Cytotoxicity testing materials

For cytotoxicity tests, materials were extracted in PBS during 16 hours. The PBS extracts were then added to the culture medium (1 3). After 3-5-72 hours, adherent cells were trypsin-digested, stained and counted by a Buiker chamber. 

Preliminary Cytotoxicity Testing. An essential first step is to carry out a preliminary study to evaluate the toxicity of the test material to the indicator cells, under the conditions of the main mutagenicity test. When selecting dose levels, the solubility of the test compound, the resulting pH of the media, and the osmolality of the test solutions all need to be considered. The latter two parameters have been known to induce false positive effects in in vitro mammalian tests (Brusick, 1986). The experimental procedure is carried out as follows. 

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. 

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. 

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. 

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. 

The cytotoxicity test described in ISO 10993-5 is a good example. It is a rapid, standardised test, very sensitive and can characterise materials and significant quantities of harmful extractables and their effect on cellular growth. Because of the high sensitivity, mouse fibroblasts L929 are used as the test cells routinely. 

Various standards and procedures exist for the evaluation of the biological and immunotoxicity response of an implant [81] from the point of view of biocompatibility. Acute toxicity screening and in vivo implantation tests are fundamental in this respect. Cytotoxicity testing to detect the biological activity of the material on a mammalian cell monolayer is often the first step in assessing biocompatibility of a device. An international standard on the biological evaluation... 

Several types of in-vitro tests are routinely applied to study the harmful effects at either cellular level (cytotoxicity tests) or on the genetic material... 

Although the correlation between low pHs (acids) and eye damage in the rabbit has not been found to be excellent, all alkalis (pH 11.5 or above) tested have been reported to produce opacities and ocular damage. Many laboratories now use pH cutoffs for testing of 2.0 or lower and 11.5 or 12.0 and higher. If a material falls outside these cutoffs (or is so identified due to other physicochemical parameters), then it is (1) not tested in the rabbit eye and is assumed to be corrosive (2) evaluated in a secondary screen such as an in vitro cytotoxicity test or primary dermal irritation test or (3) evaluated in a single rabbit before a full-scale eye irritation test is performed. It should be kept in mind that the correlation of all the... 

Crystalline silica is toxic to cells in vitro, and it is commonly used as a positive control material in cytotoxicity testing in cell culture systems. 

One significant difference between the LBT and the L-929 cytotoxicity test is the composition of the test median. The luminescent bacteria are suspended in a simple 2% sodium chloride solution, whereas the tissue culture cells require serum to supplemented their growth. Serum proteins and amino acids can complex with many chemicals, particularly metals, and thus rendo them unavailable to the test cells [27]. It was noted that for the 12 samples which were scored non-toxic with the LBT but positive to tissue culture, the Microtox F were <1.0 criteria established for a positive result. Depending upon the products or matoials under test, it may be practical and appropriate to use different F values as a failure point. These could correspond with the failure concentration established with the tissue culture or animal test for a specific class of material or product category. The highest sensitivity of the LBT should not be viewed as a cause of mme materials failing the test, but rather an q rtunity to set the failure point at a realistic and safe concentration. These correlations are discussed in depth by Jones [24]. 

A second component of selectivity concerns toxicity. Most test materials capable of enzyme induction can also induce a cytotoxic response. This leads to the concept of a chemopreventive index (Cl), in this case defined as the concentration of compound that reduced cell growth by 50% (IC50), divided by the concentration of compound required to increase enzyme activity by 2-fold (CD). In the case of sulforaphane, evaluation with cultured Hepa lclc7 cells leads to a Cl of 9.9 /iM/0.23 ixM = 42 (27). The greater the Cl value, the stronger the indication of selectivity. 

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). 

Test Materials HBsAg Inhibition (EDjo fig/ml)" Cytotoxicity (CC50 fxg/ml)- TI=CC,o/ED5o ... 

As to the biomaterial for human tissue replacement, it is necessary to demonstrate if the material has any effect on the biological properties of the tissue. Bioceramics exhibit some possible toxic reactions due to metal ions leaching from the ceramics, resulting in the tissue dying or heavy reactions. In this experiment, cytotoxicity test, hemolysis test as well as skin irritation were conducted to value the biocompatibility of the porous AI2O3 ceramics. 

Migration of substances in water can give rise to cytotoxic effects. A cytotoxicity test can provide an overall assessment of the impact of a material on water quality in addition to chemical analysis. Research has been carried out to develop a harmonized and reliable test procedure. Cytotoxicity testing is part of the assessment in some European coimtries but it is still imclear as to whether or not it will be part of the European Aeeeptance Scheme. 

ISO 10993 states Cytotoxicity tests employing cell culture techniques shall be used to determine the lysis of cells (cell death), the inhibition of cell growth, colony formation, and other effects on cells by medical devices, materials and/or their extracts.. 

In vitro cytotoxicity testing showed GEMOSIL did affect normal cell growth (Figure 3) which is proportional to the increased level of MTS absorbance. Two-way ANOVA showed that the difference was significant for both factors. Day and Material. At the beginning of the culture, the chlorhexidine increased cell growth approximately 25% but then decreased around 25% after day 7. 

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