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OCTANOL/WATER PARTITION COEFFICIENT TEST SUBSTANCE Identity:N-ethylperfluorooctasnuelfonamidoethanolm;ay alsobe referredto as N-ETFOSE Alcoholor FM-3422. (I-Octanesulfonamide,N-ethyl1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafluoro-N-(2-hydroxyethyl)-, CAS # 1691-99-2) Remarks: Materialisan off-whitew,axy solid.The testlaboratoryiden@fies the compound as "T-5874" Batch 2334. METHOD Method: OECD GLP (Y/N): Yes Year completed: Analytical: HPLC 117 & EEC-Direcbve 92/69 EEC, Annex 1994 withSpectrophotometricdetector V, PartA.8 Remarks: Testingused sixsubstanceswithknown logP. valuesas references.These substances were used tocalibrattehe elutiontime inunitsof log POW. Reference substance Benzylaicohol Toluene 1,4-dichlorobenzene Fluoranthene(98%) Dibenzyl(99%) 2,4-DDT (99%) logP. 1.1 2.7 3.4 497 4.8 6.2 A stocksolutionofthe testsubstance was prepared by dissolving165 mg in50.0 mL acetonitdieand sonicatingfor3 minutes. The stocksolutionwas diluted10 timeswith niobilephase, resultinignthe testsolution. Mobile phase isa 75125 (vtv)solutionofacetonitrileMilli-Owater respectively. RESULTS Substance Reference chemicals Benzylalcohol Toluene 1,4-dichlorobenzene Fluoranthene Dibenzyl logP. 1.1 2.7 3.4 4.7 4.8 2,4-DDT Test substance Peak 1 Peak 2 (majorcomp.) Severalsmallpeaks Interpolatferdomthe regressiolniney: 6.2 5.6* 4.4* 2.2/4.4* 0.288x- 0.730(r-0.972n,=6) CONCLUSIONS Thisstudyestimatesthe logP. forN-ETFOSE alcohol. Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St.Paul, Minnesota,55133 DATA QUALITY ReliabilityK:Jimischranking2 -perOECD. HPLC methods enables partitiocnoefficientosbe estimatedinthe logPow range between 0 and 6. -Notoxstatesimpuritieasre ofminor importanceinHPLC method, however OECD states,HPLC method islesssensitivteothepresence ofimpuritieisnthe testsubstancethanthe Shake Flaskmethod. Neverthelessi,nsome cases impuritiecsan make the interpretatioofnthe resultsdffficudlute to uncertaintiyn peak assignments. Mixtureswhich resultinan unresolvedband, upper and lowerlimitosflogP,,wshould be stated.Duplicateanalysisshould be reportedin ordertoshow confidenceinthe measurement (logP. valuesshouldfalwlithina range of+/-0.1 logunits-) dataprovidedmean valuethereforecan'tshow confidence.pH ofeluantiscriticoaflionizablceompounds. The studydidnotincludeany fluorochemicalasmong the referencesubstances. Withoutfiuorochemicarleferencecompounds, itisnotknown whether the same relationshibpetween the capacityfactork"and Kow existsforcompounds vath perfluoroalkpyolrtionsas exitsfornonfluorinaterdeferencematerialsuch as those used inthisstudy.The referencecompounds should have been also injectedindependentlytoinsureappropriateretentiotnime assignedtoeach referencecompound. Reference substanceswere injectedtwice,however only the mean retentiotnimedata isgivenforthe referencesubstancesthus repeatabiliotfythe retentiotnimesisnotclear.Itwas notdemonstratedthatthe testmaterialwas solubleintheacetonitrislteocksolutionorinthe mobilephase. No demonstrabon thatthe major components ofthetestsubstanceare detectableatUV A.