What pyrogen Tests Are Needed For FDA-Approved medical Devices?
What are endotoxins?
Endotoxins come from the cell walls of gram-negative bacteria. The endotoxins themselves are molecules with fat (also known as lipid) and complex sugar components (also known as polysaccharides). This is why endotoxins are also known in scientific literature as lipopolysaccharides (LPS). Endotoxins are also considered pyrogens because they trigger the innate immune system and produce fever when released within the human body.
What are pyrogens?
Pyrogens are molecules or substances that cause a feverous reaction when they enter the human body. Endogenous pyrogens (such as the cytokine interleukin-1) are found naturally within the human body. Endogenous pyrogens trigger fever when naturally produced by the body. Exogenous pyrogens are molecules found outside of the body, such as endotoxins from gram-negative bacteria or pyrogenic prions. For context, prions are misfolded proteins that can transmit their misfolded shape onto normal versions of the same protein. Prions are of most concern for medical products and devices that interact with brain or spinal tissues. Endotoxins are the primary pyrogens of concern for medical devices. Endotoxin-triggered fevers can be caused by triggering endogenous pyrogen production or activating toll-like receptors (TLRs) within the body. Clinically, the fever produced by endogenous cytokines is indistinguishable from the fever produced by exogenous pyrogens such as endotoxins.
Which pyrogen and endotoxin tests are needed for FDA-approved medical devices?
Interfering factors testing determines if medical device extracts contain glucans or other limulus amoebocyte lysate (LAL)-reactive components that are not endotoxins. If glucans are the only interfering factor, traditional endotoxin LAL testing can be performed with amoebocyte lysates that do not respond to glucans, or glucan-blocking agents can be used. In the rare circumstance where neutralization or dilution of a medical device extract does not eliminate the interfering substances, pyrogenicity testing from USP 151 will be required instead of traditional LAL testing.
How are medical device extracts prepared for bacterial endotoxin testing?
Medical device extracts are created by soaking the device or flushing the device’s fluid pathway with extraction fluid (often water) that has been heated to body temperature. The entire device or all device components in contact with patient tissues & fluids must contact the extraction fluid during elution. The extraction fluid is kept in contact with the device for at least an hour. Extracts from individual medical devices are often pooled for endotoxin testing.
Product families for endotoxin testing are defined by common construction materials and shared components. If material construction and components are similar enough, one device may be tested to represent all others within the product family. If a product family contains many sizes, the device with the largest surface area will be tested to represent the entire device family. When it comes to kits, each type of device must have its own product endotoxin limit and must be tested and evaluated individually.
How is bacterial endotoxin testing for medical devices performed?
A bacterial endotoxins test (BET) uses an assay known as the Limulus Amoebocyte Lysate (LAL) test. BET testing is considered a pyrogenicity test. However, do not confuse BET testing with rabbit pyrogen testing (USP 151). LAL is an extract of blood cells from the Atlantic horseshoe crab. LAL detects the lipopolysaccharide of the cell wall of gram-negative bacteria. LAL indicates LPS presence through clotting and gelling based on the amount of LPS in a sample, allowing for precise calculations to be made for endotoxin concentration.
To create a standard endotoxin stock solution for a BET, LAL is dissolved in a water or buffer solution free of endotoxins and interfering factors. All endotoxin stock solutions are calibrated to the current World Health Organization International Standard for Endotoxin. Endotoxin is expressed in Endotoxin Units (EU). One USP Endotoxin Unit (EU) is equal to one International Unit of endotoxin. Then a serial dilution of the endotoxin standard is prepared. Solutions for samples to be tested are prepared by immersing the device or dissolving the product in the same water or buffer solution used for the endotoxin stock solution. If necessary, the pH of the solution will be adjusted to fall within the range of 6.0-8.0.
A LAL gel-clot technique is used for detecting or quantifying endotoxins based on lysate reagent clotting upon endotoxin exposure. A series of tests are performed to confirm the sensitivity of the lysate solution and for any interfering factors. The sensitivity of the lysate solution is considered to be the minimum concentration of endotoxin required to cause the lysate to clot under test conditions. Both quantitative and limit testing using the LAL gel-clot technique can be performed. For brevity, only the bacterial limit test is described below.
For bacterial endotoxin limit testing of your medical device, four solutions (Solutions A, B, C, and D) are prepared. The details of these solutions are shown in Table 1 of our article on BET. Solutions B and C are positive controls with standard endotoxin solution at twice the concentration of the lysate sensitivity. Solution D is a negative control that consists of water or buffer solution. Endotoxin limits for biologics and drugs can be found HERE. Additional information endotoxin limits for combination devices can be found HERE. If endotoxin limits are exceeded for your product, ways to control endotoxins during processing and depyrogenation studies are available.
What are the endotoxin limits for medical devices marketed in the United States?
The endotoxin limit for a medical device is:
- No more than 20 USP Endotoxin Units (EU) per device.
- Not more than 2.15 USP EU for devices in contact with cerebrospinal fluid.
- Note: Intraocular devices may also require a lower endotoxin limit.
The endotoxin limit for the extracting or rinsing solution (S) is:
S = (K × N)/V
Where:
K = amount of endotoxin allowed per device
(20 Endotoxin Units per device; 2.15 for intrathecal devices)
N = number of devices tested
V = total volume of the extract or rinse
Summary
Overall, endotoxins are pyrogens (also called LPS) that come from the cell walls of gram-negative bacteria. Medical devices have endotoxin limits because high exposure to endotoxins causes fever and, in extreme cases, death. Bacterial endotoxin and pyrogen testing for medical devices differ from endotoxin tests for other products, as medical device extracts must be prepared for endotoxin testing. Medical device extracts must also pass interference testing to ensure that the extractions do not interfere with the LAL endotoxin assay. In the rare circumstance where neutralization or dilution of a medical device extract does not eliminate the interfering substances, in-vivo pyrogenicity testing from USP 151 will need to be performed instead of traditional LAL testing. When LAL bacterial endotoxin testing is performed, the endotoxin limit for a medical device must not be more than 20 USP Endotoxin Units (EU) per device and not more than 2.15 USP EU for devices in contact with cerebrospinal fluid. All in all, ensure you choose a contract testing organization that can support you with appropriate bacterial endotoxin and pyrogen testing for your unique medical device needs.
Ethide Labs is a contract testing organization that specializes in Bacterial Endotoxin Testing. Ethide Labs also offers Microbiology Testing, Bioburden Testing, Cytotoxicity Testing, Ethylene Oxide Residual Testing, Sterility Testing, Environmental Monitoring & Package Integrity Testing services for medical device companies and allied industries. Ethide is an ISO 13485 certified facility.
References
Charles A. Dinarello. Review: Infection, fever, and exogenous and endogenous pyrogens: some concepts have changed. Innate Immunity. August 1, 2004.
Galanos C. and Freudenberg M. A. Bacterial endotoxins: biological properties and mechanisms of action. Mediators of Inflammation. 1993; 2(7): S11–S16.
United States Pharmacopeial Convention. <85> Bacterial Endotoxins Test. Rockville, MD, USA. 2021. (USPC <85>).
United States Pharmacopeial Convention. <161> Medical Devices- Bacterial Endotoxin And Pyrogen Tests. Rockville, MD, USA. 2021. (USPC <161>).
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