Cytotoxicity Test — ISO 10993-5

What Is Cytotoxicity?

The cytotoxicity test evaluates in vitro whether substances released from a medical device or its material show a toxic effect on a cell culture. It is the first and most common step in biocompatibility assessment: being fast, sensitive and animal-free, it is used as a screening test.

Purpose of Cytotoxicity

The purpose is to determine whether substances migrating from the material adversely affect cell viability, proliferation or morphology, and to decide whether to proceed to further biocompatibility tests.

Which Products Is Cytotoxicity Applied To?

Surgical instruments and implants
Catheter, cannula and tubing sets
Wound dressings and bandages
Dental materials
Packaging and components in contact with the device

In Which Sectors Is Cytotoxicity Used?

Medical DeviceHealthcarePharmaceuticals

Standards Related to Cytotoxicity

ISO 10993-5in vitro sitotoksisite test
ISO 10993-1
ISO 10993-12
USP <87>
ISO 11607end product sterile barrier packaging test

Cytotoxicity Conditions

Cell line: The L929 mouse fibroblast cell line is commonly used
Method: Extract test, direct contact, or agar diffusion; chosen according to the device
Extraction: Per ISO 10993-12; common conditions are 24 hours at 37 °C or 72 hours at 50 °C
Assessment: Percentage cell viability or reactivity grade (0–4)

Equipment for Cytotoxicity

CO₂ incubator
Biosafety cabinet
Inverted phase-contrast microscope
Microplate reader (for the MTT/XTT method)
Cell-culture consumables

Sample Requirements for Cytotoxicity

The specimen must represent the final, sterilised product
Not raw material — the product that has gone through all manufacturing and sterilisation processes is submitted
The specimen quantity is determined according to the surface-area/extraction ratio prescribed by the standard
The device's type and duration of body contact must be stated
The sterilisation method and, if applicable, residue information must be shared

Duration of Cytotoxicity

Typically completed within 1–2 weeks, including extraction and cell-culture incubation.

Cytotoxicity Procedure

1The specimen is extracted at the surface-area/volume ratio prescribed by the standard.
2The cell culture is prepared and a monolayer is allowed to form.
3The extract (or, for direct contact, the specimen) is applied to the cells.
4Positive and negative controls are run under the same conditions.
5Cells are examined microscopically at the end of the specified incubation period.
6If a quantitative method is applied, cell viability is measured.
7Reactivity is graded and reported.

Cytotoxicity Acceptance Criteria

The common acceptance criterion under ISO 10993-5 is that cell viability does not fall below 70% of the control. In qualitative methods, the reactivity grade must not exceed the limit stated in the standard (commonly grade 2).

Evaluation of Results

The result is interpreted together with the controls; if the controls do not show the expected behaviour, the test is invalid. A cytotoxic finding alone does not mean the device is non-conforming — the source must be investigated (residual solvent, sterilisation residue, additive).

Cytotoxicity Report

Applied standard and revision
Specimen description, batch number and sterilisation method
Extraction conditions (solvent, temperature, duration, ratio)
Positive and negative control results
Test results and grading
Assessment and conclusion

Accreditation

EUROLAB holds internationally recognised accreditation obtained from accreditation bodies party to the IAF and ILAC multilateral recognition arrangements, under ISO/IEC 17025 for laboratory testing, ISO/IEC 17020 for inspection and surveillance, and ISO/IEC 17021-1 and ISO/IEC 17065 for system and product certification. This chain ensures the reports and certificates issued are accepted worldwide.

Whether this test can be offered within accredited scope depends on your product and the standard applied; since accreditation is method-based, the scope is confirmed in writing at the quotation stage.

Scope and authorisations

Related Regulations

MDR (EU) 2017/745
ISO 10993-1 biological evaluation plan

Medikal Cihaz Testleri — What Does This Testing Field Cover?

The section below describes the field that the Cytotoxicity test belongs to; the conditions and acceptance criteria of this particular test are given in the sections above. The scope specific to your product is confirmed in writing at the quotation stage.

Medical device testing shows that a device is safe for the patient and the user and maintains its intended performance. In this family safety is not only about avoiding harm; a device failing to perform its expected function is also treated as a risk. A monitor missing an alarm or a pump delivering the wrong dose is directly a patient safety event.

