Optimization of the Sliced Testis Steriodogenesis Assay Unaudited Final Draft Report RTI International Laboratory of Reproductive and Endocrine Toxicology Chemistry and Life Sciences Group Center for Life Sciences and Toxicology Post Office Box 12194 Research Triangle Park, NC 27709 2194 Battelle Memorial Institute 505 King Avenue Columbus, OH 43201 2693 Optimization of the Sliced Testis Steriodogenesis Assay Unaudited Draft Final Report RTI International Laboratory of Reproductive and Endocrine Toxicology Chemistry and Life Sciences Group Center for Life Sciences and Toxicology Post Office Box 12194 Research Triangle Park, NC 27709 2194 Battelle Memorial Institute 505 King Avenue Columbus, OH 43201 2693 EPA Contract No.: 68 W 01 023 ( Battelle Prime Contractor, WA 2 27) Contract No.: 08055.001.020 Study Code: Rt02 OPST Master Protocol No.: RTI 870 Table of Contents iii TABLE OF CONTENTS Section Page 1.0 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 1.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 1.2 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 2.0 MATERIALS AND CHEMICALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 2.1 Reagents and Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 2.2 Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 3.0 METHOD FOR PROTOTYPICAL ASSAY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1 4.0 METHODS FOR PHASE I   PRELIMINARY EXPERIMENTAL PHASE . . . . . . . . 4 1 4.1 Testosterone Radioimmunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 4.2 LDH Spectrophotometric Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2 4.3 Phase 1 Optimization Experiments for Media Type, Gaseous Atmosphere, Rat Age, and Storage Container Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3 4.4 Phase 1 Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 4 4.5 Phase 1 Data Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 6 5.0 RESULTS AND STATISTICAL ANALYSES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1 5.1 Testosterone RIA Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1 5.2 LDH Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2 5.3 Statistical Analysis of the Phase I Assay Optimization Experiment . . . . . . . . . 5 2 5.4 Statistical Analysis Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 4 6.0 STATISTICAL ANALYSIS OF THE PHASE IB ASSAY OPTIMIZATION EXPERIMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1 6.1 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1 6.2 Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1 6.3 Statistical Analysis Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1 7.0 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1 8.0 CONCLUSIONS FOR PHASE I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1 9.0 INCUBATION EXPERIMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1 9.1 Statistical Analysis of Phase II Incubation Optimization Experiment . . . . . . . 9 2 10 TESTES PREPARATION EXPERIMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 1 10.1 Statistical Analysis of Phase II Testis Preparation Optimization Experiment 10 1 11 EQUILIBRATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 1 12 SAMPLING TIMES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 1 13 INDUCED PARENCHYMAL FRAGEMENT INJURY . . . . . . . . . . . . . . . . . . . . . . 13 1 Table of Contents iv LIST OF FIGURES No. Page 1 Sliced Testis Steroidogenesis Assay Experimental Design Organization Diagram . . . 1 2 2 Technical Flow Illustration of the Sliced Testis Steroidogenesis Assay . . . . . . . . . . . 3 2 LIST OF TABLES No. Page 1 Summary of Experimental Factors for Phase 1 Optimization . . . . . . . . . . . . . . . . . . . 4 5 2 Factorial Test Conditions for Phase 1 Optimization Experiment . . . . . . . . . . . . . . . . . 4 5 3 Values of Standards on Testosterone RIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1 4 Testosterone RIA Intra Assay CV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2 5 Testosterone RIA Percent Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2 6 Testosterone RIA Parallelism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2 7 Factor Levels in the Phase I Assay Optimization Experiment . . . . . . . . . . . . . . . . . . . 5 6 8 Data Listing for Samples Without hCG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 7 9 Data Listing for Samples With hCG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 8 10 Summary of Data   Original Scale, Without Scale, Without hCG . . . . . . . . . . . . . . . 5 11 11 Summary of Data   Log Scale, Without hCG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 12 12 Summary of Data   Original Scale, With hCG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 15 13 Summary of Data   Log Scale, With hCG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 16 14 Summary of Statistical Analysis of Baseline Data . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 17 15 Summary of ANOCOVA Results for Individual Hour Original Scale Models . . . . . 5 18 16 Adjusted Mean Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 19 17 Summary of ANOCOVA Results for Individual Hour Log Scale Models . . . . . . . . 5 23 18 Adjusted Mean Levels Based on Log Scale Models . . . . . . . . . . . . . . . . . . . . . . . . . 5 24 19 Least Squares Means for Reduced Log scale Model: Without hCG . . . . . . . . . . . . . 5 28 20 Least Squares Means for Reduced Original scale Model: Without hCG . . . . . . . . . . 5 28 21 Least Squares Means for Reduced Log scale Model: With hCG . . . . . . . . . . . . . . . . 5 29 22 Least Squares Means for Reduced Original scale Model: With hCG . . . . . . . . . . . . 5 29 23 Analysis of Differences in Levels for With and Without hCG Stimulation Original scale Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 30 24 Adjusted Mean Differences ( With Without hCG), Based on Original Scale Models 5 30 25 Analysis of Differences in Levels for With and Without hCG Stimulation: Log Scale Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 32 26 Adjusted Mean Differences ( With Without hCG), Based on Log Scale Models . . . . 5 32 27 Phase IB Data: Sample Without hCG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 5 28 Phase IB Data: Samples With hCG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 6 29 Summary of Data   Original Scale, Without hCG . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 7 30 Summary of Data  Log Scale, Without hCG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 8 31 Summary of Data  Original Scale, With hCG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 9 Table of Contents v 32 Summary of Data   Log Scale, With hCG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 10 33 Summary of Results by Sample Condition   Original Scale, Without hCG . . . . . . . 6 11 34 Summary of Results by Sample Condition   Log Scale, Without hCG . . . . . . . . . . . 6 12 35 Summary of Results by Sample Condition   Original Scale, With hCG . . . . . . . . . . 6 13 36 Summary of Results by Sample Condition   Log Scale, With hCG . . . . . . . . . . . . . 6 14 37 Incubation Factor Settings and Coded Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3 38 Data Listing for Incubation Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 6 39 Data Listing of Coded Factor Values and Logarithm of Testosterone Concentrations 9 13 40 ANOVA Results for PROC RSREG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 19 41 Response Surface Regression Results for No hCG Stimulation . . . . . . . . . . . . . . . . . 9 20 42 Response Surface Regression Results for hCG Stimulation . . . . . . . . . . . . . . . . . . . . 9 21 43 Maximum Values for Each Time Point . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 23 44 Predicted Values for Specified Optimum Factor Combinations . . . . . . . . . . . . . . . . . 9 24 45 ANOVA Results for PROC RSREG With the Baseline Concentration Removed from Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 25 46 Response Surface Regression Results for No hCG Stimulation with Baseline Concentration Removed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 26 47 Response Surface Regression Results for hCG Stimuation with Baseline Concentration Removed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 28 48 Maximum Values for Models with Baseline Concentration Removed . . . . . . . . . . . 9 29 49 Predicted Values for Specified Optimum Factor Combinations for Models with Baseline Concentration Removed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 32 50 Testis Preparation Factor Settings and Coded Values . . . . . . . . . . . . . . . . . . . . . . . . 10 4 51 Data Listing for Incubation Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 5 52 Data Listing of Coded Factor Values and Logarithm of Testosterone Concentrations 10 7 53 ANOVA Results for PROC RSREG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 11 54 Response Surface Regression Results for No hCG Stimulation . . . . . . . . . . . . . . . . 10 12 55 Response Surface Regression Results for hCG Stimulation . . . . . . . . . . . . . . . . . . . 10 13 56 Maximum Values for Each Time Point . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 15 57 Predicted Values for Specified Optimum Factor Combinations . . . . . . . . . . . . . . . . 10 17 58 ANOVA Results for PROC RSREG with the Results Concentration Removed from Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 18 59 Response Surface Regression Results for No hCG Stimulation with Baseline Concentration Removed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 19 60 Response Surface Regression Results for hCG Stimulation with Baseline Concentration Removed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 20 61 Maximum Values for Models with Baseline Concentration Removed . . . . . . . . . . 10 21 62 Predicted Values for Specified Optimum Factor Combinations for Models with Baseline Concentration Removed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 23 63 Equilibration Data for No hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 2 64 Equilibration Data for hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 5 Table of Contents vi 65 Means of LDH Concentration ( mu/ mg) by Equilibration Time ( minutes) HCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 8 66 Standard Deviations of LDH Concentration ( mU/ mg) by Equilibration Time ( minutes) No hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 8 67 Samples Sizes of LDH Concentration ( mU/ mg) by Equilibration Time ( minutes) No hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 8 68 Coeff of Variation of LDH Concentration ( mU/ mg) by Equilibration Time ( minutes) No hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 9 69 Means of LDH Concentration ( mU/ mg) by Equilibration Time ( minutes) with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 10 70 Standard Deviations of LDH Concentration ( mU/ mg) by Equilibration Time ( minutes) with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 11 71 Sample Sizes of LDH Concentration ( mU/ mg) by Equilibration Time ( minutes) with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 12 72 Coeff of Variation of LDH Concentration ( mU/ mg) by Equilibration Time ( minutes) with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 12 73 Means of Testosterone Concentration ( ng/ mg) by Equilibration Time ( minutes) no hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 12 74 Standard Deviations of Testosterone Concentration ( ng/ mg) by Equilibration Time ( minutes) No hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 13 75 Sample Sizes of Testosterone Concentration ( ng/ mg) by Equilibration Time ( Minutes) No hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 13 76 Coeff of Variation of Testosterone Concentration ( ng/ mg) by Equilibrarion Time ( Minutes) No hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 13 77 Means of Testosterone Concentration ( ng/ mg) by Equilibration Time ( minutes) with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 14 78 Standard Deviations of Testosterone Concentration ( ng/ mg) by Equilibration Time ( minutes) with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 14 79 Sample Sizes of Testosterone Concentration ( ng/ mg) by Equilibration Time ( Minutes) with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 14 80 Coeff of Variation of Testosterone Concentration ( ng/ mg) by Equilibration Time ( minutes) with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 15 81 Summary of Equilibration Experiment: Testosterone Concentration ( ng/ mg) by Time Pont With hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 26 82 Summary of Equilibration Experiment: Testosterone Concentration ( ng/ mg) by Time Point with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 26 83 Summary of Equilibration Experiment: LDH Concentration ( mU/ mg) by Time Point with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 27 84 Summary of Equilibration Experiment: LDH Concentration ( mU/ mg) by Time Point Without hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 27 85 Sampling Time Factors Data for No hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . 12 1 86 Sampling Time Factors Data for hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 4 87 Means of LDH Concentration ( mu/ mg) by hCG Stimulation . . . . . . . . . . . . . . . . . . . 12 6 88 Standard Deviations of LDH Concentration ( mU/ mg) by hCG Stimulation . . . . . . . 12 6 89 Sample Sizes of LDH Concentration ( mU/ mg) by hCG Stimulation . . . . . . . . . . . . . 12 7 90 Coeff of Variation of LDH Concentration ( mU/ mg) by hCG Stimulation . . . . . . . . . 12 7 Table of Contents vii 91 Means of Testosterone Concentration ( ng/ mg) by hCG Stimulation . . . . . . . . . . . . . 12 8 92 Standard Deviations of Testosterone Concentration ( ng/ mg) by HCG Stimulation . . 12 8 93 Sample Sizes of Testosterone Concentration ( ng/ mg) by hCG Stimulation . . . . . . . . 12 9 94 Coeff of Variation of Testosterone Concentration ( ng/ mg) by hCG Stimulation . . . . 12 9 95 Summary of Sampling Times Experiment: Testosterone Concentration ( ng/ mg) by Sampling Time Points over 24 Hours . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 14 96 Summary of Sampling Times Experiment: LDH Concentration ( mU/ mg) by Sampling Time Points over 24 Hours . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 14 97 Induced Parenchymal Fragment Injury Data for No hCG Stimulation . . . . . . . . . . . . 13 2 98 Induced Parenchymal Fragment Injury Data for hCG Stimulation . . . . . . . . . . . . . . . 13 7 99 Means of LDH Concentration ( mU/ mg) by Type of Innjury Induced No hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 8 100 Standard Deviations of LDH Concentration ( mU/ mg) by Type of Injury Induced No hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 9 101 Sample Sizes of LDH Concentration ( mU/ mg) by Type of Injury Induced No hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 9 102 Coeff of Variation of LDH Concentration ( mU/ mg) by Type of Injury Induced No hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 9 103 Means of LDH Concentration ( mU/ mg) by Type of Injury Induced with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 10 104 Standard Deviations of LDH Concentration ( mU/ mg) by Type of Injury Induced with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 10 105 Sample Sizes of LDH Concentration ( mU/ mg) by Type of Injury Induced with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 10 106 Coeff of Variation of LDH Concentration ( mU/ mg) by Type of Injury Induced with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 11 107 Means of Testosterone Concentrationi ( ng/ mg) by Type of Injury Induced No hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 12 108 Standard Deviations of Testosterone Concentration ( ng/ mg) by Type of Injury Induced No hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 12 109 Sample Sizes of Testosterone Concentration ( ng/ mg) by Type of Injury Induced No hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 12 110 Coeff of Variation of Testosterone Concentration ( ng/ mg) by Type of Injury Induced No hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 13 111 Means of Testosterone Concentration ( ng/ mg) by Type of Injury Induced with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 13 112 Standard Deviations of Testosterone Concentration ( ng/ mg) by Type of Injury Induced with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 13 113 Sample Sizes of Testosterone Concentration ( ng/ mg) by Type of Injury Induced with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 14 114 Coeff with Variation of Testosterone Concentration ( ng/ mg) by Type of Injury Induced with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 14 115 Summary of Injury Experiment: Testosterone Concentrations ( ng/ mg) by Sampling Time Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 30 116 Summary of Injury Experiment: Testosterone Concentrations ( ng/ mg) by Sampling Time Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 30 Table of Contents viii 117 Summary of Injury Experiment: LDH Concentrations ( mU/ mg) by Sampling Time Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 31 118 Injury Experiment: LDH Concentrations ( mU/ mg) by Sampling Time Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 31 119 Vehicle and Vehicle Concentrations Data for hCG Stimulation . . . . . . . . . . . . . . . 13 32 120 Vehicle and Vehicle Concentrations Data for hCG Stimualtion . . . . . . . . . . . . . . . 13 35 121 Means of LDH Concentration ( mUmg) by Vehicle Type . . . . . . . . . . . . . . . . . . . . . 13 37 122 Standard Deviations of LDH Concentration ( mU/ mg) by Vehicle Type no hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 37 123 Sample Sizes by LDH Concentration ( mU/ mg) by Vehicle Type no hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 37 124 Coeff of Variation of LDH Concentration ( mU/ mg) by Vehicle Type no hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 37 125 Means of LDH Concentration ( mU/ mg) by Vehicle Concentration no hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 38 126 Standard Deviations of LDH Concentration ( mU/ mg) by Vehicle Concentration no hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 38 127 Sample Sizes of LDH Concentration ( mU/ mg) by Vehicle Concentration no hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 38 128 Coeff of Variation of LDH Concentration ( mU/ mg) by Vehicle Concentration no hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 38 129 Means of LDH Concentration ( mU/ mg) by Vehicle Type with hCG Stimulation . . 13 39 130 Standard Deviationss of LDH Concentration ( mU/ mg) by Vehicle Type with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 39 131 Sample Sizes of LDH Concentration ( mU/ mg) by Vehicle Type with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 39 132 Coeff of Variation of LDH Concentration ( mU/ mg) by Vehicle Type with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 39 133 Means of LDH Concentration ( mU/ mg) by Vehicle Concentration with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 40 134 Standard Deviations of LDH Concentration ( mU/ mg) by Vehicle Concentration with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 40 135 Sample Sizes of LDH Concentration ( mU/ mg) by Vehicle Concentration with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 40 136 Coeff of Variation of LDH Concentration ( mU/ mg) by Vehicle Concentration with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 40 137 Means of Testosterone Concentration ( ng/ mg) by Vehicle Type no hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 41 138 Standard Deviations of Testosterone Concentration ( ng/ mg) by Vehicle Type no hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 41 139 Sample Sizes by Testosterone Concentration ( ng/ mg) by Vehicle Type no hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 41 140 Coeff of Variation of Testosterone Concentration ( ng/ mg) by Vehicle Type no hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 41 141 Means of Testosterone Concentration ( ng/ mg) by Vehicle Concentration no hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 42 Table of Contents ix 142 Standard Deviations of Testosterone Concentration ( ng/ mg) by Vehicle Concentration no hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 42 143 Sample Sizes of Testosterone Concentration ( ng/ mg) by Vehicle Concentration no hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 42 144 Sample Sizes of Testosterone Concentration ( ng/ mg) by Vehicle Concentration no hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 42 145 Means of Testosterone Concentration ( ng/ mg) by Vehicle Type with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 43 146 Standard Deviations of Testosterone Concentration ( ng/ mg) by Vehicle Type with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 43 147 Sample Sizes of Testosterone Concentration ( ng/ mg) by Vehicle Type with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 43 148 Coeff of Variation of Testosterone Concentration ( ng/ mg) by Vehicle Type with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 43 149 Means of Testosterone Concentration ( ng/ mg) by Vehicle Concentration with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 44 150 Standard Deviations of Testosterone Concentration ( ng/ mg) by Vehicle Concentration with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 44 151 Sample Sizes of Testosterone Concentration ( ng/ mg) by Vehicle Concentration with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 44 152 Coeff of Variation of Testosterone Concentration ( ng/ mg) by Vehicle Concentration with hCG Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 44 153 Summary of Vehicle: LDH Concentrations ( mU/ mg) by Vehicle Type . . . . . . . . . 13 108 154 Summary of Vehicle Experiment: LDH Concentrations ( mU/ mg) by Vehicle Type 13 108 155 Summary of Vehicle Experiment: LDH Concentrations ( mU/ mg) by Vehicle Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 110 156 Summary of Vehicle Experiment: LDH Concentrations ( mU/ mg) by Vehicle Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 110 157 Summary of Vehicle Experiment: Testosterone Concentrations ( ng/ mg) by Vehicle Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 111 158 Summary of Vehicle Experiment: Testosterone Concentrations ( ng/ mg) by Vehicle Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 111 159 Summary of Vehicle Experiment: Testosterone Concentrations ( ng/ mg) by Vehicle Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 112 160 Summary of Vehicle Experiment: Testosterone Concentrations ( ng/ mg) by Vehicle Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 112 Section 1.0 Introduction 1 1 1.0 INTRODUCTION 1.1 Background In 1996, the Food Quality Protection Act ( FQPA) amendments were enacted by Congress to authorize the Environmental Protection Agency ( EPA) to implement an Endocrine Disruptor Screening Program ( EDSP) on pesticides and other substances found in food or water sources for endocrine effects in humans ( FQPA, 1996). In this program, comprehensive toxicological and ecotoxicological screens and tests are being developed for identifying and characterizing the endocrine effects of various environmental contaminants, industrial substances, and pesticides. A two tiered approach will be utilized. Tier 1 employs a combination of in vivo and in vitro screens, and Tier 2 involves in vivo testing methods using two generation reproductive studies. A steroidogenesis assay is proposed as one of the Tier 1 screening battery assays.

