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AR226-3129 TRADE SECRET Study Title H-24119: Local Lymph Node Assay (LLNA) Laboratory Project ID: DuPont-3324 AUTHOR: Gregory S. Ladies, Ph.D. STUDY COMPLETED ON: 10-Aug-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 NUMBEQR:.|^^^--^B1I ^^^^" SERVICE CODE NUMBER:fi--------V Page 1 of 12 Company Sanitized. Does not contain TSCA CB1 CERTIFICATION ,^. ^ ^ . ( ^ ^ ' 1 0 - ^ - 1 ^ I, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. ,'\ I Issued by Study Director: Gtfgory S. Ladies, Ph.D. Date Senior Research Toxicologist Company Sanitized. Does nol conlam TSCA CB1 H-24119: Local Lymph Node Assay (LLNA)______________________________DuPont-3324 TABLE OF CONTENTS Page CERTIFICATION.........................................................................................................................^ STUDY INFORMATION.............................................................................................................. 4 STUDY PERSONNEL................................................................................................................... 5 6 SUMMARY.................................................................................................................................... INTRODUCTION..........................................................................................................................? 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 .................................................................................................. 7 F. Statistical Analyses............................................................................................................. 8 RESULTS AND DISCUSSION..................................................................................................... 8 A. Clinical Signs ofToxicity, Body Weights, and Body Weight Gains (Tables 1-2)............. 8 B. Stimulation Index Data (Table 3)....................................................................................... 8 8 CONCLUSIONS............................................................................................................................. RECORDS AND SAMPLE STORAGE........................................................................................ 9 9 REFERENCES............................................................................................................................... TABLES ....................................................................................................................................... 10 1^ k) Company Sanitized. Does not contain TSCA CBS STUDY INFORMATION 9th Collective Nomenclature: Synonyms/Codes: H-24119 Haskell Number: 24119 CAS Registry Number: f l ^ B B ^ --M--S--D--S----N--u--m----b--er--:------------------^^^---------------- l U j m Submi tier's Notebook Nun 1111i('i) | punh-yrfl Known Impurities Physical Characteristics Stability: 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: 16-Jun-1999 / (see report cover page) In-Life Initiated/Completed: 16-Jun-1999-22-Jun-1999 Company Sanitized. Doss not contain TSCA CB1 ^ Company Sanitized. Does not contain TSCA CBf STUDY PERSONNEL Study Director: Gregory S. Ladies, Ph.D. Management: Judith C. Stadler, Ph.D. Primary Technician: Charlene Smith, S.A. Toxicology Report Preparation: Wonda K. Kelly Laboratory Veterinarian: Wanda L. West, D.V.M., A.C.L.A.M. Charles E. Cover, V.M.D. ^ / Company Sanftfzed. Does not contain TSCA CB1- SUMMARY The objective of this study was to evaluate the potential of H-24119 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), 10,25,50,75, or 100% H-24119 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 of the assay, mice received 125-Iododeoxyuridine 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 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-24119. Under the conditions of this study, H-24119 was not a dermal sensitizer. l) Company S.nl,..d. Doe, o, con,.,n TSCA CB, INTRODUCTION The purpose of this study was to examine the dermal sensitization potential of H-24119 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 of the test article was evaluated by measuring the proliferation of lymphocytes (via radiolabel uptake) obtained from the. auricular lymph nodes (i.e., the lymph nodes that drain the ears). Because H-24119 is aHUBand 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-24119 was conducted in female CBA/JHsd mice. On l-Jun-1999,47 female CBA/JHsd mice with an assigned birth date of 16-Apr-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.