Document n5QoR4Kxr9Opzv6y8dKnReL6

AR226-3119 TRADE SECRET Study Title H-23745: Local Lymph Node Assay (LLNA) Laboratory Project ID: DuPont-2642 AUTHOR: Gregory S. Ladies, Ph.D. STUDY COMPLETED ON: April 7, 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 Company Sanitized. Does not contain TSCA CBI Page 1 of 1: CERTIFICATION I, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Issued by Study Director: 6 ' ^ J ^ igory S. Laolcs, Ph.D. Senior Research Toxicologist ?-A^/^ Dale Co^nySam.lze-.Do.sno.con.alnTSCACB, 2- TABLE OF CONTENTS Page CERTIFICATION........................................................................................................................^ STUDY INFORMATION.............................................................................................................4 STUDY PERSONNEL...................................................................................................................5 SUMMARY ....................................................................................................................................6 INTRODUCTION..........................................................................................................................? MATERIALS AND METHODS ..................................................................................................7 A. Test Substance .....................................................................................................................7 B. Protocol................................................................................................................................7 C. Statistical Analyses..............................................................................................................7 RESULTS AND DISCUSSION....................................................................................................8 A. Clinical Signs ofToxicity, Body Weights, and Body Weight Gains...................................8 B. Stimulation Index Data........................................................................................................8 CONCLUSIONS............................................................................................................................8 RECORDS AND SAMPLE STORAGE ......................................................................................8 TABLES.........................................................................................................................................^ 1. MEAN BODY WEIGHTS (g)................................................................................................10 2. MEAN BODY WEIGHT GAINS (g)......................................................................................10 3. STIMULATION INDEX (SI) DATA .....................................................................................11 fe) .Company Sanitized. Does not contain TSCA CBI .3- STUDY INFORMATION Synonyms/Codes: H-23745 Haskell Number: 23745 Submitter's Notebook Number(s): Composition: Known Impurities: Physical Characteristics: Amber liquid 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: January 13, 1999 / (see report cover page) In-Life Initiated/Completed: January 13, 1999/January 18, 1999 .Company Sanitized. Does not contain TSCA CBi -4- STUDY PERSONNEL Study Director: Management: Primary Technician: Gregory S. Ladies, Ph.D. Judith C. Stadler, Ph.D. Charlene Smith, S.A. Toxicology Report Preparation: Amy L. Williamson Laboratory Veterinarian: Wanda L. West, D.V.M., A.C.L.A.M. It) Company Sanitized. Does not contain TSCA CBI .5- SUMMARY The objective of this study was to evaluate the potential ofH-23745 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, and 100% H-23745 on both ears. Acetone:olive oil (4:1 ratio) was used as the diluting vehicle. A seventh group of 6 female mice were dosed for 3 days with 25% Hexylcmnamaldehyde (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 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-23745. Under the conditions of this study, H-23745 was not a dermal sensitizer. '~T- CompanySanitized. Does not contain TSCA C8! INTRODUCTION The purpose of this study was to examine the dermal sensitization potential ofH-23745 using the mouse local lymph node assay (LLNA). fiL^NA screen with H-23745 [Haskell as conducted in female CBA/JHsd2m3ic7e4(5Ha_r_la_n_S_pr_a_gu_e_D_a_w_le_y,_F_re_d_er_ic_k_, MATERIALS AND METHODS A. Test Substance The test substance, H-23745, was supplied by the sponsor as a amber liquid. Each concentration of test article was prepared in a separate vial and vortexed to mix. 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. B. Protocol The test substance was prepared as a solution in 4:1 acetone:olive oil except the 100% concentration. Twenty-five ul of H-23745 was administered topically per ear to the dorsum of both ears of mice (6 per group) for 3 consecutive days (test days 0-2) at dosages of0% (4:1 acetone:olive oil vehicle), 10%, 25%, 50%, 75%, and 100%. One group of 6 female mice was dosed with 25% hexylcinnamaldehyde (HCA) in 4:1 acetone:olive oil as a positive control. Test days 3-4 were days of rest, followed by intravenous injection of 2 uCi of 125-Iododeoxyuridine ('"ILJdR) per mouse on the morning of test day 5. Any mouse which was not properly injected with the appropriate amount of ^lUdR 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 ~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. C. Statistical Analyses Calculations were done using the dpm data. A Stimulation Index (SI) of greater than or equal to 3.0 is considered a positive response. Data, other than for the positive control, 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. r.^ .,, finpany Sanitized. Does not contain 7- 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-23745. A stimulation index of 5.71 was observed for the positive control HCA. 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-23745. Under the conditions of this study, H-23745 was not a dermal sensitizer. 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. Company Sanitized. Does not contain TSCA C81 -8-~~~"~-- TABLES CompanySam'ffzecf. Does not contain TSCA CBf -9- H-23745: Local Lymph Node Assay (LLNA) %7 TABLE 1 MEAN BODY WEIGHTS (g) GROUP: CONCENTRATION (%): DAYS ON TEST 0 5 II 0 20.4( 0.9)3 20.9( 0.6) IV 10 20.3( 0.8) 20.8( 0.9) VI 25 20.3(0.8) 21.0(0.9) VIII 50 20.1(0.5) 20.5(1.0) a 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 * HCA (positive control) X 75 20.3( 0.9) 21.0(1.4) XII 100 20.3(0 21.0(1 TABLE 2 MEAN BODY WEIGHT GAINS (g) GROUP: CONCENTRATION (%): DAYS ON TEST 0-5 II 0 0.5(0.6)' IV 10 0.5(0.3) VI 25 0.6( 0.4) VIII 50 0.4( 0.8) a 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 * HCA (positive control) X 75 0.8( 0.6) XII 100 0.7( 0 -10- TABLE 3 STIMULATION INDEX (SI) DATA GROUP II IV VI VIII X XII xrv MATERIAL TESTED 0% (4:1 acetone:olive oil vehicle) 10%H-23745 25% H-23745 50% H-23745 75% H-23745 100% H-23745 25% HCA in 4:1 acetone:olive oil (positive control) N/A = Not Applicable SI N/A 0.82 0.82 0.82 0.24 0.53 5.71 fiempanySanitized. Does no( contain T8CA CBI