Document kaBBjk1Jzm0nD1YQ0gqgMX6wJ

Haskell Laboratory for Toxicology and Industrial Medicine January 4,2002 AR226-3215 TO: ( Milot 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 Attached is the analytical report to. for the study identified above. 'ilot Developmental Toxicity Study in Rats ANALYSIS FORI^^----BN DOSING SUSPENSIONS Medical Research Project Number: Haskell Sample Number: Analytical Report Number: SUMMARY 24516 HA-2001-045 Dosing suspensions at concentrations of 12.5, 25.0, 50.0 and 100.0 mg/mL ofH-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 stability because the other levels in this study were within the range of a previous study (M^Um^^HBB 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 ofH-24516 were submitted for concentration verification analysis on January 27,2001. On the same day, dosing suspensions at concentrations of 100.0 mg/mL ofH-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. '"limUM^^ H-24516 as used in this report refers to the active ingredient (a.i.) Process. ^^^^^^^^^^^^^^^^^h ^ Concentrations ofH-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 of 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% of 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 Co.p.nyS.n^.D""------"*""15""' 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 of the 0 mg/mL (control) sample in the study. Company Sanitized. Does not contain TSCA CBi pilot Developmental Toxicity Study in Rats HA-2001-045; Page 3 ___ SAMPLE SUBMITTAL Samples containing H-24516 at the concentrations ofO, 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 ofO, 100.0 mg/mL were collected on January 19, 2001. These samples were analyzed to determine 4-day refrigerated stability along with 5-hour (MNRfVf 8 ^ room temperature stability. The "4-day refrigerated stabiimlityy fortthheerreemmaamindder of the levels in this study had been established in a previous study SCfRRwith H-24516. Samples containing H-24516 at the concentrations ofO, 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 TSGA CB? pi^BfelP1!01 Developmental Toxicity Study in Rats fc[^A-200T"D45;Page4 ___________ 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 of 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 l.OOmin. 20.0C/min. 260C 0.00 min. 9.00 min. Company SanSteed. Does not contain TSCA CBE ( I -- A ^ilot Developmental Toxicity Study in Rats ' HHAA--220000'TT"OD4455;;:Page 5_________________ c. Calibration and Quantitation A separate sample ofH-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 of the stock were diluted with methanol to make calibration standards that bracketed the target concentration of the diluted dosing samples. A stock solution of an internal standard (1H, 1H, 2H, 2H-perfluoro-9- methyldecan-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 of 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 of these solutions were used to construct a calibration curves by least squares regression (see Figure la, Ib, and Ic for a representative curves). Measured concentrations for the dosing samples were determined by applying the peak height ratios from replicate injections of 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) of the measured concentrations in the top, middle, and bottom (T, M, and B) samples or the mean of duplicate samples for each concentration. A coefficient of 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 of the top, middle, and bottom homogeneity samples or of