Document jBR46LKkoMMp4zRG4ooVp3GDy

AR226-3112 S ABIO TIC DEG RADATIO N: H YDRO LYSIS A S A FUN C TIO N O F pH (PR ELIM INA RY TEST) Sponsor DuPont Specialty Chemicals Jackson Laboratory Chambers Works Deepwater NJ 08023 USA Research Laboratory Huntingdon Life Sciences Ltd Eye Suffolk IP23 7PX ENGLAND Draft: 23 December 1998 Final: 2 February 1999 aswpssif SanHteed. Does not e*** v sr a ' CONTENTS Page COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS............................... 3 QUALITY ASSURANCE STATEMENT....................................................................................... 4 RESPONSIBLE PERSONNEL.......................................................-................................................ 5 SUMMARY....................................................................................................................................... 6 INTRODUCTION......................................................................... :.................................................. 7 TEST SUBSTANCE.......................................................................................................................... 8 METHODS.............................. i......................................................................................................... 9 RESULTS........................................................................................................................................... H DISCUSSION..................................................................................................................................... 12 CONCLUSION................................................................................................................................... 13 SffisparayismftteecL Dees ssefierawfisSwTSfa Huntingdon Life Sciences COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS Hydrolysis as a Function of pH The study described in this report was conducted in compliance with the following Good Laboratory Practice standards and I consider the data generated to be valid. The UK Good Laboratory Practice Regulations 1997 (Statutory Instrument No 654). EC Council Directive 87/18/EEC of 18 December 1986 (Official Journal No L 15/29). OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17. A. L. Comb, B.Sc., Ph.D. Study Director Huntingdon Life Sciences Ltd. Date Sfflpewy frartteed. Dees bmM)en" TSCAW Huntingdon Life Sciences QUALITY ASSURANCE STATEMENT Hydrolysis as a Function of pH The following have been inspected or audited in relation to this study Study Phases Inspected Protocol Date of Inspection 8 August 1998 Date of Reporting 19 August 1998 Process Based Inspections Hydrolysis 2 December 1998 3 December 1998 Report 15 January 1999 15 January 1999 Protocol: An audit of the protocol for this study was conducted and reported to the Study Director and Company Management as indicated above. Process based inspections: At or about the time this study was in progress inspections and audits of routine and repetitive procedures employed on this type of study were earned out. These were conducted and reported to appropriate Company Management as indicated above. R eport Audit: This report has been audited by the Quality Assurance Department. This audit was conducted and reported to the Study Director and Company Management as indicated above. The methods, procedures and observations were found to be accurately described and the reported results to reflect the raw data. -H. Comb, B.Sc. Principal Auditor, Department o f Quality Assurance, Huntingdon Life Sciences Ltd. Date SmHntL Dees fj0eon?n TOP #*5 RESPONSIBLE PERSONNEL Huntingdon Life Sciences Hydrolysis as a Function of pH The following staff member has reviewed this report. T. C. Cowlyn, EurChem., C.Chem., M.R.S.C. (Scientific Manager, Product Characterisation, Eye) The following staff member was responsible for the conduct of the work and reporting o f the results. P. woods, H.N.C. (Scientist, Product Characterisation) @S6npe9gy9afttiaaB. 00 swseoaiftsfwwsea SUMMARY The hydrolysis o1 a function of pH was studied according to EEC guidelines. i The preliminary study showed that after 5 days at pH 4 ,7 and 9 and 50C less than 10% hydrolysis I had occurred, equivalent to half-life tunes (t*) of greater than one year under conditions more representative of the environment (25C). as found to be hydrolytically stable under acidic, neutral and basic conditions. 1I / j {I I / INTRODUCTION The purpose of this series of tests was to investigate the hydrolytic behaviour ol function of pH. a The test was conducted in accordance with the OECD Guidelines for Testing of Chemicals (1981) and the requirements of the Annex to European Commission Directive 92/69/EEC. The protocol was approved by Huntingdon Life Sciences Management on 7 July 1998, by the Sponsor on 17 July 1998 and by the Study Director on 13 August 1998. The experimental phase of the study was undertaken between 9 November 1998 and 27 November 1998. Location of study ' Huntingdon Life Sciences Ltd Eye' Suffolk IP23 7PX England Primary data from the tests performed at Huntingdon Life Sciences and a copy of the final report are stored in the archives of Huntingdon Life Sciences. . LSsm ingeeifM to TMfe a m Identity: Chemical name: Intended use: Appearance: Storage conditions: Lot number: Expiry date: Purity: Date received: TEST SUBSTANCE Pale yellow slurry Room temperature 3 2 years from date of receipt t-J 23 June 1998 t Is a suspension at room temperature and separates into its component phases. Hence on sampling tire test substance for use in this study, the material was warmed to 35 - 40C in order to yield a homogenous solution. METHODS PRELIMINARY INVESTIGATION Preparation o f buffer solutions Buffer solutions were prepared using the following volumes: pH 4.0 : Disodium hydrogen orthophosphate dodecahydrate (27.6 g) and citric acid monohydrate (12.9 g) were dissolved in purified water (1900 ml). The volume was