An international cross-laboratory survey on fish vitellogenin analysis: Methodological challenges and opportunities for best practice.

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.

Are changes in vitellogenin concentrations in fish reliable indicators of chemical-induced endocrine activity?

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.

Discovery of dysregulated circular RNAs in whole blood transcriptomes from cystic fibrosis patients - implication of a role for cellular senescence in cystic fibrosis.

BACKGROUND: A largely unexplored area of research is the identification and characterization of circular RNA (circRNA) in cystic fibrosis (CF). This study is the first to identify and characterize alterations in circRNA expression in cells lacking CFTR function. The circRNA expression profiles in whole blood transcriptomes from CF patients homozygous for the pathogenetic variant F508delCFTR are compared to healthy controls. METHODS: We developed a circRNA pipeline called circRNAFlow utilizing Nextflow. Whole blood transcriptomes from CF patients homozygous for the F508delCFTR-variant and healthy controls were utilized as input to circRNAFlow to discover dysregulated circRNA expression in CF samples compared to wild-type controls. Pathway enrichment analyzes were performed to investigate potential functions of dysregulated circRNAs in whole blood transcriptomes from CF samples compared to wild-type controls. RESULTS: A total of 118 dysregulated circRNAs were discovered in whole blood transcriptomes from CF patients homozygous for the F508delCFTR variant compared to healthy controls. 33 circRNAs were up regulated whilst 85 circRNAs were down regulated in CF samples compared to healthy controls. The overrepresented pathways of the host genes harboring dysregulated circRNA in CF samples compared to controls include positive regulation of responses to endoplasmic reticulum stress, intracellular transport, protein serine/threonine kinase activity, phospholipid-translocating ATPase complex, ferroptosis and cellular senescence. These enriched pathways corroborate the role of dysregulated cellular senescence in CF. CONCLUSION: This study highlights the underexplored roles of circRNAs in CF with a perspective to provide a more complete molecular characterization of CF.

Assessment of the bioaccumulation potential of four commonly used phenolic benzotriazoles based on in silico and experimental in vivo data.

Phenolic benzotriazoles (BTZs) are used globally as light stabilizers in various plastic products to protect them from photooxidative degradation. The same physical-chemical properties that confer their functionality, like a sufficient photostability and a high octanol-water partition coefficient, also raise concerns on their potential for environmental persistence and bioaccumulation based on in silico predictive tools. To evaluate their bioaccumulation potential in aquatic organisms, standardized fish bioaccumulation studies according to OECD TG 305 were conducted with four of the most commonly used BTZs: UV 234, UV 329, UV P, and UV 326. The resulting growth- and lipid-corrected BCF values revealed that UV 234, UV 329, and UV P were below the bioaccumulation threshold (BCF ≤ 2000), but UV 326 is considered very bioaccumulative (BCF ≥ 5000) with respect to the bioaccumulation criteria under REACH. Comparing these experimentally derived data with quantitative structure activity related or other calculated values using a logarithmic partitioning coefficient octanol-water (log Pow ) driven mathematical formula revealed significant discrepancies demonstrating the weakness of current in silico approaches for this group of substances. Furthermore, available environmental monitoring data demonstrate that these rudimentary in silico approaches can lead to unreliable bioaccumulation estimates for this chemical class due to considerable uncertainties in underlying assumptions (e.g., concentration and route of exposure). However, using more sophisticated in silico methods (i.e., CATALOGIC base-line model), the derived BCF values were better aligned with the experimentally derived ones.

Aquatic testing guidelines insufficiently control the influence of dilution water toc and hardness on cationic polymer toxicity - A proposal to improve standardized test procedures.

Cationic polymers (CPs) are widely used chemicals for wastewater treatment applications and in various "down-the-drain" household products. The aquatic toxicity of CPs results from an electrostatic interaction with negatively charged cell surfaces. These effects are greatly mitigated by the binding affinity of CPs to total organic carbon (TOC) in surface water. Consequently, baseline aquatic toxicity tests of CPs using clean lab water (TOC < 2 mg/L) typically overestimate toxicity and risk which is greatly mitigated at higher environmentally relevant OC levels. However, the point at which mitigation begins is not well defined and low-level TOC in lab water may influence the baseline toxicity outcome. Similarly, divalent cations, quantified as water hardness, may modulate the electrostatic binding between OC and CP. Although standard guidelines define limits for lab water hardness and TOC, the consequences of variability within those limits on test outcome is unknown. We investigated the impact of part-per-billion (ppb) additions of TOC to lab water at different hardness levels on CP acute toxicity to Daphnia magna and Raphidocelis subcapitata. In both species, the acute toxicities of CPs with different molecular weight and charge density varied by > 10-fold in response to slight changes in TOC and water hardness, although parameters were maintained within guideline limits. When determining the baseline aquatic toxicity of CPs, the lab water should be standardized at the lowest biologically tolerable hardness and TOC at a reliably measurable level (>1 - < 2 mg/L) to reduce variability and increase the reliability of the toxicity estimate.

