Cytotoxicity and Hemocompatibility Testing

Cytotoxicity is usually the first biocompatibility test a device team runs, and hemocompatibility is often the most demanding. Together they answer two basic questions: does anything in this device harm living cells, and how does it behave when it meets blood? For medical devices and drug-device combination products, the answers shape test plans, timelines and submissions. Here is how the standards fit together and where programs tend to stumble.

The Risk-Based Framework

ISO 10993-1 sets the overall approach. Biological safety is evaluated inside a risk management process, starting with materials, manufacturing and the nature and duration of body contact. The 2018 edition is now withdrawn. ISO published the sixth edition, ISO 10993-1:2025, in November 2025, reorganized to align with ISO 14971 and with added guidance on exposure duration, materials characterization and identification of biological hazards (ISO, 2025). TÜV NORD notes that the revision also asks manufacturers to consider reasonably foreseeable misuse and raises expectations for the Biological Evaluation Plan (TÜV NORD, 2025).

In the US, FDA’s guidance Use of International Standard ISO 10993-1 was updated in September 2023 (FDA, 2023). The update remained largely the same as the 2020 version but moved some Attachment A endpoints from suggested to required and added Attachment G, a reduced-testing pathway for certain materials that contact only intact skin (Exponent, 2023). At the time of writing, the FDA guidance page does not reference the 2025 edition, so check FDA’s recognized consensus standards database before relying on it in a submission.

Contact category drives the plan. Devices are classed by nature of contact (surface, externally communicating or implant) and duration: limited (24 hours or less), prolonged (more than 24 hours up to 30 days) or long-term (more than 30 days). Cytotoxicity is assessed for essentially every device. Hemocompatibility applies when a device contacts circulating blood, directly or indirectly.

Cytotoxicity Testing Under ISO 10993-5

ISO 10993-5:2009 describes in vitro cytotoxicity methods. Most labs use L-929 mouse fibroblasts grown as a monolayer (Nelson Labs). Three formats dominate:

• Elution (MEM extract). The device is extracted in serum-supplemented MEM at 37 °C and the extract placed on cells. It is the most common format and allows dilution series.
• Agar diffusion (overlay). The material sits on an agarose layer over the cells for 24 to 26 hours. Useful for some materials, though not every regulator favours it.
• Direct contact. The test article is placed on the cell layer. FDA suggests considering both direct contact and elution for novel materials.

Results are qualitative or quantitative. Qualitative scoring uses a 0 to 4 grade based on lysis and morphology, and a grade of 2 or lower generally passes. Quantitative tests, such as tetrazolium-based assays, report percent viability, and 70% or higher passes (Nelson Labs).

Method	Contact	Typical readout
MEM elution	Extract on cells	Grade 0–4 or % viability
Agar overlay	Indirect, through agarose	Zone of lysis, grade 0–4
Direct contact	Material on cells	Grade 0–4 around material

The test is sensitive, and not always reproducible. In an interlaboratory comparison, 52 labs tested polyethylene tubing (expected non-cytotoxic) and PVC tubing (presumed cytotoxic) using a predefined elution protocol. Only 58% identified the expected result for both materials. Serum in the extraction medium and longer incubation increased sensitivity, and the authors concluded the standard’s specifications are not explicit enough to yield comparable results (Gruber and Nickel, 2023).

“A cytotoxicity failure is a data point about your materials and process, not a verdict on your device.”

Hemocompatibility Under ISO 10993-4

ISO 10993-4:2017 classifies blood-contacting devices by intended use and contact duration and sets out structured test categories (ISO, 2017). The categories are thrombosis, coagulation, platelets, hematology (including hemolysis) and the complement system. The standard deliberately avoids detailed test requirements because knowledge of blood interactions is still limited. A fourth edition is in development; its draft closed voting in September 2026 (ISO, 2026).

