in vitro toxicity testing protocols

CHAPTER 1 Alternative Method of Assessing Toxicity Chris K Atterwill 1. Introduction The safety assessment of new chemicals and pharmaceuticals and the incorporation of these data into a human risk assessment package requires a large number of expensive, regulated tests in animal species including, in some cases, nonhuman primates (I-3). There are currently a wide range of animal replacement alternative opportunities in industrial chemical and drug development (Table 1) (4,.5). Although in vitro methodology has long been used as a basic labo- ratory tool for defining biological and toxicological processes in differ- ent cellular systems, application and use alternatives in industrial compound discovery (i.e., research and development) is slow. Coupled with a relatively low innovation rate in the design of new in vivo tests for the toxicological and safety evaluation of new compounds, this has both ethical and resource implications. From a basic scientific viewpoint in vitro toxicological models have made important contributions in elucidating, e.g., the cellular and molec- ular mechanisms involved in apoptotic and necrotic cell death and in carcinogenesis and the role of mediators, such as free radicals and oncogenes, in these processes (6). The “take-up” of in vitro systems in toxicity testing is, however, now gradually occurring in industry and resources are being invested slowly into the area for both ethical and scientific reasons. A lot of emphasis currently lies on the ethical question as public sensitivity to animal use in safety testing increases. This has occurred most significantly in the case From Methods m Molecular Wology, Vol 43 In Wtro Tox/c/ty Testmg Protocols Edited by S. O’Hare and C. K Atterwlll Copyright Humana Press Inc , Totowa, NJ 1 Atterwill Table 1 Ammal Replacement Alternatives Improved storage, exchange, and use of information, so that unnecessary repetition of experiments on animals could be avoided. Maximum use of predictions based on physical and chemical properties of molecules Mathematical modeling of quantitative structure-activity relationships. Molecular modeling and the use of computer graphics. Mathematical modeling of biochemical, physiological, pharmacological, and toxlco- logical systems and processes. The use of lower organisms not protected by legislation, including invertebrates, plants, and microbes.a The use of embryonic and larval vertebrates before they reach the developmental stage at which time they become protected by law a The use of in vitro methods including subcellular fractions, perfused organs, tissue shces, and cell suspensions; and cell organotypic cultures * Human studies, including epidemiology, postmarketing surveillance, and the properly regulated use of volunteers.a % vitro areas of cosmetic and toiletry safety assessment and the safety testing of chemi- cal intermediates used in drug synthesis, which have classically used the controversial Draize, guinea pig, and rabbit tests, and for which valid alternatives now exist. Furthermore, in drug development the classic LD,, tests for acute toxicity have not only been partially replaced by the “fixed dose” procedure, but much work is being carried out by Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) on new and rapid in vitro predictors for acute cytotoxicity using human cell lines. 2. Compound Development Utilizing Alternative Test Models The development and registration of both new drugs and chemicals currently requires the submission of a large battery of in vivo toxicity data derived from a number of species for the “risk-assessment” process (1-3). The compilation and validation of the animal batteries has been largely empirical over the years and, although being fairly well-proven for detecting toxic phenomena in animal species, can have limited pre- dictive value for human safety assessment for some of the reasons listed in Table 2 (a,b). There are large lists of chemicals with good animal- human toxic correlations, but equally, lists of compounds exist that have been withdrawn from the market because of the increasing number of clinically reported adverse reactions (ADRs). Alternative Method of Assessing Toxicity 3 Table 2a Reasons for Incorrect Predictions from Animal Toxicity Studies False negative responses Effect not looked for Use of inappropriate assay methoda Improper timing of assay Insufficient target organ exposure0 Incorrect evaluation of an experimental finding0 Failure to consider absence of preexisting pathological condition Inability to identify and measure adverse