Document y022Z5rgB0d8d6VLav8MwxVX

Haskell Laboratory for Toxicology and Industrial Medicine January 4,2002 AR226-3215 TO: ilot Developmental Toxicity Study in Rats cc: L. A. Malley FROM: J. C. Maslanka Analytical Chemist ANALYSIS FORH-24516 IN DOSING SUSPENSIONS Medical Research Project Number: Haskell Sample Number: Analytical Reference: Analytical Report Number: Service Code: Study Number: Notebook References: 24516 ^ 24516 HA-2001-045 CVS Attached is the analytical report to-for the study identified above. Company Sawnzed, iksesnwfrwftKit Cm ilot Developmental Toxicity Study in Rats ANALYSIS FOR N DOSING SUSPENSIONS Medical Research Project Number: Haskell Sample Number: Analytical Report Number: me 24516 HA-2001-045 SUMMARY Dosing suspensions at concentrations o f 12.5, 25.0, 50.0 and 100.0 mg/mL o f H-24516 were collected for homogeneity/concentration verification and 5 hour room temperature stability analysis on January 16, 2001. Subsequently on January 19, 2001, the refrigerated stability samples along with a 5-hour room temperature sample for the 100 mg/mL level from the same preparation were submitted. Only the 100.0 mg/mL level was sampled for the refrigerated stabilityjycaqge theother levels in this study were within the range o f a previous study in which the mixing and refrigerated stability had been established? In addition, 0 mg/mL (control) samples were submitted for analysis with each set of samples. Dosing suspensions at concentrations of 12.5, 25.0, and 50.0 mg/mL o f H-24516 were submitted for concentration verification analysis on January 27,2001. On the same day, dosing suspensions at concentrations of 100.0 mg/mL o f H-24516 were submitted for homogeneity/concentration verification analysis to confirm proper mixing at this level. In addition, the 0 mg/mL (control) sample was submitted for analysis. H-24516 as used in this report refers to the active ingredient (a.i.) i Process. ew Concentrations o f H-24516 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 on January 16, 2001 indicated that test substance was at the expected concentrations ( 7.5%) for all samples, homogeneously mixed in the vehicle except for the 100 mg/mL level (C.V. = 12%), and stable in the vehicle for 5 hours at room temperature. The 100 mg/mL was not sufficiently mixed before sampling for the analytical work. This was substantiated by the results for the refrigerated and room temperature stability analysis from this preparation. Results for samples collected January 19, 2001 for the 100 mg/mL indicated that the test substance was at the targeted level (86.7% o f nominal) and stable in the vehicle when held refrigerated for 4 days and followed by 5 hours at room temperature. Samples collected January 27, 2001 for the 100 mg/mL indicated that the level was Company Sanitized. Does net contain TSCA CBI homogeneously mixed (C.V. = 4%), and at the expected concentration ( 7.3%). Results for the other concentrations indicated that they were uniformly mixed (C.V.'s of 1% and 2%, respectively) and at the targeted levels ( 19%). H-24516 was not detected in any o f the 0 mg/mL (control) sample in the study. Com pany Sanitized. D o es not contain TSCA CBI Pilot Developmental Toxicity Study in Rats H A-2001-045; Page 3_______________________________ SAMPLE SUBMITTAL Samples containing H-24516 at the concentrations o f 0,12.5, 25.0, 50.0 and 100.0 mg/mL were collected on January 16,2001. These samples were analyzed to determine homogeneity/concentration verification and 5-hour room temperature stability. Samples from the same preparation at the concentrations o f 0,100.0 mg/mL