= 210 nm (Generally,UV absorbance detectorspoor choice forN-ETFOSE alcoholand relatedfluorochemicals)U.V adsorptionspectrais needed ofthe pudfiedmain components ofthe testsubstance.Assumptions are made thatthe majorpeak from the HPLC ofthetestsubstanceisthe major component. No attemptwas made toconfirmthis(e.g.Mass spec analysisto show the materialdetectedhas the appropriatemass).Itisnotknown whether the major components even come offthecolumn. Some k'valuesreportedare calculatedincorrectly. REFERENCES Study conducted atthe requestof3M.Company by NOTOX Safety& EnvironmentalResearch B.V.,The Nethedands, April7,1994. REPORT DETERMINATION OF THE PARTITION COEFFICIENT (N-OCTANOL/WATER) OF T-5874 BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) NOTOX Project 121275 NOTOX substance 38187 Page 1 of 14 - RECEIVED -9 1994 TuAiCOLOGY T-5874 NOTOX Project 121275 STATEMENT OF GLP COMPLIANCE NOTOX B.V., Is-Hertogenbosch, The Netherlands The study described in this report was conducted in compliance with the most recent edition of: The OECD Principles of Good Laboratory Practice which are essentially in conformity with: The United States Food and Drug Administration.Title 21 Code of Federal Regulations Part 58. The United States Environmental Protection Agency (F-IFRA).Title-40 Code of Federal Regulations Part 160. The United States Environmental Protection Agency (TSCA). Title 40 Code of Federal Regulations Part 792. Study Director: Drs. R. de Vries Date: ................... Page 2 T-5874 NOTOX Project 121275 QUALITY ASSURANCE STATEMENT NOTOX B.V., Is-Hertogenbosch, The Netherlands. Study procedures were subject to periodic inspections and general non study specific processes were also inspected at periodic intervals. This report was audited by the NOTOX Quality Assurance Unit and the methods and results accurately reflect the raw data. DATES OF QAU INSPECTIONS/ AUDITS REPORTING DATES 25 March 1994 28 March 1994 21 April 1994 25 March 1994 28 March 1994 21 April 1994 Quality Assurance Manager C.J. Mitchell B.Sc. Date: Oq -OL-qti Page 3 - T-5874 REPORT APPROVAL STUDY DIRECTOR: MANAGEMENT: NOTOX Project 121275 Drs. R. de Vries ctc, .......................... Date: J.A.M.W. van Helvoirt Section head Physica Chemistry qr.IlonaC. Enninga Technical Director .......................... Date: lo6 1icj94 @15 Page 4 T-5874 NOTOX Project 121275 PREFACE Sponsor Study Monitor Testing Facility Study Director Study plan TEST SUBSTANCE Identification Description Batch Purity Storage conditions Stability under storage conditions Expiry date 3M Belgium Chemical EBC Canadastraat 11 B-2070 ZWIJNDRECHT Belgium Mr. R.H. Cox NOTOX B.V. Hambakenwetering 3 5231 DD Is-Hertogenbosch The Netherlands Drs. R. de Vries Start: 30 March 1994 Completed: 07 April 1994 T-5874 Cream solid 2334 100% At room temperature in the dark Stable January 01, 1996 Paqe 5 T-5874 NOTOX Project l2jlL275 PURPOSE AND PRINCIPLE The purpose of the study was to determine the partition coefficient of the test substance between n-octanol and water. The partitioncoefficient (n-octanol/water) (Pow) is defined as the ratio of the equilibrium concentrations in a two phase system consisting of n-octanol and water. The retention time of a substance in a reversed-phase High Performance liquid Chromatography (HPLC) chromatographic system is related to its partitioncoefficient(n-octanol/water). The HPLC method is useful for log Pow values between 0 and 6. Using the HPLC method, impuritiesare of minor importance.The HPLC method must also be applied if the test substance is a mixture of compounds but is not applicable to strong