Alternative assays tests

Studies have demonstrated that one such method is to examine the effects of disinfectants on endogenous RNA-dependent DNA polymerase (i.e. reverse transcriptase) activity. In essence, HIV is an RNA virus after it enters a cell the RNA is converted to DNA under the influence of reverse transcriptase. The virus induces a cytopathic effect on T lymphocytes, and in the assay reverse transcriptase activity is determined after exposure to different concentrations of various disinfectants. However, it has been suggested that monitoring residual viral reverse transcriptase activity is not a satisfactory alternative to tests whereby infectious HIV can be detected in systems employing fresh human peripheral blood mononuclear cells. 

A complementary approach is to conduct the assays under high-throughput automated conditions. This can be either through the miniaturization of assays, that is, 96-384 plates and if possible 1536, or through the use of alternative assay technologies (e.g., microfluidics). Both scenarios require studies of equivalency testing and backwards compatibility with previous methods and results. 

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). 

The Berry spot test provides a rapid qualitative evaluation of urine. GAGs react with toluidine blue, a cationic dye, to yield a pink-colored compound. Alternative spot tests have been published but suffer from the same drawbacks regarding false-negative and false-positive specimens [2, 10, 15, 34]. Nevertheless, this fast procedure may pick up patients who have been referred to a laboratory for other metabolic examinations. For specific, initial MPS testing, the 1,9-dimethylene blue (DMB) assay, described below, is recommended. 

Mauthe RJ, Gibson DP, Bunch RT, Custer L (2001) The Syrian Hamster Embryo (SHE) cell transformation assay review of the methods and results from ILSI/HESI program on alternative carcinogenicity testing. Toxicologic Pathology 29 138-146... 

Ozolins TRS, Thomson JJ. Use of WEC in pharmaceutical screening/testing. I IP,SI (Health and Environmental Sciences Institute) Workshop on Alternative Assays for Developmental Toxicity Testing [http //www.hesiglobal.org/Coimnittees/TechnicalCommittees/DART/ HESIAltAssayPresentations.htm. Accessed May 1 2009. 

None of the in vitro alternative eye tests has proven applicable as a valid replacement for the Draize eye irritation test or has been acceptable for regulatory purposes (Table 5), though some are considered either reliable or reproducible. The most frequently used test has been the ex vivo bovine cornea opacity and permeability assay. The newer human corneal equivalents system, an in vitro culture of immortalized human corneal cells that develops into... 

Natsch A, Ryan CA, Foertsch L, Emter R, Jaworska J, Gerberick F, Kern P (2013) A dataset on 145 chemicals tested in alternative assays for skin sensitization undergoing prevalidation. J Appl Toxicol. doi 10.1002/ jat.2868. [Epub ahead of print]... 

Direct I-ELISA for antigen testing is not an available alternative since test antigen has to be mixed with pretitrated labeled antibody Thus, competitive conditions apply. One variation is that test antigen can be premixed with the labeled antibody and incubated for a period before the mixture is applied to the antigen-coated plates. In practice, this makes no difference to the assays in which antigen is added to the coated plates initially. 

Muller-Decker, K., Furstenberger, G., and Marks, F., 1992, Development of an in vitro alternative assay to the Draize skin irritancy test using human keratinocyte-derived proinflammatory key mediators and cell viability as test parameters. In Vitro Toxic. 5 191 - 209. 

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