Probing conformational dynamics of DNA binding by CO-sensing transcription factor, CooA.

The transcription factor CooA is a CRP/FNR (cAMP receptor protein/ fumarate and nitrate reductase) superfamily protein that uses heme to sense carbon monoxide (CO). Allosteric activation of CooA in response to CO binding is currently described as a series of discrete structural changes, without much consideration for the potential role of protein dynamics in the process of DNA binding. This work uses site-directed spin-label electron paramagnetic resonance spectroscopy (SDSL-EPR) to probe slow timescale (µs-ms) conformational dynamics of CooA with a redox-stable nitroxide spin label, and IR spectroscopy to probe the environment at the CO-bound heme. A series of cysteine substitution variants were created to selectively label CooA in key functional regions, the heme-binding domain, the 4/5-loop, the hinge region, and the DNA binding domain. The EPR spectra of labeled CooA variants are compared across three functional states: Fe(III) "locked off", Fe(II)-CO "on", and Fe(II)-CO bound to DNA. We observe changes in the multicomponent EPR spectra at each location; most notably in the hinge region and DNA binding domain, broadening the description of the CooA allosteric mechanism to include the role of protein dynamics in DNA binding. DNA-dependent changes in IR vibrational frequency and band broadening further suggest that there is conformational heterogeneity in the active WT protein and that DNA binding alters the environment of the heme-bound CO.

Reaction kinetics of lithium-sulfur batteries with a polar Li-ion electrolyte: modeling of liquid phase and solid phase processes.

The present investigation fits the reaction kinetics of a lithium-sulfur (Li-S) battery with polar electrolyte employing a novel two-phase continuum multipore model. The continuum two-phase model considers processes in both the liquid electrolyte phase and the solid precipitates phase, where the diffusion coefficients of the Li+ ions in a solvent-softened solid state are determined from molecular dynamics simulations. Solubility experiments yield the saturation concentration of sulfur and lithium sulfides in the polar electrolyte employed in this study. The model describes the transport of dissolved molecular and ion species in pores of different size in solvated or desolvated form, depending on pore size. The Li-S reaction model in this study is validated for electrolyte 1 M LiPF6 in EC/DMC. It includes seven redox reactions and two cyclic non-electrochemical reactions in the cathode, and the lithium redox reaction at the anode. Electrochemical reactions are assumed to take place in the electrolyte solution or the solid state and cyclic reactions are assumed to take place in the liquid electrolyte phase only. The determination of the reaction kinetics parameters takes place via fitting the model predictions with experimental data of a cyclic voltammetry cycle with in operando UV-vis spectroscopy.

Next generation risk assessment for occupational chemical safety - A real world example with sodium-2-hydroxyethane sulfonate.

Next Generation Risk Assessment (NGRA) is an exposure-led approach to safety assessment that uses New Approach Methodologies (NAMs). Application of NGRA has been largely restricted to assessments of consumer use of cosmetics and is not currently implemented in occupational safety assessments, e.g. under EU REACH. By contrast, a large proportion of regulatory worker safety assessments are underpinned by toxicological studies using experimental animals. Consequently, occupational safety assessment represents an area that would benefit from increasing application of NGRA to safety decision making. Here, a workflow for conducting NGRA under an occupational safety context was developed, which is illustrated with a case study chemical; sodium 2-hydroxyethane sulphonate (sodium isethionate or SI). Exposures were estimated using a standard occupational exposure model following a comprehensive life cycle assessment of SI and considering factory-specific data. Outputs of this model were then used to estimate internal exposures using a Physiologically Based Kinetic (PBK) model, which was constructed with SI specific Absorption, Distribution, Metabolism and Excretion (ADME) data. PBK modelling indicated a worst-case plasma maximum concentration (Cmax) of 0.8 µM across the SI life cycle. SI bioactivity was assessed in a battery of NAMs relevant to systemic, reproductive, and developmental toxicity; a cell stress panel, high throughput transcriptomics in three cell lines (HepG2, HepaRG and MCF-7 cells), pharmacological profiling and specific assays relating to developmental toxicity (Reprotracker and devTOX quickPredict). Points of Departure (PoDs) for SI ranged from 104 to 5044 µM. Cmax values obtained from PBK modelling of occupational exposures to SI were compared with PoDs from the bioactivity assays to derive Bioactivity Exposure Ratios (BERs) which demonstrated the safety for workers exposed to SI under current levels of factory specific risk management. In summary, the tiered and iterative workflow developed here represents an opportunity for integrating non animal approaches for a large subset of substances for which systemic worker safety assessment is required. Such an approach could be followed to ensure that animal testing is only conducted as a "last resort" e.g. under EU REACH.

