Overview
ISO/TR 10993-55:2023 - "Biological evaluation of medical devices - Part 55: Interlaboratory study on cytotoxicity" reports the results of an international interlaboratory study (conducted in 2006) that compared two in vitro cytotoxicity protocols. The technical report documents how neutral red uptake (NRU) and colony formation assay (CFA) protocols performed across multiple laboratories, describes test materials and extraction/dilution procedures (Annexes A and B), and explains how the findings informed the revision of ISO 10993-5 and helped define practical thresholds for cytotoxic effects.
Key topics and technical requirements
- Test methods evaluated
- Neutral Red Uptake (NRU) assay - protocol based on an ICCVAM validation study.
- Colony Formation Assay (CFA) - protocol based on Japanese guidelines for biological testing.
- Interlaboratory design
- 12 participating laboratories across six countries; 11 NRU reports and 10 CFA reports received.
- Participants included commercial CROs, manufacturer internal labs and research institutes.
- Reference materials and controls
- RM-A: segmented polyurethane with 0.1% zinc diethyldithiocarbamate (ZDEC).
- RM-B: segmented polyurethane with 0.25% zinc dibutyldithiocarbamate (ZDBC).
- RM-C: high-density polyethylene (negative reference).
- Sodium lauryl sulfate (SLS) used as a common positive control (IC50 range 22.0–85.9 µg/ml; mean ~60.7 µg/ml).
- Endpoints and metrics
- IC50 (concentration producing 50% inhibition of viability) used for NRU and CFA.
- CFA reported plating efficiency (PE) variation among labs (control PE range ~62.8%–108.7%, mean ~84.6%).
- NRU showed generally higher IC50s for RM-A than CFA (indicating lower sensitivity in NRU under shorter exposure in this study).
- Practical findings
- Document highlights interlaboratory variability, sensitivity differences between NRU and CFA, and the need for standardized protocols and reference materials to improve reproducibility.
Practical applications - who uses this standard
- Medical device manufacturers performing biological evaluation and preparing regulatory submissions.
- Contract research organizations (CROs) and testing laboratories validating cytotoxicity assays.
- Regulatory agencies and safety assessors referencing assay performance and reproducibility when assessing device biocompatibility.
- Standards developers and quality managers using historical data to refine testing criteria and acceptance thresholds.
- Relevant to anyone implementing ISO 10993 biological evaluation workflows and deciding on appropriate in vitro cytotoxicity methods.
Related standards and guidance
- ISO 10993-1 (biological evaluation process and required tests)
- ISO 10993-5 (in vitro cytotoxicity - procedures and interpretation), revised in part based on findings from this interlaboratory study
- ICCVAM validation studies and national guidance referenced for specific protocols
Keywords: ISO/TR 10993-55, cytotoxicity interlaboratory study, neutral red uptake, colony formation assay, IC50, biological evaluation of medical devices, ISO 10993-5, reference materials RM-A RM-B RM-C.