EUROPEAN COMMISSION
DIRECTORATE GENERAL JRC
JOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
European Centre for the Validation of Alternative Methods (ECVAM)

STATEMENT ON THE VALIDITY OF IN-VITRO PYROGEN TESTSAt its 24th meeting, held on 20-21 March 2006 at the European Centre for thevalidation of alternative methods (ECVAM), Ispra, Italy, the non-Commission
members of the ECVAM Scientific Advisory Committee (ESAC)1 unanimously
endorsed the following statement:
Following a review of scientific reports and peer reviewed publications on thefollowing range of in-vitro pyrogen tests:
1.
2.
3.
4.
5.

Human Whole Blood IL-1,
Human Whole Blood IL-6,
PBMC IL-6,
MM6 IL-6, andHuman Cryopreserved Whole Blood IL-1,

it is concluded that these tests have been scientifically validated for the detection ofpyrogenicity mediated by Gram-negative endotoxins, and quantification of thispyrogen, in materials currently evaluated and characterized by rabbit pyrogen tests.
These methods have the potential to satisfy regulatory requirements for the detection
and quantification of these pyrogens in these materials subject to product-specificvalidation.
The test methods have the capacity of detecting pyrogenicity produced by a wider
range of pyrogens, but the evidence compiled for, and considered within this peer
review and validation process, is not sufficient to state that full scientific validation ofthis wider domain of applicability has been demonstrated and confirmed.
Thus, the above test methods can currently be considered as full replacements for theevaluation of materials or products where the objective is to identify and evaluate
pyrogenicity produced by Gram-negative endotoxins, but not for other pyrogens.
This endorsement takes account of the dossiers prepared for peer review; the views ofindependent experts who evaluated the dossiers against defined validation criteria;
supplementary submissions made by the Management Team; and the considered viewof the Peer Review Panel appointed to oversee the process.
Thomas Hartung
Head of Unit
ECVAM
Institute for Health & Consumer Protection
Joint Research Centre
European Commission
Ispra
21 March 2006

1. The ESAC was established by the European Commission, and is composed ofnominees from the EU Members States, industry, academia and animal
welfare, together with representatives of the relevant Commission services.
This statement was endorsed by the following Members of the ESAC:
Prof Helmut Tritthart (Austria)
Dr Dagmar Jírová (Czech Republic)
Prof Elisabeth Knudsen (Denmark)Dr Timo Ylikomi (Finland)Prof André Guillouzo (France)
Dr Manfred Liebsch (Germany)
Dr Efstathios Nikolaidis (Greece)
Dr Katalin Horvath (Hungary)Prof Michael Ryan (Ireland)
Dr Annalaura Stammati (Italy)
Dr Mykolas Maurica (Lithuania)
Prof Eric Tschirhart (Luxembourg)
Dr Jan van der Valk (The Netherlands)
Dr Dariusz Sladowski (Poland)Prof Milan Pogačnik (Slovenia)
Dr Argelia Castaño (Spain)
Dr Patric Amcoff (Sweden)
Dr Jon Richmond (UK)Dr Odile de Silva (COLIPA)
Dr Julia Fentem (ECETOC)Dr Nathalie Alépée (EFPIA)Prof Robert Combes (ESTIV)Dr Maggy Jennings (Eurogroup for Animal Welfare)
Mr Roman Kolar (Eurogroup for Animal Welfare)
The following Commission Services and Observer Organisations wereinvolved in the consultation process, but not in the endorsement process itself.
Mr Thomas Hartung (ECVAM; chairman)
Mr Jens Linge (ECVAM; ESAC secretary)
Mr Juan Riego Sintes (ECB)
Ms Beatrice Lucaroni (DG Research, Unit F.5)
Mr Sylvain Bintein (DG Environment, Unit C.3)
Mr Sigfried Breier (DG Enterprise, Unit F.3)
Prof Dr Constantin Mircioiu (Romania)
Dr William Stokes (NICEATM, USA)
Prof Dr Vera Rogiers (ECOPA)

Annex
The novel pyrogen tests are based on the human fever reaction. Monocytoid cells,
either primary from human blood or as propagated cell lines, detect pyrogens ofdifferent chemical nature and respond by the release of inflammatory mediators suchas cytokines. Since lipopolysaccharides from Gram-negative bacteria are the only
type of proven pyrogen, for which an International reference material is available, thetests were standardised to detect the presence of significantly less than 0.5 Endotoxin
Units of this preparation, which is considered to be the threshold level for fever
induction in the most sensitive rabbit species according to pharmacopoeia test
procedures.
The five tests which were sufficiently reproducible and exceeded the rabbit test withregard to sensitivity and specificity for the detection of lipopolysaccharide spiked
samples, differ with regard to cell source and preparation, cryopreservation andcytokine measured. The tests have been described elsewhere (1-4). The concept ofthe validation study (5) and the international validation studies are available (6-7).
1. Poole, S., Thorpe, R., Meager, A., Hubbard, A.R., Gearing, A.J. (1988) Detectionof pyrogen by cytokine release. Lancet 8577, 130.
2. Taktak, Y.S., Selkirk, S., Bristow, A.F., Carpenter, A., Ball, C., Rafferty, B., Poole,S. (1991) Assay of pyrogens by interleukin-6 release from monocytic cell lines. J.
Pharm. Pharmacol. 43, 578.
3. Hartung, T., Wendel, A. (1996) Detection of pyrogens using human whole blood.
In Vitro Toxicol. 9, 353.
4. Schindler S, Asmus S, von Aulock S, Wendel A, Hartung T and Fennrich S. (2004)
Cryopreservation of human whole blood for pyrogenicity testing. J. Immunol. Meth.
294, 89-100.
5. Hartung, T., Aaberge, I., Berthold, S., Carlin, G., Charton, E., Coecke, S., Fennrich,S., Fischer, M., Gommer, M., Halder, M., Haslov, K., Jahnke, M., Montag-Lessing, T.,Poole, S., Schechtman, L., Wendel, A., Werner-Felmayer, G. (2001) Novel pyrogentests based on the human fever reaction. The report and recommendations of ECVAM
Workshop 43. European Centre for the Validation of Alternative Methods. Altern.
Lab. Anim. 29, 99.
6. Hoffmann S, Peterbauer A, Schindler S, Fennrich S, Poole S. Mistry Y, MontagLessing T, Spreitzer I, Loschner B, vam Aalderen M, Bos R, Gommer M, Nibbeling
R, Werner-Felmayer G, Loitzl P, Jungi T, Brcic M, Brugger P, Frey E, Bowe G,Casado J, Coecke S, de Lange J, Mogster B, Naess LM, Aaberge IS, Wendel A andHartung T. (2005) International validation of novel pyrogen tests based on the humanfever reaction. J. Immunol. Meth. 298, 161-173.
7. Schindler S, Spreitzer I, Loschner, Hoffmann S, Hennes K, Halder M, Brügger P,
Frey E, Hartung T and Montag T. (2006) International validation of pyrogen tests
based on cryopreserved human primary blood cells. J. Immunol. Meth. 316, 42-51.