Finding synergies for the 3Rs - Repeated Dose Toxicity testing: Report from an EPAA Partners' Forum.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) convened a Partners' Forum on repeated dose toxicity (RDT) testing to identify synergies between industrial sectors and stakeholders along with opportunities to progress these in existing research frameworks. Although RTD testing is not performed across all industrial sectors, the OECD accepted tests can provide a rich source of information and play a pivotal role for safety decisions relating to the use of chemicals. Currently there are no validated alternatives to repeated dose testing and a direct one-to-one replacement is not appropriate. However, there are many projects and initiatives at the international level which aim to implement various aspects of replacement, reduction and refinement (the 3Rs) in RDT testing. Improved definition of use, through better problem formulation, aligned to harmonisation of regulations is a key area, as is the more rapid implementation of alternatives into the legislative framework. Existing test designs can be optimised to reduce animal use and increase information content. Greater use of exposure-led decisions and improvements in dose selection will be beneficial. In addition, EPAA facilitates sharing of case studies demonstrating the use of Next Generation Risk Assessment applying various New Approach Methodologies to assess RDT.

Toward the definition of specific protection goals for the environmental risk assessment of chemicals: A perspective on environmental regulation in Europe.

This critical review examines the definition and implementation of environmental protection goals for chemicals in current European Union (EU) legislation, guidelines, and international agreements to which EU countries are party. The European chemical industry is highly regulated, and prospective environmental risk assessments (ERAs) are tailored for different classes of chemical, according to their specific hazards, uses, and environmental exposure profiles. However, environmental protection goals are often highly generic, requiring the prevention of "unacceptable" or "adverse" impacts on "biodiversity" and "ecosystems" or the "environment as a whole." This review aims to highlight working examples, challenges, solutions, and best practices for defining specific protection goals (SPGs), which are seen to be essential for refining and improving ERA. Specific protection goals hinge on discerning acceptable versus unacceptable adverse effects on the key attributes of relevant, sensitive ecological entities (ranging from organisms to ecosystems). Some isolated examples of SPGs for terrestrial and aquatic biota can be found in prospective ERA guidance for plant protection products (PPPs). However, SPGs are generally limited to environmental or nature legislation that requires environmental monitoring and retrospective ERA. This limitation is due mainly to the availability of baselines, which define acceptable versus unacceptable environmental effects on the key attributes of sentinel species, populations and/or communities, such as reproductive status, abundance, or diversity. Nevertheless, very few regulatory case examples exist in which SPGs incorporate effect magnitude, spatial extent, and temporal duration. We conclude that more holistic approaches are needed for defining SPGs, particularly with respect to protecting population sustainability, ecosystem function, and integrity, which are implicit in generic protection goals and explicit in the International Programme for Chemical Safety (IPCS) definition of "adverse effect." A possible solution, which the chemical industry is currently assessing, is wider application of the ecosystem services approach proposed by the European Food Safety Authority (EFSA) for the risk assessment of PPPs. Integr Environ Assess Manag 2017;13:17-37. © 2016 SETAC.

17alpha-ethinylestradiol induces an imbalance between apoptosis and cell proliferation to sex steroid disruption in a testis culture of gudgeon, Gobio gobio.

The aim of this study was to investigate the effect of the most potent xenoestrogen currently found in the environment, ethinylestradiol (EE2), on some physiological events occurring during early spermatogenesis of gudgeon (Gobio gobio), a common European fish species. Physiological pathways studies were apoptosis, cell proliferation, and steroidogenesis on sex steroids (testosterone [T], 11-ketotestosterone [11-KT], and 17beta-estradiol [E2]). Testis pieces were cultured in vitro during 21 d at 10(-4), 10(-3), 10(-2), 10(-1), 1 and 10 microg/L of EE2 as well as in positive (10(-1) microg/L of E2) and ethanol control medium. Apoptosis and cell proliferation displayed opposite responses related to the EE2 concentration. When apoptosis inhibition was observed, cell proliferation was induced at 10(-2) and 10(-1) microg/L of EE2 as well as in the positive control. In contrast, a massive cell death was detected for high EE2 concentrations (1 and 10 microg/L). Steroidogenesis was also disrupted in a dose-related manner. 11-Ketotestosterone was depressed at 10(-2) and 10(-1) microg/L of EE2 whereas E2 was detectable in the medium only at 10(-3), 10(-2), and 10(-1) microg/L of EE2. High concentrations of T were detected in the medium at 10(-3), 10(-2), and 10(-1) microg/L of EE2 but depressed at 1 and 10 microg/L of EE2. In conclusion, intermediate EE2 concentrations (10(-2) and 10(-1) microg/L) used in this experimental design have obviously disrupted early spermatogenesis, leading to an imbalance between cell death and cell proliferation in a sex steroid environment toward E2. The results of the present study could be the basis conditions for oocyte development within the testis of a common teleost fish under xenoestrogen exposure.

