EN ISO 10993-5

EN ISO 10993-5

EN ISO 10993-5 is a standard published by the International Organisation for Standardisation (ISO) and adopted by the European Committee for Standardisation (CEN), entitled ‘Biological evaluation of medical devices – Part 5: In vitro cytotoxicity tests’, i.e. ‘Biological evaluation of medical devices – Part 5: In vitro cytotoxicity tests’.

Regulatory status and version note: The base text of the standard is ISO 10993-5:2009 (third edition) and was adopted at European level as EN ISO 10993-5:2009. Amendment A11:2025 has been added to the standard, and the current consolidated version, EN ISO 10993-5:2009+A11:2025 is in force (approved by CEN on 29 January 2025, published in spring 2025)[reference:0] [reference:1]. Amendment A11 updates the standard’s status as a harmonised standard by adding Annex ZA within the framework of the MDR (2017/745). The extent of recognition by the FDA must be verified prior to submission of an application.

The basic principle of the standard is the assessment of the cytotoxic potential of medical devices or component materials in cell culture systems. This is regarded as the first and mandatory step in the biocompatibility testing sequence; it enables the toxic effect to be determined at the cellular level before proceeding to in vivo tests[reference:3].

The scope of the standard covers three basic contact models:

1. Extraction process (extract method)

The test material is extracted under specific conditions (temperature, duration, surface-to-volume ratio) in a suitable extraction medium (preferably serum-containing cell culture medium; alternatively, physiological saline, DMSO, etc.). The resulting extract is applied to the cell culture. The conditions for extract preparation are in accordance with ISO 10993-12[reference:4][reference:5]. This method is the most commonly used approach for assessing the toxic potential of substances that may be released from the product.

2. Indirect Contact method

The test material is exposed to the cell culture in such a way that it does not come into direct contact with the cells (e.g. by diffusion through a layer of agar or collagen). This method is used in particular to assess the potential of low-molecular-weight components to diffuse into the cells.

3. Direct Contact Method

The test material is applied directly to the cell layer. This method is suitable for solid materials and devices with distinct surface properties. It enables the direct interaction between the material surface and the cells to be assessed.

Cell lines used

The standard permits the use of various cell lines. Commonly used cell lines include [reference:6]:

• L929 (fibroblasts from mouse connective tissue) – the most commonly used cell line[reference:7]
• Balb/3T3 clone A31 (fibroblasts from a mouse embryo)
• V79 (Chinese hamster lung fibroblasts)
• Where appropriate, primary cells of human origin (e.g. human dermal fibroblasts) or other suitable cell lines

Methods for measuring cell viability / cytotoxicity

The standard provides various quantitative methods for assessing cell viability:

• MTT assay: A colorimetric method based on the activity of mitochondrial dehydrogenase; it measures the formation of formazan crystals in living cells[reference:8]
• NRU assay (Neutral Red Uptake): A method based on the uptake of the dye Neutral Red by the lysosomes of living cells
• CFU test (colony-forming units): Assessment of the cells’ ability to form colonies
• XTT, WST-1, LDH and other validated quantitative methods

The results are usually expressed as a percentage of cell viability and compared with the control group. The standard specifies acceptance criteria and a classification system for assessing cytotoxicity (e.g. viability below 70 per cent is classified as cytotoxic).

The key parameters reported in the study are:

• Contact model used (extract, indirect, direct) and justification
• Extract preparation conditions (in accordance with ISO 10993-12: medium, temperature, duration, surface-to-volume ratio)[reference:9]
• Cell line used and number of passages
• Cell seeding density and culture conditions
• Concentrations of extract used (usually dilutions of 100 per cent, 50 per cent, 25 per cent, etc.)
• Duration of exposure (usually 24 hours)[reference:10]
• Verwendete Methode zur Lebensfähigkeitsmessung (MTT, NRU, CFU usw.)
• Percentage cell viability (for each concentration) and IC₅₀ value
• Validity of the positive and negative controls
• Cytotoxicity classification (on a scale of 0–4 or similar)
• Data on measurement uncertainty and repeatability

Why is it carried out?

EN ISO 10993-5 is carried out for the following scientific and regulatory purposes:

• Initial biocompatibility testing: Rapid and reliable assessment of the potential of material components or extracts of a medical device to cause toxic effects at the cellular level[reference:11]
• MDR compliance: Provision of baseline data for the biological safety assessment under the EU Medical Devices Regulation (2017/745)
• Decision on the transition to in vivo testing: use of the cytotoxicity result to assess whether further biocompatibility tests (sensitisation, irritation, implantation, systemic toxicity, etc.) are required
• Material selection and development: Early identification of potentially cytotoxic materials and evaluation of alternatives as part of the product development process
• Control of the production process: Assessment of the impact of production steps such as sterilisation, coating and cleaning on the cytotoxicity profile
• Animal welfare (3Rs): Avoiding unnecessary animal testing through in vitro screening; reducing the need for animal testing
• Support with regulatory documentation: to be used as a mandatory component of the biocompatibility dossier for applications submitted to the FDA, in accordance with the EU MDR and other regulatory authorities
• Evidence-based approach: Chemical characterisation (ISO 10993-18), genotoxicity (ISO 10993-3), sensitisation (ISO 10993-10), irritation (ISO 10993-23) and other biocompatibility data contributing to the holistic assessment of the medical device

EN ISO 10993-5 does not, on its own, cover the entire biocompatibility profile of the product; local tissue effects, systemic toxicity, genotoxicity and haemocompatibility are assessed in accordance with separate standards. In vitro cytotoxicity results may not fully reflect in vivo conditions; the results should be interpreted in the context of chemical characterisation, other biological tests and clinical data.

At NanoTest, we carry out in vitro cytotoxicity assessments in accordance with EN ISO 10993-5, applying scientific rigour within the framework of our accredited laboratory infrastructure and our quality management system.

For your in vitro cytotoxicity assessments in accordance with EN ISO 10993-5, you can contact NanoTest to obtain information on the test strategy, the contact model and the selection of the cell line, extract preparation, the method for measuring cell viability, sample acceptance criteria, the test schedule and reporting procedures.