Risk Assessment Of Mixtures Research Articles

An unexpected synergistic toxicity caused by competitive bioconcentration of perfluoroalkyl acid mixtures to Daphnia magna: Further promoted by elevated temperature

The mixed pollution of the global water environment by perfluoroalkyl acids (PFAAs) and their ecological risks have aroused widespread concern. However, the relationship between the combined toxicity of PFAA mixtures and their accumulation in aquatic organisms is not well understood in the context of global warming. Here, we study the bioconcentration and combined toxicity of three PFAA mixtures (PFOA, PFDA, PFDoA) to Daphnia magna (D. magna) under different exposure concentrations and temperatures. The results show that although competitive bioconcentration exists, the combined toxicity of the PFAA mixtures to D. magna is synergistic. These contradictory phenomena occur because although the longer-chain PFDoA inhibits the bioconcentration of the shorter-chain PFOA and PFDA, the bioconcentration of PFDoA itself is promoted, and PFDoA is more toxic to D. magna than PFOA and PFDA. The toxic equivalent concentration for the PFAA mixture is 1.38–1.67 times higher than that obtained from simple addition for the three PFAAs when exposed separately. Moreover, elevated temperature promotes not only the bioconcentration of each PFAA and the competition of bioconcentration between shorter-chain and longer-chain PAFF, but also the synergistic toxicity of PFAA mixtures to D. magna. This study suggests that the effect of the interactions among different PFAAs on their bioconcentration and toxicity under different water environmental conditions, such as temperature, should be considered for ecological risk assessment of PFAA mixtures.

Roadmap for action on Risk Assessment of Combined Exposure to Multiple Chemicals (RACEMiC)

EFSA's aim by 2030, is that the Agency and its partners will be equipped for the routine implementation of human health risk assessment to multiple chemicals, across EFSA's domains of activity. To facilitate this effort, a roadmap for action has been developed by mapping the methods, data and tools that are currently available for mixture risk assessment and identifying current scientific gaps including challenges and blockers. The results shows that extensive methods, data and tools are available for dietary mixture risk assessment for pesticides, but that several scientific gaps still exist for the non-dietary mixture exposure to pesticides. For food additives and for certain contaminants, the regulatory readiness for mixture risk assessment was also found to be fairly high compared to food contact materials and cross-silo mixture risk assessment. The scientific gaps identified were prioritised according to their impact on the implementation of mixture risk assessment and, as a result, ten multi-annual project proposals were defined to address these scientific gaps on the short-term, mid-term and long-term. The roadmap also proposes and prioritises a number of working areas in the regulatory domains of pesticides, food contact materials, contaminants, food additives, as well as in the overarching domain of chemicals. Besides the scientific proposals, recommendations to improve stakeholder engagement and communication on mixture risk assessment was investigated. These included, among others, creating an online catalogue of tools, methods and data for mixture risk assessment, as well as the organisation of regular webinars/workshop to promote exchange of information between stakeholders and making more efficient use of national communication hubs for food safety in communicating with the general public.

A geospatial modeling approach to quantifying the risk of exposure to environmental chemical mixtures via a common molecular target

In the real world, individuals are exposed to chemicals from sources that vary over space and time. However, traditional risk assessments based on in vivo animal studies typically use a chemical-by-chemical approach and apical disease endpoints. New approach methodologies (NAMs) in toxicology, such as in vitro high-throughput (HTS) assays generated in Tox21 and ToxCast, can more readily provide mechanistic chemical hazard information for chemicals with no existing data than in vivo methods. In this paper, we establish a workflow to assess the joint action of 41 modeled ambient chemical exposures in the air from the USA-wide National Air Toxics Assessment by integrating human exposures with hazard data from curated HTS (cHTS) assays to identify counties where exposure to the local chemical mixture may perturb a common biological target. We exemplify this proof-of-concept using CYP1A1 mRNA up-regulation. We first estimate internal exposure and then convert the inhaled concentration to a steady state plasma concentration using physiologically based toxicokinetic modeling parameterized with county-specific information on ages and body weights. We then use the estimated blood plasma concentration and the concentration-response curve from the in vitro cHTS assay to determine the chemical-specific effects of the mixture components. Three mixture modeling methods were used to estimate the joint effect from exposure to the chemical mixture on the activity levels, which were geospatially mapped. Finally, a Monte Carlo uncertainty analysis was performed to quantify the influence of each parameter on the combined effects. This workflow demonstrates how NAMs can be used to predict early-stage biological perturbations that can lead to adverse health outcomes that result from exposure to chemical mixtures. As a result, this work will advance mixture risk assessment and other early events in the effects of chemicals.

