Innovative analytical methodologies for characterizing chemical exposure with a view to next-generation risk assessment.

The chemical burden on the environment and human population is increasing. Consequently, regulatory risk assessment must keep pace to manage, reduce, and prevent adverse impacts on human and environmental health associated with hazardous chemicals. Surveillance of chemicals of known, emerging, or potential future concern, entering the environment-food-human continuum is needed to document the reality of risks posed by chemicals on ecosystem and human health from a one health perspective, feed into early warning systems and support public policies for exposure mitigation provisions and safe and sustainable by design strategies. The use of less-conventional sampling strategies and integration of full-scan, high-resolution mass spectrometry and effect-directed analysis in environmental and human monitoring programmes have the potential to enhance the screening and identification of a wider range of chemicals of known, emerging or potential future concern. Here, we outline the key needs and recommendations identified within the European Partnership for Assessment of Risks from Chemicals (PARC) project for leveraging these innovative methodologies to support the development of next-generation chemical risk assessment.

Hydrogen inhalation: in vivo rat genotoxicity tests.

Preclinical and clinical studies have shown that molecular hydrogen (H2) has anti-oxidant, anti-inflammatory, and anti-apoptotic properties. Safety data are available in the literature and acute toxicity has been tested in isolated cells and laboratory animals. We have evaluates the genotoxicity of H2 in vivo in rats after 72 h exposure, following the International Council for Harmonization guidelines ICH S2 (R1). The study was conducted on three groups of male Wistar rats: a negative control group, a positive control group receiving methyl methanesulfonate, and a H2-treated group receiving a 3.1% H2 gas mixture for 72 h. Alkaline comet, formamidopyrimidine DNA glycosylase (Fpg)-modified comet and bone marrow micronucleus assays were performed. H2 exposure increased neither comet-tail DNA intensity (DNA damage) nor frequency of "hedgehogs" in blood, liver, lungs, or bronchoalveolar lavage fluid. No increase in Fpg-sensitive sites in lungs, no induction of micronucleus formation, and no imbalance of immature erythrocyte to total erythrocyte ratio (IME%) was observed in rats exposed to H2. The ICH S2 (R1) test-battery revealed no in vivo genotoxicity in Wistar rats after 72 h inhalation of a mixture containing 3.1% H2.

Toxic responses of metabolites produced by Ostreopsis cf. ovata on a panel of cell types.

Blooms of the dinoflagellate Ostreopsis cf. ovata are regularly associated with human intoxications that are attributed to ovatoxins (OVTXs), the main toxic compounds produced by this organism and close analogs to palytoxin (PlTX). Unlike for PlTX, information on OVTXs'toxicity are scarce due to the absence of commercial standards. Extracts from two cultures of Mediterranean strains of O. cf. ovata (MCCV54 and MCCV55), two fractions containing or not OVTXs (prepared from the MCCV54 extract) and OVTX-a and -d (isolated from the MCCV55 extract) were generated. These chemical samples and PlTX were tested on a panel of cell types from several organs and tissues (skin, intestine, lung, liver and nervous system). The MCCV55 extract, containing a 2-fold higher amount of OVTXs than MCCV54 extract, was shown to be more cytotoxic on all the cell lines and more prone to increase interleukin-8 (IL-8) release in keratinocytes. The fraction containing OVTXs was also cytotoxic on the cell lines tested but induced IL-8 release only in liver cells. Unexpectedly, the cell lines tested showed the same sensitivity to the fraction that does not contain OVTXs. With this fraction, a pro-inflammatory effect was shown both in lung and liver cells. The level of cytotoxicity was similar for OVTX-a and -d, except on intestinal and skin cells where a weak difference of toxicity was observed. Among the 3 toxins, only PlTX induced a pro-inflammatory effect mostly on keratinocytes. These results suggest that the ubiquitous Na+/K+ ATPase target of PlTX is likely shared with OVTX-a and -d, although the differences in pro-inflammatory effect must be explained by other mechanisms.

Linking nanomaterial-induced mitochondrial dysfunction to existing adverse outcome pathways for chemicals.

