Document QgeaxJGrxveyoXOeBRr8Zgme8

AR226-3150 TRADE SECRET Study Tide H-23960: Local Lymph Node Assay (LLNA) Laboratory Project ID: DuPont-4199 AUTHOR: Gregory S. Ladies, Ph.D., D.A.B.T. STUDY COMPLETED ON; May 4,2000 PERFORMING LABORATORY: E. L du Font de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 E_^^l WORK REQUEST NUMBER: fl^^B ( l^^^^^w- j SERVICE CODE NUMBERJj-- 7 Page 1 of 13 companySanitized. Does not contain TSCA CBi CERTIFICATION I, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Issued by Study Director: D.A.B.T.Date /"^W^ _) ' OStM^_____ Gregory ^/Ladies, Ph.D.. Senior Research Scientist ^- f^AY'ol^ Q -2- Doe iiO? contain TSCA CBj TABLE OF CONTENTS Page CERTIFICATION..........................................................................................................................^ LIST OF TABLES..........................................................................................................................3 STUDY INFORMATION...............................................................................................................4 STUDY PERSONNEL................................................................................................................... 5 6 SUMMARY.................................................................................................................................... INTRODUCTION...........................................................................................................................? MATERIALS AND METHODS.................................................................................................... 7 A. Test Species......................................................................................................................... 7 B. Body Weight Measurements............................................................................................... 7 C. Test Substance..................................................................................................................... 7 D. Clinical Observations..........................................................................................................7 E. Local Lymph Node Assay................................................................................................... 8 F. Statistical Analyses .............................................................................................................8 RESULTS AND DISCUSSION..................................................................................................... 9 A. Clinical Signs ofToxicity, Body Weights, and Body Weight Gains.................................. 9 B. Stimulation Index Data........................................................................................................ 9 CONCLUSIONS.............................................................................................................................9 RECORDS AND SAMPLE STORAGE......................................................................................... 9 10 REFERENCES.............................................................................................................................. 11 TABLES........................................................................................................................................ 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 -3- BaBlpany aniS?zsd. Boas not contain T8CA CBS H-23960: Local Lymph Node Assay (LLNA) STUDY INFORMATION Substance Tested: U^^^ff^ -1 Synonyms/Codes: Haskell Number: 23960 Composition DuPont-4199 Physical Characteristics: Stability7: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed. Sponsor: E. I. du Font de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Completed: April 12, 2000 / (see report cover page) In-Life Initiated/Completed: April 22, 2000 / April 28, 2000 Qt, H-23960: Local Lymph Node Assay (LLNA)______________________________DuPont-4199 STUDY PERSONNEL The following individuals participated in the conduct of 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. SUMMARY The objective of this study was to evaluate the potential ofH-23960 to produce a dermal sensitizatidn 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 90% H-23960 on the dorsal side of both ears. N,N-dimethylformamide (DMF) was used as the diluting vehicle. A seventh group of 6 female mice was dosed for 3 days with 25% Hexylcinnamaldehyde (HCA) as a positive control. On test day 5 of the assay, mice received ^-Thymidine by tail vein injection 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 oftoxicity 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 of 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 of this study, H-23960 did not produce a positive dermal sensitization response in mice. Company Sanitized. Ooss not contain TSCA CBI INTRODUCTION The purpose of this study was to examine the dermal sensitization potential ofH-23960 using the local lymph node assay (LLNA).. Following the topical application of the test article to the dorsal side of both ears, the dermal sensitization potential of the test article was evaluated by measuring the proliferation oflymphocytes (via radiolabel uptake) obtained from the^auricular qBlgnd lymph nodes (i.e., the lymph nodes that drain the ears). Because H-23960 is did 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 of 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 with this strain. Furthermore, the strain has undergone extensive interiaboratory validation with the LLNA.0'^ B. Body Weight Measurements 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 ^HHH|||BB|HBBIB> UBjThe test substance was vortexed to mix before each amount tor dosing 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. 