Quantitative comparison between in vivo DNA adduct formation from exposure to selected DNA-reactive carcinogens, natural background levels of DNA adduct formation and tumour incidence in rodent bioassays

This study aimed at quantitatively comparing the occurrence/formation of DNA adducts with the carcinogenicity induced by a selection of DNA-reactive genotoxic carcinogens. Contrary to previous efforts, we used a very uniform set of data, limited to in vivo rat liver studies in order to investigate whether a correlation can be obtained, using a benchmark dose (BMD) approach. Dose-response data on both carcinogenicity and in vivo DNA adduct formation were available for six compounds, i.e. 2-acetylaminofluorene, aflatoxin B1, methyleugenol, safrole, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and tamoxifen. BMD10 values for liver carcinogenicity were calculated using the US Environmental Protection Agency BMD software. DNA adduct levels at this dose were extrapolated assuming linearity of the DNA adduct dose response. In addition, the levels of DNA adducts at the BMD10 were compared to available data on endogenous background DNA damage in the target organ. Although for an individual carcinogen the tumour response increases when adduct levels increase, our results demonstrate that when comparing different carcinogens, no quantitative correlation exists between the level of DNA adduct formation and carcinogenicity. These data confirm that the quantity of DNA adducts formed by a DNA-reactive compound is not a carcinogenicity predictor but that other factors such as type of adduct and mutagenic potential may be equally relevant. Moreover, comparison to background DNA damage supports the notion that the mere occurrence of DNA adducts above or below the level of endogenous DNA damage is neither correlated to development of cancer. These data strongly emphasise the need to apply the mode of action framework to understand the contribution of other biological effect markers playing a role in carcinogenicit

In vivo validation of DNA adduct formation by estragole in rats predicted by physiologically based biodynamic modelling

Estragole is a naturally occurring food-borne genotoxic compound found in a variety of food sources, including spices and herbs. This results in human exposure to estragole via the regular diet. The objective of this study was to quantify the dose-dependent estragole-DNA adduct formation in rat liver and the urinary excretion of 1'-hydroxyestragole glucuronide in order to validate our recently developed physiologically based biodynamic (PBBD) model. Groups of male outbred Sprague Dawley rats (n = 10, per group) were administered estragole once by oral gavage at dose levels of 0 (vehicle control), 5, 30, 75, 150, and 300mg estragole/kg bw and sacrificed after 48h. Liver, kidney and lungs were analysed for DNA adducts by LC-MS/MS. Results obtained revealed a dose-dependent increase in DNA adduct formation in the liver. In lungs and kidneys DNA adducts were detected at lower levels than in the liver confirming the occurrence of DNA adducts preferably in the target organ, the liver. The results obtained showed that the PBBD model predictions for both urinary excretion of 1'-hydroxyestragole glucuronide and the guanosine adduct formation in the liver were comparable within less than an order of magnitude to the values actually observed in vivo. The PBBD model was refined using liver zonation to investigate whether its predictive potential could be further improved. The results obtained provide the first data set available on estragole-DNA adduct formation in rats and confirm their occurrence in metabolically active tissues, i.e. liver, lung and kidney, while the significantly higher levels found in liver are in accordance with the liver as the target organ for carcinogenicity. This opens the way towards future modelling of dose-dependent estragole liver DNA adduct formation in huma

Application of the Virtual Cell Based Assay for Simulation of in vitro Chemical fate following Acute Exposure

