In Vitro Methods in Toxicology
Horst Spielmann, M.D.
Director and Professor
Diedersdorfer Weg 1
Berlin, Germany D-12277
Alan M. Goldberg, Ph.D.
Director and Professor
Johns Hopkins Center for Alternatives to Animal Testing
111 Market Place
Baltimore, MD 21202
Lisa A. Libowitz
In Vitro Methods in Toxicology is a chapter from a textbook (in press) by Academic Press. Toxicology is scheduled for publication in 1999.
During the last century, the field of toxicology has evolved from one of describing the consequences of exposure to chemicals, to xenobiotics in animals, to mechanistically-based studies of both animals and cell or tissue cultures. During the last decade, in vitro studies of cell and tissue cultures, such as the Ames Mutagenicity Assay, have become an important approach used to understand the consequences of exposure and to assign risk.
In both U.S. and European laboratories, scientists have vigorously pursued the development of in vitro methods to advance their science. During the last 20 years, the considerable and significant advances in tissue culture methodology, the use of chemically-defined cell and tissue culture media, and the availability of human cells have transformed in vitro methods from a new technology to a valuable research tool.
In product development, drug discovery, and safety evaluation, the use of in vitro tests has become commonplace, resulting almost exclusively from the evolution of science rather than any fundamental change in philosophy. Yet all in vitro methods are alternatives to animal testing.
The Three Rs Concept of Russell and Burch
The term "alternative" emerged following the publication of a now-classic book by William Russell and Rex Burch, The Principles of Humane Experimental Technique. (1) The authors suggested that proper experimental design should consider methods that refined current techniques to lessen pain or distress, reduced the number of animals necessary for a particular test, or replaced animals with non-whole- animal models, such as in vitro cell cultures. The concept of refinement, reduction, and replacement became known as the Three Rs, and methods which incorporated one or more of the Three Rs were considered "alternative" methods.
Refinement alternatives are methods that either eliminate or minimize pain and distress or enhance the animal's well-being. The assessment of pain in animals is difficult, but generally one can recognize if an animal is in pain or predict that if a procedure would be painful to humans it would be painful to animals. Just recognizing this can change experimental design. Animal well-being can be enhanced by using appropriate handling techniques and cage sizes, as well as by environmental enrichment techniques well known to the psychological field. (2)
Reduction alternatives are those methods that provide the correct amount of data for an experimental design. It is important to recognize that statistical input must be provided at the earliest stages of experimental design and not once the data has been collected. Not only will this provide better science, it will result in using the correct number of animals -- generally, fewer than when guessing what the correct number should be.
Replacement alternatives are methods that do not use whole live animals, or that replace an animal method with physical or chemical models and/or human studies when ethically appropriate (more later). These include the use of in vitro methods. Table 1 offers examples that demonstrate how in vitro methods have replaced animals, according to the Three Rs concept, in quality control tests of hormones and drugs, as well as in toxicological research.
According to Russell and Burch, the incorporation of the Three Rs into experimental design led not only to more humane research, but also to better science. Published in 1959, The Principles of Humane Experimental Technique (1) became the foundation of all animal welfare legislation adopted in 1986 in both the United States and Europe.
In 1995 Russell and Burch reviewed the Three Rs concept with a dozen international colleagues, and confirmed its importance and usefulness. (See "The Three Rs: The Way Forward," ATLA 23, 838-866, 1995, and summarized in an editorial that appeared in Science magazine, Vol. 272, page 1403, June 1996.)
Today there are centers throughout the world that focus on the development, validation, and use of alternatives in the biomedical sciences (including toxicology). As the science behind in vitro technology advances, and as results from new methods are shown to be both transferable and reproducible, these alternatives continue to gain acceptance. Additionally, the scientific community has responded to the substantial public outcry for a decrease in animal use by considering the advantages and limitations of alternatives.
