Document gzg3906bqOLzbpXYvxwobE89

( Z A l S _ }JLHl Haskell Laboratory for Toxicology and Industrial Medicine April 5, 2002 J& M - TO: Study in Rats epeated-Dose Oral Toxicity Gavage Range Finder cc: G. A. Ladies FROM: J. C. Maslanka Analytical Chemist ANALYSIS FO R H-24691 IN DOSING SUSPENSIONS MIXING AND STABILITY STUDY Medical Research Project Number: Haskell Sample Number: Analytical Reference: Analytical Report Number: Service Code: Study Number: Notebook References: 24691 HA-2001-037 I report to satisfy protocol requirements for toxicology studies with ut may be used for other purposes. 0 0 8S not contain tSCA Company Sanitized Gavage Ran Repeated-Dose OralToxicity 'inding Study in Rats ANALYSIS FOR H-24691 IN DOSING SUSPENSIONS MIXING AND STABILITY STUDY Medical Research Project Number: Haskell Sample Number: Analytical Report Number: HA-2001-037 SUMMARY In preparation for bi-weekly mixing in this study and a 90-Day Study with H-24691, a mixing and stability study was done. Dosing suspensions at concentrations o f 1.0, 5.0, and 25.0 mg/mL o f H-24691 were prepared, collected and submitted for homogeneity/concentration verification and stability analysis (5-hour room temperature) on Test day 4. Dosing suspensions from the same preparation were collected and submitted after 7 days o f refrigeration for homogeneity (1.0 mg/mL level only), concentration verification and refrigerated stability along with a 5-hour room temperature stability. In addition, the 0 mg/mL (control) samples was collected and submitted for analysis with each set o f samples. The refrigerated samples were not administered to the animals in the study and bi-weekly mixing was not initiated in the study until the data was available from this work. H-24691 as used in this report refers to the active ingredient (a.i.)i Concentrations o f H-24691 in separate dosing suspensions were measured by gas chromatography (GC). The suspension vehicle for the study was 0.5% aqueous methylcellulose. Results from analysis o f the dosing suspensions collected for homogeneity/concentration verification and stability on Test Day 4 indicated that test substance was homogeneously mixed, at the expected concentrations (11.2% ) and stable in the vehicle for 5 hours at room temperature. Results from analysis of the dosing suspensions collected after 7 days of refrigeration for homogeneity and/or concentration verification and stability on Test Day 11 indicated that test substance was re-dispersed properly, at the expected concentrations ( 12.6%) in all levels and was stable in the vehicle for 5 hours at room temperature. H-24516 was not detected in the 0 mg/mL (control) sample. .Company afutizsd. Dobs not contain TSCA CBS HA-2001-037; Page 2 SAMPLE SUBMITTAL Samples containing H-24691 at the concentrations o f 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 o f 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 o f 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 o f 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 100C 1.00 min. 20.0C/min. 260C 0.00 min. 9.00 min. 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 o f 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 o f 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 o f 0.04 mg/mL. The ratio of the internal standard and H-24691 peak heights from replicate GC analysis o f 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 o f each sample to the calibration curve. jQpmpany Sanitized. Does not contain TSCA CBI HA-2001-037; Page 4 ANALYTICAL RESULTS A. Chromatography H-24691 eluted from the GC column as resolved peak with a retention time of approximately 3.5 minutes. The internal standard eluted from the GC column as a resolved peak with a retention time o f 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 Test Day 4 Homogeneity Test Day 11 Stability 7 Day Refrigerated' Nominal mg/mL Measured T,M,Ba mg/mL 0 NDC 1.0 0.944, 0.884, 0.836d 5.0 4.43,4.37, 4.72d 25 22.2, 22.7, 24.2 Mean (T,M,B) % Nominal -- 88.8 90.2 92.0 0 NDC 1.0 0.900, 0.864, 0.859 5.0 4.46,4.44 25 24.2, 23.9 -- 87.4 89.0 96.4 C.V. Stability5 (%) % Nominal ---- 6 85.4' 4 95.0 5 96.0 ---- 3 90.48 0 84.2B 1 84.8s 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 o f all analyses reported. e Mean result o f 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. not contain TSCA CBI HA-2001-037; Page 5 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% o f 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 o f 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% o f nominal) appeared to be less stable than expected but this can be attributed to sampling and analytical variability and not stability o f the test material in the vehicle. Test substance was not found in the 0 mg/mL samples. C. Conclusions Data from the analysis o f 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. HA-2001-037; Page 6 ACKNOWLEGEMENTS Sample analysis by Sheila A. Riley, (Chemistry Associate). SIGNATURES