Risk assessment of heavy metal and pesticide mixtures in aquatic biota using the DGT technique in sediments

Heavy metals and pesticides (HMPs) are common contaminants due to their extensive use worldwide. Diffusive gradients in thin films (DGT) are a good method for measuring the bioavailable concentration of pollutants. This study represents the first evaluation of HMP toxicity in aquatic biota using the DGT technique in sediments. Zhelin Bay was selected as the case study site because it has been contaminated by pollutants. Nonmetric multidimensional scaling (NMS) analysis reveals that a diverse range of pollutants (V, Cr, Ni, Cu, Zn, As, Se, InHg, Mo, Cd, Sb, W, Pb, CLP, PYR) are mainly influenced by sediment characteristics. Assessment of single HMP toxicity found that the risk quotient (RQ) values for Mn, Cu, inorganic Hg (InHg), chlorpyrifos (CLP) and diuron (DIU) are significantly higher than 1, indicating that the adverse effects of these single HMPs should not be ignored. The combined toxicity of HMP mixtures based on probabilistic ecotoxicological risk assessment shows that Zhelin Bay surface sediments had a medium probability (54.6%) of toxic effects to aquatic biota.

Integrated environmental risk assessment of rare earth elements mixture on aquatic ecosystems

Rare Earth elements (REE) have become essential in strategical sectors such as high- and green-technologies. Their increasing use in human activities worldwide leads to anthropogenic REE releases detectable in all compartments of the environment, transforming REE into emerging contaminants. However, their potential impacts on ecosystems are still poorly understood. In order to have a comprehensive understanding of REE ecotoxicology and to properly assess their environmental risk, we analysed the toxicity of three representative REE (neodymium Nd, gadolinium Gd, and ytterbium Yb). Following recommendations of the European Chemicals Agency, we assessed REE hazard by performing standard ecotoxicological tests on three freshwater species belonging to different trophic levels (algae, crustacean and fish). EC50 were calculated using different modes of expression of REE exposure concentration (based on nominal, measured total and dissolved concentrations) in order to more properly and accurately determine REE toxicity. In order to get closer to environmental conditions, we also tested the toxicity of REE in mixture because all of them occur naturally as such in the environment. Moreover, we added dissolved organic matter (DOM) in the test medium because DOM is ubiquitous and drives REE speciation in freshwater systems. The Results showed that DOM significantly reduced REE bioaccumulation and toxicity, probably by formation of non-bioavailable REE-DOM complexes. The algal species was the most sensitive to REE. Despite slight differences between Nd, Gd and Yb in behaviour and bioaccumulation, the three REE exhibited comparable toxicity and additive effects in mixture to all tested organisms. Thus, we considered REE as a uniform group and, for the first time, we used mixture toxicity values and environmental mixture concentrations to assess the risk of REE in freshwater (instead of considering different REE separately). The results revealed that the risk is currently limited to wastewater treatment plants, and industrial and mining activities, where released quantities of REE can induce severe damage to exposed freshwater organisms. However, the risks are likely more widespread in the future because anthropogenic REE releases are expected to increase.

Effects of consumer products chemicals ingredients and their mixtures on the estrogen receptor/androgen receptor transcriptional activation.

Unlike the environmental pollutants or industrial chemicals, the chemicals in consumer products may pose higher levels of risks, depending on how the chemicals are used in the products and how humans interact with the products. Recently, endocrine disrupting chemicals in cosmetics, personal care products, cleaners, sunscreens, and vinyl products were analytically quantified and many active chemicals including phthalates, parabens and bisphenols were detected. This indicates a wide range of exposures from common products. In this study, 35 chemicals known to be ingredients of consumer products were selected and screened for the transactivation of estrogen receptors and androgen receptors. From the results of individual chemicals, the activity of binary/ternary mixture prepared from the agonists for the ER transcription activity was measured, and compared to the predicted values obtained by the full logistic model. The measured and the predicted values were found to be very similar. This study may suggest that prediction of mixture activity by proper models would be one of the supportive tools for the risk assessment and sound regulation of chemical mixtures which have potential endocrine disrupting effects in consumer products.