Information about alternatives is widely available today through journals, databases, and sites on the World Wide Web devoted to the Three Rs. (These resources will be documented below.) However, the word "alternative" has become politicized, and for many in the scientific or animal activist community, it conjures up an image very different from the thoughtful, humane experimental science proposed by Russell and Burch. As a result, at the 1995 meeting on the Three Rs, it was decided that the word "alternative" would, when appropriate, have one of the Three Rs in front of it (i.e., replacement alternative, refinement alternative, or reduction alternative), or be used as the phrase "the Three Rs of alternatives."
Methods for increasing awareness and use of alternatives in the United States and Europe complement each other and, at the same time, demonstrate differences in the political and scientific approach to effecting change on each side of the Atlantic.
In Europe, the passage of the EU Animal Protection Guideline 86/609/EEC requires the replacement of testing on animals with non-animal procedures. The current focus is on the safety testing of cosmetics. This directive has resulted in significant governmental funding of programs to achieve its aim throughout the European Union, as well as the formation of ECVAM (European Center for the Validation of Alternative Methods) in Ispra, Italy. The basic charge of ECVAM is to:
- coordinate the validation of alternative test methods at the European Community level;
- act as a focal point for the exchange of information on the development of alternative test methods;
- establish, maintain, and manage a database on alternative procedures;
- promote dialogue among legislators, industry, biomedical scientists, consumer organizations, and animal welfare groups.
In the United States, animal welfare laws have forced institutions to establish ethical review committees and have encouraged the development of refinement procedures through these ethical committees. However, U.S. regulatory agencies such as the Food and Drug Administration (FDA) and the Environmental Protection Agency have only very recently -- and on an almost entirely voluntary basis -- begun to learn about the need to evaulate programs with regard to their use of alternatives. The FDA, through its Science Advisory Board Subcommittee on Toxicology, has taken the most active role and, as such, has truly encouraged the development and use of alternatives. In 1993, the U.S. Congress passed the NIH Revitalization Act, which resulted in the formation of the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM). ICCVAM is a consortium of 14 different federal agencies trying to help each other identify validated methods that each agency can then assess for its own individual regulatory use and acceptance. The U.S. government has provided almost no funding to this process, and most of the real advances in validation in the United States occur through the private sector. In contrast, both the private sector and European governments have provided substantial financial support to the development of alternatives in Europe (approximately $15 million annually from the governments).
Validation and Valid Methods
Political pressure to decrease animal testing has created a rush in recent years to get in vitro methods into the regulatory acceptance process. At the same time, the potential for many in vitro methods to be commerically profitable has resulted in a push to bring inadequately developed methods to the commercial market. As a result, validation has suffered from something of the "cart before the horse" phenomenon. Only recently has it been universally recognized that there was a need for a terminology of validation and a coordinated set of approaches.
The scientific community always has required that new methods be proven reproducible, published in peer-reviewed journals, and subjected to additional independent studies before becoming accepted as a new methodology for wide use, i.e., valid methods. There are, of course, different experimental approaches to prove that an in vitro method provides the same information as an established animal test. To reach international consensus with both the scientific and regulatory communities, an agreement was reached on the validation process of toxicity test procedures (3). Studies that were conducted according to these recommendations predominantly in Europe have produced significant and useful information, but have failed to provide validated methods that are accepted by the regulatory community. However, the effort provided ECVAM with valuable practical experience in creating a validation process (4).
Based on this experience, both the OECD (5) and ICCVAM (6) have developed sets of criteria that describe a necessary set of information needed before regulatory acceptance can be considered and also have provided criteria for regulatory acceptance of methodology. Once these criteria gain international acceptance within the scientific community, it is anticipated that regulatory acceptance will occur. The next stage of development is to identify the minimum set of criteria that have to be met to gain regulatory acceptance, as well as to consider approaches to implementation.
