OECD harmonised template 201: Structuring and reporting mechanistic information to foster the integration of new approach methodologies for hazard and risk assessment of chemicals.

In the European Union, the Chemicals Strategy for Sustainability (CSS) highlights the need to enhance the identification and assessment of substances of concern while reducing animal testing, thus fostering the development and use of New Approach Methodologies (NAMs) such as in silico, in vitro and in chemico. In the United States, the Tox21 strategy aims at shifting toxicological assessments away from traditional animal studies towards target-specific, mechanism-based and biological observations mainly obtained by using NAMs. Many other jurisdictions around the world are also increasing the use of NAMs. Hence, the provision of dedicated non-animal toxicological data and reporting formats as a basis for chemical risk assessment is necessary. Harmonising data reporting is crucial when aiming at re-using and sharing data for chemical risk assessment across jurisdictions. The OECD has developed a series of OECD Harmonised Templates (OHT), which are standard data formats designed for reporting information used for the risk assessment of chemicals relevant to their intrinsic properties, including effects on human health (e.g., toxicokinetics, skin sensitisation, repeated dose toxicity) and the environment (e.g., toxicity to test species and wildlife, biodegradation in soil, metabolism of residues in crops). The objective of this paper is to demonstrate the applicability of the OHT standard format for reporting information under various chemical risk assessment regimes, and to provide users with practical guidance on the use of OHT 201, in particular to report test results on intermediate effects and mechanistic information.

When Are Adverse Outcome Pathways and Associated Assays "Fit for Purpose" for Regulatory Decision-Making and Management of Chemicals?

There have been increasing demands for chemical hazard and risk assessments in recent years. Chemical companies have expanded internal product stewardship initiatives, and jurisdictions have increased the regulatory requirements for the manufacture and sale of chemicals. There has also been a shift in chemical toxicity evaluations within the same time frame, with new methodologies being developed to improve chemical safety assessments for both human health and the environment. With increased needs for chemical assessments coupled with more diverse data streams from new technologies, regulators and others tasked with chemical management activities are faced with increasing workloads and more diverse types of data to consider. The Adverse Outcome Pathway (AOP) framework can be applied in different scenarios to integrate data and guide chemical assessment and management activities. In this paper, scenarios of how AOPs can be used to guide chemical management decisions during research and development, chemical registration, and subsequent regulatory activities such as prioritization and risk assessment are considered. Furthermore, specific criteria (e.g., the type and level of AOP complexity, confidence in the AOP, as well as external review and assay validation) are proposed to examine whether AOPs and associated tools are fit for purpose when applied in different contexts. Certain toxicity pathways are recommended as priority areas for AOP research and development, and the continued use of AOPs and defined approaches in regulatory activities are recommended. Furthermore, a call for increased outreach, education, and enhanced use of AOP databases is proposed to increase their utility in chemicals management. Integr Environ Assess Manag 2019;15:633-647. © 2019 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Zürich Statement on Future Actions on Per- and Polyfluoroalkyl Substances (PFASs).

Per- and polyfluoroalkyl substances (PFASs) are man-made chemicals that contain at least one perfluoroalkyl moiety, [Formula: see text]. To date, over 4,000 unique PFASs have been used in technical applications and consumer products, and some of them have been detected globally in human and wildlife biomonitoring studies. Because of their extraordinary persistence, human and environmental exposure to PFASs will be a long-term source of concern. Some PFASs such as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) have been investigated extensively and thus regulated, but for many other PFASs, knowledge about their current uses and hazards is still very limited or missing entirely. To address this problem and prepare an action plan for the assessment and management of PFASs in the coming years, a group of more than 50 international scientists and regulators held a two-day workshop in November, 2017. The group identified both the respective needs of and common goals shared by the scientific and the policy communities, made recommendations for cooperative actions, and outlined how the science-policy interface regarding PFASs can be strengthened using new approaches for assessing and managing highly persistent chemicals such as PFASs. https://doi.org/10.1289/EHP4158.

Integrated Approaches to Testing and Assessment: OECD Activities on the Development and Use of Adverse Outcome Pathways and Case Studies.

