Endotoxin is a substance made by gram-negative bacteria or found on the outside of cell walls. Fever and headaches are frequently brought on by such endotoxins and pyrogens. There have also been reports of blood clots, kidney failure, and lung failure due to endotoxins. In the worst cases, these can trigger a full-blown chronic inflammation response within the body, which might result in multiple organ failure or death. Endotoxin testing is governed by standards from organizations like the Food and Drug Administration (FDA) and the European Medicine Agency. A product's sterility does not guarantee that it is free from endotoxin. To prevent adverse reactions in patients, it is necessary to also test drugs that are said to be sterile for pyrogens. The United States Pharmacopeia (USP), or European Pharmacopeia, determines whether endotoxins are present or absent in parenteral pharmaceutical products (EP).
Endotoxin testing is classified into three categories: the rabbit test, the limulus amoebocyte lysate test, and in vitro monocyte activation assay test (MAT).
The limulus amoebocyte lysate test (LAL test) is an assay based on the lysate of amoebocytes from horseshoe crab blood. It is the most popular technique for testing endotoxins. In a coagulation reaction, the lysate from horseshoe crab blood cells naturally interact with bacterial endotoxins. When endotoxin is detected, the clotting factor is released into the bloodstream as a defence against endotoxin. Even low quantities of bacterial endotoxin cause a coagulation cascade. This method has a high sensitivity in quantifying endotoxin's presence in blood.
The rabbit pyrogen test, which was initially reported in 1925, was the first commonly used technique to identify endotoxin in pharmaceuticals. The test liquid is injected into rabbits using this technique, and the animals are then watched for any changes in body temperature and feverish signs. The rabbit pyrogen test cannot be made quantitatively and requires a lot of time and money to do. Its use is likewise restricted to goods that do not have negative impact on test animals. The specificity of the rabbit pyrogen test is quite lesser, but it provides qualitative results. Pyrogen tolerance can be developed after receiving repeated injections which may give false results after some time.
For the detection of both endotoxin and non-endotoxin pyrogens, the Monocyte Activation Test (MAT) is an alternative method that uses animals. By co-incubating monocytes with the test sample, the monocyte activation test simulates the human immune response. Activated monocytes that are exposed to pyrogens produce the inflammatory cytokines that cause a febrile reaction. To identify the cytokines, an immunological assay (ELISA) utilizing particular antibodies and an enzymatic colour reaction are used.
Growing regulations on the use of animals for pyrogen testing are hindering the market growth of the rabbit endotoxin test and LAL test. In addition to its limited reactivity to endotoxin and endotoxin tolerance, some of the drawbacks of pyrogen testing and seasonal variation, laboratory-to-laboratory differences, type of species of rabbit, and other biological factors all have an impact on endotoxin testing. The test is also insufficient for some drug classes, such as cancer chemotherapeutic agents, radiopharmaceuticals, hypnotics and narcotics, some antibiotics, and vitamins.
In July 2021, the European Pharmacopeia released a note that they will replace rabbit pyrogen tests in upcoming years. The rabbit pyrogen test will no longer be used by the European Pharmacopoeia within the next five years, and the industry is encouraged to switch to the monocyte-activation test in its place.