Table of Contents
- What standards govern cytotoxicity testing?
- How is the extract (elution) test performed?
- How is the MTT assay used to quantify viability?
- What are the direct-contact and agar-diffusion methods?
- How is sample preparation done under ISO 10993-12?
- What do qualitative and quantitative scoring systems look like?
- How does the Chinese GB/T 16886.5 framework align?
- FAQ
- Our cytotoxicity testing service
What standards govern cytotoxicity testing?
Cytotoxicity testing is the cornerstone of the biological evaluation of medical devices, and it is usually the first biocompatibility test performed on a new device or material. The test evaluates whether a device or its leachable substances can cause harmful effects — cell death, altered metabolism, or inhibited growth — on cultured mammalian cells, using in vitro cell-culture techniques that are rapid, sensitive, and well-correlated with animal assays. Because cytotoxicity testing is the screening test that gates the downstream biocompatibility programme, its correct execution against the correct standard is the most consequential single biocompatibility decision a medical-device manufacturer makes.
The principal reference standards our laboratory works to are:
- ISO 10993-5:2009, Biological evaluation of medical devices — Part 5: Tests for in vitro cytotoxicity (ISO catalog) — the master international standard for the in-vitro cytotoxicity testing of medical devices. ISO 10993-5 defines the three categories of test (the extract test, the direct-contact test, and the indirect-contact / agar-diffusion test), the cell-culture conditions, the qualitative and the quantitative evaluation criteria, and the acceptance thresholds that distinguish the cytotoxic from the non-cytotoxic material.
- ISO 10993-12:2012 (and its revision), Sample preparation and reference materials (ISO catalog) — the companion standard that defines how the test sample is prepared, how the extract is generated (the surface-area-to-volume ratio, the extraction temperature, the extraction time, the extraction vehicle), and how the positive and the negative reference materials are used. ISO 10993-12 is the standard that makes the ISO 10993-5 result reproducible across laboratories, because the sample-preparation variables are the dominant source of the inter-laboratory variation.
- USP <87>, Biological Reactivity Tests, in vitro — the U.S. Pharmacopeial chapter that defines the in-vitro elution test for the biological reactivity of plastics (the USP plastics classes). USP <87> is aligned with ISO 10993-5, and it is the test that the U.S. Pharmacopeial classification of plastic containers and devices relies on. A device tested under USP <87> satisfies the ISO 10993-5 requirement for most regulatory pathways, but the report must name the standard it was run under.
- ISO 7405:2018, Dentistry — Evaluation of biocompatibility of medical devices used in dentistry — the dental-specific standard that defines the test procedures for the dental materials (the resin-based composites, the cements, the impression materials) whose chemistry or geometry requires a test modification beyond the general ISO 10993-5.
- MHLW Notification (Japan), Basic Principles of Biological Safety Evaluation — the Japanese Ministry of Health, Labour and Welfare notification that governs the cytotoxicity testing for the Japanese market, which specifically requires the colony-forming assay as one of the accepted methods.
- GB/T 16886.5-2017, Biological evaluation of medical devices — Part 5: Tests for in vitro cytotoxicity (std.samr.gov.cn) — the Chinese national adoption of ISO 10993-5, administered by TC248 under the NMPA. The 2017 edition replaced the 2003 edition, with the revisions to the extract preparation, the test procedure, and the result-evaluation sections.
A point worth stating plainly because it affects every project: ISO 10993-5 and USP <87> are not interchangeable in their detail. Both define the in-vitro cytotoxicity elution test, but the USP chapter is specific to the plastics classification (it tests the extract of the plastic material against the cell culture for the defined incubation period, with the pass criterion the absence of the cytotoxic response), while ISO 10993-5 is the broader standard that covers the full range of medical-device materials and the full range of test methods (extract, direct contact, agar diffusion, MTT, XTT, NRU, colony-forming). We confirm the regulatory pathway (the EU MDR, the FDA 510(k) / PMA, the NMPA registration, the Japanese MHLW) before quoting, because the test-method and the reporting requirements are driven by the pathway.
How is the extract (elution) test performed?