P15-08 PANORAMIX: Providing risk assessments of complex real-life mixtures for the protection of Europe’s citizens and the environment

P15-08 PANORAMIX: Providing risk assessments of complex real-life mixtures for the protection of Europe’s citizens and the environment

The need for environmental regulation of tires: Challenges and recommendations

The interest in tire wear particles (TWPs), generated from abrasion of tires, have gained traction over the past few years, both in regards to quantifying particulate emissions, leaching of different compounds, toxicity, and analytical methods. The life of a tire, from cradle to end-of-life, crosses over different scenarios during its lifetime and transcends environmental compartments and legislative areas, underlining the need for a collective approach. Sustainability for a tire encompasses the use of raw materials, recycling of raw materials, circular economy and material sourcing. The tire industry is currently making significant efforts towards a greener and more sustainable production considering reduction of CO2-emissions, recycling, material sources and implementing the use of biomass from plants rather than oil-derived alternatives. In this paper, we aim to analyze and discuss the need for environmental regulation of tires in order to provide a series of targeted recommendations for future legislation. Our study shows that the numerous regulations related to tires focus on chemicals, manufacturing, raw materials, use of tires on roads, waste handling, safety and polycyclic aromatic hydrocarbons (PAHs) in different life cycle stages of a tire. However, none directly addresses the contribution of TWPs to the environment. Despite the overall good intentions of the existing regulations, there is a lack of focus on the compounds that partition from the tire and disperse in the environment, their mixture effects, and the transformative products from the parent compounds in the environment. Therefore, a renewed focus is needed on risk assessment of complex mixtures like TWPs. Thus, transparency in regard to use of chemicals in TWP, mixtures, minimization of emissions, and capture of particulate pollution should be a priority.

Pesticide exposure among Czech adults and children from the CELSPAC-SPECIMEn cohort: Urinary biomarker levels and associated health risks

Current-use pesticides (CUP) are extensively applied in both agricultural and urban settings. Exposure occurs mainly via the dietary pathway; however, other pathways such as inhalation or skin contact are also important. In this study, urinary levels of 12 CUP metabolites were investigated among 110 parent-child pairs during two seasons of 2020. Metabolites of pyrethroids (3-PBA, t/c-DCCA), chlorpyrifos (TCPY), and tebuconazole (TEB-OH) were detected in more than 60% of the samples. Chlorpyrifos metabolite was found at the highest concentration and tebuconazole was detected in almost all samples. CUP urinary metabolite levels were significantly higher in children in comparison to adults, except for tebuconazole, which was similar in both groups. In children, winter samples had significantly higher concentrations of pyrethroid and chlorpyrifos metabolites in comparison to the summer samples, but in adults, only chlorpyrifos metabolite concentrations were higher in the winter. No association between CUP urinary metabolite levels and proximity/surface of agricultural areas around residences was observed. Based on our findings, we suspect that CUP exposure is mainly driven by diet and that the effect of environmental exposure is less significant. Daily Intakes were estimated with three possible scenarios considering the amount of the metabolite excreted in urine and were compared to Acceptable Daily Intake values. Using a realistic scenario, exposure to chlorpyrifos exhibited the highest health risk, but still within a safe level. The Acceptable Daily Intake was exceeded only in one child in the case of cypermethrin. The cumulative risk assessment of pesticide mixtures having an effect on the nervous system, based on the total margin of exposure calculations, did not indicate any risk. The overall risk associated with pesticide exposure in the observed population was low. However, the risk observed using the worst-case scenario suggests the need for continuous evaluation of human exposure to such compounds, especially in children.

Determination of whole mixture-based potency factors for cancer risk assessment of complex environmental mixtures by in vitro testing of standard reference materials.