Current Use of In Vitro Methods in Toxicology Testing
In vitro methods are routinely used by all industries and regulatory bodies in toxicity testing, safety assessment, and risk evaluation, and offer unique advantages (Table 2). The greatest use of in vitro methods, however, is for elucidating mechanisms of toxicity and/or demonstrating the biological process involved in a toxic response to a xenobiotic, including drugs. With the development and widespread use of the Ames Mutagenic Assay, non-animal testing became routine in both industry and regulatory laboratories. In fact, it is not uncommon to find that the only toxicity studies done and sometimes available in the public literature is an Ames test (7).
Within the cosmetics industry, the last several years have seen a remarkable change in the approach to safety evaluation. Most cosmetics now on the market have not been tested on animals, although some of their ingredients may have been. This is due, in great part, to the available databases and knowledge of chemicals used in the cosmetics industry, but also in part to the development of new methods that have been evaluated by individual companies and incorporated into their safety assessment programs. As new and never-tested chemicals are found to have potential for development into cosmetics, however, companies that do not use animal tests may find themselves uncertain about the safety of their products unless more, and more adequate, in vitro methods are developed.
Within the chemical and pharmaceutical industries, in vitro methods are used in a very different context. In vitro methods are used to screen compounds for their biological effect and to understand why a specific in vivo response has been obtained in one species and may not be seen in another. Additionally, screens are used to determine within a chemical class the presence or absence of a specific biological effect and to identify mechanism of action (either desired or toxic) of a chemical or group of chemicals. It is being recognized that with the advances and changes in chemical synthesis, e.g., combinatorial chemistry, more sophisticated and predictive in vitro tests will be required.
With the advances in genetics and genetic screening approaches, future development for these industries clearly will include the use of either genetically altered cells, or biological- and silicon-integrated chip systems and other complex and sophisticated systems yet to be envisioned.
Where Do In Vitro Methods Fit?
In the past, in vitro methodology was used as the last approach in product development to identify the underlying biology of undesired effects and, in some limited cases (such as receptor binding), to assist in product development. In the United States, the Toxic Substances Control Act (TOSCA) incorporated in vitro methods at the last stages of compound characterization. Only recently has this approach been questioned. Today, in vitro methods are used at earlier stages of chemical evaluations (8).
The Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC) has recognized that, at least in acute toxicity testing, in vitro methods can and should play a much more important role in the risk assessment process -- and, in fact, with the appropriate data in vitro methods might completely bypass animal use. In the introduction to (9), a scheme is presented that can eliminate animal testing in acute oral toxicity. It is presented here as Figure 1.
Acute Oral Toxicity (LD50)
The late Gerhard Zbinden, in his numerous and important contributions to in vitro alternatives, suggested that one did not have to find an alternative to the LD50, one just had to stop using it. In actual fact, the classical LD50 that required 60+ animals has essentially become extinct -- a fact we all should applaud. It remains necessary, however, to determine acute oral toxicity for a series of related but independent reasons. At present, the OECD has accepted two different approaches to determining acute oral toxicity: a fixed-dose procedure (FDP) and the acute toxic class method (ATC) procedure.
The fixed dose procedure, developed in Great Britain by the British Toxicological Society, has several features that demonstrate its applicability and dependence upon the Three Rs concept of alternatives. Animals are killed as soon as the first toxic signs are observed. Additionally, the experimental design reduces the number of animals considerably. The worldwide validation study of the FDP was accepted by the OECD in 1992 and is identified as OECD Guideline 420, 1992.
The ATC method has been developed and validated in Germany since 1988. It is based on a sequential dosing scheme in which one dose group is used at a time. Depending on the outcome of testing, in the next step the dose is either decreased or increased. A world-wide validation trial confirmed biostatistical prediction that the number of animals per test chemical can be reduced to an average of 7 in the ATC method without losing the accuracy of the LD-50 test. In 1996, the ATC method was accepted by the OECD (OECD Guideline 423, 1996) after it had been shown that it can be used for all types of classification schemes currently established in OECD member countries.