The extract test — also called the elution test — is the most widely applied of the ISO 10993-5 test methods, and it is the method of choice for the polymeric, the elastomeric and the high-density materials where the cytotoxic substances must first leach into a solution before they can contact the cells. The test prepares an extract of the device material, applies the extract to the cultured cells, incubates for the defined period, and evaluates the cellular response.
The extract test procedure runs as follows:
Extract preparation. The test material is incubated in the appropriate extraction vehicle — typically the cell-culture medium with serum (the Chinese GB/T 16886.5-2017 recommends the serum-containing medium as the preferred extraction vehicle), or the physiological saline — under the defined conditions. The ISO 10993-12 standard specifies the surface-area-to-volume ratio for the extraction (commonly 6 cm²/mL for the flat surfaces, or 0.2 g/mL for the irregular shapes), and the extraction conditions (commonly 37 °C for 24 ± 2 hours, or the accelerated conditions at 50 °C for 72 hours, or 70 °C for 24 hours). The extraction vehicle and the conditions are chosen to simulate the clinical exposure while maintaining the cell-culture compatibility, and the choice is documented in the test protocol because it directly affects the result.
Cell exposure. The extract is applied to the cultured cells — most commonly the L929 mouse-fibroblast cell line (the standardised cell line that has the extensive historical database and that the ISO 10993-5 and the GB/T 16886.5 recommend) — in the defined vessel (the 96-well plate for the quantitative MTT assay, the 6-well plate or the Petri dish for the qualitative microscopic evaluation). The cells are incubated with the extract under the controlled conditions (37 °C, 5 % CO₂, humidified atmosphere) for the defined exposure period (commonly 24 hours for the qualitative evaluation, 24 to 72 hours for the quantitative evaluation).
Observation and evaluation. After the incubation period, the cells are assessed for the signs of cytotoxicity. The qualitative evaluation examines the cell morphology, the cell detachment, the cell lysis, and the growth inhibition, by the light microscopy, and scores the response against the grading scale. The quantitative evaluation measures the cell viability (the metabolic activity, the membrane integrity, or the cell number) by the defined assay, and compares the result to the control. The pass criterion is the cell viability not below 70 % of the control (the threshold defined in ISO 10993-5), and the report documents the qualitative and the quantitative results alongside the extract-preparation conditions.
A point worth noting about the extract test: it is the method that best simulates the leaching exposure — the exposure that occurs when the device is in contact with the tissue fluids over time, and the cytotoxic substances gradually leach out. The extract test is therefore the method of choice for the implants, the long-term-contact devices, and the materials whose cytotoxicity is leachable rather than diffusible. For the devices where the exposure is by direct contact (the surface-contact devices, the wound dressings) or by diffusion through a barrier (the agar-diffusion setup), the alternative methods apply, and the choice of the method is documented in the protocol.
How is the MTT assay used to quantify viability?
The MTT assay is the most widely used quantitative method for the cytotoxicity evaluation under ISO 10993-5, and it is the method that the published regulatory-acceptance thresholds are most commonly expressed against. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a yellow tetrazolium salt that the viable cells with the active metabolism reduce, via the mitochondrial and the cytosolic dehydrogenases, into the purple insoluble formazan crystals. The intensity of the purple colour, measured by the spectrophotometric absorbance after the formazan solubilisation, is proportional to the number of viable cells, and the cell-viability percentage is calculated by the comparison to the control.
The MTT assay procedure:
Cell seeding. The L929 cells (or the defined alternative cell line) are seeded in the 96-well plate at the defined density (commonly 1 × 10⁴ cells per well), and incubated to the sub-confluent monolayer (commonly 24 hours).
Extract exposure. The extract (prepared per ISO 10993-12) is applied to the cells at the defined concentration (commonly the undiluted extract and the serial dilutions), and the cells are incubated for the defined exposure period (commonly 24 hours). The positive control (the cytotoxic extract, e.g. the latex or the tinuvin) and the negative control (the non-cytotoxic extract, e.g. the high-density polyethylene) are run in parallel.
MTT addition. The MTT reagent (commonly 0.5 mg/mL in the culture medium) is added to each well, and the cells are incubated for the defined period (2 to 4 hours) to allow the viable cells to reduce the MTT into the formazan.