Carbon Monoxide Poisoning: From Microbes to Therapeutics.

Carbon monoxide (CO) poisoning leads to 50,000-100,000 emergency room visits and 1,500-2,000 deaths each year in the United States alone. Even with treatment, survivors often suffer from long-term cardiac and neurocognitive deficits, highlighting a clear unmet medical need for novel therapeutic strategies that reduce morbidity and mortality associated with CO poisoning. This review examines the prevalence and impact of CO poisoning and pathophysiology in humans and highlights recent advances in therapeutic strategies that accelerate CO clearance and mitigate toxicity. We focus on recent developments of high-affinity molecules that take advantage of the uniquely strong interaction between CO and heme to selectively bind and sequester CO in preclinical models. These scavengers, which employ heme-binding scaffolds ranging from organic small molecules to hemoproteins derived from humans and potentially even microorganisms, show promise as field-deployable antidotes that may rapidly accelerate CO clearance and improve outcomes for survivors of acute CO poisoning.

Engineering neuroglobin nitrite reductase activity based on myoglobin models.

Neuroglobin is a hemoprotein expressed in several nervous system cell lineages with yet unknown physiological functions. Neuroglobin presents a very similar structure to that of the related globins hemoglobin and myoglobin, but shows an hexacoordinate heme as compared to the pentacoordinated heme of myoglobin and hemoglobin. While several reactions of neuroglobin have been characterized in vitro, the relative importance of most of those reactions in vivo is yet undefined. Neuroglobin, like other heme proteins, can reduce nitrite to nitric oxide, providing a possible route to generate nitric oxide in vivo in low oxygen conditions. The reaction kinetics are highly dependent on the nature of the distal residue, and replacement of the distal histidine His64(E7) can increase the reaction rate constants by several orders of magnitude. However, mutation of other distal pocket positions such as Phe28(B10) or Val68(E11) has more limited impact on the rates. Computational analysis using myoglobin as template, guided by the structure of dedicated nitrite reductases like cytochrome cd1 nitrite reductase, has pointed out that combined mutations of the residues B10 and CD1 could increase the nitrite reductase activity of myoglobin, by mimicking the environment of the distal heme pocket in cytochrome cd1 nitrite reductase. As neuroglobin shows high sequence and structural homology with myoglobin, we hypothesized that such mutations (F28H and F42Y in neuroglobin) could also modify the nitrite reductase activity of neuroglobin. Here we study the effect of these mutations. Unfortunately, we do not observe in any case an increase in the nitrite reduction rates. Our results provide some further indications of nitrite reductase regulation in neuroglobin and highlight the minor but critical differences between the structure of penta- and hexacoordinate globins.

Nitric oxide and thiols: Chemical biology, signalling paradigms and vascular therapeutic potential.

Nitric oxide (• NO) interactions with biological thiols play crucial, but incompletely determined, roles in vascular signalling and other biological processes. Here, we highlight two recently proposed signalling paradigms: (1) the formation of a vasodilating labile nitrosyl ferrous haem (NO-ferrohaem) facilitated by thiols via thiyl radical generation and (2) polysulfides/persulfides and their interaction with • NO. We also describe the specific (bio)chemical routes in which • NO and thiols react to form S-nitrosothiols, a broad class of small molecules, and protein post-translational modifications that can influence protein function through catalytic site or allosteric structural changes. S-Nitrosothiol formation depends upon cellular conditions, but critically, an appropriate oxidant for either the thiol (yielding a thiyl radical) or • NO (yielding a nitrosonium [NO+ ]-donating species) is required. We examine the roles of these collective • NO/thiol species in vascular signalling and their cardiovascular therapeutic potential.

Thiol-catalyzed formation of NO-ferroheme regulates intravascular NO signaling.

