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AR226-3119 DuPont-2642 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 TS CA CBi Page 1 o f 11 H-23745: Local Lymph Node Assay (LLNA) DuPont-2642 CERTIFICATION I, the undersigned, declare that this report provides an accurate evaluation o f data obtained from this study. , Issued by Study Director: Jd k d ^ f Date Company Sanitized. Does not contain TSCA CBI -2 - H-23745: Local Lymph Node Assay (LLNA) DuPont-2642 TABLE OF CONTENTS CERTIFICATION......................................................................................... STUDY INFORMATION............................................................................. STUDY PERSONNEL................................................................................... SUMMARY..................................................................................................... INTRODUCTION.......................................................................................... MATERIALS AND METHODS................................................................. A. Test Substance..................................................................................... B. Protocol................................................................................................ C. Statistical Analyses............................................................................. RESULTS AND DISCUSSION................................................. ................. A. Clinical Signs of Toxicity, Body Weights, and Body Weight Gams B. Stimulation Index Data....................................................................... CONCLUSIONS........................................................................................... RECORDS AND SAMPLE STORAGE................................................... TABLES.......................................................................................................... 1. MEAN BODY WEIGHTS (g).......................................................... *..... 2. MEAN BODY WEIGHT GAINS (g)...................................................... 3. STIMULATION INDEX (SI) DATA..................................................... Page .Company Sanitized. Does not contain TSCA CBI H-23745: Local Lymph Node Assay (LLNA) STUDY INFORMATION Haskell Number: 23745 DuPont-2642 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 Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Completed: January 13, 1999 / (see report cover page) In-Life Initiated/Comnleted: January 13, 1999 / January 18, 1999 Company Sanitized. Does not contain TSCA CBI -4 - H-23745: Local Lymph Node Assay (LLNA) STUDY PERSONNEL Study Director: Gregory S. Ladies, Ph.D. Management: Judith C. Stadler, Ph.D. Primary Technician: Charlene Smith, S.A. Toxicology Report Preparation: Amy L. Williamson Laboratory Veterinarian: Wanda L. West, D.V.M., A.C.L.A.M. DuPont-2642 Company Sanitized. Does not contain TS CA CBI -5 - H-23745: Local Lymph Node Assay (LLNA) DuPont-2642 SUMMARY The objective of this study was to evaluate the potential of H-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. Acetonerolive oil (4:1 ratio) 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-23745. Under the conditions of this study, H-23745 was not a dermal sensitizer. Company Sanitized. Does not contain TSCA CBI H-23745: Local Lymph Node Assay (LLNA) DuPont-2642 INTRODUCTION The purpose of this study was to examine the dermal sensitization potential of H-23745 using the mouse local lymph node assay (LLNA). ,TheiJ.N A screen with H-23745 [Haskell 23745 f j j ^ H l v a s conducted in female CBA/JHsd mice 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 acetoneiolive oil except the 100/o concentration. Twenty-five pi 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 of 0/o (4.1 acetoneiolive oil vehicle), 10%, 25%, 50%, 75%, and 100%. One group of 6 female mice was dosed with 25% hexylcinnamaldehyde (HCA) in 4:1 acetoneiolive oil as a positive control. Test days 3-4 were days of rest, followed by intravenous injection of 2 pCi of 125-Iododeoxyuridine (125IUdR) per mouse on the morning of test day 5. Any mouse which was not properly injected with the appropriate amount of 125IUdR 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 ~4 C 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. Company Sanitized. Does not contain -7 - ^ H-23745: Local Lymph Node Assay (LLNA) DuPont-2642 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 seen in cell proliferation measurements at any test concentration. Stimulation indexes were less than 3.0 at all concentrations of H-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 fmal report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. Company Sanitized. Does not contain TS CA CBI H-23745: Local Lymph Node Assay (LLNA) DuPont-2642 TABLES Company Sanitized. Does not contain t s o a OB* H-23745: Local Lymph Node Assay (LLNA)____________________ ________________________________________________________ DuPont-2642 TABLE 1 MEAN BODY WEIGHTS (g) GROUP: CONCENTRATION (%): DAYS ON TEST 0 5 II 0 20.4( 0.9)a 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(0.8) 21.0( 1.1) XIV* 25 20.4( 0.7) 21.3( 1.1) TABLE 2 MEAN BODY WEIGHT GAINS (g) GROUP: CONCENTRATION (%): DAYS ON TEST 0-5 II 0 0.5( 0.6)a 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( 0.4) XIV* 25 0.8( 0.8) -10- Company Sanitized. Does not contain t r c a o r H-23745: Local Lymph Node Assay (LLNA) DuPont-2642 TABLE 3 STIMULATION INDEX (SI) DATA GROUP II IV VI VIII X XII XIV 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 -11 - Company Sanitized. Does not contain TSCA CBI