The recent publication by the U.S. Food and Drug Administration (FDA) of Guidance for Industry Pyrogen and Endotoxins Testing: Questions and Answers, dated June of 2012, the Department of Health and Human Services updated the agency’s thinking regarding pyrogen and bacterial endotoxin testing requirements—and has (rightly so) prompted medical device manufacturers to take a new look at their endotoxin testing programs.
Bacterial endotoxin is the cell wall from gram-negative bacteria. The cell wall is generally composed of lipid polysaccharide (LPS) material. Medical devices that contact circulating blood or cerebrospinal fluid, or are used in intraocular products or in vitro fertilization procedures, are required to be endotoxin-free. Endotoxinfree means that the device does not contain endotoxin levels high enough to elicit a fever response in a patient. This level is generally defined as 5EU/kg.
The majority of pyrogens found on medical devices are bacterial in nature. Some medical devices that are required by the FDA to be tested for endotoxins are:
• Intravenous catheters and tubing,
• Cardiac stents and angioplasty balloons,
• Certain implants such as pacemakers/defibrillators,
• Spinal catheters,
• Transfusion catheters,
• Intraocular lens implants,
• Cranial drills,
• Perfusion tubing,
• Dialysis tubing and filters,
• Vascular grafts, and
• In vitro fertilization catheters.
Historically, the FDA has required end-product testing of medical devices to release the lot. Therefore, most quality control (QC) labs and quality assurance (QA) units would submit three to 10 samples to the lab for testing. This testing involves following a validation program that, when performed, indicates the product is compatible with the detector system (horseshoe crab blood) lysate. Some products contain chemicals that inhibit or enhance the ability of the lysate to form a clot, or in the case of turbidimetric or chromogenic testing, to develop a reaction to endotoxin. Small amounts (under ppm) of bacterial endotoxins cause the lysate to clot or create a reaction. Therefore, it was always important to test the materials for compatibility.
This procedure was more appropriately called inhibition/enhancement testing. Once validated using three lots of materials, in accordance with the FDA’s Guidance, June 2012, the products can be tested under United States Pharmacopeia (USP) 35 2012 chapter 85, Bacterial Endotoxin Test. So, the majority of medical device manufacturers would test under these requirements and release a product to market when the results indicated an acceptable response. (See Figure 1)
An acceptable response is based on the dilution factor and the sensitivity of the reagent or lysate. The current requirement under USP section 161 is 20 endotoxin units per device. Intrathecally (cerebrospinal fluid) communicating devices have a much lower limit of 2.15 EU per device. Most medical devices have some level of endotoxin, albeit a very small amount. Using this information can be helpful in controlling endotoxin levels and quality attributes.
The Sampling Plan
When establishing a sampling plan for in-process and finished products, note that the FDA leans on the recommendations in the Quality Systems Regulations’ CFR 820 current edition, sections 21 CFR 820.70 21 and 820.250 for appropriateness of the sampling plans. AANSI/ AAMI ST 72 addresses sampling plans and sampling frequency. The requirement for sampling plans is generally limited to a production batch. Alternative sampling plans besides finish product testing (i.e., pre-sterilization) are acceptable but must be validated with appropriate manufacturing controls and testing. The FDA may ask for statistical endotoxin sampling plans in future submissions. The risk assessment regarding potential for contamination in raw materials, in process materials, and finished product would be a great step forward for many. The FDA is recommending a 30-day notice for sampling plan changes. Alternative plans will require more information for wet processes. These are production processes that utilize water during the manufacturing of the product or components (i.e., extrusion/washing or in-process testing, such as leak testing for balloon catheters). The ANSI/ASTM ST72 2002/R2010, Bacterial Endotoxin Test-Test Methodologies, Routine Monitoring and Alternative to Batch Testing from the Association for the Advancement of Medical Instrumentation in Washington, DC, provides more information on alternative sampling plans.
Sample Storage and Handling
While sample storage and handing processes are certainly important, most medical device samples that come into the lab for testing are stored under room temperature conditions. In the past, the endotoxin stability of medical devices has never been questioned. Endotoxins are sticky substances and generally can be very stable once on medical devices. Liquid medical devices would be the only exceptions. Liquid medical devices are those that are used for perfusion and organ preservation. Combination products (whereby the principal mode of action is a medical device) are high risk and should be tested for endotoxin stability.
If a device company is experiencing issues with waterborne organisms (from extrusion tanks), then the source of the endotoxin and stability of that contaminate should be considered during the shipment to the lab.
Extrusion water samples should be sent to the lab controlled at 4°C to 8°C for testing. Products should be shipped to the lab under controlled conditions until data is obtained that supports the shipment conditions. Shipment temperature and humidity conditions can vary widely. Spiking samples with endotoxin and bracketing the shipment conditions may be required for future submissions.
The FDA recommends that finished product samples for analysis of bacterial endotoxin can be pooled into a composite sample. Pooled extracts can be kept at stability temperatures until tested. Generally, this temperature is required by the practitioner to validate the stability storage and handling procedures. The extracts are generally aqueous (water for injection) washings from the internal blood contact surface of intravenous or transfusion assemblies. Bacterial endotoxins are not expected to be affected by 2°C to 8°C storage. There are many references to the theory that endotoxins do not rinse off medical devices completely with water. A water rinse generally requires vigorous mixing and flushing to remove endotoxins from the polymers. The FDA recommends 30 seconds of vigorous mixing from each of the product containers. They also request that individual containers be stored and available for retest. (See Figure 2)