Document QXmxz3LJjRVJJkmBBEONj34zo

450 $#2&& - $ & & & Haskell Laboratory for Toxicology and Industrial Medicine March 13,2003 TO: MR] L Study in Rats Repeated-Dose Oral Toxicity Gavage Range Finder cc: G. A. Ladies FROM: J.C .M aslanka Analytical Chemist ANALYSIS FOR H-24691 IN DOSING SUSPENSIONS MIXING AND STABILITY STUDY REVISION No. 1 Medical Research Project Number Haskell Sample Number: Analytical Reference: Analytical Report Number: Service Code: Study Number: Notebook References: 24691 24691 HA-2001-037 Attached is the analytical report to satisfy protocol requirements for toxicology studies with fH H f lH B f lM H H B b u t may be used for other purposes. .iCcsppail Sanitized. Oses no: comain TSCA CBi REVISION No. 1 HA-2001-037; Page 2 SAMPLE SUBMITTAL Samples containing H-24691 at the concentrations of 0 ,1 .0 ,5 .0 , and, 25.0 mg/mL were collected on Test Day 4. These samples were analyzed to determine homogeneity/concentration verification and 5-room temperature stability. Samples from the same preparation were collected on Test Day 11 after 7 days of refrigeration. These samples were analyzed to determine homogeneity (1.0 mg/mL level only), concentration verification and refrigerated stability along with 5-hour room temperature stability after refrigeration. All dosing suspension samples were collected on the same day the suspensions were prepared or at the established stability time for analysis. They were analyzed when received. The suspension vehicle was 0.5% methylcellulose. METHODS 1- Analytical Methods a. Dosing Suspension Treatment Each dosing sample (3 mL) was diluted to 100 mL with methanol and mixed to dissolve the H-24691 in the suspension. The dosing samples were analyzed without further dilution or were further diluted with the 0 mg/mL sample (initial dilution) to an expected concentration of approximately 0.03mg/mL (a.i.) prior to analysis. Before all final dilutions, the internal standard diluted solution (refer to Calibration and Quantitation Section) was added to each test sample to give a final concentration of approximately 0.04 mg/mL. Each sample (final dilution) had an equivalent amount matrix (methylcellulose in methanol) when analyzed. Samples submitted for analysis were analyzed the day the suspensions were received by the testing group. b. Chromatographic Conditions Instrument: Column: Injector: Detector: Carrier Gas: Split vent: Injection Volume: Oven Program: Initial Temperature: Initial Time: Level 1 Rate: Level 1 Temperature: Level 1 Time: Total run time: Hewlett-Packard Model 6890 GC J & W, DB-1701,30 m, 0.32 mm ID, 1 um film thickness Split, 250C FID; 250C Helium (2.1 mL/min) 22.3 mL/min 2 microliter Gradient I00C 1.00 min. 20.0C/min. 260C 0.00 min. 9.00 min. C - REVISION No. 1 HA-2001-037; Page 3 c. Calibration and Quantitation A separate sample o f H-24691 was obtained to use as the analytical reference standard. A stock solution was prepared in methanol. This stock solution was sonicated to assure that all material was in solution. Before analysis, appropriate aliquots of the stock were diluted with the 0 mg/mL sample (initial dilution) to make calibration standards, which bracketed the target concentration of the diluted dosing samples. A stock solution of internal standard (1H, 1H, 2H, 2H-perfluoro-9-methyldecan-l-ol, 98% pure) was prepared in methanol and added to each calibration standard and test sample to give a final concentration of 0.04 mg/mL. The ratio of the internal standard and H-24691 peak heights from replicate GC analysis of these solutions were used to construct a calibration curve by least squares regression (see Figure 1 for a representative curve). Measured concentrations for the dosing samples were determined by applying the peak height ratios from replicate injections of each sample to the calibration curve. 