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The Rabbit and the Horseshoe Crab

23rd September 2014

The Rabbit and the Horse Shoe CrabThe LAL method for endotoxin detection was largely preceded by the Rabbit Pyrogen Testing by about 30 years.

Much information has already been provided on the procedures and details of the LAL endotoxin testing. The Rabbit Pyrogen Testing has mostly been outdated by the LAL procedure. Discussions about the Pyrogen Testing in this blog will give us a comparison and insight as to why LAL is now the preferred modality for endotoxin determination.

First we need to discuss Pyrexia. Pyrexia, or fever, is the elevation of the body temperature from the normal. Pyrogen is a substance which induces pyrexia. Endotoxin is our main pyrogen of interest due to its deleterious effects on the human body. The rabbit pyrogen test is basically a test which involves inoculating a rabbit with a product sample to determine the presence or absence of pyrogens. If a pyrogen is present in the test substance, it will cause an elevation of the animal’s body temperature from normal. Rabbits have similar temperature response to endotoxins as humans. For this reason, they are ideal for use in pyrogen detection.

The US Pharmacopeia gives a description on the administration of pyrogen testing. A test solution is administered intravenously into the animal’s ear. The rate of administration is not to exceed 10ml/kg for 10 minutes. All materials, diluents and solutions used should be sterile and pyrogen-free. Healthy, mature rabbits are selected and housed in temperatures of 20 to 30 degree Celsius. They are also kept free form any disturbance or excitement. This will later be discussed further as there are other situations which may cause increases in body temperature. A rabbit can only be subjected to a test solution once every 48 hours and well outside 2 weeks of having a body temperature greater than 0.6 degrees Celsius from its normal. The baseline temperature of the rabbit is measured not more than 30 minutes prior to the testing. It should not exceed 39.8 degrees Celsius. Rabbits used in group testing should have a variance in temperature of only 1 degree from each other. The rabbits are tested in temperatures similar to their housing. Food is withheld during the test. Water may be withheld or be made available to the test animals. Temperature is measured through rectal probes while the rabbit is restrained in its natural resting position. Test solutions are injected into the ear vein after being warmed to 35 to 39 degrees Celsius. Temperature readings are taken at 30 minute intervals for the next 1-3 hours. Three rabbits will be tested per solution.

VIEW ALSO Applications of the LAL Test in the Pharmaceutical Industry

A negative test is an elevation in the rabbit’s temperature of less than 0.5 degrees Celsius. The test solution may then be considered pyrogen free. A temperature elevation of 0.5 degrees or more will warrant a repeat of the testing in 5 more animal subjects. A negative result is an elevation in temperature of 0.5 degrees or greater in only 2 out of the 8 rabbits or if the total temperature rise in all 8 rabbits does not exceed 3.3 degrees Celsius.

The endotoxin limit is expressed as K/M. K is the threshold pyrogen dose for both humans and the rabbit animal subjects. K has a value of 5 EU/kg. M is the maximum human dose to be administered per kilogram in one hour. Intrathecal injections have a K value of 0.2 EU/kg.

There are numerous limitations in using the rabbit pyrogen testing for determining endotoxin content. Endotoxins and pyrogens are two entirely different entities. Endotoxin is a molecule contained in the cell wall of gram negative bacteria. It is a known pyrogen. Pyrogens are any substance which causes febrile responses in both humans and rabbits and it encompasses a lot of entities and situations beyond endotoxins. Mycobacteria, fungi and viruses which do not contain endotoxins may still cause febrile reactions. We have endogenous pyrogens, substances inherent to our metabolic system which can induce fever. We have interleukins which are known to raise body temperature during activation of the inflammatory pathway. Our thyroid and estrogen hormones are known to regulate body temperature and can elevate or decrease it depending on its level and activity. We have exogenous pyrogens that when introduced inside of our bodies can cause a febrile response. Bacteria, their toxins, vaccines and other microbial organisms like fungi, viruses and parasitic organisms can produce fever. Blood and blood products when transfused may cause a rise in body temperature. Medications we take in may cause different bodily reactions which may include elevated temperatures.

MORE ABOUT LAL: The Impact of Endotoxin on the Human Body

Other conditions may lead to elevations in body temperature. Exercise causes an increase in metabolic rates. The increase in blood flow and rapid cellular metabolism causes a transient fever. Emotional stress or excitation may cause a similar response. A tumor or malignancy, with its increased metabolic requirements and release of endogenous pyrogenic substances can also produce fever episodes. Temperature control of the body is regulated by the hypothalamus. Any defects or injuries to the hypothalamus and the central nervous system can alter the regulation of body temperature.

As there are so many factors which may cause pyrexia, there are also many contributors to variability in the rabbit pyrogenic testing. It is an in-vivo test, there is non-specificity for bacterial endotoxins, there is specie to specie variability and there is intra-subject variability. As rabbit pyrogen testing was developed for endotoxin determination, all the more were its limitations highlighted.

Being an in-vivo test, various studies have shown that multiple exposures to endotoxins and pyrogens produced tolerance in the test animals. Over time and after numerous testing, the febrile response of the rabbit became diminished. The bacteria Legionella pneumophilia has also been found to induce little pyrexia in rabbits but is readily detectable through the LAL procedure. In fact, a 1000 fold difference was found between the results of the two procedures in detecting Legionella endotoxin. L. pneumophilia is the bacteria responsible for causing the notorious pneumonia outbreak among the attendees of the American Legion convention at the Bellevue-Stratford Hotel in Philadelphia in July 1976. That outbreak involved 182 cases and resulted in the death of 29.

The rabbit test was determined to be inadequate for detecting pyrogenic substances in radiotherapy products, chemotherapy drugs, steroids, narcotics and other substances which had inherent probabilities to react with the human immune system and produce pyrogenic reactions even in the absence of bacterial pyrogens. This would lead to numerous false positive results. The pyrogen testing is also obviously more time consuming, rigorous and expensive. The biggest drawback of this test procedure, however, is the inability to quantify the endotoxin levels.

As the fable goes, the rabbit was fast and started early. In the comparison of the rabbit pyrogen testing with LAL, the rabbit is compared this time to the horse shoe crab, which like the turtle is a slow going resident of the sea. The pyrogen testing was adapted by the U.S. pharmacopeia in the early 1940s, predating the use of LAL endotoxin testing by 30-40 years. Due to the advantages of the LAL procedure in terms of cost, specificity, less variability, convenience and being quantifiable, the LAL test has now replaced pyrogen testing in detecting the presence of bacterial endotoxin. Thus, in our story, the horse shoe crab has undeniably outraced the rabbit.

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By: Lisa Komski In: Kit LAL