Formazan solubilisation and absorbance. The culture medium is removed, the formazan crystals are solubilised in the defined solvent (the DMSO or the isopropanol), and the absorbance is measured at the defined wavelength (commonly 570 nm). The cell-viability percentage is calculated as the absorbance of the test extract divided by the absorbance of the blank control, expressed as a percentage.
Pass criterion. The cell viability below 70 % of the control indicates the cytotoxic potential, per the ISO 10993-5 threshold. A material with the cell viability ≥ 70 % is classified as non-cytotoxic under the MTT assay; a material with the cell viability below 70 % is cytotoxic, and the report documents the dose-response if the serial dilutions were tested.
A known limitation of the MTT assay, drawn from the published cytotoxicity-research literature: the MTT is susceptible to several artefacts. The test compound or the medium component can non-specifically reduce the MTT (the false-positive overestimation of viability); the formazan-solubilisation step is an additional source of variability; the metabolic stimulation by the test material can be mistaken for the increased viability. The published best practice is to confirm the MTT result with the independent endpoint (the NRU, the LDH release, or the ATP-based luminescence) where the MTT result is borderline, and the multiparametric approach (at least two independent endpoints) is the recommended practice for the rigorous cytotoxicity evaluation.
The XTT assay is the alternative to the MTT that produces the water-soluble formazan, eliminating the solubilisation step. The XTT is preferred by some laboratories for the higher-throughput applications, and it is accepted worldwide by the regulatory authorities. The BCA (bicinchoninic acid) staining test is another quantitative alternative accepted for the Central European submissions. The colony-forming assay — the clonogenic test that quantifies the surviving cells' ability to form colonies — is specifically required by the Japanese MHLW for the Japanese-market submission, and it is the method that detects the cytostatic (the growth-inhibition) effect alongside the cytotoxic (the cell-death) effect.
What are the direct-contact and agar-diffusion methods?
The extract test is not the only method defined in ISO 10993-5, and for some device types the alternative methods are more appropriate. The standard defines three categories of the test: the extract test (described above), the direct-contact test, and the indirect-contact (agar-diffusion) test.
The direct-contact test places the test material directly onto the cultured-cell monolayer, without the intermediate extraction step. The test is designed for the materials with the flat surface that are neither the low-density nor the high-density materials (the materials that would float on the culture medium or that would crush the cells by their weight), and it evaluates the cytotoxicity under the most stringent exposure condition — the direct, continuous contact between the material surface and the cells. The test is run by placing the sterilised test-material specimen (of the defined size) directly on the cell monolayer in the defined vessel, incubating for the defined period (commonly 24 hours at 37 °C), and evaluating the cellular response under and around the material specimen by the light microscopy. The qualitative grading — the zone of the growth inhibition, the cell lysis, or the morphological change under and around the specimen — is the result.
The direct-contact test is the method of choice for the surface-contact devices (the wound dressings, the surgical drapes, the skin-contact adhesives) where the clinical exposure is the direct material-cell contact, and where the extract test may not capture the surface-specific cytotoxicity. The test has the limitation that the material specimen can mechanically damage the cells by its weight or its edge, and the test must be designed to distinguish the mechanical from the chemical cytotoxicity. The published best practice is to use the negative-control material of the same geometry to verify the absence of the mechanical effect, and the report documents the specimen geometry alongside the result.
The agar-diffusion test (the indirect-contact test) places the test material on top of the agar layer that overlays the cell culture, so that the diffusible substances from the material diffuse through the agar to reach the cells, while the non-diffusible material is physically separated. The test is the option when the test material cannot be extracted or dissolved in the culture medium (the solid, the non-extractable materials, or the materials whose extraction would change the cytotoxicity profile), and when the test material diffuses through but does not react with the agar layer. The qualitative evaluation — the zone of the decolouration (the neutral-red dye release from the dead cells), the lysis index, and the growth-inhibition zone — is the result, scored against the defined grading scale.
The agar-diffusion test requires the justification of the method choice — the sponsor must provide the rationale for the agar-diffusion method over the extract or the direct-contact method, per ISO 10993-5 and ISO 7405. The test is the method of choice for the dental materials (the resin-based composites, the cements) where the ISO 7405 standard applies, and for the materials whose cytotoxicity is diffusible rather than leachable.