'iCswpany Sanitized. Doas not containTSCA CB1 REVISION No. 1 HA-2001-037; Page 4 ANALYTICAL RESULTS A. Chromatography H-24691 eluted from the GC column as resolved peak with a retention time o f approximately 3.5 minutes. The internal standard eluted from the GC column as a resolved peak with a retention time of approximately 3.9 minutes. Representative GC chromatograms are shown in Figures 2(a - c). Test substance was not detected in the 0 mg/mL control. B . Mixing and Stability Study Analytical results from dosing suspensions collected on Test Day 4 and Test Day 11 and analyzed for homogeneity and/or concentration verification and stability are shown in Table I and Summary Table 1. The following table summarizes the results for all homogeneity and/or concentration verification and stability analyses. Test Day Sample Type Nominal mg/mL Measured TMJB* mg/mL Mean (T,M,B) %Nominal C.V. Stability1* (%) % Nominal Test Day 4 Homogeneity Test Day 11 Stability 7 Day Refrigeratedf 0 NDC 1.0 0.944,0.884,0.836"1 5.0 4.43,4.37,4.72d 25 22.2,22.7,24.2 0 NDC 1.0 0.900,0.864,0.859 5.0 4.46,4.44 25 24.2,23.9 88.8 90.2 92.0 87.4 89.0 96.4 6 85.4' 4 95.0 5 96.0 3 90.4 0 84.2* I 84.8* a Mean results for the analysis o f the top (T), middle (M) and bottom (B) samples, b Samples held 5 hours at room temperature, c Denotes none detected, d Mean result of all analyses reported. e Mean result of all analyses reported except for one duplicate repeat sample which was low due to aliquot error. f Duplicate concentration verification samples analyzed except for the 1.0 mg/mL level (homogeneity), g Mean result o f duplicate re-sampling from the original diluted sample. Original analysis was lower than expected due to aliquot error and not reported. IfafiftlzeS. cies rio! c sfrt8*81 REVISION No. 1 HA-2001-037; PageS The data for samples collected on Test Day 4 indicates that the test substance was homogeneously mixed in the vehicle at all levels (C.V.'s = 6 ,4 and 5, respectively). The test substance was at the targeted concentration in the samples ( 11.2% of nominal) and was stable in the vehicle when held 5 hours at room temperature. The data for samples collected on Test Day 11 after 7 days of refrigeration indicates that the test substance was homogeneously re-suspended in the vehicle at all levels (C.V.'s = 3 ,0 and 1, respectively). The test substance was at the targeted concentration ( 12.6% of nominal) and stable in the vehicle when held refrigerated for 7 days followed by 5 hours at room temperature. The 5-hour sample for the 25 mg/mL level (84.8% of nominal) appeared to be less stable than expected but this can be attributed to sampling and analytical variability and not stability of the test material in the vehicle. Test substance was not found in the 0 mg/mL samples. C. Conclusions Data from the analysis of the samples during the mixing study indicate that the test substance could be mixed homogeneously, was at the targeted levels and stable under the conditions necessary for mixing the dosing suspensions twice weekly for any study within the range of the concentrations tested. Test substance was not found in the 0 mg/mL samples. REVISION No. 1 HA-2001-037; Page 6 ACKNOWLEGEMENTS Sample analysis by Sheila A. Riley, (Chemistry Associate). SIGNATURES Report by: Date issued: Janet C. Maslanka Chemist ) J - //& * . - oZi53 /3 D ay/M o/Y r Sanitized, floes w ,t contain TSCA CB1 ttontpany REVISION No. 1 HA-2001-037; Page 7 Table I. Homogeneity and Stability of H-24691 in Dosing Suspensions Sample mg/mL H-24691 Type Nominal Measured Test Day 4 Homoeeneitv C o n tro l 0.00 n d (a ) Top M id d l e B ottom 1.0 1.0 1.0-0 1.0-1 1.0-2 0.944 0.884 0.809 0.860 0.840 m ean : 0.836+0.03 M ean : 0.888+0.05 C.V. 6% Top M id d l e B ottom 5.0 5.0 5 .0 -0 5.0-1 5.0-2 4.43 4.37 4.16 4.72 5.27 m ean : 4.72+0.56 M ean : 4.51+0.19 C .V .4% Top M id d l e B o tto m 25.0 22.2 25.0 22.7 25.0 24.2 M e a n 23.01.0 C.V.5% Percent Nominal -- 94.4 88.4 80.9 86.0 84.0 (83.6%) (88.8%) 88.6 87.4 83.2 94.4 105.4 (94.4%) (90.2%) 88.8 90.8 96.8 (92.0%) Stability^) 1.0-0 0.818 81.8 1.0-1 1.0-2A 1.0-2B 0.883 0.893 0.822 88.3 89.3 82.2 m ean : 0.854+0.04 (85.4%) 5.0 4.75 95.0 25.0 24.0 96.0 (A) Denotes not detected. (B) Mean result o f die original aliquot (-0 ) and duplicate aliquots from the original diluted sample (-1, -2). (C) The average measured concentration, average percent o f nominal (in parentheses), standard deviation, and coefficient o f variation of top, middle, and bottom (mean result, n=3) except for 25 mgftnL level bottom (n=l). (D) Stability samples held 5 hours a room temperature. (E) Mean result of original (-O) aliquot, one duplicate (-1) aliquot and duplicate re-analysis of the second aliquot (-2A, -2b). REVISION No. I HA-2001-037; Page 8 Table I. Homogeneity and Stability of