Biological Reactivity

USP<87> Biological Reactivity Tests – In-Vitro Laboratories providing analytical testing as per USP 87 and ISO 10993 so that biocompatibility testing of their medical devices, container-closure systems, raw materials, and drug substances are performed. While cytotoxicity testing has been a requirement for certain formats in the past, the recently revised USP 661 chapters have significantly expanded its applicability, including packaging components intended for use with solid oral dosage forms. USP 87 testing is performed to evaluate whether a product complies with specified biological reactivity or cytotoxicity requirements. The test method employed may vary, but the fundamental concept is to emulate exposure of a product, either directly or through extraction, to its end user, by exposure to mammalian cells. The reaction is then measured by grading cell viability. The methods employed may be Direct Contact, in which low density materials are directly exposed to the cells, Agar Diffusion, in which higher density materials (or their extracts) are placed on an agar overlay, or Elution, in which an extract of material is directly exposed to the cells. Cell culture assays are used to assess the biocompatibility of a material or extract through an in-vitro reaction of mammalian cells following exposure. These may be plastics or elastomers used as containers (or to seal containers) which will hold drugs or other solutions for parenteral administration (e.g. Intravenous (IV) bags, intravenous (IV) tubing), plastics that will directly contact the patient (e.g. knee brace material, dentures, contacts, catheters), materials that may be implanted (e.g. medical devices: joint replacements or cochlear implants). These techniques are useful in evaluating the potential of materials and chemicals to have toxic properties. They provide a way to screen materials prior to, or in lieu of, in-vivo tests. There are three methods outlined in USP for cytotoxicity tests: The Elution assay uses different extracting media and extraction conditions to test devices according to actual use conditions or to exaggerate those conditions. Extracts can be titrated to yield a semi-quantitative measurement of cytotoxicity. After preparation, the extracts are transferred onto a layer of cells and incubated. Following incubation, the cells are examined microscopically for malformation, degeneration and/or lysis of the cells. At least one type of cytotoxicity test should be performed on each component of any device to ensure each material, or any process which produces each component, does not imbue cytotoxic effects. The Direct Contact procedure is recommended for low-density materials, such as contact lens polymers. In this method, a piece of test material is placed directly onto cells growing on culture medium. During incubation of the cells in contact with test material, leachable chemicals in the material may diffuse into the culture medium to reach the cell layer. Reactivity of the test sample is indicated by malformation, degeneration and/or lysis of cells around the test material. The Agar Diffusion assay is appropriate for high-density materials, such as elastomeric closures. In this method, a thin layer of nutrient-supplemented agar is placed over the cultured cells. The test material (or an extract of the test material dried on filter paper) is placed on top of the agar layer and incubated. A zone of malformed, degenerated and/or lysed cells under and around the test material indicates cytotoxicity. ISO or custom methods may also be used for cytotoxicity tests, which include the tests outlined in USP, but also include materials other than polymers in scope, as well as quantitative assays that employ techniques to measure of cell viability after exposure. As referenced under USP General Chapter <661> and outlined under USP General Chapters <87> and <88>, there are a series of biological reactivity test requirements specific to plastic container components. Falling under In Vivo and In Vitro classifications, these series of qualification requirements center on culture and animal-based testing procedures. Under USP <87>, there are three distinct test methods that are assigned based upon the material of composition for the sample under test. As noted in the test list, the Agar Diffusion method is specific to rubber stoppers and the Elution Method is specific to plastic materials. The USP <88> test requirements present of series of test methods that enable a sample to be classified out to level six commonly required for use in the pharmaceutical product industry. Specific Biological Reactivity Tests: USP <87> Biological Reactivity – Direct Contact, USP <87> Biological Reactivity – Agar Diffusion (Rubber Stoppers), USP <87> Biological Reactivity – Elution Method (Plastic Materials), & USP <88> Class Six Plastic Testing. As referenced under USP General Chapter <661> and outlined under USP General Chapters <87> and <88>, there are a series of biological reactivity test requirements specific to plastic container components. Falling under In Vivo and In Vitro classifications, these series of qualification requirements center on culture and animal-based testing procedures. Under USP <87>, there are three distinct test methods that are assigned based upon the material of composition for the sample under test. As noted in the test list, the Agar Diffusion method is specific to rubber stoppers and the Elution Method is specific to plastic materials. The USP <88> test requirements present of series of test methods that enable a sample to be classified out to level six – commonly required for use in the pharmaceutical product industry. Specific Biological Reactivity Tests are USP <87> Biological Reactivity – Direct Contact, USP <87> Biological Reactivity – Agar Diffusion (Rubber Stoppers), USP <87> Biological Reactivity – Elution Method (Plastic Materials), USP <88> Class Six Plastic Testing. Biological Reactivity Testing Services include USP <88> Systemic Injection Test, USP <88> Intracutaneous Test, USP <88> Implantation Test, Biological Reactivity Testing Standards, USP <88> Biological Reactivity Tests, In Vivo, Biological Reactivity Related Services, USP Class Plastics (Medical Devices), Cytotoxicity Testing, Extractable Leachable Testing, Container Closure Testing, In Vivo Toxicology, General Safety Tests. Elastomerics, plastics or other polymeric materials with direct or indirect patient contact should be tested for adverse effects. Extracts of the materials are prepared and injected into a rodent species. The animals are monitored for any systemic or local responses. Many factors could influence the suitability of the material for a specific use. Materials composition, processing and cleaning procedures, inks, adhesives, adsorption and permeability of preservatives, sterilization method and conditions of storage could affect the performance of the material. There are three in vivo tests involved in the classification of plastics. The Systemic Injection Test and the Intracutaneous Test are designed to determine the systemic and local biological responses to plastics and other polymers by the single-dose injection of specific extracts prepared from a sample. The third test, the Implantation Test, is designed to evaluate the reaction of living tissue to a test material. The testing for the six different class plastics levels is all done using different combinations of these three tests and different extracts. USP class plastic tests are designed to assess the biological reactivity of various types of plastics materials in vivo. Originally developed to test drug containers, the class plastics tests are often performed on unmolded plastic resins as well as containers. Class plastics testing is not a substitute for medical device biocompatibility testing, medical device materials are recommended to follow ISO guidelines, but biological reactivity class plastics testing is often used by manufacturers to classify materials. The USP defines six plastics classes, from I to VI (VI being the most strict). Therefore, many plastics manufacturers find it advantageous to have their plastic resins certified as USP Class VI, especially if the resin is a likely candidate to be used in medical devices. A plastic resin material that has passed Class VI certification is assumed to be more likely to produce favorable biocompatibility results. PBL provides of certificate of analysis for all materials test at PBL that pass the USP Class VI specifications.