How is sample preparation done under ISO 10993-12?
The sample preparation — the extract generation — is the step that makes or breaks the reproducibility of the cytotoxicity test, and it is the step that ISO 10993-12 governs. The cytotoxicity result depends on the extract concentration, and the extract concentration depends on the sample-preparation variables: the surface area or the mass of the test material, the volume of the extraction vehicle, the extraction temperature, the extraction time, and the extraction-vehicle composition. ISO 10993-12 defines each of these variables, and the inter-laboratory reproducibility depends on their consistent application.
The ISO 10993-12 sample-preparation framework:
The surface-area-to-volume ratio. For the materials with a defined surface (the flat sheets, the tubes, the moulded parts), the extraction is based on the surface area, with the standardised ratio of 6 cm² of surface area per 1 mL of extraction vehicle (for the thickness up to 0.5 mm) or 3 cm²/mL (for the thickness 0.5 to 1 mm). For the irregularly-shaped materials (the powders, the fibres, the non-wovens), the extraction is based on the mass, with the standardised ratio of 0.2 g per 1 mL. For the composite devices (the multi-material, the complex geometry), the sample-preparation protocol is defined on the case-by-case basis, and the protocol is documented in the report because it directly affects the result.
The extraction conditions. ISO 10993-12 defines the standard extraction conditions: 37 ± 1 °C for 24 ± 2 hours (the physiological simulation), or the accelerated conditions of 50 °C for 72 hours, 70 °C for 24 hours, or 121 °C for 1 hour (the exaggerated conditions that simulate the accelerated leaching). The choice of the extraction condition is based on the clinical-exposure simulation and the material-chemistry compatibility, and the choice is documented in the protocol. The Chinese GB/T 16886.5-2017 recommends the 37 °C, 24-hour extraction in the serum-containing medium as the preferred condition.
The extraction vehicle. The extraction vehicle — the solution in which the test material is extracted — is chosen to simulate the clinical exposure and to maintain the cell-culture compatibility. The serum-containing culture medium (the most physiologically relevant), the physiological saline (the polar-vehicle extraction), and the vegetable oil (the non-polar-vehicle extraction) are the common choices. The polar and the non-polar vehicles are used in parallel when the cytotoxicity of both the polar and the non-polar leachables must be evaluated.
The reference materials. The positive control (the cytotoxic reference material, e.g. the latex rubber, the tinuvin, the zinc diethyldithiocarbamate) and the negative control (the non-cytotoxic reference material, e.g. the high-density polyethylene) are extracted and tested under the identical conditions, and their results verify the test-system responsiveness. A test run without the positive and the negative controls is not a valid ISO 10993-5 test, and the report documents the reference-material results alongside the test-material results.
A practical point from the published cytotoxicity-testing literature: the extraction conditions significantly affect the result. The same material extracted at 37 °C for 24 hours and at 70 °C for 24 hours can produce different cytotoxicity results, because the higher temperature releases more of the leachables. The extraction condition must be chosen to represent the clinical exposure — the 37 °C extraction for the body-temperature-contact devices, the accelerated extraction for the devices that will be heat-sterilised or that will see the elevated temperature in service. A report that does not state the extraction condition is reporting a result whose meaning depends on an unspecified variable.
What do qualitative and quantitative scoring systems look like?
ISO 10993-5 defines two evaluation approaches — the qualitative (the descriptive, microscope-based scoring) and the quantitative (the measured, assay-based scoring) — and both are reported for the comprehensive cytotoxicity evaluation.
The qualitative scoring evaluates the cellular response by the light microscopy, using the defined grading scales. The macroscopic and the microscopic observations cover:
- The cell morphology — the normal, the rounded, the shrunken, the vacuolised, the lysed cells, observed under the microscope.
- The growth inhibition — the confluent monolayer (no inhibition), the sub-confluent monolayer (partial inhibition), the sparse cells (severe inhibition), the no cells (complete inhibition), scored against the reference scale.
- The decolouration zone (the agar-diffusion test) — the zone under and around the specimen where the neutral-red-stained cells have released the dye (indicating the cell death), measured in millimetres.