A detailed review paper ( DRP) about steroidogenesis was prepared. The DRP: ( 1) summarized the state of the science of the in vivo, ex vivo, and in vitro methodologies available for measuring gonadal steroidogenesis; ( 2) for each methodology, presented a review of the individual assays and representative data generated by investigators who used the assay to evaluate a substance for steroidogenic altering activity; ( 3) provided an evaluation of the various methodologies and the assays as tools for screening substances with suspected steroidogenic activity; ( 4) recommended a particular screening method and assay as a screening tool; and ( 5) described the strengths, weaknesses, and implications for further research associated with the recommended screening assay.

The in vitro sliced testis steroidogenesis assay was selected as the most promising screening tool for identifying substances with steroidogenic altering activity. The sliced testis assay was recommended because it can be conducted at a minimal cost, quickly, and simply with standard laboratory equipment and basic laboratory training; the preparation is stable, and the parenchyma remains viable over a sufficient time period to measure changes in end product hormone production; the assay is relatively sensitive and specific; the assay uses parenchyma that maintains the cytoarchitecture of the organ; the assay uses a reduced number of animals ( up to quartered testis slices); the assay should be relatively easy to standardize ( by optimization); and the assay has a well defined endpoint in testosterone and, if desired, can be modified to include additional intermediate hormonal endpoints.

Although a promising tool, the sliced testis assay remains to be fully tested as an assay that can meet all the demands of an endocrine disruptor screening tool. Concerns raised by the EPA and Endocrine Disruptor Methods Validation Subcommittee ( EDMVS) suggested that experiments be conducted to ensure the optimization of the assay prior to more rigorous Section 1.0 Introduction 1 3 optimization was to describe in detail the experiments designed to provide data for setting in place the procedures and parameters that will optimize the performance of this assay.

1.2 Objectives The study plan for testing the factors described in the previous section involved two phases and utilized single factor and factorial experimental designs. A diagram of the experimental design for this study is illustrated in Figure 1.

The study was divided into Phases I and II. In Phase I, the Preliminary Experimental Phase, the analytical assays planned for use were verified, storage containers and lengths of storage were selected, and three factors that may affect the performance of the assay were tested. The reasoning for including these 3 factors in the preliminary phase was to establish early whether a given level of each factor was going to affect assay performance. Although any factor listed in the study plan could have been rationalized to fit such a criteria, inclusion in the preliminary phase also required that the factor be unlikely to have an interaction or, at best, a minimal interaction with another experimental factor. Although subjective, these factors were believed to best fit these criteria. Furthermore, it was believed essential to establish the optimal level for each of these factors before proceeding with the factorial experiments since an effect of one of these would require additional verification experiments after sensitivity analysis. Finally, by establishing the media type early on in the experiment, the analytical assay verification testing ( Phase I ) and Optimization of Sample Testing ( Phase II) could be initiated earlier in the study milestone schedule. Section 1.0 Introduction 1 3 The reasoning for including these 3 factors in the preliminary phase was to establish early whether a given level of each factor was going to affect assay performance. Although any factor listed in the study plan could have been rationalized to fit such a criteria, inclusion in the preliminary phase also required that the factor be unlikely to have an interaction or, at best, a minimal interaction with another experimental factor. Although subjective, these factors were believed to best fit these criteria. Furthermore, it was believed essential to establish the optimal level for each of these factors before proceeding with the factorial experiments since an effect of one of these would require additional verification experiments after sensitivity analysis. Finally, by establishing the media type early on in the experiment, the analytical assay verification testing ( Phase I ) and Optimization of Sample Testing ( Phase II) could be initiated earlier in the study milestone schedule. Section 2.0 Materials and Chemicals 2 1 2.0 MATERIALS AND CHEMICALS 2.1 Reagents and Solutions No test substances were used in this study. The chemicals used in this study were used to prepare reagents and solutions for the assay. All reagents and solutions had appropriate information documented, which included the identity, concentration, storage requirements, and expiration date. Reagents and solutions were prepared according to Standard Operating Procedures.

2.2 Standards Verification of the analytical assays required testosterone for the radioimmunoassay ( RIA) method and lactic dehydrogenase ( LDH) for the spectrophotometric assay. In addition, hCG was used as a stimulant of the sliced testis bioassay. These substances were considered standards.

2.2.1 Testosterone Chemical Name: Testosterone CAS No.: 58220 Molecular Weight: 288.4 Solubility: Clear colorless to very faint yellow solution at 100 mg/ mL in chloroform Supplier: Sigma Aldrich Chemical Company Lot No.: 6384 70 8 Purity: NLT 98% Storage Conditions: 2 year shelf life A safety protocol exists for the use of the radioactive form of testosterone.

2.2.2 Human Chorionic Gonadotropin ( hCG)

Chemical Name: hCG CAS No.: 9002 61 3 Molecular Weight: 36,700 Solubility: H 2 O Supplier: Calbiochem Lot No.: B11174, B51120 Section 2.0 Materials and Chemicals 2 2 Purity: Approx. 30% hCG by weight Storage Conditions: Freezer ( 20 ° C). Following reconstitution, aliquot and freeze ( 20 ° C). Stable for 2 years as supplied.

2.2.3 LDH Chemical Name: Lactate Dehydrogenase CAS No.: EC 1.1.127 Source: Rabbit muscle Solubility: Supplier: Sigma Aldrich Chemical Company Lot No.: 99H7480 Purity: NLT % Storage Conditions: 2 8 ° C; 1 year shelf life Section 3.0 Methods for Prototypical Assay 3 1 3.0 METHOD FOR PROTOTYPICAL ASSAY The prototypical assay describes the sliced testis assay using the conditions that are believed to be the starting conditions of the assay. This section does not describe any experiments to be conducted; rather, it describes the settings of all factors, except for the one that is being tested in order to perform the optimization experiments described in the following sections. A run is defined as a single sample vessel with assay components.

The sliced testis assay prototype uses a 15 week old male Sprague Dawley rat, which is euthanized and its testes removed. The testes are decapsulated, weighed, and placed in cold ( 4 ° C) DPBS. The media is medium 199 ( Gibco) that has added 0.71 g sodium bicarbonate, 2.1 g HEPES, 1.0 g/ L BSA, and 0.025 g/ L soybean trypsin inhibitor, and is adjusted to a pH of 7.4. The time from removal to the time of slicing is held to under 1 hour. Each testis is sliced along the longitudinal axis into approximately 4 slices. Each slice is placed in a 20 mL borosilicate scintillation vial ( loosely capped) that contains 5 mL of media ( Figure 2). The vials containing the testicular sections and media are incubated at 34 ° C on a shaker ( at 135 rpm) in 5% CO 2/ 95% air. After the first period of incubation, the media is removed and discarded. Fresh media ( 5 mL) is added to the vial and an aliquot of media ( 0.5 mL) is collected. The sample is centrifuged and the supernatant transferred to a labeled vial and stored at approximately  70 ° C in a siliconized plastic container for no more than 1 month. This sample is the baseline sample. Next, 1/ 2 of the vials are challenged with a stimulant ( e. g., hCG) and the other 1/ 2 are not. The final hCG concentration is 0.1 IU/ mL. Additional media samples are collected from the vials at 1, 2, 3, and 4 hours postchallenge. These media samples are also stored frozen for later analysis. Samples are analyzed for testosterone using an RIA method. All samples for a given day's set of runs should be analyzed in the same testosterone RIA. Section 3.0 Methods for Prototypical Assay 3 2 Figure 2. Technical Flow Illustration of the Sliced Testis Steroidogenesis Assay Section 4.0 Methods for Phase 1   Preliminary Experimental Phase 4 1 4.0 METHODS FOR PHASE I   PRELIMINARY EXPERIMENTAL PHASE Phase I is comprised of the verification experiments for the 2 analytical assay methods ( testosterone RIA and LDH spectrophotometry), the determination of the preferred storage container and storage length, and the optimization experiments for the 3 factors to be tested ( media type, incubation atmosphere, and animal age). The testosterone RIA method was verified prior to conducting any optimization assay since it was needed as an endpoint to determine optimization of the sliced testes assay. The storage container determination was also performed before any further optimization assays were run. The determination was made using standards of testosterone in the prototypical media, modified M 199 without phenol red.

4.1 Testosterone Radioimmunoassay The objective of this experiment was to verify that testosterone can be measured in the sliced testis assay media. A RIA commercial kit ( Diagnostic Products Corporation, Los Angeles, CA), that utilizes 125I testosterone and a testosterone specific antibody affixed to polypropylene culture tubes, was used to measure testosterone. The assay was verified in all 3 of the potential assay media.

Testosterone ( Sigma, St. Louis, MO; T 1500) was used for preparing the standard curve and was stored desiccated at room temperature in the RTI vault. A standard was prepared in ethanol ( 0.1 mg/ mL). Up to an 8 point standard curve ( but not less than a 4 point standard curve) was prepared using standards with concentrations of 0.07, 0.16, 0.41, 1.02 , 2.56, 6.4, 16, and 40 ng/ ml in PBS gel buffer ( 0.1 M phosphate buffered saline with 0.1% [ w/ v] sodium azide and 0.1% [ w/ v] gelatin, pH 7.4). In addition, procedural controls were included in each run. The standard curve points and the procedural controls were prepared in quadruplicate; the bioassay unknowns and the internal standards ( see below) were prepared in duplicate. The volume of all standards and controls ( including bioassay unknowns) were adjusted to 50 µ L by adding the PBS Gel Buffer. Next, 1 mL 125I testosterone was added to each antibody coated tube and mixed ( vortexed). The tubes were incubated in a 37 ° C water bath for 3 hours, during which time testosterone, whether labeled or unlabeled, competed for testosterone specific antibody binding sites. At the end of the incubation period, the free ( unbound) testosterone in the supernatant fluid of all tubes was aspirated, and tubes were wiped clean of fluid. The bound testosterone was counted in a gamma counter for 1 minute. The concentration of testosterone was estimated against the standard curve. Values were reported as a mean concentration ( ng/ mL) of duplicate analyses, unless only a single value was available. Verification of the testosterone assay involved preparation of internal standards ( at least 3) using spiked media, with concentrations ranging from 12.5 to 500 ng/ mL. Each concentration was run at each of 3 volumes   10, 25, and 50 ° L, to check for parallelism, and each sample was adjusted to 50 ° L by adding the PBS gel buffer. The Section 4.0 Methods for Phase 1   Preliminary Experimental Phase 4 2 low and high standards were analyzed in at least duplicate. Verification was based on results determined for accuracy, precision, specificity, and linearity. Accuracy is expressed as the relative error, which was determined by comparing the measured to the target concentration. Relative errors within 15% were acceptable. Precision is expressed as the relative standard deviation ( RSD) or coefficient of variance ( CV), which was determined by calculating the mean and standard deviation ( SD) of the low and high standards. A RSD or CV within 15% was acceptable. The sensitivity was acceptable if the means of the blanks and low standards were significantly different at the 5% significance level. Linear determinations of the standard curve line were made and a correlation coefficient ( r) calculated. An r of 0.90 or greater was considered acceptable.

Inter and intra assay variability was determined. The intra assay variability was determined from the precision results calculated from the results obtained by measuring the low and high standards in triplicate. The inter assay variability was determined by repeated analyses of the standards by generating a standard curve on 3 different days.

4.2 LDH Spectrophotometric Assay The objective of this experiment was to verify that LDH can be measured in the sliced testis assay media. The LDH assay measures the rate at which NADH is formed ( when NAD is reduced) when it catalyzes the oxidation of lactate to pyruvate. NADH is measured at 340 nm using a kinetic spectrophotometric method. The assay and samples are temperature sensitive. The samples should not be refrigerated or frozen. The assay has been characterized for assay conditions at 37oC. LDH activity is expressed in U/ L.

Qualification of the assay, using Media 199 without phenol red, consisted of determination of sensitivity and dilutional linearity. These were the only qualification tests specific to media use. Intra assay and inter assay imprecision were based on serum based controls normally used for instrument monitoring of quality control. Accuracy was based on the linear regression of a media sample spiked with purified LDH and diluted with media.

Intra assay imprecision was measured by assaying 10 quality control samples within a single run. The mean SD and CV were calculated. The CV should be less than 10%. Inter assay imprecision was measured by assaying 10 quality control samples over 10 separate runs. The mean SD and CV were calculated. The CV should be less than 10%.

Sensitivity of the assay or limit of detection was determined by analysis of 20 samples of media with no LDH present in the media. A low level of control was also analyzed to determine Section 4.0 Methods for Phase 1   Preliminary Experimental Phase 4 3 the lowest level of detection. Two standard deviations from the mean of the media activity were used as the limit of detection.

Dilutional linearity was used to determine accuracy and linearity limits. A sample of media was spiked with purified LDH ( Sigma # L1254). This was diluted with Media 199 without phenol red to within the linear limits of the assay. A series of dilutions by serial dilution of the spiked sample were conducted in triplicate. The linear regression of the line compared to the theoretical activity should be between 0.990 and 1.100.

All calculations were performed using EP Evaluator, Release 3.0 statistical analysis software from David Rhoads Associates, Inc., Kennett Square, PA.

4.3 Phase 1 Optimization Experiments for Media Type, Gaseous Atmosphere, Rat Age, and Storage Container Type 4.3.1 Media Type The objective was to determine the effect of different types of media ( with specified components) on testosterone production using the sliced testis assay. The prototypical assay conditions were used except with regard to the types of media.

