Hurdles and signposts on the road to virtual control groups—A case study illustrating the influence of anesthesia protocols on electrolyte levels in rats

Abstract

Introduction: Virtual Control Groups (VCGs) represent the concept of using historical control data from legacy animal studies to replace concurrent control group (CCG) animals. Based on the data curation and sharing activities of the Innovative Medicine Initiatives project eTRANSAFE (enhancing TRANSlational SAFEty Assessment through Integrative Knowledge Management) the ViCoG working group was established with the objectives of i) collecting suitable historical control data sets from preclinical toxicity studies, ii) evaluating statistical methodologies for building adequate and regulatory acceptable VCGs from historical control data, and iii) sharing those control-group data across multiple pharmaceutical companies. During the qualification process of VCGs a particular focus was put on the identification of hidden confounders in the data sets, which might impair the adequate matching of VCGs with the CCG.Methods: During our analyses we identified such a hidden confounder, namely, the choice of the anesthetic procedure used in animal experiments before blood withdrawal. Anesthesia using CO2 may elevate the levels of some electrolytes such as calcium in blood, while the use of isoflurane is known to lower these values. Identification of such hidden confounders is particularly important if the underlying experimental information (e.g., on the anesthetic procedure) is not routinely recorded in the standard raw data files, such as SEND (Standard for Exchange of Non-clinical Data). We therefore analyzed how the replacement of CCGs with VCGs would affect the reproducibility of treatment-related findings regarding electrolyte values (potassium, calcium, sodium, and phosphate). The analyses were performed using a legacy rat systemic toxicity study consisting of a control and three treatment groups conducted according to pertinent OECD guidelines. In the report of this study treatment-related hypercalcemia was reported. The rats in this study were anesthetized with isoflurane.Results: Replacing the CCGs with VCGs derived from studies comprising both anesthetics resulted in a shift of control electrolyte parameters. Instead of the originally reported hypercalcemia the use of VCG led to fallacious conclusions of no observed effect or hypocalcemia.Discussion: Our study highlights the importance of a rigorous statistical analysis including the detection and elimination of hidden confounders prior to the implementation of the VCG concept.

The road to virtual control groups and the importance of proper body-weight selection

“…The ongoing maturation of the concept of VCGs (Golden et al, 2024;Steger-Hartmann and Clark, 2023) involves the evaluation of the VCG performance on legacy studies using them as a benchmark: a virtual control group can be considered "well performing" if the legacy study results can be reproduced after the CCG was replaced by VCGs (Gurjanov et al, 2023).…”

“…HCDs are generally used for comparative purposes in regulatory toxicity studies (Kluxen et al, 2021) provided that HCD are "originating from the same laboratory, species, strain, and collected under similar conditions" (OECD, 2018). The same requirements apply for the establishment of reliable, well-performing VCGs (Steger-Hartmann et al 2020;Gurjanov et al, 2023). 28-day repeat-dose toxicity studies in rodents and non-rodents usually determine the safe starting dose for the Firstin-Man trials (ICH, 2009;OECD, 2008).…”

“…HCD was collected from these studies (Kluxen et al, 2021), where the largest amount of HCD has accumulated for rodents, since the rat is the most frequently used species (Namdari et al, 2021). Due to the abundance of HCD, first insights on the use of VCGs have been made with this species (Gurjanov et al, 2023(Gurjanov et al, , 2024Wright et al, 2023).…”

How important are concurrent vehicle control groups in (sub)chronic non-human primate toxicity studies conducted in pharmaceutical development? An opportunity to reduce animal numbers

“…When those studies are performed, relevant information about the maximum recommended starting dose in human clinical trials and information about the pharmaceutical vehicle has already been collected, and the risk for misinterpreting vehicle-related effects is low. We also specifically focused on nonclinical studies that are conducted in non-human primates (NHP), since the use of NHP is of the greatest concern to the public [ 14 ], because previous investigations on reducing control groups have mainly focused on rodents [ 15 ] and because reducing NHP use in research is a global ambition resulting from shortages in animal supply [ 16 ]. As generating virtual controls from existing databases faces numerous challenges, we did not focus on the generation of virtual controls per se but wanted to understand what the relevance is that concurrent control groups play in the determination of test article-related adverse effects.…”

Can Historical Control Group Data Be Used to Replace Concurrent Controls in Animal Studies?

“…If this is not adequately captured and considered during the selection of control data for VCGs, the identification of treatment-related findings affecting potassium or calcium levels in serum might be impaired. 1 How can it be systematically assessed that the use of VCGs does not impact the quality and the outcome of a toxicity study? Validation of a new assay or new approach methodology (NAM), which is intended to replace an in vivo study and foreseen for a use in a regulatory context is usually done with wellcharacterized reference compounds in a blinded manner.…”