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The European Partnership for Alternative Approaches to Animal Testing (EPAA) convened a 'Blue Sky Workshop' on new ideas for non-animal approaches to predict repeated-dose systemic toxicity. The aim of the Workshop was to formulate strategic ideas to improve and increase the applicability, implementation and acceptance of modern non-animal methods to determine systemic toxicity. The Workshop concluded that good progress is being made to assess repeated dose toxicity without animals taking advantage of existing knowledge in toxicology, thresholds of toxicological concern, adverse outcome pathways and read-across workflows. These approaches can be supported by New Approach Methodologies (NAMs) utilising modern molecular technologies and computational methods. Recommendations from the Workshop were based around the needs for better chemical safety assessment: how to strengthen the evidence base for decision making; to develop, standardise and harmonise NAMs for human toxicity; and the improvement in the applicability and acceptance of novel techniques. "Disruptive thinking" is required to reconsider chemical legislation, validation of NAMs and the opportunities to move away from reliance on animal tests. Case study practices and data sharing, ensuring reproducibility of NAMs, were viewed as crucial to the improvement of non-animal test approaches for systemic toxicity., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Some of the authors received reimbursement of their travel expenses by the EPAA to make their participation in the workshop possible. If deemed necessary, a list of those people who received travel expenses support can be provided., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

1. The extent of sulfoxidation of the drug, S-carboxymethyl-L-cysteine, has been shown to vary between individuals, with this phenomenon being mooted as a biomarker for certain disease states and susceptibilities. Studies in vitro have indicated that the enzyme responsible for this reaction was phenylalanine monooxygenase but to date no in vivo evidence exists to support this assumption. Using the mouse models of mild hyperphenylalaninamia (enu1 PAH variant) and classical phenylketonuria (enu2 PAH variant), the sulfur oxygenation of S-carboxymethyl-L-cysteine has been investigated. 2. Compared to the wild type (wt/wt) mice, both the heterozygous dominant (wt/enu1 and wt/enu2) mice and the homozygous recessive (enu1/enu1 and enu2/enu2) mice were shown to have significantly increased Cmax, AUC(0-180 min) and AUC(0-∞ min) values (15 - to 20-fold higher). These results were primarily attributable to the significantly reduced clearance of S-carboxymethyl-L-cysteine (13 - to 22-fold lower). 3. Only the wild type mice produced measurable quantities of the parent S-oxide metabolites. Those mice possessing one or more allelic variant showed no evidence of blood SCMC (R/S) S-oxides. These observations support the proposition that differences in phenylalanine hydroxylase activity underlie the variation in S-carboxymethyl-L-cysteine sulfoxidation and that no other enzyme is able to undertake this reaction.

Fear learning is thought to play an important role in panic disorder. Benign interoceptive sensations can become predictors (conditioned stimuli - CSs) of massive fear when experienced in the context of an initial panic attack (unconditioned stimulus - US). The mere encounter of these CSs on a later moment can induce anxiety and fear, and precipitate a new panic attack. It has been suggested that fear learning to interoceptive cues would result in unpredictable panic. The present study aimed to investigate whether fear learning to an interoceptive CS is possible without declarative knowledge of the CS-US contingency. The CS consisted of mild breathlessness (or: dyspnea), the US was a suffocation experience. During acquisition, the experimental group received six presentations of mild breathlessness immediately followed by suffocation; for the control group both experiences were always separated by an intertrial interval. In the subsequent extinction phase, participants received six unreinforced presentations of the CS. Expectancy of the US was rated continuously and startle eyeblink electromyographic, skin conductance, and respiration were measured. Declarative knowledge of the CS-US relationship was also assessed with a post-experimental questionnaire. At the end of acquisition, both groups displayed the same levels of US expectancy and skin conductance in response to the CS, but the experimental group showed a fear potentiated startle eyeblink and a different respiratory response to the CS compared to the control group. Further analyses on a subgroup of CS-US unaware participants confirmed the presence of startle eyeblink conditioning in the experimental group but not in the control group. Our findings suggest that interoceptive fear learning is not dependent on declarative knowledge of the CS-US relationship. The present interoceptive fear conditioning paradigm may serve as an ecologically valid laboratory model for unexpected panic attacks.