Rabbit pyrogen test

Historically, the rabbit pyrogen test constituted the most widely used method. This entails parenteral administration of the product to a group of healthy rabbits, with subsequent monitoring of rabbit temperature using rectal probes. Increased rabbit temperature above a certain point suggests the presence of pyrogenic substances. The basic rabbit method, as outlined in the European Pharmacopoeia, entails initial administration of the product to three rabbits. The product is considered to have passed the test if the total (summed) increase of the temperature of all three animals is less than 1.15 °C. If the total increase recorded is greater than 2.65 °C then the product has failed. However, if the response observed falls between these two limits... 

TNF-a, as previously discussed. Test methods have been designed that include incubation of a test sample with monocytes in whole blood or in cultured cell lines and analysis of a specific cytokine after a suitable time. This cell-based methodology may provide an alternative to rabbit pyrogen testing that is required for human blood products. More development is needed to determine the optimum cytokine for analysis and to assess the nature of interference conditions. 

Weary, M. The rabbit pyrogen test. In Pyrogens Marcel Dekker, Inc. New York, 1985 104—118. 

Weary M.E., Donohue G., Pearson F.C. and Story K. (1980) Relative potencies of four reference endotoxin standards as measured by the Limulus amoebocyte lysate and USP rabbit pyrogen tests. Appl. Environ. Microbiol., 40, 1148-1151. 

The classic test for measuring pyrogenicity used to be the in vivo rabbit pyrogen test (European Pharmacopoeia 2.6.8 United States Pharmacopeia <151> ISO I0993-II), which measures the rise in body temperature (an increase of 0.5°C or more indicated pyrogenicity) following an intravenous injection of the sample or the aqueous extract (typically with 40 ml water)... 

The European Pharmacopoeia requires the rabbit pyrogen test for a number of vaccines, some antibiotics, and specific excipients including glucose, if intended for the preparation of large volume parenterals (see Sect. 32.8). These products may be contaminated with pyrogens other than LPS, or are known to inhibit the LAL test. 

A third test, the monocyte activation test (MAT) is based on the in-vitro activation of human blood cells by pyrogens. This leads to the release of pro-inflammatory cytokines tumoiu necrosis factor alfa (TNF-alfa), interleukin-1 beta (BL-lbeta) and interleukin-6 (IL-6) that are determined by Enzyme-Linked Immiuio Sorbent Assay (ELISA). Consequently, the MAT will detect the presence of both exogenous and endogenous pyrogens in the test sample. The MAT is suitable, after a product-specific validation, as a replacement for the rabbit pyrogen test [50]. 

Systemic toxicity testing, such as the ISO acute systemic toxicity test, pyrogenicity test, USP/ Code of Eederal Regulations (CER) pyrogen test, ISO rabbit pyrogen test, USP systemic injection test, USP intracutaneous test, USP implant test, and USP safety test. 

LAL uses a component of the blood of horseshoe crabs. In the presence of endotoxins, the LAL will clot. Using standard curves, the amount of endotoxin present is quantifiable. The rabbit pyrogenicity test involves monitoring the temperatures of rabbits for 3 h following an intravenous injection of the test article or the saline extract of the test article. If a rabbit s temperature increases 0.5 °C following the injection, the test is considered positive. 

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