Cytotoxicity testing according to ISO 10993-5 is one of the most consistently required medical device biocompatibility tests — together with sensitization (ISO 10993-10) and irritation (ISO 10993-23), it is required for virtually all new medical devices regardless of contact site or duration.
Within the biological evaluation process, the stepwise approach outlined in ISO 10993-1 expects cytotoxicity tests and other in vitro assays to follow physical and chemical characterization but precede any animal testing.1 As they are relatively cost-effective and quick to perform, cytotoxicity assays can also be used earlier in the manufacturing process as screening tests to validate material choices or cleaning protocols.
This article provides a practical overview of cytotoxicity testing and the ISO 10993-5 standard, based on Measurlabs' several years of experience supporting medical device manufacturers with their testing needs for EU MDR and US FDA submissions.
How is cytotoxicity testing performed?
In cytotoxicity tests, a cell culture is exposed to the device or its extract, and the effects on cell viability and morphology are evaluated qualitatively and/or quantitatively:
In qualitative assays, cell morphology and viability are evaluated microscopically and rated on a numerical scale from 0 to 4. A score greater than 2 is considered a cytotoxic effect.
In quantitative assays, living cells’ ability to absorb a dye or convert a reagent into a colored product is measured photometrically. Cell viability below 70% compared to the untreated control is generally considered the threshold for cytotoxicity.
ISO 10993-5 describes several cytotoxicity testing methods and leaves it up to the testing laboratory to choose the most suitable approach for the given sample. Method selection should be guided by the physical and chemical characteristics of the medical device, as well as its intended use site and nature of the use.
Which method should be selected: extract, direct, or indirect?
Cytotoxicity testing is most often performed on a liquid extract of the device, as this allows for controlled, reproducible exposure conditions and reflects the real-world mechanism by which chemicals leach from the material. A direct test is generally more appropriate if the device comes into direct contact with tissues or cells during clinical use. Liquid and gel-like devices are also often tested by adding the material directly to the cell culture rather than preparing a separate extract.
An indirect (diffusion) method can be used if a test sample suitable for direct contact cannot be prepared, for example, because the material is too bulky or irregularly shaped. As the indirect assays described in ISO 10993-5 only allow qualitative assessment of cytotoxicity and raise concerns about whether all leachables diffuse through the agar or filter layer, a scientific justification for using them must be provided. Thus, from a regulatory perspective, opting for an extract or direct contact test is generally the safer option.2
Table 1: Summary of method selection considerations in cytotoxicity testing
Testing approach | Principle | Assessment type | Ideal use cases | Limitations |
Extract | Cells are exposed to a liquid extract of the device | Qualitative and quantitative | Preferred method for most device types | May not capture effects of direct contact with tissues |
Direct contact | Device specimen is placed directly onto the cell monolayer | Qualitative and quantitative | Devices intended for direct tissue or cell contact in clinical use; liquid and gel-like devices; soft and flexible devices such as hydrogels and wound dressings | Physical placement of specimen on the cells risks mechanical damage |
Indirect contact | Device specimen is separated from the cell layer by an agar or filter membrane | Qualitative only | Materials too bulky or irregularly shaped for direct contact testing; screening of finished devices where extract preparation is impractical | Not all leachables may diffuse through the agar or filter layer; use must be scientifically justified and regulatory acceptance is not guaranteed |
Sample preparation for extraction tests
Sample extracts for cytotoxicity assays are generally prepared in accordance with ISO 10993-12. The preferred extraction vehicle is a culture medium with serum, which supports cellular growth and extracts both polar and non-polar substances. Other vehicles, including purified water and DMSO, can be used, but this tends to introduce additional steps, such as pH adjustment, dilution, or evaporation.
When a culture medium is used, extraction is always performed at 37 ± 1 °C, because higher temperatures may compromise the stability of the medium. Extraction time is selected based on contact duration in clinical use:
Limited contact (≤24 h) with intact skin or mucosa: 4–24 h
Limited contact with other tissues: (24 ± 2) h
Prolonged (>24 h to 30 d) or long-term contact (>30 d): 72 h recommended, 24 sufficient if it can be shown that extended extraction does not release additional chemicals
The standard extraction ratio is 6 cm2 of sample surface area per milliliter of extraction vehicle for devices with a thickness of <0.5 mm and 3 cm2 for thicker items. If the surface area cannot be determined, a mass-based ratio of 0.2 g/ml for irregularly shaped solids or 0.1 g/ml for porous materials, such as textiles, is used. These ratios may be adjusted when the sample geometry or available quantity makes the standard ratio impractical, but the deviation must be justified.3
Samples should be fully submerged in the extraction medium, which means that large samples may need to be cut. However, cutting elastomers, coated materials, composites, or laminates should be avoided, as the extraction characteristics of the cut surface are likely to be different from those of the actual contact surface.
