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

PLOS ONE RESEARCH ARTICLE 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 Lars Mecklenburg ID1*, Sarah Lenz1,2, Georg Hempel2 1 Labcorp Early Development Services GmbH, Muenster, Germany, 2 Institute for Pharmaceutical and Medicinal Chemistry, Westphalian Wilhelms University, Muenster, Germany a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Mecklenburg L, Lenz S, Hempel G (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. PLoS ONE 18(8): e0282404. https://doi. org/10.1371/journal.pone.0282404 Editor: Yasmina Abd-Elhakim, Zagazig University, EGYPT Received: February 13, 2023 Accepted: July 19, 2023 Published: August 3, 2023 Copyright: © 2023 Mecklenburg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. * Abstract Safety assessment of human pharmaceuticals demands extensive animal experiments before a compound can be tested in patients or released on the market. Such experiments typically include concurrent vehicle control groups. Reconsidering the need for concurrent controls could support the strive to reduce the use of animals for scientific purposes. We reviewed reports from 20 (sub)chronic toxicity studies that were conducted in non-human primates (NHP) to characterize hazards of novel human pharmaceuticals. Firstly, we determined the toxicological endpoints that were identified to characterize the hazard. Secondly, we evaluated if the hazard could have been identified without reference to the concurrent controls. Thirdly, we employed an alternative statistical method to test for any significant change related to dose level or time. We found that toxicologically relevant hazards were identifiable without reference to concurrent controls, because individual measurements could be compared with pre-dosing values or because individual measurements could be compared to historical reference data. Effects that could not be evaluated without reference to concurrent controls were clinical observations and organ weights for which appropriate historical reference data was not available, or immune responses that could not be compared to pre-dosing measurements because their magnitude would change over time. Our investigation indicates that concurrent control groups in (sub)chronic NHP toxicity studies are of limited relevance for reaching the study objective. Under certain conditions, regulatory (sub)chronic NHP toxicity studies represent a good starting point to implement virtual control groups rather than concurrent control groups in nonclinical safety testing. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: The author(s) received no specific funding for this work. Introduction Competing interests: The authors have declared that no competing interests exist. Safety assessment of new pharmaceuticals is highly regulated, demanding that extensive animal experiments are conducted before a pharmaceutical can be tested in patients or released PLOS ONE | https://doi.org/10.1371/journal.pone.0282404 August 3, 2023 1 / 15 PLOS ONE Concurrent controls in non-human primate toxicity studies on the market [1]. In Europe for example, Directive 2001/83/EG requires that the application to obtain a marketing authorization for a medicinal product be accompanied by toxicological and pharmacological tests, which are widely referred to as “nonclinical safety studies”. Guidance on such nonclinical safety studies is given by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) and by the Organization for Economic Co-operation and Development (OECD). Nonclinical safety studies aim to characterize the hazard associated with new pharmaceuticals and to determine a No Adverse Observed Effect Level (NOAEL) which functions as a reference for determining an acceptable exposure range for humans, e.g. patients in a clinical trial. Societies across the globe strive towards reducing the use of animals for scientific purposes [2–6]. For example, the European Medicines Agency (EMA) committed to the application of replacement, reduction and refinement of animal testing as detailed in Directive 2010/63/EU [7], and in the United States of America, congress recently passed the FDA Modernization Act 2021, which allows for the use of New Approach Methodologies to evaluate the safety of drugs [8, 9]. Replacement, reduction and refinement of animal testing are widely known as the “3Rs”. This principle was originally described in 1959 by Russell and Burch [10], who published considerations on how to promote “humane” behavior towards animals in the laboratory: “Replacement means the substitution for conscious living higher animals of insentient material. Reduction means reduction in the numbers of animals used to obtain information of a given amount and precision. Refinement means any decrease in the incidence or severity of inhumane procedures applied to those animals which still have to be used.” With the aim to reduce the number of animals used in regulatory toxicity studies, the concept of virtual control groups has been introduced into nonclinical safety assessment [11]. Virtual control groups are proposed to replace concurrent control groups, which represent up to 25% of the animals in a regulatory toxicology study [12]. These animals are typically administered the vehicle, i.e. an inert medium that is used as a solvent or diluent in which the medicinally active agent is formulated. Virtual control groups are constructed from historical control data. Building adequate virtual control groups, however, is challenging and bears the risk that toxicological study outcomes are influenced, if covariates are not sufficiently controlled [13]. In consideration of the desire to replace concurrent controls with virtual controls, we aimed to evaluate the relevance of concurrent control groups in today’s pharmaceutical safety assessment. We specifically focused on those nonclinical safety studies that are conducted after the first human patients have been exposed, i.e. toxicity studies with subchronic (13 weeks) or chronic (up to 52 weeks) test article exposure. 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 (...truncated)