In Vitro Toxicology

Book Cover......Page 1
Half-Title......Page 2
Title......Page 3
Copyright......Page 4
Dedication......Page 5
Contributors......Page 6
Contents......Page 8
Preface......Page 10
1 Introduction......Page 12
1.2 LEVELS......Page 13
1.3 HISTORY......Page 15
1.3.1 The Four R’s......Page 16
1.4 DRIVING FORCES......Page 18
References......Page 21
2 General Principles for In Vitro Toxicology......Page 23
2.1 TEST SYSTEMS: CHARACTERISTICS, DEVELOPMENT, AND SELECTION......Page 24
2.1.1 Considerations in Adopting New Test Systems......Page 26
2.2 TARGET ORGAN TOXICITY MODELS......Page 29
2.3 SENSITIVITY, SPECIFICITY, AND PREDICTIVE VALUE......Page 30
2.4 PROBLEMS IN INTERPRETATION AND EXTRAPOLATION......Page 31
2.5 VALIDATION......Page 32
References......Page 33
3.1 IN VIVO IRRITATION TESTING......Page 35
3.2.1 Prediction from Preexisting Data......Page 36
3.2.2 In Vitro Approach......Page 40
3.2.3 In Vitro Models......Page 41
3.3 USING ALTERNATIVES......Page 46
3.4 VALIDATION......Page 48
References......Page 52
4.1 CURRENT IN VITRO METHODS......Page 60
4.1.1 Physicochemical Test Methods......Page 61
4.1.2 Cell Culture Techniques......Page 63
4.1.4 Human Skin Equivalents......Page 66
4.1.5 Embryonic Testing......Page 67
4.2 HUMAN VOLUNTEER STUDIES......Page 68
References......Page 69
5 Lethality Testing......Page 73
5.1.1 Primary Cultures......Page 74
5.1.3 Clonal Cell Lines......Page 75
5.1.4 Growth Conditions......Page 76
5.1.5 Metabolic Activating Systems......Page 77
5.2 ENDPOINTS......Page 78
5.3.1 Dye Exclusion/Uptake......Page 79
5.3.2 Membrane Leakage......Page 80
5.4.1 Cloning Efficiency......Page 81
5.4.3 Mitogenicity......Page 82
5.4.4 Cell-Cycle Kinetics......Page 83
5.5.2 Morphology Indicators of Cytotoxicity......Page 84
5.5.3 Quantification of Morphologic Changes......Page 85
5.6.1 Thermodynamic and Metabolic Function......Page 86
5.6.2 MTT Assay......Page 87
5.7 ASSAY VALIDATION......Page 88
5.7.1 Early Validation Studies......Page 90
5.7.2 Multicenter Studies......Page 91
5.8 THE ROLE OF PHARMACOKINETICS AND STRUCTURE-ACTIVITY RELATIONSHIPS IN MODELING......Page 95
5.8.1 Pharmacokinetics......Page 96
5.8.2 Physiologic Pharmacokinetic Modeling......Page 97
5.8.3 Computer Structure-Activity Relationships Modeling......Page 98
5.9 APPROACHES FOR EVALUATING HUMAN RISK......Page 99
References......Page 101
6 In Vitro Genetic Toxicity Testing......Page 106
6.1.1 Gene Mutations......Page 109
6.1.2 Chromosomal Mutations......Page 111
6.2 DEVELOPMENT AND APPLICATION OF GENETIC TOXICOLOGY TESTS......Page 113
6.3 HARMONIZED GUIDANCE FOR REGULATORY SUBMISSIONS......Page 119
6.3.1 Organization for Economic Cooperation and Development Screening Information Data Set Guidelines......Page 120
6.3.2 Toxic Substances Control Act Guidelines......Page 121
6.3.3 International Council on Harmonization Guidelines......Page 122
6.4 GENETIC TOXICOLOGY TESTS FOR CURRENT PRODUCT REGISTRATION......Page 123
6.4.1 Reverse Gene Mutations in Bacteria......Page 124
6.4.2 The L5178Y/Thymidine Kinase+/− Gene and Chromosomal Mutation Assay......Page 126
6.4.3 Chromosomal Aberrations: In Vitro......Page 128
6.4.4 Rodent Bone Marrow Chromosomal Effects......Page 130
