Alternatives: toxicity testing overall
Huggins J. Alternatives to animal testing: research, trends, validation, regulatory acceptance. ALTEX 2003; 20(Suppl 1): 3-61.
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Current trends and issues in the development of alternatives to the use of animals in biomedical experimentation are discussed in this position paper. Eight topics are considered and include refinement of acute toxicity assays; eye corrosion/irritation alternatives; skin corrosion/irritation alternatives; contact sensitization alternatives; developmental/reproductive testing alternatives; genetic engineering (transgenic) assays; toxicogenomics; and validation of alternative methods. The discussion of refinement of acute toxicity assays is focused primarily on developments with regard to reduction of the number of animals used in the LD50 assay. However, the substitution of humane endpoints such as clinical signs of toxicity for lethality in these assays is also evaluated. Alternative assays for eye corrosion/irritation as well as those for skin corrosion/irritation are described with particular attention paid to the outcomes, both successful and unsuccessful, of several validation efforts. Alternative assays for contact sensitization and developmental/reproductive toxicity are presented as examples of methods designed for the examination of interactions between toxins and somewhat more complex physiological systems. Moreover, genetic engineering and toxicogenomics are discussed with an eye toward the future of biological experimentation in general. The implications of gene manipulation for research animals, specifically, are also examined. Finally, validation methods are investigated as to their effectiveness, or lack thereof, and suggestions for their standardization and improvement, as well as implementation are reviewed.

1 Refinement of acute toxicity assay
Three assays have been validated and adopted as replacements for the conventional LD50 test. The assays differ primarily as to the endpoint they measure; however, all assays use fewer animals than the conventional LD50 test. The use of more humane endpoints, such as clinical signs of toxicity, rather than lethality, is perhaps the most advanced suggestion to date regarding toxicological evaluation of acute exposure. Much remains to be done, however, with regard to standardization of this approach.

2 Alternatives to eye corrosion/irritation testing in animals
Although much research has been done to date on the development of viable in vitro assays of ocular corrosion and irritancy, validation of these assays has been problematic. Several reasons have been postulated for the failure of validation efforts, the most prominent of which are the following: The in vivo test used for comparison, the Draize test, is based on subjective scoring of tissue lesions in the eye, providing variable estimates of eye irritancy; the non-animal method protocols were inadequate; the choice of test substances was not well-planned; and the statistical approaches used were not appropriate. Perhaps the most promising suggestion as to how to remedy these difficulties currently is to use complementary alternative assays in batteries to evaluate eye corrosion/irritation.

3 Alternatives to skin corrosion/irritation testing in animals
The use of alternative assays has replaced, to a large extent, the testing of corrosive or irritating substances on the skin of live animals. Examples of the assays discussed include the CorrositexÒ assay that uses no animal cells at all, the transcutaneous electrical resistance (TER) assay that uses a small section of rat skin, and several in vitro skin irritancy models that incorporate human skin in small quantities. From a scientific perspective, the replacement of the Draize test with these assays lends greater objectivity as well as more general relevance to human skin corrosion and irritation. Validation efforts utilizing these models have proven satisfactory in most instances.

4 Alternatives to skin sensitization testing in animals
Progress toward the development and refinement of alternative assays of contact sensitization is strongly dependent upon breakthroughs in our understanding of the immune processes mediating the response. Extensive efforts directed toward validation of the local lymph node assay have borne the much-needed fruit of a "stand alone" assay that incorporates elements of both refinement and reduction. However, much more basic research remains to be done before a fully validated replacement assay of contact sensitization finds regulatory support. Promising areas of research include those in which cytokine profiles associated with contact sensitization are analyzed.

5 Alternatives to developmental/reproductive toxicity testing in animals
Validation efforts are progressing well for in vitro assays of developmental/reproductive toxicity. Results from evaluations of the MM and WEC assays as well as the EST appear to be favorable; data from studies of FETAX suggest that further improvements in the assay would yield greater predictivity. Hence, our reticence to use an alternative assay to measure toxic effects on complex physiological processes such as reproduction may have to yield to the results obtained from these recent evaluations.

6 Genetic engineering methodologies
The generation and use of transgenic animals to study questions of biomedical interest have been questioned by many in view of the moral and ethical dilemmas presented by these activities. That transgenic animals may contribute to the reduction of animal use in toxicological experiments, particularly studies of carcinogenicity, is not disputed. However, advocates of replacement alternatives argue that in vitro alternatives to this type of toxicity testing have not been given adequate attention.

7 Gen chip technology as an alternative to animal testing
Gene chips (DNA microarrays) represent a technology that has already opened many doors in basic genomic research. Moreover, their value to both investigative and discovery toxicology is becoming much more apparent as more toxicology experiments are conducted using them. Use of microarrays as reduction or replacement alternatives to animal testing also holds great promise, particularly when they are used as components of prescreening batteries, and when coupled to cell culture techniques.

8 Validation of alternative methodology
Validation of alternative methods has just emerged from a rather chaotic phase in which the principles behind appropriate conduct of a validation study were defined, mainly through trial and error. Much refinement has come out of this "exploratory " phase, including recognition that validation studies should be built upon a solid platform, consisting of components such as good reference standards, reliable protocol transfer between laboratories, and appropriate application of biostatistical techniques. Efforts are now underway to apply these lessons learned to future validation studies and to harmonize validation techniques among countries in order to maximize the possibility that the data generated can be used worldwide.