The media types tested were:

? RPMI 1640 media ( without phenol red), 10% FCS, 50 ug/ mL soybean trypsin inhibitor ? Medium 199 ( Gibco), 0.71 g Na bicarbonate, 2.1 g HEPES, 1.0 g/ L BSA, 0.025 g / L soybean trypsin inhibitor, adjusted to pH 7.4 ? Eagles MEM 4.3.2 Gaseous Atmosphere The objective was to determine the effect of different types of gaseous atmospheres on testosterone production using the sliced testis assay. The prototypical assay conditions were used except with regard to the gaseous atmosphere.

The atmospheres tested were:

? 5% CO 2 / 95% air ? 5% CO 2 / 95% O 2 Section 4.0 Methods for Phase 1   Preliminary Experimental Phase 4 4 ? Air ( 3 gases)

4.3.3 Rat Age The objective will be to determine the effect of age on testosterone production using the sliced testis assay. The prototypical assay conditions will be used except with regard to the age of the rat used to obtain the testes.

The ages to be tested are:

? 11 weeks of age ? 15 weeks of age ? 22 weeks of age 4.3.4 Storage Container Type The objective was to determine the effect of storage type containers on the stability of testosterone in media. The types of containers tested were: siliconized plastic and nonsiliconized plastic.

4.4 Phase 1 Experimental Design 4.4.1 Factorial Design Experiments These experiments were conducted as a 33 full factorial design with 1 replicate per condition. The experimental factor levels are summarized in Table 1. The factorial test conditions are displayed in Table 2. The 27 factor level combinations were run in random order. Each combination was run with and without hCG stimulation for a total of 54 test runs. Each test run response ( ng T/ mL) were determined at 1, 2, 3, and 4 hours after media refreshment. Section 4.0 Methods for Phase 1   Preliminary Experimental Phase 4 5 Table 1. Summary of Experimental Factors for Phase 1 Optimization Factor Identification Units Experimental Levels Coded Experimental Levels 1 2 3 1 2 3 Media Type NA RPMI 1640 Medium 199 Eagles MEM  1 0 + 1 Gaseous Atmosphere NA 5% CO 2 ? 95% air 5% CO 2 ? 95% O 2 air  1 0 + 1 Rat Age Wks 11 15 22  1 0 + 1 NA = not applicable.

Table 2. Factorial Test Conditions for Phase 1 Optimization Experiment Media Type Gaseous Atmosphere Rat Age  1  1  1  1  1 0  1  1 + 1  1 0  1  1 0 0  1 0 + 1  1 + 1  1  1 + 1 0  1 + 1 + 1 0  1  1 0  1 0 0  1 + 1 0 0  1 0 0 0 0 0 + 1 0 + 1  1 0 + 1 0 0 + 1 + 1 + 1  1  1 + 1  1 0 + 1  1 + 1 + 1 0  1 ( Continued) Table 2. Factorial Test Conditions for Phase 1 Optimization Experiment ( Continued) Section 4.0 Methods for Phase 1   Preliminary Experimental Phase Media Type Gaseous Atmosphere Rat Age 4 6 + 1 0 0 + 1 0 + 1 + 1 + 1  1 + 1 + 1 0 + 1 + 1 + 1 4.4.2 Phase 1 Single Factor Experimental Design The objective of this set of experiments was to determine the effect of storage container type on the stability of testosterone in the media. This experimental series did not use the sliced testis assay. Stability was assessed as a function of sample handling factors. The incubation medium used will be determined in the factorial experiments ( Phase I). To conduct the stability experiments, a known amount of testosterone was added to the media to achieve a specified target concentration. The target concentration was determined from the media type experiments in Phase I and was in the range of the lowest testosterone concentrations measured in the sliced testis assay. Using this target concentration, the measured concentration was compared to the target concentration. The stability was evaluated based on the difference between the measured and target concentrations. If no statistical difference existed at the 5% level, then the sample was determined to be stable under the conditions tested.

4.5 Phase I Data Evaluation Upon completion of the Phase I optimization experiments, the results were reviewed for a possible change in the prototype conditions with regard to the 4 factors tested. A decision was made as to whether the Phase II optimization experiments would be conducted using the original prototype or modifications made to the media type, atmosphere, age of rat, and/ or storage container type used for the remaining experiments. The endpoint was the amount of testosterone released into the media during the sliced testis assay. The test conditions resulting in the highest values for testosterone were used in Phase II. Section 5.0 Results and Statistical Analyses 5 1 5.0 RESULTS AND STATISTICAL ANALYSES 5.1 Testosterone RIA Verification The testosterone RIA kit from Diagnostic Products was used to verify that the M 199 media without phenol red could be used for the sliced testis assay and provide accurate results for the RIA assay.

Table 3. Values of Standards on Testosterone RIA Value of Standard Factor Reading 8 ng/ ml 1 9.33 8 ng/ ml 1 8.70 8 ng/ ml 1 9.19 8 ng/ ml 1 8.86 8 ng/ ml 1 9.35 8 ng/ ml 2 9.63 8 ng/ ml 2 10.16 8 ng/ ml 2 10.54 8 ng/ ml 2 11.08 8 ng/ ml 2 11.86 8 ng/ ml 5 10.96 8 ng/ ml 5 11.60 8 ng/ ml 5 12.41 8 ng/ ml 5 11.56 8 ng/ ml 5 13.03 2 ng/ ml 1 2.56 2 ng/ ml 1 2.77 2 ng/ ml 1 2.56 2 ng/ ml 1 2.59 2 ng/ ml 1 2.46 0.5 ng/ ml 1 0.63 0.5ng/ ml 1 0.80 0.5ng/ ml 1 0.76 0.5ng/ ml 1 0.74 0.5ng/ ml 1 0.71 Unspiked Media Below Detection Limits of 0.04 ng/ mL Section 5.0 Results and Statistical Analyses 5 2 Table 4. Testosterone RIA Intra assay CV Number 50 ? l 25 ? l 10 ? l Unspiked M199 2 Blanks + 8 ng/ ml 10 5.24% 8.64% 7.68%

+ 2 ng/ ml 10 6.09%

+ 0.5 ng/ ml 10 13.34%

Table 5. Testosterone RIA Percent Recovery 50 ? l 25 ? l 10 ? l + 8 ng/ ml 113.8 133.3 149.0 + 2 ng/ ml 129.5 0.5 ng/ ml 146.4 Table 6. Testosterone RIA Parallelism 50 ? l 25 ? l 10 ? l + 8 ng/ mL* 9.10 ng/ mL 10.66 ng/ mL 11.92 ng/ mL * The Index between 50 and 10 ? l was 117.1%, between 25 and 10 ? l was 111.8%, and between 50 and 10 ? l was 131.0%.

5.2 LDH Verification See Appendix A.

5.3 Statistical Analysis of the Phase I Assay Optimization Experiment 5.3.1 Objectives The assay optimization experiment involves 3 factors ( media type, atmosphere, and rat age) which are run in a 33 factorial arrangement. Each of these 27 trials is run with and without hCG stimulation, and repeated measurements are taken at baseline ( time 0) and at 1, 2, 3, and 4 hours after baseline. The conditions are identified in Table 7. Objectives of the experiment are:

1. To determine the set of conditions that yields the highest estimated testosterone level, and Section 5.0 Results and Statistical Analyses 5 3 2. To determine the set of conditions that yields the largest with versus without hCG difference in testosterone levels.

5.3.2 Data Two basic SAS data sets were constructed from the raw data, and 2 fundamental types of dependent variables were used in the analyses of each type:

? Date Set 1: Cases without hCG stimulation Dependent variables: testosterone concentrations Dependent variables: ( natural) logarithm of testosterone concentrations ? Data Set 2: Cases with hCG stimulation Dependent variables: testosterone concentrations Dependent variables: ( natural) logarithm of testosterone concentrations Each data set can be viewed as consisting of 27 observations ( rows). Each observation includes dependent variable values for 4 time points and a corresponding baseline level. Each observation also includes data identifying the levels of the pertinent factors. Data are listed in Table 8 for the unchallenged samples and in Table 9 for the challenged cases.

5.4 Statistical Analysis Methods 5.4.1 Objective 1 Several statistical analysis methods were used to address the first objective. Analysis of variance ( ANOVA) and analysis of covariance ( ANOCOVA) were used to analyze the data for each individual time point ( including the baseline), and a mixed model ANOCOVA method was used to jointly analyze the data ( across time points 1 through 4). The ANOCOVA models utilized the baseline level ( or log level) as a covariate. For each type of analysis, all main effects and two factor interactions ( 2fi) of the 3 factors were initially included in the models. Tests for interactions were conducted and, where they were not detected as statistically significant ( p= 0.05), a reduced model was employed that retained the main effects, the baseline covariate ( where applicable), and only those 2fi deemed to have significant effects. Additional details are provided in the Results section.

5.4.2 Objective 2 For each of the 27 trials, differences between the with hCG and the without hCG testosterone levels were computed for each hour ( including baseline). These differences were computed on both the original and log scales. ANOVA was used to analyze these differences for each individual time point ( including the baseline). For each model and type of data, all main effects and 2fi of the 3 factors were initially included in the models. Tests for interactions were conducted and where they were not detected as statistically significant ( p= 0.05), a reduced model Section 5.0 Results and Statistical Analyses 5 4 was employed that retained the main effects and only those 2fi deemed to have significant effects. Additional details are provided in the Results section.

5.4.3 Results Overall Characterization of the Data. Table 10 provides summary statistics characterizing the testosterone levels in the non hCG stimulated data set. This summary ignores the particular experimental factors. The top portion of the table gives, by hour, the sample size ( n), the mean, standard deviation, sum, minimum, and maximum. These variables are denoted as yJ, where J denotes the hour and takes on values of 0, 1, 2, 3, and 4. The lower portion of the table gives the correlations between the hourly data. The following trends are apparent:

? the means continue to increase over time ? the standard deviations also increase over time ( i. e., as the mean level gets larger)

? the correlations are generally high and tend to be largest for adjacent hours Table 11 provides a similar summary for the log scaled data; these variables are denoted as lyJ, where J denotes the hour. Similar trends for the means and correlations are evident, but the standard deviations tend to be fairly stable across the various time points.

Table 7. Factor Levels in the Phase I Assay Optimization Experiment Factor Identification Units Factor Name Experimental Levels Coded Experimental Levels 1 2 3 1 2 3 Media Type* Z1 RPMI 1640 medium 199 Eagles MEM  1 0 + 1 Atmosphere* Z2 5% CO 2 / 95% air 5% CO 2 / 95% O 2 air  1 0 + 1 Rat Age* wks Z3 11 15 22  1 0 + 1 * Treated as a 3 level discrete factor. 5

5 Section 5 Results and Statistical Analysis Table 8. Data Listing for Samples Without hCG Animal Ear Tag Set Number Whole Testis Weight g Testis Section g z1 z2 z3 y0 = Testos. Conc. Baseline y1 = Testos. Conc. Hour 1 y2 = Testos. Conc. Hour 2 y3 = Testos. Conc. Hour 3 y4 = Testos. Conc. Hour 4 3 303 A RIGHT 1.5531 0.2331  1  1  1 0.54 3.23 5.21 6.19 6.11 5 308 B RIGHT 1.8189 0.2519  1  1 0 0.45 4.32 5.02 6.81 8.43 16 317 C LEFT 2.0203 0.2626  1  1 1 0.13 1.17 1.96 2.54 2.94 3 303 D LEFT 1.5632 0.2688  1 0  1 0.43 3.60 5.14 6.48 7.11 5 308 E LEFT 1.7979 0.2750  1 0 0 0.36 5.56 7.85 8.30 10.48 19 316 F RIGHT 2.0218 0.2524  1 0 1 0.18 2.10 3.77 5.11 6.26 2 304 G RIGHT 1.7860 0.2692  1 1  1 0.58 3.73 4.99 5.72 6.06 7 315 H RIGHT 1.7847 0.2449  1 1 0 0.57 5.17 7.01 8.39 9.17 16 317 I RIGHT 1.9804 0.2603  1 1 1 0.17 1.70 2.67 3.64 4.79 3 303 J RIGHT 1.5531 0.2742 0  1  1 0.63 5.35 6.27 7.52 8.64 5 308 K RIGHT 1.8189 0.2349 0  1 0 0.81 6.60 8.54 10.28 10.97 16 317 L LEFT 2.0203 0.2502 0  1 1 0.15 1.10 2.04 2.93 4.15 2 304 M RIGHT 1.7860 0.2668 0 0  1 0.86 6.60 9.81 11.35 13.48 5 308 N LEFT 1.7979 0.2504 0 0 0 0.49 5.58 8.02 10.00 11.23 19 316 O RIGHT 2.0218 0.2715 0 0 1 0.34 3.50 6.56 8.68 9.65 2 304 P LEFT 1.7809 0.2577 0 1  1 0.67 3.11 4.85 5.23 5.58 7 315 Q RIGHT 1.7847 0.2419 0 1 0 0.78 5.23 6.54 7.29 7.85 16 317 R RIGHT 1.9804 0.2563 0 1 1 0.13 1.31 1.82 2.46 2.83 5

6 Section 5 Results and Statistical Analysis Table 8. Data Listing for Samples Without hCG ( Continued)

Animal Ear Tag Set Number Whole Testis Teight g Testis Section g z1 z2 z3 y0 = Testos. Conc. Baseline y1 = Testos. Conc. Hour 1 y2 = Testos. Conc. Hour 2 y3 = Testos. Conc. Hour 3 y4 = Testos. Conc. Hour 4 3 303 S LEFT 1.5632 0.2651 1  1  1 0.21 3.98 6.10 7.37 10.16 5 308 T RIGHT 1.8189 0.2371 1  1 0 0.40 5.33 8.05 9.07 12.25 16 317 U LEFT 2.0203 0.2586 1  1 1 0.08 1.01 1.45 1.97 2.33 2 304 V RIGHT 1.7860 0.2741 1 0  1 0.48 3.44 5.53 6.98 8.12 5 308 W LEFT 1.7979 0.2340 1 0 0 0.69 4.91 7.36 10.05 11.72 19 316 X RIGHT 2.0218 0.2686 1 0 1 0.24 2.31 3.98 5.61 6.55 2 304 Y LEFT 1.7809 0.2681 1 1  1 0.45 4.44 5.49 6.71 6.86 7 315 Z RIGHT 1.7847 0.2560 1 1 0 0.74 5.34 6.15 7.87 8.43 16 317 AA RIGHT 1.9804 0.2408 1 1 1 0.09 0.84 1.18 1.73 1.74 5

7 Section 5 Results and Statistical Analysis Table 9. Data Listing for Samples With hCG Animal Ear Tag Set Number Whole Testis Weight g Testis Section g z1 z2 z3 yc0 = Testos. Conc. Baseline yc1 = Testos. Conc. Hour 1 yc2 = Testos. Conc. Hour 2 yc3 = Testos. Conc. Hour 3 yc4 = Testos. Conc. Hour 4 3 303 AC RIGHT 1.5531 0.2422  1  1  1 0.54 6.56 16.16 28.30 42.09 5 308 BC RIGHT 1.8189 0.2690  1  1 0 1.11 5.96 9.69 15.19 23.41 16 317 CC LEFT 2.0203 0.2680  1  1 1 0.16 2.03 4.88 8.87 15.93 3 303 DC LEFT 1.5632 0.2374  1 0  1 0.73 5.91 20.96 41.76 51.74 5 308 EC LEFT 1.7979 0.2398  1 0 0 0.43 4.39 10.84 22.92 33.45 19 316 FC RIGHT 2.0218 0.2515  1 0 1 0.26 3.80 9.94 18.38 24.77 2 304 GC RIGHT 1.7860 0.2494  1 1  1 1.29 4.71 7.14 10.26 13.39 7 315 HC RIGHT 1.7847 0.2401  1 1 0 0.62 6.35 8.09 11.90 14.64 16 317 IC RIGHT 1.9804 0.2642  1 1 1 0.10 1.30 2.74 3.63 5.13 3 303 JC RIGHT 1.5531 0.2613 0  1  1 0.72 8.26 18.51 31.30 39.20 5 308 KC RIGHT 1.8189 0.2386 0  1 0 0.47 7.84 15.75 28.83 43.60 16 317 LC LEFT 2.0203 0.2672 0  1 1 0.11 2.19 6.70 11.23 18.38 2 304 MC RIGHT 1.7860 0.2631 0 0  1 0.86 9.73 31.45 55.27 77.68 5 308 NC LEFT 1.7979 0.2461 0 0 0 0.89 7.33 18.38 33.23 59.89 19 316 OC RIGHT 2.0218 0.2516 0 0 1 0.42 4.15 10.45 18.93 27.32 2 304 PC LEFT 1.7809 0.2461 0 1  1 0.61 5.57 9.65 12.75 15.73 5