The adverse outcome pathway (AOP) framework plays a crucial role in the paradigm shift of tox­icity testing towards the development and use of new approach methodologies. AOPs developed for chemicals are in theory applicable to nanomaterials (NMs). However, only initial efforts have been made to integrate information on NM-induced toxicity into existing AOPs. In a previous study, we identified AOPs in the AOP-Wiki associated with the molecular initiating events (MIEs) and key events (KEs) reported for NMs in scientific literature. In a next step, we analyzed these AOPs and found that mitochondrial toxicity plays a significant role in several of them at the molecular and cellular levels. In this study, we aimed to generate hypothesis-based AOPs related to NM-induced mitochondrial toxicity. This was achieved by integrating knowledge on NM-induced mitochondrial toxicity into all existing AOPs in the AOP-Wiki, which already includes mitochondrial toxicity as a MIE/KE. Several AOPs in the AOP-Wiki related to the lung, liver, cardiovascular and nervous system, with extensively defined KEs and key event relationships (KERs), could be utilized to develop AOPs that are relevant for NMs. However, the majority of the studies included in our literature review were of poor quality, particularly in reporting NM physicochemical characteristics, and NM-relevant mitochondrial MIEs were rarely reported. This study highlights the potential role of NM-induced mitochondrial toxicity in human-relevant adverse outcomes and identifies useful AOPs in the AOP-Wiki for the development of AOPs for NMs.

This article investigates commonalities in the toxicity pathways of chemicals and nanomaterials. Nanomaterials have been found to affect the function of mitochondria, the powerhouses within every human cell. Mitochondrial dysfunction may cause harmful effects such as cellular damage and inflammation. By linking these findings to existing adverse outcome pathways for chemicals, the research provides valuable insights for assessing the risks associated with nanomaterial exposure. This work is crucial for understanding the potential health implications of nanomaterials and can contribute to informed decision-making in regulatory and risk assessment processes without the use of animals.

Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health.

Fungi of the genus Alternaria are ubiquitous plant pathogens and saprophytes which are able to grow under varying temperature and moisture conditions as well as on a large range of substrates. A spectrum of structurally diverse secondary metabolites with toxic potential has been identified, but occurrence and relative proportion of the different metabolites in complex mixtures depend on strain, substrate, and growth conditions. This review compiles the available knowledge on hazard identification and characterization of Alternaria toxins. Alternariol (AOH), its monomethylether AME and the perylene quinones altertoxin I (ATX-I), ATX-II, ATX-III, alterperylenol (ALP), and stemphyltoxin III (STTX-III) showed in vitro genotoxic and mutagenic properties. Of all identified Alternaria toxins, the epoxide-bearing analogs ATX-II, ATX-III, and STTX-III show the highest cytotoxic, genotoxic, and mutagenic potential in vitro. Under hormone-sensitive conditions, AOH and AME act as moderate xenoestrogens, but in silico modeling predicts further Alternaria toxins as potential estrogenic factors. Recent studies indicate also an immunosuppressive role of AOH and ATX-II; however, no data are available for the majority of Alternaria toxins. Overall, hazard characterization of Alternaria toxins focused, so far, primarily on the commercially available dibenzo-α-pyrones AOH and AME and tenuazonic acid (TeA). Limited data sets are available for altersetin (ALS), altenuene (ALT), and tentoxin (TEN). The occurrence and toxicological relevance of perylene quinone-based Alternaria toxins still remain to be fully elucidated. We identified data gaps on hazard identification and characterization crucial to improve risk assessment of Alternaria mycotoxins for consumers and occupationally exposed workers.

New methodological developments for testing the in vitro genotoxicity of nanomaterials: Comparison of 2D and 3D HepaRG liver cell models and classical and high throughput comet assay formats.