were collected on January 19, 2001. These samples were analyzed to determine 4-day refrigerated stability along with 5-hour room temperature stability. The 4-day refrigerated stability for the remgmder o f the levels in this study had been established in a previous study ( M R H B g SC V pPvw ith H-24516. Samples containing H-24516 at the concentrations o f 0,12.5, 25.0, 50.0 and 100.0 mg/mL were collected on January 27,2001. The 100.0 mg/mL samples were analyzed to determine homogeneity/concentration verification while the remaining levels were analyzed for concentration verification. All dosing suspension samples were collected on the same day the suspensions were prepared or at the prescribed protocol time for analysis. They were analyzed when received or were frozen until analyzed. The suspension vehicle was 0.5% aqueous methylcellulose. Company Sanitized. Does not contain TSCA CBf Pilot Developmental Toxicity Study in Rats )45; Page 4______________________________ METHODS 1. Analytical Methods a. Dosing Suspension Treatment Each dosing sample was diluted to 100 mL with methanol and sonicated to dissolve the H -24516 in the suspension. The dosing samples were further diluted with the 0 mg/mL sample (initial dilution) to an expected concentration o f approximately 0.09 mg/mL (a.i.) prior to analysis. An internal standard (refer to Calibration and Quantitation Section) at an equivalent amount was added to each sample before the final analysis dilution. Samples submitted for analysis were analyzed the day the suspensions were received or stored frozen until analyzed 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 urn 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. Company SanKteetf. Does not contain TSCA CBS Developmental Toxicity Study in Rats H A -200R )45; Page 5_______________________________ c. Calibration and Quantitation A separate sample o f H-24516 (-3) was obtained to use as the analytical reference standard (94.7% pure). 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 methanol to make calibration standards that bracketed the target concentration o f the diluted dosing samples. A stock solution o f an internal standard (1H, 1H, 2H, 2H-perfluoro-9methyldecan-1-01,98% pure) was prepared in methanol and added to each calibration standard and test sample to give an equivalent final concentration in all dilutions. The ratio o f the peak heights for internal standard and H-24516 at three retention times (3.5,4.17, and 4.77 minutes) from replicate GC analysis o f these solutions were used to construct a calibration curves by least squares regression (see Figure la, lb, and lc for a representative curves). Measured concentrations for the dosing samples were determined by applying the peak height ratios from replicate injections o f each sample to the respective calibration curve. Test substance homogeneity in the dosing suspensions was evaluated by calculating the coefficient of variation (C.V. = standard deviation/mean x 100) o f the measured concentrations in the top, middle, and bottom (T, M, and B) samples or the mean o f duplicate samples for each concentration. A coefficient o f variation of less than 10% is the standard criterion at Haskell Laboratory for acceptable distribution of the test substance throughout the dosing suspension. The mean result o f the top, middle, and bottom homogeneity samples or o f the duplicate concentration verification samples for each dosing level was used to determine the concentration o f the test substance for the respective dosing levels. Stability was evaluated by using the mean of the top, middle, and