Now that you have a Biological Evaluation Plan, what biological testing methods should be employed to demonstrate biocompatibility?
Biocompatibility and the necessary biological endpoints for consideration are dependent on the type of contact, the duration of contact, and the nature of contact a medical device has with the patient as outlined in ISO 10993-1:2018 Annex A. Subsequent to that, an understanding of the materials of construction and the manufacturing process and processing agents utilized can help you strategize the test methods, extraction vehicles, extraction conditions, and evaluation methods needed.
Biological evaluation methods should be aligned with core parts of the international standard ISO 10993-1. These ISO standards are internationally recognized as state of the art. Each part references the specific in vitro or in vivo test methods to be followed or references specific harmonized protocols, such as OECD (Organization for Economic Cooperation and Development), USP (US Pharmacopeia) and ASTM (American Society for Testing and Materials) standards for evaluating the biological endpoints of interest.
Depending on the intended region for submission, additional documents should be taken into consideration to account for any regulatory differences or expectations. These may include:
- Guobiao/Tuijian (GB/T 16886.1) for China, the Ministry of Health
- Labour and Welfare (MHLW)/Pharmaceutical Safety and Environmental Health Bureau (PSB)/Medical Device Evaluation Division (MDED) Notification No. 0311-1 for Japan
- Medical Device Regulation (MDR) for the European Union
- FDA Biocompatibility Guidance
The following table describes the specific biological tests associated with each relevant part of ISO 10993-1 Annex A, and the 2023 FDA Biocompatibility Guidance document, Table A.1:
| ISO 10993 | Biological Endpoint |
|---|---|
| Part 2 | Animal welfare requirements |
| Part 3 | Tests for genotoxicity, carcinogenicity and reproductive toxicity |
| Part 4 | Selection of tests for interactions with blood |
| Part 5 | Tests for in vitro cytotoxicity |
| Part 6 | Tests for local effects after implantation |
| Part 9 | Framework for identification and quantification of potential degradation products |
| Part 10 | Tests for skin sensitization |
| Part 11 | Tests for systemic toxicity |
| Part 12 | Sample preparation and reference materials |
| Part 13 | Identification and quantification of degradation products from polymeric medical devices |
| Part 14 | Identification and quantification of degradation products from ceramics |
| Part 15 | Identification and quantification of degradation products from metals and alloys |
| Part 16 | Toxicokinetic study design for degradation products and leachables |
| Part 17 | Toxicological risk assessment of medical device constituents |
| Part 18 | Chemical characterization of medical device materials within a risk management process |
| Part 19 | Physico-chemical, morphological and topographical characterization of materials |
| Part 20 | Principles and methods for immunotoxicology testing of medical devices |
| Part 22 | Guidance on nanomaterials |
| Part 23 | Tests for irritation |
Biological Testing Methods
The following table outlines the purpose of each biological testing method and the specific test methods employed as grouped by each ISO standard.
| ISO 10993 | Endpoint | Purpose | Biological Testing Methods | |
|---|---|---|---|---|
| Part 2 | Animal welfare requirements | Intended to provide provisions for humane animal care and use; to reduce overall numbers; to refine test methods; and to replace animal tests when able. | Requires justification of animal use, species and numbers used; requires trained personnel, approved protocols by an institutional animal care and use committee (IACUC), appropriate husbandry, humane endpoints, documentation of results following Good Laboratory Practices (GLP). | |
| Part 3 | Genotoxicity, carcinogenicity, reproductive toxicity | Intended to determine DNA mutations or chromosomal damage. Carcinogenicity: Genotoxic and non-genotoxic ability to affect tumorigenic potential. Reproductive/developmental toxicity: To assess impact on fertility and offspring. | No single test is capable of detecting all relevant genotoxic agents. Therefore, two tests must be considered to detect the 2 major classes of genetic damage using in-vitro bacterial cells and mammalian cells. 1. Bacterial Reverse Mutation Assay, OECD 471 AND 2. Mouse Lymphoma Assay(MLA, OECD 490) OR 3. Chromosomal Aberration Assay (OECD 473) OR 4. Mammalian cell micronucleus test (OECD 487) Because nanomaterials aren’t compatible with genotoxic bacterial assays, the recommendation is to complete the 2 mammalian assays. If results are equivocal, then it is considered non-mutagenic, as the in-vivo assay cannot meet the dosimetry limits even if concentrated for medical devices and would not be informative in any way. | |