Evaluation of the bioaccumulation of octocrylene after dietary and aqueous exposure.

Octocrylene is used as UV filter in personal care products with a high production volume and can be detected in surface water and biota. It is liquid at ambient temperature, highly lipophilic, has a high adsorption capacity to organic material and is considered as persistent in the environment. The very low water solubility complicates the evaluation of potential long-term effects in aquatic toxicity testing, since effect thresholds are often above the water solubility limit. Thus, the evaluation of the bioaccumulation potential becomes highly relevant for the assessment of long-term environmental effects. However, even the determination of the water solubility limit for a substance with such difficult properties is challenging. The following experiments are described, and results compared to available environmental monitoring data: A bioconcentration study with aqueous exposure (BCF) in zebrafish and a biomagnification study with dietary exposure (BMF) in rainbow trout, as well as supporting experiments to evaluate the water solubility. The growth and lipid corrected BCF determined by aqueous exposure was 858 L kg-1 while the corrected BMF was 0.0335. The model-based estimation of the BCF from BMF (152-1182 L kg-1) is in good agreement with the measured BCF value. Environmental monitoring data provide only limited information on the bioaccumulation potential of octocrylene, as only few investigations were made in biota and water in parallel and concentrations of octocrylene vary by several orders of magnitude during seasons. Based on the determined fish BCF data, we conclude that OCR is not bioaccumulative according to the criteria as laid down by ECHA, 2017. Furthermore, the low BMF value indicates no accumulation along the food chain.

Intrinsic Xenobiotic Metabolizing Enzyme Activities in Early Life Stages of Zebrafish (Danio rerio).

Early life stages of zebrafish (Danio rerio, zf) are gaining attention as an alternative invivo test system for drug discovery, early developmental toxicity screenings and chemical testing in ecotoxicological and toxicological testing strategies. Previous studies have demonstrated transcriptional evidence for xenobiotic metabolizing enzymes (XME) during early zf development. However, elaborate experiments on XME activities during development are incomplete. In this work, the intrinsic activities of representative phase I and II XME were monitored by transformation of putative zf model substrates analyzed using photometry and high pressure liquid chromatography techniques. Six different defined stages of zf development (between 2.5 h postfertilization (hpf) to 120 hpf) were investigated by preparing a subcellular fraction from whole organism homogenates. We demonstrated that zf embryos as early as 2.5 hpf possess intrinsic metabolic activities for esterase, Aldh, Gst, and Cyp1a above the methodological detection limit. The activities of the enzymes Cyp3a and Nat were measurable during later stages in development. Activities represent dynamic patterns during development. The role of XME activities revealed in this work is relevant for the assessing toxicity in this test system and therefore contributes to a valuable characterization of zf embryos as an alternative testing organism in toxicology.

VDJML: a file format with tools for capturing the results of inferring immune receptor rearrangements.

BACKGROUND: The genes that produce antibodies and the immune receptors expressed on lymphocytes are not germline encoded; rather, they are somatically generated in each developing lymphocyte by a process called V(D)J recombination, which assembles specific, independent gene segments into mature composite genes. The full set of composite genes in an individual at a single point in time is referred to as the immune repertoire. V(D)J recombination is the distinguishing feature of adaptive immunity and enables effective immune responses against an essentially infinite array of antigens. Characterization of immune repertoires is critical in both basic research and clinical contexts. Recent technological advances in repertoire profiling via high-throughput sequencing have resulted in an explosion of research activity in the field. This has been accompanied by a proliferation of software tools for analysis of repertoire sequencing data. Despite the widespread use of immune repertoire profiling and analysis software, there is currently no standardized format for output files from V(D)J analysis. Researchers utilize software such as IgBLAST and IMGT/High V-QUEST to perform V(D)J analysis and infer the structure of germline rearrangements. However, each of these software tools produces results in a different file format, and can annotate the same result using different labels. These differences make it challenging for users to perform additional downstream analyses. RESULTS: To help address this problem, we propose a standardized file format for representing V(D)J analysis results. The proposed format, VDJML, provides a common standardized format for different V(D)J analysis applications to facilitate downstream processing of the results in an application-agnostic manner. The VDJML file format specification is accompanied by a support library, written in C++ and Python, for reading and writing the VDJML file format. CONCLUSIONS: The VDJML suite will allow users to streamline their V(D)J analysis and facilitate the sharing of scientific knowledge within the community. The VDJML suite and documentation are available from https://vdjserver.org/vdjml/ . We welcome participation from the community in developing the file format standard, as well as code contributions.

