Regulatory assessment and risk management of chemical mixtures: challenges and ways forward.

This paper summarizes current challenges, the potential use of novel scientific methodologies, and ways forward in the risk assessment and risk management of mixtures. Generally, methodologies to address mixtures have been agreed; however, there are still several data and methodological gaps to be addressed. New approach methodologies can support the filling of knowledge gaps on the toxicity and mode(s) of action of individual chemicals. (Bio)Monitoring, modeling, and better data sharing will support the derivation of more realistic co-exposure scenarios. As knowledge and data gaps often hamper an in-depth assessment of specific chemical mixtures, the option of taking account of possible mixture effects in single substance risk assessments is briefly discussed. To allow risk managers to take informed decisions, transparent documentation of assumptions and related uncertainties is recommended indicating the potential impact on the assessment. Considering the large number of possible combinations of chemicals in mixtures, prioritization is needed, so that actions first address mixtures of highest concern and chemicals that drive the mixture risk. As chemicals with different applications and regulated separately might lead to similar toxicological effects, it is important to consider chemical mixtures across legislative sectors.

Benchmarking organic micropollutants in wastewater, recycled water and drinking water with in vitro bioassays.

Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.

Development of a panel of high-throughput reporter-gene assays to detect genotoxicity and oxidative stress.

The lack of toxicological information on many of the compounds that humans use or are exposed to, intentionally or unintentionally, poses a big problem in risk assessment. To fill this data gap, more emphasis is given to fast in vitro screening tools that can add toxicologically relevant information regarding the mode(s) of action via which compounds can elicit adverse effects, including genotoxic effects. By use of bioassays that can monitor the activation of specific cellular signalling pathways, many compounds can be screened in a high-throughput manner. We have developed two new specific reporter-gene assays that can monitor the effects of compounds on two pathways of interest: the p53 pathway (p53 CALUX) for genotoxicity and the Nrf2 pathway (Nrf2 CALUX) for oxidative stress. To exclude non-specific effects by compounds influencing the luciferase reporter-gene expression non-specifically, a third assay was developed to monitor changes in luciferase expression by compounds in general (Cytotox CALUX). To facilitate interpretation of the data and to avoid artefacts, all three reporter-gene assays used simple and defined reporter genes and a similar cellular basis, the human U2OS cell line. The three cell lines were validated with a range of reference compounds including genotoxic and non-genotoxic agents. The sensitivity (95%) and specificity (85%) of the p53 CALUX was high, showing that the assay is able to identify various types of genotoxic compound, while avoiding the detection of false positives. The Nrf2 CALUX showed specific responses to oxidants only, enabling the identification of compounds that elicit part of their genotoxicity via oxidative stress. All reporter-gene assays can be used in a high-throughput screening format and can be supplemented with other U2OS-based reporter-gene assays that can profile nuclear receptor activity, and several other signalling pathways.

Identification strategy for unknown pollutants using high-resolution mass spectrometry: androgen-disrupting compounds identified through effect-directed analysis.

Effect-directed analysis has been applied to a river sediment sample of concern to identify the compounds responsible for the observed effects in an in vitro (anti-)androgenicity assay. For identification after non-target analysis performed on a high-resolution LTQ-Orbitrap, we developed a de novo identification strategy including physico-chemical parameters derived from the effect-directed analysis approach. With this identification strategy, we were able to handle the immense amount of data produced by non-target accurate mass analysis. The effect-directed analysis approach, together with the identification strategy, led to the successful identification of eight androgen-disrupting compounds belonging to very diverse compound classes: an oxygenated polyaromatic hydrocarbon, organophosphates, musks, and steroids. This is one of the first studies in the field of environmental analysis dealing with the difficult task of handling the large amount of data produced from non-target analysis. The combination of bioassay activity assessment, accurate mass measurement, and the identification and confirmation strategy is a promising approach for future identification of environmental key toxicants that are not included as priority pollutants in monitoring programs.