Nanomaterials (NMs) are defined as materials with at least one external dimension below 100 nm. Their small size confers them interesting unique physico-chemical properties, hence NMs are increasingly used in a diversity of applications. However, the specific properties of NMs could also make them more harmful than their bulk counterparts. Therefore, there is a crucial need to deliver efficient NM hazard assessment in order to sustain the responsible development of nanotechnology. This study analysed the genotoxic potential of several NMs: one titanium dioxide (TiO2) and two zinc oxide NMs (ZnO) that were tested up to 100 µg/mL on 2D and 3D hepatic HepaRG models. Genotoxicity analysis was performed comparing the alkaline comet assay in classical and high throughput formats. Moreover, oxidative DNA lesions were investigated with the Fpg-modified comet assay. Results showed that TiO2 NMs were not cytotoxic and not genotoxic in either cell model, although a small increase in the % tail DNA was observed in 3D HepaRG cells at 100 µg/mL in the classical format. The two ZnO NMs (ZnO S. NMs a commercial suspension and NM110 provided by the European Union Joint Research Centre) induced a concentration-dependent increase in cytotoxicity that was more pronounced in the 2D (>20% cytotoxicity was observed for ZnO S. at concentrations greater than 25 µg/mL, and for NM 110 at 50 µg/mL) than in the 3D model (more than 20% cytotoxicity for ZnO S. NMs at 50 µg/mL). While ZnO S. NMs induced DNA damage associated with cytotoxicity (at 25 and 50 µg/mL in 2D and 50 µg/mL in 3D), NM110 showed a clear genotoxic effect at non-cytotoxic concentrations (25 µg/mL in 2D and at 25 and 50 µg/mL in 3D). No major differences could be observed in the comet assay in the presence or absence of the Fpg enzyme. High throughput analysis using CometChip® mostly confirmed the results obtained with the classical format, and even enhanced the detection of genotoxicity in the 3D model. In conclusion, this study demonstrated that new approach methodologies (NAMs), 3D models and the high throughput format for the comet assay, were more efficient in the detection of genotoxic effects, and are therefore promising approaches to improve hazard assessment of NMs.

An Evaluation of the Cytotoxic and Genotoxic Effects of the Marine Toxin C17-SAMT in Human TK6 and HepaRG Cell Lines.

This study investigates the genotoxicity and cytotoxicity of C17-sphinganine analog mycotoxin (C17-SAMT) using in vitro assays. C17-SAMT was previously identified as the cause of unusual toxicity in cultured mussels from the Bizerte Lagoon in northern Tunisia. While a previous in vivo genotoxicity study was inconclusive, in vitro results demonstrated that C17-SAMT induced an increase in micronucleus formation in human lymphoblastoid TK6 cells at concentrations of 0.87 µM and 1.74 µM. In addition, multiparametric cytotoxicity assays were performed in the human hepatoma HepaRG cell line, which showed that C17-SAMT induced mitochondrial dysfunction, decreased cellular ATP levels, and altered the expression of various proteins, including superoxide dismutase SOD2, heme oxygenase HO-1, and NF-κB. These results suggest that C17-SAMT is mutagenic in vitro and can induce mitochondrial dysfunction in HepaRG cells. However, the exact mode of action of this toxin requires further investigation. Overall, this study highlights the potential toxicity of C17-SAMT and the need for further research to better understand its effects.

Current status and future challenges of genotoxicity OECD Test Guidelines for nanomaterials: a workshop report.

Genotoxicity testing for nanomaterials remains challenging as standard testing approaches require some adaptation, and further development of nano-specific OECD Test Guidelines (TGs) and Guidance Documents (GDs) are needed. However, the field of genotoxicology continues to progress and new approach methodologies (NAMs) are being developed that could provide relevant information on the range of mechanisms of genotoxic action that may be imparted by nanomaterials. There is a recognition of the need for implementation of new and/or adapted OECD TGs, new OECD GDs, and utilization of NAMs within a genotoxicity testing framework for nanomaterials. As such, the requirements to apply new experimental approaches and data for genotoxicity assessment of nanomaterials in a regulatory context is neither clear, nor used in practice. Thus, an international workshop with representatives from regulatory agencies, industry, government, and academic scientists was convened to discuss these issues. The expert discussion highlighted the current deficiencies that exist in standard testing approaches within exposure regimes, insufficient physicochemical characterization, lack of demonstration of cell or tissue uptake and internalization, and limitations in the coverage of genotoxic modes of action. Regarding the latter aspect, a consensus was reached on the importance of using NAMs to support the genotoxicity assessment of nanomaterials. Also highlighted was the need for close engagement between scientists and regulators to (i) provide clarity on the regulatory needs, (ii) improve the acceptance and use of NAM-generated data, and (iii) define how NAMs may be used as part of weight of evidence approaches for use in regulatory risk assessments.

