Exposure considerations in human safety assessment: Report from an EPAA Partners' Forum.

Understanding and estimating the exposure to a substance is one of the fundamental requirements for safe manufacture and use. Many approaches are taken to determine exposure to substances, mainly driven by potential use and regulatory need. There are many opportunities to improve and optimise the use of exposure information for chemical safety. The European Partnership for Alternative Approaches to Animal Testing (EPAA) therefore convened a Partners' Forum (PF) to explore exposure considerations in human safety assessment of industrial products to agree key conclusions for the regulatory acceptance of exposure assessment approaches and priority areas for further research investment. The PF recognised the widescale use of exposure information across industrial sectors with the possibilities of creating synergies between different sectors. Further, the PF acknowledged that the EPAA could make a significant contribution to promote the use of exposure data in human safety assessment, with an aim to address specific regulatory needs. To achieve this, research needs, as well as synergies and areas for potential collaboration across sectors, were identified.

Sulfur(VI) fluorides as tools in biomolecular and medicinal chemistry.

Recent advances in the synthesis of sulfur(VI)-fluorides has enabled incredible growth in their application in biomolecular chemistry. This review aims to serve as a primer highlighting synthetic strategies toward a diversity of S(VI) fluorides and their application in chemical biology, bioconjugation, and medicinal chemistry.

Facile synthesis of sulfonyl fluorides from sulfonic acids.

Herein, we demonstrate two complementary strategies for the syntheses of sulfonyl fluorides using sulfonic acids and their salts. One strategy involves the conversion of sulfonic acid sodium salts to sulfonyl fluorides using thionyl fluoride in 90-99% yields in one hour. Lessons learned from the mechanism of this reaction also have enabled a complementary deoxyfluorination of sulfonic acids using Xtalfluor-E® - a bench stable solid - allowing for the conversion of both aryl and alkyl sulfonic acids and salts to sulfonyl fluorides in 41-94% yields. Notably, using Xtalfluor-E® enabled milder conditions and the use of both sulfonic acids and their sodium salts.

Synthesis of 2-arylpyridines by the Suzuki-Miyaura cross-coupling of PyFluor with hetero(aryl) boronic acids and esters.

The Suzuki-Miyaura cross-coupling of pyridine-2-sulfonyl fluoride (PyFluor) with hetero(aryl) boronic acids and pinacol boronic esters is reported. The reactions can be performed using Pd(dppf)Cl2 as the catalyst, at temperatures between 65 and 100 °C and in the presence of water and oxygen. This transformation generates 2-arylpyridines in modest to good yields (5%-89%).

Sulfondiimidamides unlocked as new S(VI) hubs for synthesis and drug discovery.

Despite their promise as drug targets, access to nitrogen-rich S(VI) compounds has been a significant synthetic challenge. In this issue of Chem, Zhang and Willis explore a new class of S(VI) compounds-sulfondiimidamides-providing robust strategies toward their synthesis, derivation, and promise as new sulfonamide bioisosteres.

Calcium Bistriflimide-Mediated Sulfur(VI)-Fluoride Exchange (SuFEx): Mechanistic Insights toward Instigating Catalysis.

We report a mechanistic investigation of calcium bistriflimide-mediated sulfur(VI)-fluoride exchange (SuFEx) between sulfonyl fluorides and amines. We determine the likely pre-activation resting state─a calcium bistriflimide complex with ligated amines─thus allowing for corroborated calculation of the SuFEx activation barrier at ∼21 kcal/mol, compared to 21.5 ± 0.14 kcal/mol derived via kinetics experiments. Transition state analysis revealed: (1) a two-point calcium-substrate contact that activates the sulfur(VI) center and stabilizes the leaving fluoride and (2) a 1,4-diazabicyclo[2.2.2]octane additive that provides Brønsted-base activation of the nucleophilic amine. Stable Ca-F complexes upon sulfonamide formation are likely contributors to inhibited catalytic turnover, and a proof-of-principle redesign provided evidence that sulfonamide formation is feasible with 10 mol % calcium bistriflimide.

A framework for chemical safety assessment incorporating new approach methodologies within REACH.

The long-term investment in new approach methodologies (NAMs) within the EU and other parts of the world is beginning to result in an emerging consensus of how to use information from in silico, in vitro and targeted in vivo sources to assess the safety of chemicals. However, this methodology is being adopted very slowly for regulatory purposes. Here, we have developed a framework incorporating in silico, in vitro and in vivo methods designed to meet the requirements of REACH in which both hazard and exposure can be assessed using a tiered approach. The outputs from each tier are classification categories, safe doses, and risk assessments, and progress through the tiers depends on the output from previous tiers. We have exemplified the use of the framework with three examples. The outputs were the same or more conservative than parallel assessments based on conventional studies. The framework allows a transparent and phased introduction of NAMs in chemical safety assessment and enables science-based safety decisions which provide the same level of public health protection using fewer animals, taking less time, and using less financial and expert resource. Furthermore, it would also allow new methods to be incorporated as they develop through continuous selective evolution rather than periodic revolution.

The Emerging Applications of Sulfur(VI) Fluorides in Catalysis.

