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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]

Highlights: [•] Solvent carriers are discussed in terms of the practical, statistical and regulatory considerations. [•] Analysis using a database of 141 responses (endpoints) from 73 experiments. [•] Conditional strategy to control choice is recommended. [•] Wherever possible a solvent should not be employed or at least the concentration minimized. [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]

Abstract: Exogenous treatment of fish with natural sex hormones and their mimics has been shown to influence gonadal differentiation resulting in biased phenotypic sex-ratios. This has lead to the development of the Fish Sexual Development Test (FSDT) as a method for the detection of endocrine active chemicals. Proposed test organisms include the medaka, zebrafish (ZF) and stickleback, although the guideline also allows for inclusion of species such as the fathead minnow (FHM), provided the test duration allows for sufficient sexual differentiation. However, although the processes underlying sexual differentiation are known to differ for each of these species, it is not known how, or if, these differences would influence the results of the FSDT. In the experiments reported here, responses of the ZF and FHM to prochloraz, a sterol biosynthesis inhibitor and androgen antagonist, were characterized and compared. Exposure to 320μg/L of prochloraz, from embryo until 60 (ZF) or 95–125 (FHM) days post hatch inhibited somatic growth of both species, but while a negative impact on ZF larval survival was observed (LOEC 32μg/L) there was no evidence for an effect on FHM larval survival. Prochloraz influenced sexual differentiation in both species by decreasing the proportion of females (LOEC 100μg/L (ZF), 320μg/L (FHM)) and delaying completion of sexual differentiation; manifest as an increased incidence of ovotestis in the ZF (LOEC 100μg/L) and as an increased number of fish with undifferentiated gonads in the FHM (LOEC 320μg/L). However, while exposure to 320μg/L prochloraz delayed maturation of the differentiated FHM testis, there was no such effect in the ZF. These results demonstrate that the different strategy of sexual differentiation in the ZF and FHM influences the profile of responses of their gonads to the masculinising effects of prochloraz, but does not affect their overall sensitivity. [Copyright &y& Elsevier]