acids and bases, metal complexes, substances which react with the eluent or surface-active agents. GUIDELINES The study procedure described in this report was based on the following guidelines: Organization for Economic Co-operation and Development (OECD), OECD Guideline for Testing of Chemicals, guideline no. 117: OPartition coefficient (n-octanol/water) High Performance Liquid Chromatography (HPLC) method" (adopted March 30, 1989). European Economic Community (EEC), EEC-Directive 92/69 EEC, Annex V, Part A, Methods for the determination of physico-chemical properties, A.B: "Partition coefficientn. EEC Publication no. L383, December 1992. ARCHIVING NOTOX B.V. will archive the following data for at least 10 years: protocol, report, test substance reference sample and raw data. TEST SYSTEM AND RATIONALE A High Performance Liquid Chromatograph with a spectrophotometric detector. The stationary phase was bonded silica C18. The mobile phase was 75/25 (v/v) acetonitrile/water.Both HPLC instrumentationand conditions are described in the section "method of chemical analysis". The test system was recognized by the international guidelines (OECD, EEC). - Page 6 - T-5874 NOTOX Project 121275 REFERENCE SUBSTANCES Six chemicals for which log P?W has been reported were used to calibrate the elution time in units of og Pow. The mixture of reference substances consisted of: Reference substance - benzylalcohol(p.a., Merck) - toluene-(p.a.,Merck) - 1,4-dichlorobenzene(z.s., Merck) - fluoranthene (98%, GC, Aldrich Chemie) - dibenzyl (99%, GC, Aldrich Chemie) - 2,4-ODT (99%, HPLC, Riedel de Hadn, Seelze, FRG) log P., values according to the OECO guideline. log Pow 1.1 2.7 3.4 4.7 4.8 6.2 PERFORMANCE OF THE TEST Preparation of the solutions Solutions of the reference substances (except for ethylmethylketone)were prepared in methanol (HPLC-grade, Labscan Limited Co., Dublin, Ireland) at a concentration of 1.5-2 g/l. A mixture of the reference substances was prepared by adding 125 ul of each solution to a volumetric flask. Subsequently, this flask was made up with mobile phase to a final volume of 25.0 ml. For the determination of to (retention time of the unretarded component),'a solution of formamide (p.a., Merck, Darmstadt, FRG) in methanol was prepared at a concentrationof 1.7 g/l and thereafter diluted 10 times with mobile phase. A stock solution of T-5874 was prepared by dissolving 165 mg test substance in 50.0 ml acetonitrile (3 minutes sonication).The stock solution was diluted 10 times with mobile phase, resulting in the test solution. Performance of the test The solutions were injected in the following sequence: the mixture of reference substances, mobile phase, the formamide solution, the test solution (in duplicate), mobile phase, the mixture of reference substances and the formamide solution. Temperature of measurement The temperature of the mobile phase was recorded several times during the measurements. Page 7 T-5874 NOTOX Project 121275 METHOD OF CHEMICAL ANALYSIS The conditions used for the High Performance Liquid Chromatographic method are described below: Analysis Column Mobile phase Flow Detection Injection volume LiChrospher 100 RP-18; 125 x 4 (I.D.) mm; d 5 um (Merck, Darmstadt,,FRG) 797/,*(2v5/v) acetonitrile (HPLC-grade, Labscan Limited Co., Dublin, Ireland)/Milli-Qwater (MilliporeCorp., Bedford, MA, USA) 