- The lysis index — the percentage of the lysed cells, observed microscopically, graded on the defined 0-to-5 scale.
The ISO 10993-5 reactivity grading (0 = none; 1 = slight; 2 = mild; 3 = moderate; 4 = severe) is the qualitative result, and the report documents the grade alongside the descriptive observations.
The quantitative scoring measures the cell viability by the defined assay, and reports the viability percentage. The assays include:
- MTT / XTT / MTS — the tetrazolium-reduction assays that measure the metabolic activity of the viable cells. The MTT produces the insoluble formazan (requires the solubilisation step); the XTT and the MTS produce the soluble formazan (no solubilisation required).
- NRU (neutral red uptake) — the lysosomal-activity assay that measures the viable cells' uptake of the neutral-red dye into the lysosomes. The NRU is more sensitive to the early lysosomal stress than the MTT.
- LDH release — the membrane-integrity assay that measures the lactate dehydrogenase released from the dead cells into the culture medium. The LDH is the cytotoxicity (cell-death) marker, complementing the MTT / NRU viability markers.
- ATP-based luminescence (CellTiter-Glo) — the ATP-measurement assay that lyses the cells and quantifies the released ATP by the luciferase reaction. The ATP assay is the most sensitive (detecting fewer than 100 cells per well), and it is the choice for the low-cell-number and the high-throughput applications.
- Colony-forming assay — the clonogenic test that quantifies the surviving cells' ability to form colonies, required by the Japanese MHLW.
The quantitative result — the cell-viability percentage compared to the control, with the 70 % threshold — is the pass/fail criterion under ISO 10993-5. The report documents the assay method, the measured values, the control values, the calculated viability percentage, and the pass/fail conclusion, alongside the qualitative grading. The two approaches together — the qualitative microscopic scoring and the quantitative assay measurement — provide the comprehensive cytotoxicity evaluation that the ISO 10993-5 standard requires and that the regulatory authorities expect.
How does the Chinese GB/T 16886.5 framework align?
The Chinese framework for the in-vitro cytotoxicity testing is GB/T 16886.5-2017, 医疗器械生物学评价 第5部分：体外细胞毒性试验 (Biological evaluation of medical devices — Part 5: Tests for in vitro cytotoxicity), administered by the National Technical Committee for Biological Evaluation of Medical Devices (TC248) under the NMPA. The 2017 edition replaced the 2003 edition, with the revisions to the extract-preparation section, the test-procedure section, and the result-evaluation section. GB/T 16886.5-2017 is the Chinese national adoption of ISO 10993-5:2009, and the two standards are aligned in their technical content.
The GB/T 16886.5 framework parallels the ISO 10993-5 framework in every respect:
- The three test categories (the extract test, the direct-contact test, the agar-diffusion test) are defined identically.
- The standard cell line is the L929 mouse fibroblast, which has the extensive historical database in the Chinese testing practice, and which the published GB/T 16886.5 application research uses.
- The recommended extraction conditions are the serum-containing culture medium at 37 ± 1 °C for 24 ± 2 hours, the preferred condition for the Chinese-market testing.
- The 70 % cell-viability threshold is the pass criterion, aligned with ISO 10993-5.
- The qualitative and the quantitative evaluation approaches are defined identically, with the reactivity grading (0 to 4) and the assay-based viability measurement.
A point that affects every Chinese-market project: the NMPA submission for a medical device requires the GB/T 16886.5 test report, in Chinese, for the biocompatibility section of the registration dossier. A device tested under ISO 10993-5 for the international market can usually be reported under GB/T 16886.5 for the Chinese market without the additional testing, because the technical content is aligned — but the report must be re-issued with the GB/T 16886.5 references, and the Chinese-specific extraction conditions (the serum-containing medium at 37 °C, 24 hours) must be confirmed or applied.
