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This article discusses the measurement and interpretation of vitellogenin (VTG) in fish and amphibians as a biomarker for endocrine activity. The reliability and acceptability of VTG results are important for regulatory assessments of chemicals. The article highlights the variability in VTG measurements and the need for improvement in sampling, quantification, and data handling. It proposes a decision logic to assist in the interpretation of VTG measurements and calls for the development of OECD guidance on best practices for VTG assessment. The article emphasizes the importance of considering other factors and endpoints in conjunction with VTG results to determine endocrine activity. [Extracted from the article]

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]

Although frequently examined as a target organ for non-endocrine toxicity, histopathological evaluation of the liver is becoming a routine component of endocrine disruption studies that utilize various fish species as test subjects. However, the interpretation of microscopic liver findings can be challenging, especially when attempting to distinguish adverse changes associated with endocrine disrupting substances from those caused by systemic or direct hepatic toxicity. The purpose of this project was to conduct a critical assessment of the available peer-reviewed and grey literature concerning the histopathologic effects of reproductive endocrine active substances (EAS) and non-endocrine acting substances in the livers of fish models, and to determine if liver histopathology can be used to reliably distinguish endocrine from non-endocrine etiologies. The results of this review suggest that few compound-specific histopathologic liver effects have been identified, among which are estrogen agonist-induced increases in hepatocyte basophilia and proteinaceous intravascular fluid in adult male teleosts, and potentially, decreased hepatocyte basophilia in female fish exposed to substances that possess androgenic, anti-estrogenic, or aromatase inhibitory activity. This review also used published standardized methodology to assess the credibility of the histopathology data in each of the 117 articles that reported liver effects of treatment, and consequently it was determined that in only 37% of those papers were the data considered either highly credible or credible. The outcome of this work highlights the value of histopathologic liver evaluation as an investigative tool for EAS studies, and provides information that may have implications for EAS hazard assessment. [ABSTRACT FROM AUTHOR]

ABSTRACT The ecotoxicity testing of chemicals for prospective environmental safety assessment is an area in which a high number of vertebrates are used across a variety of industry sectors. Refining, reducing, and replacing the use of animals such as fish, birds, and amphibians for this purpose addresses the ethical concerns and the increasing legislative requirements to consider alternative test methods. Members of the UK-based National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) Ecotoxicology Working Group, consisting of representatives from academia, government organizations, and industry, have worked together over the past 6 y to provide evidence bases to support and advance the application of the 3Rs in regulatory ecotoxicity testing. The group recently held a workshop to identify the areas of testing, demands, and drivers that will have an impact on the future of animal use in regulatory ecotoxicology. As a result of these discussions, we have developed a pragmatic approach to prioritize and realistically address key opportunity areas, to enable progress toward the vision of a reduced reliance on the use of animals in this area of testing. This paper summarizes the findings of this exercise and proposes a pragmatic strategy toward our key long-term goals-the incorporation of reliable alternatives to whole-organism testing into regulations and guidance, and a culture shift toward reduced reliance on vertebrate toxicity testing in routine environmental safety assessment. Integr Environ Assess Manag 2016;12:417-421. © 2015 SETAC [ABSTRACT FROM AUTHOR]

The chronic toxicity of chemicals to fish is routinely assessed by using fish early life stage (ELS) test results. Fish full life cycle (FLC) tests are generally required only when toxicity, bioaccumulation, and persistence triggers are met or when there is a suspicion of potential endocrine-disrupting properties. This regulatory approach is based on a relationship between the results of fish ELS and FLC studies first established more than 35 yrs ago. Recently, this relationship has been challenged by some regulatory authorities, and it has been recommended that more substances should undergo FLC testing. In addition, a project proposal has been submitted to the Organisation for Economic Cooperation and Development (OECD) to develop a fish partial life cycle (PLC) test including a reproductive assessment. Both FLC and PLC tests are animal- and resource-intensive and technically challenging and should therefore be undertaken only if there is clear evidence that they are necessary for coming to a regulatory decision. The present study reports on an analysis of a database of paired fish ELS and FLC endpoints for plant protection product active substances from European Union draft assessment reports and the US Environmental Protection Agency Office of Pesticide Programs Pesticide Ecotoxicity Database. Analysis of this database shows a clear relationship between ELS and FLC responses, with similar median sensitivity across substances when no-observed-effect concentrations (NOECs) are compared. There was also no indication that classification of a substance as a mammalian reproductive toxicant leads to more sensitive effects in fish FLC tests than in ELS tests. Indeed, the response of the ELS tests was generally more sensitive than the most sensitive reproduction NOEC from a FLC test. This analysis indicates that current testing strategies and guidelines are fit for purpose and that there is no need for fish full or partial life cycle tests for most plant protection product active substances. Environ Toxicol Chem 2014;33:1874-1878. © 2014 SETAC [ABSTRACT FROM AUTHOR]

