The European Pharmacopoeia (Ph. Eur.) and the World Health Organisation (WHO) require the performance of extensive quality and safety control testing before the release on the market of vaccine products for human use. Safety testing with regard to residual pertussis toxin (PT) in acellular pertussis combination vaccines is performed through assessment of fatal sensitisation of mice to histamine challenge by the vaccine product under test. Currently, use of different in-house procedures and no requirement for the inclusion of a standard reference in each assay render comparisons of results obtained for identical vaccine batches between different control laboratories very difficult. At the initiative of the European Directorate for the Quality of Medicines and HealthCare (EDQM), an international collaborative study was organised for the standardization of the Histamine Sensitizing Test (HIST) in mice and the Chinese Hamster Ovary (CHO)-cell-based assay (performed at the bulk product level) for the residual toxicity testing of acellular pertussis vaccines or acellular pertussis-based combination vaccines. The study was run under the aegis of the Biological Standardisation Programme, jointly supported by the Council of Europe and the European Commission under the project code BSP076. Ten (10) laboratories participated in the study and were requested to perform 3 independent Histamine Sensitizing Tests in mice and to report results of the lethal end-point measurement as prescribed by the Ph. Eur. monographs. Some of them also reported data from an in-house validated CHO-cell-based assay. In addition, some of the laboratories reported concomitantly data obtained by measurement of the drop in temperature induced after the histamine challenge, a method currently under investigation to be added as an alternative end-point for the HIST in the Ph. Eur. monographs for acellular pertussis-based combination vaccines in order to alleviate animal suffering (in application of the 3Rs principle). Based on the results of the collaborative study, a potency of 7500 IU/vial (International Units per vial) was assigned to the current Ph. Eur. Biological Reference Preparation (BRP) for PT. The results of the study also show that 1) intra- and inter-laboratory variations can be improved by the use of a validated standard operating procedure; 2) inclusion in each assay of a standard reference sample, calibrated in IU, can increase comparability of results among laboratories and thus help to reduce repeat testing; 3) a correlation between mortality data and temperature data was observed although, due to the limited number of data sets and the lack of a common method for the temperature end-point, further investigation of this point is required; 4) the CHO-cell-based assay did not yield comparable results and further standardisation of the assay procedure may be investigated in a follow-up project