the duplicate concentration verification samples for each dosing level was used to determine the concentration of the test substance for the respective dosing levels. Stability was evaluated by using the mean of the top, middle, and bottom homogeneity samples or of the duplicate concentration verification samples (0-day room temperature) as the baseline for comparing the corresponding room temperature and refrigerated results. Company Sanitteid. Bw net contain TSCA CBI Q 'ilot Developmental Toxicity Study in Rats HA-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 ofquantitation, resolved peaks at the reten]jpn times of approximately 3.5,4.17, and 4.77 minutes were used. The internal peak at approximately 3.99 minutes. Representative GC chromafograms 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,B mg/mL ND 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 Stability1' % Nominal -- 98.7 94.7 100.2 83.5 a Mean results for the analysis of 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% of 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. cam'"a^-^^^,,^^ Sl^ H^^ ^y rilot 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 n 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 Homogeneity" 0 ND1' -- -- 100 93.5, 74.8, 78.9 82.4 12 19-Jan-2001 Stability 4-Day Refrigerated 100 86.7'1 86.7 -- a Mean results for the analysis of the top (T), middle (M) and bottom (B) samples. b Denotes none detected. c Samples held 5 hours at room temperature. d Mean result of single sample for concentration verification/4-day refrigerated. e Mean result of single sample for 4 day-refrigerated/5 hour room temperature stability. Stability % Nominal -- 83.56 94.36 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% of nominal). Comparison of 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 Saniteedl Does not contain TSCA CB( Pilot Developmental Toxicity Study in Rats 'HA-200T"045; Page 8 The following table is included for a reference in this study totherefiigerated stability for ^UIB^^BI^ the concentration range. It summarizes the results for homogeneity/concentration verification (January 8, 2001 and January'15,To01)and the 4- day refrigerated stability analyses followed by 5 hours room temperature. Preparation Date Sample Type Nominal mg/mL Measured mg/mL Mean % Nominal C.V. (%) Stability % Nominal 8-Jan-2001 Homogeneitya 0 ND1' -- - -- 5.0 20 50 Stability 4-Day Refrigerated'1 5.0 4.53,4.25, 4.39 19.9, 17.7, 18.6 53.4, 52.6, 54.0 4.81 87.8 93.5 106.8 96.1 3 92.00 6 86.06 1 96.6C -- 97.1 4-Day Refrigerated'1 20 19.6 97.8 -- 88.2 4-Day Refrigerated1' 50 48.9 97.8 -- 95.7 15.Jan.2001 Homogeneity8 0 ND1' -- -- -- 3.33 2.88, 2.54, 2.67 81.1 Stability 4-Day Refrigerated*^ 3.33 2.94, 2.88 87.4 6 79.9 1 82.3 Mean results for the analysis of the top (T), middle (M) and bottom (B) samples. Denotes none detected. Samples held 5 hours at room temperature. Mean result of single sample for 4-day refrigerated/cone entration verification and a singIe sample for The data from MPflB^nSCf|Kamples 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 of the study. Company Sar^tesA Does not contain TSCA CB8 'ilot Developmental Toxicity Study in Rats 'rHA-200^D45;Page9_______ ____ 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 in 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 Measured3 mg/mL ND1' Mean(T,M,B) % Nominal -- C.V. (%) -- 12.5 10.7, 10.6 85.3 O.Y 25.0 20.3, 20.16 80.9 I' 50.0 46.9, 46.6C 93.5 I6 100.0 91.6,97.1,89.5 