Are Non-animal Systemic Safety Assessments Protective? A Toolbox and Workflow.

An important question in toxicological risk assessment is whether non-animal new approach methodologies (NAMs) can be used to make safety decisions that are protective of human health, without being overly conservative. In this work, we propose a core NAM toolbox and workflow for conducting systemic safety assessments for adult consumers. We also present an approach for evaluating how protective and useful the toolbox and workflow are by benchmarking against historical safety decisions. The toolbox includes physiologically based kinetic (PBK) models to estimate systemic Cmax levels in humans, and 3 bioactivity platforms, comprising high-throughput transcriptomics, a cell stress panel, and in vitro pharmacological profiling, from which points of departure are estimated. A Bayesian model was developed to quantify the uncertainty in the Cmax estimates depending on how the PBK models were parameterized. The feasibility of the evaluation approach was tested using 24 exposure scenarios from 10 chemicals, some of which would be considered high risk from a consumer goods perspective (eg, drugs that are systemically bioactive) and some low risk (eg, existing food or cosmetic ingredients). Using novel protectiveness and utility metrics, it was shown that up to 69% (9/13) of the low risk scenarios could be identified as such using the toolbox, whilst being protective against all (5/5) the high-risk ones. The results demonstrated how robust safety decisions could be made without using animal data. This work will enable a full evaluation to assess how protective and useful the toolbox and workflow are across a broader range of chemical-exposure scenarios.

Zebrafish embryo neonicotinoid developmental neurotoxicity in the FET test and behavioral assays.

The need for reliable, sensitive (developmental) neurotoxicity testing of chemicals has steadily increased. Given the limited capacities for routine testing according to accepted regulatory guidelines, there is potential risk to human health and the environment. Most toxicity studies are based on mammalian test systems, which have been questioned for low sensitivity, limited relevance for humans, and animal welfare considerations. This increased the need for alternative models, one of which is the zebrafish (Danio rerio) embryo. This study assessed selected neonicotinoids at sub-lethal concentrations for their effects on embryonic development and behavior. The fish embryo acute toxicity test (OECD TG 236) determined the lowest observable effective concentrations, which were used as the highest test concentrations in subsequent behavioral assays. In the FET test, no severe compound-induced sublethal effects were seen at < 100 µM. In the coiling assay, exposure to ≥ 1.25 µM nicotine (positive control) affected both the burst duration and burst count per minute, whereas ≥ 50 µM thiacloprid affected the mean burst duration. Exposure to ≥ 50 µM acetamiprid and imidacloprid induced significant alterations in both mean burst duration and burst count per minute. In the swimming assay, 100 µM acetamiprid induced alterations in the frequency and extent of movements, whilst nicotine exposure only induced non-significant changes. All behavioral changes could be correlated to findings in mammalian studies. Given the quest for alternative test methods of (developmental) neurotoxicity, zebrafish embryo behavior testing could be integrated into a future tiered testing scheme.

A combined in vitro approach to improve the prediction of mitochondrial toxicants.

Drug induced mitochondrial dysfunction has been implicated in organ toxicity and the withdrawal of drugs or black box warnings limiting their use. The development of highly specific and sensitive in vitro assays in early drug development would assist in detecting compounds which affect mitochondrial function. Here we report the combination of two in vitro assays for the detection of drug induced mitochondrial toxicity. The first assay measures cytotoxicity after 24h incubation of test compound in either glucose or galactose conditioned media (Glu/Gal assay). Compounds with a greater than 3-fold toxicity in galactose media compared to glucose media imply mitochondrial toxicity. The second assay measures mitochondrial respiration, glycolysis and a reserve capacity with mechanistic responses observed within one hour following exposure to test compound. In order to assess these assays a total of 72 known drugs and chemicals were used. Dose-response data was normalised to 100× Cmax giving a specificity, sensitivity and accuracy of 100%, 81% and 92% respectively for this combined approach.

Predicting in vivo phospholipidosis-inducing potential of drugs by a combined high content screening and in silico modelling approach.

Drug induced phospholipidosis (PLD) is an adverse side effect which can affect registration of new drug entities. Phospholipids can accumulate in lysosomes, organelles essential in cellular biogenesis and if compromised can lead to cellular toxicity. Drug accumulation in lysosomes (lysosomotropism) is a known mechanism leading to PLD, however phospholipidosis can also occur indirectly by altering synthesis and processing of phospholipids. Drug induced PLD can be measured in vitro using High Content Screening (HCS) approaches, by either determining accumulation of phospholipids conjugated to dyes in cells or by determining accumulation of drugs within lysosomes, by competitive loss of lysosomal dye uptake. In this study we validate two in vitro assays using HepG2 and H9c2 cells in conjunction with in silico models based on physico-chemical properties using 56 compounds (28 phospholipidogenic, 25 non-phospholipidogenic and three kidney specific). Using HCS to determine PLD and lysosomal trapping in HepG2 cells in combination with in silico modelling increase the overall prediction of PLD in vivo with a sensitivity of 96%, specificity of 92% and overall accuracy of 94%. The findings of this study demonstrate the applicability of in vitro and in silico approaches to understand the mechanism underlying PLD and the utility of these approaches as a screening strategy in the pharmaceutical industry to select drug candidates with a low in vivo PLD liability.

Diverse assessment and active student engagement sustain deep learning: A comparative study of outcomes in two parallel introductory biochemistry courses.

Although there is increasing evidence for a relationship between courses that emphasize student engagement and achievement of student deep learning, there is a paucity of quantitative comparative studies in a biochemistry and molecular biology context. Here, we present a pedagogical study in two contrasting parallel biochemistry introductory courses to compare student surface and deep learning. Surface and deep learning were measured quantitatively by a study process questionnaire at the start and end of the semester, and qualitatively by questionnaires and interviews with students. In the traditional lecture/examination based course, there was a dramatic shift to surface learning approaches through the semester. In the course that emphasized student engagement and adopted multiple forms of assessment, a preference for deep learning was sustained with only a small reduction through the semester. Such evidence for the benefits of implementing student engagement and more diverse non-examination based assessment has important implications for the design, delivery, and renewal of introductory courses in biochemistry and molecular biology.