Biodistribution of cerium dioxide and titanium dioxide nanomaterials in rats after single and repeated inhalation exposures.

BACKGROUND: Physiologically based kinetic models facilitate the safety assessment of inhaled engineered nanomaterials (ENMs). To develop these models, high quality datasets on well-characterized ENMs are needed. However, there are at present, several data gaps in the systemic availability of poorly soluble particles after inhalation. The aim of the present study was therefore to acquire two comparable datasets to parametrize a physiologically-based kinetic model. METHOD: Rats were exposed to cerium dioxide (CeO2, 28.4 ± 10.4 nm) and titanium dioxide (TiO2, 21.6 ± 1.5 nm) ENMs in a single nose-only exposure to 20 mg/m3 or a repeated exposure of 2 × 5 days to 5 mg/m3. Different dose levels were obtained by varying the exposure time for 30 min, 2 or 6 h per day. The content of cerium or titanium in three compartments of the lung (tissue, epithelial lining fluid and freely moving cells), mediastinal lymph nodes, liver, spleen, kidney, blood and excreta was measured by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) at various time points post-exposure. As biodistribution is best studied at sub-toxic dose levels, lactate dehydrogenase (LDH), total protein, total cell numbers and differential cell counts were determined in bronchoalveolar lavage fluid (BALF). RESULTS: Although similar lung deposited doses were obtained for both materials, exposure to CeO2 induced persistent inflammation indicated by neutrophil granulocytes influx and exhibited an increased lung elimination half-time, while exposure to TiO2 did not. The lavaged lung tissue contained the highest metal concentration compared to the lavage fluid and cells in the lavage fluid for both materials. Increased cerium concentrations above control levels in secondary organs such as lymph nodes, liver, spleen, kidney, urine and faeces were detected, while for titanium this was found in lymph nodes and liver after repeated exposure and in blood and faeces after a single exposure. CONCLUSION: We have provided insight in the distribution kinetics of these two ENMs based on experimental data and modelling. The study design allows extrapolation at different dose-levels and study durations. Despite equal dose levels of both ENMs, we observed different distribution patterns, that, in part may be explained by subtle differences in biological responses in the lung.

The 2022 world health organization reevaluation of human and mammalian toxic equivalency factors for polychlorinated dioxins, dibenzofurans and biphenyls.

In October 2022, the World Health Organization (WHO) convened an expert panel in Lisbon, Portugal in which the 2005 WHO TEFs for chlorinated dioxin-like compounds were reevaluated. In contrast to earlier panels that employed expert judgement and consensus-based assignment of TEF values, the present effort employed an update to the 2006 REP database, a consensus-based weighting scheme, a Bayesian dose response modeling and meta-analysis to derive "Best-Estimate" TEFs. The updated database contains almost double the number of datasets from the earlier version and includes metadata that informs the weighting scheme. The Bayesian analysis of this dataset results in an unbiased quantitative assessment of the congener-specific potencies with uncertainty estimates. The "Best-Estimate" TEF derived from the model was used to assign 2022 WHO-TEFs for almost all congeners and these values were not rounded to half-logs as was done previously. The exception was for the mono-ortho PCBs, for which the panel agreed to retain their 2005 WHO-TEFs due to limited and heterogenous data available for these compounds. Applying these new TEFs to a limited set of dioxin-like chemical concentrations measured in human milk and seafood indicates that the total toxic equivalents will tend to be lower than when using the 2005 TEFs.

Determination of in vitro hepatotoxic potencies of a series of perfluoroalkyl substances (PFASs) based on gene expression changes in HepaRG liver cells.

