Sensitization Vs. Irritation Vs. Cytotoxicity Testing For Medical Devices & Implants
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.
What is irritation?
Irritation is temporary dryness, itching, flaking, peeling, or rash at the site of product exposure. Irritation affects nearly everyone the same way. Also, once contact with an irritant is removed, the symptoms and discomfort pass.
What is irritation testing?
The animal testing for sensitization and irritation are the same since they are both forms of contact dermatitis. A patch test like the Magnusson & Kligman guinea pig maximization test is commonly used. In a patch test, a small area of bare animal skin is exposed to a product (often repeatedly) to show how the skin will respond to the product. Patch testing is a simple and effective way to evaluate your product’s ability to cause irritation or sensitization and to meet regulatory safety requirements.
What is sensitization?
Sensitization is an inflammatory immune response that occurs only in some people after repeated exposure to a product. Sensitization has symptoms similar to irritation (e.g., a red, itchy, bumpy rash). However, sensitization symptoms are caused by the body’s immune reactivity to the product (or product components). In contrast, irritation symptoms are caused by the product. A sensitization reaction is a form of allergic reaction.
What is sensitization testing?
Sensitization tests are tests in live animals. They evaluate the ability of leachables and unknown antigens to cause hypersensitivity. Sensitization tests are designed to determine if a patient will develop a reaction with repeated exposure to a medical device. A few important factors to consider when selecting a sensitivity test include the type and extent of contact with the body, the chemical composition of the product or materials, the product’s manufacturing process, and the product’s sterilization process.
How are cytotoxicity tests performed?
Most plastics (polymers) used for injectable, parenteral, and medical products will only require in-vitro cytotoxicity testing covered by USP 87. However, if material 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. The required in-vivo testing will be implantation, intracutaneous injection, or systemic injection tests. A description of all in-vitro and in-vivo cytotoxicity tests is provided below.
In-Vitro Cytotoxicity Testing (USP 87)
In-vitro tests are required to determine the biological reactivity of mammalian cells to contact with elastomeric plastics and other polymeric materials. Three in-vitro cytotoxicity tests are used to assess systemic biological reactivity. These tests are direct contact, agar diffusion, and elution assays.
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 coincide 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 to occur multiple times and under various temperature conditions.
In-Vivo Cytotoxicity Testing (USP 88)
In-vivo tests are required in certain instances to determine the systemic biological reactivity of mammalian cells to contact with elastomeric plastics and other polymeric materials. The cytotoxicity tests performed for your medical device or product depend upon the final product’s packaging, intended use, and material construction. Three in-vivo cytotoxicity tests are used to assess systemic biological reactivity. These tests are implantation, intracutaneous injection, and systemic injection studies.
How is implantation cytotoxicity testing performed?
The implantation test is designed to evaluate the reaction of living tissue to polymers by implanting the device or therapeutic product into animal tissue. Material samples must be aseptically prepared and surgically placed under aseptic conditions to ensure accurate implantation results. The factors below should be considered and tested when evaluating the cytotoxicity of implanted polymers.
Factors that can impact the suitability of a material for its intended use are:
- Material composition
- Material processing and cleaning procedures
- Contacting media(s)
- Inks
- Adhesives
- Absorption properties
- Adsorption and permeability of preservatives
- Conditions of storage
The implantation tests are performed by surgically introducing a material sample under evaluation into a test animal. There are two forms of implantation testing, intramuscular implantation in rabbits and subcutaneous implantation in rats.
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.
What tests are used for sensitization testing?
Table 1 summarizes the top nine methods used for regulatory sensitization testing. Most of these tests utilize guinea pigs for sensitivity evaluations. The Magnusson & Kligman Guinea Pig Maximization Test (GPMT) is the gold standard for sensitization testing. All other tests are alternatives to the GPMT gold standard. Although, Standard Beuhler testing is a close second to GPMT. Some sensitization tests evaluate implanted solid articles, while others only evaluate extracts of solid articles. In some medical device and implant applications, toxicology requirements may be satisfied with data available from previously marketed products.
Most of the tests referenced above are patch tests, where a small area of bare animal skin is exposed to a product (often repeatedly) to show how the skin will respond to the product. See our other articles on sensitization tests in guinea pigs, sensitization tests in mice, and Magnusson & Kligman maximization tests for additional testing details.
What are the differences between cytotoxicity and irritation-sensitization testing?
Irritation-sensitization testing primarily uses patch sampling methods to determine animal sensitivity or irritation to a product. Unlike cytotoxicity testing, irritation-sensitization testing uses live animals. While some cytotoxicity testing is in live animals, most cytotoxicity testing will use cultured cells, not live animals. Cytotoxicity testing evaluates the toxicity of product leachables, whereas irritation-sensitization testing evaluates product dose levels that create sensitivity or irritation in animals. Not all medical devices and implantable devices will need irritation-sensitization testing. However, all medical devices and implants will require in-vitro cytotoxicity testing.
Summary
Overall, cytotoxicity testing evaluates the toxicity of the polymeric materials used by medical devices and products. In contrast, sensitization tests (also termed irritation tests) are tests in live animals. Sensitization tests evaluate the ability of leachables and unknown antigens to cause hypersensitivity. The tests are designed to determine if a patient will develop a reaction with repeated exposure to a medical device. Not all medical devices and implantable devices will need irritation-sensitization testing. However, all medical devices and implants require benchtop (in-vitro) cytotoxicity testing. All in all, ensure you choose a contract testing organization that can support you with appropriate cytotoxicity testing and sensitivities testing for your unique medical device or product needs.
MycoScience is a contract manufacturing organization specializing in sterile syringe and vial filling cosmetic products or for products used in animal studies. In addition, MycoScience offers testing services, including 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
Consumer Product Testing Company. The Important Differences Between Sensitization And Irritation.
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. <1184> Sensitization Testing. Rockville, MD, USA. 2021. (USPC <1184>).