The past decade has witnessed remarkable growth of catalytic transformations in organic sulfur(VI) fluoride chemistry. This Perspective concentrates exclusively on foundational examples that utilize catalytic strategies to synthesize and react S(VI) fluorides. Key mechanistic studies that aim to provide insight toward future catalytic systems are emphasized.

Key read across framework components and biology based improvements.

At the 2019 annual meeting of the European Environmental Mutagen and Genomics Society a workshop session related to the use of read across concepts in toxicology was held. The goal of this session was to provide the audience an overview of general read-across concepts. From ECHA's read across assessment framework, the starting point is chemical similarity. There are several approaches and algorithms available for calculating chemical similarity based on molecular descriptors, distance/similarity measures and weighting schemata for specific endpoints. Therefore, algorithms that adapt themselves to the data (endpoint/s) and provide a good ability to distinguish between structural similar and not similar molecules regarding specific endpoints are needed and their use discussed. Toxico-dynamic end points are usually in the focus of read across cases. However, without appropriate attention to kinetics and metabolism such cases are unlikely to be successful. To further enhance the quality of read across cases new approach methods can be very useful. Examples based on a biological approach using plasma metabolomics in rats are given. Finally, with the availability of large data sets of structure activity relationships, in silico tools have been developed which provide hitherto undiscovered information. Automated process is now able to assess the chemical - activity space around the molecule target substance and examples are given demonstrating a high predictivity for certain endpoints of toxicity. Thus, this session provides not only current state of the art criteria for good read across, but also indicates how read-across can be further developed in the near future.

SuFEx Activation with Ca(NTf2)2: A Unified Strategy to Access Sulfamides, Sulfamates, and Sulfonamides from S(VI) Fluorides.

A method to activate sulfamoyl fluorides, fluorosulfates, and sulfonyl fluorides with calcium triflimide and DABCO for SuFEx with amines is described. The reaction was applied to a diverse set of sulfamides, sulfamates, and sulfonamides at room temperature under mild conditions. Additionally, we highlight this transformation to parallel medicinal chemistry to generate a broad array of nitrogen-based S(VI) compounds.

Utility of a next generation framework for assessment of genomic damage: A case study using the industrial chemical benzene.

We recently published a next generation framework for assessing the risk of genomic damage via exposure to chemical substances. The framework entails a systematic approach with the aim to quantify risk levels for substances that induce genomic damage contributing to human adverse health outcomes. Here, we evaluated the utility of the framework for assessing the risk for industrial chemicals, using the case of benzene. Benzene is a well-studied substance that is generally considered a genotoxic carcinogen and is known to cause leukemia. The case study limits its focus on occupational and general population health as it relates to benzene exposure. Using the framework as guidance, available data on benzene considered relevant for assessment of genetic damage were collected. Based on these data, we were able to conduct quantitative analyses for relevant data sets to estimate acceptable exposure levels and to characterize the risk of genetic damage. Key observations include the need for robust exposure assessments, the importance of information on toxicokinetic properties, and the benefits of cheminformatics. The framework points to the need for further improvement on understanding of the mechanism(s) of action involved, which would also provide support for the use of targeted tests rather than a prescribed set of assays. Overall, this case study demonstrates the utility of the next generation framework to quantitatively model human risk on the basis of genetic damage, thereby enabling a new, innovative risk assessment concept. Environ. Mol. Mutagen. 61:94-113, 2020. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

One-pot fluorosulfurylation of Grignard reagents using sulfuryl fluoride.

Herein, we report a new method for the one-pot syntheses of sulfonyl fluorides. Addition of an alkyl, aryl, or heteroaryl Grignard to a solution of sulfuryl fluoride at ambient temperature affords the desired sulfonyl fluorides in 18-78% yield. Furthermore, this method is applicable for in situ sequential reactions, whereby the Grignard reagent can be converted to the corresponding diarylsulfone, sulfonate ester, or sulfonamide in a one-pot process.

Multi-dimensional in vitro bioactivity profiling for grouping of glycol ethers.

High-content screening data derived from physiologically-relevant in vitro models promise to improve confidence in data-integrative groupings for read-across in human health safety assessments. The biological data-based read-across concept is especially applicable to bioactive chemicals with defined mechanisms of toxicity; however, the challenge of data-derived groupings for chemicals that are associated with little or no bioactivity has not been explored. In this study, we apply a suite of organotypic and population-based in vitro models for comprehensive bioactivity profiling of twenty E-Series and P-Series glycol ethers, solvents with a broad variation in toxicity ranging from relatively non-toxic to reproductive and hematopoetic system toxicants. Both E-Series and P-Series glycol ethers elicited cytotoxicity only at high concentrations (mM range) in induced pluripotent stem cell-derived hepatocytes and cardiomyocytes. Population-variability assessment comprised a study of cytotoxicity in 94 human lymphoblast cell lines from 9 populations and revealed differences in inter-individual variability across glycol ethers, but did not indicate population-specific effects. Data derived from various phenotypic and transcriptomic assays revealed consistent bioactivity trends between both cardiomyocytes and hepatocytes, indicating a more universal, rather than cell-type specific mode-of-action for the tested glycol ethers in vitro. In vitro bioactivity-based similarity assessment using Toxicological Priority Index (ToxPi) showed that glycol ethers group according to their alcohol chain length, longer chains were associated with increased bioactivity. While overall in vitro bioactivity profiles did not correlate with in vivo toxicity data on glycol ethers, in vitro bioactivity of E-series glycol ethers were indicative of and correlated with in vivo irritation scores.