8 Section 5 Results and Statistical Analysis Table 9. Data Listing for Samples With hCG ( Continued)

Animal Ear Tag Set Number Whole Testis Weight g Testis Section g z1 z2 z3 yc0 = Testos. Conc. Baseline yc1 = Testos. Conc. Hour 1 yc2 = Testos. Conc. Hour 2 yc3 = Testos. Conc. Hour 3 yc4 = Testos. Conc. Hour 4 7 315 QC RIGHT 1.7847 0.2618 0 1 0 1.38 7.03 9.69 12.35 13.47 16 317 RC RIGHT 1.9804 0.2607 0 1 1 0.15 1.03 2.14 2.92 4.01 3 303 SC LEFT 1.5632 0.2375 1  1  1 0.34 6.43 18.23 26.69 38.80 5 308 TC RIGHT 1.8189 0.2668 1  1 0 0.65 8.79 18.07 29.25 46.72 16 317 UC LEFT 2.0203 0.2683 1  1 1 0.11 1.81 4.44 7.96 12.42 2 304 VC RIGHT 1.7860 0.2593 1 0  1 0.66 5.91 15.48 24.92 32.07 5 308 WC LEFT 1.7979 0.2515 1 0 0 0.90 9.38 25.90 45.21 67.45 19 316 XC RIGHT 2.0218 0.2595 1 0 1 0.18 3.98 10.13 18.33 27.01 2 304 YC LEFT 1.7809 0.2591 1 1  1 0.43 5.17 10.59 16.60 25.20 7 315 ZC RIGHT 1.7847 0.2684 1 1 0 0.52 7.09 12.48 20.01 27.96 16 317 AAC RIGHT 1.9804 0.2459 1 1 1 0.12 2.44 5.53 9.16 13.76 5

9 Section 5 Results and Statistical Analysis Table 10. Summary of Data  Original Scale, Without hCG Simple Statistics Variable N Mean Std Dev Sum Minimum Maximum Label y0 27 0.43148 0.24012 11.65000 0.08000 0.86000 y0 =_ Testos._ Conc._ Baseline y1 27 3.72444 1.77928 100.56000 0.84000 6.60000 y1 =_ Testos._ Conc._ Hour 1 y2 27 5.30963 2.33770 143.36000 1.18000 9.81000 y2 =_ Testos._ Conc._ Hour 2 y3 27 6.52889 2.67986 176.28000 1.73000 11.35000 y3 =_ Testos._ Conc._ Hour 3 y4 27 7.55148 3.16645 203.89000 1.74000 13.48000 y4 =_ Testos._ Conc._ Hour 4 Pearson Correlation Coefficients, N = 27 Prob > | r| under H0: Rho= 0 y0 y1 y2 y3 y4 y0 y0 =_ Testos._ Conc._ Baseline 1.00000 0.83359 <. 0001 0.77555 <. 0001 0.75779 <. 0001 0.64894 0.0003 y1 y1 =_ Testos._ Conc._ Hour 1 0.83359 <. 0001 1.00000 0.96006 <. 0001 0.93735 <. 0001 0.89924 <. 0001 y2 y2 =_ Testos._ Conc._ Hour 2 0.77555 <. 0001 0.96006 <. 0001 1.00000 0.98329 <. 0001 0.96280 <. 0001 y3 y3 =_ Testos._ Conc._ Hour 3 0.75779 <. 0001 0.93735 <. 0001 0.98329 <. 0001 1.00000 0.97499 <. 0001 y4 y4 =_ Testos._ Conc._ Hour 4 0.64894 0.0003 0.89924 <. 0001 0.96280 <. 0001 0.97499 <. 0001 1.00000 5

10 Section 5 Results and Statistical Analysis Table 11. Summary of Data  Log Scale, Without hCG Simple Statistics Variable N Mean Std Dev Sum Minimum Maximum ly0 27  1.04291 0.70964  28.15853  2.52573  0.15082 ly1 27 1.15995 0.62490 31.31878  0.17435 1.88707 ly2 27 1.53822 0.57738 41.53197 0.16551 2.28340 ly3 27 1.76591 0.52363 47.67970 0.54812 2.42922 ly4 27 1.90916 0.52876 51.54720 0.55389 2.60121 Pearson Correlation Coefficients, N = 27 Prob > | r| under H0: Rho= 0 ly0 ly1 ly2 ly3 ly4 ly0 1.00000 0.90646 <. 0001 0.88113 <. 0001 0.86395 <. 0001 0.79479 <. 0001 ly1 0.90646 <. 0001 1.00000 0.97732 <. 0001 0.96441 <. 0001 0.93255 <. 0001 ly2 0.88113 <. 0001 0.97732 <. 0001 1.00000 0.99193 <. 0001 0.97266 <. 0001 ly3 0.86395 <. 0001 0.96441 <. 0001 0.99193 <. 0001 1.00000 0.98411 <. 0001 ly4 0.79479 <. 0001 0.93255 <. 0001 0.97266 <. 0001 0.98411 <. 0001 1.00000 Section 5.0 Results and Statistical Analysis 5 11 Tables 12 and 13 furnish comparable information for the hCG stimulated samples. Similar trends are evident for these data. Mean levels tend to be much higher than for the nonstimulated samples.

Analysis of Baseline Data. Since we intend to adjust for baseline ( time 0) levels for subsequent analyses of the hourly ( nonbaseline) data, it is important to understand how the experimental factors affect the baseline levels. For instance, if one of the factors does impact the baseline levels, then adjusting for baseline levels in those subsequent analyses may obscure the effect of the experimental factor. Table14 presents the results that summarize the ANOVA results for the baseline data. Initially, we fit an ANOVA model that included all mean effects ( denoted as z1, z2, and z3) and all 2 factor interactions ( denoted as z1* z2, z1* z3, and z2* z3). We examined the statistical significance of each of the interactions and reduced the model to contain only main effects and the pertinent 2fi. For 3 of the 4 cases considered, only the main effects were retained; for the log scale, without hCG case, 2 of the interaction effects were deemed significant. Among the 3 experimental factors, it is clear that the rat age ( z3) has the most pronounced effect on the baseline levels ( estimated testosterone levels are 0.54, 0.58, and 0.17 for 11 week, 15 week, and 22 week old animals). For the non hCG stimulated case, media type also impacted the baseline levels, with the highest level occurring for the z1= 0 case ( 0.54 versus 0.38 for the other two media). The lower portion of Table 14 furnishes statistics characterizing the model fit:

? R2 = the proportion of variability accounted for by the model, ? RMSE = root mean squared error = the square root of the residual variance, ? C. V. = the coefficient of variation = the RMSE divided by the mean testosterone level ( times 100%).

Analyses Directed at Objective 1. Two fundamental types of statistical analysis were used to address Objective 1 ( assessing the effects of the experimental factors on the testosterone levels)   separate analyses for each hour and a combined mixed model approach.

Individual hour analyses. These analyses involved:

? fitting the testosterone data for a given hour as a function of the experimental factors, their 2 factor interactions, and the baseline level ? examining the significance of the 2 factor interactions ( 2fi) ? choosing ( and fitting) a reduced model form by eliminating any 2fi that was not satistically significant in any of the four hourly models. 5

12 Section 5 Results and Statistical Analysis Table 12. Summary of Data  Original Scale, With hCG Simple Statistics Variable N Mean Std Dev Sum Minimum Maximum Label yc0 27 0.54667 0.36060 14.76000 0.10000 1.38000 yc0 =_ Testos._ Conc._ Baseline yc1 27 5.37556 2.48467 145.14000 1.03000 9.73000 yc1 =_ Testos._ Conc._ Hour 1 yc2 27 12.37074 7.01613 334.01000 2.14000 31.45000 yc2 =_ Testos._ Conc._ Hour 2 yc3 27 20.96852 12.82440 566.15000 2.92000 55.27000 yc3 =_ Testos._ Conc._ Hour 3 yc4 27 30.19333 18.74161 815.22000 4.01000 77.68000 yc4 =_ Testos._ Conc._ Hour 4 Pearson Correlation Coefficients, N = 27 Prob > | r| under H0: Rho= 0 yc0 yc1 yc2 yc3 yc4 yc0 yc0 =_ Testos._ Conc._ Baseline 1.00000 0.66941 0.0001 0.44159 0.0211 0.38125 0.0497 0.34634 0.0768 yc1 yc1 =_ Testos._ Conc._ Hour 1 0.66941 0.0001 1.00000 0.86213 <. 0001 0.80157 <. 0001 0.78474 <. 0001 yc2 yc2 =_ Testos._ Conc._ Hour 2 0.44159 0.0211 0.86213 <. 0001 1.00000 0.98464 <. 0001 0.96305 <. 0001 yc3 yc3 =_ Testos._ Conc._ Hour 3 0.38125 0.0497 0.80157 <. 0001 0.98464 <. 0001 1.00000 0.98016 <. 0001 yc4 yc4 =_ Testos._ Conc._ Hour 4 0.34634 0.0768 0.78474 <. 0001 0.96305 <. 0001 0.98016 <. 0001 1.00000 Section 5.0 Results and Statistical Analysis 5 13 Table 13. Summary of Data   Log Scale, With hCG Simple Statistics Variable N Mean Std Dev Sum Minimum Maximum lyc0 27  0.86822 0.80586  23.44183  2.30259 0.32208 lyc1 27 1.53650 0.61019 41.48556 0.02956 2.27521 lyc2 27 2.34072 0.64378 63.19938 0.76081 3.44840 lyc3 27 2.83766 0.70489 76.61684 1.07158 4.01223 lyc4 27 3.19518 0.71702 86.26981 1.38879 4.35260 Pearson Correlation Coefficients, N = 27 Prob > | r| under H0: Rho= 0 lyc0 lyc1 lyc2 lyc3 lyc4 lyc0 1.00000 0.84699 <. 0001 0.68581 <. 0001 0.61367 0.0007 0.54138 0.0035 lyc1 0.84699 <. 0001 1.00000 0.92139 <. 0001 0.87024 <. 0001 0.82124 <. 0001 lyc2 0.68581 <. 0001 0.92139 <. 0001 1.00000 0.98599 <. 0001 0.95954 <. 0001 lyc3 0.61367 0.0007 0.87024 <. 0001 0.98599 <. 0001 1.00000 0.98703 <. 0001 lyc4 0.54138 0.0035 0.82124 <. 0001 0.95954 <. 0001 0.98703 <. 0001 1.00000 Section 5.0 Results and Statistical Analysis 5 14 Table 14. Summary of Statistical Analysis of Baseline Data Original Scale Models Log Scale Models Without hCG Stimulation With hCG Stimulation Without hCG Stimulation With hCG Stimulation Dependent Variable: Y0 Y0 log( Y0) log( Y0)

Dependent Variable Mean: 0.431 0.547  1.043 0.868 Significance of Model Terms: z1 xx xxx z2 xx z3 xxx xxx xxx xxx z1* z2 na na xx na z1* z3 na na na na z2* z3 na na xx na R2 0.766 0.628 0.950 0.822 RMSE 0.132 0.251 0.233 0.388 C. V. 30.7 45.9   x = statistically significant effect at 0.10 level of significance. xx = statistically significant effect at 0.05 level of significance. xxx = statistically significant effect at 0.01 level of significance. na = not applicable ( effect not included in the model). 5

15 Section 5 Results and Statistical Analysis Table 15. Summary of ANOCOVA Results for Individual Hour Original Scale Models Dependent Variable: Without hCG Stimulation With hCG Stimulation Y1 Y2 Y3 Y4 Y1 Y2 Y3 Y4 Dependent Variable Mean: 3.72 5.31 6.53 7.55 5.38 12.37 20.97 30.19 Significance of Model Terms: Y0 xx xx xx z1 xx x z2 x xxx xxx xxx xx xxx xxx xxx z3 xxx xx xxx xx xxx xxx xxx xxx z1* z2 xx xx xx x na na na na z1* z3 na na na na x x R2 0.931 0.913 0.917 0.877 0.904 0.862 0.865 0.847 RMSE 0.62 0.91 1.02 1.46 1.01 3.43 6.21 9.65 C. V. 16.6 17.1 15.5 19.3 18.9 27.7 29.6 32.0 x = statistically significant effect at 0.10 level of significance. xx = statistically significant effect at 0.05 level of significance. xxx = statistically significant effect at 0.01 level of significance. na = not applicable ( effect not included in the model). Section 5.0 Results and Statistical Analysis 5 16 Table 16. Adjusted Mean Levels ( Based on Original Scale Models)

Level of Independent Variable Mean Levels of Dependent Variables: Without hCG Stimulation Mean Levels of Dependent Variables: With hCG Stimulation z1 z2 z3 Y1 Mean Y2 Mean Y3 Mean Y4 Mean Y1 Mean Y2 Mean Y3 Mean Y4 Mean  1 3.55 5.05 6.14 6.99 4.52* 10.08* 18.11 25.30 0 3.95 5.64 6.83 7.92 5.83 13.70 23.41 34.01 + 1 3.67 5.24 6.62 7.75 5.77 13.34 21.38 31.26  1 3.72 5.16* 6.31** 7.50* 5.62 12.43* 20.40** 30.39**

0 4.12 6.37 7.97 9.33 6.02 17.10 31.25 45.05 + 1 3.34* 4.40** 5.30** 5.82** 4.49** 7.59** 11.25** 15.14**

 1 3.86** 5.53* 6.59* 7.67* 6.34 16.58 28.33 38.71 0 4.89 6.58 7.99 9.56 6.91 14.50 25.61 38.99 + 1 2.43** 3.83** 5.01** 5.43** 2.88** 6.03** 8.97** 12.88**

 1  1 3.07** 4.28** 5.44** 6.01**

 1 0 4.06 6.00 7.11* 8.30  1 + 1 3.51* 4.86** 5.88** 6.65**

0  1 4.07 5.24** 6.48** 7.60*

0 0 4.85 7.63 9.43 11.03 0 + 1 2.94** 4.04** 4.57** 5.11**

+ 1  1 4.02 5.96 7.02* 8.89 + 1 0 3.44* 5.48* 7.38* 8.67 + 1 + 1 3.55* 4.29** 5.46** 5.69**

 1  1 5.43** 15.00 28.51 38.78  1 0 5.40** 9.68** 17.65** 25.55**

 1 + 1 2.73** 5.55** 8.18** 11.58**

0  1 7.68 20.02 34.14 46.02 0 0 7.05 14.90 26.88 42.62 0 + 1 2.76** 6.17** 9.21** 13.40**

+ 1  1 5.90* 14.71 22.34* 31.33*

+ 1 0 8.28 18.93 32.30 48.80 + 1 + 1 3.13** 6.37** 9.50** 13.67** Shaded cell indicates highest mean estimated level. * Indicates that the mean level is significantly lower than the cell with the maximum estimated level, p= 0.05. ** Indicates that the mean level is significantly lower than the cell with the maximum estimated level, p= 0.01. Section 5.0 Results and Statistical Analysis 5 17 Results for the original scale data are summarized in Tables 15 and 16. Table 15 provides an indication of which effects were retained in the reduced model and which of those terms were statistically significant. The lower portion of the table gives statistics characterizing the fit of the models.

Table 15 indicates that mean concentration levels increase with time. The models for the without and with hCG stimulation data are somewhat different, but both indicate statistical significance for z2 ( atmosphere type) and z3 ( rat age). For the without hCG case, the model also shows a significant effect of the covariate and of the z1* z2 ( media type by atmosphere type) interaction. For the with hCG case, the covariate was not statistically significant and there was some ( weak) indication of a z1* z3 ( media type by rat age) interaction. The RMSE values tend to increase with increasing concentration levels ( i. e., with time). The C. V. s, on the other hand, tend to be fairly stable, suggesting that a log tranform of the concentrations should result in data with approximately homogeneous variances over the various time points.