Whole mixture-based testing using in vitro new approach methodologies (NAMs) has been suggested to facilitate the hazard and risk assessment of complex environmental mixtures. Previous studies have shown that phosphorylation of DNA damage signaling proteins checkpoint kinase 1 (pChk1) and histone 2AX (γH2AX) are sensitive markers that can be used for estimating carcinogenicity potencies in vitro. Here, and with the aim to better validate the applicability, in vitro-based Mixture Potency Factors (MPFs) of Standard Reference Materials (SRMs) from environmental polycyclic aromatic hydrocarbon (PAH)-containing mixtures were determined and compared to published mutagenicity and tumorigenicity data. Also, genotoxicity was assessed by a flow cytometry-based micronucleus (MN) assay which showed that only benzo[a]pyrene (B[a]P) and coal tar SRM (SRM1597a) caused dose-dependent increases of MN formation, while extracts of diesel particulate matter (SRM1650b), diesel particulate extract (SRM1975), and urban dust (SRM1649b) did not. However, a dose-dependent activation of DNA damage signaling was observed for all PAHs and SRMs. The results demonstrated that all SRMs were more potent than B[a]P, at B[a]P-equivalent concentrations, to induce pChk1 and γH2AX, and that western blot was more sensitive than the In-Cell Western assay in detecting their activation in response to these complex mixtures. Relative MPFs, based on dose-response modelling of pChk1 and γH2AX, ranged 113 - 5270 for the SRMs, indicating several orders of magnitude higher genotoxic potential than B[a]P. Moreover, these MPFs were in good agreement with potency values based on published data from Salmonella mutagenicity and in vivo carcinogenicity studies. In conclusion, these comparisons further validate the feasibility of applying in vitro NAMs, such as whole-mixture based MPFs, in cancer risk assessment of complex mixtures.

Occupational Exposure to Hexavalent Chromium, Nickel and PAHs: A Mixtures Risk Assessment Approach Based on Literature Exposure Data from European Countries.

Hexavalent chromium (Cr(VI)), nickel (Ni) and polycyclic aromatic hydrocarbons (PAHs) are genotoxic co-occurring lung carcinogens whose occupational health risk is still understudied. This study, conducted within the European Human Biomonitoring Initiative (HBM4EU), aimed at performing a mixtures risk assessment (MRA) based on published human biomonitoring (HBM) data from Cr(VI), Ni and/or PAHs occupational co-exposure in Europe. After data extraction, Risk Quotient (RQ) and Sum of Risk Quotients (SRQ) were calculated for binary and ternary mixtures to characterise the risk. Most selected articles measured urinary levels of Cr and Ni and a SRQ > 1 was obtained for co-exposure levels in welding activities, showing that there is concern regarding co-exposure to these substances. Similarly, co-exposure to mixtures of Cr(VI), Ni and PAHs in waste incineration settings resulted in SRQ > 1. In some studies, a low risk was estimated based on the single substances’ exposure level (RQ < 1), but the mixture was considered of concern (SRQ > 1), highlighting the relevance of considering exposure to the mixture rather than to its single components. Overall, this study points out the need of using a MRA based on HBM data as a more realistic approach to assess and manage the risk at the workplace, in order to protect workers’ health.

Transgenerational effects of co-exposure to cadmium and carbofuran on zebrafish based on biochemical and transcriptomic analyses

The combined toxicity of heavy metals and pesticides to aquatic organisms is still largely unexplored. In this study, we investigated the combined impacts of cadmium (Cd) and carbofuran (CAR) on female zebrafish (F0 generation) and their following F1 generation. Results showed that mixtures of Cd and CAR induced acute synergistic effects on both zebrafish adults of the F0 generation and embryos of the F1 generation. Combined exposure to Cd and CAR could obviously alter the hepatic VTG level of females, and the individual exposures increased the relative mRNA levels of vtg1 and vtg2. Through maternal transmission, co-exposure of Cd and CAR caused toxicity to 4-day-old larvae of the F1 generation, evidenced by the significant changes in T4 and VTG levels, CYP450 activity, and the relative transcriptional levels of genes related to the hormone, oxidative stress, and apoptosis. These effects were also reflected by the global gene expression pattern to 7-day-old larvae of F1 generation using the transcriptomic analysis, and they could also affect energy metabolism. Our results provided a more comprehensive insight into the transgenerational toxic impacts of heavy metal and pesticide mixtures. These findings highlighted that it was highly necessary to consider transgenerational exposures in the ecological risk assessment of chemical mixtures.