At the centre of the field is RISK MANAGEMENT, and this is what sets medical apart from other sectors. The risk management process run under ISO 14971 also determines which tests are required. The test list is not copied from a table of standards; it is derived and justified from the device's intended use, the nature and duration of patient contact, the use environment and foreseeable misuse.

Assessment consists of several separate bodies of work, each with its own set of standards. Biocompatibility runs through the ISO 10993 series, electrical device safety through the IEC 60601 series, sterilisation through ISO 11135/11137/17665, packaging through ISO 11607, software through IEC 62304 and usability through IEC 62366. The quality system (ISO 13485) is the framework that binds these together.

Which tests are required is determined by the device's CLASS and its MODE OF CONTACT WITH THE BODY. A product in brief contact with skin and an implant entering the bloodstream do not go through the same test set; the scope is set by a biological evaluation plan prepared under ISO 10993-1. That plan justifies in writing which endpoints are required and why the others are not.

Packaging and sterilisation are taken as seriously as the device itself in this field. For a sterile device the packaging is not a transport container but the barrier that maintains sterility; if the barrier is breached, the device is no longer sterile. Packaging processes are therefore validated and shelf life is supported by real-time ageing data.

For devices containing software, assessment shifts from testing towards PROCESS assurance. IEC 62304 defines which development and verification steps must be followed according to the safety class of the software; the evidence is the traceability of the development process. Testing software does not make it safe — the process record does.

The clinical side is a separate column of the technical file. Test reports show that the device is technically safe; clinical evaluation establishes that the device delivers the intended clinical benefit and that the benefit-risk balance is acceptable. Neither replaces the other.

Method groups in this field

Biocompatibility
Cytotoxicity, irritation, skin sensitisation, systemic toxicity, haemocompatibility, genotoxicity and local effects after implantation. The required endpoints are set by the ISO 10993-1 plan.
Electrical device safety
IEC 60601-1 basic safety and essential performance; EMC, alarm, usability and environmental requirements through the collateral standards.
Sterilisation validation
Validation of sterilisation processes by ethylene oxide, radiation or moist heat; bioburden and sterility assurance level studies.
Packaging
Integrity of the sterile barrier system, seal strength, distribution simulation and shelf-life studies.
Microbiological tests
Bioburden determination, sterility testing, bacterial endotoxin and, where required, carrier validation studies.
Software and usability
IEC 62304 life cycle evidence and use-error focused evaluation under IEC 62366.

Commonly confused in this field

Cytotoxicity alone is not evidence of biocompatibility; the required set of endpoints is determined and justified according to ISO 10993-1.
An ISO 13485 certificate does not confer CE marking; classification under the MDR, the technical file and the notified body process run separately.
Accelerated ageing does not replace real-time ageing; a shelf life claim is supported by real-time data.
A change of material (supplier, pigment, sterilisation method) may require the biological evaluation to be repeated; it is not a silent change.
An assessment made without the collateral and particular standards for IEC 60601-1 is incomplete; which -1-x and -2-x standards were applied must appear in the report.
In vitro diagnostic devices fall under the IVDR rather than the MDR, and laboratory equipment is often on the IEC 61010-1 side.

Which decisions these results support

MDR/IVDR technical file and notified body assessment
Completing the biological evaluation plan
Sterilisation and packaging validation
Determining shelf life and storage conditions
Determining the scope of reassessment after a design change

Related Tests

Frequently Asked Questions About Cytotoxicity

Is passing the cytotoxicity test sufficient?

No. ISO 10993-1 requires the test set to be determined according to the device's type and duration of body contact. Cytotoxicity is a screening test; as contact duration and invasiveness increase, sensitisation, irritation, systemic toxicity and further tests come into play.

Can I send a raw-material specimen?

It should not be. Assessment is performed on the final, sterilised product; processing aids, mould-release agents and sterilisation residues directly affect the result and are not present in the raw material.

The result came back cytotoxic — what should I do?

The source must be investigated first: residual solvent, a cleaning chemical, a sterilisation residue (e.g. ethylene oxide), or a specific additive are common causes. Retesting without removing the source usually gives the same result.