92.7 4 a Mean results for the analysis of the top (T), middle (M) and bottom (B) samples or of the duplicate samples. b Denotes none detected. c C.V. for the duplicate samples used to confirm uniformity of 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% of nominal). Test substance was not found in the 0 mg/mL samples. E. Conclusions Data from the analysis of 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 SanHizad. Quss not contain TSCA CB( ^ 1Pilot Developmental 'HA\-^2O00Ol"fTfOl^5; Page 10 Toxicity Study m Rats ACKNOWLEDGMENTS Analysis of dosing samples by Sheila A. Riley (Chemistry Associate). SIGNATURES Report by: ( l ^ ^ U ^ . Janet C. Maslanka Staff Scientist ^- w-300aJ Date Date issued: 4.. 37?A/- ajsosJ Company Sanitised Does not contain TSCA CBI HA-200 ilot Developmental Toxicity Study in Rats ; Page 11 Table I. Homogeneity and Stability ofH-24516 in Dosing Suspensions Sample mg/mLH-24516 Type Nominal Measured 16-Jan-2001 Homogeneity CONTROL 0.00 NDW TOP MIDDLE BOTTOM 12.5 12.8 12.5 12.7 12.5 12.6 MeanW: 12.71,0.1 C.V.1% TOP MIDDLE BOTTOM 25.0 25.0 25.0 MeanW: 23.3 24.6 25.0 24.3^0.9 C.V.4% TOP MIDDLE BOTTOM 50.0 53.8 50.0 50.5 50.0 52.7 MeanW: 52.3fl.7 C.V.3% TOP MIDDLE BOTTOM 100.0 100.0 100.0 MeanW: 93.5 74.8(c) 78.9(c) 82.4f9.8 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 12.3 98.7 25.0 23.7 94.7 50.0 50.1 100.2 100.0 83.5 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. Company SgsfaaA Does not contain TSCA CBf HA-200 ilot Developmental Toxicity Study in Rats ;Page 12 Table II. Stability Study ofH-24516 in Dosing Suspensions Preparation Date mg/mLH-24516 Sample Type Nominal Measured Percent Nominal i W 15- Jan-200 O-DAY ROOM TEMPERATURE^) 4-DAY REFRIGERATED 4-DAY REFRIGERATED/5 HOUR RT Man^QO^) O-DAY ROOM TEMPERATURE^) 4-DAY REFRIGERATED 4-DAY REFRIGERATED/5 HOUR RT 3.33 3.33 3.33 3.33 2.70 2.94 2.88 MeanV^: 2.91f0.94 C.V.1% 2.74 5.0 4.39 5.0 4.81 5.0 4.85 81.1 88.3 86.5 (87.4%) 82.3 87.8 96.1 97.1 0-DAY ROOM TEMPERA-nWS) 4-DAY REFRIGERATED 4-DAY REFRIGERATED/5 HOUR RT 20.0 20.0 20.0 18.7 19.6 17.6 93.5 97.8 88.2 O-DAY ROOM TEMPERATURE^) 4-DAY REFRIGERATED 4-DAY REFRIGERATED/5 HOUR RT 50.0 50.0 50.0 53.4 48.9 47.9 106.8 97.8 95.7 16-Jan-200l(D) O-DAY ROOM TEMPERATURE^) 100.0 82.4 82.4 4-DAY REFRIGERATED 100.0 86.7 86.7 4-DAY REFRIGERATED/5 HOUR RT , 100.0 94.3 94.3 SamplesTorJUJIHBl (A) (B) The average measured concentratioirTOtop, middle, and bottom for the homogeneity samples (0-Day Room Temperature) used as baseline for stability comparison. (C) Reported result is the mean of the re-analysis of the duplicate original samples. Original analysis indicated aliquot error and wil^jptbe reported. Company SanHlzo& Does not eontaira TSCA CB! HA-200 ilot Developmental Toxicity Study in Rats ^45;Page 13 ___________ Table III. Homogeneity/Concentration Verification ofH-24516 in Dosing Suspensions Sample mg/mLH-24516 Type Nominal Measured 27.Jan.2001 Concentration Verification CONTROL 0.00 NT^) Percent Nominal -- #1 12.5 10.7 85.3 #2 12.5 10.6 85.1 MeanW: 10.7tP.02 (85.2%) C.V.0.2% #1 25.0 20.3 81.3 #2 25.0 20.1 80.5 MeanW: 20.2t9.14 (80.9%) C.V.1% #1 50.0 46.9 93.9 #2 50.0 46.6 93.1 MeaiiW: 46.8f0.26 (93.5%) C.V.1% Homogeneity TOP 100.0 91.6 91.6 MIDDLE 100.0 97.1 97.1 BOTTOM 100.0 Mean^: 89.5 92.7^3.9 89.5 (92.7%) 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 duplicate samples are based on measured concentration for each level. (C) 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. 