Considerations of Environmentally Relevant Test Conditions for Improved Evaluation of Ecological Hazards of Engineered Nanomaterials.

Engineered nanomaterials (ENMs) are increasingly entering the environment with uncertain consequences including potential ecological effects. Various research communities view differently whether ecotoxicological testing of ENMs should be conducted using environmentally relevant concentrations-where observing outcomes is difficult-versus higher ENM doses, where responses are observable. What exposure conditions are typically used in assessing ENM hazards to populations? What conditions are used to test ecosystem-scale hazards? What is known regarding actual ENMs in the environment, via measurements or modeling simulations? How should exposure conditions, ENM transformation, dose, and body burden be used in interpreting biological and computational findings for assessing risks? These questions were addressed in the context of this critical review. As a result, three main recommendations emerged. First, researchers should improve ecotoxicology of ENMs by choosing test end points, duration, and study conditions-including ENM test concentrations-that align with realistic exposure scenarios. Second, testing should proceed via tiers with iterative feedback that informs experiments at other levels of biological organization. Finally, environmental realism in ENM hazard assessments should involve greater coordination among ENM quantitative analysts, exposure modelers, and ecotoxicologists, across government, industry, and academia.

MSPrecise: A molecular diagnostic test for multiple sclerosis using next generation sequencing.

BACKGROUND: We have previously demonstrated that cerebrospinal fluid-derived B cells from early relapsing-remitting multiple sclerosis (RRMS) patients that express a VH4 gene accumulate specific replacement mutations. These mutations can be quantified as a score that identifies such patients as having or likely to convert to RRMS. Furthermore, we showed that next generation sequencing is an efficient method for obtaining the sequencing information required by this mutation scoring tool, originally developed using the less clinically viable single-cell Sanger sequencing. OBJECTIVE: To determine the accuracy of MSPrecise, the diagnostic test that identifies the presence of the RRMS-enriched mutation pattern from patient cerebrospinal fluid B cells. METHODS: Cerebrospinal fluid cell pellets were obtained from RRMS and other neurological disease (OND) patient cohorts. VH4 gene segments were amplified, sequenced by next generation sequencing and analyzed for mutation score. RESULTS: The diagnostic test showed a sensitivity of 75% on the RRMS cohort and a specificity of 88% on the OND cohort. The accuracy of the test in identifying RRMS patients or patients that will develop RRMS is 84%. CONCLUSION: MSPrecise exhibits good performance in identifying patients with RRMS irrespective of time with RRMS.

Adapting OECD Aquatic Toxicity Tests for Use with Manufactured Nanomaterials: Key Issues and Consensus Recommendations.

The unique or enhanced properties of manufactured nanomaterials (MNs) suggest that their use in nanoenabled products will continue to increase. This will result in increased potential for human and environmental exposure to MNs during manufacturing, use, and disposal of nanoenabled products. Scientifically based risk assessment for MNs necessitates the development of reproducible, standardized hazard testing methods such as those provided by the Organisation of Economic Cooperation and Development (OECD). Currently, there is no comprehensive guidance on how best to address testing issues specific to MN particulate, fibrous, or colloidal properties. This paper summarizes the findings from an expert workshop convened to develop a guidance document that addresses the difficulties encountered when testing MNs using OECD aquatic and sediment test guidelines. Critical components were identified by workshop participants that require specific guidance for MN testing: preparation of dispersions, dose metrics, the importance and challenges associated with maintaining and monitoring exposure levels, and the need for reliable methods to quantify MNs in complex media. To facilitate a scientific advance in the consistency of nanoecotoxicology test results, we identify and discuss critical considerations where expert consensus recommendations were and were not achieved and provide specific research recommendations to resolve issues for which consensus was not reached. This process will enable the development of prescriptive testing guidance for MNs. Critically, we highlight the need to quantify and properly interpret and express exposure during the bioassays used to determine hazard values.

OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing.

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV<30%) for most chemicals and laboratories. The reproducibility was lower (CV>30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes.

A European perspective on alternatives to animal testing for environmental hazard identification and risk assessment.

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance.

Aquatic toxicity tests with substances that are poorly soluble in water and consequences for environmental risk assessment.