The acute and chronic toxicity of chemicals to fish is routinely assessed using fish acute and early life stage (ELS) test results, usually with freshwater species. Under some regulations with certain substances, however, data on saltwater species may also be required. Evidence from earlier studies suggests that saltwater fish species are generally no more sensitive than freshwater species and that when they are more sensitive to a substance the difference in sensitivity is usually less than a factor of 10. However, most of these studies concentrated on acute lethal toxicity results for a wide range of substances and across a range of taxonomic groups. In the present study, the authors compare freshwater and saltwater acute median lethal concentration (LC50) and chronic ELS no-observed-effect concentration (NOEC) results from standardized regulatory studies specifically for fish species exposed to the same plant protection or biocidal product active substances to determine the value of testing in both freshwater and saltwater fish. The results suggest that, in most cases, use of a freshwater fish 96-h LC50 or longer-term ELS NOEC should be sufficient to protect saltwater species. In a small number of cases (12%), saltwater ELS NOECs were more sensitive by a factor >10, although differences in sensitivity were not consistent for this small number of substances when 96-h LC50 and longer-term ELS NOECs were compared. It is debatable whether such a low probability merits the additional animal use required to run saltwater fish tests, especially when onshore contaminants released to estuaries and coastal environments are likely to be diluted many-fold when compared with concentrations found in freshwaters. Environ Toxicol Chem 2014;33:703-707. © 2013 SETAC [ABSTRACT FROM AUTHOR]

Abstract: Fish in vivo screening methods to detect endocrine active substances, specifically interacting with the hypothalamic–pituitary–gonadal axis, have been developed by both the Organization for Economic Co-operation and Development (OECD) and United States Environmental Protection Agency (US-EPA). In application of these methods, i.e. regulatory testing, this paper provides a proposal on the setting of test concentrations using all available acute and chronic data and also discusses the importance of avoiding the confounding effects of systemic toxicity on endocrine endpoints. This guidance is aimed at reducing the number of false positives and subsequently the number of inappropriate definitive vertebrate studies potentially triggered by effects consequent to systemic, rather than endocrine, toxicity. At the same time it provides a pragmatic approach that maximizes the probability of detecting an effect, if it exists, thus limiting the potential for false negative outcomes. [Copyright &y& Elsevier]

The relative sensitivity of amphibians to chemicals in the environment, including plant protection product active substances, is the subject of ongoing scientific debate. The objective of this study was to compare systematically the relative sensitivity of amphibians and fish to chemicals. Acute and chronic toxicity data were obtained from the U.S. Environmental Protection Agency (U.S. EPA) ECOTOX database and were supplemented with data from the scientific and regulatory literature. The overall outcome is that fish and amphibian toxicity data are highly correlated and that fish are more sensitive (both acute and chronic) than amphibians. In terms of acute sensitivity, amphibians were between 10- and 100-fold more sensitive than fish for only four of 55 chemicals and more than 100-fold more sensitive for only two chemicals. However, a detailed inspection of these cases showed a similar acute sensitivity of fish and amphibians. Chronic toxicity data for fish were available for 52 chemicals. Amphibians were between 10- and 100-fold more sensitive than fish for only two substances (carbaryl and dexamethasone) and greater than 100-fold more sensitive for only a single chemical (sodium perchlorate). The comparison for carbaryl was subsequently determined to be unreliable and that for sodium perchlorate is a potential artifact of the exposure medium. Only a substance such as dexamethasone, which interferes with a specific aspect of amphibian metamorphosis, might not be detected using fish tests. However, several other compounds known to influence amphibian metamorphosis were included in the analysis, and these did not affect amphibians disproportionately. These analyses suggest that additional amphibian testing is not necessary during chemical risk assessment. Environ. Toxicol. Chem. 2013;32:984-994. © 2013 SETAC [ABSTRACT FROM AUTHOR]

Vitellogenin, the fish egg yolk precursor protein, is the most common biochemical endpoint in general use for the detection of (anti) estrogen active substances in fish and other oviparous species. This review aims to cover the major methods (both protein and nucleic acid) for vitellogenin determination. Comparisons are drawn between vitellogenin and other endpoint effect levels. This review highlights strengths and weaknesses of the various techniques, concentrating on practical considerations and problems in evaluating the data they generate. [ABSTRACT FROM AUTHOR]