bottom homogeneity samples or o f the duplicate concentration verification samples (0-day room temperature) as the baseline for comparing the corresponding room temperature and refrigerated results. Company SanHtzad net contain TSCA CBI ilot Developmental Toxicity Study in Rats H A-2001-045; Page 6______________________________ ANALYTICAL RESULTS A. Chromatography H -24516 eluted from the GC column as resolved peaks with retention times from 2.9 minutes to approximately 6.0 minutes. For the purpose o f quantitation, resolved peaks at the retenjjpn times o f approximately 3.5,4.17, and 4.77 minutes were used. The internal s t a n d a r d B H M H H H H H B H H H H W l ^ d as a resolved peak at approximately 3.99 minutes. Representative GC chromaiSgrams are shown in Figures 2(a - c). Test substance was not detected in the 0 mg/mL control. B. Homogeneity/Concentration Verification and Stability Samples Analytical results from dosing suspensions collected on January 16, 2001 and analyzed for homogeneity/concentration verification and stability are shown in Table I and Summary Table 1. The following table summarizes the results for homogeneity/concentration verification and stability analyses. Preparation Date 16-Jan-2001 Nominal mg/mL 0 12.5 25 50 100 Measured T,M,Ba mg/mL NDC 12.8, 12.7,12.6 23.3, 24.6,25.0 53.8, 50.5,52.7 93.5, 74.8,78.9 Mean (T,M,B) % Nominal -- 101.8 97.2 104.6 82.4 . C.V. (%) -- 1 4 3 12 Stability6 % Nominal -- 98.7 94.7 100.2 83.5 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. The data for samples collected on January 16, 2001 indicates that the test substance was homogeneously mixed in the vehicle except for the 100.0 mg/mL level (C.V. = 12). The test substance was at the targeted concentration in the samples ( 17.6% o f nominal) and was stable in the vehicle when held 5 hours at room temperature. Test substance was not found in the 0 mg/mL samples. Company ssnftfee* a s * "ofcontafnrsCACBF Pilot Developmental Toxicity Study in Rats 'HA-2001"045; Page 7 _____________________ C. Stability Study Analytical results from dosing suspensions collected on January 19,2001 and analyzed for concentration verification and stability (4 days refrigerated followed by 5 hours room temperature) are shown in Table II and Summary Table 1. The following table summarizes the results for homogeneity/concentration verification (January 16,2001) and stability analyses (January 19, 2001). Preparation Date Sample Type Nominal mg/mL Measured mg/mL Mean % Nominal C.V. (%) 16-Jan-2001 Homogeneity2 0 NDb 100 93.5, 74.8, 78.9 19-Jan-2001 Stability 4-Day Refrigerated 100 86.7d 82.4 86.7 _ 12 a Mean results for the analysis o f the top (T), middle (M) and bottom (B) samples, b Denotes none detected, c Samples held 5 hours at room temperature. d Mean result o f single sample for concentration verification/4-day refrigerated, e Mean result o f single sample for 4 day-refrigerated/5 hour room temperature stability. Stability % Nominal _ 83.5C 94.3e The data for samples collected on January 16,2001 indicates that the test substance was not homogeneously mixed in the vehicle (C.V. = 12%) but was at the targeted concentration in the samples ( 13% o f nominal). Comparison o f the data for the stability samples to the mean concentration indicates that the test substance is stable in the vehicle when held 4 days refrigerated and followed by 5 hours at room temperature. Test substance was not found in the 0 mg/mL samples. Company Sanitized Does not contain TSCA CBI Pilot Developmental Toxicity Study in Rats 'HA-200TTQ45; Page 8 The following table is included for a reference in