CYTOTOXICITY TESTING
Cytotoxicity testing can be useful as a screening tool for pharmaceuticals before more extensive toxicological testing is performed. Additionally, cytotoxicity testing can be used for quality control purposes for lot release testing of raw materials or of manufactured drug products.
Cytotoxicity tests are designed to determine the toxicity to cells of compounds either qualitatively or quantitatively. Qualitative cytotoxicity tests offered by Pacific BioLabs are the Direct Contact Test, the Indirect Contact Agar Diffusion Test, and the MEM Elution Test. In addition, Pacific BioLabs offers a very sensitive quantitative cytotoxicity test, the MTT Assay.
STANDARDS FOLLOWED
- USP <87>
- ISO 10993-5
DIRECT CONTACT
The direct contact method uses L-929 mouse fibroblasts which adhere to the bottom of a petri dish or plate. The cells are grown to subconfluency (approximately 80% confluent) and the test article is placed directly on the cells. For liquids and extracts, a piece of sterile filter paper is saturated with the test sample and the filter paper is placed directly on the cells. The cells and test article are incubated together and after 24 hours the samples are examined in an inverted phase contrast microscope. The cells are given a grade from 0 to 4 corresponding to no reactivity to severe reactivity.
This test is not recommended for very low or very high density material. Very low density material will float in the cell growth media and very high density material has the potential to damage the cells when the sample is placed on top of them. For low and high density material the Agarose Overlay (Agarose Diffusion) method is preferred.
AGAROSE OVERLAY (AGAR DIFFUSION)
Agarose overlay (also referred to as agar diffusion) is similar to the direct contact method described above except that it uses agar. The agar is poured over the cells and left to solidify. The test sample is then placed on the agar. The agar creates a “cushion” protecting the cells from materials that are dense and will also prohibit low density materials from floating in the growth media. The cells, agar and test sample are incubated for more than 24 hours and then examined under a microscope to determine the severity of toxicity to the cells based on a 0-4 grading scheme.
MEM ELUTION
Unlike the agarose diffusion and direct contact methods, the MEM Elution procedure involves applying extractables and leachables from the test article to the cells. The test article is incubated with a liquid termed the extraction vehicle. Following extraction, the test article extract is added to L-929 mouse fibroblasts. The cytotoxic effects of the extract are determined after a 48 hour incubation period. Scoring is based on conditions of the cells using a 0 to 4 grading system which is correlated the observed cytotoxic affect.
MTT ASSAY
The MTT assay is a quantitative cytotoxicity assay that uses a dye called 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (abbreviated to MTT). MTT is a yellow water soluble chemical that is cleaved by mitochondrial succinate dehydrogenase to form formazan which is violet color. This reaction will only occur in health living cells. Mitochondrial succinate dehydrogenase will not cleave MTT if the cell is dead.
The MTT assay is performed using an extract of the test article. The extract is added to the L-929 mouse fibroblasts at four different concentrations, undiluted, 1:2, 1:4, and 1:10. After 24 hours, MTT is added to the cells and the presence of violet formazan can be analyzed using a plate reader. The samples are compared to controls to determine the percentage of viable cells.