Animal testing in biological evaluations on the decline?
All medical devices that come into contact with patients – whether through the skin, mucosa, or other tissues, including wound dressings, surgical instruments, implants, or wearable sensors – must undergo rigorous safety and biocompatibility testing before clinical use. Traditionally, some of these safety assessments involve animal testing. Today, however, regulatory authorities in both the United States and Europe recognize scientifically validated laboratory methods that use reconstructed human tissue models instead of animals. These modern in vitro systems provide reliable, ethical, and regulatory-accepted alternatives.
Reduction of Animal Use according to international an European requirements
For medical devices, biocompatibility is traditionally assessed using animal studies across multiple endpoints to evaluate safety and potential biological risks. Commonly tested endpoints include:
- skin sensitization (e.g., allergic responses),
- local tissue or skin irritation (e.g., redness, itching),
- systemic toxicity – encompassing acute, subacute, subchronic, and chronic effects (e.g., organ damage, mortality),
- material-induced pyrogenicity (fever response),
- genotoxicity (DNA damage),
- carcinogenicity (cancer induction),
- reproductive toxicity (effects on fertility and reproduction),
- implantation effects (e.g., implant rejection), and
According to ISO 10993-1:2025, in vivo biological testing must be limited to what is strictly necessary and comply with ISO 10993-2:2022. More precisely the standard requires that animal use be reduced to the scientific minimum by selecting only necessary studies, avoiding duplication through existing data, applying stepwise testing strategies, and ensuring robust study design and statistical planning.
It emphasizes refinement measures to minimize pain and distress, including appropriate species selection, humane techniques, trained personnel, veterinary oversight, defined observation plans, and the use of humane endpoints to prevent unnecessary suffering. In vivo testing must not be conducted if adequate non-clinical or clinical data are already available, biological equivalence has been demonstrated, or the risk assessment indicates an unacceptable risk to animal welfare.
The standard also promotes the development and use of validated alternative methods, as well as data sharing and publication, to avoid repeated animal testing and ensure ethically and scientifically sound evaluations.
The EN ISO 10993-23:2021 standard introduces the reconstructed human epidermis (RhE) model to assess skin irritation potential of medical devices, materials, or their extracts, aiming to reduce, refine, and replace traditional in vivo testing such as the rabbit skin test. In the RhE test, human keratinocytes are cultured to form a multilayered epidermal model, exposed to test extracts, and cell viability is measured via the MTT assay to classify substances as irritant (≤50% viability) or non-irritant (>50% viability). Special irritation tests are recommended for products contacting specific tissues, such as mucosa or eyes, where alternative in vitro models or accredited methods like Hen’s Egg Test – Chorioallantoic Membrane can be applied.
ISO 18562 is an international standard series that specifies how to assess the biocompatibility of breathing gas pathways in medical devices to ensure they do not release harmful substances to patients. ISO 18562-4:2024 requires that if the exposure dose of a leachable substance exceeds the tolerable intake or threshold of toxicological concern, a margin of safety must be calculated and evaluated within a benefit–risk analysis. Biological evaluation follows the ISO 10993-1 framework, emphasizing chemical characterization and risk assessment, and where testing is necessary, it refers to in vitro methods (e.g., cytotoxicity, irritation such as RhE) rather than animal testing.
ISO 7405:2025 specifies biological test methods for dental medical devices that often include in vivo animal‑based tests, such as pulp, dentine, and endodontic usage tests, carried out under the animal welfare requirements referenced in ISO 10993‑2. While the standard encourages minimizing and refining animal use and promotes development of in vitro alternatives, there currently are no fully established in vitro replacements for all the required animal tests in dental biocompatibility evaluation.
The OECD Test Guideline 439 was updated and published in 2025 and describes a validated in vitro method using reconstructed human epidermis (RhE) models for the assessment of skin irritation potential of chemicals and mixtures. The test is primarily designed for chemical hazard classification under the UN Globally Harmonized System (GHS), specifically to identify Category 2 skin irritants* versus non-classified substances based on a defined cell viability threshold.
*Category 2 skin irritants referred to in OECD TG 439 are those chemicals that produce reversible skin irritation according to the UN GHS criteria and show ≤ 50% tissue viability in the RhE test system.
In Europe, this method is widely accepted for regulatory purposes in the context of chemical safety legislation (e.g., REACH and CLP), as OECD Test Guidelines are implemented through the OECD Mutual Acceptance of Data system. The guideline is not specifically developed for medical devices; however, it may be used to assess skin irritation potential of chemical constituents or extracts of medical devices when required under biocompatibility evaluation frameworks. Therefore, its primary regulatory application is within chemical legislation, while its use for medical devices is supportive and context-dependent rather than device-specific.
Reduction of Animal Use according to further international regulation
The FDA also emphasizes a “3R” approach to animal testing replace animal testing with validated alternatives where possible (Replacement), reduce the number of animals used (Reduction), and prioritize existing data and further analyses involving design studies to minimize pain and distress (Refinement), with full justification and documentation. The guidance further specifies quality system requirements and control measures to promptly detect and prevent biocompatibility issues in devices contacting intact skin. In general, the FDA recommends approaches that avoid or minimize animal testing when evaluating the biocompatibility of devices made from standard, well-characterized materials.
In China, medical device biocompatibility is evaluated under the GB/T 16886 series, aligned with ISO 10993, including animal welfare requirements in GB/T 16886.2 that promote reduction, refinement, and scientifically valid alternatives to animal testing.
