Inhaled multiwalled carbon nanotubes modulate the immune response of trimellitic anhydride-induced chemical respiratory allergy in brown Norway rats.

The interaction between exposure to nanomaterials and existing inflammatory conditions has not been fully established. Multiwalled carbon nanotubes (MWCNT; Nanocyl NC 7000 CAS no. 7782-42-5; count median diameter in atmosphere 61 ± 5 nm) were tested by inhalation in high Immunoglobulin E (IgE)-responding Brown Norway (BN) rats with trimellitic anhydride (TMA)-induced respiratory allergy. The rats were exposed 2 days/week over a 3.5-week period to a low (11 mg/m(3)) or a high (22 mg/m(3)) concentration of MWCNT. Nonallergic animals exposed to MWCNT and unexposed allergic and nonallergic rats served as controls. At the end of the exposure period, the allergic animals were rechallenged with TMA. Histopathological examination of the respiratory tract showed agglomerated/aggregated MWCNT in the lungs and in the lung-draining lymph nodes. Frustrated phagocytosis was observed as incomplete uptake of MWCNT by the alveolar macrophages and clustering of cells around MWCNT. Large MWCNT agglomerates/aggregates were found in granulomas in the allergic rats, suggesting decreased macrophage clearance in allergic rats. In allergic rats, MWCNT exposure decreased serum IgE levels and the number of lymphocytes in bronchoalveolar lavage. In conclusion, MWCNT did not aggravate the acute allergic reaction but modulated the allergy-associated immune response.

Evaluation of research activities and research needs to increase the impact and applicability of alternative testing strategies in risk assessment practice.

The present paper aims at identifying strategies to increase the impact and applicability of alternative testing strategies in risk assessment. To this end, a quantitative and qualitative literature evaluation was performed on (a) current research efforts in the development of in vitro methods aiming for alternatives to animal testing, (b) the possibilities and limitations of in vitro methods for regulatory purposes and (c) the potential of physiologically-based kinetic (PBK) modeling to improve the impact and applicability of in vitro methods in risk assessment practice. Overall, the evaluation showed that the focus of state-of-the-art research activities does not seem to be optimally directed at developing in vitro alternatives for those endpoints that are most animal-demanding, such as reproductive and developmental toxicity, and carcinogenicity. A key limitation in the application of in vitro alternatives to such systemic endpoints is that in vitro methods do not provide so-called points of departure, necessary for regulators to set safe exposure limits. PBK-modeling could contribute to overcoming this limitation by providing a method that allows extrapolation of in vitro concentration-response curves to in vivo dose-response curves. However, more proofs of principle are required.

Decrease of intracellular pH as possible mechanism of embryotoxicity of glycol ether alkoxyacetic acid metabolites.

Embryotoxicity of glycol ethers is caused by their alkoxyacetic acid metabolites, but the mechanism underlying the embryotoxicity of these acid metabolites is so far not known. The present study investigates a possible mechanism underlying the embryotoxicity of glycol ether alkoxyacetic acid metabolites using the methoxyacetic acid (MAA) metabolite of ethylene glycol monomethyl ether as the model compound. The results obtained demonstrate an MAA-induced decrease of the intracellular pH (pH(i)) of embryonic BALB/c-3T3 cells as well as of embryonic stem (ES)-D3 cells, at concentrations that affect ES-D3 cell differentiation. These results suggest a mechanism for MAA-mediated embryotoxicity similar to the mechanism of embryotoxicity of the drugs valproic acid and acetazolamide (ACZ), known to decrease the pH(i)in vivo, and therefore used as positive controls. The embryotoxic alkoxyacetic acid metabolites ethoxyacetic acid, butoxyacetic acid and phenoxyacetic acid also caused an intracellular acidification of BALB/c-3T3 cells at concentrations that are known to inhibit ES-D3 cell differentiation. Two other embryotoxic compounds, all-trans-retinoic acid and 5-fluorouracil, did not decrease the pH(i) of embryonic cells at concentrations that affect ES-D3 cell differentiation, pointing at a different mechanism of embryotoxicity of these compounds. MAA and ACZ induced a concentration-dependent inhibition of ES-D3 cell differentiation, which was enhanced by amiloride, an inhibitor of the Na(+)/H(+)-antiporter, corroborating an important role of the pH(i) in the embryotoxic mechanism of both compounds. Together, the results presented indicate that a decrease of the pH(i) may be the mechanism of embryotoxicity of the alkoxyacetic acid metabolites of the glycol ethers.