1 ml/min UV at X= 210 nm 10 Ill instrumentation HPLC Pump Autosampler Detector Integrator Series 410 LC (Perkin Elmer, Norwalk, CT, USA) ISS-200 (Perkin Elmer) SpectroMonitor 3100 (LDC Analytical, Riviera Beach, FL, USA) SP 4290 (Spectra Physics, San Jose, CA, USA) DATA HANDLING Using High Performance Liquid Chromatography, especially large log Pow values can be accurately determined from the chromatographic retention data. To this end, the capacity factor (k' ) was used, since it is proportional'to the partition coefficient. The capacity factor was calculated from the retention of the substance concerned (tr) and the unretarded component (to): k' = (tr- to)/to From the results of the reference substances,a plot of log Pow (x-value) versus log k' (y-value) was constructed,using linear regressionanalysis. The log k value of each component of the test substance was compared with the log k' values of the reference substances with known log Pow values. Page 8 T-5874 NOTOX Project 121275 RESULTS Representative HPLC chromatbgrams of the test solution, the blank (mobile phase), the mixture of reference substances and the formamide solution are shown in Figures 1 to 4. In the HPLC chromatograms of T-5874, using detection at 210 nm, one large peak at 4.1 minutes and a small peak at 7.7 minutes were observed. Between 1.6 and 4 minutes, several small peaks were observed. It was assumed that the large peak derived from the major component of test substance whereas the small(er) peaks derived from impurities. The to was determined to be 0.90 minutes as a mean value of both measurements (i.e 0.89 and 0.90 minutes). I The mean values of the retention times, k values, log k' values,. log P w and Pow values are summarized in Table 1. The plot of log Pow (x-Va?ue) versus log k' (y-value) from the reference.substances is shown in Figure 5. Table 1 Results of the test. Substance trIL k log k' log Pow benzylalcohol 1.14 0.274 -0.563 1.1 toluene 2.24 1.503 0.177 2.7 1,4-dichlorobenzene 2.89 2.229 0.348 3.4 fluoranthene 5.67 5.330 0.727 4.7 dibenzyl 4.07 3.547 0.550 4.8 2,4-DDT 9.12 9.190 0.963 6.2 test substance peak 1 peak 2 (major comp.) several small peaks 7.70 4.05 1.6 / 4 7.598 0.881 5.6 2 3.525 0.547 4.4 2 0.79/ 3.47 -0.10/0.54 2.2/4.4 2 2 Note Mean value of the retention times of both chromatograms. Interpolated from the regression line: y . 0.288x - 0.730 : The calculations were performed using not rounded values. (r.0.972, n-6) POW 39.8 x,04 2.5,x,04 1.50x,02/ 2.51x,04 The temperature of the mobile phase was 20.2-20.60C during the measurements. Page 9 - T-5874 I:HANNEL A NOTOX Project 121275 INJECT 07-R4-94 AR:44:59 !:,@T13RETDO BIN 53 tCq-? 71 -<7 =A 05 F7 7.6E9-'4 R8 DATA SAVED TO BIN # 53 38196/121286 FILE 1. METHOD ANALYST: KRH PERK# FiREFi% 1 2 3 4 5 6 7 8 10 -ii 13 14 i5 I IF, 17 ;O@#H'L 0.495 2.459 1.083 1 c'.7'32 1.288 2. 5 0. 4,T@=. .4 7 1 91 1 0. 6 1.:364 067 1.718 11.232 10. 