Evaluation of an alternative in vitro test battery for detecting reproductive toxicants in a grouping context.

Previously we showed a battery consisting of CALUX transcriptional activation assays, the ReProGlo assay, and the embryonic stem cell test, and zebrafish embryotoxicity assay as 'apical' tests to correctly predict developmental toxicity for 11 out of 12 compounds, and to explain the one false negative [7]. Here we report on applying this battery within the context of grouping and read across, put forward as a potential tool to fill data gaps and avoid animal testing, to distinguish in vivo non- or weak developmental toxicants from potent developmental toxicants within groups of structural analogs. The battery correctly distinguished 2-methylhexanoic acid, monomethyl phthalate, and monobutyltin trichloride as non- or weak developmental toxicants from structurally related developmental toxicants valproic acid, mono-ethylhexyl phthalate, and tributyltin chloride, respectively, and, therefore, holds promise as a biological verification model in grouping and read across approaches. The relevance of toxicokinetic information is indicated.

A high throughput screening system for predicting chemically-induced reproductive organ deformities.

There is a great need for alternative testing methods for reproductive toxicants that are practical, fast, cost-effective and easy to interpret. Previously we followed a pragmatic approach using readily available tests, which was successful in predicting reproductive toxicity of chemicals [13]. This initial battery still contained apical tests and is fairly complex and low in its throughput. The current study aimed to simplify this screening battery using a mechanistic approach and a panel of high throughput CALUX reporter gene assays. A mechanistic approach was taken to validate this high throughput test battery. To this end it was challenged with two preselected sets of chemicals addressing two major apical effect classes relevant in reproductive toxicity. We found selectivity in this battery in that 82% of the compounds inducing reproductive organ deformities were predicted correctly, while for compounds inducing neural tube defects this was the case in 47% only. This is consistent with the mechanisms of toxicity covered in the battery. The most informative assays in the battery were ERalpha CALUX to measure estrogenicity and the AR-anti CALUX assay to measure androgen receptor antagonism.

Estrogenic and esterase-inhibiting potency in rainwater in relation to pesticide concentrations, sampling season and location.

In a year-round monitoring program (1998), pesticide composition and toxic potency of the mix of pollutants present in rainwater were measured. The goal of the study was to relate atmospheric deposition of toxic potency and pesticide composition to each other and to sampling period and local agricultural activity. Rainwater was collected in 26 consecutive periods of 14 days in a background location (BACK) and in two locations representative for different agricultural practices, i.e. intensive greenhouse horticulture (HORT) and flower bulb culture (BULB). Samples were chemically analyzed for carbamate (CARB), organophosphate (OP) and organochlorine (OC) pesticides and metabolites. Esterase inhibiting potency of rainwater extracts was measured in a specially developed bio-assay with honeybee esterases and was expressed as an equivalent concentration of the model inhibitor dichlorvos. Estrogenic potency of the extracts was measured in the ER-CALUX reporter gene assay and was expressed as an equivalent concentration of estradiol. Multivariate principal component analysis (PCA) techniques proved to be valuable tools to analyze the numerous pesticide concentrations in relation to toxic potency, sampling location, and sampling season. Pesticide composition in rainwater depended much more on sampling season than on sampling location, but differences between and were mainly attributed to local differences in agricultural practice. On average, the esterase inhibiting potency exceeded the maximum permissible concentration set for dichlorvos in The Netherlands, and was significantly higher in than in and . Esterase inhibition correlated significantly with OP and CARB concentrations, as expected given the working mechanism of these insecticides. The estrogenic potency incidentally exceeded NOEC levels reported for aquatic organisms and was highest in . Although estrogenic potency of rainwater correlated with OC concentrations, the ER-CALUX responses could not be attributed to any particular pesticides. Besides, the contribution of non-analyzed xeno-estrogens as alkylphenol(-ethoxylates) and bisphenol-A to the estrogenic potency of rainwater could not be excluded. Further research should focus on the chemical identification of estrogenic compounds in rainwater. In addition, more attention should be given to the ecological consequences of atmospheric deposition of individual pesticides and of total toxic potencies that regularly exceed environmental criteria for Dutch surface waters and/or toxic threshold values for aquatic organisms.