6.4.5 Chromosomal Aberrations: In Vivo......Page 131
6.4.6 Micronucleus Tests.......Page 132
6.5 ADVANTAGES, DISADVANTAGES, AND PREDICTIVITY OF THE GENETIC TOXICOLOGY TESTS USED FOR CURRENT PRODUCT REGISTRATION......Page 133
References......Page 136
7.1.1 In Vivo......Page 139
7.1.2 In Vitro......Page 141
7.2 IRRITATION OF PARENTERALLY ADMINISTERED AGENTS......Page 146
References......Page 147
8 In Vitro Models for Evaluating Developmental Toxicity......Page 149
8.1 PRIMARY CELL CULTURE......Page 151
8.1.1 Nonmammalian Species......Page 153
8.1.2 Mammalian Species......Page 154
8.2 ESTABLISHED CELL LINES......Page 158
8.3.1 Nonmammalian Systems......Page 163
8.3.2 Mammalian Whole Embryos......Page 167
8.4 ORGAN CULTURE......Page 169
8.5 CELL-SIGNALING ASSAYS......Page 170
8.6.1 In Vitro-In Vivo Dose Conversion......Page 171
8.6.2 Limitations of In Vitro Data for Assessing Health Risks......Page 172
8.7 CONCLUSIONS......Page 173
References......Page 174
9 Neurotoxicology In Vitro......Page 197
9.2 IN VITRO SYSTEMS IN NEUROTOXICOLOGY......Page 198
9.3.1 Glia......Page 200
9.3.2 Neurons......Page 202
9.3.3 Use in Physiologic Studies......Page 205
9.4 TISSUE CULTURE METHODOLOGY......Page 207
9.5 ASSAYS OF TOXICITY: CELL CULTURE MODELS......Page 210
9.6 NEUROTOXICOLOGY......Page 212
9.7 ISOLATED TISSUE ASSAYS......Page 214
9.7.1 Electrophysiology Methods......Page 215
9.7.2 Neurochemical and Biochemical Assays......Page 216
9.9.1 Phenobarbital, Phenytoin, and Other Anticonvulsant Agents......Page 218
9.9.2 Excitotoxins......Page 220
9.9.3 Heavy Metals......Page 222
9.10 PROBLEMS......Page 223
References......Page 224
10 In Vitro Assessment of Nephrotoxicity......Page 231
10.1.1 Methodology......Page 232
10.1.2 Advantages......Page 234
10.1.3 Limitations......Page 235
10.2.1 Acetaminophen Nephrotoxicity and Metabolism in the Isolated Perfused Kidney......Page 236
10.2.2 Cisplatin Nephrotoxicity in the Isolated Perfused Kidney......Page 238
10.3.1 Methodology......Page 240
10.3.2 Advantages......Page 241
10.3.3 Limitations......Page 242
10.4.1 Biochemical Mechanisms of Nephrotoxicity: Cephalosporin Antibiotics......Page 243
10.4.2 Site-Selective Nephrotoxic Injury......Page 246
10.5 TUBULE SUSPENSIONS......Page 247
10.5.2 Advantages......Page 248
10.5.3 Limitations......Page 249
10.6.1 Comparison of Tubular Segments: Proximal Versus Distal Tubules......Page 251
10.6.2 Biochemical Mechanisms of Toxicity: 4-Aminophenol......Page 253
10.7.1 Methodology......Page 254
10.7.2 Advantages......Page 256
10.8.1 Comparison of Renal Tubular Cells: Proximal Versus Distal Tubular Cells......Page 259
10.8.2 Biochemical Mechanisms of Cytotoxicity: Halogenated Hydrocarbons......Page 261
10.9.1 Methodology......Page 264
10.9.2 Advantages......Page 265
10.9.3 Limitations......Page 267
10.10.1 Biochemical Mechanisms of Lethal Cell Injury: Halogenated Hydrocarbons......Page 269
10.10.2 Cellular Accumulation and Metabolism: Aminoglycoside Antibiotics......Page 271
10.11 CONCLUDING REMARKS......Page 274
References......Page 275
11 Primary Hepatocyte Culture as an In Vitro Toxicologic System of the Liver......Page 284
11.1 ISOLATION OF PRIMARY HEPATOCYTES......Page 287