Per- and polyfluoroalkyl substances (PFASs) are omnipresent and have been shown to induce a wide range of adverse health effects, including hepatotoxicity, developmental toxicity, and immunotoxicity. The aim of the present work was to assess whether human HepaRG liver cells can be used to obtain insight into differences in hepatotoxic potencies of a series of PFASs. Therefore, the effects of 18 PFASs on cellular triglyceride accumulation (AdipoRed assay) and gene expression (DNA microarray for PFOS and RT-qPCR for all 18 PFASs) were studied in HepaRG cells. BMDExpress analysis of the PFOS microarray data indicated that various cellular processes were affected at the gene expression level. From these data, ten genes were selected to assess the concentration-effect relationship of all 18 PFASs using RT-qPCR analysis. The AdipoRed data and the RT-qPCR data were used for the derivation of in vitro relative potencies using PROAST analysis. In vitro relative potency factors (RPFs) could be obtained for 8 PFASs (including index chemical PFOA) based on the AdipoRed data, whereas for the selected genes, in vitro RPFs could be obtained for 11-18 PFASs (including index chemical PFOA). For the readout OAT5 expression, in vitro RPFs were obtained for all PFASs. In vitro RPFs were found to correlate in general well with each other (Spearman correlation) except for the PPAR target genes ANGPTL4 and PDK4. Comparison of in vitro RPFs with RPFs obtained from in vivo studies in rats indicate that best correlations (Spearman correlation) were obtained for in vitro RPFs based on OAT5 and CXCL10 expression changes and external in vivo RPFs. HFPO-TA was found to be the most potent PFAS tested, being around tenfold more potent than PFOA. Altogether, it may be concluded that the HepaRG model may provide relevant data to provide insight into which PFASs are relevant regarding their hepatotoxic effects and that it can be applied as a screening tool to prioritize other PFASs for further hazard and risk assessment.

Internal relative potency factors based on immunotoxicity for the risk assessment of mixtures of per- and polyfluoroalkyl substances (PFAS) in human biomonitoring.

Relative potency factors (RPFs) for per- and polyfluoroalkyl substances (PFAS) have previously been derived based on liver effects in rodents for the purpose of performing mixture risk assessment with primary input from biomonitoring studies. However, in 2020, EFSA established a tolerable weekly intake for four PFAS assuming equal toxic potency for immune suppressive effects in humans. In this study we explored the possibility of deriving RPFs for immune suppressive effects using available data in rodents and humans. Lymphoid organ weights, differential blood cell counts, and clinical chemistry from 28-day studies in male rats from the National Toxicology Program (NTP) were combined with modeled serum PFAS concentrations to derive internal RPFs by applying dose-response modelling. Identified functional studies used diverse protocols and were not suitable for derivation of RPFs but were used to support immunotoxicity of PFAS in a qualitative manner. Furthermore, a novel approach was used to estimate internal RPFs based on epidemiological data by dose-response curve fitting optimization, looking at serum antibody concentrations and key cell populations from the National Health and Nutrition Examination Survey (NHANES). Internal RPFs were successfully derived for PFAS based on rat thymus weight, spleen weight, and globulin concentration. The available dose-response information for blood cell counts did not show a significant trend. Immunotoxic potency in serum was determined in the order PFDA > PFNA > PFHxA > PFOS > PFBS > PFOA > PFHxS. The epidemiological data showed inverse associations for the sum of PFOA, PFNA, PFHxS, and PFOS with serum antibody concentrations to mumps and rubella, but the data did not allow for deduction of reliable internal RPF estimates. The internal RPFs for PFAS based on decreased rat lymphoid organ weights are similar to those previously established for increased rat liver weight, strengthening the confidence in the overall applicability of these RPFs.

New approach methodologies: A quantitative in vitro to in vivo extrapolation case study with PFASs.