Adjusted means based on the models that were indicated in Table 15 are presented in Table 16. The means are those that are estimated to occur for a given level of a factor ( or given combination of factors) when other effects in the model ( e. g., the baseline level covariate) are fixed at their mean values. The 3 first columns of the table identify the factor levels ( see Table 7). Within each set of the levels ( e. g., the three rows with z1=  1, 0, and + 1), the estimated adjusted mean that is largest is highlighted. Asterisks beside the other nonhighlighted means indicate if that particular mean is deemed to be statistically significant from the one that is highlighted. For the nonstimulated data, for instance, the table indicates no significant difference among the z1 levels, although the zero level is consistently estimated to be the largest. For the atmosphere and rat age factors, the zero levels generally have the highest estimated mean testosterone concentrations and the other levels typically have significantly lower means. An exception is the rat age ( z3) factor for the hCG stimulated case, where the 11 week old and the 15 week old rats had similar adjusted mean levels. Even when interactions are considered ( lower portion of Table 16), the zero levels of all 3 factors are either estimated to have the highest adjusted means or to have adjusted means that are not significantly different from the factor combination having the highest estimated mean level.

Tables 17 and 18 show results for the log transformed data. These tables are analogous to Tables 15 and 16, respectively. The models are somewhat different than those indicated in Table 15. On the log scale, the rat age factor does not appear to be as prominent. Also, the Eagle MEM ( z1= 1) medium yields the highest mean levels, although the 199 medium ( z1= 0) levels are not significantly smaller. The air atmosphere consistently produces lower mean levels. Section 5.0 Results and Statistical Analysis 5 18 As with the original scale data, the zero levels of all 3 factors are either estimated to have the highest adjusted means or to have 5

19 Section 5 Results and Statistical Analysis Table 17. Summary of ANOCOVA Results for Individual Hour Log Scale Models Dependent Variable: Without hCG Stimulation With hCG Stimulation log( Y1) log( Y2) log( Y3) log( Y4) log( Y1) log( Y2) log( Y3) log( Y4)

Dependent Variable Mean: 1.160 1.538 1.766 1.909 1.537 2.341 2.838 3.195 Significance of Model Terms: log( Y0) xxx xxx xxx xxx xx z1 x xx x x z2 x xxx xxx xxx xx xxx xxx xxx z3 x x x z1* z2 xx xx xx xx x x z1* z3 x x xx na na na na R2 0.963 0.964 0.962 0.951 0.894 0.900 0.917 0.924 RMSE 0.185 0.167 0.156 0.181 0.261 0.268 0.268 0.260 x = statistically significant effect at 0.10 level of significance. xx = statistically significant effect at 0.05 level of significance. xxx = statistically significant effect at 0.01 level of significance. na = not applicable ( effect not included in the model) Section 5.0 Results and Statistical Analysis 5 20 Table 18. Adjusted Mean Levels Based on Log Scale Models Level of Independent Variable Mean Levels of Dependent Variables: Without hCG Stimulation Mean Levels of Dependent Variables: With hCG Stimulation z1 z2 z3 log( Y1) Mean log( Y2) Mean log( Y3) Mean log( Y4) Mean log( Y1) Mean log( Y2) Mean log( Y3) Mean log( Y4) Mean  1 1.162 1.551 1.764 1.903 1.381* 2.147* 2.669* 3.021*

0 1.085 1.456 1.679 1.834 1.527 2.362 2.836 3.190 + 1 1.232 1.608 1.855 1.990 1.702 2.513 3.008 3.374  1 1.208 1.591 1.810 1.98 1.628 2.431 2.949* 3.356*

0 1.240 1.690 1.935 2.10 1.644 2.696 3.305 3.687 + 1 1.031* 1.334** 1.552** 1.64** 1.337* 1.895** 2.259** 2.542**

 1 1.096 1.442 1.640 1.780 1.651 2.610 3.098 3.444 0 1.279 1.550 1.774 1.956 1.698 2.463 2.982 3.408 + 1 1.105 1.623 1.884 1.991 1.261 1.949* 2.433* 2.734*

 1  1 1.021* 1.406** 1.645** 1.752** 1.419 2.192* 2.730* 3.211*

 1 0 1.371 1.805 1.993 2.157 1.515 2.566 3.251 3.552  1 + 1 1.094* 1.440* 1.654* 1.801* 1.208* 1.682** 2.026** 2.302**

0  1 1.066* 1.403* 1.659* 1.857* 1.755 2.582 3.124 3.477 0 0 1.292 1.740 1.981 2.144 1.675 2.782 3.379 3.864 0 + 1 0.898** 1.225** 1.396** 1.501** 1.151* 1.723** 2.005** 2.229**

+ 1  1 1.539 1.963 2.127 2.341 1.711 2.520 2.991 3.381 + 1 0 1.057* 1.524* 1.833 2.001 1.743 2.740 3.285 3.645 + 1 + 1 1.102* 1.338** 1.606** 1.628** 1.651 2.281 2.747* 3.097**

 1  1 0.946* 1.307* 1.512* 1.587**

 1 0 1.401 1.658 1.855 2.053  1 + 1 1.139 1.686 1.925 2.070 0  1 0.982* 1.288* 1.470* 1.650*

0 0 1.213 1.470 1.685 1.821 0 + 1 1.061 1.609 1.882 2.031 + 1  1 1.359 1.730 1.939 2.104 + 1 0 1.221 1.521 1.781 1.996 + 1 + 1 1.117 1.572 1.846 1.871 Shaded cell indicates highest mean estimated level. * Indicates that the mean level is significantly lower than the cell with the maximum estimated level, p= 0.05. ** Indicates that the mean level is significantly lower than the cell with the maximum estimated level, p= 0.01. Section 5.0 Results and Statistical Analysis 5 21 adjusted means that are not significantly different from the factor combination having the highest estimated mean level.

Mixed model analyses. For each of 2 data sets and 2 types of dependent variables indicated above ( see " Data"), these analyses involved several steps. First, we employed a mixed model that included:

? the main effects of the experimental factor,

? two factor interactions,

? the baseline testosterone level,

? a linear and a quadratic time component,

? cross products of the linear and quadratic time components with the main effects, and ? cross products of the linear and quadratic time components with the 2fi.

For this " full" model, we utilized the SAS PROC MIXED procedure to determine a relevant covariance structure for the data set; in particular, we examined 10 different possible covariance structures, using maximum likelihood estimation, and selected one that appeared to be optimal or near optimal. Using that structure, we estimated fixed effects for all of the above model terms. We then reduced the model by eliminating nonsignificant higher order terms. We then re examined the covariance structure ( again selecting from among 10 possible structures) for this reduced model. Using the selected structure, we estimated the fixed effects in the reduced model ( using restricted maximum likelihood estimation) and computed adjusted means for the various factor levels, along with approximate 95% confidence intervals for the means.

The adjusted means for the various cases and factors are given in Tables 19 through 22. These are denoted as EST., where J = 1, 2, 3, or 4 denotes hour. Approximate 95% confidence limits are given in the right portion of the table. The lower and upper limits are denoted as LOWJ and HIJ, respectively, where J = 1, 2, 3, or 4 denotes hour. Unlike the individual hour analyses, these estimates ( and the interval estimates) rely on data from all 4 hours and also reflect a smoothing over time ( due to the assumed quadratic time dependence).

Analyses Directed at Objective 2. Differences in hCG stimulated and nonstimulated testosterone levels were computed for the 27 trials on an hour by hour basis. These differences were then analyzed by hour using an initial ANOVA model that included all main effects and 2fi. Section 5.0 Results and Statistical Analysis 5 22 A reduced ANOVA model was then selected by eliminating those 2fi that were not statistically significant. The original scale models are summarized in Table 23; no 2fi were deemed necessary, so that the model only includes the main effects. Both atmosphere type and rat age were judged to have impact on the testosterone concentration levels. Table 24 shows the adjusted means derived from the model. The estimated adjusted mean difference that is largest is highlighted. Asterisks beside the other nonhighlighted mean differences indicate if that particular difference is deemed to be statistically significant from the one that is highlighted. The media type appears to have little effect, but rat age and atmosphere type are significant factors affecting the with minus without hCG differences. Section 5.0 Results and Statistical Analysis 5 23 Table 19. Least Squares Means for Reduced Log scale Model: Without hCG Mean covariate value: ly0= 1.043 x1 x2 x3 EST1 EST2 EST3 EST4 LOW1 HI1 LOW2 HI2 LOW3 HI3 LOW4 HI4  1 _ _ 1.163 1.527 1.773 1.901 1.059 1.267 1.424 1.629 1.670 1.875 1.797 2.005 0 _ _ 1.116 1.471 1.708 1.828 0.942 1.290 1.298 1.644 1.536 1.881 1.655 2.002 1 _ _ 1.211 1.587 1.846 1.988 1.064 1.357 1.442 1.733 1.701 1.992 1.842 2.134 _  1 _ 1.184 1.563 1.824 1.968 1.039 1.330 1.418 1.708 1.680 1.969 1.823 2.114 _ 0 _ 1.278 1.672 1.949 2.109 1.146 1.409 1.542 1.802 1.819 2.080 1.978 2.240 _ 1 _ 1.028 1.350 1.554 1.640 0.928 1.128 1.251 1.448 1.455 1.652 1.540 1.741 _ _  1 1.137 1.472 1.683 1.771 0.901 1.374 1.236 1.708 1.448 1.919 1.534 2.007 _ _ 0 1.327 1.606 1.813 1.949 1.031 1.622 1.311 1.901 1.518 2.108 1.654 2.245 _ _ 1 1.026 1.507 1.831 1.998 0.523 1.529 1.004 2.010 1.328 2.334 1.494 2.501 Reduced model was fit using compound symmetry covariance structure. Effects retained in the reduced model were the following ( tL denote the linear time effect, tQ denotes the quadratic): ly0, z1, z2, z3, z1* z2, z1* z3, tL, tL* z1, tL* z2, tL* z3, tL* z1* z3, tQ, tQ* z3.

Table 20. Least Squares Means for Reduced Original scale Model: Without hCG Mean covariate value: y0= 0.43 x1 x2 x3 EST1 EST2 EST3 EST4 LOW1 HI1 LOW2 HI2 LOW3 HI3 LOW4 HI4  1 _ _ 3.70 5.11 6.24 7.09 2.99 4.40 4.41 5.80 5.55 6.94 6.39 7.79 0 _ _ 3.74 5.35 6.67 7.72 2.82 4.66 4.43 6.27 5.75 7.59 6.79 8.64 1 _ _ 3.76 5.40 6.75 7.82 3.05 4.48 4.69 6.10 6.04 7.46 7.11 8.53 _  1 _ 3.78 5.30 6.54 7.50 3.07 4.48 4.60 6.00 5.84 7.24 6.80 8.21 _ 0 _ 4.17 6.18 7.91 9.36 3.54 4.81 5.56 6.81 7.29 8.54 8.73 9.99 _ 1 _ 3.25 4.37 5.21 5.77 2.59 3.90 3.72 5.01 4.56 5.85 5.12 6.42 _ _  1 3.65 5.20 6.47 7.46 2.74 4.57 4.29 6.12 5.56 7.38 6.54 8.38 _ _ 0 4.45 6.30 7.87 9.15 3.29 5.62 5.14 7.46 6.70 9.03 7.99 10.32 _ _ 1 3.09 4.35 5.32 6.02 1.31 4.87 2.57 6.13 3.55 7.10 4.24 7.80 Reduced model was fit using compound symmetry covariance structure. Effects retained in the reduced model were the following ( tL denote the linear time effect, tQ denotes the quadratic): y0, z1, z2, z3, z1* z2, z1* z3, z2* z3, tL, tL* z1, tL* z2, tL* z3, tL* z1* z2, tL* z1* z3, tL* z2* z3, tQ. Section 5.0 Results and Statistical Analysis 5 24 Table 21. Least Squares Means for Reduced Log scale Model: With hCG Mean covariate value: lyc0= 0.868 x1 x2 x3 EST1 EST2 EST3 EST4 LOW1 HI1 LOW2 HI2 LOW3 HI3 LOW4 HI4  1 _ _ 1.388 2.153 2.694 3.012 1.199 1.576 1.970 2.335 2.512 2.876 2.823 3.200 0 _ _ 1.554 2.314 2.852 3.166 1.360 1.748 2.126 2.503 2.664 3.040 2.972 3.360 1 _ _ 1.668 2.471 3.051 3.408 1.469 1.867 2.278 2.665 2.858 3.244 3.209 3.607 _  1 _ 1.598 2.400 2.996 3.385 1.402 1.795 2.206 2.595 2.801 3.190 3.188 3.581 _ 0 _ 1.682 2.662 3.319 3.651 1.481 1.884 2.463 2.862 3.119 3.518 3.449 3.852 _ 1 _ 1.329 1.876 2.283 2.550 1.141 1.518 1.690 2.062 2.097 2.469 2.361 2.738 _ _  1 1.726 2.560 3.109 3.372 1.489 1.963 2.325 2.795 2.873 3.344 3.135 3.609 _ _ 0 1.793 2.443 2.951 3.317 1.530 2.056 2.182 2.704 2.690 3.212 3.054 3.580 _ _ 1 1.091 1.935 2.537 2.896 0.713 1.470 1.558 2.313 2.159 2.914 2.518 3.275 Reduced model was fit using first order autoregressive covariance structure. Effects retained in the reduced model were the following ( tL denote the linear time effect, tQ denotes the quadratic): lyc0, z1, z2, z3, z1* z2, tL, tL* z1, tL* z2, tL* z3, tL* z1* z2, tQ, tQ* z2, tQ* z3.

Table 22. Least Squares Means for Reduced Original scale Model: With hCG Mean covariate value: yc0= 0.55 x1 x2 x3 EST 1 EST 2 EST 3 EST 4 LOW 1 HI 1 LOW 2 HI 2 LOW 3 HI 3 LOW 4 HI 4  1 _ _ 5.11 12.20 20.55 30.17 4.43 5.79 10.49 13.90 17.76 23.34 26.03 34.31 0 _ _ 5.52 12.60 20.96 30.57 4.83 6.21 10.90 14.31 18.17 23.75 26.43 34.71 1 _ _ 5.59 12.68 21.03 30.65 4.89 6.29 10.97 14.39 18.24 23.82 26.51 34.79 _  1 _ 5.68 12.69 20.97 30.52 4.71 6.64 9.80 15.59 16.09 25.85 23.51 37.52 _ 0 _ 6.14 17.64 30.40 44.43 5.18 7.09 14.75 20.53 25.52 35.28 37.43 51.44 _ 1 _ 4.40 7.15 11.16 16.44 3.45 5.36 4.26 10.04 6.28 16.04 9.43 23.45 _ _  1 6.32 16.54 27.02 37.76 5.36 7.29 13.66 19.42 22.24 31.81 30.62 44.90 _ _ 0 6.78 14.01 23.91 36.48 5.79 7.78 11.12 16.90 19.12 28.70 29.34 43.63 _ _ 1 3.11 6.92 11.60 17.15 2.04 4.17 4.01 9.84 6.80 16.41 9.99 24.30 Reduced model was fit using first order autoregressive covariance structure with heterogeneous variances. Effects retained in the reduced model were the following ( tL denote the linear time effect, tQ denotes the quadratic): y0, z1, z2, z3, tL, tL* z2, tL* z3, tQ, tQ* z3. Section 5.0 Results and Statistical Analysis 5 25 Table 23. Analysis of Differences in Levels for With and Without hCG Stimulation: Original Scale Models Dependent Variable: diff( Y0) diff( Y1) diff( Y2) diff( Y3) diff( Y4)

Dependent Variable Mean: 0.12 1.65 7.06 14.44 22.64 Significance of Model Terms: z1 z2 xxx xxx xxx z3 xx xxx xxx xxx R2 0.175 0.457 0.687 0.722 0.712 RMSE 0.25 1.05 3,49 6,57 10.04 x = statistically significant effect at 0.10 level of significance. xx = statistically significant effect at 0.05 level of significance. xxx = statistically significant effect at 0.01 level of significance.