11.2 CULTURING OF ISOLATED HEPATOCYTES......Page 290
11.3.1 Phase I Oxidation: P450 Isozymes......Page 292
11.3.2 Phase II Conjugation......Page 293
11.3.3 Species Comparison Studies......Page 294
11.4 TOXICOLOGY......Page 296
11.4.1 Cytotoxicity......Page 297
11.4.2 Genotoxicity......Page 299
11.4.3 Enzyme Induction......Page 300
11.4.4 P450 Induction......Page 301
11.5 HUMAN HEPATOCYTES......Page 303
11.6 INDUSTRIAL APPLICATIONS......Page 305
11.7 CONCLUSIONS......Page 306
References......Page 307
12 Application of In Vitro Model Systems to Study Cardiovascular Toxicity......Page 314
12.1.1 Perfused Organ Preparations......Page 316
12.1.2 Isolated Muscle Preparations......Page 317
12.1.3 Organ Culture......Page 321
12.1.5 Single-Cell Suspensions......Page 322
12.1.6 Cell Culture Systems......Page 324
References......Page 328
13.1 TYPES OF GASTROINTESTINAL CELLS AND TOXICITY......Page 335
13.2 IN VITRO MODELS......Page 337
13.3.1 Irritation......Page 339
13.3.3 Malabsorption......Page 340
13.3.4 Altered Secretory Activity......Page 341
13.3.5 Altered Motility......Page 343
13.3.7 Symbiotic Population Alteration......Page 347
13.4 SPECIFIC PROBLEMS/LIMITATIONS OF IN VITRO TEST SYSTEMS......Page 349
References......Page 351
14.1 GENERAL ORGANIZATION OF THE IMMUNE SYSTEM......Page 356
14.2 TRIGGERING THE IMMUNE RESPONSE......Page 357
14.2.2 Acquired Immunity......Page 358
14.3.1 Immunosuppression......Page 359
14.3.3 Autoimmunity......Page 360
14.4.1 Origin of the Cells......Page 361
14.4.4 Conditions of Culturing Immune Cells......Page 362
14.5.1 Dendritic Cells as a Tool to Evaluate the Sensitizing Potential of Hapten......Page 363
14.5.2 Models to Assess Lymphoproliferation......Page 364
14.5.3 Cytokine Measurement to Assess T Helper or T Helper 2 Lymphocyte Differentiation......Page 366
14.5.4 Evaluation of Apoptosis Induced by Xenobiotics......Page 367
14.5.5 Models to Assess Lymphocyte Cytotoxicity (Innate or Acquired)......Page 369
14.5.6 In Vitro Antibody Production......Page 370
14.6 RELEVANCE OF IN VITRO MODELS......Page 371
References......Page 372
15 Strategy and Tactics for Employment......Page 375
15.1 DEFINING TEST OBJECTIVES......Page 377
15.1.1 Objectives Behind Data Generation and Utilization......Page 378
15.2 DESIGNING THE RESEARCH PROGRAM......Page 380
15.2.2 In Vitro Models......Page 384
15.2.4 Concept of Screens......Page 386
15.2.5 Far Horizons: How to Get Them......Page 387
15.3 CONCLUSION......Page 389
References......Page 390
16.1 SHARED GOALS: PREDICTABILITY AND RESPONSIBILITY......Page 391
16.2.1 The Drug Discovery Process......Page 392
16.2.3 Selection of Test Procedures......Page 393
16.3.1 Proactive Versus Reactive Behavior......Page 394
16.3.3 Scientific/Regulatory Collaboration......Page 395
16.4 VALIDATION......Page 396
References......Page 397
17 Safety Issues in the Use of Human Tissue by In Vitro Metabolism and Toxicology Laboratories......Page 399
17.1 SEROLOGY......Page 400
17.2 LABORATORY PROCEDURES AND GENERAL PRECAUTIONS......Page 401
17.3.1 Inhalation......Page 402
17.4.2 Ingestion......Page 403
17.4.4 Contact with Mucous Membranes/Skin......Page 404
17.6 LABORATORY CLEANUP......Page 405
References......Page 406
INDEX......Page 408