Secondary sexual characteristics (SSCs) are important features that have evolved in many fish species because of inter-individual competition for mates. SSCs are crucial not only for sexual selection, but also for other components of the reproductive process and parental care. Externally, they are especially clear in males (for instance, tubercles, fatpad, anal finnage, colouration) but are also externally present in the females (for instance, ovipositor). These characters are under hormonal control and as such there has been much interest in incorporating them as measures in fish test methods to assess the potential endocrine activity of chemicals. Here we describe the external SSCs in typical laboratory test species for endocrine testing - fathead minnow (Pimephales promelas), Japanese medaka (Oryzias latipes), zebrafish (Danio rerio) and the three-spined stickleback (Gasterosteus aculeatus L.). We also provide some examples and discuss the utility of SSC responses to the endocrine activity of chemicals in the field and the laboratory. This paper is not aimed to provide a comprehensive review of SSCs in fish but presents a view on the assessment of SSCs in regulatory testing. Due to the current regulatory importance of establishing an endocrine mode-of-action for chemicals, we also consider other, non-endocrine factors that may lead to SSC responses in fish. We conclude with recommendations for how the assessment of SSCs in fish could be usefully incorporated into the endocrine hazard and risk assessment of chemicals. [ABSTRACT FROM AUTHOR]

Highlights: [•] Feasibility of the threshold approach for formulated PPPs fish acute toxicity tests. [•] Database analysis demonstrates that a reduction of approximately 38% of the fish might be achieved. [•] Proposal for the threshold approach in the development of a regulatory dataset. [Copyright &y& Elsevier]

• The Amphibian Metamorphosis Assay (AMA) and Fish Short Term Reproduction Assay (FSTRA) are standardized regulatory tests used to determine if a chemical might affect the hypothalamic-pituitary-thyroid (HPT) axis or hypothalamic-pituitary-gonadal (HPG) axis, respectively. • Histopathology historical control data were assembled and evaluated from 55 AMA and 45 fathead minnow FSTRA studies. • Historical control data were found useful for investigating the existence of outlier results and aberrant control groups, identifying potential confounding variables, providing context for rare diagnoses, differentiating endocrine from non-endocrine effects, identifying background lesions, and helping to determine if specific improvements to established test guidance may be warranted. The Amphibian Metamorphosis Assay (AMA) is used to determine if a tested chemical has potential to impact the hypothalamic-pituitary-thyroid (HPT) axis of Xenopus laevis tadpoles, while the Fish Short Term Reproduction Assay (FSTRA) assesses potential effects on the hypothalamic-pituitary-gonadal (HPG) axis of fish such as the fathead minnow (Pimephales promelas). Several global regulatory programs routinely require these internationally validated tests be performed to determine the potential endocrine activity of chemicals. As such, they are conducted in accordance with standardized protocols and test criteria, which were originally developed more than a decade ago. Sizeable numbers of AMA and FSTRA studies have since been carried out, which allows for the mining of extensive historical control data (HCD). Such data are useful for investigating the existence of outlier results and aberrant control groups, identifying potential confounding variables, providing context for rare diagnoses, discriminating target from non-target effects, and for refining current testing paradigms. The present paper provides histopathology HCD from 55 AMA studies and 45 fathead minnow FSTRA studies, so that these data may become publicly available and thus aid in the interpretation of future study outcomes. Histopathology is a key endpoint in these assays, in which it is considered to be one of the most sensitive indicators of endocrine perturbation. In the current review, granular explorations of HCD data were used to identify background lesions, to assess the utility of particular diagnostic findings for distinguishing endocrine from non-endocrine effects, and to help determine if specific improvements to established regulatory guidance may be warranted. Knowledge gleaned from this investigation, supplemented by information from other recent studies, provided further context for the interpretation of AMA and FSTRA histopathology results. We recommend HCDs for the AMA and FSTRA be maintained to support the interpretation of study results. [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]

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: Fish bioconcentration tests are time consuming, expensive, and use many animals. Alternative methods that replace, reduce or refine the use of fish for BCF testing would therefore be of value. Test guidelines generally require that bioconcentration factors (BCFs) are determined at two exposure concentrations. However, recent revisions to the OECD Test Guideline for BCF 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. Analysis of 55 studies on plant protection products demonstrates that BCF values do not significantly differ between the two exposure concentrations. This analysis therefore provides evidence to support the revision of OECD TG 305, and in particular provides justification for using the one test concentration approach for plant protection product active substances. [Copyright &y& Elsevier]