As was pointed out earlier, acute toxicity testing as envisioned in the SGOMSEC Workshop requires that a series of physico-chemical approaches, databases, and structure-activity relationships be evaluated prior to any biological testing. During recent years, there have been remarkable and rapid changes in structure-activity relationship studies.
In the case of therapeutics, rational drug design is clearly a major approach. In the area of toxicology, there have been both commercial and non-commercial databases developed to explore structure-activity relationships. Derek and TOPKAT are but two systems based on quantitative structure-activity relationships (QSARs), and their uses and limitations have been documented extensively.
In the area of skin sensitization, incredible progress has been made. The work of Barratt and colleagues, among others, (10) (11) (12) (13) (14) (15) document the use of QSARs for corrosivity and skin sensitization testing.
Although these methods are not 100 percent predictive, in the area of corrosivity and skin sensitization studies, the success rates are quite remarkable. Coupled with other physico-chemical methodologies and in vitro studies, these QSARs clearly will eliminate the need for animal testing. Due to the availability of these methods, and an abundance of knowledge about corrosivity, it is inappropriate to use either whole animals or humans for severe corrosivity studies.
A recent paper by Goldberg and Maibach (16) has provided a review of dermal toxicity and the use of in vitro and other alternative approaches in risk assessment.
Photosensitization is used to describe the reaction of the skin to an exogenous chemical and UV or visible radiation. The term includes both phototoxic and photoallergic reactions. Phototoxicity is an acute reaction which can be induced by a single treatment with a chemical and UV or visible radiation. In contrast, the term photoirritation is used to describe phototoxic reactions in skin produced with topically applied substances combined with exposure to light. To date, no standard guideline for the testing of photoirritation potential either in vivo or in vitro has been accepted for regulatory purposes at the international level by the OECD.
In 1991, the European Commission (EC) and COLIPA (the European Cosmetics, Toiletry and Perfumery Association) established a joint EU/COLIPA program on developing and validating in vitro photoirritation tests. The first phase (1992-93) was designed as a prevalidation study to identify in vitro test procedures for a formal validation trial under blind conditions. The in vitro photoirritation assays evaluated in this trial are shown in Table 3 In the second phase (1994-95), the most promising in vitro photoirritation tests were validated with 30 carefully selected test chemicals in 11 laboratories in a blind trial, in which the 3T3 NRU PI test was carried out in nine laboratories. In this test, mouse fibroblasts, cell line 3T3, are exposed to chemicals in the absence and presence of UV-A. An increase in cytotoxicity (Neutral Red Uptake [NRU]) was used to assess the photoirritation potential of chemicals.
The results obtained in the 3T3 NRU PI test under blind conditions were reproducible, and the correlation between in vitro and in vivo data was high (17). Therefore, all participants in the study concluded that the 3T3 NRU PI test is a validated test which should be used for regulatory purposes (18) (19). All other assays are still under development and may be used only in mechanistic studies.
The general public has protested the Draize eye test more than any other procedure used on animals, so, not surprisingly, scientists in the alternatives field have devoted more time and attention to finding an alternative to this test than any other. The Draize eye test is easy for the public to understand and difficult for scientists to justify if strong irritants and corrosives are not eliminated from the testing protocol. Unfortunately, replacing the Draize test has proven to be more difficult than anticipated for several reasons, among them the lack of knowledge about the biology of eye irritation, the lack of standardized and adequate human data, and the lack of in vitro models to evaluate appropriate tests. Table 4 presents a series of alternatives to the Draize eye test that have been evaluated in Europe. None is able to replace the Draize eye test for all aspects of eye irritation and all types of chemicals.
An Integrated Strategy
Today, several organizations (SGOMSEC, CAAT, OECD, etc.) are attempting an integrated strategy. The approach, as presented by Schlede and Gerner in 1995 (Figure 2) eliminates animal testing for eye irritancy if certain structure-activity or other non-animal tests indicate the compound would be a severe irritant or corrosive. The only time that animals are to be used is when there is the expectation that the compound is not corrosive or a severe irritant. This approach first was presented in Alternative Methods in Toxicology, Volume 4, page 26, 1987, but in less detail than in Schlede and Gerner (20).