this stud^to er^ted stability for the concentration range. It summarizes the results for homogeneity/concentration verification (January 8, 2001 Sid January 15, 001) and the 4- day refrigerated stability analyses followed by 5 hours room temperature. Preparation Date Sample Type 8-Jan-2001 Homogeneity3 Stability 4-Day Refrigerated" Nominal mg/mL 0 5.0 20 50 5.0 Measured mg/mL NDb 4.53,4.25, 4.39 19.9, 17.7, 18.6 53.4, 52.6, 54.0 4.81 Mean % Nominal -- 87.8 93.5 106.8 96.1 C.V. (%) Stability % Nominal ---- 3 92.0 6 86.0C 1 96.6e -- 97.1 4-Day Refrigerated" 20 19.6 97.8 . . . 88.2 4-Day Refrigerated" 50 48.9 97.8 -- 95.7 15-Jan-2001 Homogeneity3 Stability 4-Day Refrigeratedf 0 3.33 3.33 NDb 2.88, 2.54, 2.67 2.94, 2.88 ... 81.1 87.4 ---- 6 79.9 1 82.3 a Mean results for the analysis o f the top (T), middle (M) and bottom (B) samples, b Denotes none detected, c Samples held 5 hours at room temperature. d Mean result o f single sample for 4-day refrigerated/concentration verification and a single sample for 4-day refirigerated/5 hour room temperature stability. f Mean results o f duplicate samples for 4-day refrigerated/concentration verification and a single sample for 4-day reffigerated/5 hour room temperature stability. The data from M p f i r i ^ S C ^ ^ ^ a m p l e s along with the data for the 100 mg/mL samples in this study indicate that the test substance is stable in the vehicle when held 4 days refrigerated and followed by 5 hours at room temperature for the concentration range o f the study. Company Sanftizsd. Does not contain TSCA CBi i m U 'iloi Developmental Toxicity Study in Rats 'H A -2 0 oH ) 45; Page 9 _______________________ D. Concentration Verification/Homogeneity Analytical results from dosing suspensions collected on January 27, 2001 and analyzed for homogeneity and/or concentration verification are shown in Table HI and Summary Table 1. The following table summarizes the results for homogeneity and/or concentration verification analyses. Preparation Date 27-Jan-2001 Nominal mg/mL 0 12.5 25.0 50.0 100.0 Measured3 mg/mL NDb 10.7, 10.6 20.3, 20.1c 46.9, 46.6C 91.6,97.1,89.5 Mean (T,M,B) % Nominal -- 85.3 80.9 93.5 92.7 C.V. (%) -- 0.2C Ie Ie 4 a Mean results for the analysis o f the top (T), middle (M) and bottom (B) samples or o f the duplicate samples, b Denotes none detected. c C.V. for the duplicate samples used to confirm uniformity o f mixture. The data for samples collected on January 27, 2001 indicate that the test substance was homogeneously mixed in the vehicle at all levels. The test substance was at the targeted concentration in the samples ( 20% o f nominal). Test substance was not found in the 0 mg/mL samples. E. Conclusions Data from the analysis o f the samples during the study indicate that the test substance was homogeneously mixed, at the targeted levels and was stable under all conditions used in the study. Test substance was not found in the 0 mg/mL samples. Company SsnHfredL Does not contain TSCA CBI ilot Developmental Toxicity Study in Rats 2 0 0 " 5; Page 10________________________ __ ACKNOWLEDGMENTS Analysis o f dosing samples by Sheila A. Riley (Chemistry Associate). SIGNATURES ' Janet C. Maslanka Staff Scientist Date issued: SLPO s J Date Company Ssnissd Doss not contain TSC CBI IM -- * Developmental Toxicity Study in Rats ~ HA-200 ffi4 5 ; Page 11 ____________________________ Table I. Homogeneity and Stability o f H-24516 in Dosing Suspensions Sample mg/mL H-24516 Type Nominal Measured 16-Jan-2001 Homogeneity Control 0.00 n d (a ) Top M iddle Bottom 12.5 12.8 12.5 12.7 12.5 12.6 Mean(B): 12.70.1 C.V. 1% T op M iddle B