Alternative assays validation

As noted earlier, there are several agencies or centers for alternative assay validation. Their processes are briefly summarized below. 

Three in vitro alternative assays are endorsed as scientifically validated by ECVAM in 2001 the EST, the MM, and WEC assays. The best results were obtained by combining the EST and WEC assays. The EST assay has the advantage of not using animals at all. As human cell culture technology improves, particularly regarding stem cells (8, 9), new methods will undoubtedly evolve that will enable a closer in vitro detection of in vivo human teratogenesis. 

In 2007, the DART committee held a workshop on alternative assays, which was followed up by a workshop held at the European Teratology Society Annual Meeting in 2009. These workshops focused on three alternative assays (1) whole embryo culture (WEC), (2) mouse embryonic stem cell tests (mESC), and (3) zebrafish. Each assay was presented and data from users were shared, and strengths and limitations were discussed. It should be noted that the WEC and mESC are validated by ECVAM as alternative embryotoxicity assays. Still, there are numerous research needs before even validated tests can achieve regulatory acceptance. The discussions, conclusions, and recommendations of the 2007 workshop were published by Chapin et al. (14). Bullet lists of next steps to move forward were defined for each assay (14) and are briefly summarized here ... 

In the 1990s, ECVAM held a forum to vet and evaluate new alternative assays, and developed a list of compounds for testing (24). The key driver for this activity was the fact that DART studies require large numbers of animals. The primary focus of this activity was embryo-fetal toxicity. The list generated from this forum was tested in three assays (later validated by ECVAM) (1) the micromass assay, (2) the rat WEC assay, and (3) the embryonic stem cell test (25). Compounds on the Brown list were classified as either strong, weak, or non-teratogens. The three assays successfully predicted the compound classification about 80% of the time. However, the embryonic stem cell test later performed poorly on a different group of chemicals with known in vivo activities (26). 

Accuracy is the ability of any assay to provide the correct result. Ideally, the assay should detect all of the analyte (100% recovery) and nothing else (no interference or cross-reactivity). To estimate the method accuracy, a comparison of method results with tme sample concentrations must be completed. A straightforward procedure involves the use of a standard reference material, in which the analyte concentration is known with high accuracy and precision. Standard reference materials are not generally available for biochemical analytes, however. When a reference material is not available, accuracy can be established by comparison with alternative previously validated analytical techniques, or currently accepted methods. Intralaboratory tests of matrix effects and interferences are also conducted in order to establish the accuracy of a new method. 

Implementation of new methods in regulatory work faces several critical difficulties. Among them, lack of certified reference materials, QC/QA protocol application and pending validation studies hinder the transformation of good ideas into official methods for practical regulatory work. For example, Inami et al. recently compared two immunoassays and a 5 h neuroblastoma assay with the MBA for presence/absence detection of saxitoxins in shellfish. The study concluded that a reduction in the number of mouse bioassays in the state of California could be achieved, provided that the alternative assays were applied as a screening tool. 

CTAs are possible in vitro alternatives to the standard approach for the assessment of carcinogenicity (the 2-year bioassay in rodents), which have been shown to be a multistage process able to model the most important stages of in vivo carcinogenesis [50]. CTAs are faster and more economic than in vivo assay and they could be a valid and useful screening tool for chemicals. 

NIEHS, Interagency Coordinating Committee for Validation of Alternative Methods (IC-CVAM) Peer Review Panel Evaluation of the Murine Local Lymph Node Assay (LLNA) Report, 1999. 

Needs for the Future. One significant need in the area of ocular alternatives is for the validation of current in vitro assays. The key point is that users of the assay(s) need to have confidence in their ability to interpret and reliably use the data generated. This confidence level can only be achieved by parallel testing in one s own laboratory with compounds similar to those likely to be evaluated as unknowns. The validation process will be a long, somewhat tedious project, but will be necessary before in vitro alternatives can be used responsibly... 

Shopsis, C. (1989). Validation study ocular irritancy prediction with the total cell protein, uridine uptake, and neutral red assays applied to human epidermal keratinocytes and mouse 3t3 cells. In Alternative Methods in Toxicology, Vol. 7 (Goldberg, A.M., Ed.). Mary Arm Liebert, New York, pp. 273-287. 

G. Ehling, M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. Van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, and H.-W. Vohr. An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay First round. Toxicology 212 60-68 (2005). 

In addition to identifying protein partners, yeast two-hybrid technology can be used to identify and study in detail the interaction domains between two proteins. Here, bait and/or fish truncation or deletion constructs of the parent proteins are engineered and characterized as described earlier (see 3.1 Selection and characterization of bait constructs). These are then investigated for association in a yeast two-hybrid interaction assay. Once the BD has been identified, it can be further refined by mutagenesis. The same caveat applies to these studies as for the identification of associating proteins, i.e., it is assumed that the respective fusion proteins fold and adopt the same or a similar three-dimensional conformation to the native protein. This is not always the case and results should be interpreted with caution and if possible, always validated by an alternative experimental approach. O Table 19-1 shows an example of mapping the... 

Methods have successfully been transferred to various laboratories in inter-company cross-validation exercises for a chiral separation, for an assay of the main component in a formulation and for drug stoichiometry. Revalidation is an alternative to method transfer in case of changes in product composition or analytical procedure (cf. Section I.L). Although a method transfer in CE is not a major difficulty, some aspects have to be considered, especially if a method is transferred to an instrument of another manufacturer. 

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