'ilot Developmental Toxicity Study in Rats 45; Page 14 Figure 1 Representative Analytical Calibration Curve 0.08 0.10 0.12 0.14 Concentration ing/mL (a) DM 0.10 0.12 0.14 Concentration nig/mL (b) 0.08 0.10 0.12 Concentration mg/mL (C) Figure 1: Calibration curve showing linear fit (line) to replicate peak height ratio measurements (squares) for calibration solutions ofH-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 SanStfeag. Does not corrlsfn TSCA C5! (Pilot Developmental Toxicity Study in Rats ''HA-200rV45; Page 15 ____ Figure 2 Representative High Performance Liquid Chromatographv Chromatograms FID1 B. (010116SR\017F1701.D) 50 - 40- 30 - Figure 2a: Representative GC chromatogram ofO mg/mL^control) sample with internal standard approximate^T.5,4.1, 4.7 mmiifesTtetentidlltime for internal standard is approximately 3.9 minutes. FID1 B. (010116SRW22F2t201.D) " PA 60 - 50 - 40 - 30- g : c i I ^ 20 - 10 - S 0, <0 Ul s ! 11 C1 1 L .11 J, { . \ : 0 2 4 6 8 mm Figure 2b: Representative GC chromatogram of 12.5 mg/mL H-24516 dosing solution diluted to a nominal concentration of 0.09 mg/mL. The measured concentration of the representative solution is 12.8 mg/mL. FID1 B, (010116SRV019F1901.D) pA ' 50 - 40 - s 30 - 1 0 V C0* 20 - R 01 rt M 1 ] ! 10 - s 1 ,1 ,1, k ^, s, IA.. 0 2 4 6 8 min Figure 2c: Representative GC chromatogram of 0.0919 mg/mL H-24516 analytical reference solution. SH,^.Dono.c^>l->TSCACBI Company lilot Developmental Toxicity Study in Rats 'HA-2001-045; Page 16 _____________ TABLE 1 SUMMARY OF DOSING ANALYSES Nominal: Homogeneity Samples 1-Jan-2001 Top Dosing Concentration ofH-24516 (mg/mL) 12.5 25.0 50.0 12.8 (102.4)a 23.3 (93.3) 53.8 (107.5) Middle 12.7 (101.9) 24.6 (98.4) 50.5 (101.0) Bottom Average Measured Cone.1' Average Percent Nominal b Standard Deviation Coefficient of Variation'' 12.6 (101.1) 12.7 (101.8) 0.1 1% 25.0 (100.0) 24.3 (97.2) 0.9 4% 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 12.3 (98.7) 23.7 (94.7) 50.1 (100.2) 83.5 (83.5) 27-Jan-2001 Concentration Verification' Top Middle Bottom b Average Measured Cone. Average Percent Nominal b Standard Deviation Coefficient of Variation 10.7 (85.3) 10.6 (85.1) 10.7 (85.2) 0.02 0.2% 20.3 (81.3) 20.1 (80.5) 20.2 (80.9) 0.14 1% 46.9 (93.9) 46.6 (93.1) 46.8 (93.5) 0.26 1% 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. JDonipanySssrffe^- &&s- we?ess^Se? TgA GBS 'ilot Developmental Toxicity Study in Rats HA-2001-045; Page 17 TABLE 1 (continued) SUMMARY OF DOSING ANALYSES Stability Study Nominal: Homogeneity Samples Dosing Concentration ofH-24516 (mg/mL) 3.33" 5.0t 20.0" 50.0' 100.0' Top Middle Bottom Average Measured Cone.e Average Percent Nominal Standard Deviatione 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 Temperature*^ 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) 19.6 (97.8) 48.9 (97.8) 86.7 (86.7) 4-Day Refrigerated/5 hr. 2.74 (82.3) 17.6 (88.2) 47.9 (95.7) 94.3 (94.3) ^flHBI^BtfBlf Samples prepared January 15, 2001 Samples prepared January 8,2001 ^^^^^^^^^^^jl Samples prepared January 27,2001 foi^UIBIUm Numbers in parentheses are the respecnrc percent of nominal values. Statistics based on the average measured concentration (ppm) of the top middle and bottom of each dosing level. The mean measured values from analysis of homogeneity samples (considered fresh/0-day room temperature samples) were used as baselines for comparison with the respective stability samples. Company Sanitized. Does no? contsh TSCA CB("