Cytotoxicity endpoint

Garrett NE, Lewtas J. 1983. Cellular toxicity in Chinese hamster ovary cell cultures I. Analysis of cytotoxicity endpoints for twenty-nine priority pollutants. Environ Res 32 455-465. 

Fish cell lines have been used to screen and rank ecotoxicants for their basal cytotoxicity. A variety of cytotoxic endpoints as outlined in section III. 1 have been used. The number of compounds that have been examined is large and individual compounds are listed in other reviews184. Here some of the ecotoxicant classes investigated and the cell lines used are described. Metals have been the most intensively studied and with the largest number of cell lines. These include BB, BF-2,... 

Lu S, lessen B, Strock C, Will Y (2012) The contribution of physicochemical properties to multiple in vitro cytotoxicity endpoints. Toxicol In Vitro 26, 613-20. 

CYP3A4 level is comparable to primary cells (University of Liverpool and the IMI MIP-DILI consortium, unpublished data), and these cells are generally less sensitive to DILI compounds than PHHs, based on cytotoxicity endpoints. 

Tissue Culture Assay. Kogure et al. (48) report a novel tissue culture assay for detecting several types of sodium channel blockers. The mouse neuroblastoma cell line ATCC CCL 131 is grown in RPMI 1640 supplemented with 13.5% fetal bovine serum and 100 pg/ml gentamycin, in an atmosphere of 5% C0 95% air at 37 C. Ninety-six well plates are seeded with 1 x 10 cells in 200 pi of medium containing 1 mM ouabain and 0.075 mM veratridine. Veratridine and ouabain cause neuroblastoma cells to round-up and die. In the presence of sodium channel blockers (e.g., TTXs or STXs), the lethal action of veratridine is obviated and cells retain normal morphology and viability. An important feature of this assay is that a positive test for sodium channel blockers results in normal cell viability. Since bacterial extracts can contain cytotoxic components, this assay offers an advantage over tests that use cell death as an endpoint. The minimum detectable level of TTX is approximately 3 nM, or approximately 1/1000 mouse unit. 

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 mechanism by which the cytotoxic agents were approved is of inferest. Many were approved on fhe basis of response rafe as a surrogafe for survival (e.g., via the accelerated approval path). - Of nofe is the development of fhe cytotoxic agents, capecitabine received initial accelerated approval on the basis of fhe surrogafe endpoint of response rate in patients with refractory breast cancer. This was later followed up with a randomized Phase 111 trial of docetaxel wifh or withouf capecifabine in patienfs with refractory breast cancer with this trial demonstrating an improvement in survival for the combination of docetaxel + capecitabine vs. docetaxel alone. ... 

A much more conventional approach was required for the approval of pemefrexed for the treatment of patients with advanced mesothelioma. In a trial of cisplafinum plus pemetrexed vs. cisplafinum alone, there was a significant improvement in patient survival (a median of 13 vs. 10 months). In addition to an improvement in survival, the trial also had patient-oriented endpoints such as amount of dyspnea and an improvemenf in vital capacity (e.g., clinical benefit). There is no doubt that future clinical trials should build upon these important patient-oriented endpoints, particularly trials in which response (tumor shrinkage) may not be as commonly seen as the response with cytotoxic agents. ... 

Viral and cytotoxicity titers will be expressed as —loglO of the 50 percent titration endpoint for infectivity (TCID50) or cyctotoxicity (TCD50), respectively, as calculated by the method of Spearman Karber. 

Endpoint assays such as proliferation or cytotoxicity assays are routinely used for functional assessments. For these assessments, primary cells, transformed cells, or cells transfected with the target receptor are exposed to range of concentrations of the test article. Proliferation or cytoxicity is then measured using a variety of methods such as crystal violet vital dye staining, MTT/MTS incorporation, or a luminescence readout like ATP lite. In addition, assays that analyze phosphorylation of specific transcription factors, or release of specific cytokines and chemokines, are also common. Figure 9.5 illustrates the measure of functional consequences of receptor-test article interaction by quantifying cytokine release. Cells from the species under evaluation were cultured in the presence of serial dilutions of the test article or control reagents, and supernatants harvested for determination of cytokine levels by ELISA (i.e.,... 

Riss, T.L. and Moravec, R.A. 2004. Use of multiple assay endpoints to investigate the effects on incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays. Assay Drug Dev. Technol. 2, 51-62. 

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