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AR226-3135 DuPont-3548 TRADE SECRET Study Title H-24256: Local Lymph Node Assay (LLNA) Laboratory Project ID: DuPont-3548 Author: Gregory S. Ladies, Ph.D., D.A.B.T. Study Completed on: November 15,1999 Performing Laboratory: E. I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Work Request Number: J Service Code Number:* 3 Company Sanitized. Does not contain TSCA CBI Page 1 of 13 H-24256: Local Lymph Node Assay (LLNA) DuPont-3548 CERTIFICATION I, the undersigned, declare that this report provides an accurate evaluation o f data obtained from this study. Issued by Study Director: Date Company Sanitized. Does not contain TSCA CBi -2- H-24256: Local Lymph Node Assay (LLNA) TABLE OF CONTENTS DuPont-3548 Page CERTIFICA TIO N ................................................................................................................................. 2 LIST OF TABLES................................................................................................................................3 STUDY INFORMATION.................................................................................................................... 4 STUDY PERSONNEL........................................................................................................................ 5 SUMMARY.......................................................................................................................................... 6 INTRODUCTION................................................................................................................................. 7 MATERIALS AND METHODS.........................................................................................................7 A. Test Species............................................................................................................................... 7 B. Test Substance...........................................................................................................................7 C. Body Weight Measurements....................................................................................................7 D. Clinical Observations............................................................................................................... 7 E. Local Lymph Node Assay........................................................................................................ 8 F. Statistical Analyses.................................................................................................................. 8 RESULTS AND DISCUSSION.......................................................................................................... 9 A. Clinical Signs o f Toxicity, Body Weights, and Body Weight Gains....................................9 B. Stimulation Index Data............................................................................................................. 9 CONCLUSIONS................................................................................................................................... 9 RECORDS AND SAMPLE STORAGE..............................................................................................9 REFERENCES.................................................... :..............................................................................10 TABLES............................................................................................................................................... 11 LIST OF TABLES Page TABLE 1 MEAN BODY WEIGHTS (g )....................................................................................... 12 TABLE 2 MEAN BODY WEIGHT GAINS (g )............................................................................12 TABLE 3 STIMULATION INDEX (SI) DATA............................................................................13 Company Sanitized. Doas not contam TSCA -3 - H-24256: Local Lymph Node Assay (LLNA) Haskell Number: 24256 DuPont-3548 Stability: The test substance appeared to be stable under the conditions o f the study; no evidence o f instability was observed. Sponsor: E. I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Comoleted: October 27,1999 / (see report cover page) In-Life Initiated/Completed: October 27,1999 / November 2, 1999 Company Sanitized. Does not contain TSCA CE -4- H-24256: Local Lymph Node Assay (LLNA) STUDY PERSONNEL Study Director: Gregory S. Ladies, Ph.D., D.A.B.T. Management: Judith C. Stadler, Ph.D. Primary Technician: Charlene Smith, S.A. Laboratory Veterinarian: Wanda L. West, D.V.M., A.C.L.A.M. DuPont-3548 Company Sanitized. Does not contain TSCA CB! -5- H-24256: Local Lymph Node Assay (LLNA) DuPont-3548 SUMMARY The objective of this study was to evaluate the potential of H-24256 to produce a dermal sensitization response in mice using the local lymph node assay (LLNA). Six groups of female CBA/JHsd mice were dosed for 3 days with 0 (vehicle), 5%, 10%, 25%, 50%, or 100% H-24256 on both ears. Dimethylsulfoxide was used as the diluting vehicle. A seventh group of 6 female mice were dosed for 3 days with 25% Hexylcinnamaldehyde (HCA) as a positive control. On day 5 o f the assay, mice received 125-Iododeoxyundine and were sacrificed 5 hours later. The cell proliferation in the draining auricular lymph nodes of the ears was then evaluated and compared to control. There were no clinical signs of toxicity observed in the study. There were no statistically significant differences in mean body weights and mean body weight gains between the vehicle control group and any test substance group. No statistically significant differences were seen in cell proliferation measurements at any test concentration. Stimulation indexes were less than 3.0 at all concentrations. A 25% concentration o f the positive control, HCA, produced a positive dermal sensitization response in mice. Therefore, the LLNA test system was valid for this study with H-24256. Under the conditions o f this study, H-24256 was not a dermal sensitizer. Company Sanitized. Does not contain TSCA CBI -6- H-24256: Local Lymph Node Assay (LLNA) DuPont-3548 INTRODUCTION The purpose o f this study was to examine the dermal sensitization potential o f H-24256 using the mouse local lymph node assay (LLNA). Following the topical application of the test article to the dorsal side of both ears, the dermal sensitization potential o f the test article was evaluated by measuring the proliferation o f lymphocytes (via radiolabel uptake) obtained fronUhe auricular lymph nodes (i.e., the lymph nodes that drain the ears). Because H-24256 is a f l^ B a n d did not appear to have severe skin irritating capability (pH ~8), the 100% concentration was used as the high dose. For subsequent dilutions, the test article was found to be soluble in dimethlysulfoxide (DMSO). MATERIALS AND METHODS A. Test Species The Local Lymph Node Assay screen with H-24256 was conducted in female CBA/JHsd mice. On 19-Oct-1999,47 female CBA/JHsd mice with an assigned birth date o f 27-Aug-1999, were received from Harlan Sprague Dawley, Frederick, Maryland. The CBA/JHsd mouse was selected to conduct the LLNA because Haskell Laboratory has an extensive historical database with this strain. Furthermore, this strain has undergone extensive interlaboratory validation with the LLNA.(1,2'3) B. Test Substance The test substance, H-24256, was supplied by the sponsor a s ; _______ The test substance was mixed before each dose was removed. The test substance appeared to be" stable under the conditions of the study. No evidence of instability, such as a change in color or physical state, was observed. C. Body W eight Measurements All mice were weighed on test day 0 and prior to sacrifice on test day 5. D. Clinical Observations Cage-site examinations to detect moribund or dead mice and abnormal behavior and appearance among mice were conducted at least once daily throughout the study. At every weighing, each mouse was individually handled and examined for abnormal behavior and appearance. Company Sanitized. Does not contain TSCA CB! -7 - H-24256: Local Lymph Node Assay (LLNA) DuPont-3548 E. Local Lymph Node Assay The test substance was prepared as a solution in DMSO except the 100% concentration. Twenty-five fil of H-24256 was administered topically to the dorsum of each ear of mice (6 per group) for 3 consecutive days (test days 0-2) at dosages o f 0% (DMSO vehicle), 5%, 10%, 25%, 50%, and 100%. One group o f 6 female mice was dosed with 25% hexylcinnamaldehyde (HCA) as a positive control. Test days 3-4 were days of rest, followed by intravenous injection o f 2 /iCi of 125-Iododeoxyuridine (125IudR) per mouse on the morning o f test day 5. Approximately 5 hours later, animals were sacrificed, draining auricular lymph nodes were removed, and single cell suspensions were prepared. The single cell suspensions were then incubated at ~4C overnight. Counts per minute (cpm) data were obtained on test day 6 by counting the single cell suspensions on a gamma counter, and the cpm data were converted to disintegrations per minute (dpm) data. F. Statistical Analyses Significance will be judged at p < 0.05 except for Jonckheere-Terpstra trend test which will be judged at p < 0.01. Parameter Body Weight Body Weight Gain Lymph Node DPM Dataab Preliminary Test Levene's test^ for homogeneity and Shapiro-Wilk test^ for normality6 Method of Stalistical Analysis If preliminary test is not significant If preliminary test is significant One-way Analysis of Variance;^ followed, Kruskal-Wallis test;w followed, with Dunn's with Dunnett's test^ test^ and/or Sequential application^10^of the Jonckheere-Terpstra trend test^11* a If more than one mouse was eliminated from a group, statistics were not done on the data for that group, b DPM Positive control data was not included in the statistical analysis. c If the Shapiro-Wilk test was