To accomplish international recognition of the RTgill-W1 cell line assay for predicting acute toxicity to fish, the project aims to initiate a round-robin trial in order to test the robustness of the established methodology. In particular, the team aims to evaluate the transferability of the RTgill-W1 cell line assay from the lead laboratory (Eawag) to other laboratories, and assess the within- and between-laboratory reproducibility.
The following steps are proposed and shall be supervised by K. Schirmer and a researcher from the Schirmer lab:
Step 1: Identify four laboratories able to participate in the round-robin test
Several laboratories have already, in principle, expressed their interest in participating in the round-robin test. One infrastructural requirement for participation is access to a fully functional cell culture laboratory.
Detailed lists of resource and infrastructure needs for setting up the RTgill-W1 cell line assay have been prepared and will be distributed and discussed with the laboratories who have expressed their interest in participation of the round-robin test. These should help potential participants to evaluate their capacities and needs in participating.
Step 2: Select chemicals and set-up infrastructure for analysis
Chemical selection (aiming at 6 substances) will be based on the CEllSens chemical list (Schirmer et al., 2008), considering chemicals of different physico-chemical properties (lipophilicity, volatility) and modes of toxic action. Another criterion will be the availability of established quantification protocols, likewise derived from the CEllSens project (work performed by Joop Hermens’ laboratory). At least for the participating laboratories in Europe, chemical analysis shall be carried out centrally in Joop Hermens’ laboratory. If shipment of samples from the US proves difficult, we will provide analytical protocols to P&G. Based on the results of the CEllSens project, participating laboratories will be instructed on the chemical source and relevant concentration range. Preparation of chemical stock solutions, application to cell cultures, and preparation of samples for shipment are detailed in SOPs developed in the frame of the CEllSens project.
Step 3: Transfer protocols to participating laboratories
All participating laboratories will be provided with the CEllSens Quality Management Handbook (QMH), which contains all necessary SOPs. At a set time after sending out this information, we will organize a teleconference to discuss all details. RTgill-W1 cell cultures will subsequently be shipped from the Schirmer lab to the participating laboratories (live cultures will be shipped to save time). Upon establishment of the cell cultures in all labs, we will organize hands-on training geared toward the toxicity testing procedure, either by a training course done at Eawag or by visiting the partners on-site.
Step 4: Testing phase
During the testing phase, we will establish a “hot-line”, i.e. provide continuous support and advise to the participating labs to ensure that potential problems can be identified and solved in a timely manner. Raw data will be entered in the standardized evaluation sheets developed within the CEllSens project. Samples for chemical quantification will be shipped by the partners frozen to Joop Hermens’ laboratory where they will be collected for large batch analysis.
Step 5: Evaluation phase
All data will be collected and compiled by Eawag and appropriately evaluated. The results will be discussed thoroughly with all partners. The minimum final outcome will be a report that can be used to guide further validation studies (e.g., in the frame of OECD/EURL ECVAM). Beyond the report, we aim at a joint publication in a peer-reviewed journal.