Table 24. Adjusted Mean Differences ( With Without hCG), Based on Original Scale Models Level of Independent Variable Mean Levels of Dependent Variables z1 z2 z3 Mean diff ( Y0) Mean diff ( Y1) Mean diff ( Y2) Mean diff ( Y3) Mean diff ( Y4)

 1 0.20 1.16 5.20 12.00 18.13 0 0.08 1.64 7.59 15.67 24.99 + 1 0.06 2.16 8.40 15.64 24.80  1 0.09 1.98 7.53 14.77* 23.84*

0 0.14 1.89 10.61 22.93 35.20 + 1 0.12 1.09 3.04** 5.62** 8.89**

 1 0.15 2.31 10.53 20.48 29.31 0 0.19 1.79 7.15 15.65 26.67 + 1 0.01 0.85** 3.50** 7.19** 11.94**

Shaded cell indicates highest mean estimated level. * Indicates that the mean level is significantly lower than the cell with the maximum estimated level, p= 0.05. ** Indicates that the mean level is significantly lower than the cell with the maximum estimated level, p= 0.01. Section 5.0 Results and Statistical Analysis 5 26 Tables 25 and 26 show comparable results for the differences of the log transformed data. In this case, rat age appears less important and atmosphere type is the most dominant factors.

Table 25. Analysis of Differences in Levels for With and Without hCG Stimulation: Log Scale Models Dependent Variable: diff ( log( Y0)) diff ( log( Y1)) diff ( log( Y2)) diff ( log( Y3)) diff ( log( Y4))

Dependent Variable Mean: 0.175 0.377 0.802 1.072 1.286 Significance of Model Terms: z1 x z2 xx xxx xxx z3 R2 0.142 0.305 0.528 0.585 0.503 RMSE 0.393 0.289 0.313 0.335 0.405 x = statistically significant effect at 0.10 level of significance. xx = statistically significant effect at 0.05 level of significance. xxx = statistically significant effect at 0.01 level of significance.

Table 26. Adjusted Mean Differences ( With Without hCG), Based on Log Scale Models Level of Independent Variable Mean Levels of Dependent Variables z1 z2 z3 Mean diff log( Y0) Mean diff log( Y1) Mean diff log( Y2) Mean diff log( Y3) Mean diff log( Y4)

 1 0.282 0.289 0.659** 0.963 1.166 0 0.079 0.307 0.732 0.995 1.197 + 1 0.164 0.533 1.017 1.278 1.496  1 0.186 0.493 0.956 1.248 1.486 0 0.242 0.380 0.931 1.298 1.503 + 1 0.096 0.257 0.520** 0.670** 0.869**

 1 0.236 0.446 0.957 1.259 1.447 0 0.243 0.274 0.637* 0.948 1.170 + 1 0.045 0.410 0.814 1.008 1.241 Shaded cell indicates highest mean estimated level. * Indicates that the mean level is significantly lower than the cell with the maximum estimated level, p= 0.05. ** Indicates that the mean level is significantly lower than the cell with the maximum estimated level, p= 0.01. Section 6.0 Statistical Analysis of the Phase IB Assay Optimization Experiment 6 1 6.0 Statistical Analysis of the Phase IB Assay Optimization Experiment 6.1 Objectives Since the gaseous atmosphere of 5% CO 2 / 95% O 2 was optimal, and it was thought that most laboratories would not have incubators to accommodate this mixture, a comparison was made between incubated samples and those in media that had be gassed with the mixture.

The Phase IB assay optimization experiment involved assessing the effect of a single experimental factor   using gassed or incubated samples. All other factors were held fixed. Ten trials of each condition were run both with and without hCG stimulation. For each trial, repeated measurements are taken at baseline ( time 0) and at 1, 2, 3, and 4 hours after baseline. The objective of the experiment was to assess whether the treatments differed in terms of the resultant testosterone levels.

6.2 Data Two basic SAS data sets were constructed from the raw data , and 2 fundamental types of dependent variables were used in the analyses of each type:

? Date Set 1: Cases without hCG stimulation Dependent variables: testosterone concentrations Dependent variables: ( natural) logarithm of testosterone concentrations ? Data Set 2: Cases with hCG stimulation Dependent variables: testosterone concentrations Dependent variables: ( natural) logarithm of testosterone concentrations Each data set can be viewed as consisting of 20 observations ( rows). Each observation includes dependent variable values for 4 time points and a corresponding baseline level. Each observation also includes data identifying the levels of the pertinent factors. Data are listed in Table 27 for the unchallenged samples and in Table 28 for the challenged cases.

6.3 Statistical Analysis Methods ANOVA was used to analyze the data for each individual time point ( including the baseline). ANOVAs were performed for both original scale data and log scaled data ( natural logarithm). ANOCOVA models utilizing the baseline level ( or log level) as a covariate were Section 6.0 Statistical Analysis of the Phase IB Assay Optimization Experiment 6 2 also employed. Section 6.0 Statistical Analysis of the Phase IB Assay Optimization Experiment 6 3 6.3.1 Results Overall Characterization of the Data. Table 29 provides summary statistics characterizing the testosterone levels in the non hCG stimulated data set. This summary ignores the experimental factor. The top portion of the table gives, by hour, the sample size ( n), the mean, standard deviation, sum, minimum, and maximum. These variables are denoted as yJ, where J denotes the hour and takes on values of 0, 1, 2, 3, and 4. The lower portion of the table gives the correlations between the hourly data. The following trends are apparent:

? the means continue to increase over time, ? the standard deviations also increase over time ( i. e., as the mean level gets larger), and ? the correlations are generally high and tend to be largest for adjacent hours.

Table 30 provides a similar summary for the log scaled data; these variables are denoted as lyJ, where J denotes the hour. Similar trends for the means and correlations are evident, but the standard deviations tend to be fairly stable across the various time points.

Tables 31 and 32 furnish comparable information for the hCG stimulated samples. Similar trends are evident for these data. Mean levels tend to be much higher than for the nonstimulated samples.

Analysis of Variance and Covariance. Means, by hour and sample condition, are presented in Table 33 for the non hCG stimulated original scale data. The table gives the number of observations, the approximate 95% confidence limits for the mean, the estimated mean, and standard deviation. The last column gives the mean ( for hours 1, 2, 3, and 4) adjusted for the baseline level, as determined from the ANOCOVA. Tables 34, 35, and 36 give corresponding results for the log scaled data and the hCG stimulated cases. The table below summarizes the findings detailed in Tables 33 through 36. For the most part, the ANOVAs and ANOCOVAs of the hourly data did not detect significant differences between the testosterone levels of the gassed and incubated samples. If the 0.05 significance level is used to judge statistical significance, then only the baseline case of Table 33 yielded a significant difference. Section 6.0 Statistical Analysis of the Phase IB Assay Optimization Experiment 6 4 Source Type Data Analyzed ANOVA Results ANOCOVA Results Table 33 Original scale, non hCG stimulated Baseline gassed samples have lower testosterone mean ( p= 0.05); all other hours not significantly different at 0.10 level. Hour 1 and hour 4 gassed samples have higher testosterone means ( p= 0.08 and 0.07, respectively); other hours not significantly different at 0.10 level.

Table 34 Log scale, non hCG stimulated Baseline gassed samples have lower testosterone mean ( p= 0.06); all other hours not significantly different at 0.10 level. No significant differences at 0.10 level.

Table 35 Original scale, hCG stimulated No significant differences at 0.10 level. No significant differences at 0.10 level.

Table 36 Log scale, hCG stimulated No significant differences at 0.10 level. No significant differences at 0.10 level. 6

5 Section 6 Statistical Analysis of the Phase IB Assay Optimization Experiment Table 27. Phase IB Data: Sample Without hCG Ear Tag Body Weight g Testis Wt g Section Wt g Run Number x0 y0 = Testos. Conc. Baseline y1 = Testos. Conc. Hour 1 y2 = Testos. Conc. Hour 2 y3 = Testos. Conc. Hour 3 y4 = Testos. Conc. Hour 4 320 361.74 RIGHT  1.5641 0.2413 1 1 0.35 3.49 5.46 6.66 8.05 321 361.74 LEFT  1.5303 0.2657 2 1 0.38 3.92 4.95 6.88 8.27 321 365.84 RIGHT  1.8176 0.2623 3 1 0.36 4.66 6.52 7.88 8.53 321 365.84 LEFT  1.8273 0.2463 4 1 0.34 3.94 4.94 6.30 7.71 321 365.84 LEFT  1.8273 0.2389 5 1 0.32 4.53 6.36 7.56 7.73 322 372.13 RIGHT  1.5835 0.2341 6 1 0.14 3.30 4.34 5.65 6.40 322 372.13 LEFT  1.5263 0.2436 7 1 0.18 1.67 2.47 3.16 3.63 323 365.46 RIGHT  1.7040 0.2722 8 1 0.45 3.87 5.41 6.06 7.90 324 356.94 RIGHT  1.4908 0.2626 9 1 0.42 5.32 7.49 9.71 11.01 323 365.46 LEFT  1.6760 0.2711 10 1 0.35 5.32 7.07 8.83 10.17 320 361.74 RIGHT  1.5641 0.2713 11 2 0.49 5.06 7.37 9.13 10.97 320 361.74 LEFT  1.5303 0.2543 12 2 0.38 2.94 4.36 5.44 6.45 321 365.84 RIGHT  1.8176 0.2610 13 2 0.38 4.76 6.78 8.51 9.21 321 365.84 LEFT  1.8273 0.2408 14 2 0.36 3.64 5.37 6.34 6.87 322 372.13 RIGHT  1.5835 0.2472 15 2 0.33 3.38 4.87 5.53 6.41 322 372.13 LEFT  1.5263 0.2499 16 2 0.46 4.45 5.56 6.85 8.15 323 365.46 RIGHT  1.7040 0.2474 17 2 0.40 4.26 6.30 7.99 9.13 323 365.46 LEFT  1.6760 0.2630 18 2 0.39 3.56 5.63 6.78 7.71 324 356.94 RIGHT  1.4908 0.2365 19 2 0.37 2.62 3.69 4.82 5.84 324 356.94 LEFT  1.4136 0.2684 20 2 0.49 4.56 5.85 6.55 9.02 x0 = sample condition: 1 = gassed, 2 = incubated 6

6 Section 6 Statistical Analysis of the Phase IB Assay Optimization Experiment Table 28. Phase IB Data: Samples With hCG Ear Tag Body Weight g Testis Wt g Section Wt g Run Number x0 y0 = Testos. Conc. Baseline y1 = Testos. Conc. Hour 1 y2 = Testos. Conc. Hour 2 y3 = Testos. Conc. Hour 3 y4 = Testos. Conc. Hour 4 320 361.74 RIGHT  1.5641 0.2387 1 1 0.36 9.56 20.60 32.84 41.13 320 361.74 LEFT  1.5303 0.2727 2 1 0.52 6.31 13.49 22.25 29.13 321 365.84 RIGHT  1.8176 0.2518 3 1 0.40 5.12 13.29 18.32 25.33 321 365.84 LEFT  1.8273 0.2748 4 1 0.48 7.57 14.92 23.79 30.83 321 365.84 LEFT  1.8273 0.2681 5 1 0.43 4.84 9.66 13.47 18.59 322 372.13 RIGHT  1.5835 0.2429 6 1 0.28 8.73 18.74 30.71 45.84 322 372.13 LEFT  1.5263 0.2523 7 1 0.23 4.18 9.38 12.68 18.38 323 365.46 RIGHT  1.7040 0.2550 8 1 0.42 5.95 12.41 21.00 30.28 324 356.94 RIGHT  1.4908 0.2489 9 1 0.12 5.09 11.99 17.25 28.43 323 365.46 LEFT  1.6760 0.2513 10 1 0.21 3.40 8.04 13.78 19.14 320 361.74 RIGHT  1.5641 0.2451 11 2 0.34 6.71 17.75 29.37 39.76 320 361.74 LEFT  1.5303 0.2409 12 2 0.38 5.07 12.01 17.63 23.46 321 365.84 RIGHT  1.8176 0.2471 13 2 0.24 7.90 17.49 27.99 39.46 321 365.84 LEFT  1.8273 0.2695 14 2 0.41 7.52 19.79 30.29 43.69 322 372.13 RIGHT  1.5835 0.2452 15 2 0.37 4.24 10.43 14.70 19.41 322 372.13 LEFT  1.5263 0.2635 16 2 0.31 5.06 11.70 19.98 29.90 323 365.46 RIGHT  1.7040 0.2615 17 2 0.44 5.71 13.87 21.35 30.16 323 365.46 LEFT  1.6760 0.2328 18 2 0.72 6.93 21.00 31.05 40.41 324 356.94 RIGHT  1.4908 0.2632 19 2 0.81 5.73 13.45 23.01 34.28 324 356.94 LEFT  1.4136 0.2491 20 2 0.31 6.14 16.68 26.65 33.43 x0 = sample condition: 1 = gassed, 2 = incubated 6

7 Section 6 Statistical Analysis of the Phase IB Assay Optimization Experiment Table 29. Summary of Data   Original Scale, Without hCG Simple Statistics Variable N Mean Std Dev Sum Minimum Maximum Label Y0 20 0.36700 0.08652 7.34000 0.14000 0.49000 y0 =_ Testos._ Conc._ Baseline Y1 20 3.96250 0.92905 79.25000 1.67000 5.32000 y1 =_ Testos._ Conc._ Hour 1 Y2 20 5.53950 1.26145 110.79000 2.47000 7.49000 y2 =_ Testos._ Conc._ Hour 2 Y3 20 6.83150 1.57510 136.63000 3.16000 9.71000 y3 =_ Testos._ Conc._ Hour 3 Y4 20 7.95800 1.76957 159.16000 3.63000 11.01000 y4 =_ Testos._ Conc._ Hour 4 Pearson Correlation Coefficients, N = 20Prob > | r| under H0: Rho= 0 Y0 Y1 Y2 Y3 Y4 Y0y0 =_ Testos._ Conc._ Baseline 1.00000 0.56839 0.0089 0.57435 0.0081 0.51050 0.0215 0.65502 0.0017 Y1y1 =_ Testos._ Conc._ Hour 1 0.56839 0.0089 1.00000 0.95710 <. 0001 0.94357 <. 0001 0.94257 <. 0001 Y2y2 =_ Testos._ Conc._ Hour 2 0.57435 0.0081 0.95710 <. 0001 1.00000 0.97439 <. 0001 0.94352 <. 0001 Y3y3 =_ Testos._ Conc._ Hour 3 0.51050 0.0215 0.94357 <. 0001 0.97439 <. 0001 1.00000 0.95462 <. 0001 Y4y4 =_ Testos._ Conc._ Hour 4 0.65502 0.0017 0.94257 <. 0001 0.94352 <. 0001 0.95462 <. 0001 1.00000 6

8 Section 6 Statistical Analysis of the Phase IB Assay Optimization Experiment Table 30. Summary of Data   Log Scale, Without hCG Simple Statistics Variable N Mean Std Dev Sum Minimum Maximum ly0 20  1.03875 0.30294  20.77491  1.96611  0.71335 ly1 20 1.34511 0.27439 26.90217 0.51282 1.67147 ly2 20 1.68265 0.26153 33.65299 0.90422 2.01357 ly3 20 1.89300 0.25530 37.86004 1.15057 2.27316 ly4 20 2.04705 0.25043 40.94100 1.28923 2.39880 Pearson Correlation Coefficients, N = 20Prob > | r| under H0: Rho= 0 ly0 ly1 ly2 ly3 ly4 ly0 1.00000 0.5842 20.0068 0.60978 0.0043 0.55761 0.0106 0.65998 0.0015 ly1 0.58422 0.0068 1.00000 0.96741 <. 0001 0.95797 <. 0001 0.95831 <. 0001 ly2 0.60978 0.0043 0.96741 <. 0001 1.00000 0.97796 <. 0001 0.95576 <. 0001 ly3 0.55761 0.0106 0.95797 <. 0001 0.97796 <. 0001 1.00000 0.96540 <. 0001 ly4 0.65998 0.0015 0.95831 <. 0001 0.95576 <. 0001 0.96540 <. 0001 1.00000 6

