.8 163.2 146.0
249.9 + 230.2 + 180.8 + 139.8 +
71.3 23.0 16.0 22.9
65.6 90.3 559.5 323.1 150.4 119.6 97.5 75.3
8 Day Group 229.6 579.0 329.0 184.6 142.8 99.4 68.7 44.2
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
1906.0
16 Day Group
1797.8
1782.6 1828.8 _+ 67.3
2085.0 93.0
32 Day Group 1513.8 295.8
1757.6 1785.5 + 286.6 81.7 156.8 + 120.5
-- Data are expressed as pg N-ethyl FOSE-14C equivalents/sample collection period.
Notebook References: NB-51806-50, NB-55673-30-33
C0*i325
Appendix 5 - Table 2
Total Carbon-14 in Urine After an Oral Dose of N-Ethyl FOSE-Hc in Feed to Rats (Mean Dose, 10.13 mgAg)
Collection Period (Days)
Rat Identification
AB
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
1 Day Group
3.15^
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
16 Day Group
70.34
35.46
32 Day Group
60.36
85.44
2.11 2.66 + 0.52
2.65 2.69 + 0.60 2.18 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 ng N-ethyl FOSE-14C eguivalents/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 F0SE-1*C in Feed to Rats (Mean Dose, 10.13 mg/kg)
Rat Identification
Time PostDose (Hours)
Digestive Tract (plus contents)
Feces
1A 1B 1C 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 . 6 7 + 2.35 83.89 + 36.44
-- Data are expressed as yg N-ethyl FOSE-**C 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 (FC-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.
t-0oO)3(S
Appendix 7 - Table 1 Comparative' Data Showing Normal Fecal Excretion for Rats
Collection Period (Days)
x +_ S.D. of 3 rats from the present study
x _+ S.D. of 5 control rats frcm
FC-Exp. 8
x S.D. of 5 control rats from
FC-Exp. 9
0-1
8.0 + 2 . 6 8 ^
10.29 + 3.68
1-2 9.47 + 2.9< 7.79 + 1.58
2-3 10.79 + 1.14^ 10.27 + 2.04
3-4 11.61 + 0.91- 10.33 + 2.22
4-5 8.92 + 2.47- 8.51 +_ 2.63
5-6 9.78 Hh 2.17- 9.11 +_ 1.79
6-7 10.69 + 1.27^.
8.65 + 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
Data are expressed as -- This data is from the -- This data is from the
grams of feces 2 Day Group of 8 Day Group of
excreted percollection period. this study. this study.
Notebook References: NB-56531-16u-16v and 16dd-16hhf and NB-53102-29t-29bb and 42q
CO < 3 5 2 9
i V
Appendix B
Carbon-14 Content in Tissues After an Oral Dose of N-Ethyl FOSE-14C in Feed to Rats Qtean Dose, 10.13 stg/kg)
Rat
Red Blood
Bone Digestive
Subcut. Abdom.
Identification Liver-- Spleen^- Kidney5. LungsS- Celled Plasma^ Marrow^. Tract-- Carcass^- Fati Fat-- Muscle^-
1A IB 1C
Mean
32.55 49.58 24.05
35.39
6.77 7.00 4.80
6.19
8.93 14.65
6.76
10.11
8.13 11.89
6.16
8.73
21.95 27.73 20.35
23.34
7.36 10.61
6.13
8.03
12.40 15.00
8.73
12.04
11.85 31.98
8.54
17.46
2.29 3.23 1.61
2.38
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
Mean
4A 4B 4C
Mean
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
5.69 6.54 9.51
7.25
8.30 9.08 7.81
8.40
18.83 21.44 19.90
20.06
7.76 9.10 7.92
8.26
10.72 10.89 11.41
11.01
8.51 12.37
8.12
9.67
8.21 10.07
8.92
9.07
_b
-- --
1.47 1.98 2.40
1.95
4.07 4.30 3.97
4.11
3.35 4.10 4.30
3.92
1.13 1.65 1.88
1.55
__ b -- --
----,
2.21 3.07 1.87
2.38
2.14 1.80 1.79
1.91
1.63 1.40 1.36
1.46
BA 8B 8C
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 0.23 0.25 -- 0.71 0.48 0.98
w e 0.75 0.36 0.52
16A 16B 16C
Mean
32A 32B 32C
Mean
20.97 28.67 27.09
25.58
0.83 0.87 1.11
0.94
20.25 33.78 20.95
24.99
0.76 1.37 0.64
0.92
1.93 2.05 3.07
2.35
1.86 3.57 1.80
2.41
1.47 1.81 2.36
1.88
1.66 2.45 1.36
1.82
2.24 3.04 3.25
2.84
1.75 2.19 2.92
2.29
0.83 2.65 0.89
1.46
1.95 2.87 1.59
2.14
0.99 1.01 1.52
1.17
-- --
0.69 1.50 0.63
0.94
-- --
--
0.03 0.04 0.16
-- 0.13 0.01 0.16
0.36
0.03
0.21
-- 0.17 0.03 0.18
Ml 0.08 0.00 0.13 -- 0.28 0.00 0.29 -- 0.06 0.00 0.09
-- 0.14 0.00 0.17
g- Data are expressed as pg N-ethyl FOSE-1*C equivalents/g. -- Sample was not taken.