effect0 False negative and false positive responses Failure to consider differences in metabolic activation and detoxrfrcationa Disregard of anatomrcal and physiological differences between species“ Inability of animals to express human-specific reaction pattern9 Table 2b Conversion Factors for Predictmg from Animal Studies to Individuals at Risk Toxic effects From experiment to humans! From humans to individuals at risk Molecular Bioavailability Healthy men and women Subcellular Pharmacokinetics Babies and children Cellular Protein bindinga Pregnant women Tissue Metabolism0 Elderly Organ Dose and time People at genetic risk Whole animal Concentrationa Diabetics Receptor sensitivitya People with infections Anatomical characteristics Immunosuppressed individuals Physiological characteristicsa Alcohol or drug abusers Repau mechanismsa Smokers Species-specific responsea Patients with organic disease Mechanisms of actiona Occupationally exposed indivrduals OAreas in which alternatlves tests can have an impact. (Data taken from table compiled by G Zbinden, personal communication.) It is also well accepted that during the development of a “safe” and effective pharmaceutical (or agrochemical agent) there is a large attrition rate throughout the safety assessment process with massive financial implications. When one superimposes on this the varying worldwide regulatory requirements for administration of a new compound to humans, one can see a number of important reasons for developing in vitro toxicity testing systems either as prescreens or as adjuncts to cur- rent in vivo test packages. This, together with harmonization of the cur- 4 Atterwill rent regulatory requirements, will hopefully improve the sensitivity and specificity of animal tests (3). 3. Summary of Gains 1. Financial gains: Reduce attrition rate by use of prescreening strategies prior to full regulatory animal study packages and develop better predictors of human toxic phenomena. 2. Scientific gains: Describe more effectively the lesions seen m vivo from regulatory studies and give better definition of safe concentration and clinical dose. Define “direct toxicant effects” on target organs as opposed to indirect effects, and give human reaction mdication using primate cells. “Adjunct” studies will improve sensitivity and specificity of ani- mal studies. 3. Ethical gains: Implementation of 3R strategy (reduce, refine, replace am- ma1 testing). Supplement and reduce current m vivo toxicity tests, particu- larly those involving distressing procedures and the use of large mammals and primates. 4. Principles, Aims, and Types of In Vitro Toxicity Testing The general advantages and disadvantages of in vitro testing for toxic- ity are described in Table 3. 5. Validation The successful use and industrial and regulatory acceptance of a new in vitro test model depends on a certain degree of validation (7,s). Detailed validation is generally required if the in vitro test is to replace an in vivo test or is to be used as a prescreen where financial factors are critical. However, when the result from an in vitro procedure is submitted to a regulatory authority along with that from an in vivo test package in order to explain a lesion, then full validation is not formally necessary as long as good laboratory practice (GLP) procedures have been adopted in the execution of that test and the test and endpoints have acceptable rele- vance. For example, the gradual replacement of the Draize procedure by tests such as the EYETEX or SKIN2 tests has required extensive valida- tion of that test. So, for example, would the use of a prescreen for a new immunosuppressant with adrenal toxicity where there were a limited number of available backup compounds or they were very expensive to synthesize. On the other hand, if a drug company were trying to confirm to the Food and Drug Administration (FDA) that a particular drug had no Alternative Method of Assessing Toxicity 5 Table 3 Advantages and Disadvantages of In Vitro Systems for Detection of Xenobiotic-Mediated Toxicity Advantages (general) Detect direct (vs Indirect) toxrc/cytotoxic effects on target organ. Use controlled conditions of exposure-concentration of toxrcant known. Study parent compound vs metabolite (rt liver S9 metabolizing fractions from different species) Study effects on cells vs subcellular organelles. Has resource implications (time, animals, number of compounds tested). Disadvantages (general) Systems not always representative of mature, differentiated target organs (cells dedifferentiated in cell lines?). Xenobiotic concentrations not