9 Section 6 Statistical Analysis of the Phase IB Assay Optimization Experiment Table 31. Summary of Data   Original Scale, With hCG Simple Statistics Variable N Mean Std Dev Sum Minimum Maximum Label YC0 20 0.38900 0.16189 7.78000 0.12000 0.81000 yc0 =_ Testos._ Conc._ Baseline YC1 20 6.08800 1.58725 121.76000 3.40000 9.56000 yc1 =_ Testos._ Conc._ Hour 1 YC2 20 14.33450 3.87932 286.69000 8.04000 21.00000 yc2 =_ Testos._ Conc._ Hour 2 YC3 20 22.40550 6.48074 448.11000 12.68000 32.84000 yc3 =_ Testos._ Conc._ Hour 3 YC4 20 31.05200 8.66685 621.04000 18.38000 45.84000 yc4 =_ Testos._ Conc._ Hour 4 Pearson Correlation Coefficients, N = 20Prob > | r| under H0: Rho= 0 YC0 YC1 YC2 YC3 YC4 YC0yc0 =_ Testos._ Conc._ Baseline 1.00000 0.14785 0.5339 0.24875 0.2903 0.25182 0.2841 0.19761 0.4037 YC1yc1 =_ Testos._ Conc._ Hour 1 0.14785 0.5339 1.00000 0.88953 <. 0001 0.90763 <. 0001 0.87874 <. 0001 YC2yc2 =_ Testos._ Conc._ Hour 2 0.24875 0.2903 0.88953 <. 0001 1.00000 0.97501 <. 0001 0.93343 <. 0001 YC3yc3 =_ Testos._ Conc._ Hour 3 0.25182 0.2841 0.90763 <. 0001 0.97501 <. 0001 1.00000 0.96812 <. 0001 YC4yc4 =_ Testos._ Conc._ Hour 4 0.19761 0.4037 0.87874 <. 0001 0.93343 <. 0001 0.96812 <. 0001 1.00000 6

10 Section 6 Statistical Analysis of the Phase IB Assay Optimization Experiment Table 32. Summary of Data   Log Scale, With hCG Simple Statistics Variable N Mean Std Dev Sum Minimum Maximum lyc0 20  1.02614 0.42653  20.52279  2.12026  0.21072 lyc1 20 1.77407 0.26184 35.48142 1.22378 2.25759 lyc2 20 2.62709 0.27611 52.54184 2.08443 3.04452 lyc3 20 3.06710 0.30327 61.34207 2.54003 3.49165 lyc4 20 3.39608 0.29444 67.92156 2.91126 3.82516 Pearson Correlation Coefficients, N = 20Prob > | r| under H0: Rho= 0 lyc0 lyc1 lyc2 lyc3 lyc4 lyc0 1.00000 0.2590 10.2702 0.29545 0.2060 0.3062 40.1891 0.21198 0.3696 lyc1 0.2590 10.2702 1.00000 0.91838 <. 0001 0.91443 <. 0001 0.89007 <. 0001 lyc2 0.29545 0.2060 0.91838 <. 0001 1.00000 0.96817 <. 0001 0.93546 <. 0001 lyc3 0.30624 0.1891 0.91443 <. 0001 0.96817 <. 0001 1.00000 0.97426 <. 0001 lyc4 0.21198 0.3696 0.89007 <. 0001 0.93546 <. 0001 0.97426 <. 0001 1.00000 6

11 Section 6 Statistical Analysis of the Phase IB Assay Optimization Experiment Table 33. Summary of Results by Sample Condition   Original Scale, Without hCG x0 N Obs Variable Lower 95% CL for Mean Upper 95% CL for Mean Mean Std Dev ANOCOVA Adjusted Mean 1 10 Y0 Y1 Y2 Y3 Y4 0.26 3.23 4.45 5.57 6.51 0.40 4.77 6.55 8.17 9.37 0. 33 4.00 5.50 6.87 7.94 0.101.08 1.47 1.82 2.00 4.30 5.89 7.32 8.57 2 10 Y0 Y1 Y2 Y3 Y4 0.37 3.34 4.79 5.80 6.82 0.44 4.51 6.36 7.79 9.13 0.41 3.92 5.58 6.79 7.98 0.06 0.81 1.10 1.39 1.62 3.62 5.19 6.34 7.34 x0 = sample condition: 1 = gassed, 2 = incubated Bolded entries are statistically significant at the 0.10 level. Bolded and underlined entries are statistically significant at the 0.05 level. 6

12 Section 6 Statistical Analysis of the Phase IB Assay Optimization Experiment Table 34. Summary of Results by Sample Condition   Log Scale, Without hCG x0 N Obs Variable Lower 95% CL for Mean Upper 95% CL for Mean Mean Std Dev ANOCOVA Adjusted Mean 1 10 ly0 ly1 ly2 ly3 ly4  1.433 1.105 1.437 1.667 1.820  0.898 1.583 1.893 2.110 2.252  1.165 1.344 1.665 1.889 2.036 0.374 0.334 0.318 0.309 0.302 1.426 1.743 1.961 2.118 2 10 ly0 ly1 ly2 ly3 ly4  1.008 1.190 1.553 1.751 1.914  0.816 1.502 1.848 2.044 2.202  0.912 1.346 1.700 1.897 2.058 0.134 0.218 0.206 0.205 0.202 1.264 1.623 1.825 1.976 x0 = sample condition: 1 = gassed, 2 = incubated Bolded entries are statistically significant at the 0.10 level. Bolded and underlined entries are statistically significant at the 0.05 level. 6

13 Section 6 Statistical Analysis of the Phase IB Assay Optimization Experiment Table 35. Summary of Results by Sample Condition   Original Scale, With hCG x0 N Obs Variable Lower 95% CL for Mean Upper 95% CL for Mean Mean Std Dev ANOCOVA Adjusted Mean 1 10 YC0 YC1 YC2 YC3 YC4 0.25 4.65 10.39 15.60 22.10 0.44 7.50 16.12 25.62 35.31 0.35 6.08 13.25 20.61 28.71 0.13 1.99 4.00 7.00 9.23 6.14 13.44 20.94 29.02 2 10 YC0 YC1 YC2 YC3 YC4 0.30 5.27 12.82 20.13 27.80 0.57 6.93 18.01 28.28 38.99 0.43 6.10 15.42 24.20 33.40 0.18 1.16 3.62 5.69 7.82 6.03 15.22 23.87 33.09 x0 = sample condition: 1 = gassed, 2 = incubated Bolded entries are statistically significant at the 0.10 level. Bolded and underlined entries are statistically significant at the 0.05 level. 6

14 Section 6 Statistical Analysis of the Phase IB Assay Optimization Experiment Table 36. Summary of Results by Sample Condition   Log Scale, With hCG x0 N Obs Variable Lower 95% CL for Mean Upper 95% CL for Mean Mean Std Dev ANOCOVA Adjusted Mean 1 10 lyc0 lyc1 lyc2 lyc3 lyc4  1.474 1.524 2.331 2.737 3.085  0.818 1.989 2.757 3.214 3.538  1.146 1.757 2.544 2.975 3.312 0.458 0.325 0.297 0.334 0.317 1.776 2.562 2.995 3.323 2 10 lyc0 lyc1 lyc2 lyc3 lyc4  1.176 1.652 2.539 2.978 3.295  0.637 1.931 2.881 3.340 3.665  0.906 1.792 2.710 3.159 3.480 0.377 0.195 0.239 0.253 0.259 1.772 2.693 3.139 3.469 x0 = sample condition: 1 = gassed, 2 = incubated Bolded entries are statistically significant at the 0.10 level. Bolded and underlined entries are statistically significant at the 0.05 level. Section 7.0 Discussion 7 1 7.0 DISCUSSION The optimization of the sliced testis assay is necessary in order to proceed to the prevalidation and validation stages of the testing of the assay for use in the Tier I tests for screening of substances for potential as endocrine disruptors. The Phase I studies have contributed to the initial portion of this optimization. These factors will be used for the rest of the optimization in Part II. Without these initial studies, we would not have used the best gaseous atmosphere for optimal testosterone concentrations from the testicular tissues. Section 9.0 Incubation Experiment 8 1 8.0 CONCLUSIONS FOR PHASE I The testosterone RIA and the LDH assay can both be verified with M 199 without phenol red. Both were validated and show the characteristics necessary for use to optimize the sliced testis assay.

There were certain factors in the initial Phase I experiments that definitely were not beneficial to use in the assay. For instance, 22 week old rats do not show the responsiveness in their testicular tissue that is necessary for an optimal assay. The air atmosphere was also not a favorable condition for the assay. The prototypical assay media, Media 199 without phenol red, was equal to any of the others tested. Statistical analysis was necessary to show that the atmosphere of 5% CO 2/ 95% O 2 was optimal, and that rats of the 11 to 15 week old range could be used for the assay. From these conclusions, we were ready to advance to the Phase II experiments.

Media 199 without phenol red will be used after it is gassed with the 5 % CO 2/ 95% O 2 mixture and pH adjusted to 7.4 for testicular tissues from 11 to 15 week old rats for the Phase II experimental studies. Section 9.0 Incubation Experiment 9 1 PHASE II Phase II Experiments consisted of several groups of studies. The multifactorial studies were for the incubation conditions of the assay and the testes slice preparation factors of the assay. After the optimal factors were determined, the LDH experiments were performed to determine LDH and testosterone concentrations under various conditions.

9.0 INCUBATION EXPERIMENT 9.1 Statistical Analysis of Phase II Incubation Optimization Experiment 9.1.1 Objectives The objectives of the incubation optimization experiment were to determine the effect of various incubation factor settings on testosterone production using the sliced testis assay and to identify the incubation factor settings that maximize the amount of testosterone production. These experiments tested the effect( s) of six factors: incubation temperature, incubation vessel type, incubation shaker speed, incubation media volume, hCG concentration, and testicular fragment size. Table 37 presents the incubation factor settings used in this experiment. Each trial in the experiment was run with and without hCG stimulation, repeated measurements were taken at baseline ( time 0) and at 1, 2, 3, and 4 hours after baseline.

9.1.2 Data A SAS data set was constructed from the raw Excel data files, and 2 fundamental types of dependent variables were used in the analyses: the original testosterone concentrations and the ( natural) logarithm of the testosterone concentrations. Each observation includes dependent variable values for 4 time points and a corresponding baseline level. Each observation also includes data identifying the levels of the pertinent factors. The data used in the analysis are displayed in Tables 38 and 39.

9.1.3 Statistical Analysis Methods In order to assess the effects of each incubation factor on the amount of testosterone production, ANOVA models were fit to the data separately for the trials with and without hCG stimulation. For each hCG stimulation type, the ANOVA models were fit to each individual time point. All main effects and 2 factor interactions ( 2fi) of the 6 factors were initially included in the models. Tests for interactions were conducted, and where they were not detected as statistically significant ( p= 0.10), a reduced model was employed that retained the main effects, Section 9.0 Incubation Experiment 9 2 the baseline covariate, and only those 2fi's deemed to have significant effects. Once the final ANOVA model was determined, the data were fit using a response surface regression analysis to determine the maximum predicted value of testosterone and the incubation factor levels associated with the maximum. Additional details are provided in the Results portion of this section.

9.1.4 Results To assess the effects of each incubation factor on testosterone production, an ANOVA model was fit separately to the set of data for each hCG stimulation type and each time point beyond the baseline.

Table 37. Incubation Factor Settings and Coded Values Factor Identification Units Variable Name Factor Name Experimental Levels Coded Experimental Levels 1 2 3 4 5 1 2 3 4 5 Incubation Temperature oC Temp X1  34*  37    1  + 1

Incubation Vessel Type NAa Vessel X2 scintillation vial* test tube     1 + 1

Incubation Shaker Speed NA Speed X3  none low high*    1 0 + 1

Incubation Media Volume ml Inc_ vol X4  2.5 5* 10    1 0 + 1

hCG Concentration IU/ ml hCG_ co nc X5 0.001 0.01 0.1* 1 10  2  1 0 + 1 + 2 Fragment Size mg mg_ tiss ue X6 25 50 125 250*   0.8  0.6 0 + 1

* Prototypical value. a NA  not applicable. Note: Shaker speed was treated as a continuous factor.

The ANOVA models containing all main effects and 2fi's were fit to the original testosterone concentrations and the logarithms of testosterone concentrations using PROC GLM. The logarithm of the baseline testosterone concentration ( T0) was also added to the model as a dependent variable. Upon examining the residuals from fitting the full ANOVA models, the residuals for the original testosterone concentrations showed signs of heteroskedasticity. The residuals for the logarithms of testosterone concentrations appear homoskedastic and randomly distributed around zero, thereby satisfying the assumptions necessary for valid ANOVA modeling results. The logarithms of the testosterone concentrations were therefore chosen as the more appropriate dependent variables for the analysis of the incubation experiment data. Section 9.0 Incubation Experiment 9 3 For the full ANOVA models using the logarithm of testosterone concentration as the dependent variable, we examined the t tests for the significance of each factor in the model. In the first round of model evaluation, we examined the t test results for the 2fis to see if any of those terms could be removed from the models. Any 2fis with p values greater than 0.1 were removed from the models. The reduced models, one model for the data without hCG stimulation and one model for the data with hCG stimulation, were fit to the logarithms of testosterone concentration again and the t tests of the model terms were re evaluated for significance. After 2 rounds of removing insignificant 2fis, we arrived at the final reduced models containing only significant 2fis and first order factors.

To obtain the optimal combination of factors for each time point, the data were fit using response surface regression models and the SAS procedure RSREG. ( The PROC GLM modeling results were used to determine which factors and 2fis needed to remain in the models. Any factors which did not have any associated 2fis in the final models were treated as covariates in the response surface regression models.) The ANOVA results for the response surface regressions are summarized in Table 40. The parameter estimates from the response surface regression models for the data without hCG stimulation are presented in Table 41, and the parameter estimates for the data with hCG stimulation are presented in Table 42. Because the vessel type is a categorical variable, we needed to run separate models for each vessel type in PROC RSREG. Differentiation of the prediction equation is used to determine the location of the stationary point; in most cases the stationary point was not a point of maximal response. Under these circumstances, the RIDGE technique in SAS is used to find a point of maximal response within the experimental region. Starting at the center of the experimental region, a hypersphere having a given radius is considered, and the point on the hypersphere with the maximal response is determined. The radius is then incremented and the maximum on that hypersphere is determined. This process, when repeated, traces out a " ridge of maximal response." The iterations are terminated when one of the factors reaches the extremity of its range, as defined by the levels used in the experiment. We determined the factor combination that produced the maximum predicted value of logarithm of testosterone concentration for each time point at each combination of with/ without hCG stimulation and for each vessel type. The optimal value factor combinations were plugged back into the response surface regression models to obtain predicted values for the optimal factor combinations at all time points. For example, we used the RIDGE regression results to determine the optimal factor combinations for the first time point ( T1) for the scintillation vial without hCG stimulation. We then used the optimal T1 combination to obtain the predicted values for logarithm of testosterone concentration and their standard errors for the scintillation vial without hCG stimulation for that time point and for all the other time points ( T2, T3 and T4). The optimal factor results, presented in Table 43, show the effects of the optimal factors for 1 time point on the other time points in evaluating the best combination of factors to use for further experimentation. Section 9.0 Incubation Experiment 1 This suggests that the baseline levels are affected by ( some of) the factors and that their resultant effect on later times is diminished if adjustments for baseline levels are made.

2 This indicates that baseline adjustments for the latter cases were unnecessary, as was also apparent from Table 4 ( since baseline effect was not significant).

9 4 After examining the possible optimal factors for each time point, vessel type, and hCG stimulation, the chosen optimal values and their predicted values and standard errors are presented in Table 44.

In order to assess the effects of the baseline testosterone concentrations on the effects of factors, the same analysis steps that were performed above were also repeated on the logarithms of testosterone concentration but with the baseline concentration removed from the models. The ANOVA results for the final reduced model from PROC RSREG are shown in Table 45.