The Organisation for Economic Co-operation and Development (OECD) works with member countries and other stakeholders to improve and harmonize chemical assessment methods. In 2012, the OECD Adverse Outcome Pathways (AOPs) Development Programme started. The Programme has published six AOPs thus far and more than 60 AOPs are under various stages of development under the Programme. This article reviews recent OECD activities on the use of AOPs in developing Integrated Approaches to Testing and Assessments (IATAs). The guidance document for the use of AOPs in developing IATA, published in 2016, provides a framework for developing and using IATA and describes how IATA can be based on an AOP. The guidance document on the reporting of defined approaches to be used within IATA, also published in 2016, provides a set of principles for reporting defined approaches to testing and assessment to facilitate their evaluation. In the guidance documents, the AOP concept plays an important role for building IATA approaches in a science-based and transparent way. In 2015, the IATA Case Studies Project was launched to increase experience with the use of IATA and novel hazard methodologies by developing case studies, which constitute examples of predictions that are fit-for-regulatory use. This activity highlights the importance of international collaboration for harmonizing and improving chemical safety assessment methods.

The adverse outcome pathway concept: A basis for developing regulatory decision-making tools.

The Adverse Outcome Pathway (AOP) concept is expected to guide risk assessors in their work to use all existing information on the effects of chemicals on humans and wildlife, and to target the generation of additional information to the regulatory objective. AOPs will therefore be used in the Organisation for Economic Co-operation and Development (OECD) chemical safety programme, as underlying scientific rationales for the development of alternative methods for hazard assessment, such as read-across, in vitro test methods or the development of integrated testing strategies that have the potential to replace animal tests. As a proof-of-concept, the OECD has developed an AOP for skin sensitisation, and as a follow-up has: a) implemented the AOP into the OECD QSAR Toolbox, so that information related to the Key Events (KEs) in the AOP can be used to group chemicals that are expected to act by the same mechanism and hence have the same skin sensitisation potential; b) developed alternative test methods for the KEs, so that ultimately chemicals can be tested for skin sensitisation without the use of animal tests. The development of integrated testing strategies based on the AOP is ongoing. Building on this proof-of-concept, the OECD has launched an AOP development programme with a first batch of AOPs published in 2016. A number of IT tools, which together form an AOP Knowledge Base, are at various stages of development, and support the construction of AOPs and their use in the development of integrated approaches for testing and assessment. Following the publication of the first batch of AOPs, OECD member countries will decide on priorities for their use in supporting the development of tools for regulatory use.

Health effects of toxicants: Online knowledge support.

Research in toxicology generates vast quantities of data which reside on the Web and are subsequently appropriated and utilized to support further research. This data includes a broad spectrum of information about chemical, biological and radiological agents which can affect health, the nature of the effects, treatment, regulatory measures, and more. Information is structured in a variety of formats, including traditional databases, portals, prediction models, and decision making support tools. Online resources are created and housed by a variety of institutions, including libraries and government agencies. This paper focuses on three such institutions and the tools they offer to the public: the National Library of Medicine (NLM) and its Toxicology and Environmental Health Information Program, the United States Environmental Protection Agency (EPA), and the Organisation for Economic Co-operation and Development (OECD). Reference is also made to other relevant organizations.

The influence of an intramolecular hydrogen bond in differential recognition of inhibitory acceptor analogs by human ABO(H) blood group A and B glycosyltransferases.

Human ABO(H) blood group glycosyltransferases GTA and GTB catalyze the final monosaccharide addition in the biosynthesis of the human A and B blood group antigens. GTA and GTB utilize a common acceptor, the H antigen disaccharide alpha-l-Fucp-(1-->2)-beta-d-Galp-OR, but different donors, where GTA transfers GalNAc from UDP-GalNAc and GTB transfers Gal from UDP-Gal. GTA and GTB are two of the most homologous enzymes known to transfer different donors and differ in only 4 amino acid residues, but one in particular (Leu/Met-266) has been shown to dominate the selection between donor sugars. The structures of the A and B glycosyltransferases have been determined to high resolution in complex with two inhibitory acceptor analogs alpha-l-Fucp(1-->2)-beta-d-(3-deoxy)-Galp-OR and alpha-l-Fucp-(1-->2)-beta-d-(3-amino)-Galp-OR, in which the 3-hydroxyl moiety of the Gal ring has been replaced by hydrogen or an amino group, respectively. Remarkably, although the 3-deoxy inhibitor occupies the same conformation and position observed for the native H antigen in GTA and GTB, the 3-amino analog is recognized differently by the two enzymes. The 3-amino substitution introduces a novel intramolecular hydrogen bond between O2' on Fuc and N3' on Gal, which alters the minimum-energy conformation of the inhibitor. In the absence of UDP, the 3-amino analog can be accommodated by either GTA or GTB with the l-Fuc residue partially occupying the vacant UDP binding site. However, in the presence of UDP, the analog is forced to abandon the intramolecular hydrogen bond, and the l-Fuc residue is shifted to a less ordered conformation. Further, the residue Leu/Met-266 that was thought important only in distinguishing between donor substrates is observed to interact differently with the 3-amino acceptor analog in GTA and GTB. These observations explain why the 3-deoxy analog acts as a competitive inhibitor of the glycosyltransferase reaction, whereas the 3-amino analog displays complex modes of inhibition.

