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AR226-3150 DuPont-4199 TRADE SECRET Study Title H-23960: Local Lymph Node Assay (LLNA) Laboratory Project ID: DuPont-4199 A u t h o r : Gregory S. Ladies, PhD ., D.A.B.T. S t u d y C o m p l e t e d o n : May 4,2000 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 W ork Request Num ber: I] im m jS e r v ic e C o d e N u m b e r Page 1 of 13 Company Sanitized. Does not contain TSC CBS H-23960: Local Lymph Node Assay (LLNA) DuPont-4199 C E R T IF IC A T IO N I, the undersigned, declare that this report provides an accurate evaluation o f data obtained from this study. Issued by Study Director: Senior Research Scientist Li ~ l i A y - ^ o Date -2Swnpgrty SsoiflzedL Doe; ofcontain TSCA C l H-23960: Local Lymph Node Assay (LLNA) DuPont-4199 TABLE OF CONTENTS Page CERTIFICATION......................................... 2 LIST OF TABLES............................................................................................................................. 3 STUDY INFORMATION..................................................................................................................4 STUDY PERSONNEL...................................................................................................................... 5 SU M M A R Y ....................................................................................... 6 INTRODUCTION.............................................................................................................................. 7 MATERIALS AND METHODS.......................................................................................................7 A. Test Species................... 7 B. Body Weight M easurements..................................................................................................7 C. Test Substance........................................................................................................................ 7 D. Clinical Observations.............................................................................................................7 E. Local Lymph Node Assay......................................................................................................8 F. Statistical A nalyses................................................................................................................8 RESULTS AND DISCUSSION.............................................................................................................9A. Clinical Signs o f Toxicity, Body Weights, and Body Weight Gains...................................9 B. Stimulation Index Data...........................................................................................................9 CONCLUSIONS................................................................................................................................ 9 RECORDS AND SAMPLE STORAGE........................................................................................... 9 R EFER EN C ES.................................................................................................................................. 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 -3Company Sanitized. Does not contain TSCA CBf H-23960: Local Lymph Node Assay (LLNA) STUDY INFORMATION Substance Tested: l DuPont-4199 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: April 12, 2000 / (see report cover page) In-Life Initiated/Comoleted: April 22, 2000 / April 28, 2000 -4 - GC; t" :yt. :'V H-23960: Local Lymph Node Assay (LLNA) DuPont-4199 STUDY PERSONNEL The following individuals participated in the conduct o f this study: Study Director: Gregory S. Ladies, Ph.D., D.A.B.T. Management: Judith C. Stadler, Ph.D., D.A.B.T. Primary Technician: Charlene Smith, S.A. Toxicology Report Preparation: Brenda Tiffin Laboratory Veterinarian: Wanda L. West, D.V.M., A.C.L.A.M. H-23960: Local Lymph Node Assay (LLNA) DuPont-4199 SUMMARY The objective o f this study was to evaluate the potential o f H-23960 to produce a dermal sensitization response in mice using the local lymph node assay (LLNA). Six groups o f female CBA/JHsd mice were dosed for 3 days with 0 (vehicle), 5%, 10%, 25%, 50%, or 90% H-23960 on the dorsal side o f both ears. N,N-dimethylformamide (DMF) was used as the diluting vehicle. A seventh group o f 6 female mice was dosed for 3 days with 25% Hexylcinnamaldehyde (HCA) as a positive control. On test day 5 o f the assay, mice received 3H-Thymidine by tail vein injection and were sacrificed 5 hours later. The cell proliferation in the draining auricular lymph nodes o f the ears was then evaluated and compared to control. 