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Vitellogenin (VTG), a biomarker for endocrine activity, is a mechanistic component of the regulatory assessment of potential endocrine-disrupting properties of chemicals. This review of VTG data is based on changes reported for 106 substances in standard fish species. High intra-study and inter-laboratory variability in VTG concentrations was confirmed, as well as discrepancies in interpretation of results based on large differences between fish in the dilution water versus solvent control, or due to the presence of outlier measurements. VTG responses in fish were ranked against predictions for estrogen receptor agonist activity and aromatase inhibition from bioactivity model output and ToxCast in vitro assay results, respectively. These endocrine mechanisms explained most of the VTG responses in the absence of systemic toxicity, the magnitude of the VTG response being proportional to the in vitro potency. Interpretation of the VTG data was sometimes confounded by an alternative endocrine mechanism of action. There was evidence for both false positive and negative responses for VTG synthesis, but overall, it was rare for substances without endocrine activity in vitro to cause a concentration-dependent VTG response in fish in the absence of systemic toxicity. To increase confidence in the VTG results, we recommend improvements in the VTG measurement methodologies and greater transparency in reporting of VTG data (including quality control criteria for assay performance). This review supports the application of New Approach Methodologies (NAMs) by demonstrating that endocrine activity in vitro from mammalian cell lines is predictive for in vivo VTG response in fish, suggesting that in vitro mechanistic data could be used more broadly in decision-making to help reduce animal testing. [Display omitted] • Vitellogenin (VTG) concentrations in unexposed fish show high intra-study and inter-laboratory variability. • VTG responses in fish are mainly due to estrogen receptor agonist activity or aromatase inhibition. • VTG responses did not always detect endocrine activity (false positive and negative cases). • It is rare for substances without endocrine activity in vitro to cause a concentration-dependent VTG response in fish. • Confidence in the VTG result can be improved by greater consistency in methodology and reporting. [ABSTRACT FROM AUTHOR]

A novel perspective on defining black holes designed to be more broadly applicable outside of asymptotically flat spacetimes, in the context of classical general relativity, is presented, discussed, and characterized. The construction formalizes the heuristic idea that black holes are the "past Cauchy development of the set of singularities". As such, the formulation depends in a critical way on the identification of singularities, which has been treated in the literature through various boundary constructions. While many of the known boundary constructions (e.g.Schmidt's b-boundary or Geroch's g-boundary) could in principal be cited as underlying one's notion of a black hole, well-known topological concerns leads us to take the perspective that Scott and Szekeres' abstract boundary provides the most natural choice. The framework is utilized to put forward a general, non-IVP formulation of the Weak Cosmic Censorship Conjecture. • Intuitively developed a novel, general, and global definition of classical black holes. • Captures global nature of a black hole without any asymptotic requirement. • Demonstrated definitions in several classic black hole spacetimes. • Rigorously affirmed several key intuitive features of the definitions. • Used this to pose a general non-IVP formulation of weak cosmic censorship. [ABSTRACT FROM AUTHOR]

Endocrine screening assays not only provide mechanistic information on the potential of a substance to interact with the endocrine system, but also data potentially relevant for risk assessment. However, these screening assays have a number of limitations that should be considered before the direct use of such data for risk assessment purposes. This paper discusses the limitations that should be considered for both human and environmental risk assessment. A proposal is made to provide an objective and transparent process in order to consider which endpoint(s) might be incorporated into a risk assessment, and when more definitive studies may be of value. The proposal is complemented with an easy-to-follow flowchart to aid industry scientists and regulators when evaluating the relevance of these data. Such an approach is necessary to ensure the appropriate use of screening data to further our understanding of the eco/toxicological profile of substances undergoing screening. [ABSTRACT FROM AUTHOR]