adjusted to 2000 ml with purified water. pH 7.0 : Potassium dihydrogen orthophosphate (6.81 g) was dissolved in purified water (1900 ml), 1M sodium hydroxide (30 ml) was added and the pH was adjusted to 7.0 with 1M hydrochloric acid. The volume was adjusted to 2000 ml with purified water. pH 9.0 : Disodium tetraborate decahydrate (33.1 g) and potassium dihydrogen orthophosphate (3.60 g) were dissolved in purified water (1900 ml) and the pH was adjusted to 9.0 with 1M hydrochloric acid. The volume was adjusted to 2000 ml with purified water. The pH of the buffer solutions were measured using a pH meter. PROCEDURE Aliquots (100 ml) o f eacji buffer solution (previously equilibrated at 50C) were measured into reagent bottles containing^^jH H ^H ^^approxim ately 180 mg). Due to the acidic nature of the test substance, the pHs o f the resulting solutions were adjusted with 1M sodium hydroxide to within 0.05 of the intended pH of the respective buffer. The bottles were purged with nitrogen, sealed and placed in a thermostatically controlled water bath at 50C in the dark. They were held at 50C until sampling was required (after short incubation period of approximately 10 minutes, and then after 2.4, 24 and 120 hours (5 days)). The water bath was monitored during the period of the test to ensure that the test temperature was maintained. The test was performed in duplicate at each pH value. At each sampling time portions (10 ml) of the test solutions were removed and cooled. Aliquots (1 ml) of the samples were pipetted into separate 10 ml volumetric flasks, to which purified water (6 ml) and 0.2M aqueous sodium hydroxide (0.5 ml) were added before diluting to volume with acetonitrile. The final solutions were then analysed by ion chromatography. The pH values of the solutions were measured over the period of the test. IO N CHROMATOGRAPHY CONDITIONS Instrument: Dionex GP40 Pump Dionex PED-2 Pulsed Electrochemical Detector Perkin-Elmer ISS 200 Autosampler Dionex ASRS-II Membrane Suppressor Column: PLRP-S (25 cm x 4.6 mm internal diameter) Column temperature: Mobile phase composition: Ambient Aqueous solution containing 2mM ammonium hydroxide and ImM sodium carbonate/ acetonitrile (75:25 v/v) Regenerant solution 50mN aqueous sulphuric acid Flow rate: 1 ml/min Injection volume: 100 pi Detector: Conductivity mode Retention time: Approximately 6 minutes The peak observed at 6 minutes corresponds to the perfluorobexylethylsulphcnate ion which, from information supplied by fee Sponsor, represents 92 % of the active ingredient. Consequently the levels o f l l ^ H H H A n the water solubility test samples will be quantified relative to this component only. ^ * PREPARATION OF CALIBRATION A stock calibration solution of concentration 132.5 mg/l of active ingredient was prepared by weighing test substance (106.0 mg) into a 100 ml volumetric flask and dissolving in and diluting to volume with purified water. A sub-sample (50 ml) of the stock solution was transferred to a 100 ml volumetric flaskJ to which 0.2M aqueous sodium hydroxide (5 ml) and purified water (20 ml) were added prior to diluting to volume with acetonitrile. Calibration solutions in the range 13.25 to 132.5 me/1 were prepared by dilutions of the final solution with mobile phase. CALCULATIONS The concentrations o J j H B B H S D die analysed solutions (CA) were calculated from standards introduced before and zmer samples (bracketing standards) by the following equation: sample peak area x standard concentration (mg/l) mean peak area of bracketing standards The concentrations o M i ,, the test solutions (CJ were calculated from the following equation: Ct(mg/1) = CA(mg/l) x dilution factor Ssnffisa. See* nelcontain C0f RESULTS L in ea rity The detector calibration was found to be linear over the range 0 to 132.5 mg/1 of standard -solutions in mobile phase with a regression coefficient of 0.9988 (Table 1, Figure 1). Prelim inary investigation Results from tte^relim inai^ii^estigation showed that there was no significant change in the concentration o i { H | | | | 0 B j v h e n incubated in pH 4, 7 and 9 buffer solutions at 50C (see Table 2). Less than 10% hydrolysis had occurred, equivalent to half-life times (t**) of greater than one year under conditions more representative of the environment (25C). pH o f reaction solutions The changes in pH of the buffer solutions over the period of the test are presented in Table 3. The results show that there were no significant changes in pH of the buffer solutions with time. I. ^ eswSatnTSCA CBS ) I DISCUSSION Results from the preliminary study showed that the hydrolysis rates at pH 4, 7 and 9 and 50C were such that less than 10% hydrolysis was observed in each case after 5 days. This is equivalent to an environmental half-life of greater than 1 year under each condition investigated. i CONCLUSION The preliminary investigation indicated that neutral and basic conditions. is hydrolytically stable under acidic, RW I TABLEI Standard calibration foi >y ion chrom atography Standard concentration (mg/1) 13.25 26.51 53.01 79.25 106.0 132.5 Linear regression (including x = 0, y = 0) x = concentration y = peak area y - 561x + 1460 r = 0.9988 Peak area 8238 16812 32982 47741 60304 74469 . m TSeA FIGURE 1 Standard calibration foi y ion chrom atography p a 3 S A MIBU03 50 88 00 "P 8 SiP * SS TABLE 2 Prelim inary investigation results for hydrolysis ol pH 4 Urne (hours) 0 2.4 24 120 pH 7 Time (hours) 0 2.4 24 120 pH 9 Time (hours) 0 2.4 24 14M Concentration, Ct (mg/1) Sample A Sample B 488.7 500.3 487.9 492.1 508.5 519.3 501.6 500.2 Sample A 483.3 492.5 503.8 492.8 Concentration, Ct (mg/1) Sample B 503.6 507.8 513.3 508.3 Concentration, Ct (mg/1) Sample A Sample B 529.9 537.4 529.3 532.2 538.1 544.8 542.2 533.4 <SoMM s a lito 0 *' TABLE 3 pH during hydrolysis tests Nominal pH 4 7 9 Test Preliminary Preliminary Preliminary Temperature (C) 50 50 50 Initial P i _________________ Final 3.96 4.02 7.03 7.02 9.05 8.98 <D