Expert survey on identification of gaps in available test methods for evaluation of endocrine disruptors

According to the 2012 WHO/UNEP publication 'State of the Science of Endocrine Disrupting Chemicals' research into endocrine disrupting chemicals over the last decade has indicated that, despite the progress achieved in development and validation of test methods for evaluation of endocrine disruptors, there are still several gaps that need to be addressed. Considering the expected significant amount of work needed to fill the gaps and the limited resources available, it will be important to set priorities for the upcoming period (next 5-10 years) for the development and validation of test methods. Thus there is a need to focus the European input to the OECD test guideline programme to effectively enhance the identification of chemical substances with endocrine disrupting properties whilst making best use of existing resources. With this objective in mind, DG Environment, supported by JRC, is organising a European expert workshop on setting priorities for further development and validation of test methods for evaluating endocrine disruption. The workshop will take place on 30 May - 01 June 2017 in Brussels. The deliberations will focus on what is necessary and achievable in the context of resources, timescales and animal welfare considerations. In preparation for the workshop, JRC has drawn up a questionnaire to gather input from experts in the field on key issues to be used as a basis for the further discussions at the workshop. An online survey with the title "Identifying gaps in available test methods for evaluation of endocrine disruptors" was performed on the EU Survey platform and open for commenting from 19/05/2015 until 15/06/2015. A selected group of experts (EFSA Scientific Committee and WG on EDs, ECHA ED WG and RAC, WNT (European members from OECD webpage), Experts identified in Annex 3 of the "Roadmap for setting priorities for further development and validation of test methods and testing approaches for evaluating endocrine disruptors") was invited to participate in the survey. Experts were asked to rank endocrine related diseases/disorders regarding the possibility to predict them with existing test methods (TMs). They were further asked to rank diseases/disorders regarding the need to develop new test methods to better cover those. Experts were then requested to provide their views on including further tests based on those discussed in the OECD (2012) "Detailed Review Paper on the state of the science on novel in vitro and in vivo screening and testing methods and endpoints for evaluating endocrine disruptors" and their views on the current OECD Conceptual Framework and proposals for improvements. Forty experts representing 15 countries and different stakeholder groups (authorities; academia; civil society organisation; industry) replied. The purpose of this report is to present the detailed survey results. Multiple choice questions were evaluated and where possible quantitative rankings were performed. In addition, the survey respondents provided a lot of valuable information in numerous free text comments. Those are included in the report in tables as they were received, without editing them, unless personal information had to be removed. Brief summaries of the main points raised are added after each section.JRC.F.3-Chemicals Safety and Alternative Method

Identification of NF-κB Modulation Capabilities within Human Intestinal Commensal Bacteria

The intestinal microbiota plays an important role in modulation of mucosal immune responses. To seek interactions between intestinal epithelial cells (IEC) and commensal bacteria, we screened 49 commensal strains for their capacity to modulate NF-κB. We used HT-29/kb-seap-25 and Caco-2/kb-seap-7 intestinal epithelial cells and monocyte-like THP-1 blue reporter cells to measure effects of commensal bacteria on cellular expression of a reporter system for NF-κB. Bacteria conditioned media (CM) were tested alone or together with an activator of NF-κB to explore its inhibitory potentials. CM from 8 or 10 different commensal species activated NF-κB expression on HT-29 and Caco-2 cells, respectively. On THP-1, CM from all but 5 commensal strains stimulated NF-κB. Upon challenge with TNF-α or IL-1β, some CM prevented induced NF-κB activation, whereas others enhanced it. Interestingly, the enhancing effect of some CM was correlated with the presence of butyrate and propionate. Characterization of the effects of the identified bacteria and their implications in human health awaits further investigations

Screening methodology to identify potential endocrine disruptors according to different options in the context of an impact assessment

Several pieces of EU legislation regulate the marketing and use of chemical substances. While several regulations, including the regulations on Plant Protection Products (PPPR), Biocidal Products (BPR) and Chemicals (REACH), include provisions for endocrine disrupting substances (EDs), objective scientific criteria are lacking. In order to evaluate the potential health, socio-economic and environmental impacts of applying four different options for criteria defining EDs across these pieces of legislation, the Commission initiated an Impact Assessment (IA). This IA has been supported by two studies, focusing on (a) selection of substances for the IA and the screening of their potential for identification as EDs according to different options for defining criteria for identification of endocrine disruptors and (b) the potential impacts of various policy options on health, environment, trade, agriculture and socio-economy. This report describes a screening methodology that has been developed by the JRC to support the first study which has assessed all pesticide and biocide active ingredients and a selection of substances falling under REACH, the Cosmetic Products Regulation and the Water Framework Directive. This screening methodology is not intended to replace an in-depth risk assessment process, and the results obtained are not intended to pre-empt regulatory conclusions that may eventually be made under different pieces of EU legislation.JRC.I.5-Systems Toxicolog