Prioritization of mycotoxins based on mutagenicity and carcinogenicity evaluation using combined in silico QSAR methods.

Mycotoxins and their metabolites are a family of compounds that contains a great diversity of both structure and biological properties. Information on their toxicity is spread within several databases and in scientific literature. Due to the number of molecules and their structure diversity, the cost and time required for hazard evaluation of each compound is unrealistic. In that purpose, new approach methodologies (NAMs) can be applied to evaluate such large set of molecules. Among them, quantitative structure-activity relationship (QSAR) in silico models could be useful to predict the mutagenic and carcinogenic properties of mycotoxins. First, a complete list of 904 mycotoxins and metabolites was built. Then, some known mycotoxins were used to determine the best QSAR tools for mutagenicity and carcinogenicity predictions. The best tool was further applied to the whole list of 904 mycotoxins. At the end, 95 mycotoxins were identified as both mutagen and carcinogen and should be prioritized for further evaluation.

Fate and PPARγ and STATs-driven effects of the mitochondrial complex I inhibitor tebufenpyrad in liver cells revealed with multi-omics.

The biological effects of the pesticide and mitochondrial complex I inhibitor tebufenpyrad (TEBU) on liver cells were investigated by combining proteomics and metabolomics. Both cell culture media and cellular lysates were analyzed in dose-response and kinetic experiments on the HepaRG cell line. Responses were compared with those obtained on primary human and rat hepatocytes. A multitude of phase I and II metabolites (>80) mainly common to HepaRG cells and primary hepatocytes and an increase in metabolization enzymes were observed. Synthesis of mitochondrion and oxidative phosphorylation complex constituents, fatty acid oxidation, and cellular uptake of lipids were induced to compensate for complex I inhibition and the decrease in ATP intracellular contents caused by TEBU. Secretion of the 20 S circulating proteasome and overall inhibition of acute inflammation followed by IL-6 secretion in later stages were observed in HepaRG cells. These effects were associated with a decrease in STAT1 and STAT3 transcription factor abundances, but with different kinetics. Based on identified TEBU targets, docking experiments, and nuclear receptor reporter assays, we concluded that liver cell response to TEBU is mediated by its interaction with the PPARγ transcription factor.

Investigation of the Genotoxic Potential of the Marine Toxin C17-SAMT Using the In Vivo Comet and Micronucleus Assays.

The contaminant responsible for the atypical toxicity reported in mussels from Bizerte Lagoon (Northern Tunisia) during the last decade has been characterized as C17-sphinganine analog mycotoxin (C17-SAMT). This neurotoxin showed common mouse toxic symptoms, including flaccid paralysis and severe dyspnea, followed by rapid death. For hazard assessment on human health, in this work we aimed to evaluate the in vivo genotoxic effects of this marine biotoxin using the classical alkaline and modified Fpg comet assays performed to detect DNA breaks and alkali-labile sites as well as oxidized bases. The micronucleus assay was used on bone marrow to detect chromosome and genome damage. C17-SAMT induces a statistically insignificant increase in DNA tail intensity at all doses in the duodenum, and in the spleen contrary to the liver, the percentage of tail DNA increased significantly at the mid dose of 300 µg/kg b.w/d. C17-SAMT did not affect the number of micronuclei in the bone marrow. Microscopic observations of the liver showed an increase in the number of mitosis and hepatocytes' cytoplasm clarification. At this level of study, we confirm that C17-SAMT induced DNA damage in the liver but there was no evidence of effects causing DNA oxidation or chromosome and genome damage.

Analytical and toxicological aspects of nanomaterials in different product groups: Challenges and opportunities.