ottom 25.0 23.3 25.0 24.6 25.0 25.0 Mean(B): 24.3+0.9 C.V. 4% Top M iddle B ottom 50.0 53.8 50.0 50.5 50.0 52.7 Mean(B); 52.3+1.7 C.V.3%o Top M iddle Bottom 100.0 100.0 100.0 Mean(B): 93.5 74.8(c > 78.9(c ) 82.49.8 C.V. 12% Percent Nominal -- 102.4 101.9 101.1 (101.8%) 93.3 98.4 100.0 (97.2%) 107.5 101.0 105.3 (104.6%) 93.5 74.8 78.9 (82.4%) Stability^) 12.5 25.0 50.0 100.0 12.3 23.7 50.1 83.5 98.7 94.7 100.2 83.5 (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 are based on measured concentration for each level. (C) Mean result of the original analysis and duplicate re-analysis of original sample reported. (D) Stability samples held 5 hours a room temperature. . Compaq Does not contain TSCA CBf P ^ ^ H P i l o t Developmental Toxicity Study in Rats HA-2Q0TO45; Page 12_____________________________ Table II. Stability Study o f H-24516 in Dosing Suspensions Preparation Date mg/mL H-24516 Sample Type Nominal Measured Percent Nominal 15-Jan-200 l( A ) 0-Day Room T emperature^ ) 4-Day Refrigerated 4-Day Refrigerated/5 Hour RT 3.33 2.70 3.33 2.94 3.33 2.88 Mean(Q ; 2.91+0.04 C.V. 1% 3.33 2.74 81.1 88.3 86.5 (87.4%) 82.3 8-Jan-200l(A ) 0-D a y R o o m T em pera tu re() 4-D ay refrigerated 4-Day Refrigerated/5 H our RT 5.0 5.0 5.0 4.39 87.8 4.81 96.1 4.85 97.1 0-Day Room Temperature(b ) 4-Day Refrigerated 4-Day Refrigerated/5 Hour RT 0-D a y R o o m T em pera tu re^6 ) 4-Day Refrigerated 4-Day R efrigerated/5 Hour RT 20.0 20.0 20.0 50.0 50.0 50.0 18.7 93.5 19.6 97.8 17.6 88.2 53.4 106.8 48.9 97.8 47.9 95.7 16-J an -200l(D ) 0-D a y R o o m T em pera tu re^6 ) 100.0 82.4 82.4 4-D ay Refrigerated 100.0 86.7 86.7 4-Day Refrigei^ ted/5 H our RT ^ 100.0 94.3 94.3 (A) Samples frori_ (B) The average measured concentration^ top, middle, and bottom for the homogeneity samples (0-Day Room Temperature) used as baseline for stability comparison. (C) Reported result is the mean o f the re-analysis o f the duplicate original samples. Original analysis indicated aliquot error and w ilbipt be reported. (D ) Samples fron 'J Company Sanitized Does not contain TSCA CS! f l .ilt Developmental Toxicity Study in Rats H A -200 RFPJ45f ; Page 13 ______ Table III. Homogeneity/Concentration Verification o f H-24516 in Dosing Suspensions Sample mg/mL H -24516 Type Nominal Measured 27-Jan-2001 Concentration Verification Control 0.00 n d (a ) Percent Nominal -- #1 12.5 10.7 85.3 #2 12.5 10.6 85.1 Mean(P): 10.7+0.02 (85.2%) C.V. 0.2 % #1 25.0 20.3 81.3 #2 25.0 20.1 80.5 Mean(B): 20.2+0.14 (80.9%) C.V.l%o #1 ' 50.0 46.9 93.9 #2 50.0 46.6 93.1 Mean(B): 46.8+0.26 (93.5%) C.V. 1% Homogeneity T op 100.0 91.6 91.6 M iddle Bottom 100:0 100.0 97.1 89.5 Mean(c X- 92.7+3.9 C.V. 4% 97.1 89.5 (92.7%) (A) Denotes not detected. (B) The average measured concentration, average percent o f nominal (in parentheses), standard deviation, and coefficient of variation of duplicate samples are based on measured concentration for each level. (C) The average measured concentration, average percent o f nominal (in parentheses), standard deviation, and coefficient of variation o f top, middle, and bottom are based on measured concentration for each level. Company SanMzetf, Dost not cantato TSA B i ( j/ m A ilot Developmental Toxicity Study in Rats ^HA-200 H^0k45; Page 14 Figure 1 Representative Analytical Calibration Curve (a) (b) Concentration mg/mL (C) Figure 1: Calibration curve showing linear fit (line) to replicate peak height ratio measurements (squares) for calibration solutions of H-24516 diluted over a concentration range of 0.0510 to 0.1531 mg/mL. [(a) Retention time = 3.5 minutes, (b) Retention = 4.17 