WP4: New cell systems and new endpoints

 

WP4 Results pdf 83 kB, download the entire summary.

In WP4 different alternative ways have been evaluated to improve the prediction of cell-based cytotoxicity assays by incorporating more specific end-point parameters to already used human cell lines, as well as cell models from the human haematopoietic system in the testing strategy. After evaluation, the following methods and endpoints were selected for further testing.

 

New cell systems

Cytokine (IL-5) secretion in human blood-derived mononuclear cells by ELISA technique and Flow Cytomix (11-plex) determination
The inhibitory effect on cytokine production for 21 of the reference chemicals have been analyzed by ELISA technique and compared with the in vitro toxicity data from 3T3 cells (see WP2) and with in vivo toxicity data (see WP1).

 

The capacity of the assays to determine EC50 or IC50 values of the ACuteTox test chemicals was 17/21 (IFN-  and IL-5) and 16/21 (TNF- ). The cytokine secretion assays had very good correlations (R2 around 0.85) with rodent toxicity data (LD50 values for rat and mouse). The correlation with 3T3 cells was less good, with R2 values lower than 0.7.

 

CFU-GM assays on cell differentiation in human cord blood-derived cells
The effect on cell differentiation for the first 21 reference chemicals, as well as four additional outliers, has been analyzed with CFU-GM assays. The capacity of the assays to determine IC50 values of the ACuteTox test compounds was 20/25.

 

Correlations with rodent toxicity data gave a very good coefficient of correlations (R2 above 0.85). In general, the compounds showed a higher effect in CFU-GM assays compared to 3T3 cells. A comparison of the CFU-GM assay with the 3T3 toxicity assay had a coefficient of correlations of 0.65.

 

New endpoints

Cytotoxicity screening by flow cytometric assays with three different human cell lines, A704, HepG2 and SH-SY5Y cells have been evaluated.  Cytomic assays, including Multiparameter FCM assays of Calcium/Mitochondrial membrane potential/Superoxide production in A704, HepG2 and SH-SY5Y cell lines, as well as the novel High-Content Analysis (HCA) by Bioimage Analysis of 8-oxoG in A704 and SH-SY5Y cells showed best correlation when compared with in vitro cytotoxicity (3T3 cells), in vivo rodent toxicity, and in vivo human toxicity, and hence were selected for the additional testing.