Abstract Next-Generation Risk Assessment is defined as an exposure-led, hypothesis-driven risk assessment approach that integrates new approach methodologies (NAMs) to assure safety without the use of animal testing. These principles were applied to a hypothetical safety assessment of 0.1% coumarin in face cream and body lotion. For the purpose of evaluating the use of NAMs, existing animal and human data on coumarin were excluded. Internal concentrations (plasma Cmax) were estimated using a physiologically based kinetic model for dermally applied coumarin. Systemic toxicity was assessed using a battery of in vitro NAMs to identify points of departure (PoDs) for a variety of biological effects such as receptor-mediated and immunomodulatory effects (Eurofins SafetyScreen44 and BioMap Diversity 8 Panel, respectively), and general bioactivity (ToxCast data, an in vitro cell stress panel and high-throughput transcriptomics). In addition, in silico alerts for genotoxicity were followed up with the ToxTracker tool. The PoDs from the in vitro assays were plotted against the calculated in vivo exposure to calculate a margin of safety with associated uncertainty. The predicted Cmax values for face cream and body lotion were lower than all PoDs with margin of safety higher than 100. Furthermore, coumarin was not genotoxic, did not bind to any of the 44 receptors tested and did not show any immunomodulatory effects at consumer-relevant exposures. In conclusion, this case study demonstrated the value of integrating exposure science, computational modeling and in vitro bioactivity data, to reach a safety decision without animal data.

Risk assessment methodologies in toxicology have remained largely unchanged for decades. The default approach uses high dose animal studies, together with human exposure estimates, and conservative assessment (uncertainty) factors or linear extrapolations to determine whether a specific chemical exposure is 'safe' or 'unsafe'. Although some incremental changes have appeared over the years, results from all new approaches are still judged against this process of extrapolating high-dose effects in animals to low-dose exposures in humans. The US National Research Council blueprint for change, entitled Toxicity Testing in the 21st Century: A Vision and Strategy called for a transformation of toxicity testing from a system based on high-dose studies in laboratory animals to one founded primarily on in vitro methods that evaluate changes in normal cellular signalling pathways using human-relevant cells or tissues. More recently, this concept of pathways-based approaches to risk assessment has been expanded by the description of 'Adverse Outcome Pathways' (AOPs). The question, however, has been how to translate this AOP/TT21C vision into the practical tools that will be useful to those expected to make safety decisions. We have sought to provide a practical example of how the TT21C vision can be implemented to facilitate a safety assessment for a commercial chemical without the use of animal testing. To this end, the key elements of the TT21C vision have been broken down to a set of actions that can be brought together to achieve such a safety assessment. Such components of a pathways-based risk assessment have been widely discussed, however to-date, no worked examples of the entire risk assessment process exist. In order to begin to test the process, we have taken the approach of examining a prototype toxicity pathway (DNA damage responses mediated by the p53 network) and constructing a strategy for the development of a pathway based risk assessment for a specific chemical in a case study mode. This contribution represents a 'work-in-progress' and is meant to both highlight concepts that are well-developed and identify aspects of the overall process which require additional development. To guide our understanding of what a pathways-based risk assessment could look like in practice, we chose to work on a case study chemical (quercetin) with a defined human exposure and to bring a multidisciplinary team of chemists, biologists, modellers and risk assessors to work together towards a safety assessment. Our goal was to see if the in vitro dose response for quercetin could be sufficiently understood to construct a TT21C risk assessment without recourse to rodent carcinogenicity study data. The data presented include high throughput pathway biomarkers (p-H2AX, p-ATM, p-ATR, p-Chk2, p53, p-p53, MDM2 and Wip1) and markers of cell-cycle, apoptosis and micronuclei formation, plus gene transcription in HT1080 cells. Eighteen point dose response curves were generated using flow cytometry and imaging to determine the concentrations...