German regulatory authorities and the U.S. EPA developed a step-wise strategy for eye irritation testing which takes into account the escape clause of OECD Guideline 405 for eye irritation testing. In this procedure, a sequence of physico-chemical data as well as results obtained in toxicity testing in vivo and in vitro is taken into consideration to evaluate the eye irritation potential of new chemicals (Figure 2). In this procedure, only chemicals which are positive may be classified and labeled "risk of severe damage to eyes." All chemicals which are found negative have to undergo in vivo testing in the Draize eye test in rabbits. Progress in tissue culture technique and in cell and molecular biology may then allow scientists to replace the Draize eye test with non-animal methods. This approach is used in several EU member states according to experience gained in validation trials at the national level but not yet in the United States and other OECD member countries.
The following journals, reports, books, and other resources provide the latest information about in vitro alternatives in toxicology:
Journals devoted to the biology of systems with a focus on molecular understandings include Cell -- the Journal of Biological Chemistry, among others. These are not cited here, as they do not focus on the field of toxicology, but clearly their literature is of utmost importance.
In the field of toxicology, journals devoted to in vitro methods include:
- In Vitro Animal: Cellular & Developmental Biology, published by the Society forIn Vitro Biology, Largo, Maryland
- Toxicology In Vitro,Elsevier Science Ltd., Oxford, United Kingdom
- In Vitro Toxicology,Mary Ann Liebert, Inc., Larchmont, New York
- ATLA, (Alternativesto Laboratory Animals), published by Fund for the Replacementof Animals in Medical Experiments, Nottingham, England
ECVAM Workshop Reports:
The European Centre for the Validation of Alternative Methods (ECVAM) has published 28 reports.
These reports are available online through Altweb: The Alternatives to Animal Testing Web Site, or in printed format from ECVAM: ECVAM, TP 580, JRC Environmental Institute, Ispra (VA), Italy.
- The Practical Applicabilityof Hepatocyte Cultures in Routine Testing, B.J. Blaauboer,et al.
- In Vitro PhototoxicityTesting, H. Spielmann, et al.
- In Vitro NeurotoxicityTesting, C. Atterwill, et al.
- Alternatives to Animal Testingin the Quality Control of Immunobiologicals: Current Statusand Future Prospects, C. Hendriksen, et al.
- Practical Aspects of theValidation of Toxicity Test Procedures, M. Balls, etal.
- A Prevalidation Study onIn Vitro Skin Corrosivity Testing, P. Botham, etal.
- Development and Validationof Non-animal Tests and Testing Strategies: the Identificationof a Coordinated Response to the Challenge and the OpportunityPresented by the Sixth Amendment to the Cosmetics Directive(76/768.EEC)
- The Integrated Use of AlternativeApproaches for Predicting Toxic Hazard, M. Barratt, etal.
- Safety and Efficacy Testingof Hormones and Related Products, B. Garthoff, et al.
- Nephrotoxicity Testing inVitro, G. Hawksworth, et al.
- The Three Rs: The Way Forward,M. Balls, et al.
- Screening Chemicals forReproductive Toxicity: The Current Alternatives, N. Brown,et al.
- Methods for Assessing PercutaneousAbsorption, D. Howes, et al.
- The Use of In VitroSystems for Evaluating Haematotoxicity, L. Gribaldo, etal.
- The Use of Biokinetics andIn Vitro Methods in Toxicological Risk Evaluation,B. Blaauboer, et al.
- Acute Toxicity Testing InVitro and the Classification and Labelling of Chemicals,H. Seibert, et al.
- Alternatives to the AnimalTesting of Medical Devices, O. Svendsen, et al.
- In Vitro Tests forRespiratory Toxicity, C. Lambre, et al.