Performance of the flow cytometric E-screen assay in screening estrogenicity of pure compounds and environmental samples.

In vitro estrogenicity screens are believed to provide a first prioritization step in hazard characterization of endocrine disrupting chemicals. When applied to complex environmental matrices or mixture samples, they have been indicated valuable in estimating the overall estrogen-mimicking load. In this study, the performance of an adapted format of the classical E-screen or MCF-7 cell proliferation assay was profoundly evaluated to rank pure compounds as well as influents and effluents of sewage treatment plants (STPs) according to estrogenic activity. In this adapted format, flow cytometric cell cycle analysis was used to allow evaluation of the MCF-7 cell proliferative effects after only 24 h of exposure. With an average EC(50) value of 2 pM and CV of 22%, this assay appears as a sensitive and reproducible system for evaluation of estrogenic activity. Moreover, estrogenic responses of 17 pure compounds corresponded well, qualitatively and quantitatively, with other in vitro and in vivo estrogenicity screens, such as the classical E-screen (R(2)=0.98), the estrogen receptor (ER) binding (R(2)=0.84) and the ER transcription activation assay (R(2)=0.87). To evaluate the applicability of this assay for complex samples, influents and effluents of 10 STPs covering different treatment processes, were compared and ranked according to estrogenic removal efficiencies. Activated sludge treatment with phosphorus and nitrogen removal appeared most effective in eliminating estrogenic activity, followed by activated sludge, lagoon and filter bed. This is well in agreement with previous findings based on chemical analysis or biological activity screens. Moreover, ER blocking experiments indicated that cell proliferative responses were mainly ER mediated, illustrating that the complexity of the end point, cell proliferation, compared to other ER screens, does not hamper the interpretation of the results. Therefore, this study, among other E-screen studies, supports the use of MCF-7 cell proliferation as estrogenicity screen for pure compounds and complex samples.

Bcl-2 and Caspase 3 mRNA levels in the testes of gudgeon, Gobio gobio, exposed to ethinylestradiol (EE2).

Apoptosis inhibition has been reported in the male reproductive tract of teleost fish exposed to 17beta-estrogen or estrogen-like compounds. In order to understand the molecular mechanisms of cell death inhibition, this study examined 2 genes involved in the apoptotic pathway, Bcl-2 and Caspase 3, an anti-apoptotic and a pro-apoptotic genes, respectively. Partial cDNA sequences of Bcl-2 and Caspase 3 were cloned from gudgeon (Gobio gobio), a common European cyprinid fish. To follow mRNA levels of Bcl-2 and Caspase 3 under xenoestrogen exposure, we first performed an in vitro experiment on fish testis exposed to the most potent xenoestrogen found in the environment, ethinylestradiol (EE2). We further studied mRNA expression of both genes in the testis of fish exposed to xenoestrogens in situ. In the in vitro experiment, fragments of gudgeon testis were exposed for 21 days to 10(-3), 10(-2), 10(-1), 1 and 10 microg/L of EE2, as well as to positive (10(-1) microg/L of E2) and ethanol control medium. Results showed a significant induction of Bcl-2 mRNA at 10(-1) microg/L (p<0.05). Surprisingly, Caspase 3, a cell death effector, displayed the same profile as observed for the anti-apoptotic gene Bcl-2. In the experiment on wild gudgeon exposed from birth to an estrogenic sewage treatment plant effluent, the mRNA expression of Bcl-2 and Caspase 3 in feminized fish (ovotestis) was not significantly different due to high variability of expression between individuals. At the current state of knowledge on spermatogenesis disruption in teleost fish, in vitro studies seem better adapted than in situ investigations to enlighten the molecular pathway of apoptosis inhibition in testis exposed to xenoestrogens.