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. 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-23960. Under the conditions o f this study, H-23960 did not produce a positive dermal sensitization response in mice. -6 - Company Sanitized. Does not contain TSC CBS H-23960: Local Lymph Node Assay (LLNA) DuPont-4199 INTRODUCTION The purpose o f this study was to examine the dermal sensitization potential o f H-23960 using the local lymph node assay (LLNA). Following the topical application o f the test article to the dorsal side of both ears, die dermal sensitization potential o f the test article was evaluated by measuring the proliferation o f lymphocytes (via radiolabel uptake) obtained fron^hw uricular lymph nodes (i.e., the lymph nodes that drain the ears). Because H-23960 is not appear to have severe skin irritating capability at the 90% test concentration (pH ~3), the 90% concentration was used as the high dose. For subsequent dilutions, the test article was found to be soluble in N,N-dimethylformamide (DMF). MATERIALS AND METHODS A. Test Species The Local Lymph Node Assay with H-23960 was conducted in female CBA/JHsd mice. On March 16, 2000,48 female mice, with an assigned birth date o f January 28, 2000, were received from Harlan Sprague Dawley, Fredrick, Maryland. The CBA/JHsd mouse was selected to conduct the LLNA because Haskell Laboratory has an extensive database w ith this strain. Furthermore, the strain has undergone extensive interlaboratory validation with the LLNA.(1"3) B. Body W eight M easurem ents All mice were weighed on test day 0 and prior to sacrifice on test day 5. C. Test Substance The test substance, H-23960, was supplied by the sponsor as _ ---------- M H B lfh e test substance was vortexed to mix before each amount tor dosing was removed. The test substance appeared to be stable under the conditions o f the study. No evidence o f instability, such as a change in color or physical state, was observed. D. Clinical O bservations 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. 7 SanFtlxsiS. Or-s-p r-r*^^.e^air tij-t's ; /B> H-23960: Local Lymph Node Assay (LLNA) DuPont-4199 E. Local Lym ph Node Assay The test substance was prepared as a solution in DMF. Twenty-five il o f H-23960 was administered topically to the dorsum o f each ear o f mice (6 per group) for 3 consecutive days (test days 0-2) at dosages o f 0% (DMF vehicle), 5%, 10%, 25%, 50%, and 90%. One group o f 6 female mice was dosed with 25% hexylcinnamaldehyde (HCA) as a positive control. Test days 3-4 were days o f rest followed by intravenous injection o f 20/iCi o f H-Thymidine per mouse on the morning o f test day 5. One mouse in the 5% dose group was not properly injected with the appropriate amount o f 3H-Thymidine and was excluded from study. Approximately 5 hours later, anim als were sacrificed, draining auricular lymph nodes were removed, and single cell suspensions were prepared. The single cell suspensions were then incubated at 2-8C overnight. Disintegrations per minute (dpm) data were obtained on test day 6 by counting the single cell suspensions on a beta counter. A stimulation index (SI) was derived for each experimental group by dividing the mean dpm of the experimental group by the mean dpm o f the vehicle control group. Statistical significance in the test article groups compared to control and/or Sis o f greater than or equal to 3.0 indicated a positive response. F. Statistical Analyses Significance was judged at p < 0.05 except for dpm data which was judged at p <0.01. Parameter Body Weight Body Weight Gain Lymph Node DPM Data3'" Preliminary Test Test for lack o f trend Levene's test for homogeneity and Shapiro-Wilk test for normalityc,d Method o f Statistical Analvsis If preliihinary test is If preliminary test is not significant significant Sequential Preliminary tests for application(1) o f the pairwise comparison