Document 6wjZgjLjxxJVppo88Lwqg9QY1

AR226-3129 DuPont-3324 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 Nem ours and Com pany Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Work Request Number: Service Code N u M B E R :Q d ^ 3 Company Sanitized. Does not contain TSA CB1 Page 1 of 12 H-24119: Local Lymph Node Assay (LLNA) CERTIFICATION DuPont-3324 I, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Issued by Study Director: gory S. Ladies, Ph.D. Senior Research Toxicologist ISi Date Company Sanitized. Does not contain TSC A CB1 2 H -24119: Local Lymph Node Assay (LLNA) TABLE OF CONTENTS DuPont-3324 Page C E R T IF IC A T IO N ............................................................................................................................................2 STU D Y IN FO R M A T IO N ..............................................................................................................................4 STU D Y P E R S O N N E L ....................................................................................................................................5 SU M M A R Y ....................................................................................................................................................... 6 IN T R O D U C T IO N ............................................................................................................................................7 M A TERIALS AN D M ETH O D S...................................................................................................................7 A. T est S p e c ie s......................................................................................................................................... 7 B. T est S u b stan c e .....................................................................................................................................7 C. Body W eight M easurem ents............................................................................................................. 7 D . Clinical O bservations..........................................................................................................................7 E. Local Lym ph N ode A s s a y ................................................................................................................ 7 F. Statistical A n aly ses............................................................................................................................. 8 RESU LTS AND D ISC U SS IO N .................................................................................................................... 8 A. Clinical Signs of Toxicity, B ody W eights, and B ody W eight Gains (Tables 1-2)............... 8 B. Stim ulation Index D ata (Table 3 ) .................................................................................................... 8 CO N CLU SIO N S................................................................................................................................................8 RECO RD S AND SA M PLE S T O R A G E ..................................................................................................... 9 R E F E R E N C E S .................. 9 T A B L E S ........................................................................................................................................................... 10 Company Sanitized. Does not contain TSCA CB! 3 H-24119: Local Lymph Node Assay (LLNA) STUDY INFORMATION DuPont-3324 Haskell Number: 24119 CAS Registry Number: M SD S Number: ___ Submitter's Notebook Number(s): Purity 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 Pont 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 4 H-24119: Local Lymph Node Assay (LLNA) DuPont-3324 Company Sanitized. Does not contain TSCA CBf 5 H-24119: Local Lymph Node Assay (LLNA) STUDY PERSONNEL Study Director: Gregory S. Ladies, Ph.D. M anagem ent: Judith C. Stadler, Ph.D. Primary Technician: Charlene Smith, S.A. Toxicology Report Preparation: W onda K. Kelly Laboratory Veterinarian: W anda L. West, D.V.M., A.C.L.A.M. Charles E. Cover, V.M.D. DuPont-3324 Company Sanitized. Does not contain TSCA C B f 6 H -24119: Local Lymph Node Assay (LLNA) DuPont-3324 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), 1 0 ,2 5 ,5 0 ,7 5 , or 100% H -24119 on both ears. D im ethylsulfoxide was used as the diluting vehicle. A seventh group o f 6 fem ale mice were dosed for 3 days with 25% Hexylcinnamaldehyde (HCA) as a positive control. On day 5 of the assay, m ice 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. Stim ulation 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. T