EN ISO 10993-12

EN ISO 10993-12 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 12: Sample preparation and reference materials”, i.e. “Biological evaluation of medical devices – Part 12: Sample preparation and reference materials”

Regulatory status and version note: The base text of the standard is ISO 10993-12:2021 (fifth edition) and was adopted at European level as EN ISO 10993-12:2021. Amendment 1 (A1:2025) has been added to the standard; the current consolidated version is EN ISO 10993-12:2021+A1:2025; the German version was published as DIN EN ISO 10993-12:2026-02[reference:0]. The standard has been recognised as a harmonised standard under the EU Medical Devices Regulation (MDR 2017/745) (Annex ZA)[reference:1]. The scope of recognition by the FDA must be verified prior to submission of an application.

The main purpose of the standard is to set out requirements and guidelines for the selection, preparation and extraction of samples, as well as for the selection of reference materials for biological testing, as defined in other parts of the ISO 10993 series[reference:2]. In other words, the standard provides the basic framework for sample preparation in biological testing; the reliability and reproducibility of the test results depend to a large extent on the correct application of this standard.

The main focus of the 2021 revision: ISO 10993-12:2021 has clearly distinguished the recommendations for sample preparation for biological testing from those for chemical testing (which are covered in detail in ISO 10993-18:2020) distinct from sample preparation for chemical testing (which is covered in detail in ISO 10993-18:2020) defines[reference:3]. This revision emphasises that, given the increasing complexity of products and materials, sample preparation must be designed on a product-specific basis and taking into account the clinical conditions of use[reference:4].

The main subject areas covered by the standard are as follows[reference:5]:

Selection of test samples
A selection of representative components from the medical device
Preparation of test samples and reference materials
Experimental controls
Selection and requirements for reference materials
Preparation of extracts (extraction vessels, conditions and methods; specific conditions for in situ polymerising materials)
Records

Extraction conditions and choice of solvents

The extraction conditions defined in Section 10.3 form the core of the standard. The basic principle is that the extract obtained must not be any less aggressive than the conditions encountered during clinical use [Reference:6].

Solvents (extraction agents): In general, the use of both polar (e.g. water, physiological saline, cell culture medium) and non-polar (e.g. vegetable oil, n-hexane) solvents is recommended[Reference:7]. However, under instrument-specific conditions (e.g. with a pre-filled syringe that comes into contact exclusively with saline solution), extraction using a single solvent may be sufficient [Reference:8]. The selected solvents must be compatible with the material and must not damage it [Reference:9].

Temperature and duration: The extraction conditions vary depending on the duration of contact with the device (limited, long-term, continuous). For devices with limited contact, (37 ± 1) °C for (24 ± 2) hours is typically recommended for the cytotoxicity test [Reference:10]. For devices with longer and continuous contact times, however, an extraction period of 72 hours is recommended, as a 24-hour extraction may not reflect the chemicals released after the 24-hour period has elapsed. However, if data are available showing that a 24-hour extraction period is sufficient and that extending it to 72 hours does not result in any additional release of chemicals, then 24 hours is sufficient[Reference:11].

Surface-to-volume ratio: For solid devices, the ratio of surface area to extraction volume (e.g. 3 cm²/ml or 6 cm²/ml) is used; for irregularly shaped or mass-based devices, however, the mass-to-volume ratio (e.g. 0.2 g/ml) is used[Reference:12].

The key parameters reported in the study are as follows:

Selection of the test sample and definition of the representative parts of the device
Sample preparation method (cutting, grinding, surface area measurement, etc.)
Extraction solvent used (polar/non-polar) and the reason for its selection
Extraction temperature and extraction time
Area/volume or mass/volume ratio
Extraction vessel and conditions
If reference material has been used: description and certification status
Description of the experimental controls
Specific extraction conditions for in situ polymerised materials (where applicable)
Description of records and traceability

Why is it carried out?

EN ISO 10993-12 is used for the following scientific and regulatory purposes:

Establishing a basis for the validity of biological tests: The results of all other biological tests in accordance with ISO 10993 (cytotoxicity, sensitisation, irritation, implantation, systemic toxicity, etc.) depend directly on the quality of sample preparation as defined in this standard
Obtaining extracts that reflect clinical use: ensuring that the extraction conditions are no less aggressive than the actual conditions under which the product is used[reference:13]
Ensuring reproducibility and comparability: achieving consistent, comparable results across different laboratories and studies
MDR compliance: Documentation of appropriate sample preparation and the use of reference materials for the biological safety assessment under the EU Medical Devices Regulation (2017/745)[reference:14]
Ensuring experimental control through reference materials: verifying that the test system is functioning correctly and ensuring comparability between laboratories
Device-specific scientific rationale: In cases where standard approaches are insufficient for complex devices, the development of specialised sample preparation strategies based on the material and clinical application conditions [reference:15]
Documentation and traceability: ensuring that the sample preparation process is transparent and verifiable during regulatory inspections

EN ISO 10993-12 is not a stand-alone test standard, but rather a standard covering procedures and infrastructure. The results must be interpreted in the context of the biological test itself and the chemical characterisation of the medical device (ISO 10993-18).

As NanoTest, we carry out sample preparation and reference material processes in accordance with EN ISO 10993-12 with scientific rigour, drawing on our accredited laboratory infrastructure and our quality management system.

Please feel free to contact NanoTest for information on sample selection, extraction strategies, the choice of solvents and conditions, the procurement of reference materials, and the documentation and reporting processes involved in your sample preparation and reference material procedures in accordance with EN ISO 10993-12.

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