ALTEX :: Alternatives to Animal Experiments

1998, Supplement

A New Test for the Detection of Pyrogens in Pharmaceutical Products: Examinations for the Validation of the Human Whole Blood Assay

Markus Weigandt, Peter Lexa, and Hans-Guenther Sonntag

Hygiene-Institut der Universitaet Heidelberg, D-Heidelberg

SUMMARY

The human whole blood assay utilises the natural fever response to detect pyrogens by determination of the release of IL-1β. In order to replace the official method, the rabbit pyrogen test, a validation of the whole blood assay is necessary. A comparison of the results obtained from many blood samples has revealed the following:

  1. Blood not stimulated by LPS does not produce IL-1β
  2. Stimulation by LPS induces a concentration-dependent release of IL-1β beginning at a concentration of between 2-5 pg/mL LPS.
  3. The amount of IL-1β released varies greatly between samples obtained from different individuals.
  4. Storing blood samples results in a right shifted LPS/IL-1β curve with a steeper gradient and higher maximum value of IL-1β

In this paper we suggest an experimental method for the determination of pyrogens based on the established semi-quantitative LAL gelation method as detailed in the European Pharmacopoeia. Using this methodology, we were able to show that the amount of endotoxin in a number of different infusion solutions was below the LAL-endotoxin limit concentration. LPS was quantitatively determined from spiked samples.

Keywords: human whole blood, alternative method, interleukin-1β, drug release, pyrogen test