We prove new incidence bounds between a plane point set, which is a Cartesian product, and a set of translates H of the hyperbola x y = λ ≠ 0 , over a field of asymptotically large positive characteristic p. They improve recent bounds by Shkredov, which are based on using explicit incidence estimates in the early terminated procedure of repeated applications of the Cauchy-Schwarz inequality, underlying many qualitative results related to growth and expansion in groups. The improvement – both quantitative, plus we are able to deal with a general H , rather than a Cartesian product – is mostly due to a non-trivial "intermediate" bound on the number of k -rich Möbius hyperbolae in positive characteristic. In addition, we make an observation that a certain energy-type quantity in the context of H can be bounded via the L 2 -moment of the Minkowski distance in H and can therefore fetch the corresponding estimates apropos of the Erdős distinct distance problem. [ABSTRACT FROM AUTHOR]

Fish toxicity testing has been conducted since the 1860's in order to help define safe levels of chemical contaminants in lakes, rivers and coastal waters. The historical emphasis on acute lethality testing of chemicals has more recently focussed on long term sublethal effects of chemicals on fish and their prey species. Fish toxicity testing is now embedded in much environment legislation on chemical safety while it is recognized that animal use should be Replaced, Reduced and Refined (the 3Rs) where possible. The OECD Fish Toxicity Testing Framework provides a useful structure with which to address the needs of environmental safety assessment whilst implementing the 3Rs. This commentary aims to promote the implementation of the recommendations of the OECD Fish Toxicity Testing Framework. [ABSTRACT FROM AUTHOR]

The fish short-term reproduction assay (FSTRA) is an in vivo screen to assess potential interactions with the fish endocrine system. After a 21-day exposure period vitellogenin (VTG) and secondary sexual characteristics are measured in males and females. Egg production and fertility are also monitored daily throughout the test. This paper presents data from 49 studies performed to satisfy test orders from the United States Environmental Protection Agency's Endocrine Disruptor Screening Program. Data Evaluation Records were used to collate the typical control variability and performance of test parameters in FSTRAs conducted in different laboratories with fathead minnow (Pimephales promelas). We also examine the statistical power of FSTRA endpoints and assess whether available historical control data (HCD) assist evidence-based interpretation of the endpoints. Statistically significant inter-laboratory differences were found for all endpoints except survival. HCD could therefore be usefully developed on a laboratory-by-laboratory basis to aid interpretation of new study data. Reliable HCD ranges could be developed for survival, body weight/length, gonadal somatic index, fertilisation success, and male tubercle score, and used in association with stated test acceptability criteria to interpret FSTRA data. In contrast, high intra- and inter-laboratory control variability for VTG and fecundity means that HCD for these endpoints are of limited use during study interpretation. • Most fish short-term reproduction endpoints differ significantly between laboratories. • Useful Historical Control Data (HCD) ranges are calculable for most endpoints. • High variability precludes HCD calculation for vitellogenin and fecundity. [ABSTRACT FROM AUTHOR]

Revised information requirements for endocrine disruptor (ED) assessment of chemicals under the European Union's Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) Regulation have been proposed. Implementation will substantially increase demands for new data to inform ED assessment. This article evaluates the potential animal use and financial resource associated with two proposed ED policy options, and highlights areas where further clarification is warranted. This evaluation demonstrates that studies potentially conducted to meet the proposed requirements could use tens of millions of animals, and that the approach is unlikely to be feasible in practice. Given the challenges with implementing either policy option and the need to minimise the reliance on animal testing, further consideration and clarification is needed on several aspects prior to implementation of the requirements. This includes how testing will be prioritised in a proportionate approach; how to harness new approach methodologies to waive higher-tier animal testing; and need for provision of clear guidance particularly in applying weight-of-evidence approaches. There is now a clear opportunity for the European Commission to lead the way in developing a robust and transparent ED assessment process for industrial chemicals which fully implements replacement, refinement, and reduction of the use of animals (the 3Rs). • New endocrine disruptor assessment data requirements have been proposed for EU REACH. • This analysis estimates the potential animal use and resource needed to meet these. • The required studies could use millions of animals and be unachievable in practice. • Further consideration/clarification is needed on many aspects before implementation. • Opportunities exist for robust ED assessments that fully implement the 3Rs. [ABSTRACT FROM AUTHOR]