representative of those in vivo (e.g., plasma protein bindmg factors). Biological barriers absent (e.g., blood-brain barrier in neural cultures of CNS). Metabolite profiles differ Difficulty of culturing/maintaining certain target organs in vitro. direct neurotoxic effect in humans despite some minor behavioral changes detected in the rat, then submission of data from cultured human exposed neurons would be acceptable, probably without full validation of that particular culture model. Accepted validation criteria for an in vitro system are described in Table 4 and include definition of the specificity, sensitivity, and predic- tive value of such a test. The validation parameters are obtained by con- ducting blind validation trials. It is my belief that the requirements for in vitro test validation can be summarized as follows: 1. Full validation involving multicenter coordination: To support in vrvo test replacement. 2. In-house validation: In vivo test reduction, to support, e.g., development of a prescreen, 3. Limited inter- or intralaboratory: To support refinement or supplementa- tion of in vivo toxicity test data, to develop adjunct tests; use of the model to define basic scientific toxic phenomena. It is believed that validation should not be used as an excuse for nonadoption, nondevelopment, or nonacceptance of in vitro methodol- ogy. Sadly, and largely for political reasons, this scenario still exists in many companies and countries. Atterwill Table 4 Validation Criteria for In Vitro Test Models A formal validation study will require: Careful selection of chemicals (mimmum 20-40?) Use of chemical pairs Toxicological classification from m vivo data “Blind” testing to be performed Method for evaluation of test outcome (absolute values) Method for evaluation of test performance Methods of expressing test performance Other points Are there good in vivo comparative data for compounds chosen? Which kind of in vivo assay trying to emulate/evaluate in vitro? Agree with collaborating centers in validation trial at beginning who will be organizing and collatmg data. 6. Spectrum of Available In Vitro Toxicity Tests The currently available models in In Vitro Toxicology (Table 5) (5-8) span six main areas: reproductive toxicity, mutagenicity, irritancy test- ing, immunotoxicity, target organ toxicity (including endocrine and neu- rotoxicity), and ecotoxicity involving the use of fish, invertebrates, and so on. Within these main areas there are also important subareas. As alluded to above there are various modes in which to operate these tests in an industrial setting and generally the mode predominance varies significantly according to both scientific area and whether or not one is operating in the drug, chemical, or cosmetic industry. For example, a test system might progress from unvalidated use in the fine description of a pathologically identified lesion for a lead development compound, to the subsequent, semivalidated use of this system in a prescreening mode for second-generation drug candidate compounds. Alternatively, the agro- chemical industry has developed a tiered in viva/in vitro hierarchical model for the labeling of industrial chemicals as skin irritants. This latter development was performed under the auspices of the British Toxico- logical Society, showing how the scientific and industrial communities can interact so well on such issues. Here, a chemical for irritancy classi- fication would proceed from tests on isolated skin or cells in vitro to tests in a limited number of animals in vivo depending on negative or positive outcomes in the initial in vitro tests. Alternative Method of Assessing Toxicity 7 Table 5 In Vttro Models Currently Available in Toxicology Mutagenicity testing Irritancy testing Reproductive toxicity testing Quality-Structure Activity Relationship (QSAR) Target organ toxicity Immunotoxicity Hemtc system Endocrine toxicity Neurotoxtctty Acutelcytotoxicity testing 7. Recent Successes and Developments in In Vitro Toxicity Testing It is refreshing to observe the momentum that is now gathering in this field (see Table 6) and the way in which “in the face of adversity” some tests are being accepted as full replacement alternatives. It is noteworthy to say that a lot of this energy has been provided by the public and by academic research centers. Apart from the mutagenicity test area where many innovations con- tinue to occur, some of the following recent developments in other areas warrant attention. 1. Eyetex, Skmtex, and Corrosrtex tests for eye and skin irritancy (Ropak Corporation Ltd) and the SKIN2 Model (Advanced Tissue Sciences). More recently, the Ropak Solatex system for predicting photoirritation in vitro. 