The GB/T 16886 series — the Chinese adoption of the full ISO 10993 series — covers the complete biological-evaluation framework (Part 1 the evaluation-and-testing framework, Part 5 the cytotoxicity, Part 10 the sensitisation, Part 11 the systemic toxicity, and so on), and the cytotoxicity test (Part 5) is the first test in the GB/T 16886 biological-evaluation programme for the Chinese-market device. The cytotoxicity test gates the downstream biological evaluation — a cytotoxic material is not tested further, because the cytotoxicity is the disqualifying result — and the correct execution of the GB/T 16886.5 test is the most consequential biocompatibility decision for the Chinese-market device.
FAQ
Which standard should my device be tested to?
ISO 10993-5 (the international standard) for most regulatory pathways; USP <87> for the U.S. Pharmacopeial plastics classification; GB/T 16886.5-2017 for the Chinese NMPA submission; ISO 7405 for the dental materials. The companion standard ISO 10993-12 (sample preparation) applies to all. We confirm the regulatory pathway before quoting, because the test-method and the reporting requirements are driven by the pathway.
What cell line is used?
The L929 mouse fibroblast is the standardised cell line for the ISO 10993-5 / GB/T 16886.5 testing, recommended for its extensive historical database and its reproducibility. Other cell lines (the V79, the BALB/3T3, the primary cells) can be used where scientifically justified, but the L929 is the default, and the regulatory authorities expect the L929 unless the rationale for the alternative is documented.
What is the pass criterion?
The cell viability ≥ 70 % of the control, under the quantitative MTT or equivalent assay. A material with the viability ≥ 70 % is non-cytotoxic; a material with the viability below 70 % is cytotoxic, and the dose-response (the serial-dilution result) documents the cytotoxic potency. The qualitative grading (0 to 4 reactivity) is reported alongside the quantitative result.
Why does the extraction condition matter?
Because the extraction condition (the temperature, the time, the vehicle, the surface-area-to-volume ratio) directly determines the concentration of the leachables in the extract, and the extract concentration directly determines the cytotoxicity result. The same material extracted at 37 °C for 24 hours and at 70 °C for 24 hours can produce different results, and the ISO 10993-12 extraction condition must be chosen to represent the clinical exposure and documented in the report.
My material is cytotoxic — what next?
The cytotoxicity is a disqualifying result for the biocompatibility programme, and the material cannot proceed to the further biological evaluation without the resolution. The resolution path is the material modification (the change of the cytotoxic component, the reduction of the leachable, the surface treatment), the extract-condition verification (the confirmation that the cytotoxicity is real and not an extraction-condition artefact), and the re-testing. The cytotoxicity test is the screening test, and the positive result is the early warning that prevents the costly downstream failure of the biologically incompatible material.
Our cytotoxicity testing service
Our laboratory provides cytotoxicity testing across the full ISO 10993-5 method range — the extract (elution) test, the direct-contact test, and the agar-diffusion test — with the quantitative assays (MTT, XTT, NRU, LDH, ATP-luminescence, colony-forming) and the qualitative microscopic grading, under the ISO 10993-12 sample-preparation framework. For the Chinese market, we test under GB/T 16886.5-2017 and report in Chinese for the NMPA submission. Each project begins with a scoping step that confirms the device type, the material, the intended clinical exposure, the target market and the regulatory pathway, so the report you receive answers the question your regulator, your clinical team or your quality system will actually ask.
We prepare the extracts under the ISO 10993-12 / GB/T 16886.5 conditions (the defined surface-area-to-volume ratio, the defined extraction temperature and time, the defined vehicle), run the test with the L929 cells (or the justified alternative), apply the qualitative grading (0 to 4 reactivity) and the quantitative viability assay (with the 70 % threshold), and run the positive and the negative controls under the identical conditions. For the Japanese-market submission, we run the colony-forming assay. Reports are issued with the standard, the method, the extraction conditions, the qualitative grade, the quantitative viability percentage, the control results, and the pass/fail conclusion explicitly stated, with the microscopic images and the absorbance data included where the result depends on them, in a format suitable for the EU MDR technical documentation, the FDA 510(k) / PMA submission, the NMPA registration dossier or the internal quality audit.
To start a project, send us the device type, the material, the intended clinical exposure (the contact duration, the contact tissue), the target market, the regulatory pathway, and the test method you believe applies (or let us confirm it). We will return a project scope, sample requirement, schedule and quotation, and begin testing on your confirmation.