Comparing the results in Table 45 with those in Table 40 ( results for models with baseline concentration in the models) indicates that there are many more significant terms for the models without the baseline concentrations. 1 The root mean square errors ( RMSEs) in Table 45 are higher than Table 40 for the cases with no hCG stimulation, whereas the RMSEs in Table 45 are lower than Table 40 for the cases with hCG stimulation. 2 The response surface regression results from PROC RSREG are presented in Tables 46 and 47. The optimal values for each time point are shown in Table 48. The factor combinations that produced the optimal values for each time point in Table 48 are very similar to the combinations in Table 43. The standard errors of the predicted values for Table 48 are higher for the data without hCG stimulation than Table 43, whereas the standard errors for Table 48 are lower than Table 43 for the data with hCG stimulation. The differences in the standard errors between the two types of models ( with and without baseline concentrations) are consistent with the results of the RMSEs in Tables 40 and 45. The predicted values for the chosen optimal combination of factors are presented in Table 49. The predicted values in Table 49 are very similar to the predicted values in Table 44. The standard errors for the cases without hCG stimulation are similar between the two model types; however the standard errors for the cases with hCG are lower in Table 13 than those in Table 8. 9

5 Section 9 Phase II Table 38. Data Listing for Incubation Experiment Run Temp Vessel Speed Inc Vol hCG Conc mg Tissue Weight Test 0 Test 1 Test 2 Test 3 Test 4 M 37 test tube high 2.5 0.010 250 0.2606 7.061 24.751 30.890 65.349 87.145 MC 37 test tube high 2.5 0.010 250 0.2666 7.877 65.791 272.168 N 37 test tube high 2.5 1.000 50 0.0569 5.800 22.671 25.308 30.404 33.919 NC 37 test tube high 2.5 1.000 50 0.0494 7.490 18.016 44.939 59.717 78.138 O 37 test tube high 10.0 0.010 50 0.0554 3.971 12.274 14.079 18.412 18.051 OC 37 test tube high 10.0 0.010 50 0.0480 16.667 35.833 57.500 75.000 P 37 test tube high 10.0 1.000 250 0.2736 0.365 9.064 11.367 13.852 16.228 PC 37 test tube high 10.0 1.000 250 0.2654 0.414 24.265 65.825 93.331 109.570 13 37 vial high 2.5 0.010 50 0.0509 30.452 69.745 59.332 55.010 50.098 13C 37 vial high 2.5 0.010 50 0.0506 59.486 103.557 206.522 175.692 13* 37 vial high 2.5 0.010 50 0.0488 38.934 45.082 51.639 47.746 50.205 13C* 37 vial high 2.5 0.010 50 0.0492 19.715 24.390 26.829 14 37 vial high 2.5 1.000 250 0.2653 35.733 46.702 41.425 40.709 39.540 14C 37 vial high 2.5 1.000 250 0.2569 24.251 240.132 378.786 407.513 635.072 14* 37 vial high 2.5 1.000 250 0.2621 27.890 90.729 70.927 59.634 51.354 14C* 37 vial high 2.5 1.000 250 0.2512 39.411 238.774 245.900 502.906 458.718 15 37 vial high 10.0 0.010 250 0.2580 27.209 28.605 28.488 31.085 15C 37 vial high 10.0 0.010 250 0.2411 4.977 42.555 110.162 134.343 216.715 15* 37 vial high 10.0 0.010 250 0.2606 8.711 8.941 15.349 15.503 15C* 37 vial high 10.0 0.010 250 0.2569 34.761 88.945 134.877 156.637 16 37 vial high 10.0 1.000 50 0.0490 3.265 3.469 2.245 2.041 1.837 16C 37 vial high 10.0 1.000 50 0.0535 1.682 2.617 4.486 5.421 6.729 16* 37 vial high 10.0 1.000 50 0.0506 2.767 5.138 5.336 5.138 5.336 16C* 37 vial high 10.0 1.000 50 0.0566 2.473 6.184 13.428 17.668 24.205 S 37 test tube low 5.0 0.100 125 0.1236 1.375 6.068 9.709 14.968 16.100 SC 37 test tube low 5.0 0.100 125 0.1282 4.134 7.098 12.637 15.679 19 37 vial low 5.0 0.100 125 0.1296 6.096 44.830 59.182 72.068 75.540 9

6 Table 38. Data Listing for Incubation Experiment ( Continued)

Run Temp Vessel Speed Inc Vol hCG Conc mg Tissue Weight Test 0 Test 1 Test 2 Test 3 Test 4 19C 37 vial low 5.0 0.100 125 0.1158 5.534 43.178 61.658 74.180 94.732 I 37 test tube none 2.5 0.010 50 0.0503 4.374 11.332 16.700 21.272 23.658 IC 37 test tube none 2.5 0.010 50 0.0544 4.044 19.669 39.338 40.625 57.169 J 37 test tube none 2.5 1.000 250 0.2430 2.469 5.802 7.284 10.329 13.951 JC 37 test tube none 2.5 1.000 250 0.2592 2.160 8.642 12.539 13.927 18.519 K 37 test tube none 10.0 0.010 250 0.2405 2.204 7.318 9.813 11.268 10.894 KC 37 test tube none 10.0 0.010 250 0.2496 1.482 4.888 7.973 7.652 9.014 L 37 test tube none 10.0 1.000 50 0.0473 3.383 7.400 9.091 8.457 LC 37 test tube none 10.0 1.000 50 0.0569 1.933 3.163 4.042 4.394 9 37 vial none 2.5 0.010 250 0.2510 3.705 14.980 20.478 24.462 27.410 9C 37 vial none 2.5 0.010 250 0.2499 3.601 15.846 23.609 35.214 10 37 vial none 2.5 1.000 50 0.0504 8.929 55.556 58.532 70.437 77.579 10C 37 vial none 2.5 1.000 50 0.0554 14.801 73.466 96.570 129.422 172.924 11 37 vial none 10.0 0.010 50 0.0474 3.797 13.502 16.245 19.198 21.730 11C 37 vial none 10.0 0.010 50 0.0515 2.330 1.553 1.748 12 37 vial none 10.0 1.000 250 0.2467 0.365 4.945 7.053 9.360 12.282 12C 37 vial none 10.0 1.000 250 0.2341 5.083 6.835 10.423 17.044 E 34 test tube high 2.5 0.010 50 0.0499 9.619 9.018 10.621 9.018 10.220 EC 34 test tube high 2.5 0.010 50 0.0514 12.257 41.829 57.004 82.101 97.082 F 34 test tube high 2.5 1.000 250 0.2463 9.013 18.270 23.752 30.694 46.041 FC 34 test tube high 2.5 1.000 250 0.2440 8.893 17.254 22.131 30.574 36.352 G 34 test tube high 10.0 0.010 250 0.2534 8.011 13.062 15.746 16.338 17.285 GC 34 test tube high 10.0 0.010 250 0.2503 2.797 5.473 8.670 10.228 10.907 H 34 test tube high 10.0 1.000 50 0.0506 3.755 7.708 12.451 13.636 16.206 HC 34 test tube high 10.0 1.000 50 0.0493 6.085 19.270 21.704 19.675 20.081 U 34 test tube high 5.0 0.100 125 0.1225 8.000 13.551 16.653 20.898 22.286 UC 34 test tube high 5.0 0.100 125 0.1269 4.649 13.002 14.106 20.016 24.665 U* 34 test tube high 5.0 0.100 125 0.1254 6.380 16.667 21.372 27.193 22.488 UC* 34 test tube high 5.0 0.100 125 0.1282 6.786 12.871 18.487 20.827 7.878 9

7 Section 9 Phase II Table 38. Data Listing for Incubation Experiment ( Continued)

Run Temp Vessel Speed Inc Vol hCG Conc mg Tissue Weight Test 0 Test 1 Test 2 Test 3 Test 4 5 34 vial high 2.5 0.010 50 0.0546 11.172 11.538 11.722 12.271 9.890 5C 34 vial high 2.5 0.010 50 0.0457 13.786 10.284 15.536 15.536 12.473 5* 34 vial high 2.5 0.010 50 0.0520 4.615 4.615 3.654 4.808 6.154 5C* 34 vial high 2.5 0.010 50 0.0505 7.723 11.485 11.683 8.713 7.921 6 34 vial high 2.5 1.000 50 0.0481 15.800 12.266 12.474 13.721 16.632 6C 34 vial high 2.5 1.000 50 0.0508 34.449 91.142 153.346 206.693 197.441 6* 34 vial high 2.5 1.000 50 0.0485 14.639 32.371 36.907 42.474 50.928 6C* 34 vial high 2.5 1.000 50 0.0454 16.520 14.978 20.044 20.264 26.432 7 34 vial high 10.0 0.010 50 0.0498 3.614 7.028 9.438 10.241 15.462 7C 34 vial high 10.0 0.010 50 0.0541 2.218 2.403 1.664 1.848 7* 34 vial high 10.0 0.010 50 0.0530 2.642 3.396 4.340 4.717 5.094 7C* 34 vial high 10.0 0.010 50 0.0472 2.331 2.542 1.907 8 34 vial high 10.0 1.000 250 0.2520 2.341 12.143 13.373 13.651 14.921 8C 34 vial high 10.0 1.000 250 0.2685 4.469 22.868 37.877 54.562 70.689 8* 34 vial high 10.0 1.000 250 0.2441 6.145 7.948 8.726 9.586 10.651 8C* 34 vial high 10.0 1.000 250 0.2455 5.662 31.772 53.931 98.737 142.729 21 34 vial high 5.0 0.100 125 0.1196 8.528 8.612 9.448 6.773 9.532 21C 34 vial high 5.0 0.100 125 0.1289 4.888 40.962 86.656 209.930 324.438 21* 34 vial high 5.0 0.100 125 0.1227 7.742 8.883 7.661 7.579 9.128 21C* 34 vial high 5.0 0.100 125 0.1266 8.610 24.329 38.468 48.578 68.325 Q 34 test tube low 5.0 0.100 125 0.1256 0.717 4.697 8.599 9.475 11.226 QC 34 test tube low 5.0 0.100 125 0.1214 6.096 6.260 9.967 9.720 10.626 Q* 34 test tube low 5.0 0.100 125 0.1288 8.463 9.084 8.929 11.102 10.093 QC* 34 test tube low 5.0 0.100 125 0.1320 5.909 13.712 15.833 18.561 21.136 R 34 test tube low 5.0 0.100 125 0.1302 2.995 4.378 9.908 15.131 14.132 RC 34 test tube low 5.0 0.100 125 0.1256 5.494 6.210 11.306 13.217 12.341 V 34 test tube low 2.5 0.100 125 0.1232 1.948 7.143 13.555 16.883 19.643 VC 34 test tube low 2.5 0.100 125 0.1269 2.837 11.348 20.331 31.678 49.724 W 34 test tube low 10.0 0.100 125 0.1237 7.842 10.024 12.369 16.006 18.432 9

8 Table 38. Data Listing for Incubation Experiment ( Continued)

Run Temp Vessel Speed Inc Vol hCG Conc mg Tissue Weight Test 0 Test 1 Test 2 Test 3 Test 4 WC 34 test tube low 10.0 0.100 125 0.1270 2.441 3.858 5.669 7.559 8.583 X 34 test tube low 5.0 0.001 125 0.1262 7.369 11.094 14.659 16.640 18.463 XC 34 test tube low 5.0 0.001 125 0.1258 6.200 12.480 13.752 17.647 19.873 Y 34 test tube low 5.0 10.000 125 0.1263 2.692 6.809 8.947 11.718 12.827 YC 34 test tube low 5.0 10.000 125 0.1247 2.165 8.340 14.354 17.562 21.010 Z 34 test tube low 5.0 0.100 25 0.0223 3.587 ZC 34 test tube low 5.0 0.100 25 0.0235 12.766 23.404 31.064 49.787 AA 34 test tube low 5.0 0.100 250 0.2450 1.143 4.082 6.898 8.286 10.490 AAC 34 test tube low 5.0 0.100 250 0.2530 1.502 3.162 4.862 7.747 8.814 17 34 vial low 5.0 0.100 125 0.1254 20.335 24.402 21.132 50.718 53.907 17C 34 vial low 5.0 0.100 125 0.1275 45.333 28.941 40.314 17* 34 vial low 5.0 0.100 125 0.1223 20.687 28.373 21.096 50.286 54.538 17C* 34 vial low 5.0 0.100 125 0.1323 11.413 13.983 12.472 34.618 46.712 18 34 vial low 5.0 0.100 125 0.1294 27.821 36.012 23.416 56.260 62.906 18C 34 vial low 5.0 0.100 125 0.1290 17.674 20.388 18.527 48.372 63.256 22 34 vial low 2.5 0.100 125 0.1262 2.853 22.979 30.349 40.254 49.366 22C 34 vial low 2.5 0.100 125 0.1288 4.581 37.112 55.745 86.568 125.000 23 34 vial low 10.0 0.100 125 0.1298 2.234 9.014 11.325 13.482 14.946 23C 34 vial low 10.0 0.100 125 0.1255 0.637 9.004 14.821 19.602 26.056 24 34 vial low 5.0 0.001 125 0.1226 3.507 23.002 31.403 37.847 43.312 24C 34 vial low 5.0 0.001 125 0.1238 1.858 12.278 15.670 20.679 24.717 25 34 vial low 5.0 10.000 125 0.1285 0.623 12.918 19.377 22.957 24.358 25C 34 vial low 5.0 10.000 125 0.1235 2.429 18.462 21.862 31.741 50.526 26 34 vial low 5.0 0.100 25 0.0274 7.664 6.204 7.299 7.299 26C 34 vial low 5.0 0.100 25 0.0272 27 34 vial low 5.0 0.100 250 0.2647 3.060 12.694 17.265 22.289 22.818 27C 34 vial low 5.0 0.100 250 0.2537 2.877 25.778 46.078 90.579 121.167 A 34 test tube none 2.5 0.010 250 0.2435 7.885 18.070 22.300 27.556 36.468 AC 34 test tube none 2.5 0.010 250 0.2606 7.866 14.275 19.916 22.909 26.592 9

9 Section 9 Phase II Table 38. Data Listing for Incubation Experiment ( Continued)

Run Temp Vessel Speed Inc Vol hCG Conc mg Tissue Weight Test 0 Test 1 Test 2 Test 3 Test 4 B 34 test tube none 2.5 1.000 50 0.0523 13.767 15.488 17.400 19.503 BC 34 test tube none 2.5 1.000 50 0.0533 18.949 30.769 32.645 35.835 37.148 C 34 test tube none 10.0 0.010 50 0.0452 9.513 15.708 18.142 16.372 19.027 CC 34 test tube none 10.0 0.010 50 0.0516 12.984 24.225 34.884 45.543 48.062 D 34 test tube none 10.0 1.000 250 0.2408 7.766 7.226 9.053 9.925 12.209 DC 34 test tube none 10.0 1.000 250 0.2406 6.151 7.606 11.887 14.630 16.417 T 34 test tube none 5.0 0.100 125 0.1254 7.257 16.188 18.979 16.108 27.193 TC 34 test tube none 5.0 0.100 125 0.1287 4.895 15.540 28.594 36.752 T* 34 test tube none 5.0 0.100 125 0.1253 6.305 11.333 19.154 24.900 18.037 TC* 34 test tube none 5.0 0.100 125 0.1296 3.858 11.960 17.515 19.444 23.843 1 34 vial none 2.5 0.010 50 0.0476 15.336 86.134 120.588 135.294 150.420 1C 34 vial none 2.5 0.010 50 0.0448 18.750 41.071 55.580 62.500 78.125 2 34 vial none 2.5 1.000 250 0.2439 12.423 30.217 53.424 61.337 71.628 2C 34 vial none 2.5 1.000 250 0.2345 19.446 49.595 88.913 134.371 3 34 vial none 10.0 0.010 250 0.2314 1.642 6.785 14.866 11.841 16.854 3C 34 vial none 10.0 0.010 250 0.2462 3.249 7.433 13.891 11.779 16.288 4 34 vial none 10.0 1.000 50 0.0521 2.495 7.294 9.405 10.749 10.940 4C 34 vial none 10.0 1.000 50 0.0528 4.167 10.795 16.288 20.644 24.621 20 34 vial none 5.0 0.100 125 0.1239 4.358 18.402 27.603 32.849 37.530 20C 34 vial none 5.0 0.100 125 0.1263 1.504 30.404 37.609 42.439 47.348 20* 34 vial none 5.0 0.100 125 0.1242 18.438 23.108 31.481 33.253 20C* 34 vial none 5.0 0.100 125 0.1224 26.389 34.967 42.157 44.444 9