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Tackling ISO 10993-12 Biocompatibility

May 4, 2021
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Compliance & Regulatory: MedTech
Guidelines & Standards
Clear Up The Confusion Of Medical Device Biocompatibility Testing Requirements By Discovering The 4 Step Process Of ISO 10993-12.

In our previous post ( “Understanding Your Biocompatibility Testing Requirements Part 1 - ISO 10993-1”) we discussed how to perform a risk analysis for your device and determine the appropriate endpoints needed for market approval. Now, rather than moving on to test procedures and acceptance criteria, we are first going to jump to ISO 10993-12: Biological evaluation of medical devices—Part 12: Sample preparation and reference materials. We realize it may seem odd to discuss Part 12 as the second part of our series, but the method used for selecting and preparing your device materials for testing is critical for each biocompatibility endpoint moving forward.  

By the end of this post, you will understand how to select and prepare test samples and reference materials for your biological evaluations per ISO 10993-12, allowing you to move confidently into the biological testing phase.  To understand and navigate the complexities of ISO 10993-12, we’ve broken it down into 4 simple steps:

  1. Sample Selection
  1. Reference of Materials and Controls
  1. Sample Preparation
  1. Preparation of Extracts and Samples

If some of these terms are unfamiliar to you, that is a common response! Continue diving into this post to learn more about what ISO 10993-12 entails and how preparing your testing samples can impact your medical device’s biocompatibility endpoints.

Step 1 - Sample Selection

Biocompatibility testing of your device can be performed on the following:

  • Final product
  • Representative sample from the final product (composition and surface characteristics)
  • Materials processed in the same manner as the final product
  • Appropriate extracts of any of these

Except for select tests (e.g., implantation), ISO 10993-12 recommends testing be performed with the device in its final form whenever possible. If the final form of your device is unavailable, your choice of test sample should be justified. This may involve an additional risk assessment demonstrating how the biocompatibility is not impacted by the test sample’s differences and how your sample mirrors the device in its finished form with respect to its formulation, manufacturing processes, sterilization, geometry, etc. These elements matter because, when considering biocompatibility, you’re not just assessing the constituent materials of your device, you’re also testing whether your selected manufacturing and processing impact your device’s biocompatibility. Keep this in mind as you consider all changes moving from prototype or component materials to final product, and it’ll mitigate biocompatibility hiccups in the future.  

Additionally, if your entire device can’t be tested as one, each individual material in the final product should be proportionally represented in the test sample. This helps maximize the exposure of the test system to the components of the device that are known to have potential for a biological response. For example, test samples of surface-coated devices should include both the coating material and the substrate. Moreover, if adhesives and/or seals are used in the manufacturing process, test samples should include a representative portion of joints and/or seals that come into contact with the patient.  

Step 2 - Reference Materials and Controls

With your test sample selected, you can now determine the most appropriate reference materials (RMs) for your endpoint evaluation. RMs are established in-house by individual laboratories after undergoing extensive chemical, physical, and biological characterization to ensure their reproducibility under specified test conditions. Alternatively, certified reference materials (CRMs) are available for use. CRMs have undergone collaborative testing in three or more laboratories to characterize their critical chemical, physical, and biological characteristics under specified test conditions and can be simply purchased for your biological safety testing. Many companies select CRMs for their higher purity, critical characteristics, suitability for intended use, and general availability.

RMs and CRMs can be used as positive and/or negative control materials to demonstrate the suitability of a procedure to yield a reproducible response. Additionally, the RM/CRM is characterized/certified for each biological test for which the material is desired. This means, if a material is only characterized/certified for one reference test method or response (e.g., delayed-type hypersensitivity), then it can’t be used as an RM for another (e.g., cytotoxicity) without additional validation.  

RMs and CRMs can also be used as experimental controls. When used this way, the RM/CRM should be the same type of material (e.g., polymer, ceramic, metal, colloid, etc.) as your test sample. However, for mechanistically based test procedures (e.g., genotoxicity and immune delayed-type hypersensitivity assays), pure chemicals can be used as experimental controls. Examples of available RMs and controls can be found in Table 1 below.