PER: and polyfluoroalkyl substances (PFASs) have been associated with increased blood lipids in humans. Perfluorooctanoic acid (PFOA) has been also linked with elevated alanine transferase (ALT) serum levels in humans, and in rodents the liver is a main target organ for many PFASs. With the focus on New Approach Methodologies, the chronic oral equivalent effect doses were calculated for PFOA, PFNA (perfluorononanoic acid), PFHxS (perfluorohexanesulfonic acid) and PFOS (perfluorooctane sulfonic acid) based on in vitro effects measured in the HepaRG cell line. Selected in vitro readouts were considered biomarkers for lipid disturbances and hepatotoxicity. Concentration-response data obtained from HepaRG cells on triglyceride (TG) accumulation and expression changes of 12 selected genes (some involved in cholesterol homeostasis) were converted into corresponding human dose-response data, using physiologically based kinetic (PBK) model-facilitated reverse dosimetry. Next to this, the biokinetics of the chemicals were studied in the cell system. The current European dietary PFASs exposure overlaps with the calculated oral equivalent effect doses, indicating that the latter may lead to interference with hepatic gene expression and lipid metabolism. These findings illustrate an in vitro-in silico methodology, which can be applied for more PFASs, to select those that should be prioritized for further hazard characterization.

A congener-specific modelling approach for the transfer of polychlorinated dibenzo-p-dioxins and dibenzofurans and dioxin-like polychlorinated biphenyls from feed to eggs of laying hens.

Calibration of a kinetic model for the transfer of PCDD/Fs and dl-PCBs from feed to the hen's body and eggs was thus far restricted to the total TEQ concentration, i.e. the summed concentrations of PCDD/Fs and dl-PCBs expressed in terms of equivalents of 2,3,7,8-TCDD. However, this approach may lead to over- or underestimation of the transfer if the mixture contains congeners with kinetic characteristics which differ considerably from those used in such a model. This paper extends a previous transfer model of PCDD/Fs and dl-PCBs from feed to egg yolk fat and abdominal fat of high production laying hens, based on the total TEQ approach, to the level of individual congeners. Both modelling approaches are compared and the new approach is presented as a webtool application. This congener-specific approach enabled the calibration of 25 of the 29 relevant PCDD/F and dl-PCB congeners with respect to their individual transfer characteristics to body fat and egg yolk fat and their clearance from the body. Limitations of the available experimental data prevented the calibration of 1,2,3,4,6,7,8-HpCDD, OCDD, OCDF and PCB 123. The fraction transferred to egg yolk fat after long-term daily intake of contaminated feed was found to be at least 0.78 for 2,3,7,8-TCDD, 0.75 for PeCDD, 0.42-0.61 for HxCDDs, 0.70 for 2,3,7,8-TCDF, 0.71 for PeCDF, 0.54-0.60 for HxCDFs, 0.18-0.24 for HpCDFs and 0.89-1.00 for dl-PCBs. Various experimental and feed incident mixtures were used to compare the total TEQ- model with the congener-specific approach. An overestimation of the transfer by the total TEQ method was shown in particular for mixtures with a substantial contribution of hexa-, hepta- and octa-PCDD/Fs to the total TEQ level.

PACEMweb: a tool for aggregate consumer exposure assessment.

BACKGROUND: To ascertain the safe use of chemicals that are used in multiple consumer products, the aggregate human exposure, arising from combined use of multiple consumer products needs to be assessed. OBJECTIVE: In this work the Probabilistic Aggregate Consumer Exposure Model (PACEM) is presented and discussed. PACEM is implemented in the publicly available web tool, PACEMweb, for aggregate consumer exposure assessment. METHODS: PACEM uses a person-oriented simulation method that is based on realistic product usage information obtained in surveys from several European countries. PACEM evaluates aggregate exposure in a population considering individual use and co-use patterns as well as variation in product composition. Product usage data is included on personal care products (PCPs) and household cleaning products (HCPs). RESULTS: PACEM has been implemented in a web tool that supports broad use in research as well as regulatory risk assessment. PACEM has been evaluated in a number of applications, testing and illustrating the advantage of the person-oriented modeling method. Also, PACEM assessments have been evaluated by comparing its results with biomonitoring information. SIGNIFICANCE: PACEM enables the assessment of realistic aggregate exposure to chemicals in consumer products. It provides detailed insight into the distribution of exposure in a population as well as products that contribute the most to exposure. This allows for better informed decision making in the risk management of chemicals. IMPACT: Realistic assessment of the total, aggregate exposure of consumers to chemicals in consumer products is necessary to guarantee the safe use of chemicals in these products. PACEMweb provides, for the first time, a publicly available tool to assist in realistic aggregate exposure assessment of consumers to chemicals in consumer products.