Nitric oxide (NO) is an endogenously produced signaling molecule that regulates blood flow and platelet activation. However, intracellular and intravascular diffusion of NO are limited by scavenging reactions with several hemoproteins, raising questions as to how free NO can signal in hemoprotein-rich environments. We explore the hypothesis that NO can be stabilized as a labile ferrous heme-nitrosyl complex (Fe2+-NO, NO-ferroheme). We observe a reaction between NO, labile ferric heme (Fe3+) and reduced thiols to yield NO-ferroheme and a thiyl radical. This thiol-catalyzed reductive nitrosylation occurs when heme is solubilized in lipophilic environments such as red blood cell membranes or bound to serum albumin. The resulting NO-ferroheme resists oxidative inactivation, is soluble in cell membranes and is transported intravascularly by albumin to promote potent vasodilation. We therefore provide an alternative route for NO delivery from erythrocytes and blood via transfer of NO-ferroheme and activation of apo-soluble guanylyl cyclase.

ADME characterization and PBK model development of 3 highly protein-bound UV filters through topical application.

Estimating human exposure in the safety assessment of chemicals is crucial. Physiologically based kinetic (PBK) models which combine information on exposure, physiology, and chemical properties, describing the absorption, distribution, metabolism, and excretion (ADME) processes of a chemical, can be used to calculate internal exposure metrics such as maximum concentration and area under the concentration-time curve in plasma or tissues of a test chemical in next-generation risk assessment. This article demonstrates the development of PBK models for 3 UV filters, specifically octyl methoxycinnamate, octocrylene, and 4-methylbenzylidene camphor. The models were parameterized entirely based on data obtained from in vitro and/or in silico methods in a bottom-up modeling approach and then validated based on human dermal pharmacokinetic (PK) data. The 3 UV filters are "difficult to test" in in vitro test systems due to high lipophilicity, high binding affinity for proteins, and nonspecific binding, for example, toward plastic. This research work presents critical considerations in ADME data generation, interpretation, and parameterization to assure valid PBK model development to increase confidence in using PBK modeling to help make safety decisions in the absence of human PK data. The developed PBK models of the 3 chemicals successfully simulated the plasma concentration profiles of clinical PK data following dermal application, indicating the reliability of the ADME data generated and the parameters determined. The study also provides insights and lessons learned for characterizing ADME and developing PBK models for highly lipophilic and protein-bound chemicals in the future.

A novel co-culture model of human prostate epithelial and stromal cells for androgenic and antiandrogenic screening.

The risk assessment of endocrine-disrupting chemicals (EDCs) greatly relies on in vitro screening. A 3-dimensional (3D) in vitro prostate model that can reflect physiologically-relevant prostate epithelial and stromal crosstalk can significantly advance the current androgen assessment. This study built a prostate epithelial and stromal co-culture microtissue model with BHPrE and BHPrS cells in scaffold-free hydrogels. The optimal 3D co-culture condition was defined, and responses of the microtissue to androgen (dihydrotestosterone, DHT) and anti-androgen (flutamide) exposure were characterized using molecular and image profiling techniques. The co-culture prostate microtissue maintained a stable structure for up to seven days and presented molecular and morphological features of the early developmental stage of the human prostate. The cytokeratin 5/6 (CK5/6) and cytokeratin 18 (CK18) immunohistochemical staining indicated epithelial heterogeneity and differentiation in these microtissues. The prostate-related gene expression profiling did not efficiently differentiate androgen and anti-androgen exposure. However, a cluster of distinctive 3D image features was identified and could be applied in the androgenic and anti-androgenic effect prediction. Overall, the current study established a co-culture prostate model that provided an alternative strategy for (anti-)androgenic EDC safety assessment and highlighted the potential and advantage of utilizing image features to predict endpoints in chemical screening.

Carbon Monoxide-Sensing Transcription Factors: Regulators of Microbial Carbon Monoxide Oxidation Pathway Gene Expression.

