Integrated strategy of miniaturization cytomic in vitro assay and data minig for prediction of acute toxicity to humans: application to risk assessment and toxic classification of chemicals

HERRERA G; DÍAZ L; GOMES A; PINTO S; ALVAREZ S; ALGUERÓ S; CALLAGHAN RC; O´CONNOR JE

Workshop; 13th Zytomik Leipziger Workshop; 2008

Backgorund: European Union has informed REACH policy for registration, Evaluation and Athoritation of Chemicals. REACh requires toxicity assessment of about 30,000 chemicals . In view of the huge amount of animal testing that this involves, REACh urges novel in vitro methods. We have developed for REACh-focused projects novel in vitro cytomic methods based upon flow cytometry (FCM) and High-Content Analysis(HCA). Material and Methods: we have set up 96 well format assays grouped as Cytomic Panel for Cytometry Screening (CPCS) and Cytomic Panel for Oxidative Stress Screening (CPOSS) with three human cell lines. CPCS includes FCM assays of viability, intracellular calcium and mitochondria and plasma membrane potentials. CPOSS includes FCM analysis os superoxide anion and peroxides and HCA assay of oxidative damage to genomic and mitochondrial DNA. We have determined IC50, EC20 or EC50 in vitro values to 58 chemicals of references plus controls. To asses the predictive value of the in vigor assays, IC50 or EC50 were correlated with reported in vivo LC50 or LD50 in humans, rat and mouse. All the in vivo LC50/LD50 and in vitro IC50/EC50 obtained were log transformed and normalized for cluster analysis and hierarchization. The results were then compared with the toxics classes of the Global Harmonised Systems of Classification and Labelling of Chemicals (GHS). Results: most of the cytomic assays in CPSS and COPSS have excellent correlation coefficient with the in vivo human toxicity. The assays do not separate perfectly compounds belonging to the mid-toxic GHS classes but identify compounds labeled as non-toxic by GHS and provide alerts for specific toxicity. Discusuion and conclusion: Cytomic analysis assays and data mining by array analysis seem useful in chemical hazard assessment.