Cytotoxicity assay materials

The test material, test cells, used, method of treatment, harvesting of cells, cytotoxicity assay, and so on, should be clearly stated as well as the statistical methods used. Richardson et al. (1989) recommend that comparison be made between the frequencies in control cells and at each dose level using Fisher s Exact Test. 

Additionally, the test materials used in the validation process should be as closely related as possible to the characteristics of the unknowns to be tested. It is clear from the literature, for instance, that many cytotoxicity assays give good correlations with the in vivo ocular irritancy data for surfactants, but the correlations fail when compounds from other chemical classes are tested. Since any particular assay may be used differently by individual safety assessment programs, users must evaluate potential methods under conditions likely to be encountered in their own situations. 

This as yet unidentified plant has yielded a small amount of a potently active compound in the 1138 yeast bioassay this has been submitted to the National Cancer Institute for bioassay in the 60-cell line assay. The active material has been identified as the known alkaloid cryp-tolepine (30) cryptolepine had an IC50 value of 5.8 fig/ml in the M109 cytotoxicity assay. 

The results of the cytotoxicity assay are shown in table 10.5. No optimization of the supercritical fluid extraction was carried out with respect to pressure, temperature, total amount of gas, etc. However, the data in table 10.5 show that all the supercritical fluid extracts yielded positive cytotoxicity results. In fact, for one of the plant materials, B638786, the supercritical fluid extract was substantially more active (i.e., it had a much lower ED50) than the standard ethanol extract. 

Cytotoxicity Assay. Test materials were evaluated for cytotoxicity by measuring cell survival at the end of the each experiment using the sulforhodamine B staining method (8). 

The in vitro biocompatibility of CPs has been generally investigated by cell viability, proliferation and cytotoxicity assays of various cell types, including rat pheochromocy-toma cells (PC 12), rat Schwann cells, cardiac myoblasts, astrocytes, and various neural tissue explants [ 13,27,43,47]. Studies on a wide variety of CPs have indicated good in vitro cell responses, with minimal cytotoxicity and, in several cases, preferential adherence of cells to the CP surface, compared to conventional implant materials. However, the majority in vitro studies examine the passive or nonelectrochemically activated state of CPs. Due to the application-specific nature of biocompatibility, passive CP assessment, although a useful preliminary study, is an imperfect characterization of CP biological performance for most implant applications. 

Caution must be applied in the use of ISO 10993 and its various parts. Device developers and regulators alike default to using Part 1 of this standard as a checklist . Not all assays described in the various parts are applicable to all device materials. Sometimes the form of the material can lead to false positives in certain studies. Not all parts of the standard provide sufficient detail to allow the uninformed to appreciate that various labs may claim to conduct testing to the standard but use widely varying protocols. Cytotoxicity assays quite commonly result in positive findings, as has occurred in testing silver-coated textiles and materials with hydroxylapatite... 

The antioxidant and anticancer evaluation of Scindapsus officinalis (Roxb.) Schott fruits has been attempted to investigate its antitumor activity [29]. The collection and authentication of the plant material, mainly fruits, and their various extractions were done. Identification of plant s active constituents by preliminary phytochemical screening was carried out. An in vitro cytotoxic assay using the brine shrimp lethality assay with brine shrimp eggs (Artemia salina) at a dose of 1-10 pg/ml with the fmit extract was performed. 

The third mandated test according to ISO 10993-1 is Irritation. Irritation is defined as a localized inflammatory response to single repeated, or continuous applications of the test substance without involvement of an immunological mechanism. ISO 10993-10 purposes a four-tier approach to assessing the potential of a material to cause irritation. A device manufacturer should first conduct a review of the literature to determine whether others have reported that the chemical or material under consideration, or structurally related chemicals or materials, can cause irritation. It is essential that the chemical or material of interest already be sufficiently characterized that it can be correlated to those described in the literature. The second step is to use available, validated in vitro tests (such as cytotoxicity assays using mammalian cells in culture) to identify, whenever possible, severely irritating materials without using test animals. 

ISO 10993 standard family regulates the testing required for a determination of biocompatibility for tissue contact devices. Cytotoxicity testing is a basic requirement for the biocompatibility assessment. The cytotoxicity assay requires the elution of the sensor with physiological saline and the evaluation of the effect of the eluate on living cells. This can be critical for enzymes which are not properly immobilized, plasticizers in substrates or membrane layers, and the often-used reference electrode material silver/silver chloride. Also relevant for CGM sensors, but not addressed by the elution test, is the effect of the enzymatic reaction in the sensor in the absence of polarization of the sensor. First-generation sensors which... 

Experimental controls namely, negative contfols, blanks, and positive controls should be used in all biological evaluations to accurately validate a certain procedure and compare the results between materials. Some examples of materials that may be used as positive and negative controls for cytotoxicity assays are listed in Table 7.5. 

Table 7.5 Materials that have been used as negative and positive controls for cytotoxicity assays according to ISO 10993-12 (International Oi anization tor Standardization, 2004)...

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 principle of the human skin model test is that the test material is apphed topically for up to 4h to a three-dimensional human skin model, comprising at least a reconstructed epidermis with a functional stratum comeum (outermost layer of the skin). The human skin models can come from various sources, but they must meet certain criteria. Corrosive materials are identified by their abdity to produce a decrease in cell viabdity (as determined, e.g., by using a dye reduction assay) below defined threshold levels at specified exposure periods. The principle of the test is in accordance with the hypothesis that corrosive chemicals are able to penetrate the stratum comeum (by diffusion or erosion) and are sufficiently cytotoxic to cause cell death in the underlying cell layers. 

MTT assay is a standard colorimetric assay used to determine cytotoxicity of potential medicinal agents and other toxic materials. It is based on the reduction of the tetrazolium salt MTT by viable cells. A mitochondrial dehydrogenase enzyme is able to cleave the tetrazolium rings of the pale yellow MTT and form dark purple formazan crystals, which are largely impermeable to cell membranes resulting in the accumulation of these crystals within healthy cells. Solubilization of the cells by the addition of a detergent results in the liberation of crystals, which are solubilized. The metabolic activity of cells is directly proportional to the concentration of the created formazan product (22), whose color is quantified in a colorimetric assay. 

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