Carbon monoxide (CO) serves as a source of energy and carbon for a diverse set of microbes found in anaerobic and aerobic environments. The enzymes that bacteria and archaea use to oxidize CO depend upon complex metallocofactors that require accessory proteins for assembly and proper function. This complexity comes at a high energetic cost and necessitates strict regulation of CO metabolic pathways in facultative CO metabolizers to ensure that gene expression occurs only when CO concentrations and redox conditions are appropriate. In this review, we examine two known heme-dependent transcription factors, CooA and RcoM, that regulate inducible CO metabolism pathways in anaerobic and aerobic microorganisms. We provide an analysis of the known physiological and genomic contexts of these sensors and employ this analysis to contextualize known biochemical properties. In addition, we describe a growing list of putative transcription factors associated with CO metabolism that potentially use cofactors other than heme to sense CO.

Characterization and utility of two monoclonal antibodies to cholera toxin B subunit.

Cholera toxin B subunit (CTB) is a potent immunomodulator exploitable in mucosal vaccine and immunotherapeutic development. To aid in the characterization of pleiotropic biological functions of CTB and its variants, we generated a panel of anti-CTB monoclonal antibodies (mAbs). By ELISA and surface plasmon resonance, two mAbs, 7A12B3 and 9F9C7, were analyzed for their binding affinities to cholera holotoxin (CTX), CTB, and EPICERTIN: a recombinant CTB variant possessing mucosal healing activity. Both 7A12B3 and 9F9C7 bound efficiently to CTX, CTB, and EPICERTIN with equilibrium dissociation constants at low to sub-nanomolar concentrations but bound weakly, if at all, to Escherichia coli heat-labile enterotoxin B subunit. In a cyclic adenosine monophosphate assay using Caco2 human colon epithelial cells, the 7A12B3 mAb was found to be a potent inhibitor of CTX, whereas 9F9C7 had relatively weak inhibitory activity. Meanwhile, the 9F9C7 mAb effectively detected CTB and EPICERTIN bound to the surface of Caco2 cells and mouse spleen leukocytes by flow cytometry. Using 9F9C7 in immunohistochemistry, we confirmed the preferential localization of EPICERTIN in colon crypts following oral administration of the protein in mice. Collectively, these mAbs provide valuable tools to investigate the biological functions and preclinical development of CTB variants.

Solubility and dissolution kinetics of sulfur and sulfides in electrolyte solvents for lithium-sulfur and sodium-sulfur batteries.

In this study, we monitor the dissolution of sulfur and sulfides in electrolyte solvents for lithium-sulfur (Li-S) and sodium-sulfur (Na-S) batteries. The first aim of this research is to assemble a comprehensive set of data on solubilities and dissolution kinetics that may be used in the simulation of battery cycling. The investigation also offers important insights to address key bottlenecks in the development and commercialization of metal-sulfur batteries, including the incomplete dissolution of sulfur in discharge and insoluble low-order sulfides in charge, the probability of shuttling of soluble polysulfides, and the pausing of the redox reactions in precipitated low order sulfides depending on their degree of solid state. The tested materials include sulfur, lithium sulfides Li2Sx, x = 1, 2, 4, 6, and 8, and sodium sulfides Na2Sx, x = 1, 2, 3, 4, 6, and 8, dissolved in two alternative electrolyte solvents: DOL:DME 1:1 v/v and TEGDME. The determined properties of the solute dissolution in the solvent include saturation concentration, mass transfer coefficient, and diffusion coefficient of the solvent in the solid solute. In general, the DOL:DME system offers high solubility in Li-S batteries and TEGDME offers the highest solubility in Na-S batteries. Low solubility sulfides are Li2S2 and Li2S for the Li-S batteries, and Na2S3, Na2S2, and Na2S for the Na-S batteries. However, it is noted that Na2S3 dissolves fast in TEGDME and also TEGDME diffuses fast into Na2S3, offering the possibility of a swollen Na2S3 structure in which Na+ ions might diffuse and continue the redox reactions in a semisolid state.

Thiol catalyzed formation of NO-ferroheme regulates canonical intravascular NO signaling.

