With the release of the landmark report Toxicity Testing in the 21st Century: A Vision and a Strategy, the U.S. National Academy of Sciences, in 2007, precipitated a major change in the way toxicity testing is conducted. It envisions increased efficiency in toxicity testing and decreased animal usage by transitioning from current expensive and lengthy in vivo testing with qualitative endpoints to in vitro toxicity pathway assays on human cells or cell lines using robotic high-throughput screening with mechanistic quantitative parameters. Risk assessment in the exposed human population would focus on avoiding significant perturbations in these toxicity pathways. Computational systems biology models would be implemented to determine the dose-response models of perturbations of pathway function. Extrapolation of in vitro results to in vivo human blood and tissue concentrations would be based on pharmacokinetic models for the given exposure condition. This practice would enhance human relevance of test results, and would cover several test agents, compared to traditional toxicological testing strategies. As all the tools that are necessary to implement the vision are currently available or in an advanced stage of development, the key prerequisites to achieving this paradigm shift are a commitment to change in the scientific community, which could be facilitated by a broad discussion of the vision, and obtaining necessary resources to enhance current knowledge of pathway perturbations and pathway assays in humans and to implement computational systems biology models. Implementation of these strategies would result in a new toxicity testing paradigm firmly based on human biology.

Based on existing data and previous work, a series of studies is proposed as a basis toward a pragmatic early step in transforming toxicity testing. These studies were assembled into a data-driven framework that invokes successive tiers of testing with margin of exposure (MOE) as the primary metric. The first tier of the framework integrates data from high-throughput in vitro assays, in vitro-to-in vivo extrapolation (IVIVE) pharmacokinetic modeling, and exposure modeling. The in vitro assays are used to separate chemicals based on their relative selectivity in interacting with biological targets and identify the concentration at which these interactions occur. The IVIVE modeling converts in vitro concentrations into external dose for calculation of the point of departure (POD) and comparisons to human exposure estimates to yield a MOE. The second tier involves short-term in vivo studies, expanded pharmacokinetic evaluations, and refined human exposure estimates. The results from the second tier studies provide more accurate estimates of the POD and the MOE. The third tier contains the traditional animal studies currently used to assess chemical safety. In each tier, the POD for selective chemicals is based primarily on endpoints associated with a proposed mode of action, whereas the POD for nonselective chemicals is based on potential biological perturbation. Based on the MOE, a significant percentage of chemicals evaluated in the first 2 tiers could be eliminated from further testing. The framework provides a risk-based and animal-sparing approach to evaluate chemical safety, drawing broadly from previous experience but incorporating technological advances to increase efficiency.

The question of how animal studies should be designed, conducted, and analyzed remains underexposed in societal debates on animal experimentation. This is not only a scientific but also a moral question. After all, if animal experiments are not appropriately designed, conducted, and analyzed, the results produced are unlikely to be reliable and the animals have in effect been wasted. In this article, we focus on one particular method to address this moral question, namely systematic reviews of previously performed animal experiments. We discuss how the design, conduct, and analysis of future (animal and human) experiments may be optimized through such systematic reviews. In particular, we illustrate how these reviews can help improve the methodological quality of animal experiments, make the choice of an animal model and the translation of animal data to the clinic more evidence-based, and implement the 3Rs. Moreover, we discuss which measures are being taken and which need to be taken in the future to ensure that systematic reviews will actually contribute to optimizing experimental design and thereby to meeting a necessary condition for making the use of animals in these experiments justified.

SummaryCarbon dioxide (CO 2 ) is the most commonly used agent for euthanasia of laboratory rodents, used on an estimated tens of millions of laboratory rodents per year worldwide, yet there is a growing body of evidence indicating that exposure to CO 2 causes more than momentary pain and distress in these and other animals. We reviewed the available literature on the use of CO 2 for euthanasia (as well as anaesthesia) and also informally canvassed laboratory animal personnel for their opinions regarding this topic. Our review addresses key issues such as CO 2 flow rate and final concentration, presence of oxygen, and prefilled chambers (the animal is added to the chamber once a predetermined concentration and flow rate have been reached) versus gradual induction (the animal is put into an empty chamber and the gas agent(s) is gradually introduced at a fixed rate). Internationally, animal research standards specify that any procedure that would cause pain or distress in humans should be assumed to do so in non-human animals as well (Public Health Service 1986, US Department of Agriculture 1997, Organization for Economic Cooperation and Development 2000). European Union guidelines, however, specify a certain threshold of pain or distress, such as 'skilled insertion of a hypodermic needle', as the starting point at which regulation of the use of animals in experimental or other scientific procedures begins (Biotechnology Regulatory Atlas n.d.). There is clear evidence in the human literature that CO 2 exposure is painful and distressful, while the non-human literature is equivocal. However, the fact that a number of studies do conclude that CO 2 causes pain and distress in animals indicates a need for careful reconsideration of its use. Finally, this review offers recommendations for alternatives to the use of CO 2 as a euthanasia agent.Keywords Carbon dioxide; euthanasia; pain; distress; anaesthesia; welfare; rodents Carbon dioxide (CO 2 ) is commonly used for euthanasia and anaesthesia of laboratory rodents, largely because of its ease of use, relative safety, and low cost, as well as its capacity to euthanize large numbers of animals in a short time span (Ambrose et al. 2000). In large institutions and those with significant rodent-breeding programmes, large numbers of rodents are often euthanized in a short time (Kline et al. 1963) and an appropriate gas agent is often the best way to approach such a challenge. However, CO 2 is not physiologically inert and the published evidence on whether or not CO 2 administration causes pain or distress in animals raises questions about its routine use. This paper reviews this published evidence and includes information on the effects of CO 2 in both humans and nonhumans. Methodological details are included when possible in order to provide a clear