New in vitro dermal absorption database and the prediction of dermal absorption under finite conditions for risk assessment purposes.

Most QSARs for dermal absorption predict the permeability coefficient, K(p), of a molecule, which is valid for infinite dose conditions. In practice, dermal exposure mostly occurs under finite dose conditions. Therefore, a simple model to predict finite dose dermal absorption from infinite dose data (K(p) and lag time) and the stratum corneum/water partition coefficient (K(SC,W)) was developed. To test the model, a series of in vitro dermal absorption experiments was performed under both infinite and finite dose conditions using acetic acid, benzoic acid, bis(2-ethylhexyl)phthalate, butoxyethanol, cortisone, decanol, diazinone, 2,4-dichlorophenol, ethacrynic acid, linolenic acid, octylparaben, oleic acid, propylparaben, salicylic acid and testosterone. For six substances, the predicted relative dermal absorption was not statistically different from the measured value. For all other substances, measured absorption was overpredicted by the model, but most of the overpredictions were still below the European default absorption value. In conclusion, our finite dose prediction model provides a useful and cost-effective estimate of dermal absorption, to be used in risk assessment for non-volatile substances dissolved in water at non-irritating concentrations.

Relative absorption and dermal loading of chemical substances: Consequences for risk assessment.

Quantification of skin absorption is an essential step in reducing the uncertainty of dermal risk assessment. Data from literature indicate that the relative dermal absorption of substances is dependent on dermal loading. Therefore, an internal exposure calculated with absorption data determined at a dermal loading not comparable to the actual loading may lead to a wrong assessment of the actual health risk. To investigate the relationship between dermal loading and relative absorption in a quantitative manner, 138 dermal publicly available absorption experiments with 98 substances were evaluated (87 in vitro, 51 in vivo; molecular weight between 40 and 950, logP between -5 and 13), with dermal loading ranging mostly between 0.001 and 10mg/cm(2). In 87 experiments (63%) an inverse relationship was observed between relative dermal absorption and dermal loading, with an average decrease of factor 33+/-69. Known skin irritating and volatile substances less frequently showed an inverse relationship between dermal loading and relative absorption.

Quercetin increases the bioavailability of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rats.

This study investigates whether the previous observation that quercetin increases the transport of PhIP through Caco-2 monolayers in vitro could be confirmed in an in vivo rat model. Co-administration of 1.45 micromol PhIP/kg bw and 30 micromol quercetin/kg bw significantly increased the blood AUC(0-8h) of PhIP in rats to 131+/-14% of the AUC(0-8h) for rats dosed with PhIP alone. Significantly increased blood PhIP levels were detected at 15, 30, 45 and 180 min. At 4 and 8h post-dosing a difference in the PhIP levels in the blood between the two treatment groups was no longer observed. In vitro and in silico modeling of PhIP transport using Caco-2 cells and a previously described kinetic model for PhIP transport revealed that the relative increase in PhIP transport caused by quercetin is dependent on the concentration of the two compounds. When substituting the PhIP and quercetin concentrations used in the in vivo experiment in the kinetic model, an effect of quercetin on PhIP transport was predicted that matches the actual effect of 131% observed in vivo. It is concluded that quercetin increases the bioavailability of the pro-carcinogen PhIP in rats pointing at a potential adverse effect of this supposed beneficial food ingredient.

Risk analysis and technology assessment in support of technology development: Putting responsible innovation in practice in a case study for nanotechnology.

