Too advanced for assessment? Advanced materials, nanomedicine and the environment.

Advanced materials, and nanomaterials, are promising for healthcare applications and are in particular in the spotlight of medical innovation since rapidly developed nano-formulated vaccines provide relief in the SARS-CoV-2 pandemic. Further increased rapid growth is to be expected as more and more products are in development and reach the market, beneficial for human health. However, the human body is not a dead end and these products are likely to enter the environment, whereas their fate and effects in the environment are unknown. This part of the life-cycle of advanced medicinal products tends to be overlooked, if the perspective is human-centered and excludes the connectedness of human activity with, and consequences for our environment. Gaps are reviewed that exist in awareness, perspective taking, inclusion of environmental concerns into research and product development and also in available methodologies and regulatory guidance. To bridge these gaps, possible ways forward start to emerge, that could help to find a more integrative way of assessing human and environmental safety for advanced material medicinal products and nanomedicines.

Environmental Risk Assessment of Nanomaterials in the Light of New Obligations Under the REACH Regulation: Which Challenges Remain and How to Approach Them?

Within the European regulation on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH, EC No 1907/2006) specific provisions for nanomaterials were included, which have become effective on 1 January 2020. Although knowledge on the peculiarities of testing and assessing fate and effects of nanomaterials in the environment strongly increased in the last years, uncertainties about how to perform a reliable and robust environmental risk assessment for nanomaterials still remain. These uncertainties are of special relevance in a regulatory context, challenging both industry and regulators. The present paper presents current challenges in regulatory hazard and exposure assessment under REACH, as well as classification of nanomaterials, and makes proposals to address them. Still, the nanospecific considerations made here are expected to also be valid for environmental risk assessment approaches in other regulations of chemical safety. Inter alia, these proposals include a way forward to account for exposure concentrations in aquatic toxicity test systems, a discussion of how to account for availability of dissolving nanomaterials in aquatic test systems, and a pragmatic proposal to deduce effect data for soil organisms. Furthermore, it specifies how to potentially deal with nanoforms under the European regulation on Classification, Labelling and Packaging of substances and mixtures (CLP) and outlines the needs for proper exposure assessments of nanomaterials from a regulatory perspective. Integr Environ Assess Manag 2020;16:706-717. © 2020 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Nanopharmaceuticals: Tiny challenges for the environmental risk assessment of pharmaceuticals.

Many new developments and innovations in health care are based on nanotechnology. The field of nanopharmaceuticals is diverse and not as new as one might think; indeed, nanopharmaceuticals have been marketed for many years, and the future is likely to bring more nanosized compounds to the market. Therefore, it is time to examine whether the environmental risk assessment for human pharmaceuticals is prepared to assess the exposure, fate, and effects of nanopharmaceuticals in an adequate way. Challenges include the different definitions for nanomaterials and nanopharmaceuticals, different regulatory frameworks, the diversity of nanopharmaceuticals, the scope of current regulatory guidelines, and the applicability of test protocols. Based on the current environmental risk assessment for human medicinal products in the European Union, necessary adaptations for the assessment procedures and underlying study protocols are discussed and emerging solutions identified.

Approach on environmental risk assessment of nanosilver released from textiles.

Based on the increased utilization of nanosilver (silver nanomaterials=AgNM) as antibacterial agent, there is the strong need to assess the potential environmental implication associated with its new application areas. In this study an exemplary environmental risk assessment (ERA) of AgNM applied in textiles was performed. Environmental exposure scenarios (via municipal sewage treatment plant (STP)) with wastewater supply from domestic homes) were developed for three different types of textiles equipped with AgNM. Based on these scenarios predicted environmental concentrations (PECs) were deduced for STPs and for the environmental compartments surface water, sediment as well as soil. These PECs were related to PNECs (predicted no effect concentrations). PNECs were deduced from results of ecotoxicity tests of a selected AgNM (NM-300K). Data on ecotoxicology were derived from various tests with activated sludge, cyanobacteria, algae, daphnids, fish, duckweed, macrophytes, chironomids, earthworms, terrestrial plants as well as soil microorganisms. Emission data for the AgNM NM-300K from textiles were derived from washing experiments. The performed ERA was based on the specifications defined in the ECHA Guidances on information requirements and chemical safety assessment. Based on the chosen scenarios and preconditions, no environmental risk of the AgNM NM-300K released from textiles was detected. Under conservative assumptions a risk quotient for surface water close to 1 indicated that the aquatic compartment may be affected by an increased emission of AgNM to the environment due to the high sensitivity of aquatic organisms to silver. Based on the successful retention of AgNM in the sewage sludge and the still ongoing continual application of sewage sludge on farmland it is recommended to introduce a threshold for total silver content in sewage sludge into the respective regulations. Regarding potential risk mitigation measures, it is emphasized to preferably directly introduce AgNM into the textile fiber since this will strongly minimize the release of AgNM during washing. If this is not possible due to technical limitations or other reasons, the introduction of a threshold level controlling the release of AgNM from textiles is suggested. It has to be noted that this study is a case study which is only valid for the investigated NM-300K and its potential application in textiles.

The zebrafish embryo model in environmental risk assessment--applications beyond acute toxicity testing.