^'2'^ B. Test Substance ^U^HB^lLF^6 The test substance, H-24119, was supplied by the sponsor ^^^^^^^^^l_ test substance was inverted to mix before each amount for 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. C. Body Weight 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. E. Local Lymph Node Assay The test substance was prepared as a solution in DMSO except the 100% concentration. Twenty-five /xl of H-24119 was administered topically to the dorsum of each ear of mice (6 per Sanitized. Does not contain TSCA CB1 _________________________________3?mpany H-24119: Local Lymph Node Assay (LLNA)____________________________DuPont-3324 group) for 3 consecutive days (test days 0-2) at dosages of 0% (DMSO vehicle), 10%, 25%, 50%, 75%, and 100%. One group of 6 female mice was dosed with 25% hexylcinnamaldehyde (HCA) as a positive control. Test days 34 were days of rest, followed by intravenous injection of 2 fid of 125-Iododeoxyuridine ('^ludR) per mouse on the morning of 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. The cell proliferation data of one mouse in the 100% test article group which was not properly injected with the appropriate amount of ^ludR was excluded from study. F. Statistical Analyses Calculations were done using the dpm data. Any mouse with a dpm value less than or equal to 0 was eliminated from the study. A Stimulation Index (SI) of greater than or equal to 3.0 is considered a positive response. Cell Proliferation data were also analyzed for statistical significance using Jonckheere's Trend Test when a monotone dose response was evident. If a monotone dose response was not evident, either Dunnett's or Dunn's Multiple Comparison Procedure was used. Body weight and body weight gain data were analyzed using a one-way Analysis of Variance and Dunnett's test. The significance level was judged at p < 0.05. RESULTS AND DISCUSSION A. Clinical Signs of Toxicity, Body Weights, and Body Weight Gains (Tables 1-2) 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. 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-24119. Under the conditions of this study, H-24119 was not a dermal sensitizer. CONCLUSIONS A 25% concentration of HCA produced a positive dermal sensitization response in mice. Therefore, the LLNA test system was valid for this study with H-24119. Under the conditions of this study, H-24119 was not a dermal sensitizer. Company Sanitized. Does nol contain TSCA CBI 9 H-24119: Local Lymph Node Assay (LLNA)______________________________DuPont-3324 RECORDS AND SAMPLE STORAGE Specimens (if applicable), raw data, and the final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. 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 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 Procedures. Toxicology 103:63-73. 3. Kimber, I., Hilton, J., Dearman, R.J., 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 InterLaboratory Exercise. J. Toxicol. Environ.. Health, Part A 53(7):563-579. '"'ampany Sanitized. Dca" not contain TSCA f.R^ 10 TABLES - . ^mpany Sanitized. Does not contain TSCA CBi Tl ^ H-24119: Local Lymph Node Assay (LLNA) TABLE 1 MEAN BODY WEIGHTS (g) GROUP: CONCEISTTRATION (%): NUMBER OP ANDMALS/GROUP DAYS ON TEST 0 5 n 0 6 20.8 (1.6)* 21.4(1.8) IV 10 6 21.1(1.7) 22.2(1.5) \ /I 2 5 6 21.1 (1 .1) 21.2 (1 .1) vin 50 6 20.8 (1.7) 21.2(1.6) - K 7 5 6 20.8 (1.8) 20.7 (1.4) * Standard deviation is reported in parentheses. Statistical methods: One-way Analysis of Variance and Dunnett's tests were performed on data There were no statis tically significant differences at p > 0.05 xn 10 6 20.6( 20.7 TABLE 2 GROUP: CONCENTRATION (%): NUMBER OF ANIMALS/GROUP DAYS ON TEST 0-5 MEAN BODY WEIGHT GAINS (g) n IV VI vm 0 10 25 50 6 6 6 6 0.7 (0.4)* 1.1 (0.6) 0.0 (0.6) 0.5 (0.6) X x 75 1 6 6 -0.1 (0.5) 0.0 * Standard deviation is reported in parentheses. 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 Company Sanitize 11 TABLES GROUP n IV VI Vffl X xn xrv STIMULATION INDEX (SI) DATA n MATERIAL TESTED 6 0% (DMSO) 6 10%H-24119 6 25%H-24119 6 50%H-24119 6 75%H-24119 5 100%H-24119 6 25% HCA in DMSO (positive control) N/A = Not Applicable n = Number of animals per group There were no statistical differences at p > 0.05. SI N/A 0.79 0.38 0.65 0.83 0.54 7.81 C"miiany Sanitized, Do-ss not conlam T3CA CBI 12