Governments invest in "key enabling technologies," such as nanotechnology, to solve societal challenges and boost the economy. At the same time, governmental agencies demand risk reduction to prohibit any often unknown adverse effects, and industrial parties demand smart approaches to reduce uncertainties. Responsible research and innovation (RRI) is therefore a central theme in policy making. Risk analysis and technology assessment, together referred to as "RATA," can provide a basis to assess human, environmental, and societal risks of new technological developments during the various stages of technological development. This assessment can help both governmental authorities and innovative industry to move forward in a sustainable manner. Here we describe the developed procedures and products and our experiences to bring RATA in practice within a large Dutch nanotechnology consortium. This is an example of how to put responsible innovation in practice as an integrated part of a research program, how to increase awareness of RATA, and how to help technology developers perform and use RATA. Integr Environ Assess Manag 2018;14:9-16. © 2017 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Xenobiotic metabolism in human skin and 3D human skin reconstructs: a review.

In this review, we discuss and compare studies of xenobiotic metabolism in both human skin and 3D human skin reconstructs. In comparison to the liver, the skin is a less studied organ in terms of characterising metabolic capability. While the skin forms the major protective barrier to environmental chemical exposure, it is also a potential target organ for adverse health effects. Occupational, accidental or intended-use exposure to toxic chemicals could result in acute or delayed injury to the skin (e.g. inflammation, allergy, cancer). Skin metabolism may play a role in the manifestation or amelioration of adverse effects via the topical route. Today, we have robust testing strategies to assess the potential for local skin toxicity of chemical exposure. Such methods (e.g. the local lymph node assay for assessing skin sensitisation; skin painting carcinogenicity studies) incorporate skin metabolism implicitly in the in vivo model system used. In light of recent European legislation (i.e. 7(th) Amendment to the Cosmetics Directive and Registration Evaluation and Authorisation of existing Chemicals (REACH)), non-animal approaches will be required to reduce and replace animal experiments for chemical risk assessment. It is expected that new models and approaches will need to account for skin metabolism explicitly, as the mechanisms of adverse effects in the skin are deconvoluted. 3D skin models have been proposed as a tool to use in new in vitro alternative approaches. In order to be able to use 3D skin models in this context, we need to understand their metabolic competency in relation to xenobiotic biotransformation and whether functional activity is representative of that seen in human skin.

Myricetin stimulates the absorption of the pro-carcinogen PhIP.

The effect of the flavonoid myricetin on the transport of the pro-carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) through differentiated Caco-2 monolayers, a model for the intestinal epithelium, is described. Myricetin causes an increase of the transport of PhIP from the apical to the basolateral compartment. This effect was observed at physiologically relevant concentrations of PhIP and myricetin. Cyclosporin A (MRP2 inhibitor) but not PSC833 (P-gp inhibitor) showed a similar effect on PhIP transport. The results indicate that myricetin induces an increased basolateral uptake of the pro-carcinogen PhIP, in part through inhibition of the MRP2 mediated excretion of PhIP from the intestinal cells back to the lumen.

Prediction of in vivo embryotoxic effect levels with a combination of in vitro studies and PBPK modelling.

The new EU legislations for chemicals (Registration, Evaluation and Authorization of Chemicals, REACH) and cosmetics (Seventh Amendment) stimulate the acceptance of in vitro and in silico approaches to test chemicals for their potential to cause reproductive effects. In the current study seven compounds with known in vivo developmental effects were tested in the embryonic stem cell test (EST). The EST correctly classified 5-fluorouracil, methotrexate, retinoic acid, 2-ethoxyacetic acid and 2-methoxyacetic acid for their in vivo embryotoxic potential. The toxicity of 2-methoxyethanol and 2-ethoxyethanol was underestimated due to a lack of metabolic capacity in the EST. This study further investigated the possibility to use in silico techniques to extrapolate in vitro effect concentrations determined in the EST to in vivo exposure levels. This approach was evaluated by comparing in silico predicted in vivo effect levels with effect levels measured in rodents. The in vivo effect levels of 2-methoxyethanol, 2-ethoxyethanol, methotrexate and retinoic acid were correctly predicted with in silico modelling. Contrary, in vivo embryotoxicity of 5-fluorouracil was overestimated following this approach. It is concluded that a combination of in vitro and in silico techniques appears to be a promising alternative test method for risk assessment of embryotoxic compounds.