herefore, the LLN A test system was valid for this study with H -24119. U nder the conditions o f this study, H -24119 was not a dermal sensitizer. Company Sanitized. Does not contain TSCA CBI 7 H -24119: Local Lymph Node Assay (LLNA) DuPont-3324 INTRODUCTION The purpose of this study was to exam ine the derm al sensitization potential o f 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 fr^ ^ d ^ a u ric u la r lymph nodes (i.e., the lymph nodes that drain the ears). Because H -24119 is a H ^ 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 Lym ph N ode Assay screen with H -24119 was conducted in female CBA /JH sd 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 m ouse 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 LLN A .(1,2,3) B. Test Substance The test substance, H -2 4 1 19, was supplied by the sponsor as 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 W eight Measurements All m ice were w eighed 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 DM SO except the 100% concentration. Tw enty-five /xl of H -24119 was administered topically to the dorsum of each ear of m ice (6 per Company Sanitized. Does not contain TSCA CB1 8 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 3-4 were days of rest, followed by intravenous injection o f 2 iCi of 125-Iododeoxyuridine (125IudR) per m ouse on the m orning 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. The cell proliferation data of one mouse in the 100% test article group w hich was not properly injected w ith the appropriate am ount o f 125IudR was excluded from study. F. Statistical Analyses Calculations were done using the dpm data. A ny 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 m onotone dose response was evident. If a m onotone dose response was not evident, either D unnett's or D unn's M ultiple Com parison Procedure was used. Body weight and body weight gain data were analyzed using a one-way Analysis of Variance and D unnetf s test. The significance level was judged at p < 0.05. RESULTS AND DISCUSSION A. Clinical Signs of Toxicity, Body Weights, and Body W eight 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. Stim ulation indexes were less than 3.0 at all concentrations o f H -24119. U nder the conditions o f this study, H -24119 w as not a dermal sensitizer. - CONCLUSIONS A 25% concentration o f HCA produced a positive dermal sensitization response in mice. Therefore, the LLN A test system was valid for this study with H -24119. U nder the conditions of this study, H -24119 was not a dermal sensitizer. Company Sanitized. Does not contain TSCA CBI 9 H-24119: Local Lymph Node Assay (LLNA) RECORDS AND SAMPLE STORAGE DuPont-3324 Specimens (if applicable), raw data, and the final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, W ilmington, 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 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 M urine Local Lymph N ode Assay and Com parison o f M odified Procedures. Toxicology 103:63-73. 3 K im ber I. H ilton, J., Dearm an, 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 M edicaments Using The Local Lymph Node Assay: An Inter Laboratory Exercise. J. Toxicol. Environ.. Health, Part A 53(7).563-579. "'.ompany Sanitized. Dess not contain t s c a CBk 10 H-24119: Local Lymph Node Assay (LLNA) DuPont-3324 TABLES ompany Sanitized. Does not contain TSCA CBS H -24119: Local Lymph Node Assay (LLNA) DuPont-3324 TABLE 1 M EAN BO D Y W EIGHTS (g) GROUP: CO NCENTRATION (%): NUM BER OF ANIM ALS/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) VI 25 6 21.1 (1.1) 21.2(1.1) vrn 50 6 20.8 (1.7) 21.2(1.6) X 75 6 20.8 (1.8) 20.7 (1.4) * Standard deviation is reported in parentheses. Statistical m ethods: O ne-w ay A nalysis o f V ariance and D u n n e tt's tests w ere perform ed on data There were no statistically significant differences at p > 0.05 xn XIV 100 25 66 20.6(1.5) 20.7 (1.5) 20.5 (1.5) 20.9(1.1) TABLE 2 GROUP: CONCENTRATION (%): NUM BER OF ANIM ALS/GROUP DAYS ON TEST 0-5 M EAN BODY W EIGHT GAINS (g) II IV VI vm 0 10 25 50 6 666 0.7 (0.4)* 1.1 (0.6) 0.0 (0.6) 0.5 (0.6) X xn 75 100 66 -0.1 (0.5) 0.0 (0.4) XIV 25 6 0.4 (0.5) * Standard deviation is reported in parentheses. Statistical m ethods: O ne-w ay A nalysis o f V ariance and D u n n e tt's tests w ere perform ed on data There were no statistically significant differences at p > 0.05 Company aSaanniittfirzeed. Poeo nof_ 11 contain TSCA CBI H -24119: Local Lymph Node Assay (LLNA) DuPont-3324 TABLE 3 GROUP n TV VI vm 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 = N um ber 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'Tttipany Sanitized. Dc?s not contain TSCACW 12