Challenges in Predicting the Change in the Cumulative Exposure of New Tobacco and Related Products Based on Emissions and Toxicity Dose-Response Data.

Many novel tobacco products have been developed in recent years. Although many may emit lower levels of several toxicants, their risk in the long term remains unclear. We previously published a method for the exposure assessment of mixtures that can be used to compare the changes in cumulative exposure to carcinogens among tobacco products. While further developing this method by including more carcinogens or to explore its application to non-cancer endpoints, we encountered a lack of data that are required for better-substantiated conclusions regarding differences in exposure between products. In this special communication, we argue the case for more data on adverse health effects, as well as more data on the composition of the emissions from tobacco products. Such information can be used to identify significant changes in relevance to health using the cumulative exposure method with different products and to substantiate regulatory decisions.

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 Exposure to Multiple Mycotoxins: An Example of Using a Tiered Approach in a Mixture Risk Assessment.

Humans are exposed to mycotoxins on a regular basis. Exposure to a mixture of mycotoxins may, therefore, result in a combination of adverse effects, or trigger the same effects. This should be accounted for when assessing the combined risk of multiple mycotoxins. Here, we show the outcome of using different approaches in assessing the risks related to the combined exposure to mycotoxins. We performed a tiered approach using assessment groups with a common target organ (kidney, liver and haematologic system), or a common adverse effect (phenomenon) (reduced white blood cell count), to combine the exposure to mycotoxins. The combined exposure was calculated for the individuals in this assessment, using the Monte Carlo Risk Assessment (MCRA) tool. The risk related to this combined exposure was assessed using toxicological reference values, e.g., health based guidance values. We show that estimating the combined risk by adding the single compounds' risk distributions slightly overestimates the combined risk in the 95th percentile, as compared to combining the exposures at an individual level. We also show that relative potency factors can be used to refine the mixture risk assessment, as compared to ratios of toxicological reference values with different effect sizes and assessment factors.

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.

Development of a quantitative chemical risk assessment (QCRA) procedure for contaminants of emerging concern in drinking water supply.

The uncertainties on the occurrence, fate and hazard of Contaminants of Emerging Concern (CECs) increasingly challenge drinking water (DW) utilities whether additional measures should be taken to reduce the health risk. This has led to the development and evaluation of risk-based approaches by the scientific community. DW guideline values are commonly derived based on deterministic chemical risk assessment (CRA). Here, we propose a new probabilistic procedure, that is a quantitative chemical risk assessment (QCRA), to assess potential health risk related to the occurrence of CECs in DW. The QCRA includes uncertainties in risk calculation in both exposure and hazard assessments. To quantify the health risk in terms of the benchmark quotient probabilistic distribution, the QCRA estimates the probabilistic distribution of CECs concentration in DW based on their concentration in source water and simulating the breakthrough curves of a granular activated carbon (GAC) treatment process. The model inputs and output uncertainties were evaluated by sensitivity and uncertainty analyses for each step of the risk assessment to identify the most relevant factors affecting risk estimation. Dominant factors resulted to be the concentration of CECs in water sources, GAC isotherm parameters and toxicological data. To stress the potential of this new QCRA approach, several case studies are considered with focus on bisphenol A as an example CEC and various GAC management options. QCRA quantifies the probabilistic risk, providing more insight compared to CRA. QCRA proved to be more effective in supporting the intervention prioritization for treatment optimization to pursue health risk minimization.

Pulmonary toxicity and gene expression changes after short-term inhalation exposure to surface-modified copper oxide nanoparticles.

