Animal testing in vitro assays

Animal Tests, In Vitro Assays, and In Silico Methods 158... 

ANIMAL TESTS, IN VITRO ASSAYS, AND IN SILICO METHODS... 

There is a continuing interest in the development of biomarker assays for use in environmental risk assessment. As discussed elsewhere (Section 16.6), there are both scientific and ethical reasons for seeking to introduce in vitro assays into protocols for the regulatory testing of chemicals. Animal welfare organizations would like to see the replacement of toxicity tests by more animal-friendly alternatives for all types of risk assessment—whether for environmental risks or for human health. 

The last step of the drug discovery process involves the testing of lead compounds to address issues such as efficacy, bioavailability, and safety. Testing may include in vitro assays but ultimately would require a suitable disease model and studies in animals. Many compounds may need to be designed and synthesized to identify the one compound with all the desired properties. Such a compound can be advanced to preclinical studies and eventually to the clinic. 

As a general rule, in vivo assays are more challenging than in vitro assays because the matrices for the samples are more complex. The most common use for in vivo assays is to measure the concentration of NCE dosed into a laboratory animal by collecting multiple sample time points, one can use the analytical results to plot the PK profile of the NCE and also obtain various PK parameters that help determine a test compound s PK properties. Preclinical PK parameters of a test compound are then used to predict its human PK parameters. Another use of in vivo assays is combining the results with pharmacodynamic (PD) observations to perform PK/PD modeling.77 82 PK/PD modeling is an important aspect of new drug discovery because it can be used to predict the exposures and durations required to determine clinical efficacy of a NCE. 

In recent years, much attention and effort have been directed toward the search for non-whole-animal tests to predict ocular irritation by drugs and chemicals. A variety of in vitro assays, as well as nonexperimental approaches, have been proposed. These model systems run the gamut of responses observed in vivo using biological... 

The in vitro battery would ideally include measures of opacity, cytotoxicity, and inflammation. The actual test method(s) will vary depending upon the experience of the laboratory, types of compounds to be tested, and so on. If the measured endpoint(s) indicates that the test material is approximately equipotent with known irritants, one would presume the unknown to be an irritant and further testing would not generally be required. One should keep in mind, however, that in many cases in vitro assays are more sensitive than whole-animal testing, so a positive response in vitro may not always indicate an in vivo irritant. If the assays give equivocal results or responses similar to those seen with non- or mild irritants, some type of animal testing may be indicated as confirmation. 

The designation potential endocrine disrupter has been proposed for chemical products with an endocrine-disruption ability that is demonstrated in an in vitro assay but not confirmed in an in vivo animal model. To date, most of the available information on chemical products with endocrine disrupter activity has been generated by in vitro experiments [10]. Various existing tests and bioassays of very different types have been proposed by distinct international bodies to identify hormonal... 

The use of animals for pharmacological and toxicological studies has yielded invaluable information for drug development. However, many drug candidates failed in Phase I and II clinical trials because the animal models were insufficient to represent human systems and functions for some drugs. Efficacy and acceptable toxicities derived from animal models were not replicated in humans (Exhibit 5.8). In recent years, the direction in development of drugs has shifted toward the use of ex vivo, in vitro assays and even in silico methods. Nevertheless, some tests must stiU be confirmed in animals. 

In vitro assays are increasingly being used. Some of the reasons are cost, availability of more rapid results, and avoidance of negative publicity. Assays such as cytochrome P-450 enzymes, the Ames test, and the mouse lymphoma tk test are in vitro methods. For absorption studies, Caco-2 (Exhibit 5.9) and Madin-Darby canine kidney cell assays are now routinely used. Hepatocyte cell lines with metabolism capacity are being developed to test drug metabolism and toxicity. All these examples show that, where possible, pharmaceutical firms are gradually dispensing with animal studies. 

Although chemical sensitization is a more complex process than immunosuppression, by far most of the efforts on developing in vitro assays are in this field. An important reason for this is that from the various fields of immunotoxicity, most of the animals are used for sensitization testing. In fact the number of animals required for sensitization is second only after developmental toxicity testing. 

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