Should DTPA, an Aminocarboxylic acid (ethylenediamine-based) chelating agent, be considered a developmental toxicant?

Aminocarboxylic acid (ethylenediamine-based) chelating agents, such as DTPA and EDTA, are widely used in a variety of products and processes. Recently the European RAC proposed to classify DTPA as a developmental toxicant Category 1B according to CLP. This paper provides unequivocal and significant evidence that developmental effects cannot be considered an intrinsic property of the chelating substances themselves since: (1) animals fed a zinc deficient diet during gestation exhibit developmental toxicity of a similar nature and severity to that observed in studies involving such chelates, (2) sufficient supplementation of zinc in the diet, or administration of zinc bound chelates, completely negates the developmental effects. Moreover, the bioavailability of DTPA is very low with >95% of oral doses excreted unchanged via the feces within 24 h. If DTPA would possess the intrinsic property to be developmentally toxic, simple zinc supplementation should not be sufficient to negate these effects. Furthermore, the relevance of classification is highly questionable since worker or consumer exposure could not lead to a scenario whereby sufficient zinc deficiency would manifest itself. Therefore classification of DTPA for such effects is not protective of human health; instead it leads to onerous and disproportionate restrictions being placed on this substance.

Sulfonamide Synthesis via Calcium Triflimide Activation of Sulfonyl Fluorides.

A method using calcium triflimide [Ca(NTf2)2] as a Lewis acid to activate sulfonyl fluorides toward nucleophilic addition with amines is described. The reaction converts a wide array of sterically and electronically diverse sulfonyl fluorides and amines into the corresponding sulfonamides in good yield.

Global regulatory requirements for mutagenicity assessment in the registration of industrial chemicals.

Mutagenicity is an important toxicological endpoint that requires thorough evaluation during the industrial chemical registration process. Regulatory requirements for mutagenicity assessment in registration of industrial chemicals vary in geographic regions (and in some cases by intended application). Here we compile the mutagenicity testing requirements for registration of industrial chemicals from representative geographic regions (in alphabetical order), that is Australia, Brazil, Canada, China, European Union (EU), India, Japan, South Korea, Taiwan, and United States (US). We further discuss the challenges that industry is facing to meet global regulations, for example, different testing requirements among geographic regions, different strategies in follow-up tests to in vitro positive findings, no-observed-adverse-effect-levels in genetic toxicity testing, and human relevance of mutagenicity. Environ. Mol. Mutagen. 58:345-353, 2017. © 2017 Wiley Periodicals, Inc.

Use of genetic toxicity data in GHS mutagenicity classification and labeling of substances.

One of the key outcomes of testing the potential genotoxicity or mutagenicity of a substance is the conclusion on whether the substance should be classified as a germ cell mutagen and the significance of this for other endpoints such as carcinogenicity. The basis for this conclusion are the criteria presented in classification and labelling systems such as the Globally Harmonized System for classification and labeling (GHS). This article reviews the classification criteria for germ cell mutagenicity and carcinogenicity and how they are applied to substances with evidence of mutagenicity. The implications and suitability of such a classification for hazard communication, risk assessment, and risk management are discussed. It is proposed that genotoxicity assessments should not focus on specifically identifying germ cell mutagens, particularly given the challenges associated with communicating this information in a meaningful way. Rather the focus should be on deriving data to characterize the mode of action and for use in the risk assessment of mutagens, which could then feed into a more robust, risk based management of mutagenic substances versus the current more hazard based approaches. Environ. Mol. Mutagen. 58:354-360, 2017. © 2017 Wiley Periodicals, Inc.

Pd-Catalyzed Conversion of Aryl Iodides to Sulfonyl Fluorides Using SO2 Surrogate DABSO and Selectfluor.

A one-pot Pd-catalyzed conversion of aryl iodide to aryl sulfonyl fluorides using DABSO and Selectfluor has been developed generating aryl sulfonyl fluorides in good to excellent yields. The reaction results in the generation of electronically and sterically diverse sulfonyl fluorides. Additionally, sulfonyl fluorides can be converted to aryl sulfonamides and sulfonic esters using Cs2CO3 under mild conditions.

Validation of Computational Methods.

In this chapter, we provide an overview of how (Quantitative) Structure Activity Relationships, (Q)SARs, are validated and applied for regulatory purposes. We outline how chemical categories are derived to facilitate endpoint specific read-across using tools such as the OECD QSAR Toolbox and discuss some of the current difficulties in addressing the residual uncertainties of read-across. Finally we put forward a perspective of how non-testing approaches may evolve in light of the advances in new and emerging technologies and how these fit within the Adverse Outcome Pathway (AOP) framework.