- Alternative Methods forSkin Sensitisation Testing, O. de Silva, et al.
- The Use of Tissue Slicesfor Pharmacotoxicology Studies, P. Bach, et al.
- Production of Avian (EggYolk) Antibodies: IgY, R. Schade, et al.
- Pharmacokinetics in EarlyDrug Research, D. Leahy, et al.
- Monoclonal Antibody Production,U. Marx, et al.
- The Development and Validationof Expert Systems for Predicting Toxicity, J. Dearden, etal.
- Current Status and FutureDevelopments of Databases on Alternative Methods, A. Janusch,et al.
- Genetically Engineered CellLines: Characterisation and Applications in Toxicity Testing,F. Wiebel, et al.
- Issues Relating to the Releaseof Proprietary Information and Data for Use in the Validationof Alternative Methods, M. Todd, et al.
- The Use of Transgenic Animalsin the European Union, T.B. Mepham, et al.
CAAT Technical Workshop Reports:
The Johns Hopkins Center for Alternatives to Animal Testing (CAAT) has published eight technical reports.
The following reports are available online through Altweb or in printed format from CAAT: CAAT, 111 Market Place, Suite 840, Baltimore, MD, 21202
- Technical Problems Associatedwith In Vitro Testing Systems, May 17-18, 1989. Editors:John M. Frazier and June A. Bradlaw.
- Structure-Activity Relationshipsin Predictive Toxicology, June 21- 22, 1990. Prepared by:Shelley S. Sehnert.
- Report and Recommendationsof the CAAT/ERGATT Workshop on the Validation of ToxicityTest Procedures. Reprinted from ATLA 19, 1990, FRAME, Nottingham,England, Balls, et al.
- Cell Culture Systems andIn Vitro Toxicity Testing, June 13-15, 1990. Preparedby J. Bradlaw, O. Flint, et al.
- The International Statusof Validation of In Vitro Toxicity Tests, June 16-20,1991. Edited by John M. Frazier.
- Final Scientific Reportsof CAAT Grantees, 1994. Prepared by: T. Baumann et al.
- Molecular and Cellular Approachesto Extrapolation for Risk Assessment, 1994. Editor: ThomasR. Sutter
- Alternatives in MonoclonalAntibody Production, 1997. Editor: Joanne Zurlo.
Alternative Methods in Toxicology: Volumes 1 - 11:
- Goldberg, A.M. (Ed.): TheJohns Hopkins Center for Alternatives to Animal Testing.IN: Alternative Methods in Toxicology. Vol. 1. ProductSafety Evaluation. Mary Ann Liebert, Inc., New York,1983.
- Goldberg, A.M. (Ed.) AlternativeMethods in Toxicology. Vol. 2. Acute Toxicity Testing:Alternative Approaches. Mary Ann Liebert, Inc., NewYork, 1984.
- Goldberg, A.M. (Ed.) AlternativeMethods in Toxicology. Vol. 3. In Vitro Toxicology.Mary Ann Liebert, Inc., New York, 1985.
- Frazier, J.M., Gad, S.,Goldberg, A.M., and McCulley, J.P.: Alternative Methodsin Toxicology Vol. 4. A Critical Evaluation of AlternativesTo Acute Ocular Irritation Testing. Mary Ann Liebert,Inc., New York, 1987.
- Goldberg, A.M. (Ed.): AlternativeMethods in Toxicology, Vol. 5. In Vitro Toxicology -Approaches to Validation. Mary Ann Liebert, Inc., NewYork, 1987.
- Goldberg, A.M. (Ed.): AlternativeMethods in Toxicology, Vol. 6. Progress In Vitro Toxicology.Mary Ann Liebert, Inc., New York, 1988.
- Goldberg, A.M. (Ed.): AlternativeMethods in Toxicology Vol. 7. In Vitro Toxicology - NewDirections. Mary Ann Liebert, Inc., New York, 1989.