FDA’s 2023 guidance is more specific (FDA, 2023):

• Direct blood contact. Consider hemolysis, complement activation and thrombogenicity unless the risk assessment already addresses them.
• Indirect blood contact. Hemolysis is generally enough, with some in vitro thrombogenicity work for novel cardiovascular materials or devices that release chemicals into blood.
• Hemolysis method. For direct-contact devices, run both direct and extract methods per ASTM F756 or an equivalent.
• Waivers. Justify any omitted test by comparing materials, processing and geometry to a legally marketed device with similar blood contact.

ASTM F756 reports express results as a hemolytic index. Contract lab reports commonly grade 0 to 2% as non-hemolytic, 2 to 5% as slightly hemolytic and above 5% as hemolytic. Complement is often measured as SC5b-9, and coagulation by partial thromboplastin time. Because acceptance limits for many hemocompatibility endpoints are not fixed, a marketed comparator device makes results far easier to interpret.

Extraction Conditions Matter

Most of these tests depend on extracts prepared under ISO 10993-12. Its standard conditions include 37 °C for 24 or 72 hours, 50 °C for 72 hours, 70 °C for 24 hours and 121 °C for 1 hour (Nelson Labs, 2025). For cytotoxicity, FDA recommends extracting at 37 °C for 24 to 72 hours in culture medium with 5 to 10% serum, which pulls out both polar and non-polar constituents (FDA, 2023). Higher temperatures can alter culture medium or degrade materials such as absorbables. FDA also expects testing on the final finished device, sterilized, or a justified representative article.

When a Test Fails

A cytotoxic or hemolytic result calls for an investigation, not an immediate retest. FDA asks sponsors to explain the likely source of a finding such as grade 2 cytotoxicity and to weigh it within the overall benefit-risk assessment. For inherently cytotoxic materials, dilution series show where toxicity stops, and that level is compared with the clinical dose and duration.

• Check the test first. Controls, extraction ratio, vehicle, pH and osmolality shifts.
• Look at the process. Residual processing aids, mould release agents, cleaning residues and sterilant residuals are frequent culprits.
• Characterize the chemistry. Extractables data often identify the cause faster than repeat biology.
• Document under change control. Root cause, corrective action and any retest rationale belong in the quality record.

Chemical Characterization as a Partner

ISO 10993-18:2020 takes a risk-based approach to identifying chemicals in or released from a device. Compounds above the analytical evaluation threshold then go to toxicological risk assessment under ISO 10993-17:2023 (NAMSA, 2026). FDA accepts that chemical analysis of the final device can support waiving some tests. It also warns that chemistry alone rarely suffices, because it does not capture surface properties or geometry that affect thrombogenicity. The same rigour you would apply to compendial method verification applies to the analytical methods behind E&L studies.

Combination Products and GLP

For drug-device combination products, FDA says general biocompatibility principles apply but extra or modified testing may be needed, and sample preparation may depend on the product type and endpoint. Sponsors should consult the lead FDA Center and review division early. FDA also expects biological safety studies to follow Good Laboratory Practice under 21 CFR Part 58, with a compliance statement or a detailed account of deviations. FDA does not accept ISO/IEC 17025 accreditation as a substitute.

What This Means for Device and Combination Product Teams

• Write the Biological Evaluation Plan first. Let the risk assessment, not habit, choose the endpoints.
• Plan for the 2025 edition. Map gaps against ISO 10993-1:2025 and confirm regulator recognition status.
• Control extraction choices. Specify vehicle, temperature, time and serum content and justify each.
• Use a comparator for blood tests. A marketed device anchors hemocompatibility interpretation.
• Treat failures as CAPA events. Investigate under your quality system and link to supplier and process controls.

Terraforme Biosciences helps device and combination product teams build biological evaluation plans, manage contract lab studies and investigate failures within a compliant quality system. Learn more about our regulatory guidance services, or explore our quality management systems support.

Conclusion

Cytotoxicity and hemocompatibility testing are early signals of how a device will behave in the body. Run them inside a risk-based plan, control the extraction and test variables, and pair them with chemistry. Then a surprise result becomes a solvable problem rather than a submission delay.