Pyrogen testing of lipidic parenterals with a novel in vitro test--application of the IPT based on cryopreserved human whole blood.

The European Pharmacopoeia has made the testing of small volume parenterals (< 15) obligatory since 2004. This concerns many formulations, e.g. vitamins, steroids and hormones, many of which are applied intramuscularly using a lipidic carrier. Lipopolysaccharides, the best established endotoxins from Gram-negative bacteria, bind strongly to lipophilic substances, which mask them in Limulus amebocyte lysate assays. End-product testing, therefore, can only be carried out in rabbit pyrogen tests. This will lead to a pronounced increase in animal experiments if no alternative procedures become available. We have described a novel in vitro pyrogen test (IPT) based on human whole blood, which has recently been validated in a collaborative study including the European Pharmacopoeia. Here, the utility of the IPT for lipophilic substances and lipid-containing end-products was assessed. For a variety of lipids commonly added to formulations of injectable endproducts, namely peanut oil, sesame oil, miglyol and paraffin, a protocol which allows interference-free testing was established applying the pharmacopoeial criterion of 50 to 200% retrieval of an LPS spike. Furthermore, end-product testing for three sample formulations was possible. In all, a method could be established which allows the determination of given or calculated ELC (endotoxin limit concentrations) according to Pharmacopoeia. It is concluded that monocytes do react to lipid-bound LPS, indicating that immune responses to contaminated endproducts must be anticipated, and that the IPT is suitable for endproduct control of these formulations.