What Is Agar Diffusion Test For Cytotoxicity?
What is cytotoxicity testing, and why is it essential for your medical device or product?
Cytotoxicity testing is a series of in-vitro tests that determine the biological reactivity of mammalian cell cultures to contact with elastomeric plastics and other polymeric materials that will come in direct or indirect patient contact during medical device or product use. The agar diffusion test is one of three standard tests for cytotoxicity. The types of tests to perform for your medical device or product depend upon the final product, the final product’s intended use, and the materials the final product is made of and packaged within. Reusable device processing and cleaning procedures are also essential considerations for cytotoxicity assessment. Reusable devices may require cytotoxicity testing for initial use and device use after recommended reprocessing.
What do agar diffusion tests assess?
Elastomeric closures are packaging closures made from materials that can resume their original shape after experiencing tension or compression. Agar diffusion tests are designed for assessing the cytotoxicity of elastomeric closures. In these tests, the agar layer acts as a cushion. The agar protects the cells from any mechanical damage and allows any leachable chemicals to diffuse from the product or packaging samples. The cells are then evaluated to determine the toxicity of the samples. Material extracts can also be assessed for cytotoxicity using the agar diffusion test by applying material extracts to a piece of filter paper.
How are agar diffusion tests performed?
For agar diffusion test’s sample preparation, material or medical device extracts are prepared using a 0.9% sodium chloride injection or a mammalian cell culture media. For samples that aren’t extracts, portions of the test samples with flat surfaces not less than 100 square millimeters (mm) in surface area are used for the agar diffusion assay. Polyurethane film containing either zinc diethyldithiocarbamate (ZDEC)2 or zinc dibutyl dithiocarbamate (ZDBC) is used as a positive control.
Next, L-929 mammalian fibroblast cells are grown in a serum-supplemented minimum essential medium (MEM) to greater than 80% confluence. The culture medium is then aspirated, and a solution of not more than 2% agar is added to each 60 mm diameter plate. The agar layer must be thin enough to allow for the diffusion of leached chemicals from the samples. Finally, samples, positive controls, and negative controls (or their extracts) are added on top of the solidified agar surface. All samples and controls are cultured in duplicate with no more than three specimens per prepared plate. Then all of the cultures are incubated at 37 ± 1°C in a humidified incubator containing 5 ± 1% of carbon dioxide.
After incubation, cells exposed to samples, positive controls, and negative controls are stained or evaluated without staining under the microscope. The biological reactivity of the cells exposed to the sample or sample extracts is rated on a scale of 0-4 (see Table 1 below). The biological reactivity is determined by assessing the nonlethal injury of the cells (cellular degeneration) and any structural defects (malformations) the cells have. The agar diffusion test is valid if the observed responses to the negative controls are grade 0 and the Positive controls are all at least grade 3. The sample meets the requirements of the agar diffusion test if the biological responses of the samples are not greater than grade 2.
Summary
Overall, cytotoxicity testing evaluates the toxicity of the polymeric materials used by medical devices and products. The agar diffusion test is one of three standard tests for cytotoxicity. Agar diffusion tests are designed for assessing the cytotoxicity of elastomeric closures. In these tests, the agar layer acts as a cushion. The agar protects the cells from any mechanical damage and allows any leachable chemicals to diffuse from the product or packaging samples. The cells are then evaluated to determine the toxicity of the samples. The types of cytotoxicity testing to perform for your medical device or product depend upon the final product, the final product’s intended use, and the materials the final product is made of and packaged within. Make sure you choose a contract testing organization that can support you with appropriate cytotoxicity testing for your unique medical device or product needs.
Ethide Labs is a contract testing organization that specializes in Cytotoxicity Testing. Ethide Labs provides in-vitro cytotoxicity tests in-house and outsources in-vivo cytotoxicity work for toxicity testing of medical devices, products, and drugs. Ethide Labs also offers Bacterial Endotoxin Testing, Sterilization Validations, Bioburden Testing, Microbiology Testing, Environmental Monitoring, EO Residual Testing & Package Integrity Testing services for medical device companies and allied industries. Ethide is an ISO 13485 certified facility.
References
United States Pharmacopeial Convention. <87> Biological Reactivity Tests, In Vitro. Rockville, MD, USA. 2021. (USPC <87>).