minutes, (c) Retention time = 4.77 minutes.] Company Sanitised. Does not co n lsin TSC A CBS Pilot Developmental Toxicity Study in Rats 45; Page 15 Figure 2 Representative High Performance Liquid Chromatography Chrom atogram s FID1 B. (010116SRW17F1701.D) Figure 2a: Representative GC chromatogram o f 0 mp/mT,icontroll sample with internal standard pdded. Retention time for H-24516 is 'approximately 3.5,4.1, 4.7 minutes. KetentnJftime for internal standard is approximately 3.9 minutes. FIDI B. (010116SR\022F2201.D) pA ' 60 - Figure 2b: Representative GC chromatogram o f 12.5 mg/mL H-24516 dosing solution diluted to a nominal concentration o f 0.09 mg/mL. The measured concentration o f the representative solution is 12.8 mg/mL. FI0 1 B , (010116$R\019F1901.D) pA " 60 50 40 30 : 20: 10 ___ Q. Figure 2c: Representative GC chromatogram o f 0.0919 mg/mL H-24516 analytical reference solution. s -- teed. Dow n* .M T S C A C B ! Company __lilot Developmental Toxicity Study in Rats 'H A -2001-045; Page 16 TABLE 1 SUMMARY OF DOSING ANALYSES Nominal: Homogeneity Samples 1-Jan-2001 Top Middle Bottom Average Measured Conc.b Average Percent Nominal Standard Deviation b Coefficient of Variation b Dosing Concentration ofH -24516 (mg/mL) 12.5 25.0 50.0 12.8 (102.4)a 12.7 (101.9) 12.6 (101.1) 12.7 (101.8) 0.1 1% 23.3 (93.3) 24.6 (98.4) 25.0 (100.0) 24.3 (97.2) 0.9 4% 53.8 (107.5) 50.5 (101.0) 52.7 (105.3) 52.3 (104.6) 1 .7 3% 100.0 93.5 (93.5) 74.8 (74.8) 78.9 (78.9) 82.4 (100.0) 9 .8 12% Stability Samples 5-Hour Room Temperature 27-Jan-2001 Concentration Verification* Top Middle Bottom Average Measured Cone. b Average Percent Nominal Standard Deviation b Coefficient of Variation b 12.3 (98.7) 10.7 (85.3) 10.6 (85.1) C 10.7 (85.2) 0.02 0.2% 23.7 (94.7) 20.3 (81.3) 20.1 (80.5) C 20.2 (80.9) 0.14 1% 50.1 (100.2) 46.9 (93.9) 46.6 (93.1) C 46.8 (93.5) 0.26 1% 83.5 (83.5) 91.6 (91.6) 97.1 (97.1) 89.5 (89.5) 92.7 (92.7) 3 .9 4% Numbers in parentheses are the respective percent of nominal values. Statistics based on the average measured concentration (ppm) of the top, middle and bottom of each dosing level or duplicate samples. Samples not required for study. fSompanf S u f& re- S e s TSCA 81 Pilot Developmental Toxicity Study in Rats H A -2001-045; Page 17 TABLE 1 (continued) SUMMARY OF DOSING ANALYSES Stability Study Nominal: Homogeneity Samples Dosing Concentration o f H-24516 (mg/mL) 3.33 3 20.0 b 50.0 b 100.0c Top Middle Bottom Average Measured C one.e Average Percent Nominal Standard D eviatione Coefficient of Variatione 2.88 (86.5)d 2.54 (76.3) 2.67 (80.2) 2.70 (81.1) 0.2 6% 4.53 (90.6) 4.25 (85.0) 4.39 (87.8) 4.39 (87.8) 0.14 3% 19.9 (99.5) 17.7 (88.5) 18.6 (93.0) 18.7 (93.5) 1.1 6% 53.4 (106.8) 52.6 (105.2) 54.0 (108.0) 53.4 (106.8) 0 .7 1% 93.5 (93.5) 74.8 (74.8) 78.9 (78.9) 82.4 (82.4) 9 .8 12% Stability Samples 0-Day Room Temperaturef 2.70 (81.1) 4.39 (87.8) 18.7 (93.5) 53.4 (106.8) 82.4 (82.4) 4-Day Refrigerated 2.91 (87.4) 4.81 (96.1) 19.6 (97.8) 48.9 (97.8) 86.7 (86.7) 4-Day Refrigerated/5 hr. 2.74 4.85 17.6 47.9 94.3 (82.3) (97.iL (88.2) (95.7) (94.3) a Samples prepared January 15, 2001 foa b Samples prepared January 8,2001 fo ri c Samples prepared January 27,2001 fon d Numbers in parentheses are the respect ^percent o f nominal values. e Statistics based on the average measured concentration (ppm) o f the top middle and bottom o f each dosing level. f The mean measured values from analysis o f homogeneity samples (considered fresh/0-day room temperature samples) were used as baselines for comparison with the respective stability samples. Company Sanitized. Does not contain TSCA CBf