| Part 4 | Hemocompatibility | These tests are intended to evaluate the interactions between blood and blood components with the device. Only direct or indirect blood contacting components should be tested. Per the 2023 FDA Biocompatibility Guidance Document, “For devices having indirect contact with circulating blood (regardless of contact duration), we recommend that you consider only hemolysis testing as complement activation and in vivo thrombogenicity testing are generally not needed for indirect blood-contacting devices.” | Hemolysis (in-vitro): 1. Material-induced (ASTM F756-17): Testing by direct and indirect (extract) contact with blood should be considered. 2. Mechanically-induced (ASTM 1841): Determines the effects of devices with mechanical operation and/or complicated flow paths to cause red blood cell membrane rupture. Thrombosis: 1. Partial Thromboplastin Time (ASTM F2382-18): An in vitro assayevaluating the potential to cause an effect on the coagulation cascade via the intrinsic coagulation pathway. 2. Platelet and Leukocyte Count Assay (ASTM F2888-19): Intended to evaluate the potential of leachables/extractables to affect platelets and leukocytes and therefore provide an estimate of the surface’s thrombogenic potential. 3. Complement Activation Assay (SC5b-9): An in vitrotest intended to evaluate the potential of the device to activate the complement system. SC5b-9 is generally considered the optimal test. 4. In-vivo Thrombogenicity: Intended to evaluate thrombogenic properties following intravascular placement in the test system. | |
| Part 5 | Cytotoxicity | Determines the in-vitro biological response of established mammalian cells. Qualitative – morphology, vacuolization, detachment, lysis, membrane integrity, reactivity; Quantitative – cell death, inhibition of cell growth, cell proliferation or colony formation | 1. Direct Contact: Ideal for devices/materials that contact tissue directly. 2. Device Extracts: Measures leachable substances that diffuse from the device (e.g. Elution, MTT, XTT, Neutra Red Uptake). 3. Indirect Contact: Measures leachable substances through a barrier (e.g. Agar diffusion or Filter diffusion). Preferred culture medium should contain serum, to support cellular growth and extracts using both polar and non-polar substances. | |
| Part 6 | Implantation | Determines the local and systemic effect following implantation of the test article. | 1. Implant into the most clinically relevant location (subcutaneous, intramuscular, bone, brain). 2. If non-absorbable, duration of testing should be based off intended clinical use/exposure time. 3. If absorbable, duration of testing should include early, mid, and long-term tissue response and amount of material remaining/tissue reactivity at each time point; long term duration should be beyond the expected point of full absorption. 4. Comparison of tissue response to control. 5. Evaluation of macroscopic and microscopic effects. | |
| Part 9 | Degradation Products | Determines an in vitro or in vivo strategy to quantify potential substances or by-products for resorbable or degradable devices and evaluate the biological hazard from these degradation products. | If there is insufficient degradation data of the material, then refer to Part 13, 14 or 15. | |
| Part 13 | Polymers | Two methods to generate degradation products from polymers: – Accelerated (screening method) – Real-time (simulated environment) | 1. Test solution should be as similar to intended clinical use environment as possible. 2. Duration of testing is selected based on intended clinical use. 3. Accelerated degradation test or real-time in simulated environment. 4. Evaluates change in mass balance and a change in molecular mass distribution. | |
| Part 14 | Ceramics | Two methods to quantify degradation products from ceramics (including glasses). | 1. In-vitro testing by chemical dissociationin an aqueous environment. 2. Test in extreme solution at low pH or simulation of frequently encountered in-vivo pH. | |
| Part 15 | Metals/alloys | Determining degradation products generated by chemical alteration. | 1. Electrochemical tests 2. Immersion tests Both tests report any significant changes to the surface. | |
| Part 10 | Sensitization | Determines the potential of a medical device to cause an allergic reaction (Type IV hypersensitivity reaction). | 1. Guinea Pig Maximization Test (GPMT): Intradermal and topical exposure of extracts. 2. Buehler Test: Topical exposure of extracts or device materials in guinea pigs. 3. Murine Local Lymph Node Assay (LLNA): Quantifies lymphocyte proliferation following topical exposure of test extracts. In vitro sensitization testing has only been validated for neat chemicals and not yet for medical devices. | |
| Part 11 | Systemic Toxicity | Determines the potential of a device or material to cause systemic toxicity over a specified duration of time: Acute: Within 72 hours after single, multiple or continuous exposures to the test article extract for 24 hours. Subacute: Repeated or continuous administration of the test article or extract between 24 hours and 28 days. Subchronic: Repeated or continuous administration of the test article or extract (typically 90 days). Chronic: Repeated or continuous administration of test article or extract for 6-12 months. | 1. The route of administration chosen should be most clinically relevant. 2. Negative, vehicle or sham-treated controls should be employed. 3. Acute test – single dose 4. Other test durations, e.g. repeat dose daily for duration of testing. 5. Conduct serial evaluation of body weights, clinical observations, clinical chemistry; plus gross pathology, histopathology of organ systems. | |