Grouping of PFAS for human health risk assessment: Findings from an independent panel of experts

An expert panel was convened to provide insight and guidance on per- and polyfluoroalkyl substances (PFAS) grouping for the purposes of protecting human health from drinking water exposures, and how risks to PFAS mixtures should be assessed. These questions were addressed through multiple rounds of blind, independent responses to charge questions, and review and comments on co-panelists responses. The experts agreed that the lack of consistent interpretations of human health risk for well-studied PFAS and the lack of information for the vast majority of PFAS present significant challenges for any mixtures risk assessment approach. Most experts agreed that “all PFAS” should not be grouped together, persistence alone is not sufficient for grouping PFAS for the purposes of assessing human health risk, and that the definition of appropriate subgroups can only be defined on a case-by-case manner. Most panelists agreed that it is inappropriate to assume equal toxicity/potency across the diverse class of PFAS. A tiered approach combining multiple lines of evidence was presented as a possible viable means for addressing PFAS that lack analytical and/or toxicological studies. Most PFAS risk assessments will need to employ assumptions that are more likely to overestimate risk than to underestimate risk, given the choice of assumptions regarding dose-response model, uncertainty factors, and exposure information.

Competitive Metabolism of Polycyclic Aromatic Hydrocarbons (PAHs): An Assessment Using In Vitro Metabolism and Physiologically Based Pharmacokinetic (PBPK) Modeling.

Humans are routinely exposed to complex mixtures such as polycyclic aromatic hydrocarbons (PAHs) rather than to single compounds, as are often assessed for hazards. Cytochrome P450 enzymes (CYPs) metabolize PAHs, and multiple PAHs found in mixtures can compete as substrates for individual CYPs (e.g., CYP1A1, CYP1B1, etc.). The objective of this study was to assess competitive inhibition of metabolism of PAH mixtures in humans and evaluate a key assumption of the Relative Potency Factor approach that common human exposures will not cause interactions among mixture components. To test this objective, we co-incubated binary mixtures of benzo[a]pyrene (BaP) and dibenzo[def,p]chrysene (DBC) in human hepatic microsomes and measured rates of enzymatic BaP and DBC disappearance. We observed competitive inhibition of BaP and DBC metabolism and measured inhibition coefficients (Ki), observing that BaP inhibited DBC metabolism more potently than DBC inhibited BaP metabolism (0.061 vs. 0.44 µM Ki, respectively). We developed a physiologically based pharmacokinetic (PBPK) interaction model by integrating PBPK models of DBC and BaP and incorporating measured metabolism inhibition coefficients. The PBPK model predicts significant increases in BaP and DBC concentrations in blood AUCs following high oral doses of PAHs (≥100 mg), five orders of magnitude higher than typical human exposures. We also measured inhibition coefficients of Supermix-10, a mixture of the most abundant PAHs measured at the Portland Harbor Superfund Site, on BaP and DBC metabolism. We observed similar potencies of inhibition coefficients of Supermix-10 compared to BaP and DBC. Overall, results of this study demonstrate that these PAHs compete for the same enzymes and, at high doses, inhibit metabolism and alter internal dosimetry of exposed PAHs. This approach predicts that BaP and DBC exposures required to observe metabolic interaction are much higher than typical human exposures, consistent with assumptions used when applying the Relative Potency Factor approach for PAH mixture risk assessment.

The toxic effects of combined exposure of chlorpyrifos and p, p'-DDE to zebrafish (Danio rerio) and tissue bioaccumulation.

Pesticides are widely used and frequently detected in the environment. The evaluation on the toxic effects of the co-exposure of two or more pesticides or related metabolites could reflect the real situation of the exposing risks. In this study, zebrafish was used as a model to investigate the potential toxic interactions of chlorpyrifos and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) on the survival rate, oxidative stress response and neurotoxicity, as well as their bioaccumulation and distribution in tissues. Co-exposure of chlorpyrifos and p,p'-DDE resulted in significant additive acute toxic effects on adult zebrafish with model deviation ratio (MDR)=1.64. Both 7-day short-term at 1% LC50 and 35-day long-term at 0.5% LC50 co-exposure of chlorpyrifos with p,p'-DDE (50 and 100µg/L) significantly reduced the survival rate of zebrafish colony to 75 and 82.5%. Co-exposure of chlorpyrifos and p,p'-DDE contributed to increased activity of antioxidant enzyme CAT, SOD and GST and excessive MDA generation, and decreased activity of CarE, CYP450 and AChE, compared with either single exposure of them. In co-exposure, the bioaccumulation of chlorpyrifos and p,p'-DDE was significantly different from the single exposure group. Overall, this study unraveled the potential toxic interaction of chlorpyrifos and p,p'-DDE on zebrafish and provided reference for environmental risk assessment of pesticide mixture.