Table 1: Examples of available RMs and controls

Step 3 - Sample Preparation

Now that you’ve selected samples and RMs, you will need to take precautions to avoid contamination during sample and RM preparation, even if your device is not sterile when used. This includes handling samples aseptically and cleaning cutting tools after each use when test samples need to be sliced into pieces. Additionally, during sample preparation, sterile-use devices that are supplied non-sterile and require sterilization prior to use or cleaning prior to sterilization should follow manufacturer sterilization and cleaning recommendations, respectively.  

Step 4 - Preparations of Extracts of Samples

Sometimes your device and/or test procedure will require that an extract of your device be used to evaluate biological endpoints. This method of sample preparation is especially important to understand because most biocompatibility problems arise from toxins leaching out of the device into surrounding tissues.  

Containers: Extraction should be performed in clean, chemically inert, closed containers with minimum dead space as to avoid contamination. Commonly used are borosilicate glass tubes with caps having an inert liner, like PTFE. In determining the appropriate extraction container, conditions, and methods for the nature and use of your final product, consider the properties of your device’s material, leachables, and residues.

Conditions: Based on historical, standardized practice, extraction should be performed under one of the following conditions:

  • (37 ± 1) °C for (72 ± 2) h;  
  • (50 ± 2) °C for (72 ± 2) h;  
  • (70 ± 2) °C for (24 ± 2) h;  
  • (121 ± 2) °C for (1 ± 0.1) h.

The goal is to mimic the conditions that the device or material will undergo during sterilization and/or clinical use. Understanding the properties of your material is important, as you will need to understand the temperatures at which the material may melt or fuse and avoid exposing the product to temperatures in that range.  Extraction for cytotoxicity testing (ISO 10993-5) is typically accepted at (37 ± 1) °C for (24 ± 2) h in tissue culture media as to not alter the chemistry and stability of the media. Additionally, for medical devices with short-term skin or mucosa contact, extraction times of less than 24 h, but not less than 4 h, are typically acceptable.  

Methods: The standard surface area can be used to determine the volume of extraction vehicle needed. When the surface area of your device cannot be determined, a mass/volume of extraction fluid must be used.  (See Table 2 for more details.) The device material is often cut into small pieces to enhance extraction; however, elastomers, coated materials, composites, laminates, etc. should not be sliced for extraction because there may be differences in extraction characteristics between the intact and cut surfaces.  

Table 2: Standard surface areas and extract liquid volumes

Extraction is conducted with both polar and non-polar extraction vehicles under agitation to stimulate the release of water-soluble and lipid-soluble chemicals, respectively. During extraction, physiological saline is often used as the polar vehicle and vegetable oil is used as the non-polar vehicle. Once the liquid extractions vehicles are prepared, they should be used immediately to prevent sorption to the container and changes in chemical composition. Also, extraction vehicles should be used without adjusting their pH and without routine filtration and/or processing to remove particulate unless rationale is otherwise documented.

Below are examples of polar and non-polar extraction vehicles:

  • Polar vehicles: water, physiological saline, culture media without serum
  • Non-polar vehicles: freshly refined vegetable oil of pharmacopoeias quality
  • Additional vehicles: ethanol/water, ethanol/saline, polyethylene glycol 400 (diluted to a physiological osmotic pressure), dimethyl-sulfoxide, and culture media with serum (cytotoxicity testing)

In ISO 10993-12, special attention is paid to various types of polymer products/materials and how to appropriately perform their extraction. If your product is designed to polymerize in situ, test samples are obtained to represent the intended clinical conditions to fully capture the potential toxicity of the reacting polymer components. This means your test extracts should be based on the kinetics of polymerization after mixing the components and the expected cure time. Additionally, if your device is composed of a polymeric material not expected to dissolve or resorb, then your test sample should experience no more than slight softening (<10% dissolution) in your selected extraction vehicle. This could induce extractables and leachables that would not be experienced physiologically and skew your biocompatibility results. Conversely, if your material is soluble, consider the following:

  • Factors such as test system compatibility, route of administration, and extent of dissolution
  • If the material dissolves completely, the resulting solution can be evaluated
  • If the material is an aqueous solution and used in this form, it can be tested directly and not extracted
  • OECD Guidelines for the Testing of Chemicals can be used to determine the maximum concentrations of test substances for specific test methods  

Now that you know how to select and prepare your test samples and RMs, you’re ready to embark on biocompatibility testing! Look out for the next installment of this blog series as we explore ISO 10993-3: Biological evaluation of medical devices—Part 3: Tests for genotoxicity, carcinogenicity, and reproductive toxicity.