Monolayers of IEC-18 cells as an in vitro model for screening the passive transcellular and paracellular transport across the intestinal barrier: comparison of active and passive transport with the human colon carcinoma Caco-2 cell line.

PURPOSE: previous studies have shown that the rat small intestinal cell line IEC-18 provides a size-selective barrier for paracellularly transported hydrophilic macromolecules. In order to determine the utility of IEC-18 cells as an in vitro model to screen the passive paracellular and transcellular components of the intestinal transport of nutrients and drugs, we have now examined the transport of GlySar (H(+)-coupled di/tripeptide carrier), O-methyl-d-glucose (glucose carrier), vincristine and rhodamine 123 (P-glycoprotein), and calcein and DNPSG (MRPs) and the bidirectional transport of paracellularly transported compounds. Transport of these compounds across the filter grown IEC-18 cells was compared with transport across the human colon carcinoma Caco-2 cells. RESULTS: in IEC-18 cells, transepithelial transport of GlySar and methylglucose was as fast as the transport of mannitol, which is transported passively via the paracellular route. Whereas in Caco-2 cells, mannitol transport was much slower than the transport of GlySar and methylglucose. In contrast to Caco-2 cells, no H(+)-coupled transport of GlySar could be measured in IEC-18 cells. P-Glycoprotein-mediated transport was characterised in Caco-2 cells by an enhanced transport of vincristine and rhodamine 123 in the basolateral to apical direction and by the inhibition of this transport by verapamil. In IEC-18 cells, permeability of vincristine and rhodamine 123 was similar in both directions and verapamil had no effect on the transport of these compounds. Both IEC-18 and Caco-2 cells efflux the organic anions calcein and DNPSG to the apical and basolateral compartments, and this efflux could be inhibited by probenecid. CONCLUSIONS: in conclusion, no carrier-mediated transport of GlySar, methylglucose, vincristine and rhodamine 123 could be determined in IEC-18 cells in contrast to Caco-2 cells. However, both IEC-18 and Caco-2 cells showed MRP-mediated eflux system(s) in the apical and basolateral membrane. Monolayers of IEC-18 cells appear to be more suitable than monolayers of Caco-2 cells as an in vitro system to screen the passive component of the intestinal transport in a deconvoluted screening regimen, where passive transport is represented by the IEC-18 monolayer permeability and active transport is represented by monolayers of cells expressing the transport proteins heterologously.

Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation. The European Centre for the Validation of Alternative Methods Skin Irritation Task Force report 2.

The European Centre for the Validation of Alternative Methods (ECVAM) Skin Irritation Task Force was established in 1996, to review the status of the development and validation of alternative tests for skin irritation and corrosion, and to identify appropriate non-animal tests for predicting human skin irritation that were sufficiently well-developed to be prevalidated and validated by ECVAM. The EpiDerm method, based on a reconstituted human skin model, was proposed as being sufficiently well advanced to enter a prevalidation (PV) study. Based on a review of test protocols, prediction models (PMs), and data submitted by test developers on ten specified chemicals, with 20% sodium lauryl sulphate as a reference standard, the task force recommended the inclusion of four other tests: EPISKIN and PREDISKIN, based on reconstituted human epidermis or on human skin; the non-perfused pig-ear test, based on pig skin; and the skin integrity function test (SIFT), with ex vivo mouse skin. The prevalidation study on these methods was funded by ECVAM, and took place during 1999-2000. The outcome of the PV study was that none of the methods was ready to enter a formal validation study, and that the protocols and PMs of the methods had to be improved in order to increase their predictive abilities. Improved protocols and PMs for the EpiDerm and EPISKIN methods, the pig ear test, and the SIFT were presented at an extended Task Force meeting held in May 2001. It was agreed that, in the short term, the performance of the revised and harmonised EpiDerm and EPISKIN methods, as well as the modified SIFT, should be evaluated in a further study with a new set of 20 test chemicals. In addition, it was decided that the SIFT and the pig ear test would be compared to see if common endpoints (transepidermal water loss, methyl green-pyronine stain) could be identified.