Quantitative chemical risk assessment for mixtures: Application to alkylphenol mixtures and phthalate mixtures in tap and bottled water.

The occurrence and hazard risks of mixtures of Contaminants of Emerging Concern (CECs) in drinking water (DW) lead to serious consideration regarding the possible impacts on public health. Consequently, there is ongoing research, development and empowerment of risk assessment procedures to get more toxicological insight. For instance, alkylphenols and phthalates have been frequently reported to be present both in bottled and tap water, affecting different human endpoints. Currently, deterministic chemical risk assessment (CRA) is used to evaluate the compounds' mixture health risk. However, CRA deals just qualitatively with sources of uncertainty, which may lead to erroneous assessment of risks. Here, a new procedure for quantitative chemical risk assessment of CEC mixtures (QCRAMIX) is proposed. Its potential is illustrated by a case study where the risks related to the presence of mixtures of alkylphenols or phthalates in tap versus bottled DW are compared. Uncertainties in both exposure and hazard assessment steps of the procedure are included to calculate a probabilistic mixture Benchmark Quotient (BQMIX). The QCRAMIX procedure highlighted the non-negligible health risks posed by those compounds in both DW sources based on overall water consumption. In fact, DW consumers' behaviour in 13 different countries, in terms of total DW consumption and fraction of bottled and tap water consumed, were considered to evaluate the influence on health risk. For alkylphenols, the total water consumption was found to be the most relevant factor in increasing the health risk, while for phthalates the risk was found to be mainly influenced by the percentage of bottled water consumed. Hence, the proposed QCRAMIX procedure can be a valuable tool for prioritization of CECs to be included in DW regulations which aim to minimize the overall risk, accounting for actual DW consumption.

Internal Relative Potency Factors for the Risk Assessment of Mixtures of Per- and Polyfluoroalkyl Substances (PFAS) in Human Biomonitoring.

Background:In human biomonitoring, blood is often used as a matrix to measure exposure to per- and polyfluoroalkyl substances (PFAS). Because the toxicokinetics of a substance (determining the steady-state blood concentration) may affect the toxic potency, the difference in toxicokinetics among PFAS has to be accounted for when blood concentrations are used in mixture risk assessment.Objectives:This research focuses on deriving relative potency factors (RPFs) at the blood serum level. These RPFs can be applied to PFAS concentrations in human blood, thereby facilitating mixture risk assessment with primary input from human biomonitoring studies.Methods:Toxicokinetic models are generated for 10 PFAS to estimate the internal exposure in the male rat at the blood serum level over time. By applying dose–response modeling, these internal exposures are used to derive quantitative internal RPFs based on liver effects.Results:Internal RPFs were successfully obtained for nine PFAS. Perfluorobutanoic acid (PFBA), perfluorohexanoic acid (PFHxA), perfluorononanoic acid (PFNA), perfluorododecanoic acid (PFDoDA), perfluorooctane sulfonic acid (PFOS), and hexafluoropropylene oxide-dimer acid (HFPO-DA, or GenX) were found to be more potent than perfluorooctanoic acid (PFOA) at the blood serum level in terms of relative liver weight increase, whereas perfluorobutane sulfonic acid (PFBS) and perfluorohexane sulfonic acid (PFHxS) were found to be less potent. The practical implementation of these internal RPFs is illustrated using the National Health and Nutrition Examination Survey (NHANES) biomonitoring data of 2017–2018.Discussion:It is recommended to assess the health risk resulting from exposure to PFAS as combined, aggregate exposure to the extent feasible. https://doi.org/10.1289/EHP10009

Combined exposures to bisphenols, polychlorinated dioxins, paracetamol, and phthalates as drivers of deteriorating semen quality.