The Xenopus Eleutheroembryonic Thyroid Assay (XETA) was recently published as an OECD Test Guideline for detecting chemicals acting on the thyroid axis. However, the OECD validation did not cover all mechanisms that can potentially be detected by the XETA. This study was therefore initiated to investigate and consolidate the applicability domain of the XETA regarding the following mechanisms: thyroid hormone receptor (THR) agonism, sodium-iodide symporter (NIS) inhibition, thyroperoxidase (TPO) inhibition, deiodinase (DIO) inhibition, glucocorticoid receptor (GR) agonism, and uridine 5′-diphospho-glucuronosyltransferase (UDPGT) induction. In total, 22 chemicals identified as thyroid-active or -inactive in Amphibian Metamorphosis Assays (AMAs) were tested using the XETA OECD Test Guideline. The comparison showed that both assays are highly concordant in identifying chemicals with mechanisms of action related to THR agonism, DIO inhibition, and GR agonism. They also consistently identified the UDPGT inducers as thyroid inactive. NIS inhibition, investigated using sodium perchlorate, was not detected in the XETA. TPO inhibition requires further mechanistic investigations as the reference chemicals tested resulted in opposing response directions in the XETA and AMA. This study contributes refining the applicability domain of the XETA, thereby helping to clarify the conditions where it can be used as an ethical alternative to the AMA. • The Xenopus Eleutheroembryonic Thyroid Assay (XETA) was recently developed to screen for thyroid activity of chemicals. • XETA results were compared to Amphibian Metamorphosis Assay (AMA) outcomes for the detection of thyroid-active chemicals. • Results were concordant for all selected mechanisms, but the XETA did not detect inhibition of the sodium-iodide symporter. • Thyroperoxidase inhibition requires further mechanistic investigations due to equivocal responses in the XETA. • The XETA and the AMA consistently identified uridine 5′-diphospho-glucuronosyltransferase inducers as thyroid-inactive. [ABSTRACT FROM AUTHOR]

Vitellogenin (VTG) is a biomarker for possible endocrine activity of chemicals acting via the estrogen, androgen, or steroidogenesis pathways. VTG is assessed in standardised fish guideline studies conducted for regulatory safety assessment of chemicals. VTG data can be highly variable leading to concerns for potential equivocal, false positive and/or negative outcomes. Consequently, additional fish testing may be required to address uncertainties in the VTG response, and possibly erroneous/missed identification of endocrine activity. To better understand the technical challenges of VTG assessment and reporting for regulatory purposes, a survey was sent to 27 testing laboratories performing these analyses. The survey results from 16 respondents (6 from the UK, 3 from the USA, and 7 from the EU) were analysed and discussed in a follow-up webinar. High variability in background VTG concentrations was widely acknowledged and thought to be associated with fish batch, husbandry, laboratory practices, and several methodological aspects. These include sample collection and storage, VTG quantification, data handling, and the benchmarks used for data acceptability. Information gathered in the survey provides a basis for improving and harmonizing the measurement of VTG in fish, and an opportunity to reassess the suitability of current acceptability criteria in test guidelines. • Vitellogenin (VTG) is an endocrine activity biomarker in fish regulatory studies. • A survey of laboratories revealed technical challenges for VTG assessment. • These include methodological, data handling and data acceptability issues. • The results provide a basis for improving and harmonizing the measurement of VTG. • Implementation of the recommendations will support decision-making processes. [ABSTRACT FROM AUTHOR]

Abstract The endocrine system is responsible for growth, development, maintaining homeostasis and for the control of many physiological processes. Due to the integral nature of its signaling pathways, it can be difficult to distinguish endocrine-mediated adverse effects from transient fluctuations, adaptive/compensatory responses, or adverse effects on the endocrine system that are caused by mechanisms outside the endocrine system. This is particularly true in toxicological studies that require generation of effects through the use of Maximum Tolerated Doses (or Concentrations). Endocrine-mediated adverse effects are those that occur as a consequence of the interaction of a chemical with a specific molecular component of the endocrine system, for example, a hormone receptor. Non-endocrine-mediated adverse effects on the endocrine system are those that occur by other mechanisms. For example, systemic toxicity, which perturbs homeostasis and affects the general well-being of an organism, can affect endocrine signaling. Some organs/tissues can be affected by both endocrine and non-endocrine signals, which must be distinguished. This paper examines in vitro and in vivo endocrine endpoints that can be altered by non-endocrine processes. It recommends an evaluation of these issues in the assessment of effects for the determination of endocrine disrupting properties of chemicals. This underscores the importance of using a formal weight of evidence (WoE) process to evaluate potential endocrine activity. [ABSTRACT FROM AUTHOR]

