Study Outline for Cytotoxicity/MEM/Agar Overlay
Devices/materials are generally extracted in serum-supplemented mammalian cell culture media (MEM) 37 ± 1ºC. After extraction, the extracts are placed in contact with a monolayer of L-929 cells (mouse fibroblasts). The cells are then allowed to grow in the extraction fluid for a specified amount of time before the cells are evaluated using either qualitative or quantitative methods.
Qualitative evaluation involves observing the cells under a microscope and assigning a cytotoxic grade (0-4). The grade is based on an estimated percent lysis (death) and on the morphology (appearance) of the cells. Test materials pass the assay if the cytotoxic score is ≤ 2 (≤ 50% lysis).
Quantitative evaluation utilizes a tetrazolium dye which is used to assess the metabolic activity of cells. The results are reported as percent viability (% living cells). Test materials pass the assay if the percent viability if ≥ 70%. Multiple dilutions of the test article extract are generally included with each assay.
Materials are generally tested by surface area using at least 100 mm2 of material per well. Weight may also be used for liquids, gels, or powders using at least 100 mg per well. The material is placed on a layer of agarose that is placed on top of a monolayer of L-929 cells and incubated for 24 – 26 hours at 37 ± 1ºC in 5 ± 1% CO2. After incubation, the zone of cell destruction is measured and scored based on a 0 – 4 scale listed in AAMI/ANSI/ISO 10993-5. The material meets ISO requirements if the cytotoxic response is not greater than grade 2 (mildly reactive). Although Agar Overlay has been historically an accepted test method it may not be appropriate when submitting to certain regulatory bodies.
Occasionally, a device or material will exhibit a level of cytotoxic reactivity that is higher than what is allowed by the ANSI/AAMI/ISO 10993-5 standard and will result in a failed test. Although your product may have failed a cytotoxicity test, it does not necessarily mean that your device or material is unsafe for clinical use. It simply means that you are obligated to identify the source of failure and assess any toxicological risks.
Our Experts Can Help
There are several possible causes for cytotoxicity failure, and Nelson Labs offers services to help you identify and assess the failure. From there, our experts can assess any potential toxicological risk to patient safety.
We evaluate the materials and processes associated with the device. Based on this information, our experts will often recommend targeted analytical chemistry to determine the cause of the failure. We can then work with clients to justify the failure through testing and assessment.
A discussion with our experts to identify the cause of cytotoxicity failure and develop an action plan often leads to manufacturers successfully submitting their device for regulatory approval without any further action.
If you have additional questions about Cytotoxicity, or would like to consult with the experts at Nelson Labs, just send us a request or call us at +1 (801) 290-7500.