Semen quality in men continues to decline in Western countries, but the contours of the issue remain obscure, in relation to contributing chemicals. To obtain more clarity about the chemicals that drive the deterioration of semen quality, we conducted a mixture risk assessment based on European exposures. We included chemicals capable of affecting semen quality after prenatal exposures, among them androgen receptor antagonists, substances that disrupt prostaglandin signalling, suppress testosterone synthesis, inhibit steroidogenic enzymes or activate the aryl hydrocarbon receptor. We employed the Hazard Index approach (HI), based on risk quotients of exposures in Europe and reference doses for reductions in semen quality. By summing up the risk quotients of the 29 chemicals included in the assessment we examined fold-exceedances of "acceptable" mixture exposures relative to an index value of 1. For bisphenols A, F, S, phthalates DEHP, DnBP, BBzP, DiNP, n-butyl paraben and paracetamol we relied on biomonitoring studies in which these 9 chemicals were measured together in the same subjects. This allowed us to construct personalised Hazard Indices. Highly exposed subjects experienced combined exposures to the 9 chemicals that exceeded the index value of 1 by more than 100-fold; the median was a 17-fold exceedance. Accounting for median background exposures to the remaining 20 chemicals added a Hazard Index of 1.39. Bisphenol A made the largest contribution to the HI, followed by polychlorinated dioxins, bisphenols S and F and DEHP. Eliminating bisphenol A alone would still leave unacceptably high mixture risks. Paracetamol is also a driver of mixture risks among subjects using the drug. Tolerable exposures to substances associated with deteriorations of semen quality are exceeded by a large margin. Bisphenols, polychlorinated dioxins, phthalates and analgesics drive these risks. Dedicated efforts towards lowering exposures to these substances are necessary to mitigate risks.

MRA Toolbox v. 1.0: a web-based toolbox for predicting mixture toxicity of chemical substances in chemical products

The chemical risk assessment paradigm is shifting from “substance-based” to “product/mixture-based” and from “animal testing” to “alternative testing” under chemical regulations. Organisms and the environment may be exposed to mixtures rather than a single substance. Conducting toxicity tests for all possible combinations is impractical due to the enormous combinatorial complexity. This study highlights the development and application case studies of Mixture Risk Assessment Toolbox, a novel web-based platform that supports mixture risk assessment through the use of different prediction models and public databases. This integrated framework provides new functional values for assessors to easily screen and compare the toxicity of mixture products using different computational techniques and find strategic solutions to reduce the mixture toxicity in the product development process. The toolbox (https://www.mratoolbox.org) includes four additive toxicity models: two conventional (Concentration Addition; and Independent Action) and two advanced (Generalized Concentration Addition; and Quantitative Structure–Activity Relationship-based Two-Stage Prediction) models. We demonstrated the multiple functions of the toolbox using three cases: (i) how it can be used to calculate the mixture toxicity, (ii) those for which safety data sheet (SDS) only indicating representative toxicity values (EC50; and LC50), and (iii) those comprising chemicals with low toxic effects.

Environmental Risk Assessment of Dexamethasone Sodium Phosphate and Tocilizumab Mixture in Zebrafish Early Life Stage (Danio rerio).

Pharmaceuticals are widely regarded as a menace to the aquatic environment. The constant consumption of biologically active chemicals for human health has been matched by an increase in the leaking of these compounds in natural habitats over the last two decades. This study was aimed to evaluate the molecular pathway underling the developmental toxicity of exposure in the ecological environment. Zebrafish embryos were exposed at doses of dexamethasone sodium phosphate (DEX) 1 μmol/L, tocilizumab 442.1 μmol/L and dexamethasone + tocilizumab (1 μmol/L and 442.1 μmol/L, respectively) from 24 h post-fertilization (hpf) to 96 hpf. This study confirmed that DEX exposure in association with tocilizumab 442.1 μmol/L at 1 μmol/L (non-toxic concentration) affected the survival and hatching rate, morphology score, and body length. Additionally, it significantly disturbed the antioxidant defense system in zebrafish larvae. Furthermore, a DEX 1 μmol/L and tocilizumab 442.1 μmol/L association also increased the production of apoptosis-related proteins (caspase-3, bax, and bcl-2).