BACKGROUND, AIM, AND SCOPE: The use of fish embryos is not regulated by current legislations on animal welfare and is therefore considered as a refinement, if not replacement of animal experiments. Fish embryos represent an attractive model for environmental risk assessment of chemicals since they offer the possibility to perform small-scale, high-throughput analyses. MAIN FEATURES: Beyond their application for determining the acute toxicity, fish embryos are also excellent models for studies aimed at the understanding of toxic mechanisms and the indication of possible adverse and long-term effects. Therefore, we have reviewed the scientific literature in order to indicate alternative applications of the fish embryo model with focus on embryos of the zebrafish. RESULTS AND DISCUSSIONS: The analysis of the mode of action is important for the risk assessment of environmental chemicals and can assist in indicating adverse and long-term effects. Toxicogenomics present a promising approach to unravel the potential mechanisms. Therefore, we present examples of the use of zebrafish embryos to study the effect of chemicals on gene and protein patterns, and the potential implications of differential expression for toxicity. The possible application of other methods, such as kinase arrays or metabolomic profiling, is also highlighted. Furthermore, we show examples of toxicokinetic studies (bioconcentration, ABC transporters) and discuss limitations that might be caused by the potential barrier function of the chorion. Finally, we demonstrate that biomarkers of endocrine disruption, immune modulation, genotoxicity or chronic toxicity could be used as indicators or predictors of sub-acute and long-term effects. CONCLUSIONS: The zebrafish embryo represents a model with an impressive range of possible applications in environmental sciences. Particularly, the adaptation of molecular, system-wide approaches from biomedical research is likely to extend its use in ecotoxicology. RECOMMENDATIONS AND PERSPECTIVES: Challenges for future research are (1) the identification of further suitable molecular markers as indicators of the mode of action, (2) the establishment of strong links between (molecular) effects in short-term assays in embryos and long-term (toxic) effects on individuals, (3) the definition of limitations of the model and (4) the development of tests that can be used for regulatory purposes.

Eya1 and Six1 promote neurogenesis in the cranial placodes in a SoxB1-dependent fashion.

Genes of the Eya family and of the Six1/2 subfamily are expressed throughout development of vertebrate cranial placodes and are required for their differentiation into ganglia and sense organs. How they regulate placodal neurogenesis, however, remains unclear. Through loss of function studies in Xenopus we show that Eya1 and Six1 are required for neuronal differentiation in all neurogenic placodes. The effects of overexpression of Eya1 or Six1 are dose dependent. At higher levels, Eya1 and Six1 expand the expression of SoxB1 genes (Sox2, Sox3), maintain cells in a proliferative state and block expression of neuronal determination and differentiation genes. At lower levels, Eya1 and Six1 promote neuronal differentiation, acting downstream of and/or parallel to Ngnr1. Our findings suggest that Eya1 and Six1 are required for both the regulation of placodal neuronal progenitor proliferation, through their effects on SoxB1 expression, and subsequent neuronal differentiation.

The role of cyp1a and heme oxygenase 1 gene expression for the toxicity of 3,4-dichloroaniline in zebrafish (Danio rerio) embryos.

Expression profiling of exposed cells or organisms can reveal genes sensitive to environmental contaminants or toxic compounds. However, the mechanistic relevance of altered gene expression often remains to be elucidated. Toxicant-dependent differential gene expression may indicate protection to or mediation of toxicity. Previous studies revealed a number of differentially transcribed genes in zebrafish embryos exposed to the model compound 3,4-dichloroaniline (3,4-DCA). To evaluate the significance of two of the most sensitive genes, cytochrome P 450 1a (cyp1a) and heme oxygenase 1 (hmox1), for 3,4-DCA toxicity, RNA interference-mediated knockdown and overexpression studies have been conducted. Knockdown of gene transcription by siRNA for cyp1a and hmox1 enhanced the frequency of developmental disorders in embryos exposed to 3,4-DCA. Vice versa, injection of cyp1a and hmox1 mRNA reduced the number of disorders. The opposite effects of siRNA and mRNA injection clearly indicate a protective role of the corresponding proteins. Functional studies such as the one presented could be applied to a wide variety of genes. They would be ideally suited to study the role of genes identified from toxicogenomic studies in the zebrafish embryo model.

Differential gene expression as a toxicant-sensitive endpoint in zebrafish embryos and larvae.

The zebrafish (Danio rerio) embryo toxicity test (DarT) is under consideration as an alternative to the acute fish toxicity test. Microscopically visible developmental disorders or death are the endpoints used to report on toxicity in DarT. These endpoints are easily observed. They, however, rarely reveal mechanisms leading to a toxic effect and are relatively insensitive compared to chronic toxic effects. We hypothesized that, by using gene expression profiles as an additional endpoint, it may be possible to increase the sensitivity and predictive value of DarT. Therefore, as a proof of principle, we exposed zebrafish embryos to the reference compound 3,4-dichloroaniline (3,4-DCA) and analyzed gene expression patterns with a 14k oligonucleotide array. Important stress response genes not included in the microarray were additionally quantified by reverse transcriptase polymerase chain reaction. Six genes involved in biotransformation (cyp1a, ahr2), stress response (nfe212, maft, hmox1) and cell cycle control (fzr1) were significantly regulated. With the exception of fzr1, these genes proved to be differentially expressed in post hatch life stages as well. The identified genes point toward an aryl hydrocarbon receptor-mediated response. Differential gene expression in embryos exposed for 48 h was observed at 3,4-DCA concentrations as low as 0.78 microM, which is more than 10-fold below the concentrations that elicited visible toxic effects. Upon exposure for 5 days, differential expression was detected at concentrations as low as 0.22 microM of 3,4-DCA, which was close to the lowest observed effect concentration (0.11 microM) in the 30-day early life stage test. This study therefore indicates that gene expression analysis in DarT is able to reveal mechanistic information and may also be exploited for the development of replacement methods for chronic fish tests.