SIGNATURES Report by: Date issued: Janet C. Maslanka Chemist 5~- O fiA-jU --9 .0 0 o d Day/Mo/Yr $ n p an y Sanitized. Does not contain tso a r.m HA-2001-037; Page 7 Table I. Homogeneity and Stability o f H-24691 in Dosing Suspensions Sample mg/mL H-24691 Type Nominal Measured Test Day 4 Homoeeneitv Control 0.00 n d (a ) Top M iddle 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(B ): 0.836+0.03 M e a n (c ); 0.8880.05 C.V. 6% Top M iddle B ottom 5.0 5.0 5.0-0 5.0-1 5.0-2 m ean(B ): 4.43 4.37 4.16 4.72 5.27 4.72+0.56 M ea n (c ): 4.51+0.19 C.V. 4% Top M iddle Bottom 25.0 22.2 25.0 22.7 25.0 24.2 M ean(c ): 23.0+1.0 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 0.883 88.3 1.0-2 A 0.893 89.3 1.0-2B 0.822 82.2 m ean(B\- 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 the original aliquot (-O) and duplicate aliquots from the original diluted sample (-1, -2). (C) The average measured concentration, average percent of nominal (in parentheses), standard deviation, and coefficient o f variation o f top, middle, and bottom (mean result, n=3) except for 25 mg/mL 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). c HA-2001-037; Page 8 Table I. Homogeneity and Stability o f H-24691 in Dosing Suspensions (continued) Test Day mg/mL H-24691 Sample Type Nominal Measured Test Day 11 Homoeeneity/Stability Control 0.00 n d (a ) Percent Nominal -- Top M iddle Bottom Concentration Verification/Stability #1 #2 #1 #2 1.0 0.900 1.0 0.864 1.0 0.859 M ean(B ): 0.874+0.02 C .V .3 % 5.0 4.46 5.0 4,44 M e a it^ ); 4.45+0.01 C.V. 0% 25.0 24.2 25.0 23.9 M e a n ^ ) : 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-Day Refrigerated/5 H our RT 1.0 1.0 0.955 0.852 95.5 85.2 M e ttiti) : 0.904+0.07 (90.4% ) C.V. 8% 7-Day Refrigerated/5 H our RT 5.0 5.0 4.18 83.6 4.23 84.6 M e a n V ): 4.21+0.04 (84.2% ) C.V. 1% 7-Day Refrigerated/5 H our RT 25.0 25.0 20.5 82.0 21.8 87.2 M e a n ^ ) : 21.2+0.92 (84.8% ) C.V. 4% (A) Denotes not detected. (B) The average measured concentration, average percent of nominal (in parentheses), standard deviation, and coefficient of variation of top, middle, and bottom. (C) The average measured concentration, average percent of nominal (in parentheses), standard deviation, and coefficient of variation o f duplicate samples. (D) The average measured concentration, average percent of nominal (in parentheses), standard deviation, and coefficient of variation of duplicate samples. Mean result of duplicate re-analysis of the original sample. Original sample analysis was lower than expected not reported. Peak Height Ratio HA-2001-037; Page 9 Figure 1 Representative Analytical Calibration Curve Figure 1: Calibration curve showing linear fit (line) to replicate peak height ratio measurements (squares) for calibration solutions of H-24691 diluted over a concentration range of 0.0156 to 0.0468 mg/mL. The 0 mg/mL control diluted sample was used as the zero. sannr<l. Boas con'" TSCACB1 Company HA-2001-037; Page 10 Figure 2 Representative High Performance Liquid Chromatography Chromatograms Figure 2a: Representative HPLC chromatogram o f 0 mg/mL (control) sample with internal standard (lH,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 HPLC chromatogram o f 25 mg/mL H-24691 dosing solution diluted to a nominal concentration o f 0.03 mg/mL. The measured concentration o f the representative solution is 24.2 mg/mL Figure 2c: Representative HPLC chromatogram o f 0.0312 mg/mL H -24691 analytical reference solution. Oaes not contato 7 SLA Cto Company Sanitized. HA-2001-037; Page 11 TABLE 1 SUMMARY OF DOSING MIXING AND STABILITY ANALYSES Nominal: Homogeneity Samples Test Day 4 Top ______Dosing Concentration o f H-24691 (mg/mL) 1.0 5.0 25.0 0.944 (94.4) a 4.43 (88.6) 22.2 (88.8) Middle Bottom Average Measured C one.c Average Percent Nominal Standard D eviationc Coefficient of Variation c 0.884 (88.4) 0.836 b (83.6) 0.888 (88.8) 0.05 6% 4.37 (87.4) 4.72 b (94.4) 4.51 (90.2) 0.19 4% 22.7 (90.8) 24.2 (96.8) 23.0 (92.0) 1 .0 5% Stability Samples 5 Hour Room Temperature Test Day 11 (7-Day Refrigerated) Top Middle Bottom Average Measured Cone.c Average Percent Nominal Standard Deviationc Coefficient of Variation c 0.854 d (85.4) 0.900 (90.0)a 0.864 (86.4) 0.859 (85.9) 0.874 (87.4) 0.02 3% 4.75 (95.0) 4.46 e (89.2) 4.44 (88.8) -- 4.45 (89.0) 0.01 03% 24.0 (96.0) 24.2e (96.8) 23.9 (95.6) -- 24.1 (96.4) 0.21 1% 5 Hour Room Temperature 0.904f (90.4) 4.21f (84.2) 21.2f (84.8) " Numbers in parentheses are the respective percent of nominal values. b Mean result of the original aliquot and duplicate aliquots from the original diluted sample. l: Statistics based on the average measured concentration (ppm) o f the top, middle and bottom of each dosing level. 11 Mean result of original aliquot, one duplicate aliquot and re-analysis of the second duplicate aliquot. * Duplicate concentration verification samples reported for uniformity o f mixing. 1 Mean result of duplicate re-analysis of the original sample.