For registration with the National Medical Products Administration, manufacturers must submit biocompatibility data (e.g., cytotoxicity, irritation, sensitization) in accordance with applicable Chinese standards, typically generated by qualified testing centers as part of the safety dossier.
However, it must be stated that for the time being both the FDA as well as the Chinese NMPA still quarrel with excepting in vitro test data.
GLP Compliance and Non-Animal Testing in Medical Device Biocompatibility
The European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) promotes, validates, and standardizes non-animal testing methods to reduce reliance on animal experiments while supporting regulatory safety assessments. In GLP-certified laboratories, reconstructed human epidermis (RhE) models such as EpiDerm™ and EPISKIN-SM™ are routinely used to test skin irritation of medical device materials. Results are documented in formal GLP-compliant reports to support regulatory submissions, including FDA filings and CE conformity assessments, demonstrating that validated in vitro methods can ensure safety without animal testing.
The Paul-Ehrlich-Institut encourages for example replacing the traditional Rabbit Pyrogen Test (RPT), which uses thousands of rabbits annually, with validated non-animal methods such as the Monocyte Activation Test (MAT) or recombinant factor C assays. These in vitro tests are now included in the European Pharmacopoeia, offering a humane and sustainable alternative while maintaining test quality and regulatory compliance.
The U.S. FDA requires that all in vitro and in vivo biocompatibility studies submitted for medical devices be conducted under GLP according to 21 CFR Part 58, with a clear GLP compliance statement. Non-GLP studies must be justified, and it is explicitly stated that ISO/IEC 17025 accreditation cannot replace GLP compliance. Test reports must provide sufficient detail – including methods, results, deviations, and statistical analyses – to allow FDA to critically evaluate the study while meeting all referenced ISO 10993-1 requirements.
Worldwide regulatory frameworks, including ISO 10993, FDA guidance, and European standards, increasingly prioritize minimizing, refining, and replacing animal testing in medical device biocompatibility, encouraging validated in vitro alternatives and ethically sound approaches wherever scientifically feasible.
Experience from a laboratories stand point
Hohenstein Medical can illustrate the sensitization potential of individual test substances in vitro using three key events according to DIN EN ISO 10993-10 Annex C
The offer to reduce or replace the use of animal in toxicity testing has led to the development of various non-animal methods for risk assessment.
Assays for the testing of skin sensation
Direct Peptide Reactivity Assay (DPRA)
- Key event 1: Covalent binding to skin proteins
The DPRA is an in chemico method that quantifies the reactivity of a test chemical or a product extract through its depletion of synthetic peptides containing cysteine or lysine. The percentage peptide depletion values for cysteine and lysine are determined and assigned to one of four reactivity classes in a predication model, where the tested product is classified as sensitizing or non-sensitizing to the skin.
IL-18 RhE Assay
- Key event 2: Keratinocyte response
Skin sensitization is assessed in vitro by measuring the basal release of interleukin 18 (IL-18) after application of a product extract to reconstructed human epidermis (RhE). IL-18 is quantified in the culture medium of the RhE using the ELISA method. In parallel, the viability of the cells is measured by MTT test. (The assay can be combined with the test for skin irritation according to DIN EN ISO 10993-23).
U937 CELL LINE ACTIVATION TEST (U-SENS™)
- Key event 3: Activation of dendritic cells
U-SENS™ method is an in vitro assay that quantifies changes of CD86 cell surface marker expression by flow cytometry on a human histiocytic lymphoma cell line (U937 cells).
BACKGROUND – Adverse Outcome Pathway for skin sensitization: The prediction of skin sensitization in humans relies on combining several individual tests in experimental strategies, as none of the validated animal-free assays alone can fully reproduce the complex network of underlying mechanisms. The OECD has described the sequential events that lead to a skin sensitizing effect. This series of events is referred to as the AOP (Adverse Outcome Pathway).
The AOP for skin sensitization describes four key events, whereby key events 1 to 3 are described in the risk assessment in accordance with DIN EN ISO 10993-10 Annex C as alternative methods not involving animal testing.
Key event 1– Covalent binding to skin proteins: the molecular initiating event after penetration of the stratum corneum is the irreversible formation of the hapten-protein complex. Key event 2– Keratinocyte response: this key event involves the activation of biochemical pathways in the keratinocytes and includes inflammatory mediator responses as well as gene expression changes associated with cell signaling pathways such as the formation of interleukin (IL-)18. Key event 3– Activation of dendritic cells: the detailed biochemical events after formation of the hapten-protein complex have not been fully clarified. Effects at the cellular and tissue levels are also not completely known but involve epidermal responses which include: 1) immune recognition of chemical allergens by keratinocytes, specialized epidermal dendritic cells (i.e. Langerhans cells) and dermal dendritic cells; 2) Cellular responses manifested as expression of specific cell surface markers like e.g. CD86 in U937 cells. Key event 4– T-cell proliferation: at the organ level (lymph nodes and skin), the responses are: 1) dendritic cell migration to the lymph node, where the antigen is presented to activate naive T-lymphocytes (T-cells), and 2) differentiation and proliferation of T-cells into allergen-specific effector and memory T-cells.
Customer benefit
Validated non-animal and in vitro methods can support regulatory compliance and provide evidence for CE marking under the MDR. A risk-based approach strengthens the biological evaluation while avoiding unnecessary or duplicate animal studies.
Early identification of material-related risks supports product optimisation and more efficient development. Reliable testing helps protect patients and users by detecting irritation, sensitisation, toxicity and other biological hazards. Addressing these risks before market launch can reduce complaints, adverse reactions and corrective actions.
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