Unifying the Effort Behind In Vitro Alternative Method Development

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Emerging Technologies

Unifying the Effort Behind In Vitro Alternative Method Development

Frank A. Barile, St. John’s University College of Pharmacy

Published: June 4, 2008

About the Author(s)
The author is Professor of Pharmaceutical Sciences, Toxicology Division, Department of Pharmaceutical Sciences, at St. John’s University College of Pharmacy and Allied Health Professions, New York.

Dr. Barile has authored and co-authored approximately 75 papers in peer-reviewed biomedical and toxicology journals, as well as three books (latest, Principles of Toxicology Testing, Informa HC, 2007). He contributed original in vitro toxicology data to the international Multicenter Evaluation for In Vitro Cytotoxicity (MEIC) program (1990-2000).

He is currently President of the In Vitro & Alternative Methods (IVAM) Specialty Section of the U.S. Society of Toxicology, and a member of the Scientific Advisory Committee for Alternative Toxicological Methods (SACATM), a public advisory committee to the NIEHS. Dr. Barile continues fundamental research on the development of alternative models for cytotoxicity testing of environmental chemicals and therapeutic drugs on cultured mouse embryonic stem cells.

Frank A. Barile, Ph.D.
Professor of Pharmaceutical Sciences
St. John’s University College of Pharmacy
8000 Utopia Parkway
Queens, NY 11439
Email: barilef@stjohns.edu

No single method is expected to substitute for the complexity of systemic or local toxicity in humans or animals. The response of the mammalian organism to a chemical is known to involve various physiologic targets and a variety of complex toxicokinetic factors. In addition, humans interact simultaneously with a variety of toxic insults, logarithmically increasing the toxicologic outcomes in vivo. Thus, many questions have emerged as a result of historical and current attempts at developing in vitro and alternative models for the prediction of systemic and local toxicity.

For instance, why should an in vitro test be expected to respond to the same number of toxic insults as the animal model, particularly when an animal test is not called upon to explain the myriad effects from the combination of several chemicals? Thus the answers to these queries are not limited by the biotechnology available now for the development of alternative models, but by the lack of a cohesive effort to implement the objectives of the original “3R” plan (reduction, refinement, replacement). That is:

  1. Are alternative methods capable of fulfilling the need to model different types of quantitative general toxicity, such as acute systemic toxicity or local irritancy?
  2. Can the information obtained from in vitro models be used as a paradigm for predicting, supplementing, replacing or confirming animal toxicology data?

These two questions may require an assortment of clever approaches. For instance, it is possible to apply the 3Rs to animal toxicology testing1 of existing chemicals with the results generated from in vitro tier testing. This goal is achievable with an amalgamation of results obtained from reliable chemical and analytical measurements of different samples—for example, information obtained from human and animal blood and tissue concentrations of the same chemicals, from human volunteer testing of dermal and ocular irritancy, or through the tabulation of databases with clinically relevant human toxicity information.

Based on observable trends and practical experience of many laboratories and regulatory agencies with in vitro cytotoxicology programs, it is conceivable and realistic that toxicology testing in the foreseeable future will be regularly performed with cell culture models. Furthermore, such testing will be more efficient and more predictive of human toxicity than the current animal tests, given the amount of time, resources, and variability of information associated with animal testing.

With the realization of this objective comes the benefit that many new types of in vitro toxicology and toxicokinetic tests will be available, while many of the current methods will be validated in refined programs. These programs are conducted as refined multi-factorial model systems of large in vitro databases to account for human toxicity, including computerized physiologically based kinetic modeling. Simultaneously, the gradual acceptance of new in vitro methods should not only depend on formal validation programs, but may be a consequence of the parallel experience of various academic, government, regulatory and industrial efforts using both in vitro and in vivo methods. Finally, these achievements are based not only on political or societal attempts to reduce, refine, or replace the use of animals in research, but on the complementary approaches of efficient and reliable systems already in place for protecting the public interest.