Copper oxide nanoparticles (CuO NPs) have previously been shown to cause dose-dependent pulmonary toxicity following inhalation. Here, CuO NPs (10 nm), coated with polyethylenimine (PEI) or ascorbate (ASC) resulting in positively or negatively charged NPs, respectively, were evaluated. Rats were exposed nose-only to similar exposure dose levels of ASC or PEI coated CuO NPs for 5 consecutive days. On day 6 and day 27 post-exposure, pulmonary toxicity markers in bronchoalveolar lavage fluid (BALF), lung histopathology and genome-wide transcriptomic changes in lungs, were assessed. BALF analyses showed a dose-dependent pulmonary inflammation and cell damage, which was supported by the lung histopathological findings of hypertrophy/hyperplasia of bronchiolar and alveolar epithelium, interstitial and alveolar inflammation, and paracortical histiocytosis in mediastinal lymph nodes for both types of CuO NPs. Transcriptomics analysis showed that pathways related to inflammation and cell proliferation were significantly activated. Additionally, we found evidence for the dysregulation of drug metabolism-related genes, especially in rats exposed to ASC-coated CuO NPs. Overall, no differences in the type of toxic effects and potency between the two surface coatings could be established, except with respect to the (regional) dose that initiates bronchiolar and alveolar hypertrophy. This disproves our hypothesis that differences in surface coatings affect the pulmonary toxicity of CuO NPs.

Risk Assessment of Per- and Polyfluoroalkyl Substance Mixtures: A Relative Potency Factor Approach.

Per- and polyfluoroalkyl substances (PFAS) often occur together as contamination in exposure media such as drinking water or food. The relative potency factor (RPF) methodology facilitates the risk assessment of mixture exposure. A database of liver endpoints was established for 16 PFAS, using data with the same species (rat), sex (male), and exposure route (oral) and comparable exposure duration (42-90 d). Dose-response analysis was applied to derive the relative potencies of 3 perfluoroalkyl sulfonic acids (perfluorobutane sulfonic acid, perfluorohexane sulfonic acid, perfluorooctane sulfonic acid), 8 perfluoroalkyl carboxylic acids (perfluorobutanoic acid, perfluorohexanoic acid, perfluorononanoic acid, perfluoroundecanoic acid, perfluorododecanoic acid, perfluorotetradecanoic acid, perfluorohexadecanoic acid, perfluorooctadecanoic acid), 2 perfluoroalkyl ether carboxylic acids (tetrafluoro-2-[heptafluoropropoxy]propanoic acid, 3H-perfluoro-3-[(3-methoxy-propoxy)propanoic acid]), and 2 fluorotelomer alcohols (6:2 FTOH, 8:2 FTOH) compared to perfluorooctanoic acid (PFOA), based on liver effects. In addition, the RPFs of 7 other perfluoroalkyl acids were estimated based on read-across. This resulted in the relative potencies of 22 PFAS compared to the potency of index compound PFOA. The obtained RPFs can be applied to measured PFAS quantities, resulting in the sum of PFOA equivalents in a mixture. This sum can be compared with an established PFOA concentration limit (e.g., in drinking water or food) or an external health-based guidance value (e.g., tolerable daily intake, acceptable daily intake, or reference dose) to estimate the risk resulting from direct oral exposure to mixtures. Assessing mixture exposure is particularly relevant for PFAS, with omnipresent exposure in our daily lives. Environ Toxicol Chem 2021;40:859-870. © 2020 SETAC.

Culture Conditions Affect Chemical-Induced Developmental Toxicity In Vitro: The Case of Folic Acid, Methionine and Methotrexate in the Neural Embryonic Stem Cell Test.