2. The use of hepatocyte “couplets” for in vitro investigation of xenobiotic effects on bile flow. Together with measurements of hepatotoxicity and fatty acid accumulation by these cells, rt may now be possible to obtain a complete hepatoxicological profile in one in vitro model. 3. Luminescent bacteria (Microtox test) for measuring the ecotoxic potential of industrial effluent. 4. More sensitive in vitro toxicity measurements using, for example, the mito- chondrial MTT test for succurate dehydrogenase activity. This test gives a more sensitive and earlier prediction of toxicity than classical LDH or neu- tral red measurements. Atterwill Table 6 Orgamzatlons Involved m the Development of Alternative Testing Bodies for promotion of alternatlve nonanimal testing FRAME-Fund for Replacement of Animals in Medical Experiments ERGAT-European Research Group for Alternative Testing EURONICHE-European group for alternatlve methods for biology teaching CAAT-Center for Alternatives to Animal Testings (Johns Hopkins Medical School, Baltimore, MD) Dr. Hadwen Trust-Nonammal research and testing strategies (UK-based) Societies, conferences, and journals advancing alternative testing PIVT-Practical In Vitro Toxicology conference IVTS-In Vitro Toxicology Society (UK) Scandinavian Cellular Toxicology Society FRAME Toxicity Committee and Conference TIV-Toxicology In Vitro, Journal ATLA-Alternatwes to Laboratory Animals, Journal Hildegard Doerenkamp and Gerhard Zbinden Foundation for Reahstlc Animal Protection and Scientific Research, Switzerland 5. Measurements of calcium accumulation in single cultured neurons for the measurement of neurotoxicity. 6. Tlered tests involving both simple and organotyplc organ systems; hierar- chical proceeds involving a battery of m vitro and in vivo models. 8. Conclusions Industry and academia have come far in developing in vitro alterna- tives, and bodies such as Fund for Replacement of Animals in Medical Experiments (FRAME), European Centre for Validation in Alternative Methods (ECVAM), and Center for Alternatives to Animal Testing (CAAT) (USA), have simultaneously enhanced public and regulatory awareness (Table 6). The regulatory and industrial acceptance of new alternative tests depends on proper, well-coordinated validation trials at a level befitting the intended use of the alternative test. This has started through FRAME and European Community (EC) initiatives and good examples have been set by the cosmetics and toiletry industries. More commercial “takeup” is still required for these new tests at the toxico- logical prescreening and in vivo adjunct testing level. Regulatory har- monization of in vivo animal testing is occurring for both ethical and resource reasons. The gradual replacement of the LD,, test by the “Fixed Alternative Method of Assessing Toxicity 9 Dose” procedure for acute toxicity testing and the realization that 6 mo chronic testing (I) is sufficient to identify the most important pathology (excluding carcinogenicity) are most welcome changes. The potential risks for humans in adopting alternative toxicity tests are few and ben- efits great if the data generated is used correctly. The imminent replace- ment of all in vivo tests is unlikely but in the future may gradually occur. In the meantime in vitro tests will continue to supplement the somewhat “impirical” animal tests for human toxicity. References 1. Volans, G. N., Sims, J., Sullivan, F. M., and Turner, P. (eds.) (1989) Basic Science in Toxzcology, V International Congress of Toxicology (ICTV), Taylor & Francis. 2 Poole, A. and Leslie, G. B , eds. (1989) A Practical Approach to Toxological Investigations, Cambridge University Press. 3. Lumley, C., Parkmson, C, and Walker, S. R. (1992) An international appraisal of the minimum duration of chronic animal toxicity studies. Hum. Exp. Toxicol. 11, 155-162 4. Parish, W. E. and Hard, G. C. (eds.), Toxicology In Vitro. Proceedings of Second International Conference on Practical In Vitro Toxicology 4/5. 5. Atterwill, C. K. and Steele, C. E., eds. (1987) In-Vitro Methods in Toxicology, Cambridge University Press, Cambridge. 6. Walum, E., Stenberg, K., and Jenssen, D. (1990) Understanding Cell Toxicology- Principles and Practice, Ellis Horwood, New York, London, Sydney. 7. OECD Environment Monograph No. 36. Scientific Criteria for Validation of In Vitro Toxicity Tests, Sept. 1990. 8. FRAME 21st Anniversary Issue (1990) ATLA, 18. [...]