  1. Can the results generated from these methods be used to compile a set of standardized tests which together can substitute as the predictive battery?

This goal may require several relevant systems, such as human hepatocytes, heart, kidney, lung, nerve cells, and other cell lines of applicable and relevant importance. Twenty years ago, the fledgling perception that a standardized battery of in vitro tests could be developed for tier testing of acute local and systemic toxicity, was ambitious. Progress in sensitivity, reliability, and technical feasibility of testing protocols, supported by current validation studies, are a testament to the realization of those original objectives.

Certain methods assigned to the battery are used for systematic mapping of the different effects attributed to general systemic cytotoxicity. The mechanisms underlying cytotoxic phenomena explain how these effects are influenced by the physicochemical properties of the molecules and how the knowledge is useful for the interpretation of results in cellular testing. Alternative cell based models are applied on actions of chemicals known to be cytotoxic to animals or humans by comparing the in vitro and in vivo concentrations of the same chemicals.

Figure 1 summarizes the organization and applications of in vitro cytotoxicity testing and assigns a position for systemic cytotoxicity, organ-specific cytotoxicity, and organizational cytotoxicity within the general scheme. According to the diagram, carcinogenicity and mutagenicity data contributes to cytotoxicology evaluation but this information can be circumvented. Note also that in vitro toxicokinetic studies contribute to the establishment of the model. Together with in vitro concentrations derived from the tests, a model for cytotoxicity is formulated. Overall, the objective is to arrive at human toxic blood concentrations to assist in the prediction of human toxicity and evaluation of risk assessment.

Figure 1

Figure 1. Summary of the organization and applications of in vitro cytotoxicity testing with assignment of positions for systemic, organ-specific, and organizational cytotoxicology within the scheme of risk assessment.

Accordingly, a battery of selected protocols functions as a primary screening tool before the implementation of routine animal testing. Subsequent animal tests are then performed to confirm the results from the tier testing, as well as to detect outliers that elude the screening protocols. Upon completion of a specific and limited number of animal tests to confirm the in vitro data, a summary report of all available information is used as a basis for screening and/or predicting risk to humans.

With the refinement of general in vitro/in vivo testing and the progress afforded such studies with time, certain tests will be employed with greater confidence. This is forecasted as it becomes apparent that the battery reflects the acute or chronic toxicity of certain groups of substances, resulting in the inevitable elimination of particular aspects of animal testing.

  1. Are quick, simple, and economic in vitro cell systems capable of sustaining the regulatory standards demanded by public health needs?

Together with genotoxic and mutagenic tests, in vitro cellular test batteries are applied as biological markers of risks induced by chemicals, whether synthetic or naturally occurring. Such risks include biomonitoring of food and food additives and water and air analysis for environmental toxicants. If similar batteries of protocols are validated for their ability to screen for or predict human toxic effects, these measurements will establish a significant frame of reference for monitoring of environmental, occupational and commercial toxic threats.

Although much of the mystery originally surrounding the techniques has waned, the doctrine underlying the establishment of the methodology remain. For instance, improvement in serum requirements in media has fostered continued excitement in the role of growth factors and cell differentiation. In addition, had it not been for these techniques, the recent discoveries in stem cell biology may not have been realized. Consequently, the discipline of cell culture, and its application to in vitro toxicology and biology, represents a tool poised to answer scientific questions in the biomedical sciences that can only be addressed with the proliferation of isolated cells, without the influence of other organ systems. With this understanding, the use of cell culture does not purport to represent the whole human organism, but can significantly contribute to our understanding of the workings of its components.