Alternative methods for regulatory toxicology – a state-of-the-art review

This state-of-the art review is based on the final report of a project carried out by the European Commission’s Joint Research Centre (JRC) for the European Chemicals Agency (ECHA). The aim of the project was to review the state of the science of non-standard methods that are available for assessing the toxicological and ecotoxicological properties of chemicals. Non-standard methods refer to alternatives to animal experiments, such as in vitro tests and computational models, as well as animal methods that are not covered by current regulatory guidelines. This report therefore reviews the current scientific status of non-standard methods for a range of human health and ecotoxicological endpoints, and provides a commentary on the mechanistic basis and regulatory applicability of these methods. For completeness, and to provide context, currently accepted (standard) methods are also summarised. In particular, the following human health endpoints are covered: a) skin irritation and corrosion; b) serious eye damage and eye irritation; c) skin sensitisation; d) acute systemic toxicity; e) repeat dose toxicity; f) genotoxicity and mutagenicity; g) carcinogenicity; h) reproductive toxicity (including effects on development and fertility); i) endocrine disruption relevant to human health; and j) toxicokinetics. In relation to ecotoxicological endpoints, the report focuses on non-standard methods for acute and chronic fish toxicity. While specific reference is made to the information needs of REACH, the Biocidal Products Regulation and the Classification, Labelling and Packaging Regulation, this review is also expected to be informative in relation to the possible use of alternative and non-standard methods in other sectors, such as cosmetics and plant protection products.JRC.I.5-Systems Toxicolog

Adverse Outcome Pathway: Peroxisome Proliferator-Activated Receptor α Activation and Reproductive Toxicity—Development and Application in Assessment of Endocrine Disruptors/Reproductive Toxicants

In the process of a paradigm shift in toxicity testing, many efforts have focused on how to integrate and interpret information for biological events occurring at molecular and cellular level to be predictive of adverse effects at organism or population level to be useful, for example, for regulatory decision-making. The adverse outcome pathway (AOP) concept provides such a framework of knowledge-based safety assessment of chemicals that links mechanistic information with an apical endpoint. Here we outline an AOP that links the activation of peroxisome proliferator-activated receptor α (PPARα) to reproductive tract malformations and impaired fertility in males. The development of this AOP relies on evidence collected from rodent models and incorporates human mechanistic and epidemiological data. Interest in PPARα action as a mechanistic basis for effects on the reproductive system arises from the relationships between activation of this receptor and impairment of steroidogenesis leading to reproductive toxicity. The PPARα-initiated AOP is a first step for structuring current knowledge about mode of action (MoA), which is neither androgen receptor-mediated nor via direct aromatase inhibition. In the current form, the pathway lays a strong basis for linking an endocrine MoA with an adverse effect, a prerequisite requirement for the identification of endocrine disrupting chemicals.JRC.F.3-Chemicals Safety and Alternative Method

A robust and adaptable high throughput screening method to study host-microbiota interactions in the human intestine

The intestinal microbiota has many beneficial roles for its host. However, the precise mechanisms developed by the microbiota to influence the host intestinal cell responses are only partially known. The complexity of the ecosystem and our inability to culture most of these micro-organisms have led to the development of molecular approaches such as functional metagenomics, i.e. the heterologous expression of a metagenome in order to identify functions. This elegant strategy coupled to high throughput screening allowed to identify novel enzymes from different ecosystems where culture methods have not yet been adapted to isolate the candidate microorganisms. We have proposed to use this functional metagenomic approach in order to model the microbiota's interaction with the host by combining this heterologous expression with intestinal reporter cell lines. The addition of the cellular component to this functional metagenomic approach introduced a second important source of variability resulting in a novel challenge for high throughput screening. First attempts of high throughput screening with various reporter cell-lines showed a high distribution of the response and consequent difficulties to reproduce the response, impairing an easy and clear identification of confirmed hits. In this study, we developed a robust and reproducible methodology to combine these two biological systems for high throughput application. We optimized experimental setups and completed them by appropriate statistical analysis tools allowing the use this innovative approach in a high throughput manner and on a broad range of reporter assays. We herewith present a methodology allowing a high throughput screening combining two biological systems. Therefore ideal conditions for homogeneity, sensitivity and reproducibility of both metagenomic clones as well as reporter cell lines have been identified and validated. We believe that this innovative method will allow the identification of new bioactive microbial molecules and, subsequently, will promote understanding of host-microbiota interactions