In vitro tests are increasingly applied in chemical hazard assessment. Basic culture conditions may affect the outcome of in vitro tests and should be optimised to reduce false predictions. The neural embryonic stem cell test (ESTn) can predict early neurodevelopmental effects of chemicals, as it mimics the differentiation of stem cells towards the neuroectodermal lineage. Normal early neural differentiation depends crucially on folic acid (FA) and methionine (MET), both elements of the one-carbon (1C) cycle. The aim of this study was to assess the concentration-dependent influence of FA and MET on neural differentiation in the ESTn, and its consequences for assay sensitivity to methotrexate (MTX), a compound that interferes with the 1C cycle. Neural differentiation was inhibited below 0.007 mM and above 0.22 mM FA, while both stem cell viability (< 0.097 mM, > 1.52 mM) and neural differentiation (< 0.181 mM, > 1.35 mM) were affected when changing MET concentrations. A 10-day exposure to 13 nM MTX inhibited neural differentiation, especially in FA- and MET-deficient conditions. However, a 24-hour exposure to 39 nM MTX decreased neural cell and neural crest cell differentiation markers only when the concentration of FA in the medium was three times the standard concentration, which was expected to have a protective effect against MTX. These results show the importance of nutrient concentrations, exposure scenarios and timing of read-outs for cell differentiation and compound sensitivity in the ESTn. Caution should be taken when interpreting results from a single in vitro test, especially when extrapolating to effects on complex morphogenetic processes, like neural tube development.

Probabilistic approach for assessing cancer risk due to benzo[a]pyrene in barbecued meat: Informing advice for population groups.

BACKGROUND: Consumption of meat prepared by barbecuing is associated with risk of cancer due to formation of carcinogenic compounds including benzo[a]pyrene (BaP). Assessment of a population's risk of disease and people's individual probability of disease given specific consumer attributes may direct food safety strategies to where impact on public health is largest. The aim of this study was to propose a model that estimates the risk of cancer caused by exposure to BaP from barbecued meat in Denmark, and to estimate the probability of developing cancer in subgroups of the population given different barbecuing frequencies. METHODS: We developed probabilistic models applying two dimensional Monte Carlo simulation to take into account the variation in exposure given age and sex and in the individuals' sensitivity to develop cancer after exposure to BaP, and the uncertainty in the dose response model. We used the Danish dietary consumption survey, monitoring data of chemical concentrations, data on consumer behavior of frequency of barbecuing, and animal dose response data. FINDINGS: We estimated an average extra lifetime risk of cancer due to BaP from barbecued meat of 6.8 × 10-5 (95% uncertainty interval 2.6 × 10-7 - 7.0 × 10-4) in the Danish population. This corresponds to approximately one to 4,074 extra cancer cases over a lifetime, reflecting wide uncertainty. The impact per barbecuing event on the risk of cancer for men and women of low body weight was higher compared to higher bodyweight. However, the difference due to sex and bodyweight between subgroups are dwarfed by the uncertainty. INTERPRETATION: This study proposes a model that can be applied to other substances and routes of exposure, and allows for deriving the change in risk following a specific change in behaviour. The presented methodology can serve as a valuable tool for risk management, allowing for the formulation of behaviour advice targeted to specific sub-groups in the population.

Modelling the long-term feed-to-fillet transfer of leuco crystal violet and leuco malachite green in Atlantic salmon (Salmo salar).