The widespread integration of engineered nanomaterials into consumer and industrial products creates new challenges and requires innovative approaches in terms of design, testing, reliability, and safety of nanotechnology. The aim of this review article is to give an overview of different product groups in which nanomaterials are present and outline their safety aspects for consumers. Here, release of nanomaterials and related analytical challenges and solutions as well as toxicological considerations, such as dose-metrics, are discussed. Additionally, the utilization of engineered nanomaterials as pharmaceuticals or nutraceuticals to deliver and release cargo molecules is covered. Furthermore, critical pathways for human exposure to nanomaterials, namely inhalation and ingestion, are discussed in the context of risk assessment. Analysis of NMs in food, innovative medicine or food contact materials is discussed. Specific focus is on the presence and release of nanomaterials, including whether nanomaterials can migrate from polymer nanocomposites used in food contact materials. With regard to the toxicology and toxicokinetics of nanomaterials, aspects of dose metrics of inhalation toxicity as well as ingestion toxicology and comparison between in vitro and in vivo conclusions are considered. The definition of dose descriptors to be applied in toxicological testing is emphasized. In relation to potential exposure from different products, opportunities arising from the use of advanced analytical techniques in more unique scenarios such as release of nanomaterials from medical devices such as orthopedic implants are addressed. Alongside higher product performance and complexity, further challenges regarding material characterization and safety, as well as acceptance by the general public are expected.

Toxicity of palytoxin, purified ovatoxin-a, ovatoxin-d and extracts of Ostreopsis cf. ovata on the Caco-2 intestinal barrier model.

Human intoxications in the Mediterranean Sea have been linked to blooms of the dinoflagellate Ostreopsis cf. ovata, producer of palytoxin (PlTX)-like toxins called ovatoxins (OVTXs). Exposure routes include only inhalation and contact, although PlTX-poisoning by seafood has been described in tropical regions. To address the impact of OVTXs on the intestinal barrier, dinoflagellate extracts, purified OVTX-a and -d and PlTX were tested on differentiated Caco-2 cells. Viability, inflammatory response and barrier integrity were recorded after 24 h treatment. OVTX-a and -d were not cytotoxic up to 20 ng/mL but increased IL-8 release, although to a lesser extent compared to PlTX. While PlTX and OVTX-a (at 0.5 and 5 ng/mL respectively) affected intestinal barrier integrity, OVTX-d up to 5 ng/mL did not. Overall, OVTX-d was shown to be less toxic than OVTX-a and PlTX. Therefore, oral exposure to OVTX-a and -d could provoked lower acute toxicity than PlTX.

Chronic effects of two rutile TiO2 nanomaterials in human intestinal and hepatic cell lines.

BACKGROUND: TiO2 nanomaterials (NMs) are present in a variety of food and personal hygiene products, and consumers are exposed daily to these NMs through oral exposition. While the bulk of ingested TiO2 NMs are eliminated rapidly in stool, a fraction is able to cross the intestinal epithelial barrier and enter systemic circulation from where NMs can be distributed to tissues, primarily liver and spleen. Daily exposure to TiO2 NMs, in combination with a slow rate of elimination from tissues, results in their accumulation within different tissues. Considerable evidence suggests that following oral exposure to TiO2 NMs, the presence of NMs in tissues is associated with a number of adverse effects, both in intestine and liver. Although numerous studies have been performed in vitro investigating the acute effects of TiO2 NMs in intestinal and hepatic cell models, considerably less is known about the effect of repeated exposure on these models. In this study, we investigated the cytotoxic effects of repeated exposure of relevant models of intestine and liver to two TiO2 NMs differing in hydrophobicity for 24 h, 1 week and 2 weeks at concentrations ranging from 0.3 to 80 µg/cm2. To study the persistence of these two NMs in cells, we included a 1-week recovery period following 24 h and 1-week treatments. Cellular uptake by TEM and ToF-SIMS analyses, as well as the viability and pro-inflammatory response were evaluated. Changes in the membrane composition in Caco-2 and HepaRG cells treated with TiO2 NMs for up to 2 weeks were also studied. RESULTS: Despite the uptake of NM-103 and NM-104 in cells, no significant cytotoxic effects were observed in either Caco-2 or HepaRG cells treated for up to 2 weeks at NM concentrations up to 80 µg/cm2. In addition, no significant effects on IL-8 secretion were observed. However, significant changes in membrane composition were observed in both cell lines. Interestingly, while most of these phospholipid modifications were reversed following a 1-week recovery, others were not affected by the recovery period. CONCLUSION: These findings indicate that although no clear effects on cytotoxicity were observed following repeated exposure of differentiated Caco-2 and HepaRG cells to TiO2 NMs, subtle effects on membrane composition could induce potential adverse effects in the long-term.