Butyrate produced by commensal bacteria potentiates phorbol esters induced AP-1 response in human intestinal epithelial cells

The human intestine is a balanced ecosystem well suited for bacterial survival, colonization and growth, which has evolved to be beneficial both for the host and the commensal bacteria. Here, we investigated the effect of bacterial metabolites produced by commensal bacteria on AP-1 signaling pathway, which has a plethora of effects on host physiology. Using intestinal epithelial cell lines, HT-29 and Caco-2, stably transfected with AP-1-dependent luciferase reporter gene, we tested the effect of culture supernatant from 49 commensal strains. We observed that several bacteria were able to activate the AP-1 pathway and this was correlated to the amount of short chain fatty acids (SCFAs) produced. Besides being a major source of energy for epithelial cells, SCFAs have been shown to regulate several signaling pathways in these cells. We show that propionate and butyrate are potent activators of the AP-1 pathway, butyrate being the more efficient of the two. We also observed a strong synergistic activation of AP-1 pathway when using butyrate with PMA, a PKC activator. Moreover, butyrate enhanced the PMA-induced expression of c-fos and ERK1/2 phosphorylation, but not p38 and JNK. In conclusion, we showed that SCFAs especially butyrate regulate the AP-1 signaling pathway, a feature that may contribute to the physiological impact of the gut microbiota on the host. Our results provide support for the involvement of butyrate in modulating the action of PKC in colon cancer cells

Commensal gut bacteria modulate phosphorylation-dependent PPARγ transcriptional activity in human intestinal epithelial cells

In healthy subjects, the intestinal microbiota interacts with the host’s epithelium, regulating gene expression to the benefit of both, host and microbiota. The underlying mechanisms remain poorly understood, however. Although many gut bacteria are not yet cultured, constantly growing culture collections have been established. We selected 57 representative commensal bacterial strains to study bacteria-host interactions, focusing on PPARγ, a key nuclear receptor in colonocytes linking metabolism and inflammation to the microbiota. Conditioned media (CM) were harvested from anaerobic cultures and assessed for their ability to modulate PPARγ using a reporter cell line. Activation of PPARγ transcriptional activity was linked to the presence of butyrate and propionate, two of the main metabolites of intestinal bacteria. Interestingly, some stimulatory CMs were devoid of these metabolites. A Prevotella and an Atopobium strain were chosen for further study, and shown to up-regulate two PPARγ-target genes, ANGPTL4 and ADRP. The molecular mechanisms of these activations involved the phosphorylation of PPARγ through ERK1/2. The responsible metabolites were shown to be heat sensitive but markedly diverged in size, emphasizing the diversity of bioactive compounds found in the intestine. Here we describe different mechanisms by which single intestinal bacteria can directly impact their host’s health through transcriptional regulation.ISSN:2045-232

Edge effect reduction on cellular growth applied to the HT-29-PPARγ reporter cell-line.

<p>A Parental HT-29 cells were homogeneously seeded using a pipetting robotic workstation and pre-incubated at room temperature for the indicated time (0, 0.5 or 1 h) prior to 37°C incubation for 24 h. Cellular monolayer homogeneity was monitored using cell staining with crystal violet and quantified by absorption measurement at 595 nm. Left panel is a surface plot of a representative set of plates at increasing times of room temperature pre-incubation. Boxplot representation (right panels) of the OD 595 values for the border compared to the core of the respective representative plates. Mean p-values for different cell-lines tested are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105598#pone-0105598-t001" target="_blank">Table 1</a>. B HT-29-PPARγ reporter cells were homogeneously seeded using a pipetting robotic workstation and pre-incubated at room temperature for the indicated time (0 or 1 hour) then at 37°C for 24 h prior to sodium butyrate (2 mM) activation. Luciferase activity (RLU) was quantified after 24 h of activation. Graph (left panels) shows two representative plates with different pre-incubation times at room temperature. Boxplots (right panels) represent the border and core values of the surface plots. Mean p-values for different cell-lines tested are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105598#pone-0105598-t002" target="_blank">Table 2</a>.</p

Different normalizations of assay plates.

<p>Different mathematical approaches for normalization were applied to the dataset from the initial HT-29-PPARγ screening. A shows all data-points as dots ordered by activity-rank whereby the red dots are points on the plate’s cores and the green points are well on the borders. Dark green areas show overlapping of red and green dots. B shows ranked Z-score normalization of the results. (red dots are points on the cores and the green points are well on the borders. Dark green areas show overlapping of red and green dots. C. shows the ranked B-score values for the same data set. (red dots are points on the cores and the green points are well on the borders. Dark green areas show overlapping of red and green dots.</p