Novel Pyrogen Tests Based on the Human Fever Reaction

The Report and Recommendations of ECVAM Workshop 431,2

Reprinted with minor amendments from ATLA 29: 99-123

Appendix 1

Summary of EU project: Human(e) Pyrogen Test (QLRT-1999-00811)

In February 2000, an international joint validation study on advanced pyrogen tests was started. The overall aim of this project, sponsored by the Fifth Framework Programme of the European Commission, is to develop, evaluate and validate a method based on the human fever reaction to replace the rabbit pyrogen test and the Limulus amoebocyte lysate (LAL) test. The network brings together the most promising test systems developed within recent years in Europe, for transnational comparison and subsequent validation of the most promising models. This precompetitive development should initiate further applications and exploitations for new fields of pyrogen testing, such as cellular therapies, medical devices and pyrogen pollution control in the workplace. A method for introduction into the European Pharmacopoeia (Ph. Eur.) will be developed as a replacement for the rabbit pyrogen test for end-product control. This effort was,accomplished by the Ph. Eur., by establishing a group of experts in order to prepare a monograph.

Objectives of the Four Work Packages (WPI-MT4) of the Study

WPI: Evaluation of tests

  1. Drafting of Standard Operation Procedures (SOPs).
  2. Evaluation of test performance and intralaboratory variances.

WP2: Comparison of Tests I

  1. Establishment of each test in two to three partner laboratories.
  2. Determination of interlaboratory variation of individual tests; comparison of tests.
  3. Selection of the most promising model based on previous results.

WP3: Pre-validation

  1. Establishment of the consensus test in all partner laboratories.
  2. Prevalidation, development of a prediction model, evaluation of applications.
  3. Refinement of the SOP with regard to the analysis procedure; deduction of a prediction model for the rabbit pyrogen test (Partner 8).

WP4: Validation

  1. Validation of the test and the prediction model.
  2. Definition of applications and limitations of the test; dissemination of results, proposal for a monograph in the Ph. Eur., adoption to industrial need for non-regulated applications.

Project Workplan

The project consists of four work packages, which relate to the four phases of the project (Table I).

Table I: Summary of work packages

Work package
Phase 1 2 3 4
Duration 6 months 12 months 8 months 10 months
Number of tests 6 4-6 1-2 1-2
Tests per group 1 2-3 1-2 1(-2)
Number of samples 6 10 20 10 (blind)
Refinement Protocol Exchange of endpoints Analysis procedure
Deliverable SOP
Intralaboratory variation
(reduction of tests)		 Transferability
Interlaboratory variation
(optimisation of tests)
Reduction of tests		 Reproducibility
SOP analysis
procedure
Prediction model		 Validation
GLP concordance
Draft monograph
Milestone Optimised protocols; evaluation Comparison and selection of tests Prevalidation Validation

SOP = Standard Operating Procedure, GLP = Good Laboratory Practice.

WP1/Phase 1: Evaluation of tests

In Europe, in recent years, a number of alternative cellular assays have been developed, which aim to exploit the human primary fever reaction in order to replace the animal test (rabbit pyrogen test) and offer testing in the relevant species (man). Partners 1 to 7 have developed such tests. All of these test systems are based upon the response of human leucocytes (principally monocytes), which release inflammatory mediators (endogenous pyrogens) in response to pyrogenic contamination (exogenous pyrogens). However, the cell-based in vitro assay systems differ with regard to the cells employed (isolated primary blood leucocytes or whole-blood or immortal monocytic cell lines), the mediator determined (interleukin-1, interleukin-6, tumour necrosis factor, neopterin, or nitric oxide), and the precise set-up of the test. Standard protocols for each test are developed or refined and checked by ECVAM for consistency. A set of six test samples is sent to each of the laboratories involved, to compare test performance and intralaboratory variation. Test failures would be investigated at this stage.

WP2/Phase 2: Comparison and selection of tests

Protocols are refined again including exchange of endpoints. Each test is transferred to two or three partner laboratories, with documentation of the transfer and checking of transferability. A second set of six test samples is sent to each of the laboratories involved to compare test performance and interlaboratory variation. The most promising test(s) will be selected.

WP3/Phase 3: Prevalidation

The selected test(s) is (are) first transferred to an independent laboratory (P9) which was not involved in the development, to test assay transferability and performance without bias. Then the test is established in each of the partner laboratories. A set of 20 test samples is sent to each of the partner laboratories, to permit comparison of this (these) test(s). Minor refinements of the protocol might be considered (1).

WP4/Phase 4: Validation

Under blind conditions, a set of ten test samples is sent to each of the partners, and an independent analysis of raw data is performed by Partner 8 according to the method described by Balls & Karcher (2). Based on these results, a final protocol will be published and a method for the Ph. Eur. will be proposed.

Network partners and tasks: project leaders and members of the Project Management Committee

Partner 1: Dr T. Hartung, Coordinator
Steinbeis Transfer Centre For In Vitro Pharmacology and Toxicology at the University of Konstanz, Germany
Transfer of methodology to industry, Grampositive and fungal reference materials.

Partner 2: Dr S. Poole
NIBSC, London, UK
Test based on isolated primary human blood cells or MonoMac cell lines and IL6 release, Gram-negative reference materials.

Partner 3: Dr T. Montag-Lessing
Paul-Ehrlich-Institut, Langen, Germany
Test based on human whole blood and IL-1 release.

Partner 4: Dr A.M. Gommer
RIVM, Bilthoven, The Netherlands
Test based on a human cell line (MonoMac-6) and IL-6 release.

Partner 5: Dr G. Werner-Felmayer
University of Innsbruck, Austria
Test based on a human leucocyte cell line (THP-1) and neopterin or NO release.

Partner 6: Dr T. Jung
University of Bern, Switzerland

Test based on a human cell line (THP-1) and TNF release.

Partner 7: Dr P. Brügger
Novartis, Basle, Switzerland
Test based on human isolated blood leucocytes and IL-1 release.

Partner 8: Dr S. Coecke
ECVAM, IHCP, JRC, EC, Italy
Independent biometrical analysis, check of standard protocols, GLP compliance.

Partner 9: Dr I. Aaberge
NIPH, Oslo, Norway
Independent evaluation of test performance in phase 3 and 4.

Associate Contractor 1: Dr S. Berthold
DPC Biermann, Bad Nauheim, Germany
Provision of ELISAs, kit development.

References

  1. Balls, M., Blaauboer, B.J., Fentem, J.H., Bruner, L., Combes, R.D., Ekwall, B., Fielder, R.J., Guillouzo, A., Lewis, R.W., Lovell, D.P., Reinhardt, C.A., Repetto, G., Sladowski, D., Spielmann, H. & Zucco, F. (1995). Practical aspects of the validation of toxicity test procedures. The report and recommendations of ECVAM workshop 5. ATLA 23: 129-147.
  2. Balls, M. & Karcher, M. (1995). The validation of alternative test methods. ATLA 23: 884-886.

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