Pyrogenic synthetic amorphous silica (NM-203): Genotoxicity in rats following sub-chronic oral exposure.

The genotoxicity of nano-structured synthetic amorphous silica (SAS), a common food additive, was investigated in vivo in rats. A 90-day oral toxicity study was performed according to OECD test guideline 408 and the genotoxicity of pyrogenic SAS nanomaterial NM-203 was assessed in several organs, using complementary tests. Adult Sprague-Dawley rats of both sexes were treated orally for 90 days with 0, 2, 5, 10, 20, or 50 mg SAS/kg bw per day. Dose levels were selected to approximate expected human dietary exposures to SAS. DNA strand breaks were evaluated by the comet assay in blood, bone marrow, liver, and spleen according to OECD test guideline 489; mutations induced in bone marrow precursors of erythrocytes were assessed by the Pig-a assay and chromosome/ genome damage by the micronucleus assay in blood (OECD test guideline 474) and colon. No treatment-related increases of gene (Pig-a) or chromosome/genome (micronucleus) mutations were detected in the blood. The percentage of micronucleated cells was not increased in the colon of treated rats. Among the organs analyzed by the comet assay, the spleen was the only target showing a weak but biologically relevant genotoxic effect.

Genotoxic impact of aluminum-containing nanomaterials in human intestinal and hepatic cells.

Exposure of consumers to aluminum-containing nanomaterials (Al NMs) is an area of concern for public health agencies. As the available data on the genotoxicity of Al2O3 and Al0 NMs are inconclusive or rare, the present study investigated their in vitro genotoxic potential in intestinal and liver cell models, and compared with the ionic form AlCl3. Intestinal Caco-2 and hepatic HepaRG cells were exposed to Al0 and Al2O3 NMs (0.03 to 80 µg/cm2). Cytotoxicity, oxidative stress and apoptosis were measured using High Content Analysis. Genotoxicity was investigated through γH2AX labelling, the alkaline comet and micronucleus assays. Moreover, oxidative DNA damage and carcinogenic properties were assessed using the Fpg-modified comet assay and the cell transforming assay in Bhas 42 cells respectively. The three forms of Al did not induce chromosomal damage. However, although no production of oxidative stress was detected, Al2O3 NMs induced oxidative DNA damage in Caco-2 cells but not likely related to ion release in the cell media. Considerable DNA damage was observed with Al0 NMs in both cell lines in the comet assay, likely due to interference with these NMs. No genotoxic effects were observed with AlCl3. None of the Al compounds induced cytotoxicity, apoptosis, γH2AX or cell transformation.

Role of enteric glial cells in the toxicity of phycotoxins: Investigation with a tri-culture intestinal cell model.

Lipophilic phycotoxins are secondary metabolites produced by phytoplankton. They can accumulate in edible filtering-shellfish and cause human intoxications, particularly gastrointestinal symptoms. Up to now, the in vitro intestinal effects of these toxins have been mainly investigated on simple monolayers of intestinal cells such as the enterocyte-like Caco-2 cell line. Recently, the combination of Caco-2 cells with mucus secreting HT29-MTX cell line has been also used to mimic the complexity of the human intestinal epithelium. Besides, enteric glial cells (EGC) from the enteric nervous system identified in the gut mucosa have been largely shown to be involved in gut functions. Therefore, using a novel model integrating Caco-2 and HT29-MTX cells co-cultured on inserts with EGC seeded in the basolateral compartment, we examined the toxicological effects of two phycotoxins, pectenotoxin-2 (PTX2) and okadaic acid (OA). Cell viability, morphology, barrier integrity, inflammation, barrier crossing, and the response of some specific glial markers were evaluated using a broad set of methodologies. The toxicity of PTX2 was depicted by a slight decrease of viability and integrity as well as a slight increase of inflammation of the Caco-2/HT29-MTX co-cultures. PTX2 induced some modifications of EGC morphology. OA induced IL-8 release and decreased viability and integrity of Caco-2/HT29-MTX cell monolayers. EGC viability was slightly affected by OA. The presence of EGC reinforced barrier integrity and reduced the inflammatory response of the epithelial barrier following OA exposure. The release of GDNF and BDNF gliomediators by EGC could be implicated in the protection observed.