| Part 12 | Sample preparation and reference materials | Provides guidance on test article selection, test article preparation, experimental controls, reference materials, preparation of extracts, and extraction conditions and methods based on device type, intended use, and duration of exposure, applicable to all biological testing conducted. | N/A | |
| Part 16 | Toxicokinetics | To evaluate the absorption, distribution, metabolism and excretion of substances of time in the body. | Consideration of study and design should be conducted on a case-by-case basis. Consider if chemical characterization data identifies leachables or degradation products at a level that has the potential of being toxic; if the device is a permanent implant; or if the device undergoes bioresorption. Toxicokinetics may be covered with appropriately designed implantation studies to determine amount of material remaining at specific time points concurrently with scoring of reactivity of cells on histopathology to determine steady state, and histopathology on additional organs for systemic effect. | |
| Part 17 | Toxicological Risk Assessment of chemical characterization data | Provides guidance on establishing exposure limits to chemicals to evaluate toxicological risk to specific patient populations for the device. | 1. Use the dose-based threshold for the device for calculation of the Analytical Evaluation Threshold (AET). 2. Determine maximum patient exposure of a detected extractable. 3. Determine a tolerable intake level of a constituent over a specified time period. 4. Determine a worst-case estimated exposure dose for each constituent. 5. Comparison to toxicological screening limits or calculated margin of safety for chemicals to which a human may be exposed. 6. Determine threshold of toxicological concern for cancer and non-cancer effects. 7. Conduct a toxicological evaluation of results. | |
| Part 18 | Chemical Characterization | The device is subjected to a variety of tests to evaluate the amount, identity, and characteristics of the extractables. | Common methods include a range of orthogonal tests following simulated, exaggerated, or exhaustive extractions in a polar, semi-polar, and non-polar vehicle. These include: – Total Non-Volatile Residue (NVR) – Fourier Transform Infrared (FTIR) spectroscopy of residues obtained from the test article extracts and observed particulates. – Gas Chromatography with Mass Spectrometry (GC-MS) for detection of Semi-Volatile Organic Compounds (SVOC) from the test article extracts. – Ultra Performance Liquid Chromatography – Ultraviolet Spectrometry – Mass Spectrometry (UPLC‑UV-MS) for detection of Non-Volatile Organic Compounds (NVOC) from the test article extracts. The expectation is that this is performed in both positive (+) and negative (-) ionization modes. – Headspace GC-MS (HS-GC-MS) analysis for detection of Volatile Organic Compounds (VOC) from the polar extract. – Determination of trace metals using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) from the polar extract. – Ion Chromatography (IC) from the polar extract. | |
| Part 20 | Immunotoxicology | To study the adverse health effects that result, directly or indirectly, from the interaction of xenobiotics with the immune system. | Evaluation of materials, processing agents, results of genotoxicity testing, chemical characterization data and the toxicological evaluation of results, GPMT, material mediated pyrogenicity testing, and evaluation of the immune system/tissues/clinical pathology from results of implantation studies. An evaluation is indicated only where circumstances suggestive of possible immunotoxicity apply. | |
| Part 22 | Nanomaterials | Evaluation of medical devices that contain, generate or are composed of nanomaterials (dimensions between 1 nm and 100 nm), as these can elicit different biological effects than constituents >1 micron. | Evaluate physico-chemical properties, morphological features, biomolecular adsorption, device design, intended clinical use, route of exposure, and dose. | |
| Part 23 | Irritation | This in vivo test is intended to evaluate the dermal irritation potential of a device after a short-term exposure or repeated-dose exposure. | In vivo topical for powder, liquid, or solid test articles with semi-occlusive or occlusive bandages. In vivo intracutaneous (intradermal) typically employed for medical devices contacting breached or compromised surface, externally communicating, or implants. Single exposure and repeated exposure strategies based on the most appropriate use of the device. Repeated exposure should be for those devices with repeated use and cumulative long-term duration. NOTE: The in vitro reconstructed human epidermis model is not yet recognized by the FDA. | |
8 Additional Considerations for the Evaluation of Biological Testing Methods
- Test the final finished device – test only direct or indirect patient contacting components; remove non-contacting components.
