Biocompatibility assessment of MD (ISO/TR 10993-55:2023)
Assessing the biocompatibility of medical devices is of major importance as it ensures the biological safety of products with regards to the patients and/or the users. It is mainly governed according to the standards of ISO 10993 series “Biological evaluation of medical devices”.
ISO 10993-1 presents the biological endpoints considered relevant for the examination of each category of medical device. Cytotoxicity assessment is recommended for all types of medical device (Class I to Class III), and for all types of contact with the patient and/or user.
ISO 10993-5 sets out test methods for cytotoxicity in vitro models. These tests are carried out on mammalian cell line cultures, to identify whether or not a medical device has cytotoxic potential.
From an animal ethics perspective, the in vitro cytotoxicity test allows an initial estimation of the toxicity of a medical device in compliance with the requirements of ISO 10993-2 (Biological evaluation of medical devices – Part 2: Animal welfare requirements). It is therefore perfectly in line with the 3Rs strategy (Replace, Reduce, Refine) aiming to replace and reduce animal experiments whenever possible.
To enable the design of in vitro cytotoxicity tests, and to be freely adapted to the clinical application scenario and the type of medical device, testing specifications to be performed are not well-defined in ISO 10993-5 standard. In fact, since the first edition of the standard in 1992, ISO 10993-5 only provides little detail regarding how to proceed, as well as for interpreting the results. This has the effect of covering the diversity of medical devices, but also leading to a certain variability in results and a potential lack of reliability.
Interlaboratory variability according to ISO/TR 10993-55
ISO/TR 10993-55 “Interlaboratory study on cytotoxicity”, published in February 2023, describes the results of an international study conducted by twelve laboratories in 2006. The aim of the study was to evaluate the performance of two protocols for assessing the cytotoxicity of medical devices: the NRU (Neutral Red Uptake) assay and the CF (Colony Forming) assay.
Despite variations in the results obtained between different laboratories, the findings of this study have validated the two protocols, and therefore their integration into the 2009 version of ISO 10993-5.
The threshold value for cell viability reduction of 30%, introduced in the 2009 version of ISO 10993-5, was also determined in this study. A reference material (material C: high-density polyethylene sheet), recognized for its non-cytotoxicity, was extracted, and a range of dilutions tested (0, 25, 50, 75 and 100%). After incubation with the cells, the cytotoxic potential of the samples was assessed using the colony-forming assay. This same procedure was carried out by ten laboratories participating in the study. The material proved to be non-cytotoxic for all the laboratories.
For one laboratory, the results showed a difference in cell viability (plating efficiency = PE) close to 30% between the 0% concentration (PE = 77.8%) and the 100% concentration (PE = 100%). As the material tested is not cytotoxic, the results should theoretically have been identical for the entire concentration range tested. This variation therefore highlighted a certain inherent uncertainty in the test itself, unrelated to the tested material.
A threshold value was introduced in the ISO 10993-5 standard of 2009: only a reduction of more than 30% in the measurement value is considered as a cytotoxic effect. A reduction of less than 30% is therefore considered to be due to method uncertainty.
This interlaboratory study, including an evaluation of the samples using the neutral red method, also revealed variations in the results obtained between different laboratories performing the same test protocol. These variations were shown to be dependent on the cytotoxicity grade of the materials tested. For example, reference material B (polyurethane film containing 0.25% zinc dibutyldithiocarbamate, a cytotoxic material) showed a greater variation in results between the different participating laboratories than reference material A (segmented polyurethane film containing 0.1% zinc dibutyldithiocarbamate, a cytotoxic material), as indicated by the results presented below. The IC50 (%) corresponds to the extract concentration inducing a 50% reduction in cell viability.
|Average IC50 (%)|
|Test protocol||Reference material-A||Reference material-B|
|Neutral Red Uptake (NRU) assay||17.0 ± 5.5||59.9 ± 24.4|
|Colony Forming (CF) assay||0.87 ± 0.42||52.2 ± 16.5|
In this way, for all participants, reference materials A and B were significantly cytotoxic. However, this cytotoxic effect was weaker for reference material B: the IC50 values obtained for reference material A are lower than those of reference material B. The results obtained for reference material B, which is less cytotoxic, show a much greater interlaboratory variability.
This study shows that the laboratories have correctly and consistently identified the various reference materials given the inherent variability of the tests. However, there is some variability in results depending on the material tested, which could lead to false-negative or false-positive results in certain borderline cases.
Variability between different extraction/incubation conditions.
Another interlaboratory study was published in 2023 by Angela Nickel and Sarah Gruber from the Johner Institute in Germany. This study was based on the results of 52 laboratories to whom the same samples were provided for cytotoxicity assessment. The choice of test method remained unrestrained in order to assess the latitude given to laboratories by ISO 10993-5. This also enabled to identify the parameters most likely to vary the results of a cytotoxicity test.
One of the key findings of this study is that only 58% of the participating laboratories were able to correctly identify the cytotoxic potential of all tested materials. This clearly demonstrates that the choice of test method and parameters according to the materials are essential elements to obtain consistent results.
The results also indicate that two parameters are particularly critical in performing cytotoxicity tests: the percentage of serum supplemented to the culture medium, and the contact time between the cells and the extract tested (known as the incubation period):
- Serum is an essential component for cell culture. It is a blood derivative that is rich in growth factors. Typically, culture medium supplementation with serum ranges from 0 to 20% and must be adapted based on the cell type and culture conditions. Nickel & Gruber’s study showed that only 16% of tests performed with 10% fetal calf serum (FCS) supplementation (a percentage frequently used in cell culture) led to misidentification of the cytotoxic sample. In contrast, all of the tests performed with a different or unknown serum concentration failed.
- Regardless of other parameters and the test type, the results showed that longer incubation time result in greater test sensitivity. The standard recommends a minimum incubation time of 24 hours; however, only 44% of laboratories that followed this duration were able to identify the cytotoxic sample. On the other hand, longer incubation period clearly allowed a better identification of the cytotoxic potential of the materials.
To minimize this variability, it is essential for the laboratory to apply quality control measures, such as regular calibration of equipment, use of standardized protocols and periodic validation of test results.
Consequently, many factors could be responsible for the variability described above:
- Cell culture environment (incubation temperature, humidity, percentage of CO2),
- Quality of culture media,
- Quality of serum batches used,
- Inter-operator variability.
In order to limit variability, it is strongly recommended to:
- Identify the specificities of the medical device being studied that may influence the results of a cytotoxicity test,
- Define the test conditions precisely before starting the test. Laboratories offer standard protocols that can be adapted and customized to the type of medical device and associated risks.
EFOR group provides expertise in validating the biocompatibility of medical devices in accordance with ISO 10993 through a team of biocompatibility experts and toxicology specialists. We support the entire medical device biological evaluation project, from strategy definition to biological and chemical test analysis.
We support you in setting up cytotoxicity test protocols, in collaboration with laboratories and according to the specific features of the medical device concerned. For example, particles generated by coated implants, or plasticizers used in PVC, need to be considered when launching a cytotoxicity test. Our experts can advise you on the selection of testing laboratories. Our team is also trained in the selection of test samples, test follow-up and the management of any non-conformities linked to cytotoxicity tests, often requiring detailed analysis to identify their origin.
Our technical management teams are available to assist you in your projects and can be contacted at email@example.com.
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