Alternatives To LAL Pyrogen Testing
What are pyrogens and endotoxins?
Endotoxins come from the cell walls of gram-negative bacteria. The endotoxins themselves are molecules with both fat components and complex sugar components (also known as polysaccharides). The presence of fat components and complex sugars are 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. Endotoxins are the most ubiquitous pyrogenic contaminants for medical devices or products. However, there are other types of pyrogens. This article covers traditional bacterial endotoxin tests, LAL test procedures, and in-vivo pyrogen tests like rabbit pyrogen tests.
Why are endotoxins harmful, and how do endotoxins cause pyogenesis?
The lipopolysaccharide components that endotoxins are composed of are foreign to the human body. Thus, our immune system identifies LPS as something foreign that it should get rid of quickly. Compare our body to a house and our immune system to the inhabitants of a home. In this situation, the innate immune system treats LPS like an uninvited guest (such as a burglar or a rodent). The innate immune system entered does its best to get the uninvited guest (LPS) out of the house (the human body).
Though the immune system is commonly referred to as a single entity, there are two types of immunity, our innate immune system, and our adaptive immune system. The innate immune system is the immune system we have at birth. The natural immune system is general and non-specific. In reference to the example above, the innate immune system can identify an uninvited guest but cannot determine that the uninvited guest is a rodent or a wasp. When activated by something foreign to the human body, the innate immune system has a single mode of action. The immune system will raise the body temperature and release signaling proteins called chemokines and cytokines to increase circulation and recruit immune cells to the site of infection. This results in the pain, warmth, swelling, and redness experienced when you cut your finger or the fever and chills you experience when you have the flu. The adaptive immune system is specific, and we develop it over time. The adaptive immune system is what we activate when we vaccinate our body against specific bacterial or viral strains.
When LPS is detected within the human body, the innate immune system is activated. The activation of the innate immune system by these endotoxins results in a fever. Endotoxin’s ability to produce a fever upon exposure in the human body is why endotoxins are called pyrogens.
If endotoxins run unchecked within the human body, an individual can become septic and die. Thus, appropriate bacterial endotoxin testing for medical devices and products is vitally important to patient safety. Endotoxin testing is performed on raw materials to create pharmaceuticals, the final pharmaceutical formulation, and medical device products.
What is an in-vitro pyrogen test?
A bacterial endotoxins test (BET) uses an assay known as the Limulus Amoebocyte Lysate (LAL) test. BET testing is an in-vitro pyrogenicity test. However, do not confuse BET testing with rabbit pyrogen testing, which we will be described below. LAL is an extract of blood cells from the Atlantic horseshoe crab. LAL tests detect the LPS of the cell wall of gram-negative bacteria, even if these bacteria are dead. LAL tests detect LPS through clotting and gelling in the presence of LPS, allowing for precise calculations to be made as to the concentration of endotoxins in a sample.
What is an in-vivo pyrogen test, and when is it important?
In-vivo pyrogen tests are designed to test whether a product will produce a fever in patients when administered. An in-vivo pyrogen test determines fever production by measuring the rise in temperature of rabbits following an intravenous injection of a test solution. This in-vivo test is not required for most medical products or medical devices, as LAL tests are the gold standard. In-vivo pyrogen tests are only designed for products that the rabbit animal model can tolerate in a dose not exceeding 10 milliliters per kilogram injected intravenously within not more than 10 minutes. Where appropriate, a validated and equivalent in-vitro pyrogen or BET may be used in place of the in-vivo rabbit pyrogen test.
How is in-vivo pyrogen testing performed?
Testing preparation
For in-vivo pyrogen testing, all syringes, needles, and glassware must be pyrogen-free through heating at 250°C for not less than 30 min or by any other suitable method. Ensure all diluents and solutions for washing and rinsing of devices or parenteral injection assemblies such that they remain sterile and pyrogen-free. Control pyrogen tests will be performed using pyrogen-free diluents and solutions. Clinical thermometers, thermistor probes, or other temperature sensing devices with an accuracy of ±0.1°C are used for temperature monitoring of the rabbits. For these temperature-sensing devices, a full temperature reading must be reached in less than five minutes. Temperature-sensing probes are to be used in the rectum of the test rabbits.
