WP4: New cell systems and new endpoints
WP4 results, download the entire summary: 
Results WP4.pdf
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 the 57 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 of the 57 reference chemicals has been analyzed with CFU-GM 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 and oxidative stress screening by using flow cytometric assays with three different human cell lines, A704, HepG2 and SH-SY5Y cells have been evaluated.
The cytomic panel for cytotoxicity screening including:
o Intracellular Ca2+ (Fluo-4 probe)
o Mitochondrial membrane potential (rhodamine123)
o Plasma membrane potential (DIBAC probe)
o Intracellular lipid content (BODIPY probe)
The cytomic panel for oxidative stress screening including:
o Intracellular peroxides
o Mitochondrial generation of superoxide
o Intracellular levels of the oxidized DNA base 8-oxo-guanine
In summary, the cytomic assays correlated excellently with in vivo human toxicity, lower with in vitro and very poor with rodent toxicities. The suitability of these assays for classification, according to the Global Harmonization System (GHS) has also been assessed. The result showed that the cytomic assays do not separate clearly compounds belonging to toxic classes (GHS classes 1-5) but seem to reveal compounds labeled as non-toxic by GHS. It was concluded that cytomics is a promising analytical system for ACuteTox and for similar in vitro cell-based toxicological studies.