A significant advance in the field of early medicine was Paracelsus’ recognition that all compounds have the capacity to be poisonous depending upon dosage [1
]. This observation makes toxicity testing a necessary and critical industry practice to identify and define safety thresholds for all new potential chemotherapeutics. The advent of in vitro
cytotoxicity testing has greatly streamlined this process and is now considered to be a nearly compulsory activity starting at target validation and continuing through medicinal modification.
Unlike animal-based toxicology testing, there are clearer definitions and greater agreement for what constitutes cytotoxicity in vitro
. Classically speaking, a compound or treatment is considered to be cytotoxic if it prevents cellular attachment, causes dramatic morphological changes, adversely affects replication rate, or leads to a reduction in overall viability [2
]. It should be noted that the manifestation of these effects is greatly dependent on length of compound exposure and mechanism of cytotoxicity [3
A host of new assays have been described and utilized which measure biomarkers of cellular stress or specific signaling events more proximal to initial cytotoxic insult (i.e. glutathione, caspases) [4
]. These methods offer early indication of potential cytotoxicity, but are typically relegated to secondary screening because they are more difficult to employ as endpoint assays due to the transient nature of the biomarker and kinetic differences associated with cell death progression [5
]. Therefore assay chemistries predicated upon the detection of changes in membrane integrity remain the gold standard for in vitro
Many methods exist for the assessment of membrane integrity, including several classic dye inclusion, exclusion and lysosomal accumulation techniques [7
]. Although well validated, these methods are poorly suited for HTS implementation due to low sensitivity, multiple processing steps or miniaturization problems associated with higher plate densities [9
]. Recent advances in reagent formulations accommodate these miniaturized formats and are fully compatible with automated dispensing systems and integrated detection instruments. These reagents deliver the linearity, sensitivity, and robustness (Z’
Values) necessary for properly interrogating large chemical libraries for cytotoxic risk. The most acceptable assay format is known as “add-mix-measure”, whereby reagent chemistry is delivered directly to the test well.