Leuco crystal violet (LCV) and leuco malachite green (LMG) are the main metabolites of two dyes that are forbidden for use in food production, but can be present at low background concentration in novel Atlantic salmon feed ingredients such as processed animal proteins (animal by-product [ABP]). In this study, the potential transfer of dietary LCV or LMG to the fillet of farmed Atlantic salmon was investigated. The uptake and elimination rate kinetics were determined in seawater-adapted Atlantic salmon (initial weight 587 ± 148 g) fed two levels of either LCV- or LMG-enriched diets (~500 and 4000 µg kg-1, respectively) for 40 days, followed by a 90-day depuration period with feeding on control diets (<0.15 µg kg-1 LCV and LMG). A three-compartmental model was developed, based on a fillet fat, fillet muscle and a central body compartment comprising all other tissues. Model calibrations showed a good fit with measured values during overall uptake and elimination period; however, the model poorly predicted the short-term (days) peak measured values at the end of the exposure period. The model was used to simulate the long-term (>16 months) LCV and LMG feed-to-fillet transfer in Atlantic salmon under realistic farming conditions such as the seasonal fluctuations in feed intake, growth and fillet fat deposition. The model predictions gave highest expected LCV and LMG fillet concentrations of approximately 0.12 and 0.45 µg kg-1, depending on the dietary levels of ABP and background level of LCV and LMG contamination. These levels are under the reference point for action of 2 µg kg-1 for the sum of MG and LMG that EFSA assessed as adequate to protect public health. However, for LCV, the predicted highest levels exceed the analytical decision limit (CCα) of 0.15 µg kg-1 for the method used in this paper.

APROBA-Plus: A probabilistic tool to evaluate and express uncertainty in hazard characterization and exposure assessment of substances.

To facilitate the application of probabilistic risk assessment, the WHO released the APROBA tool. This tool applies lognormal uncertainty distributions to the different aspects of the hazard characterization, resulting in a probabilistic health-based guidance value. The current paper describes an extension, APROBA-Plus, which combines the output from the probabilistic hazard characterization with the probabilistic exposure to rapidly characterize risk and its uncertainty. The uncertainty in exposure is graphically compared with the uncertainty in the target human dose, i.e. the dose that complies with the specified protection goals. APROBA-Plus is applied to several case studies, resulting in distinct outcomes and illustrating that APROBA-Plus could serve as a standard extension of routine risk assessments. By visualizing the uncertainties, APROBA-Plus provides a more transparent and informative outcome than the more usual deterministic approaches, so that risk managers can make better informed decisions. For example, APROBA-Plus can help in deciding whether risk-reducing measures are warranted or that a refined risk assessment would first be needed. If the latter, the tool can be used to prioritize possible refinements. APROBA-Plus may also be used to rank substances into different risk categories, based on potential health risks without being compromised by different levels of conservatism that may be associated with point estimates of risk.

Impact of serum as a dispersion agent for in vitro and in vivo toxicological assessments of TiO2 nanoparticles.

Nanoparticles (NP) have a tendency to agglomerate after dispersion in physiological media, which can be prevented by the addition of serum. This may however result in modification of the toxic potential of particles due to the formation of protein corona. Our study aimed to analyze the role of serum that is added to improve the dispersion of 10 nm TiO2 NPs on in vitro and in vivo effects following the exposure via the respiratory route. We characterized NP size, surface charge, sedimentation rate, the presence of protein corona and the oxidant-generating capacity after NP dispersion in the presence/absence of serum. The effect of serum on NP internalization, cytotoxicity and pro-inflammatory responses was assessed in a human pulmonary cell line, NCI-H292. Serum in the dispersion medium led to a slower sedimentation, but an enhanced cellular uptake of TiO2 NPs. Despite this greater uptake, the pro-inflammatory response in NCI-H292 cells was lower after serum supplementation (used either as a dispersant or as a cell culture additive), which may be due to a reduced intrinsic oxidative potential of TiO2 NPs. Interestingly, serum could be added 2 h after the NP treatment without affecting the pro-inflammatory response. We also determined the acute pulmonary and hepatic toxicity in vivo 24 h after intratracheal instillation of TiO2 NPs in C57BL/6N mice. The use of serum resulted in an underestimation of the local acute inflammatory response in the lung, while a systemic response on glutathione reduction remained unaffected. In conclusion, serum as a dispersion agent for TiO2 NPs can lead to an underestimation of the acute pro-inflammatory response in vitro and in vivo. To avoid potential unwanted effects of dispersants and medium components, we recommend that the protocol of NM preparation should be thoroughly tested, and reflect as close as possible realistic exposure conditions.