Notebook Referencesi NB-51806-47-49, NB-53102-43-44, 47-52, and 54, NB-56531-11-12
c0^530
Fbrm 6747-1VA
Appendix 9
TECHNICAL REPORT SUMMARY
TO: TECHNICAL COMMUNICATIONS CENTER - 201-2CN portant - U n p o rt it printed on both ados ofpopor, tond two copis to TCC.i
Ost June 9, 1981
DNIelon
Dept. Number
CENTRAL RESEARCH LABORATORIES. Anslytlcsl and Properties Research Laboratory 0502
Project
PrelectNumber
Service to Riker - Isolation of Trace Fluorochemicals
Report Tme
Perfluorooctane Sulfonic Acid - A Rat-Liver
AR No. 7474 - Metabolite of FM-3422 - June 9. 1981 To
A000007 noport Mumhtr
474
S. J. Gibson. J. D. Johnson - 218-2-02 Author!)
employ* Numbirw
S. Ve Pathre Notebook Reference
security p
Open
B Owed
(Company Confidential) (Spacial Authoritation)
KEYWORDS: (Salact tarmi from 3 M Theiaurut. Suggettother applicable tarmi.I
CU R R E N T OBJECTIVE: Request No. C57427
m CHEMICAL fc, REGISTRY "
233150 No. of Pepe IncludineCovermeet
3
New ChamlealiReportad
Yae
O No
"
CRLAP Analytical Report
Project No. 91S0S026 Requestor - S. J. Gibson, J. D. Johnson
Chemical Analysis
RESORT ABSTRACT: (300*260word] Thii abitract information itdiitributadby the Technical Communication Center to alert3M'era to Com pany RAD. ItiiCom pany confidential malarial.
A rat-liver metabolite Is identified as perfluorooctane sulfonic a d d by *'r-NM*.
Information Liaiion Initiai:
003531
3M CONFIDENTIAL
CENTRAL ANALYTICAL LABORATORY
Report No. . . . __ J42_____ Dot................. A V i j - i H i
Subject: Perfluorooctane Sulfonic Acid - A Rat-Liver Metabolite of FM-3422
S # J Gibsoo
R equestorPa.ItfU M PP ..
D ept. N a m e ____ BA tar...........
P r o j. No.91505026
R equ est No. . . Q A 7 ^ 2 Z _ . D a t e d . . 1 0 . . J3SQ----
Report:
Two metabolites isolated from the liver of a rat administered ^C-labeled FM-3422 were submitted for spectroscopic analysis. These two metabolites were labeled as I-CHjCl-MeOH eluted and II-CHj eluted.
flfeCHi
C F j CF2 -C F 2 (C F 2 ) j - C F 2 CF2 N-CH2 -CH2 OH
FM-3422
Experimental
Both samples were reconstituted in CDjOD. The 1*F-NMR spectra on these samples were obtained on the Varlan XL-100 and XL-200 NMR spectrometers.
Metabolite I **F NMR 13875N Metabolite II **F NMR 30190X
Results
The chemical shifts (ppm upfield from CFClj) of the major peaks in the fluorine spectra are given below. The peak frequencies are normalized to (CFi) * 81.0 ppm.
I
81.0
114.3
120.4
121.5
122.4
126.0
II
81.0
112.9
120.2
121.8
122.8
126.2
Discussion
Both spectra were typical of perfluorooctane sulfonyl derivatives. The metabolite I was identical to perfluorooctane sulfonic acid as determined by comparing the reference '*F NMR (11252X) of the latter with that of I.
CFj -- CF2 -- CF2 --
81.0
126.0 122.4
(CF2 )j -- CF2 -- CF2 --
121.5
120.4 114.3
S O sH
(I)
The spectrum of the metabolite II was very similar to that of I except the chemical shift of the fluoromethylene alpha to the sulfonyl group. It is observed at 112.9 ppm inII, 1.4 ppm upfield from that in I. The 112.9 ppm peakt
AR No. 7474 June 9 1981 Page 2
although not unambiguously, can be assigned to the alpha fluorooethylene of the sulfonamide (-S02N H 2) group:
CFj --
CFa -- CFj --
(CF2)$ --
CF2 -- CF2 -- 112.9
S 0 2NH2
(II)
Conclusion
The liver metabolite labeled I-CHClj-MeOH Is Identified as perfluorooctane sulfonic a d d and that labeled II-CHClj la suggested as perfluorooctane sulfanamide.
S. V. Pathre SVP/rs
C03533