not significant but Levene's test was significant, a robust version of Dunnett's test was used. Company Sanitized. Does not contain TSCA CBI -8- H-24256: Local Lymph Node Assay (LLNA) DuPont-3548 RESULTS AND DISCUSSION A. Clinical Signs of Toxicity, Body W eights, and Body W eight Gains (Tables 1-2) There were no clinical signs o f toxicity observed in the study. There were no statistically significant differences in mean body weights and mean body weight gains between the vehicle control group and any test substance group. B. Stimulation Index Data (Table 3) No statistically significant differences were observed in cell proliferation measurements at any test concentration. Stimulation indexes were less than 3.0 at all concentrations of H-24256. Under the conditions o f this study, H-24256 was not a dermal sensitizer. CONCLUSIONS A 25% concentration o f HCA produced a positive dermal sensitization response in mice. Therefore, the LLNA test system was valid for this study with H-24256. Under the conditions of this study, H-24256 was not a dermal sensitizer. RECORDS AND SAMPLE STORAGE The raw data and final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. Company Sanitized. Does not contain TSCA CB1 -9- H-24256: Local Lymph Node Assay (LLNA) DuPont-3548 REFERENCES 1. Loveless, S.E., Ladies, G.S., Gerberick, G.F., Ryan, C.A., Basketter, D.A., Scholes, E.W., House, R.V., Hilton, J., Dearman, R.J., and Kimber, I. (1996). Further Evaluation o f the Local Lymph Node Assay in the Final Phase of an International Collaborative Trial. Toxicology 108:141-152. 2. Kimber, I., Hilton, J., Dearman, R.J., Gerberick, G.F., Ryan, C.A., Basketter, D.A., Scholes, E.W., Ladies, G.S., Loveless, S.E., House, R.V., and Guy, A. (1995). An International Evaluation o f the Murine Local Lymph Node Assay and Comparison o f Modified Procudures. Toxicology 103:63-73. 3. Kimber, I., Hilton, J., Dearman, RJ., Gerberick, G.F., Ryan, C.A., Basketter, D.A., Lea, L., House, R.V., Ladies, G.S., Loveless, S.E., and Hastings, K.L. (1998). Assessment o f The Skin Sensitization Potential of Topical Medicaments Using The Local Lymph Node Assay: An Inter-Laboratory Exercise. J. Toxicol. Environ. Health, Part A 53(7):563-579. 4. Levene, H. (1960). Robust test for equality o f variances. In Contributions to Probability and Statistics (I. Olkin, ed.), 278-292. Stanford University Press, Palo Alto, CA. 5. Shapiro, S.S. and M.B. Wilk, (1965). An analysis o f variance test for normality (complete samples). Biometrika 52: 591-611. 6. Snedecor, G. W. and W. G. Cochran, (1967). Statistical Methods, 6th ed. The Iowa State University Press, Iowa, pp. 246-248, 349-352. 7. Dunnett, C.W., (1955). A multiple comparison procedure for comparing several treatments with a control. J. Amer. Statist. Assoc. 50: 1096-1121. 8. Kruskal, W.H. and W.A. Wallis, (1952). Use o f ranks in one-criterion analysis of variance. J. Amer. Statist. Assoc. 47: 583-621. 9. Dunn, O.J., (1964). Multiple contrasts using rank sums. Technometrics 6: 241-252. 10. Selwyn, M. R., (1995). "The Use o f Trend Tests to Determine a No-Observable-Effect Level in Animal Safety Studies," Journal o f the American College o f Toxicology, 14(2): 158-168. 11. Jonckheere, A. R., (1954). "A Distribution-Free K-Sample Test Against Ordered Alternatives," Biometrika 41:133-145. Company Sanitized. Does not contain TSCA CBI -10- H-24256: Local Lymph Node Assay (LLNA) DuPont-3548 TABLES -11 - Company Sanitized. Does not contain TSCA CB1 H-24256: Local Lymph Node Assay (LLNA) DuPont-3548 TABLE 1 MEAN BODY WEIGHTS (g) GROUP: n IV VI VIII X CONCENTRATION (%): 0 5 10 25 50 DAYS ON TEST 0 21.6 (1.7)a 21.6(1.8) 22.0(1.1) 21.6 (0.5) 22.2(1.3) 5 22.0(1.7) 22.2(1.8) 22.4(1.0) 22.6 (0.9) 22.6(1.1) a Standard Deviation is reported in parenthesis. Statistical methods: One-way Analysis of Variance and Dunnett's tests were performed on data There were no statistically significant differences at p > 0.05 * HCA (positive control) XII XIV 100 25* 22.6(1.6) 22.1 (1.5) 23.0(1.5) 23.0(1.8) TABLE 2 MEAN BODY WEIGHT GAINS (g) GROUP: n IV VI VIU X XII CONCENTRATION (%): 0 5 10 25 50 100 DAYS ON TEST 0-5 0.4 (0.6)a 0.7 (0.4) 0.4 (0.5) 1.1 (0.7) 0.5 (0.8) 0.4 (0.6) a Standard Deviation is reported in parenthesis. Statistical methods: One-way Analysis o f Variance and Dunnett's tests were performed on data There were no statistically significant differences at p > 0.05 * HCA (positive control) XIV 25* 0.8 (0.6) -12- Company Sanitized. Does not contam T S C A C B l H-24256: Local Lymph Node Assay (LLNA) TABLE 3 STIMULATION INDEX (SI) DATA GROUP II IV VI VIII X XII xrv MATERIAL TESTED 0% (DMSO) 5% H-24256 10% H-24256 25% H-24256 50% H-24256 100% H-24256 25% HCA in DMSO (positive control) SI N/A 2.00 0.75 0.25 1.00 0.00 3.50 N/A = Not Applicable n = Number of animals per group. There were no statistically significant differences at p > 0.05 DuPont-3548 -13- Company Sanitized. Does not contain TSCA CBI