Interfacility variations in blood chemistry parameters and their implications for virtual control group construction

• Interfacility variations in rat blood chemistry were systematically explored. • Significant differences persisted despite standardized study protocols. • Variability mainly stemmed from sample handling and measurement methods. • Metadata recording and harmonization identified as critical for data integrity. • Correction factors and Z-score standardization proposed for robust VCG creation. The implementation of virtual control groups (VCGs) using historical control data (HCD) offers a practical approach to reduce animal use in non-clinical toxicology studies, in line with the 3Rs principle. However, the scientific validity of VCGs largely depends on the comparability of data across facilities, particularly when an HCD integrated from multiple facilities is used to create VCGs. This study investigated interfacility variations in blood chemistry parameters using control group data from 28-d repeated-dose toxicity studies in rats sourced from the Open TG-GATEs database. Despite the standardized study protocols, significant interfacility differences were observed in most parameters, including total bilirubin, albumin, and alkaline phosphatase. These variations are primarily attributed to differences in sample handling and measurement methods. To address these challenges, we examined potential mitigation strategies, including correction factors and statistical standardization approaches, such as Z-score transformation, to improve data alignment across facilities. Our findings highlight the importance of recording and unifying metadata related to the study design and analytical procedures. Establishing the minimum alignment criteria is essential for advancing VCGs as scientifically robust tools for ethical and efficient safety assessments. As a practical next step, it is desirable to evaluate the utility of VCGs in non-good laboratory practice (GLP) studies and accumulate supporting evidence, followed by promoting their application in GLP studies.