Jonckheere-Terpstra trend test AND/OR One-way repeated Kruskall-Wallis test measure analysis o f followed with Dunn's variance;*-5-*followed, test with Dunnett's test a If more than 1 mouse was eliminated from a group, statistics were not done on the data for that group. b Positive control dpm data was not included in the statistical analysis. c Preliminary test not required when running only the Jonckheere-Terpstra trend test. d If the Shapiro-Wilk test was not significant but Levene's test was significant, a robust version of Dunnett's test was used. -8iji h " - - H-23960: Local Lymph Node Assay (LLNA) DuPont-4199 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. Stim ulation Index D ata (Table 3) No statistically significant differences were seen in cell proliferation measurements at any test concentration. Stimulation indexes were less than 3.0 at all concentrations o f H-23960. CONCLUSIONS A 25% concentration o f HCA produced a positive dermal response in mice. Therefore, the LLNA test system was valid for this study with H-23960. Under the conditions o f this study, H-23960 did not produce a positive dermal sensitization response in mice. RECORDS AND SAMPLE STORAGE The raw data, and the final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. -9 Company Sanitized. Does not contain TSCA CBI H-23960: Local Lymph Node Assay (LLNA) DuPont-4199 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 o f 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 o f Topical Medicaments Using The Local Lymph Node Assay: An Inter-Laboratory Exercise. J. Toxicol. Environ. Health, P a rt A 53(7), 563-579. 4. 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. 5. Jonckheere, A. R., (1954). "A Distribution-Free K-Sample Test Against Ordered Alternatives," Biometrika 41,133-145. 6. Levene, H. (1960). Robust test for equality o f variances. In Contributions to Probability and Statistics (I. Olkin, ed.), pp 278-292. Stanford University Press, Palo Alto, CA. 7. Shapiro, S.S. and M.B. Wilk, (1965). An analysis o f variance test for normality (complete samples). Biometrika 52, 591-611. 8. Snedecor, G. W. and W. G. Cochran, (1967). Statistical Methods, 6th ed., pp. 246-248, 349 352. The Iowa State University Press, Iowa. 9. Dunnett, C.W., (1955). A multiple comparison procedure for comparing several treatments with a control. J. Amer. Statist. Assoc. 50,1096-1121. 10. Kruskal, W.H. and W.A. Wallis, (1952). Use of ranks in one-criterion analysis o f variance. J. Amer. Statist. Assoc. 47, 583-621. 11. Dunn, O.J., (1964). Multiple contrasts using rank sums. Technometrics 6, 241-252. - 10- D i e s n o ! eoR iiK r' Mi H-23960: Local Lymph Node Assay (LLNA) DuPont-4199 TABLES - 11 - Company Sanitized. Does not contain TSCA CBS H-23960: Local Lymph Node Assay (LLNA) DuPont-4199 TABLE 1 MEAN BODY WEIGHTS(g) GROUP: II IV VI VIII X XII XIV CONCENTRATION (%): 0 5 10 25 50 90 25* DAYS ON TEST 0 20.3 (1.0) 20.4(1.5) 20.4(1.7) 20.2(1.0) 20.1 (1.0) 20.2 (0.9) 20.2(1.0) 5 21.3 (1.0) 21.1 (1.3) 21.1 (1.0) 21.0(1.5) 20.4 (1.1) 20.9 (0.6) 21.3 (0.9) Standard deviation is reported in parenthesis. Statistical methods: Jonckheere-Terpstra trend test was performed on data There were no statistically significant differences at p > 0.05 HCA (positive control). TABLE 2 MEAN BODY WEIGHT GAINS(g) GROUP: CONCENTRATION (%): DAYS ON TEST 0-5 II 0 1.0 (0.7) IV 5 0.6 (0.5) VI 10 0.7 (0.8) VIII 25 0.8 (0.7) X 50 0.3 (0.3) XII 90 0.7 (0.7) XIV 25* 1.1 (0.4) Standard deviation is reported in parenthesis. 0, Statistical methods: Jonckheere-Terpstra trend test was performed on data There were no statistically significant differences at p > 0.05 JC4-0? * HCA (positive control). o r\ - 12- H-23960: Local Lymph Node Assay (LLNA) DuPont-4199 TABLE 3 GROUP n IV VI vm X xn XIV STIMULATION INDEX (SI) DATA MATERIAL TESTED 0% (DMF) 5% H-23960 10% H-23960 25% H-23960 50% H-23960 90% H-23960 25% HCA in DMF (positive control)* n 6 5 6 6 6 6 6 SI N/A 1.11 1.00 1.20 0.77 0.87 4.62 N/A = Not applicable n --Number o f animals per group. There were no statistically significant differences at p>0.01 by Jonckheere-Terpstra trend test and Dunnett's Test. * = DPM Positive control data was not included in the statistical analysis. - 13- Sanitized. Dees m i contain TSC cbf