VP16 is an essential structural protein of herpes simplex virus. It plays important roles in immediate-early transcriptional regulation, in the modulation of the activities of other viral components, and in the pathway of assembly and egress of infectious virions. To gain further insight into the compartmentalization of this multifunctional protein we constructed and characterized recombinant viruses expressing VP16 linked to the green fluorescent protein (GFP). These viruses replicate with virtually normal kinetics and yields and incorporate the fusion protein into the virion, resulting in autofluorescent particles. De novo-synthesized VP16-GFP was first detected in a diffuse pattern within the nucleus. Nuclear VP16-GFP was progressively recruited to replication compartments, which coalesced into large globular domains. By 10 to 12 h after infection additional distinct foci containing VP16-GFP could be seen, almost exclusively located at the periphery of the replication compartments. At the same time pronounced accumulation was observed in the cytoplasm, first in a diffuse pattern and then accumulating in vesicle-like compartments which were concentrated in an asymmetric fashion reminiscent of the Golgi. Inhibition of DNA replication resulted in prolonged diffuse nuclear distribution with minimal cytoplasmic accumulation. Treatment with brefeldin disrupted the cytoplasm vesicular pattern, resulting in redistributed large foci. Time-lapse microscopy demonstrated various dynamic features of infection, including the active induction of very long cellular projections (up to 100 M). Vesicular clusters containing VP16 were transported within projections to the termini, which developed bulbous ends and appeared to embed into the membranes of adjacent uninfected cells.The herpesvirus tegument is defined as the unstructured proteinaceous layer between the viral capsid and the envelope incorporating, in the case of herpes simplex virus (HSV), at least 15 or more virus-encoded proteins in differing copy number and accounting for approximately 50% of the volume of the virion (8, 17). Although only a subset of HSV tegument proteins is absolutely essential for replication in tissue culture, it is becoming clear that these proteins play important and diverse structural and functional roles during infection. Recent data on the pathway of maturation of HSV indicate that the virus acquires a primary envelope at the inner nuclear membrane but that this is followed by fusion at the outer nuclear envelope and reenvelopment in a Golgi-related compartment (12,26,40). However, the site(s) and mechanism(s) of recruitment of tegument proteins remain unclear.One of the most-abundant tegument proteins is VP16 (18), the product of the UL48 gene (6) of HSV, and it is one of the subset of essential tegument proteins (43). VP16 plays important roles in the transcriptional regulation of immediate-early (IE) genes, in the modulation of the activities of other viral components, and in the assembly and egress of infectious virions. VP16 is assemb...

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 three 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 parameterised. 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 (e.g., drugs that are systemically bioactive) and some low risk (e.g., 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.

The bacterial reverse mutation assay (Ames test) is a biological assay used to assess the mutagenic potential of chemical compounds. In this paper approaches for the development of an in silico mutagenicity screening tool are described. Three individual in silico models, which cover both structure activity relationship methods (SARs) and quantitative structure activity relationship methods (QSARs), were built using three different modelling techniques: (1) an in-house alert model: which uses SAR approach where alerts are generated based on experts judgements; (2) a kNN approach (k-Nearest Neighbours), which is a QSAR model where a prediction is given based on outcomes of its k chemical neighbours; (3) a naive Bayesian model (NB), which is another QSAR model, where a prediction is derived using a Bayesian formula through preselected identified informative chemical features (e.g., physico-chemical, structural descriptors). These in silico models, were compared against two well-known alert models (DEREK and ToxTree) and also against three different consensus approaches (Categorical Bayesian Integration Approach (CBI), Partial Least Squares Discriminate Analysis (PLS-DA) and simple majority vote approach). By applying these integration methods on the validation sets it was shown that both integration models (PLS-DA and CBI) achieved better performance than any of the individual models or consensus obtained by simple majority rule. In conclusion, the recommendation of this paper is that when obtaining consensus predictions for Ames mutagenicity, approaches like PLS-DA or CBI should be the first choice for the integration as compared to a simple majority vote approach.