  1. Is the collaborative effort primed to secure a significant impact on the 3Rs?

Historically, it was discovered that a tissue or organ that was part of an intact biological specimen, when isolated, would exhibit a breakdown in the supporting matrix, followed by migration of individual cells from the specimen as a consequence. With the addition of serum or whole blood, the resulting clot formed a hanging drop, making it possible to look at these cells through an ordinary light microscope. With some refinement, the “explants” demonstrated that cells migrate out of the dissected specimen. Common knowledge dictates that organs are composed of tissues, and cells are their framework. This decades-old axiom was re-discovered when it became possible to cultivate individual cells in small glass tubes while bathed in plasma or embryonic serum extract. Advances in aseptic techniques allowed cells to be maintained in culture for longer periods of time in the absence of microbiological contamination. Further developments in synthetic media, sterile supplies and equipment, and understanding of extracellular interactions were driven by the recognition that this technology was not only scientifically necessary, but that the contributions to public health would be unmistakably promoted.

Today, the same idealistic motivation presides over the toxicologic scientific goal as it did years ago—that is, can one or a few in vitro tests mimic the in vivo response. The test(s) would require the following:

  • sensitive indicators capable of discriminating among results to avoid false positives and contribute an explanation toward the target mechanism (sensitivity testing);
  • the cell system would need to integrate the essence of visceral organs to express the myriad of possible responses (testing for relevance);
  • the length of time of in vitro exposure would be equivalent to in vivotime periods (testing for reliability); and,
  • the model system should generate toxicokinetic and toxicodynamic interactions reproducible in many laboratories (reliability).

To date, many proposed alternative in vitro toxicology tests in the U.S. have accumulated in the scientific literature and regulatory platforms without the benefit of arriving at general acceptance. The situation is due in part to the difficulty associated with introduction of experimental systems from individual laboratories. Thus the organization of multilaboratory validation programs is prompted by in vitro toxicologists as a possible remedy for the problem. For example, public interest groups, academic institutions, corporate industries, and government agencies in the European Union (E.U.) and U.S. must be actively engaged in promoting the development of in vitro toxicology tests by organizing well planned programs.

Contemporary validation studies can be facilitated through collaborative international cooperation, particularly through the efforts of government sponsored and independent organizations, such as the U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), U.S. National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), the European Centre for Validation of Alternative Methods (ECVAM), and the Center for Documentation and Evaluation of Alternative Methods to Animal Experiments (ZEBET, Germany). These alliances provide a foundation for validating alternative methods along with promotion and encouragement for the harmonization of scientific approaches to validation and review. In addition, both ICCVAM and ECVAM have outlined key objectives: to stimulate development of test methods and testing strategies in prioritized areas by individual laboratories and to facilitate the nomination of promising test methods for appropriate regulatory translation.

Concluding Remarks

Current and future validation should not be hampered by theoretical considerations of the best way to achieve regulatory acceptance. The refinement of the validation programs should be approached as a set of generally applicable rules. The most effective future validation will probably require studies directed at all levels, including the efforts of individual laboratories, reliability-oriented multilaboratory programs, and multiple-method multilaboratory perspectives focused on cell based toxicology. In addition, methods used extensively in past years should not be treated with the same approach as newly developed, untried procedures. This perspective wastes time and resources.

It is important to understand that the progress of in vitro cytotoxicology relies on the continuous development of better strategies to evaluate methodologies rather than as a set of inflexible rules for the inclusion or exclusion of protocols. More importantly, efforts to evaluate the scientific integrity of methods for validation purposes should be managed, guided and administered by independent investigative groups rather than government or industry sponsored organizations. The groups must be free of vested interests, with their services directed toward the development of scientifically valid programs. Consequently, the rate-limiting step is not dependent on the available biotechnology but relies on a cohesive effort among the competing entities to discover the most efficient means to this end.


Research performed in this laboratory is supported in part by grants from the International Foundation for Ethical Research (IFER, Chicago, IL, USA) and the Alternatives Research & Development Foundation (ARDF, Jenkintown, PA, USA).
©2008 Frank Barile

Parts of this essay are adapted from:

Barile, F.A. (2007). Standardization and Validation of Alternative Methods, in Principles of Toxicology Testing. 259-272, Taylor & Francis LLC, Boca Raton, FL, USA.