The total injected volume per rabbit is not less than 1 ml and not more than 10 ml of solution. In 2008, iccvam evaluated the status of five proposed in vitro test methods, all types of mat, to replace the rabbit pyrogen test, and concluded that none of the proposed test methods could completely replace the rabbit pyrogen test to detect gramnegative endotoxin, a common pyrogenic substance. The lal limulus amebocyte lysate testing, also known as bacterial endotoxin testing, is an in vitro assay used to detect the presence and concentration of bacterial endotoxins in drugs and biological products, and is an important part of pharmaceutical microbiology. For the rabbit pyrogen test, inject a minimum of 10 times this dose per kg of body weight into each rabbit. These should be washed with water for injection and then heated at 260c for two hours. The pyrogen test is designed to limit the risk of a febrile reaction following parenteral administration of drugs. Pdf limitations of the rabbit pyrogen test for assessing. Materialmediated pyrogens in medical devices altex. The in vivo rabbit pyrogen test rpt is considered to be the gold standard for. As the administration of substances containing pyrogens can lead to shock, fever or death, parenteral products are required to undergo testing to confirm the absence of these agents. The pyrogen test is designed to limit the risk of febrile reaction following parentral administration of drugs. Therefore, 0 iukg batch release, 5iukg and 10iukg were test ed in the rabbit pyrogen test, whereas 0 iuml, 0. The lal tests in general were 3 to 300 times more sensitive than was the usp rabbit pyrogen test method.
Do not use a rabbit for pyrogen testing more frequently than once every 48 hours, nor prior to 2 weeks following a maximum rise of its temperature of 0. Pyrogen testing is performed to detect pyrogenic or fever responses following exposure to a medical device, biomaterial, or their extracts. Bioreliance also offers a general safety test to detect extraneous toxic contaminants. The rabbit pyrogen test was first described by florence seibert in 1925 6. Questions and answers as an alternative to the rabbit pyrogen test which should be validated according to usp.
O a pyrogen is a foreign substance that causes a fever temperature elevation in an animals body. Pyrogen test definition of pyrogen test by medical dictionary. In three parts, this page overviews the history of pyrogen testing, what pyrogens are and finally the different assays that are commercially available to test for them as well as an evaluation of their strengths and weaknesses. The test involves the measurement of rise in body temperature of rabbits following iv injection of sterile solution of. Rabbit pyrogen test was the most commonly used pyrogen testing method.
Since its conception alternative methods with improved sensitivity, relevancy. Second, the rabbit may be used for the pyrogen test only when the body temperature in all 8 mea. The pyrogenic response may be due to endotoxin contamination or a materialmediated response associated with nonendotoxin materials that can cause a fever response. The rabbit pyrogen test rpt has been used since the 1940s. The lal and the usp rabbit pyrogen test data correlated well when the endotoxin in a relatively pure and undegraded form was examined. Jan 20, 2014 do not use a rabbit for pyrogen testing more frequently than once every 48 hours 0. Two widely used pyrogen test methods are the rabbit pyrogen test rpt and the bacterial endotoxin test bet based on limulus amebocyte lysate lal. Pyrogens are feverproducing agents such as bacterial endotoxins and chemicals. Low endotoxin recovery please provide evidence that the drug product formulation does not interfere with endotoxin recovery in the lal test. The rabbit test is no longer widely used and has been largely been replaced, for the testing of parenteral drug products, by the lal test, particularly within europe under the requirements of the european pharmacopoeia commission 7 and the european medicines agency. The rabbit test is no longer widely used and has been largely been replaced, for the testing of parenteral drug products, by the lal test, particularly within europe under the requirements of the european pharmacopoeia commission 7 and the european medicines agency 8. Endotoxins, which are a type of pyrogen, are lipopolysaccharides present in the. Comparative study ofrabbit pyrogen testandhuman whole. Comparison of limulus amebocyte lysates and correlation with.
Contact our experienced scientific staff to learn about more about rabbit pyrogen testing. Thehumanwhole blood assaywas performed as described previously hartung, t. The rabbit pyrogen test is used, both by vaccine manufacturers and national control laboratories, to assess a number of vaccines for their pyrogen content, including the multivalent diphtheria, tetanus and hepatitis b vaccine, hepatitis b, rabies, tick borne encephalitis, and those based on meningococcal outer membrane vesicles omvs. Oct 11, 2015 rabbit pyrogen test preliminary test sham test intravenous injection of sterile pyrogenfree saline solution warm the pyrogen free solution up to 38. The rabbit pyrogen testing method surfaced in the 1940s after some patients became ill from intravenous drugs. Additionally, the usp pyrogen test mentions that, a validated, equivalent in vitro pyrogen or bacterial endotoxin test may be used in place of the in vivo rabbit. The pyrogen test on rabbits is based on the measurement of the increase in the rabbits temperature upon being injected with a product that could contain a contaminant of the pyrogen type. The required high number of pyrogen tests on rabbits and the variable sensitivity of that test system made the development of alternative tests highly necessary. Thesensitivity andrelative simplicity ofthe limulus amoebocyte lysate lal test for the detection ofbacterial endotoxinindicate thatit can be used to replace the moreelaborate, ex.