Nitric oxide (NO) is an endogenously produced physiological signaling molecule that regulates blood flow and platelet activation. However, both the intracellular and intravascular diffusion of NO is severely limited by scavenging reactions with hemoglobin, myoglobin, and other hemoproteins, raising unanswered questions as to how free NO can signal in hemoprotein-rich environments, like blood and cardiomyocytes. We explored the hypothesis that NO could be stabilized as a ferrous heme-nitrosyl complex (Fe 2+ -NO, NO-ferroheme) either in solution within membranes or bound to albumin. Unexpectedly, we observed a rapid reaction of NO with free ferric heme (Fe 3+ ) and a reduced thiol under physiological conditions to yield NO-ferroheme and a thiyl radical. This thiol-catalyzed reductive nitrosylation reaction occurs readily when the hemin is solubilized in lipophilic environments, such as red blood cell membranes, or bound to serum albumin. NO-ferroheme albumin is stable, even in the presence of excess oxyhemoglobin, and potently inhibits platelet activation. NO-ferroheme-albumin administered intravenously to mice dose-dependently vasodilates at low- to mid-nanomolar concentrations. In conclusion, we report the fastest rate of reductive nitrosylation observed to date to generate a NO-ferroheme molecule that resists oxidative inactivation, is soluble in cell membranes, and is transported intravascularly by albumin to promote potent vasodilation.

Cell-free and alkylated hemoproteins improve survival in mouse models of carbon monoxide poisoning.

I.v. administration of a high-affinity carbon monoxide-binding (CO-binding) molecule, recombinant neuroglobin, can improve survival in CO poisoning mouse models. The current study aims to discover how biochemical variables of the scavenger determine the CO removal from the RBCs by evaluating 3 readily available hemoproteins, 2,3-diphosphoglycerate stripped human hemoglobin (StHb); N-ethylmaleimide modified hemoglobin (NEMHb); and equine myoglobin (Mb). These molecules efficiently sequester CO from hemoglobin in erythrocytes in vitro. A kinetic model was developed to predict the CO binding efficacy for hemoproteins, based on their measured in vitro oxygen and CO binding affinities, suggesting that the therapeutic efficacy of hemoproteins for CO poisoning relates to a high M value, which is the binding affinity for CO relative to oxygen (KA,CO/KA,O2). In a lethal CO poisoning mouse model, StHb, NEMHb, and Mb improved survival by 100%, 100%, and 60%, respectively, compared with saline controls and were well tolerated in 48-hour toxicology assessments. In conclusion, both StHb and NEMHb have high CO binding affinities and M values, and they scavenge CO efficiently in vitro and in vivo, highlighting their therapeutic potential for point-of-care antidotal therapy of CO poisoning.

Ready for regulatory use: NAMs and NGRA for chemical safety assurance.

New approach methodologies (NAMs) that do not use experimental animals are, in certain settings, entirely appropriate for assuring the safety of chemical ingredients, although regulatory adoption has been slow. In this opinion article we discuss how scientific advances that utilize NAMs to certify systemic safety are available now and merit broader acceptance within the framework of next generation risk assessments (NGRA).

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.

Impact of glycoengineering and antidrug antibodies on the anticancer activity of a plant-made lectin-Fc fusion protein.

Plants are an efficient production platform for manufacturing glycoengineered monoclonal antibodies and antibody-like molecules. Avaren-Fc (AvFc) is a lectin-Fc fusion protein or lectibody produced in Nicotiana benthamiana, which selectively recognizes cancer-associated high-mannose glycans. In this study, we report the generation of a glycovariant of AvFc that is devoid of plant glycans, including the core α1,3-fucose and ß1,2-xylose residues. The successful removal of these glycans was confirmed by glycan analysis using HPLC. This variant, AvFcΔXF , has significantly higher affinity for Fc gamma receptors and induces higher levels of luciferase expression in an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assay against B16F10 murine melanoma cells without inducing apoptosis or inhibiting proliferation. In the B16F10 flank tumour mouse model, we found that systemic administration of AvFcΔXF , but not an aglycosylated AvFc variant lacking affinity for Fc receptors, significantly delayed the growth of tumours, suggesting that Fc-mediated effector functions were integral. AvFcΔXF treatment also significantly reduced lung metastasis of B16F10 upon intravenous challenge whereas a sugar-binding-deficient mutant failed to show efficacy. Lastly, we determined the impact of antidrug antibodies (ADAs) on drug activity in vivo by pretreating animals with AvFcΔXF before implanting tumours. Despite a significant ADA response induced by the pretreatment, we found that the activity of AvFcΔXF was unaffected by the presence of these antibodies. These results demonstrate that glycoengineering is a powerful strategy to enhance AvFc's antitumor activity.