Systematic reviews, pioneered in the clinical field, provide a transparent, methodologically rigorous and reproducible means of summarizing the available evidence on a precisely framed research question. Having matured to a well-established approach in many research fields, systematic reviews are receiving increasing attention as a potential tool for answering toxicological questions. In the larger framework of evidence-based toxicology, the advantages and obstacles of, as well as the approaches for, adapting and adopting systematic reviews to toxicology are still being explored. To provide the toxicology community with a starting point for conducting or understanding systematic reviews, we herein summarized available guidance documents from various fields of application. We have elaborated on the systematic review process by breaking it down into ten steps, starting with planning the project, framing the question, and writing and publishing the protocol, and concluding with interpretation and reporting. In addition, we have identified the specific methodological challenges of toxicological questions and have summarized how these can be addressed. Ultimately, this primer is intended to stimulate scientific discussions of the identified issues to fuel the development of toxicology-specific methodology and to encourage the application of systematic review methodology to toxicological issues.

A for Injection (1), and the most promising approaches for their validation. Experts from industry, regulatory authorities, German ministries, academia, research, national and international validation centres, and animal welfare organisations, were invited to actively participate in the meeting. The objective of the Expert Meeting was to review available alternative methods for BoNT potency testing, and to formulate recommendations for making progress toward implementing the Three Rs, i.e. Refinement, Reduction, and Replacement, in BoNT potency testing. In addition, ways in which communication on BoNT issues between manufacturers, researchers and regulators could be encouraged, and how improvements in regulatory harmonisation between different countries and continents could be achieved, were discussed. The meeting started with presentations by the individual participants, giving an overview on the regulatory and scientific status of alternative methods to the LD50 test for BoNT potency testing. Afterwards, the participants were divided into two separate break-out groups. Break-out Group 1 discussed the regulatory requirements for BoNT potency testing and the validation and implementation of alternative methods. Break-out Group 2 discussed the developed and available alternative methods and their suitability for reducing, refining or replacing the LD50 potency test.

20 th century. Relevant techniques include (among others) organson-a-chip (microdevices containing cells and fluids intended to simulate physiological processes in organs); organoids (three-dimensional spheroids containing multiple cell types and intended to simulate physiological processes); high-throughput systems (rapid screening of large numbers of chemicals for biological activity against panels of different cells or biomolecules); induced pluripotent stem cells (adult cells that have been genetically reprogrammed to an embryonic stem cell-like state); and computational modeling (using computation to study the behavior of complex systems).In our view, these methods (and no doubt others in various stages of development) have the potential to replace the use of animals as the default option in both safety testing and biomedical research. That is, these methods will come to comprise the rule, with animal experiments being the exception. This is consistent with Dutch efforts to expeditiously end animal experi- IntroductionThe Principles of Humane Experimental Technique, the landmark book that gave us the 3Rs framework of replacement, reduction, and refinement, turns 60 this year. First published in 1959, Principles was the outcome of a project spearheaded by the Universities Federation for Animal Welfare (UFAW), overseen by a committee that included future Nobel Prize-winning scientist Peter Medawar, and carried out by the British scientists William Russell and Rex Burch (Russell and Burch, 1959). The 3Rs framework helped to inspire and guide humane progress in experimental technique during the second half of the 20 th century and beyond (Stephens and Mak, 2013;Balls et al., 2019).The 60 th anniversary of Principles falls in the midst of substantial developments in non-animal methods, i.e., potential replacement technology. Indeed, scientific experimentation is at the cusp of a new era of techniques hardly imagined in the mid-Food for Thought …

Assessments of methodological and reporting quality are critical to adequately judging the credibility of a study's conclusions and to gauging its potential reproducibility. To aid those seeking to assess the methodological or reporting quality of studies relevant to toxicology, we conducted a scoping review of the available guidance with respect to four types of studies: in vivo and in vitro, (quantitative) structure-activity relationships ([Q]SARs), physico-chemical, and human observational studies. Our aims were to identify the available guidance in this diverse literature, briefly summarize each document, and distill the common elements of these documents for each study type. In general, we found considerable guidance for in vivo and human studies, but only one paper addressed in vitro studies exclusively. The guidance for (Q)SAR studies and physico-chemical studies was scant but authoritative. There was substantial overlap across guidance documents in the proposed criteria for both methodological and reporting quality. Some guidance documents address toxicology research directly, whereas others address preclinical research generally or clinical research and therefore may not be fully applicable to the toxicology context without some translation. Another challenge is the degree to which assessments of methodological quality in toxicology should focus on risk of bias - as in clinical medicine and healthcare - or be broadened to include other quality measures, such as confirming the identity of test substances prior to exposure. Our review is intended primarily for those in toxicology and risk assessment seeking an entry point into the extensive and diverse literature on methodological and reporting quality applicable to their work.