... are incubated for 48 h The cultures are then rinsed and incubated for 3 h in medium containing Neutral Red that is taken up by viable cells After rinsing, the dye present in the cell population is liberated and the amount is quantified using a spectrophotometer, in order to obtain an indication of cell number Comparison of the number of cells in control and test cultures provides an index of cytotoxicity... and C K Atterwill Vol 43: In Vitro Toxicity Testing Protocols Copynght 33 Humana Press Inc., Totowa, NJ Bidey 34 replicate cultures may be maintained, has made the TFC monolayer the model of choice for the quantitative assessment of the agents interfering with or modifying TSH-receptor interaction, transmembrane iodide movement, or cell proliferation The most widely adopted in vitro functional markers... hemispheres using fine forceps 5 The cortical tissue is chopped into about eight pieces These are placed in a sterile tube containing 10 mL of Trypsin/EDTA solution in BME, then are covered and incubated in a water bath at 37°C for 25 min 3.1 Production and Maintenance 20 Cookson, MeClean, and Pentreath 6 The cells are then triturated using a sterile Pasteur pipet and centrifuged at 1000 r-pm in a benchtop... plate for protein determination and treated as in Section 3.3.5 BSA standards are made up in 0.2M NaCl The remaining solution is transferred to a miniature scintillation vial containing 3 mL scintillation cocktail A serial dilution of [3H]2-DG (0.50.0005 l&i) is made up in parallel to act as a standard Samples are shaken and left overnight in the dark at 4OC Samples are counted on a liquid scintillation... LLC-RKl Cell Screening for Nephrotoxicity David X&t J White and Chris Seaman 1 Introduction In this test, kidney-derived cells are cultured in the presence of test compounds whose cytotoxicity is then determined by the Neutral Red method, and serves as an indicator of potential nephrotoxicity (1) Healthy LLC-RKl cells (an established cell line, ATCC CCL) maintained in culture continuously divide and... Tox/c/ty Testing Protocols Copyright Humana Press Inc , Totowa, NJ 11 12 White and Seaman system As a result of this, one-way transport “blisters” are formed in the monolayer, a feature in common with primary kidney cells and other cell lines in culture (2) The application of such cultures to determine nephrotoxicity may potentially allow the rapid, highly reproducible testing of many chemicals on a routine... retain certain characteristics of kidney cells in vivo (2) Compounds that have a deleterious effect on these cells may, therefore, be considered as potential in vivo nephrotoxins In this test system, harmful effects on cell viability are determined by monitoring the uptake of the vital dye Neutral Red into the lysosomes of healthy cells LLC-RKl cells are maintained in culture and exposed to varying... neurotoxic-induced injury can be found in ref 1 The preparation and use of astrocytes is a well established and documented procedure for which there is general consensus regarding the principal steps (see refs 2,3) There are, however, many variations in the From- Methods m Molecular B/ology, Edited by: S O’Hare and C K Atterwill Vol 43 In Vitro Toxrcity Testing Protocols Copyright Humana Press Inc , Totowa,... The remaining cells are treated for protein determination as in Section 3.4., steps 4 and 5 and scmtillation counting as in Section 3.4., step 6 4 Notes 1 Suitable antibiotics are either penicillin/streptomycin (stock 10,000 p,Penicillin G, 10 mg streptomycm/mL) or gentamycin (10 mg/mL stock solution) We commonly use the latter 2 Multiwell dishes are convenient for toxicological evaluations since one... free balanced salt solution (BSS) containing 200 IU/mL penicillin, 200 pg/rnL streptomycin, and 40 IU/mL gentamicin Growth medium: 500 mL Eagle’s medium, 2 mM L-glutamine, 20% v/v lamb serum, 0.1 ug/mL hydrocortisone, 10 mL U/n-& insulin, 1 @4 potassium iodide, 20 IU/mL penicillin, 20 pg/mL streptomycin, 4 IU/mL gentamicin, 1 pg/rnL fungizone, 40 mL U/r& thyrotrophin, 12.5 mL 1M HEPES buffer 3 Methods . oncogenes, in these processes (6). The “take-up” of in vitro systems in toxicity testing is, however, now gradually occurring in industry and resources are being invested slowly into the area. irritancy test- ing, immunotoxicity, target organ toxicity (including endocrine and neu- rotoxicity), and ecotoxicity involving the use of fish, invertebrates, and so on. Within these main areas there. Acutelcytotoxicity testing 7. Recent Successes and Developments in In Vitro Toxicity Testing It is refreshing to observe the momentum that is now gathering in this field (see Table 6) and the way in