To exclude hazards to users and patients, manufacturers must test their medical devices for biocompatibility. Biocompatibility tests are required in regulatory acts such as the Medical Device Regulation (MDR, Regulation (EU) No. 745/2017) which replaced the Medical Devices Directives 93/42 EEC and 90/385/EEC in May 2021. To meet this requirement, manufacturers and test laboratories have sometimes had to resort to in-vivo test methods.
According to the German Federal Institute for Risk Assessment manufacturing and quality control of medical devices accounted for 19% of the animal tests performed in Germany in 2020. However, in accordance with some parts of the ISO 10993 series and some national animal welfare acts, animal testing shall only be permitted if there is no reliable alternative test method.
New: Validated In-Vitro Test Methods
Up to late 2020, the ISO 10993 series of standards (“Biological evaluation of medical devices”) comprised a total of 21 parts. In January 2021 two new parts were added to the series, including ISO 10993-23 (“Tests for irritation”). This new part evolved out of ISO 10993-10, which has been exclusively applicable to tests for skin sensitization since late 2021.
As the most important change, the ISO 10993-23 sets out the first definition of in-vitro test methods that present viable alternatives to animal experiments in many cases. While in-vitro tests in Petri dishes had previously been known, they had never been adequately validated. In the past, extracts from medical devices often had to be tested by subcutaneous injection of rabbits. However, tests for irritation can now be performed on reconstructed human epidermis (RHE).
Advantages and Limits of RHE Models
RHE is grown from human keratinocytes. It is commercially available from various suppliers at different degrees of differentiation. Grown from cultured keratinocytes, reconstructed human epidermis (RHE) is fully formed, essentially viable and mimics the cell morphology of native human skin, with dorsal, basal, and granular layers. Together with its uppermost layer, the stratum corneum, the tissue protects the organism against rapid penetration by toxic substances.
To test for the potential to produce irritation, the test extract and MTT dye—a yellow tetrazolium salt—are applied to the RHE model and incubated for a certain time. The percentage of living cells is then determined by means of an MTT assay. Determination relies on the reduction of the yellow dye in the mitochondria to the bluish-purple colored formazan. The change in color is quantified by means of UV/VIS spectroscopy and converted into a value for cell viability. If this value falls under a defined threshold, generally 50 per cent, the tested substance is classed as a skin irritant. However, the in-vitro method only works for substances that can be extracted with polar or non-polar solvents. It has not been validated for insoluble solids.
In-vitro testing has benefits that extend beyond animal welfare. As cell reactions in the RHE model are faster than in the cutaneous rabbit model, in-vitro testing takes less time – good for both manufacturers and test laboratories. In addition, in-vitro tests do not require registration or approval and incur no costs or efforts for animal procurement, housing, or care.
Four Steps of Risk Analysis
Step 1 precedes the selection of the test methods and involves identification and characterisation of all substances used in the medical device in accordance with ISO 10993-18. This also includes any process aids adhering to the product and any additives used in manufacturing.
The 2020 recast of the standard calls for the definition and application of an analytical evaluation threshold (AET). In the EU, compliance with this requirement is verified by the notified bodies. The AET indicates the value above which a toxicological risk assessment must be performed. AET identification for all substances frequently involves major efforts. To speed up the approval process, manufacturers are recommended to call in a third-party testing laboratory.
Step 2 of the process is dedicated to a literature review of the chemical and physical properties of the substances identified in step 1 and their potential to produce irritation. The procedure for performance and documentation of this research is set forth in ISO 10993-17. Databases and the results of past examinations can also be used in the assessment. Specialized databases containing computer-aided data can prove helpful for substances for which no valid information on risk assessment is available.
Step 3 then clarifies whether an in-vitro method is suitable for determining the potential for irritation of these remaining substances. Only where this is impossible are in-vivo tests then considered in step 4.
Conclusion and Outlook
RHE models are now validated for use in the hazard identification of chemicals by the OECD. After publication of the ISO 10993-23 standard, these models can now also be applied in testing medical devices for their potential to produce irritation. By using the models, manufacturers and test laboratories can already replace many animal tests by in-vitro tests today.
This does not apply to sensitization tests in accordance with ISO 10993-10, however. A recast of this standard is expected in a few years at the earliest. Yet, third-party test laboratories like TÜV SÜD can support manufacturers by helping them to fulfill all regulatory requirements with the lowest possible amount of animal testing and to minimize delays in approval due to missing data.