Healthy, mature rabbits housed individually and in environments free of disturbances will be used for experimentation. It is important that these rabbits are housed in an environment with a temperature between 20° and 23°C with a variance in temperature of not more than ±3°C from the selected temperature. Before using a new rabbit in a pyrogen test, condition the rabbit not more than seven days before the pyrogenicity experiment with a sham test that includes all of the steps described in the procedure below except the injection. Rabbits are not to be used for pyrogen testing more frequently than once every 48 hours or within 2 weeks following a pyrogen test that rises its temperature to 0.6°C or more.
Testing procedure
Testing is performed in a designated pyrogen testing area under the same environmental housing conditions for the rabbits as described in “testing preparation.” Food is withheld from the rabbits during the testing period, but access to water is allowed. Use light-fitting neck stocks for rabbits with rectal temperature probes so that the rabbits can assume a natural resting position. Within 30 minutes before injection, determine the “control temperature” of each rabbit. The “control temperature” is the baseline temperature for determining any temperature increases after injection of test solutions. In any one group of test rabbits, use only those rabbits whose control temperatures do not vary by more than 1° from each other. Any rabbit with a temperature exceeding 39.8°C is excluded from the experiment. After the initial preparations described above, inject three rabbits’ ear veins with 10 milliliters of the test solution per kilogram of body weight. Each ear vein injection is to be finished within 10 minutes after the start of the injection. The test solution is either the product itself or washes of medical device surfaces that contact patient tissues during use. All test solutions are protected from microbial or other contamination before injection and prepared using aseptic techniques. Injections are performed after warming the test solutions are warmed to a temperature of 37 ± 2°C. After rabbits are injected, their body temperature is recorded at 30-minute intervals for one to three hours.
Interpretation of test results
Any temperature decreases during pyrogen testing are a zero rise in temperature. If no rabbit shows an increase in temperature of 0.5°C or above its control temperature, the product meets the requirements for the pyrogen absence. If a rabbit shows an individual temperature rise of 0.5°C or more, the pyrogen test is continued using five other rabbits. If not more than three of the eight rabbits show individual rises in temperature of 0.5°C or more. If the sum of the maximum temperature rises of the eight rabbits does not exceed 3.3°C, the product meets the requirements for the pyrogen test.
Summary
Overall, pyrogen testing can be performed in-vitro or in-vivo. In most cases, an in-vitro BET test will be sufficient to prove that your medical device or product meets the requirements for pyrogenicity. However, if in-vitro BET testing cannot be determined to be equivalent to in-vivo pyrogenicity testing, in-vivo pyrogenicity testing will need to be performed. In-vivo pyrogenicity testing evaluates the pyrogenic response of a product through measuring the rise in body temperature of rabbits when given an intravenous injection of the medical product or medical device extract. In general, if all rabbits do not have a 0.5°C or more increase in body temperature following injection, the product passes the pyrogenicity test. When outsourcing regulatory testing, ensure you choose a contract testing organization that can support you with appropriate pyrogen testing for your unique medical device or product needs.
Ethide Labs is a contract testing organization that specializes in pyrogen testing like Bacterial Endotoxin Testing. Ethide Labs also offers Environmental Monitoring, Microbiology Testing, Sterilization Validations, Bioburden Testing, Package Integrity Testing, Cytotoxicity Testing, and EO Residual Testing services for medical device companies and allied industries. Ethide is an ISO 13485 certified facility.
References
United States Pharmacopeial Convention. <151> Pyrogen Test. Rockville, MD, USA. 2021. (USPC <151>).
United States Pharmacopeial Convention. <85> Bacterial Endotoxins Test. Rockville, MD, USA. 2021. (USPC <85>).