Sample preparation for direct contact tests
When cytotoxicity testing is conducted on a solid medical device directly rather than on its extract, the test item should have at least one flat surface, as sharp edges may cause physical damage to the cells during testing and thus lead to a false positive result. The sample material should cover approximately one-tenth of the cell culture surface, so small pieces (~0.25 cm2) should be prepared.
Quantitative cytotoxicity assays: NRU, MTT, and XTT
Once suitable samples have been obtained, they are added to the cell culture and incubated at (37 ± 1) °C for a minimum of 24 hours. Three replicates of each test sample, positive control, and negative control are run in parallel.
The cytotoxic effects are then evaluated, preferably using one of the three quantitative methods described in ISO 10993-5:
The neutral red uptake (NRU) assay measures lysosomal membrane integrity through viable cells’ capacity to absorb and retain neutral red dye in their lysosomes.
The MTT assay measures mitochondrial activity by tracking the conversion of a yellow reagent into purple formazan crystals.
The XTT assay works on the same principle as MTT but produces an orange water-soluble formazan instead of the purple crystals.
Cell viability is measured photometrically in all three assays, and the measured color intensity correlates directly with the number of viable cells. If cell viability is at least 70% compared to the untreated control, it can be concluded that the medical device is not cytotoxic.
ISO 10993-5 does not specify a preference for any assay, which means that method selection can be based on practical considerations. The most important of these is possible interference with the dye or reagent; MTT and XTT can be affected by colored or turbid extracts, and copper compounds specifically interfere with formazan detection in the MTT assay. In the NRU assay, compounds that interact with lysosomal function can lead to an overestimation of toxicity,4 but such compounds are rarely present in conventional medical device materials. For this reason, Measurlabs uses NRU as the default method unless another method is specifically requested or clearly more suitable for the test material.
When are qualitative cytotoxicity assays used?
ISO 10993-5 expresses a preference for quantitative evaluation of cytotoxicity and states that qualitative means are “appropriate for screening purposes”.5 However, despite this preference, qualitative microscopic evaluation of cell morphology is routinely used for regulatory submissions. Qualitative cytotoxicity assays are also useful in complementing quantitative results, particularly in borderline cases where measured cell viability is close to the 70% threshold.
What to do if the results show a cytotoxic effect?
A positive cytotoxicity result may result from incorrect test condition or method selection, such as the device extract interfering with the reagent, or a gel-form sample suffocating the cells in a direct contact test. To avoid such mistakes, a biological evaluation plan (BEP) outlining the most suitable test conditions for the specific device should ideally be in place before testing begins.
If the positive result is not explained by test design, changes may need to be made to the manufacturing process. It is sometimes enough to improve the cleaning process, as cleaning agent residues may cause a cytotoxic result. In other cases, material choices may need to be reconsidered. In some devices, such as dental acid etchants or devices that contain uncured polymer resins, cytotoxicity may be acceptable if the clinical benefit outweighs the risks.6
One partner for all your biocompatibility testing needs
Measurlabs offers cytotoxicity testing using several methods described in the ISO 10993-5 standard:
We also offer a comprehensive range of other ISO 10993 biocompatibility tests, as well as support for drafting the biological evaluation plan (BEP) and the biological evaluation report (BER). Our experts can help tailor a testing plan to both EU MDR and FDA submission requirements, and the tests can be performed under ISO 17025 accreditation and GLP certification, where required.
References:
1 The stepwise approach to biocompatibility testing is described in Section 4.4, Animal Welfare, of ISO 10993-1:2025.
2 See Räägel et al. (2021) Medical Device Industry Approaches for Addressing Sources of Failing Cytotoxicity Scores in Biomedical Instrumentation & Technology. The authors point out that “regulatory agencies have expressed reluctance to accept the agar diffusion assay” due to concerns about the limited diffusion of leachables. The preference for quantitative methods is also directly stated in Section 8.5 of ISO 10993-5:2009.
3 Section 10.3.3 of ISO 10993-12:2021
4 See Choi et al. (2022), Interfering with Color Response by Porphyrin-Related Compounds in the MTT Tetrazolium-Based Colorimetric Assay in International Journal of Molecular Sciences and Cudazzo et al. (2019), Lysosomotropic-related limitations of the BALB/c 3T3 cell-based neutral red uptake assay and an alternative testing approach for assessing e-liquid cytotoxicity in Toxicology in Vitro
5 Section 8.5.1 of ISO 10993-5:2009
6 Section VI.A. Cytotoxicity of the FDA Guidance Document on the Use of International Standard ISO 10993-1 mentions dental acid etchants and devices containing uncured polymer resins as examples of devices that may be legally marketed despite showing cytotoxic effects. Comparative testing with a device already on the market may be required.