Functionality Testing Vs. Cytotoxicity Testing For Injectables
How are plastic materials used in injectable products?
Many product-packaging systems use medical-grade polymers and elastomeric materials. Elastomeric materials are plastics that can resume their original shape after experiencing tension or compression. Plastics are used in injectable products such as vials, bottles, prefilled syringes, flexible bags, and blow-fill-seal containers. Stoppers, cap liners, plungers, needle shields, tip caps, seal liners, and injection ports are all examples of injectable product components that utilize plastics. Any plastic with direct or transient contact with a pharmaceutical product or medical product formulation must pass certain regulatory requirements before being used as a device or packaging material. Polymer coatings also fall under the assessment criteria for elastomeric materials. Information on these polymers’ composition, manufacturing, and use can be found in USP 1381.
What are functionality tests?
Functionality tests are a subset of package integrity tests. Package integrity testing assesses how well a package stays complete and undivided to keep wrapped objects protected and sterile. Package integrity and container-closure integrity are terms that are used interchangeably. These terms are the same, depending upon the definition of container-closure integrity. In the past, container–closure integrity referred to a package that had passed or could pass a microbiological challenge test. However, package integrity and container-closure integrity are synonymous under the USP 1207 definition of container-closure integrity. In the context of package integrity and package integrity testing, a product package is considered both the product packaging and the product. Package integrity testing includes leak testing, seal quality testing, and other assessments such as the functionality testing described below.
What are cytotoxicity and cytotoxicity testing?
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. 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. 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. In-vitro USP 87 methods of cytotoxicity testing include direct contact, agar diffusion, and elution testing. In-vivo USP 88 methods of cytotoxicity testing include intracutaneous injection, systemic, and implantation testing. Most medical devices and products will only require in-vitro cytotoxicity testing.
What are the regulatory tests needed for medical-grade packaging used in injectables?
As mentioned earlier, medical-grade plastics in direct or indirect contact with a medical or pharmaceutical product must be assessed for safety before use. Plastic materials can vary widely in their purity, meaning that intentionally or unintentionally added elements exist in manufactured plastics. Thus, physiochemical and cytotoxicity requirements for medical-grade plastics must be met, in addition to functional requirements. Depending upon the elastomeric material, USP 1663 extractables testing may be needed. Overall, safety tests for elastomeric plastics fall under two primary categories: package integrity testing (e.g., functionality, seal quality, and leak testing) and cytotoxicity testing (e.g., cytotoxicity and physiochemical testing).
What are the functionality tests pharmaceutical-grade plastic materials undergo?
Functionality tests are performed on plastic closures that a hypodermic needle will pierce. These tests ensure that the plastic closures can adequately seal product containers and provide effective product delivery through needles. Self-sealing functionality tests are needed only for multi-dose containers. The elastomeric plastic closures being tested should mimic those used in the final product-package system, including any terminal sterilization processes (e.g., steam, ethylene oxide) or surface modifications (e.g., siliconization, fluoropolymer coatings). Multiple 21-gauge hypodermic needles with a 12° long-bevel are used for the following functionality tests.
#1: Penetrability Testing
Procedure: Ten vials are filled with water and fit with elastomeric closures. Closures are then examined by measuring the force required to pierce the plastic closure with a sterile hypodermic needle repeatedly.
Acceptance criteria: Piercing force is no greater than ten Newtons for each closure.
#2: Fragmentation Testing
Procedure: Fragmentation testing for dry preparations involves first fitting twelve clean vials with plastic closures. For liquid preparations, twelve vials are filled with water within four milliliters of their capacity before being fit with closures. All vials stand for sixteen hours after preparation. Next, a hypodermic needle is used to inject one milliliter of water and remove one milliliter of air. The water injection and air removal process is completed four times for each elastomeric closure, piercing in a new location with each injection and using a new needle for each closure. Once all four piercings are complete, vial liquid is filtered through a membrane with a pore size of half a micron or less. Rubber fragments visible to the naked eye are then counted after filtration for each of the twelve vials.
Acceptance criteria: No more than five fragments with a diameter greater than 50 microns (μm) are visible. Particles can be examined microscopically for size verification.
#3: Self-Sealing Capacity Testing
Procedure: Ten vials are filled with water and fit with elastomeric closures. Closures are then pierced ten times each with a new hypodermic needle. Each needle piercing is at a different location on each plastic closure. Next, all ten vials are immersed in a 0.1% (1 gram/Liter) methylene blue solution for forty minutes. The external pressure is reduced by 27 kilo Pascals for the first ten minutes before being restored to atmospheric pressure for the remaining thirty minutes of immersion. Finally, the vials are removed, rinsed, and visually inspected for any trace of blue within the filled vials.
