[History of the LAL-test: validation and regulatory acceptance]

Bacterial endotoxins gram-negative bacteria are the most relevant substances inducing hyperthermia in humans (pyrogens). Since endotoxins may contaminate pharmaceutical preparations during production process, purity is assured by monitoring the increase in body temperature of rabbits exposed to such preparations. This bioassay can be replaced by the LAL-test in which the clotting reaction of blood cells of the horseshoe crab limulus polyphemus is measured after contact with bacterial endotoxins. Although this reaction is significantly more sensitive to endotoxins than hyperthermia in rabbits, the LAL-test had to undergo 25-30 years of validation to achieve regulatory acceptance. Although endotoxin induced blood clotting in Limulus is quite similar to the same reaction in humans, acceptance of the assay as an alternative to testing in rabbits was delayed, since chemicals present in pharmaceutical preparations may interfere with the LAL clotting reaction. In addition, fever can be induced also by substances other than endotoxins. Therefore it has to be proven for each new preparation that the LAL-test can replace the rabbit pyrogen test in a case by case validation according to guidelines, as e.g. the German guideline, which was released in 1993. During the past decade some laboratories of the pharmaceutical industry in Germany were able to replace 95% of the rabbit pyrogen tests by the LAL-test without increasing the risk for humans.