The use of in vitro toxicity data and physiologically based kinetic modeling to predict dose-response curves for in vivo developmental toxicity of glycol ethers in rat and man.

At present, regulatory assessment of systemic toxicity is almost solely carried out using animal models. The European Commission's REACH legislation stimulates the use of animal-free approaches to obtain information on the toxicity of chemicals. In vitro toxicity tests provide in vitro concentration-response curves for specific target cells, whereas in vivo dose-response curves are regularly used for human risk assessment. The present study shows an approach to predict in vivo dose-response curves for developmental toxicity by combining in vitro toxicity data and in silico kinetic modeling. A physiologically based kinetic (PBK) model was developed, describing the kinetics of four glycol ethers and their embryotoxic alkoxyacetic acid metabolites in rat and man. In vitro toxicity data of these metabolites derived in the embryonic stem cell test were used as input in the PBK model to extrapolate in vitro concentration-response curves to predicted in vivo dose-response curves for developmental toxicity of the parent glycol ethers in rat and man. The predicted dose-response curves for rat were found to be in concordance with the embryotoxic dose levels measured in reported in vivo rat studies. Therefore, predicted dose-response curves for rat could be used to set a point of departure for deriving safe exposure limits in human risk assessment. Combining the in vitro toxicity data with a human PBK model allows the prediction of dose-response curves for human developmental toxicity. This approach could therefore provide a means to reduce the need for animal testing in human risk assessment practices.

Relative developmental toxicity of glycol ether alkoxy acid metabolites in the embryonic stem cell test as compared with the in vivo potency of their parent compounds.

The embryonic stem cell test (EST) has been proposed as an in vitro assay that might reduce animal experimentation in regulatory developmental toxicology. So far, evaluation of the EST was not performed using compounds within distinct chemical classes. Evaluation within a distinct class of chemically related compounds can define the usefulness of the assay for the chemical class tested. The aim of the present study was to evaluate the relative sensitivity of the EST for a selected series of homologous compounds and to compare the data to the relative developmental toxicity of the compounds in vivo. To this end a series of proximate developmentally toxic glycol ether alkoxy acid metabolites was tested in the EST. All glycol ether alkoxy acid metabolites tested showed a concentration-dependent inhibition of cardiomyocyte differentiation at noncytotoxic concentrations, with methoxyacetic acid as the most potent compound followed by ethoxyacetic acid, butoxyacetic acid, and phenoxyacetic acid, respectively. The potency ranking of the compounds in the EST corresponds with the available in vivo data. The relative differences between the potencies of the compounds appeared more pronounced in the in vivo studies than in the EST. A possible explanation for this discrepancy could be the difference in the kinetics of the compounds in vivo as compared with their in vitro kinetics. This study illustrates that the EST can be used to set priorities for developmental toxicity testing within classes of related compounds.

From dermal exposure to internal dose.

Exposure scenarios form an essential basis for chemical risk assessment reports under the new EU chemicals regulation REACH (Registration, Evaluation, Authorisation and restriction of Chemicals). In case the dermal route of exposure is predominant, information on both exposure and dermal bioavailability is necessary for a proper risk assessment. Various methodologies exist to measure dermal exposure, providing quantitative or semiquantitative information. Although these studies may provide very specific and relevant information, it should be realized that case by case in-depth exposure assessment would be a very expensive process. Dermal bioavailability data are most often obtained from in vitro studies or animal experiments. For the design of studies, which generate data relevant for chemical risk assessment, detailed information on the exposure conditions is crucial (skin surface exposed, exposure duration, dose and physical state of the chemical). Results from non-testing methods for skin absorption, such as (Q)SARs, have been used only to a very limited extent for regulatory purposes. Suggestions are made in order to extend the use these methods to dermal risk assessment of chemical substances, thereby improving the practicability of REACH.