Occurrence of glucocorticogenic activity in various surface waters in The Netherlands.

Considering the important role that surface waters serve for drinking water production, it is important to know if these resources are under the impact of contaminants. Apart from environmental pollutants such as pesticides, compounds such as (xeno)estrogens have received al lot of research attention and several large monitoring campaigns have been carried out to assess estrogenic contamination in the aquatic environment. The introduction of novel in vitro bioassays enables researchers to study if - and to what extent - water bodies are under the impact of less-studied (synthetic) hormone active compounds. The aim of the present study was to carry out an assessment on the presence and extent of glucocorticogenic activity in Dutch surface waters that serve as sources for drinking water production. The results show glucocorticogenic activity in the range of

Trigger values for investigation of hormonal activity in drinking water and its sources using CALUX bioassays.

To screen for hormonal activity in water samples, highly sensitive in vitro CALUX bioassays are available which allow detection of estrogenic (ERα), androgenic (AR), progestagenic (PR), and glucocorticoid (GR) activities. This paper presents trigger values for the ERα, AR, PR, and GR CALUX bioassays for agonistic hormonal activities in (drinking) water, which define a level above which human health risk cannot be waived a priori and additional examination of specific endocrine activity may be warranted. The trigger values are based on 1) acceptable or tolerable daily intake (ADI/TDI) values of specific compounds, 2) pharmacokinetic factors defining their bioavailability, 3) estimations of the bioavailability of unknown compounds with equivalent hormonal activity, 4) relative endocrine potencies, and 5) physiological, and drinking water allocation factors. As a result, trigger values of 3.8ng 17ß-estradiol (E2)-equivalents (eq)/L, 11ng dihydrotestosterone (DHT)-eq/L, 21ng dexamethasone (DEX)-eq/L, and 333ng Org2058-eq/L were derived. Benchmark Quotient (BQ) values were derived by dividing hormonal activity in water samples by the derived trigger using the highest concentrations detected in a recent, limited screening of Dutch water samples, and were in the order of (value) AR (0.41)>ERα (0.13)>GR (0.06)>PR (0.04). The application of trigger values derived in the present study can help to judge measured agonistic hormonal activities in water samples using the CALUX bioassays and help to decide whether further examination of specific endocrine activity followed by a subsequent safety evaluation may be warranted, or whether concentrations of such activity are of low priority with respect to health concerns in the human population. For instance, at one specific drinking water production site ERα and AR (but no GR and PR) activities were detected in drinking water, however, these levels are at least a factor 83 smaller than the respective trigger values, and therefore no human health risks are to be expected from hormonal activity in Dutch drinking water from this site.

Sample preparation for combined chemical analysis and in vitro bioassay application in water quality assessment.

The combination of in vitro bioassays and chemical screening can provide a powerful toolbox to determine biologically relevant compounds in water extracts. In this study, a sample preparation method is evaluated for the suitability for both chemical analysis and in vitro bioassays. A set of 39 chemicals were spiked to surface water, which were extracted using Oasis MCX cartridges. The extracts were chemically analyzed by liquid chromatography linear ion trap Orbitrap analysis and recoveries appeared to be on average 61% Compounds with logK(ow) values in the range between 0 and 4 are recovered well using this method. In a next step, the same extracts were tested for genotoxic activity using the Comet assay and Ames fluctuation test and for specific endocrine receptor activation using a panel of CALUX assays, for estrogenic (ER), androgenic (AR), glucocorticoid (GR), progestagenic (PR), and thyroidogenic (TR) agonistic activities. The results of the genotoxicity assays indicated that spiked genotoxic compounds were preserved during sample preparation. The measured responses of the GR CALUX and ER CALUX assays were similar to the predicted responses. The measured responses in the AR CALUX and PR CALUX assays were much lower than expected from the analytical concentration, probably due to antagonistic effects of some spiked compounds. Overall, the presented sample preparation method seems to be suitable for both chemical analysis and specific in vitro bioassay applications.