Read-across and new approach methodologies applied in a 10-step framework for cosmetics safety assessment - A case study with parabens.

Parabens are esters of para-hydroxybenzoic acid that have been used as preservatives in many types of products for decades including agrochemicals, pharmaceuticals, food and cosmetics. This illustrative case study with propylparaben (PP) demonstrates a 10-step read-across (RAX) framework in practice. It aims at establishing a proof-of-concept for the value added by new approach methodologies (NAMs) in read-across (RAX) for use in a next-generation risk assessment (NGRA) in order to assess consumer safety after exposure to PP-containing cosmetics. In addition to structural and physico-chemical properties, in silico information, toxicogenomics, in vitro toxicodynamic, toxicokinetic data from PBK models, and bioactivity data are used to provide evidence of the chemical and biological similarity of PP and analogues and to establish potency trends for observed effects in vitro. The chemical category under consideration is short (C1-C4) linear chain n-alkyl parabens: methylparaben, ethylparaben, propylparaben and butylparaben. The goal of this case study is to illustrate how a practical framework for RAX can be used to fill a hypothetical data gap for reproductive toxicity of the target chemical PP.

Quaternary Structure and Deoxyribonucleic Acid-Binding Properties of the Heme-Dependent, CO-Sensing Transcriptional Regulator PxRcoM.

RcoM, a heme-containing, CO-sensing transcription factor, is one of two known bacterial regulators of CO metabolism. Unlike its analogue CooA, the structure and DNA-binding properties of RcoM remain largely uncharacterized. Using a combination of size exclusion chromatography and sedimentation equilibrium, we demonstrate that RcoM-1 from Paraburkholderia xenovorans is a dimer, wherein the heme-binding domain mediates dimerization. Using bioinformatics, we show that RcoM is found in three distinct genomic contexts, in accordance with the previous literature. We propose a refined consensus DNA-binding sequence for RcoM based on sequence alignments of coxM-associated promoters. The RcoM promoter consensus sequence bears two well-conserved direct repeats, consistent with other LytTR domain-containing transcription factors. In addition, there is a third, moderately conserved direct repeat site. Surprisingly, PxRcoM-1 requires all three repeat sites to cooperatively bind DNA with a [P]1/2 of 250 ± 10 nM and an average Hill coefficient, n, of 1.7 ± 0.1. The paralog PxRcoM-2 binds to the same triplet motif with comparable affinity and cooperativity. Considering this unusual DNA binding stoichiometry, that is, a dimeric protein with a triplet DNA repeat-binding site, we hypothesize that RcoM interacts with DNA in a manner distinct from other LytTR domain-containing transcription factors.

Beyond AOPs: A Mechanistic Evaluation of NAMs in DART Testing.

New Approach Methodologies (NAMs) promise to offer a unique opportunity to enable human-relevant safety decisions to be made without the need for animal testing in the context of exposure-driven Next Generation Risk Assessment (NGRA). Protecting human health against the potential effects a chemical may have on embryo-foetal development and/or aspects of reproductive biology using NGRA is particularly challenging. These are not single endpoint or health effects and risk assessments have traditionally relied on data from Developmental and Reproductive Toxicity (DART) tests in animals. There are numerous Adverse Outcome Pathways (AOPs) that can lead to DART, which means defining and developing strict testing strategies for every AOP, to predict apical outcomes, is neither a tenable goal nor a necessity to ensure NAM-based safety assessments are fit-for-purpose. Instead, a pragmatic approach is needed that uses the available knowledge and data to ensure NAM-based exposure-led safety assessments are sufficiently protective. To this end, the mechanistic and biological coverage of existing NAMs for DART were assessed and gaps to be addressed were identified, allowing the development of an approach that relies on generating data relevant to the overall mechanisms involved in human reproduction and embryo-foetal development. Using the knowledge of cellular processes and signalling pathways underlying the key stages in reproduction and development, we have developed a broad outline of endpoints informative of DART. When the existing NAMs were compared against this outline to determine whether they provide comprehensive coverage when integrated in a framework, we found them to generally cover the reproductive and developmental processes underlying the traditionally evaluated apical endpoint studies. The application of this safety assessment framework is illustrated using an exposure-led case study.