Spleen immune status is affected after acute handling stress but not regulated by cortisol in Eurasian perch, Perca fluviatilis.

The effects of acute stress on immune status and its regulation by cortisol/corticosteroid receptors have received little attention in percids. To address that question, we investigated the physiological and immune responses of Eurasian perch, Perca fluviatilis to acute stress. We exposed immature perch to an 1-min exondation and measured at 1 h, 6 h, 24 h and 72 h post-stress: (1) stress-related parameters including plasma cortisol and glucose levels, (2) immune parameters in the plasma and in the spleen (complement, respiratory burst and lysozyme activity, total immunoglobulins; gene expression of lysozyme, complement unit 3, apolipoprotein A1 and 14 kDa, hepcidin and chemotaxin) (3) the corticosteroid receptors gene expression in the spleen after having cloned them. In addition, the in vitro effects of cortisol on the spleen immune parameters were also investigated. Plasma cortisol and glucose levels increased markedly 1h post-stress and returned at basal levels after 24 h. P. fluviatilis mineralocorticoid receptor, but not glucocorticoid receptors, was significantly up-regulated both in vivo after the stress and in vitro by cortisol at a physiological concentration (100 ng/ml). The plasma immune parameters were not significantly affected by the stress. In contrast, spleno-somatic index, spleen lysozyme activity, lysozyme and hepcidin gene expression were depleted and total immunoglobulins increased along the whole time-course (1-72 h). But, these immune parameters were not regulated in vitro by cortisol at physiological or supra-physiological doses. Our results indicate that handling stress may affect spleen antibacterial defences without clear effects on circulating immune compounds and that the elevation of plasma cortisol after handling stress may not be related to the regulation of this splenic response.

Understanding the gap between the estrogenicity of an effluent and its real impact into the wild.

To study the reliability between in vitro and in vivo data collected downstream 2 sewage treatment plants (STP) as well as from bleached kraft mill industry (BKME), 5 rivers (3 impacted and 2 references) were investigated in the Walloon region (southern of Belgium). For the in vitro part of the work, water samples were collected to measure the estrogenicity of the 'out' effluent compared to reference sample point by MCF-7 assay. Results indicated significant estrogenicity of effluents from STP and BKME and a weak estrogenicity in reference sites. However, estradiol equivalents (EEQ) estimated into rivers were probably too low to impact wild population. Chemical analysis of 13 compounds of interest indicated that extraction procedure used in this study gave low recoveries of estrogen-like xenobiotics, leading to probably under-estimated MCF-7 responses. Surprisingly, a full scan mode has revealed an unexpected compound in the sample of BKME which was: 7-isopropyl-1,1,4a-trimethyl-1,2,3,4a,9,10,10a-octahydrophenanthrene, a product of pulp mill manufacture. In parallel to in vitro, in vivo assessment of estrogenic impact of effluent was followed on the gudgeon (Gobio gobio). Samples were achieved during 2 different periods of the reproductive cycle, resting period (RP) and pre-spawning period (pSP). Unspecific physiological parameters to estrogenic exposure (gonadosomatic index and systematic testis cell counting) displayed no significant differences related to endocrine disruption of the reproductive tract, only differences were correlated with the reproductive state of fish (RP versus pSP). Concerning the potent biomarker of estrogen exposure, vitellogenin (vtg), only basal induction was revealed but not related to estrogenic exposure. Nevertheless, vtg over-expression was found for male fish presenting a feminization of the reproductive tract captured downstream the STP station of Wégnez in the Vesdre River. Intersexuality, another indicator of the estrogenicity impact in fish, was observed in every site. Actually, ovotestis was systematically formed by protoplasmic oocyte observed in low percentage in every group analysed (impacted and references). Moreover, in fish captured in Wégnez, oocyte diameter was significantly higher compared to the other groups. In this study, only moderate to none impact in population of gudgeon was noticed. Moreover, in this case no discrepancy between in vitro and vivo was viewed although both approaches revealed gaps in monitoring effluent incidence into the environment. We should remain careful in the interpretation when only partial approaches are used in order to characterize impact in the aquatic milieu.