Endotoxin Testing

Endotoxin testing, or a bacterial endotoxin test (BET), such as LAL (limulus amebocyte lysate), is an in vitro assay used to detect bacterial endotoxins. The BET uses the lysate in blood cells from horseshoe crabs to detect bacterial endotoxins, thus warning quality control microbiologists of possible bacterial endotoxin contamination in their samples.

A bacterial endotoxin is a type of pyrogen (a fever-causing agent) and is found in the cell wall of Gram-negative bacteria. If certain amounts of endotoxin are introduced into the bloodstream or spinal fluid via a parenteral drug or medical device, fever, septic shock, organ failure, or death can occur. Due to the possibility of serious illness or death, all injectable pharmaceutical products and implantable medical devices that come into contact with the bloodstream or spinal fluid are tested for endotoxins to ensure patient safety. These products and devices are tested for endotoxins with a bacterial endotoxin test (BET). The most reliable and accurate BET test utilizes LAL (limulus amebocyte lysate) to detect bacterial endotoxins in final product water from pharmaceutical water systems, and raw materials. Charles River offers a variety of bacterial endotoxin test FDA-licensed LAL reagents including the exclusive Endosafe^® LAL cartridge technology, a rapid, simplified FDA licensed LAL test.

All injectable pharmaceutical products and implantable medical devices, nuclear medicine and compounded parenterals need to be tested to ensure there is no presence of endotoxin, which can lead to a pyrogenic response (fever) and symptoms of septic shock. Endotoxins can be detected in these products and devices through bacterial endotoxin testing (BET). The most reliable and accurate bacterial endotoxin test uses LAL (limulus amebocyte lysate) to test for bacterial endotoxins. Charles River offers FDA-licensed LAL reagents for gel-clot, kinetic chromogenic, and kinetic turbidimetric BET test methods including the proprietary Endosafe LAL cartridge technology.

The rabbit pyrogen test (RPT) is performed to check for the presence of pyrogens in products that could contain a Gram-negative or Gram-positive bacteria. Rabbits are used because their body temperature increases when they are injected with a pyrogenic product. From 1942 to 1983, the rabbit pyrogen test was the standard for pyrogen testing; however, it was labor-intensive, lengthy, and expensive. In 1983, the FDA and US Pharmacopeia adopted the bacterial endotoxin test utilizing Limulus amebocyte.

As an endotoxin is a type of pyrogen, both assays test for harmful Gram-negative bacteria that can produce life-threatening symptoms if introduced into the bloodstream or spinal fluid. The rabbit pyrogen test is an in vivo (performed inside of a living organism) test to detect pyrogens. If a rabbit is injected with a pyrogenic product, its body temperature will increase making it possible to determine the presence of pyrogens. However, the RPT is prone to errors and can result in the rabbit's death.

A bacterial endotoxin test (BET), such as LAL (limulus amebocyte lysate), is an in vitro assay used to detect bacterial endotoxins. The bacterial endotoxin test uses the lysate from blood cells from horseshoe crabs to detect bacterial endotoxins. As the BET is an in vitro (performed outside of a living organism; not a destructive assay) assay, the three principles of Replace, Reduce, and Refine was a primary consideration when developing the test.

Endotoxin testing is important due to the risk of patient safety by not testing for endotoxins. If certain amounts of endotoxin are introduced into the bloodstream or spinal fluid via a parenteral drug or medical device, fever, septic shock, organ failure, or death can occur. It’s important to conduct testing in-process, as well as during final product testing, to ensure the safety of your product and patients. Learn more about endotoxin contamination control strategies in GMP Annex 1. 

Endotoxin testing is required for any product that is being intravenously entered into the human body. All injectable pharmaceutical products and implantable medical devices must be tested for the presence of endotoxins. If endotoxins are present, it can lead to a pyrogenic response (fever) or symptoms of septic shock. It’s safe to reference your local regulatory requirements for endotoxin testing, such as USP, FDA and EP chapters. 

Several endotoxin test methods can be utilized, such as gel-clot, kinetic chromogenic, and kinetic turbidimetric bacterial endotoxin test, as well as the proprietary Endosafe LAL cartridge technology test method. The primary and most trusted method by both regulators, pharmaceutical manufacturers, and the general patient population is the LAL method. 

The most popular bacterial endotoxin test uses LAL (limulus amebocyte lysate) to test for bacterial endotoxins with over 70 million tests performed annually. Charles River offers FDA-licensed LAL reagents for gel-clot, kinetic chromogenic, and kinetic turbidimetric BET test methods including the proprietary Endosafe LAL cartridge technology.