Ursel liideritzpiichel and thomas montag paulehrlichinstitut, dlangen summary a comparative study of rabbitpyrogen test and human whole blood assay wasperformed onreleasedpreparations ofhuman serum albumin. Rabbit pyrogen tests applied and environmental microbiology. The principal problem dealt with is determining an endotoxin limit for the lal assay. It produces results that are both reliable and reproducible, which are. Limitations of the rabbit pyrogen test for assessing meningococcal. The pyrogens, as their name suggests, refer to all the substances that cause an increase in fever, also known as pyrexia. The us interagency coordinating committee for the validation of alternative test methods iccvam declared that mat is suitable after a productspecific validation as a replacement for the rabbit pyrogen test rpt iccvam, 2008. The equipment, injectors and needles used in the test should be pyrogen free. This is significant because the lal test is more sensitive than the usp rabbit pyrogen test, and because, as comparative lal and rabbit assays indicate. Pdf the rabbit pyrogen test was developed in the early 1900s to detect contaminating pyrogens in parenteral medicines. Demonstrated that gram negative bacteria were pyrogenic whereas.
Pyrogen testing is based on either a 100 yearold rabbit pyrogen test rpt, the most widely used limulus amoebocyte lysate lal test or the broad spectrum and biologically relevant monocyte. Withhold food from the rabbits overnight and until the test is completed. Pdf the rabbit pyrogen test and limulus amoebocyte lysate lal assay have been used to detect endotoxins in vaccines, but interactions between the. Pyrogen testing should be done to every batch of pharmaceutical product particularly parenterals for which water is the usual vehicle. Comparing the lal method with the pyrogen test on rabbits. Pdf comparison of the rabbit pyrogen test and limulus. A newer test uses blood from the horseshoe crab to test for toxins. Pyrogen testing has been occurring for the best part of 100 years. Pyrogen testing market size, share global industry report. The replacement of the rabbit pyrogen test by the lal test gelclot method is possible when the endotoxin limit for the observed drug product is defined, the set maximal endotoxin concentration level in such material is acceptable and standardised test procedures and validation techniques are established. Measuring the change in body temperature following injection of test solution on three rabbits. The pyrogen test on rabbits is based on the measurement of the increase in the rabbit s temperature upon being injected with a product that could contain a contaminant of the pyrogen type. O usp xii rabbit test approved method 1942 to 1980 pyrogen. The current status of the limulus amebocyte lysate lal test for final release of drug products and devices in the united states and abroad is discussed.
Rabbits have a similar pyrogen tolerance to humans, so by observing a change in body temperature in rabbits it is possible to make a determination of the presence of pyrogens. In this test, rabbits are restrained and injected with a test substance while their body temperature is monitored for changes that suggest the substance might be contaminated with pyrogens. The rabbit pyrogen test requires the injection of a small amount of batched test material into a rabbits blood stream, and monitoring for temperature increases. The equipment, injectors and needles used in the test should be pyrogenfree. When is the usp chapter pyrogenicity test the rabbit pyrogen test appropriate. An obsolete test of the adequacy of the hypothalamicadenohypophysisadrenocortical axis, in which timed doses of bacterial pyrogen evoke an increase in plasma cortisol in normal subjects. In 1912, the rpt was included in the british pharmacopoeia as a recommended method for detecting pyrogens in injectable. It is intended to be used for liquid products that can be tolerated by the test rabbit in a dose of 10 ml per kg, injected intravenously, generally within a period of not more than 4 minutes. Detection of pyrogens in albumin 20% solution with the. The in vitro monocyte activation test for pyrogen detection offers a valuable alternative to the rabbit test. Kojima, in biocompatibility and performance of medical devices, 2012. Detection of both endotoxin and nonendotoxin pyrogenic contaminants. The rabbit pyrogen test was developed in the early 1900s to detect contaminating pyrogens.
The rabbit pyrogen test in an in vivo test to detect pyrogens qualitatively. The test involves measuring the rise in temperature of rabbits following the intravenous injection of a test solution and is designed for products that can be tolerated. Differences, up to 100fold, exist in the sensitivity of the various lal preparations to the same endotoxin. The rabbit pyrogen test should be done at least once with 3 lots of finished drug products to demonstrate that the product does not contain pyrogenic substances other than bacterial endotoxin. The rpt can miss humanspecific pyrogens, does not allow for quantification and requires the use of animals. Comparative study ofrabbit pyrogen testandhuman whole bloodassay onhuman serumalbumin ingo spreitzer, matthias fischer, katja hartrsch. The rabbit pyrogen test was developed in the early 1900s to detect contaminating pyrogens in parenteral medicines. Pdf highly sensitive pyrogen detection on medical devices. This method can detect nonbacterial endotoxin pyrogens as well as bacterial endotoxins. Pyrogens include endotoxins toxins derived from gramnegativebacteria and nonendotoxic pyrogens substances derived from microorganisms other than gramnegativebacteria, or from chemical substances. In vitro pyrogen test methods national toxicology program. However, the increasing application for animalfree tests is a predominant factor that could hinder the use of this test type to some extent in near future. Alternate pyrogen tests peta international science consortium, ltd.864 1615 1440 339 328 1218 712 1640 1395 145 1355 507 1611 524 881 160 1303 134 655 1205 1390 491 1607 322 176 1490 682 346 1310 367 1499 565 1228 56 1393 1443 1135 997 1114 93 1400 1401 1113 786