Avian reproduction studies are a regulatory requirement for pesticides in many regions. The data often require careful interpretation due to the nature of the study design. Here we present the historical control dataset for bobwhite quail and mallard duck reproduction studies performed at the Evans Analytical Group LLC avian toxicology laboratory over the period 1985–2016. The analysis demonstrates the stability of reproductive parameters over time and good agreement to normal control ranges as required by the regulatory test guidelines. The major source of variation is shown to be within study variation. Power analyses confirm the generally good power properties of the test design. Recommendations for the use of historical control data for the interpretation of avian reproduction studies are made. We believe the analysis and evaluation presented here can facilitate the development of practical guidance that can be implemented in regulatory programmes requiring this test. [ABSTRACT FROM AUTHOR]

A Weight-of-evidence (WoE) evaluation should be applied in assessing all the available data for the identification of endocrine disrupting (ED) properties of chemicals. The European Commission draft acts specifying criteria under the biocidal products and plant protection products regulations require that WoE is implemented for the assessment of such products. However, only some general considerations and principles of how a WoE should be conducted are provided. This paper reviews WoE approaches to distil key recommendations specifically for the evaluation of potential ED properties of chemicals. In a manner, which is consistent with existing, published WoE frameworks, the WoE evaluation of ED properties can be divided into four phases: 1) Definition of causal questions and data gathering and selection, 2) Review of individual studies, 3) Data integration and evaluation, and 4) Drawing conclusions based on inferences. Recommendations are made on how to conduct each phase robustly and transparently to help guide the WoE evaluation of potential endocrine disrupting properties of chemicals within a European regulatory context. [ABSTRACT FROM AUTHOR]

Photocatalytically generated gold on carbon and TiO 2 composites were used for acetylene hydrogenation in excess ethylene, in a way close to current industrial practices. The crystalline effect of TiO 2 and the addition of Pd to supported Au catalysts on acetylene hydrogenation were investigated. Carbon and amorphous TiO 2 supported Au were highly active, selective, and stable and possessed the highest ethylene yield of 83% at 210 °C with a H 2 /C 2 H 2 ratio of 2/1 for selective hydrogenation of acetylene to ethylene. With further addition of Pd to the supported Au catalyst, ethylene yield reached a maximum value of 57% at 30 °C with a H 2 /C 2 H 2 ratio of 2/1. The enhanced performance originated from small neutral Au nanoparticles and the formation of bimetallic Au–Pd catalyst. This report opens up promising possibilities for the design of highly efficient heterogeneous supported gold catalysts. [ABSTRACT FROM AUTHOR]

Amphibians are currently the most threatened and rapidly declining group of vertebrates and this has raised concerns about their potential sensitivity and exposure to plant protection products and other chemicals. Current environmental risk assessment procedures rely on surrogate species (e.g. fish and birds) to cover the risk to aquatic and terrestrial life stages of amphibians, respectively. Whilst a recent meta-analysis has shown that in most cases amphibian aquatic life stages are less sensitive to chemicals than fish, little research has been conducted on the comparative sensitivity of terrestrial amphibian life stages. Therefore, in this paper we address the questions “What is the relative sensitivity of terrestrial amphibian life stages to acute chemical oral exposure when compared with mammals and birds?” and “Are there correlations between oral toxicity data for amphibians and data for mammals or birds?” Identifying a relationship between these data may help to avoid additional vertebrate testing. Acute oral amphibian toxicity data collected from the scientific literature and ecotoxicological databases were compared with toxicity data for mammals and birds. Toxicity data for terrestrial amphibian life stages are generally sparse, as noted in previous reviews. Single-dose oral toxicity data for terrestrial amphibian life stages were available for 26 chemicals and these were positively correlated with LD50 values for mammals, while no correlation was found for birds. Further, the data suggest that oral toxicity to terrestrial amphibian life stages is similar to or lower than that for mammals and birds, with a few exceptions. Thus, mammals or birds are considered adequate toxicity surrogates for use in the assessment of the oral exposure route in amphibians. However, there is a need for further data on a wider range of chemicals to explore the wider applicability of the current analyses and recommendations. [ABSTRACT FROM AUTHOR]

