Sample representativeness and quantity determination for test articles have been discussed in my previous posts. Continuing on the topic of what to consider when performing biological testing, this post will cover the extraction conditions to be applied for testing. Extraction conditions are also important when completing the submission form for the test facility. Different tests as well as test articles can warrant different parameters. As such, referencing ISO 10993-12 is essential to ensure testing is performed appropriately and accurately.
In extraction-based testing, the intent is to create conditions that cause extractables to be released from the test article and into the test extract, which are then tested for potential toxicity. It is recommended that the testing parameters used are for harsher conditions than the conditions the test article would be exposed to in a clinical setting so as to evaluate worst-case exposure while not so extreme they cause significant degradation. Per Annex C of ISO 10993-12, “Material should be extracted at temperatures which provide maximum extractables without material degradation.” Dissolution of the test article can create challenges for biological testing during dosing of the animals, as the test extracts may have been altered in viscosity, osmolality, or pH. These may cause failures not representative of the test article itself. Therefore, the extraction conditions should be exaggerated to provide a sufficient hazard potential from the test article without causing significant damage, such as severe swelling or degradation.
Regarding the polar and non-polar extractions for biological testing, ISO 10993-12, Clause 10.3.1 provides a list of acceptable extraction conditions for biological testing based on historical precedent. The list of acceptable extraction conditions is as follows:
- 37°C for 24 hours
- 37°C for 72 hours
- 50°C for 72 hours
- 70°C for 24 hours
- 121°C for 1 hour
For cytotoxicity testing 37°C for 24 hours is acceptable to evaluate devices and/or materials that are categorized as limited contact. This is because temperatures exceeding 37°C can adversely impact the chemistry or stability of the culture medium used in the testing. For devices and/or materials that are intended for prolonged or long-term contact, the temperature of 37°C for cytotoxicity is still applicable, but a 72-hour extraction time is strongly recommended. If these parameters are not followed and a strong rationale is not provided, there is a high risk of the device file being flagged in a submission.
For extraction-based testing outside of cytotoxicity the 50°C for 72 hours parameter is the most commonly used and accepted by the FDA and other notified bodies to provide sufficient exaggerated conditions. As extraction parameters are material dependent, there may be instances where other parameters can be used. However, since other parameters are less common, they will most likely be flagged by the reviewer. Therefore, selecting other parameters, while they are listed in ISO 10993-12, requires a clear justification.
Examples of when 37°C may be considered would be for surface devices which make brief or intact skin contact, as 37°C is the average body temperature. The same principle applies to externally communicating devices, provided it can be shown that they are not exposed to temperatures higher than 37°C (e.g., delivery systems). The temperature of 121°C should only be used if the materials of the test article are confirmed to remain stable. Examples include devices made of ceramic or metal which have been approved for steam sterilization at this temperature. Note that these are general examples; when it comes to specific test articles, testing conditions and extraction parameters are to be evaluated on a case-by-case basis. Since 50°C for 72 hours is accepted as the most widely used parameter, it is considered the first choice, provided the test article is expected to remain stable under these conditions. If it has been observed that 50°C causes excessive degradation of the test article or there is a material with a known glass transition near this temperature, this can be used as justification as to why 37°C may be used. An example of this occurrence is the testing of an absorbable suture. While the device is intended to degrade over time, the temperature of 50°C causes the device to break down in a manner that is not clinically representative. Therefore, the use of 37°C as an extraction temperature would be more relevant.
Biocompatibility continues to be a complex subject with many contingencies. Should further investigation or assistance be needed on your biological testing, do not hesitate to contact Nelson Labs Expert Advisory Services at [email protected]. The team will be happy to assist you.