- Goldberg, A.M.(Ed.): AlternativeMethods in Toxicology Vol. 8. Mechanism and New Technology.Mary Ann Liebert, Inc., New York, 1991.
- Goldberg, A.M.(Ed.): AlternativeMethods in Toxicology Vol. 9 In Vitro Toxicology - TenthAnniversary Symposium of CAAT. Mary Ann Liebert, NewYork, 1993.
- Rougier, A, Goldberg, A.M.,Maibach, H. (Ed.) Alternative Methods in Toxicology, Vol.10 In Vitro Skin Toxicology. Mary Ann Liebert, Inc.,New York, 1994.
- Goldberg, A.M., Van Zutphen,L.F.M., Alternative Methods in Toxicology, Vol. 11 WorldCongress Proceedings. Mary Ann Liebert, Inc., New York,1995.
Books, Symposium Proceedings, etc.:
- Atterwil, C.K. and Steele,C.E. (Eds.) In Vitro Methods in Toxicology. CambridgeUniversity Press, Cambridge UK, 1987.
- Balls, M., Bridges, J. andSouthee, J. (Eds.) Animals and Alternatives in Toxicology:Present Status and Future Prospects. Macmillan Academicand Professional Ltd., London, 1991
- Frazier, J.M. (Ed.) InVitro Toxicity Testing, Applications to Safety Evaluation.Marcel Dekker Inc. New York, 1992.
- Gad, S.C. (Ed.) In VitroToxicology. Raven Press, Neew York, 1994.
- O'Hare, S. and Atterwil,C.K. (Eds.) In Vitro Toxicity Testing Protocols.Humana Press, Totowa, NJ, 1995
- Castell, J.V. and Gomez-Lechon,M.J. In Vitro Methods in Pharmceutical Research.Academic Press, San Diego (CA), 1997.
The Internet/World Wide Web
A growing number of Web sites are devoted to the field of alternatives to the use of animals in toxicology (or related subjects). In particular, Altweb: The Alternatives to Animal Testing Web Site is designed as an international clearinghouse for information and resources on alternatives. See Figure 3 for a complete list of related Web sites accessible through Altweb.
- Russell, W.M.S.and Burch, R.L. (1959). The Principles of Humane Experimental Technique. Methuen & Co. Ltd., London.
- Recognition andAlleviation of Pain and Distress in Laboratory Animals,National Research Council Report, Published by NationalAcademy Press, 1992.
- Balls, M., Blaauboer,B., Brusik, D., Frazier, J., Lamb, D., Pemberton, M., Reinhardt,C., Roberfroid, M., Rosenkranz, H., Schmid, B., Spielmann,H., Stammati, A.L., and Walum, E. (1990). Report and recommendationsof the CAAT/ERGATT workshop on the validation of toxicitytest procedures. ATLA 18: 313-337.
- Balls, M., Blaauboer,B.J., Fentem, J., Bruner, L., Combes, R.D., Ekwal, B., Fielder,R.J., Guillouzo, A., Lewis, R.W., Lovell, D.P., Reinhardt,C.A., Repetto, G., Sladowski, D. Spielmann, H., and Zucco,F. (1995). Practical aspects of the validation of toxicitytest procedures. The report and recommendations of ECVAMWorkshop 5. ATLA 23: 129-147.
- OECD (Organisationfor Economic Cooperation and Development). Final Reportof the OECD Workshop on Harmonisation of Validation andAcceptance Criteria for Alternative Toxicological Test Methods.ENV/MC/CHEM/TG(96)9, OECD Publications Office, Paris, 1996.
- NIEHS Validationand Regulatory Acceptance of Toxicological Test Methods:A Report of the Ad Hoc Interagency Coordinating Committeeon the Validation of Alternative Methods. NIH PublicationNo. 97-3981, NIEHS, Research Triangle Park, North Carolina,1997.