High-resolution mass spectrometric identification and quantification of glucocorticoid compounds in various wastewaters in the Netherlands.

In the past two decades much research effort has focused on the occurrence, effects, and risks of estrogenic compounds. However, increasing emissions of new emerging compounds may also affect the action of hormonal pathways other than the estrogenic hormonal axis. Recently, a suite of novel CALUX bioassays has become available that enables looking further than estrogenic effects only. By employing these bioassays, we recently showed high glucocorticogenic activity in wastewaters collected at various sites in The Netherlands. However, since bioassays provide an integrated biological response, the identity of the responsible biological compounds remained unknown. Therefore, our current objective was to elucidate the chemical composition of the wastewater extracts used in our previous study by means of LC-high-resolution Orbitrap MS/MS and to determine if the compounds quantified could account for the observed glucocorticoid responsive (GR) CALUX bioassay response. The mass spectrometric analysis revealed the presence of various glucocorticoids in the range of 13-1900 ng/L. In extracts of hospital wastewater-collected prior to sewage treatment-several glucocorticoids were identified (cortisol 275-301 ng/L, cortisone 381-472 ng/L, prednisone 117-545 ng/L, prednisolone 315-1918 ng/L, and triamcinolone acetonide 14-41 ng/L) which are used to treat a great number of human pathologies. A potency balance calculation based on the instrumental analyses and relative potencies (REPs) of the individual glucocorticoids supports the conclusion that triamcinolone acetonide (REP = 1.3), dexamethasone (REP = 1), and prednisolone (REP = 0.2) are the main contributors to the glucocorticogenic activity in the investigated wastewater extracts. The action of these compounds is concentration additive and the overall glucocorticogenic activity can be explained to a fairly large extent by their contribution.

Detection of multiple hormonal activities in wastewater effluents and surface water, using a panel of steroid receptor CALUX bioassays.

It is generally known that there are compounds present in the aquatic environment that can disturb endocrine processes, for example via interaction with the endogenous hormone receptors. Most research so far has focused on compounds that bind to the estrogen and/or androgen receptor, but ligands for other hormone receptors might also be present. In this study, a newly completed panel of human cell derived CALUX reporter gene bioassays was utilized to test water extracts for estrogen (ER), as well as androgen (AR), progesterone (PR), and glucocorticoid (GR) receptor mediated transactivation activity. Effluents from industry, hospital, and municipal sewage treatment plants, as well as tap water and different sources of surface water were tested. The CALUX reporter gene panel showed high sensitivity and specificity to known agonists, enabling discrimination between different receptor based endocrine responses present in the aquatic environment. Our results clearly showed the presence of agonistic activity on the ER, as well as on the AR, PR, and GR in the raw and wastewater and surface water extracts. However, no hormone receptor-mediated transactivation was detected in the drinking water or in the blank water. The levels of estrogenic activity were 0.2-0.5 ng E2-equiv/L for surface water and 0.4-1.0 ng E2-equiv/L for municipal effluents, which was consistent with previous studies. Surprisingly, the other hormonal activities were found to be present in similar or much higher levels. Most notably, glucocorticoid-like activity was detected in all samples, at surprisingly high levels ranging from 0.39-1.3 ng Dex-equiv/L in surface water and 11-243 ng Dex-equiv/L in effluents. When regarding the fact that dexamethasone in the GR CALUX bioassay is a factor 12 more potent than the natural hormone cortisol, results expressed as cortisol equivalents would range up to 2900 ng cortisol equiv/L. Further studies are needed to establish the identity of the active compounds and to understand the significance of the level of activities with regard to human and ecotoxicological risks.