Biocompatibility Testing of Medical Devices

The ISO 10993-5 standard defines in vitro cytotoxicity tests performed to determine whether medical device materials or extracts that may be released from the device cause toxic effects on cells. This test is one of the fundamental and most commonly applied steps of biocompatibility evaluation. In the tests, the L929 fibroblast cell line or similar cell lines are generally used. The material is brought into contact with the cells via direct contact, agar diffusion, or extract methods.

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At the end of the test process, cell viability, metabolic activity, or cell morphology is measured. Cell metabolism is evaluated and the percentage of viability is calculated using colorimetric and fluorimetric assays such as MTT, XTT, WST-1, and Alamar Blue. Morphological changes in cells are examined microscopically to determine cytotoxic effects such as necrosis, apoptosis, or membrane damage.

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This test is of vital importance as an initial screening test, especially for products that come into long-term contact with the body, and is mandatory in CE certification processes.

ISO 10993-3 aims to identify genotoxic or mutagenic components that may be released from medical devices. Genotoxicity analyses performed at the cellular level, such as the Ames test (bacterial reverse mutation test), in vitro micronucleus test, or chromosomal aberration tests, are recommended. When necessary, carcinogenicity and reproductive toxicity evaluations should be conducted through long-term animal studies.

Devices that come into contact with blood require evaluation in terms of hemolysis, coagulation, platelet activation, complement activation, and thrombosis risk. The interaction of the device with blood is analyzed using methodologies such as in vitro hemolysis tests (ASTM F756) and platelet adhesion and aggregation tests.

This test determines whether medical device materials cause inflammation, fibrosis, necrosis, or foreign body reactions in body tissues. Histopathological examination performed using the subcutaneous implantation method in rabbits or rats reports the severity of the reaction through a scoring system.

It determines the limit values of EtO, Ethylene Chlorohydrin (ECH), and Ethylene Glycol (EG) residuals in devices sterilized with Ethylene Oxide. Measurements are performed using analytical methods such as GC-MS (Gas Chromatography–Mass Spectrometry). Exposure calculations are conducted based on the patient’s body weight.

The sensitization test applied within the scope of the ISO 10993-10 standard aims to evaluate the potential of a medical device or material to induce delayed-type allergic reactions in the immune system. Using the most commonly applied method, the Guinea Pig Maximization Test (GPMT), the test material is administered to guinea pigs with the aid of a specific adjuvant to induce sensitization, and a possible allergic response is then observed during the challenge phase. Alternatively, the Local Lymph Node Assay (LLNA) method is used to measure lymph node cell proliferation in mice. As a result of the tests, reactions such as erythema or edema on the skin are evaluated. The sensitization test is particularly critical for products that come into prolonged contact with the skin and is an important biocompatibility parameter for determining allergic risks to human health.

It is evaluated whether the material or its extract causes systemic toxic effects when administered via intravenous, intraperitoneal, or oral routes. Acute, subacute, subchronic, and chronic toxicity tests may be performed. The evaluation is completed through clinical observation, hematology, biochemistry, and histopathology.

It evaluates the products generated by the degradation of polymeric medical devices through hydrolysis, oxidation, or enzymatic processes. Characterization is performed using advanced techniques such as GPC (Gel Permeation Chromatography), LC-MS, or GC-MS.

The dissolution rate, ion release, and structural stability of ceramic-based devices are evaluated. It is particularly important for bone grafts and dental applications. The biological effects of the released ions are also analyzed.

The potential of the device to stimulate, suppress, or trigger allergic reactions in the immune system is tested. Analyses such as cytokine measurements (IL-6, TNF-α), lymphocyte proliferation tests, and flow cytometry are applied.

Metal ions released from metal devices due to corrosion, wear, or dissolution are detected. The soluble metal content is determined using the ICP-MS (Inductively Coupled Plasma Mass Spectrometry) method. The limits of allergenic and toxic metals such as nickel, chromium, and cobalt are evaluated.

The ISO 10993-17 standard defines the principles for quantitatively evaluating the potential toxic effects on human health of chemical substances that may be released from medical devices. Within the scope of this standard, a Tolerable Intake (TI) is calculated for each chemical compound released from the device and compared with the Exposure Dose to determine the Margin of Safety (MoS). In the risk assessment, toxicological effects such as chronic, subchronic, and acute toxicity, carcinogenicity, genotoxicity, and reproductive toxicity are taken into account. In addition, a risk–benefit analysis is performed by considering dose calculations based on body weight, duration of use, and route of exposure. This process requires scientific expertise to ensure biological safety and to reduce unnecessary animal testing.

The ISO 10993-18 standard aims to provide a detailed identification of chemical compounds present in the material structure of a medical device or that may be released during production, sterilization, and use. This analysis includes the determination of extractable and leachable substances from the device. Chemical characterization is performed using advanced analytical techniques such as Gas Chromatography–Mass Spectrometry (GC-MS), Liquid Chromatography–Mass Spectrometry (LC-MS), and Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The resulting chemical profile provides data for toxicological risk assessment within the scope of ISO 10993-17. This standard is of critical importance for ensuring the material safety of medical devices and for forming the basis of biocompatibility testing.

The ISO 10993-23 standard defines biological tests performed to determine the local irritation potential of medical devices or materials and was published in 2021, separating it from ISO 10993-10. This standard focuses on evaluating acute or delayed irritative effects, depending on the duration of use, of medical devices that come into direct contact with tissues such as skin, mucosa, and eyes.

ISO 10993-23 includes in vitro, ex vivo, and in vivo test methods; however, in line with the 3R principles (Replace, Reduce, Refine), it particularly encourages the use of in vitro skin model systems and cell-based tests aimed at reducing animal use. Among the most commonly used in vitro human skin models are artificial skin systems such as EpiDerm™, SkinEthic™, and EpiSkin™, which assess irritation potential based on the ET50 (Effective Toxic Dose 50) value.

During testing, effects such as inflammatory responses, cellular damage, and disruption of tissue integrity in epithelial tissues resulting from chemical, physical, or biological interactions originating from the device or its extract are investigated. The release of pro-inflammatory cytokines such as IL-1α and IL-8 may be measured as indicators of tissue damage.

ISO 10993-23 emphasizes that irritation tests should be selected in accordance with the chemical characterization of the material and the route of exposure. Within the CE certification process of medical devices, validated tests under this standard are required, particularly for products that pose a risk of dermal, ocular, and mucosal irritation.