The European Plant Protection Products Regulation 1107/2009 requires that registrants establish whether pesticide metabolites pose a risk to the environment. Fish acute toxicity assessments may be carried out to this end. Considering the total number of pesticide (re-) registrations, the number of metabolites can be considerable, and therefore this testing could use many vertebrates. EFSA's recent “Guidance on tiered risk assessment for plant protection products for aquatic organisms in edge-of-field surface waters” outlines opportunities to apply non-testing methods, such as Quantitative Structure Activity Relationship (QSAR) models. However, a scientific evidence base is necessary to support the use of QSARs in predicting acute fish toxicity of pesticide metabolites. Widespread application and subsequent regulatory acceptance of such an approach would reduce the numbers of animals used. The work presented here intends to provide this evidence base, by means of retrospective data analysis. Experimental fish LC 50 values for 150 metabolites were extracted from the Pesticide Properties Database ( http://sitem.herts.ac.uk/aeru/ppdb/en/atoz.htm ). QSAR calculations were performed to predict fish acute toxicity values for these metabolites using the US EPA's ECOSAR software. The most conservative predicted LC 50 values generated by ECOSAR were compared with experimental LC 50 values. There was a significant correlation between predicted and experimental fish LC 50 values (Spearman r s = 0.6304, p < 0.0001). For 62% of metabolites assessed, the QSAR predicted values are equal to or lower than their respective experimental values. Refined analysis, taking into account data quality and experimental variation considerations increases the proportion of sufficiently predictive estimates to 91%. For eight of the nine outliers, there are plausible explanation(s) for the disparity between measured and predicted LC 50 values. Following detailed consideration of the robustness of this non-testing approach, it can be concluded there is a strong data driven rationale for the applicability of QSAR models in the metabolite assessment scheme recommended by EFSA. As such there is value in further refining this approach, to improve the method and enable its future incorporation into regulatory guidance and practice. [ABSTRACT FROM AUTHOR]

Fish bioconcentration test guidelines generally require that bioconcentration factors (BCFs) are determined at two exposure concentrations. However, recent revisions to the OECD test guideline for bioconcentration testing (TG 305) provide the option to use only one exposure concentration, when justification is provided, although two concentrations may still be required for some regulatory purposes. Recently, this justification has been demonstrated for plant protection product active ingredients. To determine whether this justification has a broader validity for general chemicals, an analysis of 236 BCF studies on general chemicals was conducted. The results presented here again demonstrate that BCF values do not significantly differ between concentrations when more than one concentration is used. This relationship is particularly strong for BCFs ⩾1000 L/kg, which is beneficial, since only chemicals with BCFs >2000 L/kg may require regulatory action. This analysis therefore provides a data-driven rationale for using the one test concentration approach for general chemical substances and thus could contribute to a substantial reduction in the use of fish in bioconcentration tests. [ABSTRACT FROM AUTHOR]

