Alternative Tests for Animal Welfare
Researchers at the Paul-Ehrlich-Institut establish new methods of pyrogen testing in the European Pharmacopoeia – and thus offer an alternative to animal testing with rabbits and horseshoe crabs.
As well as other types of test, the European Pharmacopoeia also prescribes animal testing for the quality control of medicinal products. For many years, the Paul-Ehrlich-Institut has been conducting research into the development of alternative test methods. A team led by Dr Ingo Spreitzer from the Microbiological Safety section of the Microbiology division has now achieved another breakthrough. In January 2021, a revision of the Pharmacopoeia came into force which makes pyrogen testing possible entirely without animals.
Pyrogens are impurities that cause fever and shock-like conditions after an injection. In particular, the triggers are lipopolysaccharides (endotoxins) that are contained in the membrane of gram-negative bacteria such as E. coli. There are two traditional test methods for ruling out pyrogens: the Rabbit Pyrogen Test (RPT) and the LAL (Limulus Amoebocyte Lysate) test.
The in vitro MAT and rFC tests are included in the European Pharmacopoeia as an alternative to animal testing. It is now a question of adapting the monographs of the individual medicinal products. We have already succeeded in doing that for the TBE vaccine.
Dr Ingo Spreitzer , Deputy Head of the Microbiological Safety Section
From 2014 to 2017, more than 6,000 rabbits a year were required in Germany for the RPT. If the rabbits develop a fever, this immune response indicates the presence of pyrogens. The Paul-Ehrlich-Institut was involved in the development of the Monocyte Activation Test (MAT) as an alternative to the RPT, and has been using it for official batch testing since its accreditation in 2014. It is based on human blood cells, so its results are predictive for patients. In 2010, with the involvement of the PEI, the in vitro test was included in the Pharmacopoeia as a General Chapter and has since provided an alternative to testing on rabbits.
Endotoxins can be detected and also quantified with blood components from the horseshoe crab, Limulus polyphemus. The protein factor C (FC) contained in the blood cells (amoebocytes) of the horseshoe crab reacts to endotoxins by binding the bacteria. To obtain the protein, blood is taken from this endangered species, which was living on the seabed as long as 150 million years ago. These living fossils are then returned to the sea – but an estimated 8-15 percent of the animals die. The blood withdrawal from the crabs and their numbers are strictly monitored, but they cannot permanently meet the world's increasing demand for LAL reagents.
An alternative was implemented in January 2021 as a new General Chapter in the Pharmacopeia: rFC tests produced by combinant methods. Dr Spreitzer has actively been promoting its implementation for several years.
Committees
- Dr Ingo Spreitzer, European Pharmacopoeia: Working Party BET Bacterial endotoxins test (Chair) - EDQM
Literature
Bolden J, Knutsen C, Levin J, Milne C, Morris T, Mozier N, Spreitzer I, von Wintzingerode F (2020): Currently available recombinant alternatives to horseshoe crab blood lysates: Are they comparable for the detection of environmental bacterial endotoxins? A Review.
PDA J Pharm Sci Technol 74: 602-611.
Etna MP, Giacomini E, Rizzo F, Severa M, Ricci D, Shaid S, Lambrigts D, Valentini S, Galli Stampino L, Alleri L, Gaggioli A, Von Hunolstein C, Spreitzer I, Coccia EM (2020): Optimization of the monocyte-activation-test for evaluating pyrogenicity of tick-borne encephalitis virus vaccine.
ALTEX 37: 532-544.