A strategy towards the generation of testable adverse outcome pathways for nanomaterials.

Manufactured nanomaterials (NMs) are increasingly used in a wide range of industrial applications leading to a constant increase in the market size of nano-enabled products. The increased production and use of NMs are raising concerns among different stakeholder groups with regard to their effects on human and environmental health. Currently, nanosafety hazard assessment is still widely performed using in vivo (animal) models, however the development of robust and reg­ulatory relevant strategies is required to prioritize and/or reduce animal testing. An adverse outcome pathway (AOP) is a structured representation of biological events that start from a molecular initiating event (MIE) leading to an adverse outcome (AO) through a series of key events (KEs). The AOP framework offers great advancement to risk assessment and regulatory safety assessments. While AOPs for chemicals have been more frequently reported, the AOP collection for NMs is limited. By using existing AOPs, we aimed to generate simple and testable strategies to predict if a given NM has the potential to induce a MIE leading to an AO through a series of KEs. Firstly, we identified potential MIEs or initial KEs reported for NMs in the literature. Then, we searched the identified MIE or initial KEs as keywords in the AOP-Wiki to find associated AOPs. Finally, using two case studies, we demonstrate how in vitro strategies can be used to test the identified MIE/KEs.

Permeability of the Cyanotoxin Microcystin-RR across a Caco-2 Cells Monolayer.

Microcystins (MCs) are toxins produced by several cyanobacterial species found worldwide. While MCs have a common structure, the variation of two amino acids in their structure affects their toxicity. As toxicodynamics are very similar between the MC variants, their differential toxicity could rather be explained by toxicokinetic parameters. Microcystin-RR (MC-RR) is the second most abundant congener and induces toxicity through oral exposure. As intestinal permeability is a key parameter of oral toxicokinetics, the apparent permeability of MC-RR across a differentiated intestinal Caco-2 cell monolayer was investigated. We observed a rapid and large decrease of MC-RR levels in the donor compartment. However, irrespective of the loaded concentration and exposure time, the permeabilities were very low from apical to basolateral compartments (from 4 to 15 × 10-8 cm·s-1) and from basolateral to apical compartments (from 2 to 37 × 10-8 cm·s-1). Our results suggested that MC-RR would be poorly absorbed orally. As similar low permeability was reported for the most abundant congener microcystin-LR, and this variant presented a greater acute oral toxicity than MC-RR, we concluded that the intestinal permeability was probably not involved in the differential toxicity between them, in contrast to the hepatic uptake and metabolism.

In vitro investigation of the genotoxicity of portimine, a cyclic imine toxin produced by the dinoflagellate Vulcanodinium rugosum, on human hepatic HepaRG cells.

Portimine, a recently identified cyclic imine produced by the dinoflagellate Vulcanodinium rugosum, has been described as a potent apoptotic agent in contrast to most of the cyclic imines that are well-known to be neurological toxins. As apoptosis can be a consequence of a high level of DNA lesions, we investigated the responses of portimine on several endpoints aimed at detecting DNA damage in the hepatic cell line HepaRG. Portimine induced phosphorylation of H2AX, which could possibly be consistent with the previously published induction of apoptosis with this toxin. In addition, detection of apoptosis through the activation of caspase-3, the induction of strand breaks detected by the comet assay as well as chromosome and genome mutations using the micronucleus assay were addressed. Surprisingly, portimine treatment resulted in increases in only γH2AX in differentiated HepaRG cells whereas no effects on the other endpoints were detected. These increases in γH2AX in the absence of genotoxic effects in the other tests could indicate that portimine could possibly induce a DNA replication stress and/or that the compound can be detoxified by the HepaRG cells.