Many of the gold standard methods for assessing medical device biocompatibility have traditionally been based on using live animals as test subjects. This has understandably raised concerns about animal welfare, but also about the representativeness of small animals – usually rodents – as human models.
In recent years, regulators and standardization bodies have increasingly started working towards phasing out animal testing where appropriate without compromising patient safety. Both the US Food and Drug Administration (FDA) and the European Union have expressed support for the “3Rs” principle to replace, reduce, and refine animal testing where feasible. In addition, the International Standardization Organization (ISO) has included information on non-animal methods in the most recent versions of standards that previously featured animal methods only.1
This article provides an overview of recent developments in alternative test methods, such as in vitro assays, that may be used to address specific biocompatibility endpoints. Examples of situations where animal testing may be minimized or avoided entirely when bringing new medical devices to the market are also discussed.
What are the alternatives?
Potential alternatives to animal testing (also known as in vivo assays) in medical device biocompatibility assessments include the following:
In chemico tests, which are used to study the reactions between chemicals found in medical devices and biological molecules.
In vitro assays, which are performed on isolated cells or tissues outside of the living organism to assess the effects of medical device materials on the viability of biological systems.
In silico assays, which use computational modeling to predict the reactions medical devices are likely to cause when in contact with human tissues.
In chemico and in vitro methods are already widely used in medical device testing to evaluate certain biocompatibility endpoints, such as chemical safety and cytotoxicity. Together with in silico methods and existing information on the assessed material, they may also be used to address endpoints more often covered by in vivo tests, such as skin sensitization.2
ISO 10993 standards & alternatives to animal testing
ISO 10993 standards are used in numerous jurisdictions, including the US and the EU, to evaluate the biocompatibility of medical devices. All new devices regardless of the type and duration of contact with the body usually require the following ISO 10993 biocompatibility tests:
There are several testing options for addressing each of these biocompatibility endpoints. The evaluation should proceed in a step-wise manner, beginning with physical and chemical characterization of the device, continuing with in vitro tests, and only including in vivo tests when strictly required.3
Chemical characterization and cytotoxicity – no animal testing needed
Due to the step-wise approach, biocompatibility testing generally commences with chemical characterization and continues with cytotoxicity testing, both of which are based on non-animal methods.
Chemical characterization is performed using standard chemical analysis methods, such as GC-MS, LC-MS, and ICP-MS, depending on the target chemicals. Cytotoxicity is assessed using a variety of in vitro methods, such as the neutral red uptake method (NRU) and the minimum essential medium (MEM) elution, both of which are based on exposing a cell culture to medical device material extracts.
Skin sensitization – in vitro methods available
Among the most commonly required biocompatibility tests, ISO 10993-10 for assessing skin sensitization is the one that most often requires some animal testing. In the most recent version of the standard from 2023, the fully validated testing options include the murine local lymph node assay, the guinea pig maximization test, and the Buehler test, all of which are performed on live animals.
Non-animal alternative methods are described and discussed in Annex C of the standard. These include the DPRA (direct peptide reactivity assay) by OECD test guideline (TG) 442C and the KeratinoSens™ assay by OECD TG 442D. While a total of six OECD assays have been validated for identifying the skin-sensitization potential of chemicals, they may not be directly applicable to medical device materials, which is why ISO does not list them on an equal footing with in vivo methods.4
Based on our experience, a combination of in vitro skin sensitization test results and supporting information may be accepted for regulatory submission in Europe for low-risk devices. It may also be possible to waive skin sensitization tests for FDA approval in specific circumstances. This requires that the medical device only comes into contact with intact skin and is composed of certain synthetic polymers or natural fabrics.5
Irritation – in vitro often sufficient for low-risk devices
The most recent version of the ISO 10993-23 standard contains in vitro testing options performed on a reconstructed human epidermis (RhE) model. When devices come into contact with intact skin only, in vitro test results are generally enough for submission to EU officials. Contact with other tissues tends to necessitate animal testing.
The FDA does not recognize the clauses of ISO 10993-23 that describe in vitro tests.6 Still, it may be possible to waive animal tests for certain polymers and fabrics in a similar fashion to skin sensitization tests.
Non-animal methods for evaluating further biocompatibility endpoints
More extensive testing is required when medical devices are used in contact with tissues other than intact skin. The following are some examples of in vitro tests for addressing further biocompatibility endpoints:
Monocyte activation test (MAT) for material-mediated pyrogenicity testing
Hemolysis and complement, platelet, and coagulation activation tests for assessing hemocompatibility
Bacterial reverse mutation tests for genotoxicity testing
For some endpoints, no in vitro tests are currently mentioned in the corresponding ISO 10993 standards. These include implantation effects and acute, subacute, subchronic, and chronic systemic toxicity.
Steps to minimize animal testing
As animal methods are still the default for addressing several biocompatibility endpoints, manufacturers wishing to avoid animal testing will have to justify waiving these tests or using alternative methods. The key to this is producing a comprehensive set of information on the medical device, the materials it consists of, and its physical and mechanical properties. A detailed Biological Evaluation Plan (BEP) is also needed to describe the biocompatibility assessment procedure and to provide the reasoning for waiving animal tests.
You can order the BEP as well as a wide range of medical device tests from Measurlabs. Do not hesitate to reach out to our experts through the form below to ask for more information and to request a quote.
References
1 See footnote 22 on page 9 of FDA’s 2023 guidance document, Use of International Standard ISO 10993-1, "Biological evaluation of medical devices - Part 1: Evaluation and testing within a risk management process" and the Animals in science section on the European Commission website for their stances on the 3Rs principle.
2 Integrated approaches to testing and assessment are discussed as alternatives to animal testing in section C.1.3 of the ISO 10993-10:2023 standard.
3 See Annex B.1.1 of ISO 10993-1, where it is stated that testing should be primarily based “upon physical and chemical characterization and in vitro testing, with in vivo testing only being carried out where these are required to fill gaps in our understanding.”
4 See Annex C of ISO 10993-10:2023 for a discussion of non-animal methods for skin sensitization testing.
5 The polymers and fabrics that may not require testing are listed in Annex G of FDA’s 2023 guidance document Use of International Standard ISO 10993-1, "Biological evaluation of medical devices - Part 1: Evaluation and testing within a risk management process".
6 FDA database on recognized consensus standards for medical devices: ISO 10993-23 First edition 2021-01.