- Testing must be conducted according to 21 CFR Part 58 Good Laboratory Practice (GLP).
- Coupons of the final finished test article(s) may be used if they have undergone identical manufacturing process and materials and are provided in the same ratio as the final finished device. Per ISO 10993-1:2018, “testing shall be performed on the final medical device, or representative samples from the medical device or materials processed in the same manner as the final medical device (including sterilization, if needed).”
- Extraction conditions in duration and/or temperature for any test article should attempt to simulate or exaggerate the clinical use conditions, without destruction or degradation of the device, to determine a worst-case scenario of potential toxicological hazards. Extraction vehicles should be chosen for their ability to extract both polar and non-polar substances.
- Understand the materials of construction: For example, for polymeric test samples, the extraction temperature should not exceed the glass transition temperature as it may adversely impact test article composition; for biologically-derived devices, an understanding of their decomposition temperature should be taken into consideration.
- Positive and negative controls should be employed for all tests (or historical controls for positive reference materials).
- Vertical standards will provide additional technical considerations for the non-clinical assessment of specific medical device types and will be expected to be known, understood and applied as appropriate. Examples of additional vertical standards include those designated for evaluation of dental devices (ISO 7405), neurological devices that contact the cerebral spinal fluid or nervous tissue (ASTM F2901-19), ophthalmic implants (ISO 11979), breathing gas pathways (ISO 18562), or absorbable devices (ISO/TS 37137) to name a few. These standards may include alternative test methods or additional biological tests to include in the overall risk assessment.
- Additional testing requirements may be incorporated with reprocessed devices.
Additional Considerations for the Evaluation of Results
For any unexpected or unusual findings, a root cause analysis should be performed. Some examples include the following:
Visual appearance of extracts
- If particulates are observed in any of the extracts, these should be further identified to ensure they are not due a manufacturing issue or inappropriate extraction conditions; the test method most commonly employed is Fourier Transform Infrared Spectroscopy (FTIR).
Colorants
- If colorants are part of direct or indirect patient contacting components of the medical device, identification of the chemical should be disclosed; if the device has brief tissue contact or limited contact, disclosure is not required if extracts from cytotoxicity, sensitization, and irritation testing are colorless, clear and without particulates.
Unfavorable results
- Although unfavorable results may be of toxicological concern, the relevance of the results, potential root causes, and overall risk-based evaluation of the results should be performed. In evaluating the biocompatibility of a device, biological safety is determined based on an evaluation of all the available data (overall weight of the evidence) rather than the findings for any individual effect.
Frequently Asked Questions (FAQ)
Are you required to conduct all recommended testing for your medical device based on the nature of contact and contact duration?
No. Certain tests may be able to be waived or rationalized based on scientific justification, sufficient data from the literature, clinical data, results from prior biological testing, or a combination of these. For example, if a device only has contact with intact skin, the 2023 FDA Guidance Document provides a least burdensome approach whereby if the patient contacting materials (including processing chemicals and additives) are included in this list. They are considered to “pose a very low biocompatibility risk because they have a long history of safe use in legally marketed medical devices that contact skin.” In these cases, testing is not warranted.
In another example, if chemical characterization has been conducted, along with a toxicological evaluation of results, this may allow for waiving of acute, subacute, subchronic, and chronic systemic toxicity testing, as well as genotoxicity, and reproductive/developmental toxicity testing.
What are some common deficiencies seen in the biological testing methods employed for certain medical devices?
Common feedback from the FDA or regulatory bodies may include a lack of consideration of specific vertical standards, a lack of identification of extract particulates, incomplete discussion on the relevance of unfavorable test results, an incomplete identification of colorants used in direct or indirect patient contacting components, or submission of older testing without a gap analysis of the methods as compared to current standards. A thorough assessment of all methods and results is necessary to determine the overall risk.
What does it mean if you have an unfavorable result?
Just because there may be an unfavorable biological test result does not mean that the device is not considered biologically safe. It is important to determine potential root causes and discuss these in the assessment. For example, the observed effects may be attributed to the test method, which may interact with device materials in ways not clinically intended; or test conditions may be overchallenging and not considered clinically relevant. The purpose of the risk assessment is to evaluate the overall weight of evidence before forming a conclusion. A risk/benefit analysis can also be incorporated if the patient population has no other treatment options.