- Toxicity Testing:Strategies to Determine Needs and Priorities, National ResearchCouncil Report. Published by National Academy Press, Washington,D.C., 1984.
- Screening ExistingTSCA Inventory Chemicals for Neurotoxicity, Workshop onTesting and Screening Technologies for Review of Chemicalsin Commerce, U.S. Congressional Office of Technology Assessment.Chapter by Jean Harry, John Donoghue and Alan M. Goldberg,Washington DC, 1995.
- Alternative TestingMethodologies, Scientific Group on Methodologies for theSafety Evaluation of Chemicals (SGOMSEC), Joint ResearchCentre, Ispra, Italy, 1997.
- Basketter, D.A.,Scholes, E.W., Chamberlain, M., and Barratt, M.D. (1995).An alternative strategy to use guinea pigs for the identificationof skin sensitization hazard. Food and Chemical Toxicology33: 1051-1056.
- Barratt, M.D. (1996a).Quantitative Structure-Activity Relationships (QSARs) forSkin Corrosivity of Organic Acids, Bases and Phenols: PrincipalComponents and Neural Network Analysis of Extended Datasets.Toxicology In Vitro 10: 85-94.
- Barratt, M.D. (1996b).Quantitative Structure-Activity Relationships for Skin Irritationand Corrosivity of Neutral and Electophilic Organic Chemicals,Toxicology In Vitro, 10:247-256.
- Basketter, D.A.,Scholes, E.W., Chamberlain, M., and Barratt, M.D. (1995).An Alternative Strategy to the Use of Guinea Pigs for theIdentification of Skin Sensitization Hazard, Volume 33,#12, pages 1051-1056, December, 1995.
- Magee, P.S., Hostynek,J.T., and Maibach, H.I. (1994). Modeling Allergic ContactDermatitis, Alternative Methods in Toxicology, Volume 10,Ed. Rougier, A., Goldberg, A.M., Maibach, H.I., In VitroSkin Toxicology, Mary Ann Liebert, Inc. Publishers, NewYork, pages 281-291.
- Rougier, A., Goldberg,A.M., Maibach, H.I., Eds. Alternative Methods in Toxicology,Volume 10, In Vitro Skin Toxicology, Irritation,Phototoxicity, Sensitization, Mary Ann Liebert, Inc. Publishers,New York, 1994.
- Goldberg, A.M.,Maibach, H.I., Environmental Health Perspectives, Volume106, Supplement 2, April 1998.
- Spielmann, H.,Balls, M., Dupuis, J., Pape, W.J.W., Pechovitch, G., deSilva, O., Holzhuetter, H.G., Clothier, R., Desolle, P.,Gerberick, F., Liebsch, M., Lovell, W.W., Maurer, T., Pfannenbecker,U., Potthast, J.M., Sladowski, D., Steiling, W. and Brantom,P. (1998). The international EU/COLIPA in vitro phototoxicityvalidation study: results of phase II (blind trial): the3T3 NRU phototoxicity test. Toxicology in Vitro 12:305-327, 1998.
- European CommissionStatement on the Scientific Validity of the 3T3 NRU PT Test(an in vitro test for phototoxic potential). JointResearch Centre, Environment Institute, Ispra, Italy, 1997.
- Balls, M., andCorcelle, G. (1998). ECVAM News and Views: Statement onthe Scientific Validity of the 3T3 NRU PT Test (An InVitro Test for Phototoxic Potential). ATLA 26:7-8.
- Frazier, J., Gad,S., Goldberg, A.M., McCulley, J., Eds. A Critical Evaluationof Alternatives to Acute Ocular Irritation Testing, AlternativeMethods in Toxicology, Volume 4, Mary Ann Liebert, Inc.Publishers, New York, 1994.
New ALTEX: 1/2017
January 8-13, 2017
Various locations throughout Europe
Beginning April 2017
June 14-16, 2017
SAVE THE DATE:
10th World Congress on Alternatives and Animal Use in the Life Sciences
August 20-24, 2017