4--@5 @-8.458 2. OIA It :i 07-04-94 08:44:55 0. RUN 10 INDEX 10 RT ARER BC 0.15 C4.25 0. -24'@ 0.53 0.79 -1@ 0-t 7@ q -i.e. @.71 -i.9i -,7 2 7@".19 7,.51 3. E'fl 4.05 .7.69 1399 02 6951 A? 0 6 1 02 53119 08 194A A5 7179 Rn 1 -@";:1:7@A--@ 7000 02 5588 02 1797 03 5268 02 5842 03 4857 02 3750 02 29495 02 108707 03 5694 01 'c:'S E.5 -.H= nR" PS= I. BIN 53 Figure I HPLC chromatogram of the test solution of 330 mg/l T-5874 in mobile phase. Column Mobile phase Flow Detection Injection volume LiChrospher 100 RP-18; 125x4 (I.D.) mm; dp=5 Um 75/25 (v/v) acetonitrile/Milli-O water 1 ml/min UV, at X = 210 nm 10 ul Note: The peaks between 0 and 1.6 minutes were al3o observed in the chromatograms of the blank (see Figure 2). Therefore it was assumed that these peaks did not derive from the test substance. - Page 10 - T-5874 NOTOX Project 121275 I:HRNNEL A INJECT 07-04-9r4 A9:14:04 R-@TOREDTO SIH 56 Ql. L@7 .52 7 DATA SRYED TO BIN ER 0 56 38196/121286 FILE i. METHOD RNRLYST: KRH -AK# ARERY. 1 1.543 2 10.515 85 4 55.335 5 7.768 6 6.901 .7 2.509 8 11.576 TOTAL ioo. 07-04-94 09:34:04 0. RUN 13 INDEX 13 RT t-41.5 0.25 Cl. 4 C4.52 @4.i;7 0.99 1.18 l@.Fl AREA BC 077 F12 5976 AP e11190 pi2 -7144q O@O 4415 03 3922 02 1426 03 9579 01 56834 CH= nall PS= i. BIN 56 Figure 2 HPLC chromatogram of a blank (mobile phase). Column Mobile phase Flow Detection Injection volume LiChrospher 100 RP-18; 125x4 (I.D.) mm; dp=5 pm 75/25 (v/v) acetonitrile/Milli-wQater 1 ml/min UV, at X = 210 nm 10-iil - Page 11 T-5874 CHANNEL P NOTOX Project 121.275 INJECT 07-04-q4 r4;:.ciPI:1'7;'TCIRET1O) -BIN 57 -.98 4. Li ER 0 DATA SAYED TO BIN 57 38196/121286 FILE 1. METHOD ANRLYST: KRH PEAK# ARERY. 1 0.023 2 0.:145 3 0. 0 F-; 4 r-4.,0-,79 5 0.073 C4.I-.@9 7 8. '3 7 0.112 11. -S:17 i Cl.0 1 --< ii :IC4.5E.7 12 15.524 13 38.584 14 0. --1<6 15 17-1.2 7 TOTRL ciCl. 07-04-94 -A-9:5R-:i-? 0. RLIN 14 INDEX 14 RT RRFR BC 0.14 0.25 0.34 0.53 0.79 ci@96 1.14 24 ZI. C@9 4.rlR. 5. FS 0-;9.8 9.13 1131 02 7039 02 ;-@917 e2 42751 08 3542 05 6771 06 4-<9774 03 5460 Ot 550940 Al All @-2-4 514173 O-S 755334 01 18773--<9 01 15361 01 645770 01 -A*865613 rH= uR." PS= 1. BIN 57 Figure 3 HPLC chromatogram of the mixture of reference substances in mobile phase. Column Mobile phase Flow Detection Injection volume LiChrospher 100 RP-18; 125x4 (I.D.) mm; dp=5 um 75/25 (v/v) acetonitrile/Milli-Qwater 1 ml/min UV, at X = 210 nm 10 ul - Page 12 - T-5874 NOTOX Project 121275 ,--HANNEL A lt4JECT 07-04-q4 R7:37:44 ;70RED TO BIN 49 1. 4':1.87 2.97 3:.I6-5s DATA SAVED TO BIN 49 38196/12128@ FILE i. METHOD ANRLYST: KRH PEAK# Fi.REFi% 0. --<5 1. 1 P-.9 0.79 4 10.44 5 1.884 6 54.548 7 F--2.4 6 8 0.12 9 0'-4; 10 0.424 915 TOTAL C40. C47-04-94 07:17:44 0. RUN 6 INDEX 6 RT 0. J.6 L4. -..55 0.78 A-.89 -t--ti 1 87 97 iF5, RREP BC 2624 02 8926 02 5930 02 78363 08 14143 06 40q4<7 08 -t79 05 Pqq 05 23224 02 3125 03 1.5F:qR9 01 7cir45'39 .......... ............ rH= "A". PS= 1. BIN 49 Figure 4 HPLC chromatogram of a formamide solution of 0.2 g/l in mobile phase. Column Mobile phase Flow Detection Injection volume LiChrospher 100 RP-18; 125x4 (I.D.) mm; dp=5 um 75125 (v/v) acetonitrile/Milli-Qwater I ml/min UV, at X 210 nm 10 ul Page 13 T-5874 NOTOX Project 121275 13/04/94 TRJ Pow 1.000 ----------------------------------------------------------------------:-o-------------- 0.800 ---------------------------------------------------------------------- 0 0 0.600 o: 9 ----------.-........-.-.--------r---------4---------- ---------r----------------- k 0.400 ------------------:-----------------o--------------:-------------------------- 0.200 --------------------------------------------------------I------------------- o.ooo 0.0 1.0 2.0 3.0 4.0 5.0 6.0 log Pow Figure 5 Plot of log Pow (x-value)versus log k' (y-value)from the reference substances. Page 14