Drug metabolism can result in the formation of highly reactive metabolites that 
are known to play a role in toxicity resulting in a significant proportion of 
attrition during drug development and clinical use. Thus, the earlier such 
reactivity was detected, the better. This review summarizes our multi-year 
project, together with pertinent literature, to examine a battery of in vitro 
tests capable of detecting the formation of reactive metabolites. Principal 
prerequisites for such tests were delineated: chemicals known/not known to cause 
tissue injury and produce reactive metabolites, activation system (mainly 
human-derived), small- and large-molecular targets (small-molecular trappers, 
peptides, proteins), analytical techniques (mass spectrometry), and cellular 
toxicity biomarkers. The current status of in vitro tools to detect reactive 
intermediates is the following: 1. Small-molecular trapping agents such 
glutathione or cyanide detect the production of reactive species with high 
sensitivity by proper MS technique. However, it seems that also putative 
"negatives" give rise to corresponding adducts. 2. Results from peptide and dG 
(DNA targeting) trapper studies are generally in line with those of 
small-molecular trappers, although also important differences exist. These two 
trapping platforms do not overlap. 3. It is anticipated that the in vitro adduct 
studies could be fully interpreted only in conjunction with toxicity biomarker 
(such as the Nrf2 pathway) information from whole cells or tissues. However, 
while there are tools to characterize the chemical liability and there are 
correlation between individual/integrated endpoints and toxicity, there are 
still severe gaps in understanding the mechanisms behind the link between 
reactive metabolites and adverse effects.