Abstract: The European regulation on plant protection products (1107/2009) (), the revisions to the biocides Directive (COM[2009]267) (), and the regulation concerning chemicals (Regulation (EC) No. 1907/2006 ‘REACH’) (EC.2006) only support the marketing and use of chemical products on the basis that they do not induce endocrine disruption in humans or wildlife species. In the absence of agreed guidance on how to identify and evaluate endocrine activity and disruption within these pieces of legislation a European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) task force was formed to provide scientific criteria that may be used within the context of these three legislative documents. The resulting ECETOC technical report () and the associated workshop () presented a science-based concept on how to identify endocrine activity and disrupting properties of chemicals for both human health and the environment. The synthesis of the technical report and the workshop report was published by the ECETOC task force (). Specific scientific criteria for the determination of endocrine activity and disrupting properties that integrate information from both regulatory (eco)toxicity studies and mechanistic/screening studies were proposed. These criteria combined the nature of the adverse effects detected in studies which give concern for endocrine toxicity with an understanding of the mode of action of toxicity so that adverse effects can be explained scientifically. A key element in the data evaluation is the consideration of all available information in a weight-of-evidence approach. However, to be able to discriminate chemicals with endocrine properties of low concern from those of higher concern (for regulatory purposes), the task force recognised that the concept needed further refinement. Following a discussion of the key factors at a second workshop of invited regulatory, academic and industry scientists (), the task force developed further guidance, which is presented in this paper. For human health assessments these factors include the relevance to humans of the endocrine mechanism of toxicity, the specificity of the endocrine effects with respect to other potential toxic effects, the potency of the chemical to induce endocrine toxicity and consideration of exposure levels. For ecotoxicological assessments the key considerations include specificity and potency, but also extend to the consideration of population relevance and negligible exposure. It is intended that these complement and reinforce the approach originally described and previously published in this journal (). [Copyright &y& Elsevier]

Abstract: The European legislation on plant protection products (Regulation (EC) No. 1107/2009) and biocides (Directive 98/8/EC), as well as the regulation concerning chemicals (Regulation (EC) No. 1907/2006 ‘REACH’) only support the marketing and use of chemical products on the basis that they do not induce endocrine disruption in humans or non-target species. However, there is currently no agreed guidance on how to identify and evaluate endocrine activity and disruption. Consequently, an ECETOC task force was formed to provide scientific criteria that may be used within the context of these three legislative documents. Specific scientific criteria for the determination of endocrine disrupting properties that integrate information from both regulatory (eco)toxicity studies and mechanistic/screening studies are proposed. These criteria combine the nature of the adverse effects detected in studies which give concern for endocrine toxicity with an understanding of the mode of action of toxicity so that adverse effects can be explained scientifically. The criteria developed are presented in the form of flow charts for assessing relevant effects for both humans and wildlife species. In addition, since not all chemicals with endocrine disrupting properties are of equal hazard, assessment of potency is also proposed to discriminate chemicals of high concern from those of lower concern. The guidance presented in this paper includes refinements made to an initial proposal following discussion of the criteria at a workshop of invited regulatory, academic and industry scientists. [ABSTRACT FROM AUTHOR]

The European Chemical Agency and European Food Safety Authority recommend that gross pathology findings, from avian reproduction studies, be used to support assessment of potential endocrine disrupting properties of active pesticidal and biocidal substances. In open literature, little information is available on the utility of gross pathology data for informing endocrine evaluations. Here the gross pathology data from historical control groups of 51 northern bobwhite and 51 mallard reproduction tests is analyzed to evaluate the utility of such information. Incidence of gross morphology findings in untreated birds may aid the interpretation of some gross abnormalities, potentially indicative of an endocrine interaction (e.g. reproductive condition). Statistical analysis of the historical control data indicates that gross pathology is not likely to be useful for detecting endocrine effects as abnormalities with relatively high increases in prevalence (more than 20–30%, depending on prevalence in controls) are reliably interpreted as a treatment response. Gross pathology changes are only indicative and not diagnostic of endocrine interactions making it difficult to distinguish gross pathology abnormalities, due to endocrine-mediated effects, from systemic toxicity. This work demonstrates the utility of using historical control analyses to establish the value and properties of selected endpoints for regulatory applications. • Limitations of gross pathology findings to indicate endocrine effects. • Historical control data sets establish useful baseline information of pathology findings. • Data should not be over interpreted in weight of evidence for potential endocrine effects. [ABSTRACT FROM AUTHOR]