Acceptance criteria: All vials contain no trace of blue solution.
What types of plastic (elastomeric) materials are used in injectable products?
Plastics used for injectables come in two primary classifications: type I and type II elastomeric components. Type I elastomeric closures are preferred and meet specific appearance, absorbance, and reducing substance requirements. Type II elastomeric closures are suitable for special uses (e.g., repeated hypodermic needle piercings for syringe refiling). However, type II elastomers do not meet type I criteria. Instead, type II materials meet alternative criteria for their intended use.
What are the cytotoxicity tests for medical-grade plastic materials?
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 direct contact testing?
Direct contact cytotoxicity tests can evaluate nearly all materials. Additionally, sample extraction and testing of a sample’s leachable chemicals can occur simultaneously with direct contact testing. Direct contact methods cannot assess very low density or extremely high-density materials that could cause mechanical damage to cultured live cells.
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.
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 multiple times and under various temperature conditions.
What is in-vivo systemic injection testing for cytotoxicity?
A systemic injection is an injection into the circulatory system. Systemic injection testing determines the local biological responses of animals (mice) to plastic extracts injected into the bloodstream. Systemic injection testing and intracutaneous testing may be performed using the same extracts. Extracts are prepared depending on the heat resistance of the material being assessed. Thus, extracts are prepared at 50°, 70°, or 121°C. Natural elastomers are tested in sodium chloride injection and vegetable oils only. Sample sizes for elastomer extract preparations are twenty-five centimeters squared worth of combined surface area per twenty milliliters of extract medium. Elastomers used for extracts must remain uncut. USP 88 provides additional details on extract preparations for intracutaneous testing.
Systemically injected mice are assessed immediately after injection and at 4, 24, 48, and 72 hours after injection. Each study uses ten mice. All mice injected with polymer extracts must show the same or less reactivity as controls injected with blanks to pass this examination.
What is in-vivo intracutaneous testing for cytotoxicity?
An intracutaneous injection is an injection between the layers of the skin. Intracutaneous testing determines the local biological responses of animals (rabbits or guinea pigs) to plastic extracts injected under the skin. Systemic injection testing and intracutaneous testing may be performed using the same extracts. Extracts are prepared depending on the heat resistance of the material being assessed. Thus, extracts are prepared at either 50°, 70°, or 121°C. Natural elastomers are tested in sodium chloride injection and vegetable oils only. USP 88 provides additional details on extract preparations for intracutaneous testing.
For each plastic sample, two animals are intracutaneously injected. After injection, the injection sites are assessed for evidence of any tissue reaction such as erythema, edema, and necrosis. All animals are observed at 24, 48, and 72 hours after injection for tissue reaction. The average erythema and edema scores for the control injection sites are also assessed at 24, 48, and 72 hours. Scoring systems and pass/fail criteria for this test can be found in USP 88.
Summary
Overall, stoppers, cap liners, plungers, needle shields, tip caps, seal liners, and injection ports are all examples of injectable product components that utilize medical-grade plastics. Any plastic with direct or transient contact with a pharmaceutical product or medical product formulation must pass certain regulatory requirements before being used as a device or packaging material. Safety tests for medical-grade plastics fall under two primary categories: package integrity testing (e.g., functionality, seal quality, and leak testing) and cytotoxicity testing (e.g., biological reactivity and physiochemical testing). This article describes three functionality tests and five cytotoxicity tests performed on plastic closures. These functionality tests are penetrability, fragmentation, and self-sealing capacity testing. Self-sealing capacity testing is only needed for multi-dose containers. The cytotoxicity tests are direct contact, agar diffusion, elution, systemic injection, and intracutaneous testing. However, agar diffusion testing is likely the only cytotoxicity test needed for medical-grade plastics. All in all, ensure you choose a contract manufacturing organization that can support you with appropriate package integrity and toxicity testing for your unique implantable device or injectable needs.
MycoScience is a contract manufacturing organization specializing in sterile syringe and vial filling. MycoScience also offers Preservative Efficacy Testing, Cytotoxicity Testing, Bioburden Testing, Cleaning Validations, Microbial Aerosol Challenge Testing, Accelerated Aging, Microbiology Testing, EO Residual Testing, Bacterial Endotoxin Testing, Package Integrity Testing, Sterilization Validations & Environmental Monitoring services medical devices and allied industries. MycoScience 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>).
United States Pharmacopeial Convention. <381> Elastomeric Components In Injectable
Pharmaceutical Product Packaging/Delivery Systems (USPC <381>).
United States Pharmacopeial Convention. <1207> Package Integrity Evaluation- Sterile Products. Rockville, MD, USA. 2021. (USPC <1207>).