H-24691 in Dosing Suspensions (continued) Test Day mg/mL H-24691 Sample Type Nominal Measured Test Day 11 Homoeeneitv/Stabilitv Control 0.00 NDW Percent Nominal -- Top M id d l e Bottom Concentration Verification/Stabilitv #1 #2 #1 #2 1.0 0.900 1.0 0.864 1.0 0.859 M ean& h 0.874+0.02 C.V. 3% 5.0 4.46 5.0 4.44 M eaniO; 4.45+0.01 c .v .o % 25.0 24.2 25.0 23.9 Mean(P>: 24.1+0.21 C.V. 1% 90.0 86.4 85.9 (87.4%) 89.2 88.8 (89.0%) 96.8 95.6 (96.4%) Stability 7-D a y R efr ig er a ted /5 H o u r R T 1.0 0.955 1.0 0.852 Mean&X- 0.904+0.07 C.V. 8% 95.5 85.2 (90.4%) 7 -D a y R efr ig er a ted /5 h o u r R T 5.0 4.18 5.0 4.23 M ean& h 4.21+0.04 C.V. 1% 83.6 84.6 (84.2%) RT7 - D a y R e f r i g e r a t e d / 5 H o u r 25.0 20.5 25.0 21.8 M ean^X-21.2+0.92 C.V.4% 82.0 87.2 (A) Denotes not detected. (B) The average measured concentration, average percent of nominal (in parentheses), standard deviation, and coefficient o f variation o f top, middle, and bottom. (C) The average measured concentration, average percent of nominal (in parentheses), standard deviation, and coefficient o f variation of duplicate samples. (D) The average measured concentration, average percent of nominal (in parentheses), standard deviation, and coefficient o f variation of duplicate samples. Mean result o f duplicate re-analysis o f the original sample. Original sample analysis was lower than expected not reported. REVISION No. I HA-2001-037; Page 9 Figure 1 Representative Analytical Calibration Curve 0.00 0.01 0.02 0.03 0.04 0.05 Concentration, m g/m L Figure 1: Calibration curve showing linear fit (line) to replicate peak height ratio measurements (squares) for calibration solutions o f H-24691 diluted over a concentration range o f 0.0156 to 0.0468 mg/mL. The 0 mg/mL control diluted sample was used as the zero. REVISION No. I HA-2001-037; Page 10 Figure 2 Representative Gas r.hrnmatngraphv C hrom atogram s Figure 2a: Representative GC chromatogram of 0 mg/mL (control) sample with internal standard (1H,lH,2H,2H-perfluoro-9-methyldecan-l-ol) added. Retention time for H-24691 is approximately 3.5 minutes. Retention time for internal standard is approximately 3.9 minutes. Figure 2b: Representative GC chromatogram of25 mg/mL H-24691 dosing solution diluted to a nominal concentration o f 0.03 mg/mL. The measured concentration of foe representative solution is 24.2 mg/mL Figure 2c: Representative GC chromatogram of 0.0312 mg/mL H-24691 analytical reference solution. 'Compatf Sanitized. U rns not contain TSCA CB? REVISION No. 1 HA-2001-037; Page H TABLE 1 SUMMARY OF DOSING MIXING AND STABILITY ANALYSES Nominal: Homogeneity Samples Test Day 4 Top Dosing Concentration of H-24691 (mg/mL) LO 5 J 25.0 0.944 (94.4)* 4.43 (88.6) 22.2 (88.8) Middle 0.884 (88.4) 4.37 (87.4) 22.7 (90.8) Bottom Average Measured Cone.* Average Percent Nominal Standard Deviation ' Coefficient o f Variationc 0.836" (83.6) 0.888 (88.8) + 0.05 6% 4.72" (94.4) 4.51 (90.2) 0 .1 9 4% 24.2 (96.8) 23.0 (92.0) 1 .0 5% Stability Samples 5 Hour Room Temperature Test Day 11 (7-Day Refrigerated) Top 0.854" (85.4) 0.900 (90.0)* 4.75 (95.0) 4.46* (89.2) 24.0 (96.0) 24.2* (96.8) Middle 0.864 (86.4) 4.44 (88.8) 23.9 (95.6) Bottom Average Measured Cone.c Average Percent Nominal Standard Deviation* Coefficient of Variation* 0.859 (85.9) 0.874 (87.4) 0 .0 2 3% -- 4.45 (89.0) 0 .0 1 03% -- 24.1 (96.4) 0.21 1% 5 Hour Room Temperature 0.904r 4.21r 21.2f (90.4) (84.2) (84.8) * Numbers in parentheses are the respective percent of nominal values. b Mean result o f the original aliquot and duplicate aliquots from the original diluted sample. c Statistics based on die average measured concentration (ppm) of the top, middle and bottom of each dosing level. " Mean result of original aliquot, one duplicate aliquot and re-analysis of the second duplicate aliquot * Duplicate concentration verification samples repotted for uniformity of mixing. f Mean result of duplicate re-analysis of the original sample. REVISION No. 1 HA-2001-037; Page 12 Reason For Revision No. 1 The report was revised to reflect a change in Figure 2 (page 10) of the report. The change was to correct the header and all references to "High Performance Liquid Chromatography or HPLC" to "Gas Chromatography Chromatograms or GC". SIGNATURES Report by: Chemist not contain TSCA CBt Company Sanitized. Does