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. H-23960: Local Lymph Node Assay (LLNA) DuPont-4199 E. Local Lymph Node Assay The test substance was prepared as a solution in DMF. Twenty-five ^il of H-23960 was administered topically to the dorsum of each ear of mice (6 per group) for 3 consecutive days (test days 0-2) at dosages of0% (DMF vehicle), 5%, 10%, 25%, 50%, and 90%. One group of 6 female mice was dosed with 25% hexylcinnamaldehyde (HCA) as apositive control. Test days 3-4 were days of rest followed by intravenous injection of20jxCi of H-Thymidine per mouse on the morning of test day 5. One mouse in the 5% dose group was not properly injected with the appropriate amount of^-Thymidine and was excluded from study. 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 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 of the vehicle control group. Statistical significance in the test article groups compared to control and/or SIs of 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 Data8'" Preliminary Test Test for lack of trend Levene's test for homogeneity^ and Shapiro-Wilktest^ for normality0111 Method of Statistical Analysis If preliminary test is If preliminary test is not significant significant Sequential application^ of the Preliminary tests for pairwise comparison Jonckheere-Terpstra trend test^ AND/OR One-way repeated measure analysis of variance;^ followed, Kruskall-Wallis test^ followed with Dunn's test^ with Dunnett's test If more than 1 mouse was eliminated from a group, statistics were not done on the data for that group. Positive control dpm data was not included in the statistical analysis. Preliminary test not required when running only the Jonckheere-Terpstra trend test. If the Shapiro-Wilk test was not significant but Levene's test was significant, a robust version ofDunnett's test was used. r-ip K--!^,--.'--'-,-'-'' n'- RESULTS AND DISCUSSION A. Clinical Signs of Toxicity, Body Weights, and Body Weight Gains (Tables 1-2) There were no clinical signs oftoxicity 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 seen in cell proliferation measurements at any test concentration. Stimulation indexes were less than 3.0 at all concentrations ofH-23960. CONCLUSIONS A 25% concentration ofHCA produced a positive dermal response in mice. Therefore, the LLNA test system was valid for this study with H-23960. Under the conditions of 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 CB8 H-23960: Local Lymph Node Assay (LLNA) DuPont-4199 REFERENCES 1. Loveless, S.E., Ladies, G.S., Gerberick, G.F., Ryan, C.A., Basketter, DA., Scholes, E.W., House, R.V., Hilton, J., Dearman, R.J., and Kimber, L (1996). Further Evaluation of 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 of the Murine Local Lymph Node Assay and Comparison of 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 of The Skin Sensitization Potential of Topical Medicaments Using The Local Lymph Node Assay: An mter-Laboratory Exercise. J. Toxicol. Environ. Health, Part A 53(7), 563-579. 4. Selwyn, M. R., (1995). The Use of Trend Tests to Determine a No-Observable-Effect Level in Animal Safety Studies. Journal of the American College of 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 of variances. In Contributions to Probability and Statistics (I. OUdn, ed.), pp 278-292. Stanford University Press, Palo Alto, CA. 7. Shapiro, S.S. and M.B. Wilk, (1965). An analysis of 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, 349352. 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 of variance. J. Amer. Statist. Assoc. 47, 583-621. 11. Dunn, O.J., (1964). Multiple contrasts using rank sums. Technometrics 6, 241-252. -10- ^orOT^w^emiSizcj, O-ag no'; eoni' H-23960: Local Lymph Node Assay (LLNA) DuPont-4199 TABLES -11- 'Company Sanitized. Does not contain TSCA CSi H-23960: Local Lymph Node Assay (LLNA) TABLE 1 MEAN BODY WEIGHTS(g) GROUP: CONCENTRATION (%): DAYS ON TEST 0 5 II 0 20.3 (1.0) 21.3 (1.0) IV VI vni X xn X 5 10 25 50 90 2 20.4(1.5) 20.4(1.7) 20.2(1.0) 20.1 (1.0) 20.2 (0.9) 20.2 21.1 (1.3) 21.1 (1.0) 21.0(1.5) 20.4(1.1) 20.9 (0.6) 21.3 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 VIII 10 25 0.7 (0.8) 0.8 (0.7) X XII X 50 90 2 0.3 (0.3) 0.7 (0.7) 1.1 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). -12- H-23960: Local Lymph Node Assay (LLNA) DuPont-4199 TABLES STIMULATION INDEX (SI) DATA GROUP MATERIAL TESTED n SI n 0% (DMF) 6 N/A IV 5% H-23960 5 1.11 VI 10% H-23960 6 1.00 vm 25% H-23960 x 50% H-23960 6 1.20 6 0.77 xn 90% H-23960 XIV 25%HCAinDMF 6 0.87 6 4.62 (positive control)* N/A == Not applicable n = Number of animals per group. There were no statistically significant differences at p>0.01 by Jonckheere-Terpstra trend test and Dmmett's Test. * = DPM Positive control data was not included in the statistical analysis. -13- , not contain TSCA CB?