A single point mutation reverses the donor specificity of human blood group B-synthesizing galactosyltransferase.

Blood group A and B antigens are carbohydrate structures that are synthesized by glycosyltransferase enzymes. The final step in B antigen synthesis is carried out by an alpha1-3 galactosyltransferase (GTB) that transfers galactose from UDP-Gal to type 1 or type 2, alphaFuc1-->2betaGal-R (H)-terminating acceptors. Similarly the A antigen is produced by an alpha1-3 N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine from UDP-GalNAc to H-acceptors. Human alpha1-3 N-acetylgalactosaminyltransferase and GTB are highly homologous enzymes differing in only four of 354 amino acids (R176G, G235S, L266M, and G268A). Single crystal x-ray diffraction studies have shown that the latter two of these amino acids are responsible for the difference in donor specificity, while the other residues have roles in acceptor binding and turnover. Recently a novel cis-AB allele was discovered that produced A and B cell surface structures. It had codons corresponding to GTB with a single point mutation that replaced the conserved amino acid proline 234 with serine. Active enzyme expressed from a synthetic gene corresponding to GTB with a P234S mutation shows a dramatic and complete reversal of donor specificity. Although this enzyme contains all four "critical" amino acids associated with the production of blood group B antigen, it preferentially utilizes the blood group A donor UDP-GalNAc and shows only marginal transfer of UDP-Gal. The crystal structure of the mutant reveals the basis for the shift in donor specificity.

A beta-helical antifreeze protein isoform with increased activity. Structural and functional insights.

The insect spruce budworm (Choristoneura fumiferana)(Cf) produces a number of isoforms of its highly active antifreeze protein (CfAFP). Although most of the CfAFP isoforms are in the 9-kDa range, isoforms containing a 30- or 31-amino acid insertion have also been identified. Here we describe the functional and structural analysis of a selected long isoform, CfAFP-501. X-ray crystal structure determination reveals that the 31-amino acid insertion found in CfAFP-501 forms two additional loops within its highly regular beta-helical structure. This effectively extends the area of the two-dimensional Thr array and ice-binding surface of the protein. The larger isoform has 3 times the thermal hysteresis activity of the 9-kDa CfAFP-337. As well, a deletion of the 31-amino acid insertion within CfAFP-501 to form CfAFP-501-Delta-2-loop, results in a protein with reduced activity similar to the shorter CfAFP isoforms. Thus, the enhanced antifreeze activity of CfAFP-501 is directly correlated to the length of its beta-helical structure and hence the size of its ice-binding face.

Elevated temperature and tyrosine iodination aid in the crystallization and structure determination of an antifreeze protein.

Production and refolding of recombinant Choristoneura fumiferana antifreeze protein (CfAFP) leads to a disulfide-bonded product containing dynamic conformational microheterogeneity. Difficulties in the crystallization of this protein arising from its microheterogeneity were overcome by screening of crystallization conditions at various temperatures and finally using a temperature of 318 K to obtain diffraction-quality crystals. In addition, heavy-atom derivatization of this protein required the iodination of a specific tyrosine residue, leading to the successful single anomalous scattering (SAS) structure determination. The techniques of higher temperature screening, to reduce dynamic conformational microheterogeneity, and defined tyrosine iodination, for specific heavy-atom incorporation, are methods which can be employed with other proteins to aid in structure determination.

Crystal structure of beta-helical antifreeze protein points to a general ice binding model.

Reported here is the 2.3 A resolution crystal structure of spruce budworm (Choristoneura fumiferana) antifreeze protein (CfAFP), solved by single anomalous scattering. The structure reveals an extremely regular left-handed beta-helical platform consisting of 15-amino acid loops with a repetitive Thr-X-Thr motif displayed on one of the helix's three faces. This motif results in a two-dimensional array of threonine residues in an identical orientation to those in the nonhomologous, right-handed beta-helical beetle AFP from Tenebrio molitor (TmAFP). The CfAFP structure led us to reevaluate our ice binding model, and the analysis of three possible modes of docking gives rise to a binding mechanism based on surface complementarity. This general mechanism is applicable to both fish and insect AFPs.