Collaborative study for the validation of cell line assays for in-process toxicity and antigenicity testing of Clostridium septicum vaccine antigens - Part 1.

Large numbers of mice are used in testing during the production of Clostridial vaccines. Previous work has indicated that cell line assays could replace mouse tests for certain aspects of this testing. Replacement assays have been developed for the testing of the toxins and toxoids of several clostridial species but none of these assays have been assessed in an international collaborative study. Under the common aegis of the European Partnership for Alternative Approaches to Animal Testing (EPAA) and of the European Directorate for the Quality of Medicines & HealthCare (EDQM), collaborative study BSP130 was initiated to evaluate Vero cell based alternative methods to the current mouse tests used to measure the toxicity of Clostridium septicum toxin (the minimum lethal dose (MLD) test), the freedom from toxicity of C. septicum toxoid (the MLD test) and the antigenicity of C. septicum toxoid (the total combining power (TCP) test). The principal aims of BSP130 were to determine the repeatability and reproducibility of the in vitro assays and to demonstrate concordance of the proposed in vitro and current in vivo TCP and MLD tests. 11 laboratories from 7 countries participated in the collaborative study and each tested 6 toxins and 6 toxoids. The participants' Vero cell lines were up to 1 000 times more sensitive than the mouse strains. The MLD assay in mice and on Vero cells generally ranked the toxins in a similar order in most of the laboratories. The TCP assay in mice and on Vero cells also generally ranked the toxoids in a similar order in most of the laboratories. The results demonstrate that the repeatability and reproducibility of the in vitro Vero cell based assays are no worse than that of the in vivo assays and that they are easily transferable to other laboratories. The concordance correlations between the in vivo and in vitro methods were for the MLD assays ρc=0.961 (log-transformed values) and ρc=0.921 (non-log-transformed values) and for the TCP assays ρc=0.968 (log-transformed values) and ρc=0.980 (non log-transformed values). These correlations are excellent showing that the Vero cell assays can be used as alternatives to the mouse tests for the assessment of C. septicum toxin MLD and toxoid TCP values. This study can be used by vaccine manufacturing companies as a guide for applying the same approach to other clostridial toxins and toxoids.