Aquatic toxicity tests with substances that are poorly soluble in water have been conducted using different methods, and estimates of toxicity have varied accordingly. The present study illustrates differences in toxicity values resulting from variation in test designs and solution preparation methods, and offers guidance on the best way to conduct these tests. Consequences for environmental risk assessment and classification are also discussed. The present study mainly considers active ingredients of plant protection products, but is also considered relevant to other chemicals. It is recommended that toxicity tests be conducted only up to the saturation limit, dispersants avoided, and solvents used only if necessary to support handling and speed of dissolution. Analytical measurements of exposure concentrations should reflect what organisms are exposed to. If acute toxicity testing at the saturation limit yields no adverse effects, further testing should not normally be required; the toxicity value of the endpoints should be considered as the saturation limit and adverse classification should not be required. Chronic testing, if required, should then be conducted at the practical saturation limit as this is the most realistic worst-case exposure scenario. If no adverse effects occur, the risk should be acceptable because higher aqueous exposure cannot occur. This could be substantiated by testing additional species. Assessment factors on no observed effect concentration (NOEC) values at the saturation limit require careful consideration in the risk assessment to avoid unnecessarily low regulatory acceptable concentrations.

Acute and chronic effects of nano- and non-nano-scale TiO(2) and ZnO particles on mobility and reproduction of the freshwater invertebrate Daphnia magna.

Among the emerging literature addressing the biological effects of nanoparticles, very little information exists, particularly on aquatic organisms, that evaluates nanoparticles in comparison to non-nanocounterparts. Therefore, the potential effects of nano-scale and non-nano-scale TiO(2) and ZnO on the water flea, Daphnia magna, were examined in 48-h acute toxicity tests using three different test media, several pigment formulations--including coated nanoparticles--and a variety of preparation steps. In addition, a 21-d chronic Daphnia reproduction study was performed using coated TiO(2) nanoparticles. Analytical ultracentrifugation analyses provided evidence that the nanoparticles were present in a wide range of differently sized aggregates in the tested dispersions. While no pronounced effects on D. magna were observed for nano-scale and non-nano-scale TiO(2) pigments in 19 of 25 acute (48-h) toxicity tests (EC50>100 mg L(-1)), six acute tests with both nano- and non-nano-scale TiO(2) pigments showed slight effects (EC10, 0.5-91.2 mg L(-1)). For the nano-scale and non-nano-scale ZnO pigments, the acute 48-h EC50 values were close to the 1 mg L(-1) level, which is within the reported range of zinc toxicity to Daphnia. In general, the toxicity in the acute tests was independent of particle size (non-nano-scale or nano-scale), coating of particles, aggregation of particles, the type of medium or the applied pre-treatment of the test dispersions. The chronic Daphnia test with coated TiO(2) nanoparticles demonstrated that reproduction was a more sensitive endpoint than adult mortality. After 21d, the NOEC for adult mortality was 30 mg L(-1) and the NOEC for offspring production was 3 mg L(-1). The 21-d EC10 and EC50 values for reproductive effects were 5 and 26.6 mg L(-1), respectively. This study demonstrates the utility of evaluating nanoparticle effects relative to non-nano-scale counterparts and presents the first report of chronic exposure to TiO(2) nanoparticles in D. magna.

Lignosulfonic acid blocks in vitro fertilization of macaque oocytes when sperm are treated either before or after capacitation.

Lignin-derived macromolecules (LDMs) are biologically active compounds that affect a variety of cell-to-cell interactions including the inhibition of fertilization and embryo development in a number of nonmammalian species. The effect of ligno-sulfonic acid (LSA), a highly sulfonated LDM, on cynomolgus macaque sperm-oocyte interaction was evaluated with a zona pellucida binding assay and by in vitro fertilization (IVF). Sperm were treated with LSA (1.5 mg/mL) either before washing or after capacitation. Capacitation included centrifugation through 80% Percoll followed by 2 consecutive washes with medium, overnight incubation, and activation with dibutyryl cyclic adenosine monophosphate and caffeine. The zona binding assay was performed using immature oocytes that had adhered to the center of glass "binding chambers." The number of capacitated sperm that attached to the zona over a 3-minute period was recorded. Sperm attachment was significantly inhibited by LSA as compared to controls whether treatment occurred after capacitation (92.5%; P <.001) or before washing (82.5%; P <.001). When sperm were treated similarly with fucoidin, a sulfated polysaccharide known to inhibit sperm-oocyte interaction, sperm-zona binding was significantly inhibited by postcapacitation treatment but not by prewash treatment. Treatment of sperm with LSA consistently blocked fertilization over 4 IVF cycles both before washing and after capacitation. Fertilization rate for controls was 65% +/- 17%. No LSA-treated sperm were observed on the surface of lightly rinsed oocytes after 4 hours of coincubation. Localization of biotinylated LSA showed labeling over the entire sperm surface with the greatest intensity observed over the head and midpiece. LSA treatment had no effect on the percentage of motile sperm or quality of sperm motility. Due to the antifertility properties of this nontoxic molecule, LSA appears to have potential as a vaginal contraceptive.