One of the emerging concepts on the reduction of animal use in non-clinical studies is the use of virtual control group (VCG) to replace concurrent control group (CCG). The VCG involves the generation of a control data from historical control data to match a specific study design. This review focuses on two recently published proof-of-concept (POC) studies conducted in rats. One major issue that was consistently seen across these POC studies was the non-reproducibility of some quantitative endpoints between the CCG and the VCG, with clinical pathology parameters being the most affected. The inconsistencies observed with the clinical pathology parameters when using VCGs may lead to: (1) misconception about the accuracy and sensitivity of traditional clinical pathology biomarkers and its implications on safety monitoring in the clinic
(2) inability to correctly identify and characterize organ dysfunctions
(3) interference with the weight-of-evidence approach used in identifying hazards in toxicologic clinical pathology and toxicology studies at large
and (4) wrong interpretations and data reproducibility issues. Other alternatives to reduce animal use in toxicology studies are also discussed including blood microsampling for toxicokinetics, scientifically justified use of recovery animals, and appropriate use and continuous investments in new alternative methods.