Cytotoxicity Testing Vs. Physiochemical Testing For Medical-Grade Plastics
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. The composition, manufacturing, and use of these polymers can be found in USP 1381.
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 physiochemical tests?
In many ways, physiochemical testing is a cytotoxicity pre-test for plastic materials. Certain plastics can be excluded from medical use due to their physicochemical properties. Thus, physiochemical tests can save time and money on cytotoxicity testing. Physiochemical tests for type I and type II elastomers are described below.
What are the regulatory tests needed for the medical-grade plastics used in injectables?
As mentioned earlier, 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 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).
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 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 at 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.
What types of plastic 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 physiochemical tests do pharmaceutical-grade plastics undergo?
Sample Preparation: First, whole elastomeric components with a 100 ± 10 square centimeters surface area are placed into a wide-necked glass flask. If the surface area mentioned above is not possible with uncut elastomers, use uncut elastomeric components that will most closely approximate 100 ± 10 square centimeters. Next, the elastomeric samples are covered with water so that the volume of purified water is approximately two milliliters of water per centimeter squared of the elastomer’s surface area. Then weigh the water-covered elastomers in the flask. Next, cover the glass flask and heat the immersed elastomers in an autoclave so that a temperature of 121 ± 2°C is reached and held for thirty minutes. Then slowly cool the immersed elastomers to room temperature. Purified water is then added to bring the immersed elastomers to their original mass. This solution is shaken and used for the physiochemical tests described below. Purified water is used as a negative control for these tests.
#1: Appearance (Turbidity & Opalescence) Testing
Procedure: Turbidity can be determined visually or with a calibrated turbidimeter. Particle-free water must be used to create all solutions for appearance testing. A formazin stock solution is made by combining a hydrazine sulfate and a hexamethylenetetramine solution. The formazin stock solution must stand for two days before use. A formazin standard suspension is made by diluting fifteen milliliters of the formazin stock suspension with particle-free water until a volume of one liter is reached. Use the formazin stock suspension to prepare reference suspensions A-D. Details on reference suspension preparations can be found in Table 1 of USP 381.
For testing, six identical test tubes made of transparent glass are filled with the solutions listed below:
- Sample solution
- Particle-free water
- Reference suspension A
- Reference suspension B
- Reference suspension C
- Reference suspension D
For evaluation, compare the solutions in diffuse daylight against a black background or a turbidimeter with a particle-free water blank.
Acceptance criteria: For type I elastomers, the sample solution should not be more opalescent than reference suspension B. Alternatively, the measured turbidity of the sample solution should not be more than reference suspension B (6 nephelometric turbidity units (NTU) or formazin turbidity units (FTU)).
For type II elastomers, the sample solution should not be more opalescent than reference suspension C. Alternatively, the turbidity of the sample solution should not be more than reference suspension C (18 NTU/FTU). Note that reference suspension A and reference suspension D should be 3 NTU and 30 NTU, respectively.
#2: Color Testing
Procedure: Prepare a color standard by diluting 3.0 milliliters of matching fluid O with 97.0 milliliters of diluted hydrochloric acid (10 ± 0.5%). Next, fill two clear, identical tubes: one tube with sample solution and the second tube with the color standard. Compare the liquids in daylight against a white background.
Acceptance criteria: The sample solution must not be more intense (in color) than the color standard.
#3: Acidity/Alkalinity Testing
Procedure: Acidity and alkalinity are determined using bromothymol blue solution. The bromothymol blue solution is made with bromothymol blue powder, 0.02 M sodium hydroxide, alcohol, and purified water. Twenty milliliters of prepared sample solution and 0.1 milliliters of Bromothymol blue solution are combined for testing. If the solution is yellow, the test solution is titrated with 0.01 N sodium hydroxide until a blue endpoint is reached. If the solution is blue, the test solution is titrated with 0.01 N hydrochloric acid until a yellow endpoint is reached. If the solution is green, it is already at a pH of seven, and no titration is needed. Use purified water as a blank. If the purified water is not green, correct the sample test results by subtracting or adding the volume of titrant required for the purified water.
Acceptance criteria: Green indicates a neutral solution. Not more than 0.3 milliliters of 0.01 N sodium hydroxide produces a blue color, while not more than 0.8 milliliters of 0.01 N hydrochloric acid produces a yellow color. Generally, elastomers with high acidity or alkalinity should be avoided.
#4: Absorbance Testing
Procedure: Absorbance testing must be performed within five hours of preparing the elastomeric sample solution. Once the sample solution is made, it is passed through a 0.45-micron filter. The absorbance of the filtrate is evaluated in a spectrophotometer at wavelengths between 220 to 360 nanometers. Purified water is used as a blank.
Acceptance criteria: For type I elastomers, the acceptance criterium is not more than 0.2. Whereas for type II, it is not more than 4.0.
#5: Reducing Substances Testing
Procedure: Absorbance testing must be performed within four hours of preparing the elastomeric sample solution. First, one milliliter of diluted sulfuric acid is added to twenty milliliters of sample solution and twenty milliliters of 0.002 M potassium permanganate. This solution is then boiled and allowed to cool. Then a single gram of potassium iodide is added before titrating the solution with 0.01 M sodium thiosulfate. Finally, perform a titration using twenty milliliters of purified water and record the difference in the volume of the 0.01 M sodium thiosulfate solution required to reach a neutral pH. A quarter milliliter of starch solution TS is the indicator for both titrations.
Acceptance criteria: For type I elastomers, the difference between titration volumes must be not more than three milliliters of 0.01 molar sodium thiosulfate. For type II elastomers, the difference between titration volumes must not be more than seven milliliters of 0.01 molar sodium thiosulfate.
#6: Volatile Sulfides Testing
Procedure: Elastomeric samples are processed differently for this physiochemical test. Instead of using the sample stock solution, elastomeric components (cut or uncut) with a total surface area of 20 ± 2 centimeters square are placed in a 100-mL flask. Then fifty milliliters of a 20-gram/liter citric acid solution is added. A control solution is made by dissolving 0.154 milligrams of sodium sulfide in fifty milliliters of a 20-gram/liter citric acid solution. Place a piece of lead acetate paper over the mouth of the sample and control solutions, and weigh it down. Heat the flasks in an autoclave to 121°C and hold for thirty minutes. Cool to room temperature before evaluating for stains.
Acceptance criteria: A black stain on the paper produced by the test solution is not more intense than any stain produced by the control solution.
#7: Ammonium Testing
Procedure: Prepare an alkaline potassium tetraiodomercurate solution in water. Then mix one volume of this solution with an equal volume of a sodium hydroxide solution. Dilute five milliliters of elastomer sample solution with water to fourteen milliliters. Then add 0.3 milliliters of alkaline potassium tetraiodomercurate solution. Wait five minutes before assessing the sample solution with an ammonium standard solution.
Acceptance criteria: After five minutes, the yellow color in the test solution is no darker than the Ammonium standard solution.
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 five cytotoxicity tests and seven physiochemical tests performed on plastic closures. Direct contact, agar diffusion, elution, systemic injection, and intracutaneous testing are the cytotoxicity tests. However, agar diffusion testing is likely the only cytotoxicity test needed for medical-grade plastics. The physiochemical tests described are appearance, color, acidity-alkalinity, absorbance, reducing substances, volatile sulfides, and ammonium testing. All in all, ensure you choose a contract manufacturing organization that can support you with appropriate toxicity testing for your unique implantable device or injectable 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>).
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>).
