Elution Vs. Agar Diffusion Cytotoxicity Testing
What is cytotoxicity?
Cytotoxicity refers to molecules and compounds that are poisonous to living cells. Cytotoxins are often chemical but can also be from natural or biological sources.
What is cytotoxicity testing?
Cytotoxicity testing evaluates the biological reactivity of mammalian cells and tissues to contact with elastomeric plastics, excipients, and other materials that will come in direct or indirect patient contact during medical product use. Thus, sometimes cytotoxicity testing is referred to as biological reactivity testing. Cytotoxicity is significant as it evaluates the biological effects of a sample’s leachable chemicals. 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.
How are cytotoxicity tests for elastomeric plastics performed?
The following cytotoxicity tests cover both types I and II elastomeric plastics. Nearly all plastics used for injectable, parenteral, and medical products will only require in-vitro cytotoxicity testing covered by USP 87. However, if elastomeric components do not meet the requirements of USP 87 direct contact, agar diffusion, and elution testing, in-vivo cytotoxicity testing outlined in USP 88 will be needed. Either intracutaneous or systemic injection tests can be used for in-vivo testing of elastomers.
What is in-vitro elution testing?
Elution tests are designed for evaluating extracts from plastic materials. Elution tests for cytotoxicity are beneficial for assessing high-density materials and evaluating dose-response in-vitro. Elution testing methods allow sample extraction to occur multiple times and under various temperature conditions.
What is in-vitro agar diffusion testing?
Agar diffusion tests are beneficial 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 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 is elution testing performed?
For elution testing, product or material extracts are prepared using a 0.9% sodium chloride injection or mammalian cell culture media. Often 0.1 g of elastomeric materials or 0.2 g of other plastic material is used per milliliter of extraction medium. 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), and a cell suspension is prepared. Equal amounts of the L-929 cell suspension are added to culture plates with a 35-millimeter diameter to create a single-layer cell culture. After L-929 cells have been cultured to reach the appropriate confluence, the cell culture medium is aspirated from the plates and replaced with extracts from material samples, positive controls, and negative controls. Extracts from cell culture media remain without dilutions. Extracts prepared with the Sodium Chloride Injection are diluted to a 25% extract concentration with a serum-supplemented cell culture medium. All samples and controls are cultured in duplicate or triplicate 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 here for details). The biological reactivity is determined by assessing the nonlethal injury of the cells (cellular degeneration) and any structural defects (malformations) the cells have. The elution 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 elution test’s requirements if the samples’ biological responses are not greater than grade 2.
How are agar diffusion tests performed?
For agar diffusion testing, materials 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 here for details). 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 agar diffusion test’s requirements if the samples’ biological responses are not greater than grade 2.
What are the differences between elution and agar diffusion testing?
Unlike elution tests, agar diffusion tests utilize an agarose layer for cytotoxicity assessment. Further, agar diffusion tests are best for assessing the cytotoxicity of elastomeric closures. In contrast, elution tests evaluate the cytotoxicity of any sample liquid extracts and do not involve the use of agarose. L-929 fibroblast cells are used to evaluate cytotoxicity for both agar diffusion and elution tests. Further, agar diffusion and elution tests use the same scoring system for cytotoxicity evaluation.
Summary
Overall, cytotoxicity testing evaluates the toxicity of the polymeric materials used by medical devices and products. This article compares two in-vitro cytotoxicity tests: agar diffusion and elution testing. Only in-vitro (benchtop) testing will be necessary to evaluate cytotoxicity in nearly all circumstances. All in all, ensure you choose a contract manufacturing organization that can support you with appropriate cytotoxicity testing for your unique medical device or product needs.
Ethide Labs is a contract testing organization specializing in Cytotoxicity Testing. Ethide Labs also offers Microbiology Testing, Bioburden Testing, EO Residual Testing, Bacterial Endotoxin Testing, Sterility Testing, Environmental Monitoring & Package Integrity Testing services for medical device companies and allied industries. Ethide is an ISO 13485 certified facility.
References
Michael J. Akers. Sterile Drug Products Formulation, Packaging, Manufacture, and Quality. Drugs and the Pharmaceutical Sciences. Informa Healthcare. 2010.
United States Pharmacopeial Convention. <87> Biological Reactivity Tests, In Vitro. Rockville, MD, USA. 2021. (USPC <87>).
United States Pharmacopeial Convention. <88> Biological Reactivity Tests, In Vivo. Rockville, MD, USA. 2021. (USPC <88>).