In order to reliably assess the risk of adverse systemic effects of chemicals by using in vitro methods, there is a need to simulate their absorption, distribution, metabolism, and excretion (ADME) in vivo to determine the target organ bioavailable concentration, and to compare this predicted internal concentration with an effective internal concentration. The effective concentration derived from in vitro toxicity studies should ideally take into account the fate of chemicals in the in vitro test system, since there can be significant differences between the applied nominal concentration and the in vitro bioavailable concentration. Whereas PBK models have been developed to simulate ADME properties in vivo, the Virtual Cell Based Assay (VCBA) has been developed to simulate in vitro fate. In this project, the VCBA model in R code, was applied to better interpret previously obtained in vitro acute toxicity data and study how they can be compared to results from acute toxicity in vivo. For 178 chemicals previously tested in vitro with the 3T3 BALB/c cell line using the Neutral Red Uptake cytotoxicity assay, physicochemical parameters were retrieved and curated. Of these chemicals, 83 were run in the VCBA to simulate a 96-well microplate set up with 5% serum supplementation, and their no effect concentration (NEC) and killing rate (Kr) optimized against the experimental data. Analyses of results of partitioning of the chemicals show a strong relation with their lipophilicity, expressed here as the logarithm of the octanol/water partitioning coefficient, with highly lipophilic chemicals binding mostly to medium lipid. Among the chemicals analysed, only benzene and xylene were modelled to evaporate by more than 10 %, and these were also the chemicals with highest degradation rates during the 48 hours assay. Chemical degradation is dependent not only on the air and water degradation rates but also on the extent of binding of the chemical. Due to the strong binding of some chemicals to medium lipids and proteins we analysed the impact of different serum supplementations (0%, 5% and 10%) on the chemical dissolved concentrations. As expected, for the more lipophilic chemicals, different serum levels result in different dissolved concentrations, with lipid and protein binding reducing chemical loss by evaporation. Still the lack of saturation modelling might mislead the 0 % supplementation since the lipids coming solely from cells exudates are able to sequester chemical to a large extent, eg. after 48 hours, 63% (1.2E-5 M) of dimethyldioctadecylammonium chloride was bound to lipid from the cells. Although highly lipophilic chemicals have a very small bioavailable fraction, cellular uptake rate is also dependent on logKow, which compensates for this lack of bioavailability to some extent. Based on the relevance of lipophilicity on in vitro chemical bioavailability, we have developed an alert system based on logKow, creating four classes of chemicals for the experimental condition with 10% serum supplementation: logKow 5- 10 (A), logKow <5 (B), logKow <2.5 (C), and logKow <2 (D). New chemicals from Classes A and B, which will in the future be tested in vitro, were run first on the VCBA, without considering toxicity (NEC and Kr set to 0). VCBA simulations indicated that these chemicals are more than 50% bound to medium proteins, lipids and plastic. Therefore, for chemicals with logKow falling in these classes, special care should be taken when extrapolating the obtained in vitro toxic concentrations to in vivo relevant doses. A comparison of the VCBA-predicted dissolved concentrations corresponding to nominal IC50 values with the available rat oral LD50 values did not improve the previously obtained correlations. This is probably because other in vivo kinetic processes play an important role but were not considered in this in vitro-in vivo extrapolation. The comparison of the VCBA predicted IC50 dissolved concentrations with the available rat oral LD50 values, did not improve the previously obtained correlations. Nevertheless, other in vivo kinetic processes that are not modelled may play an important role. They should be considered in the in vitro-in vivo extrapolations. A local sensitivity analysis showed the relative low impact of Molar Volume and Molecular Diffusion Volume on the final dissolved concentration, supporting the use of approximated values obtained through the herein created QSARs. The logkow and Henry Law Constant showed, as expected, a high impact in partitioning. Killing rate was shown to also have a relative low impact in the final chemical concentration, indicating that although its optimization is important, finding the Kr that leads to the absolute best correlation between experimental and predicted concentration-viability curves, is not imperative. The VCBA can be applied to virtually any chemical as long as the physicochemical data (for the fate model) and the experimental toxicity data (that include cell growth/death) are available. However, being such a generic model, several assumptions had to be made: i) no distinction of chemical classes (inorganic, polar organic chemicals), ii) no consideration of metabolism, iii) saturation kinetics and iv) external in vitro conditions. The advantages of having a generic model are that the VCBA can fit several experimental set ups and should be used in an exploratory manner, to help refinement of experimental conditions. The herein obtained VCBA results should be double checked experimentally the partition with a set of chemical compounds to better understand to what extent VCBA represents chemicals of different properties. In future developments, it would be important to reduce the uncertainties of the model such as binding-saturation and consider inclusion of other endpoints such as metabolic activity.JRC.F.3-Chemicals Safety and Alternative Method

Use of Physiologically Based Biokinetic (PBBK) Modeling to Study Estragole Bioactivation and Detoxification in Humans as Compared with Male Rats

The extent of bioactivation of the herbal constituent estragole to its ultimate carcinogenic metabolite 1′-sulfooxyestragole depends on the relative levels of bioactivation and detoxification pathways. The present study investigated the kinetics of the metabolic reactions of both estragole and its proximate carcinogenic metabolite 1′-hydroxyestragole in humans in incubations with relevant tissue fractions. Based on the kinetic data obtained a physiologically based biokinetic (PBBK) model for estragole in human was defined to predict the relative extent of bioactivation and detoxification at different dose levels of estragole. The outcomes of the model were subsequently compared with those previously predicted by a PBBK model for estragole in male rat to evaluate the occurrence of species differences in metabolic activation. The results obtained reveal that formation of 1′-oxoestragole, which represents a minor metabolic route for 1′-hydroxyestragole in rat, is the main detoxification pathway of 1′-hydroxyestragole in humans. Due to a high level of this 1′-hydroxyestragole oxidation pathway in human liver, the predicted species differences in formation of 1′-sulfooxyestragole remain relatively low, with the predicted formation of 1′-sulfooxyestragole being twofold higher in human compared with male rat, even though the formation of its precursor 1′-hydroxyestragole was predicted to be fourfold higher in human. Overall, it is concluded that in spite of significant differences in the relative extent of different metabolic pathways between human and male rat there is a minor influence of species differences on the ultimate overall bioactivation of estragole to 1′-sulfooxyestragol

Matrix Modulation of the Bioactivation of Estragole by Constituents of Different Alkenylbenzene-containing Herbs and Spices and Physiologically Based Biokinetic Modeling of Possible In Vivo Effects

The alkenylbenzene estragole is a constituent of several herbs and spices. It induces hepatomas in rodents at high doses following bioactivation by cytochrome P450s and sulfotransferases (SULTs) giving rise to the ultimate carcinogenic metabolite 1'-sulfooxyestragole which forms DNA adducts. Methanolic extracts from different alkenylbenzene-containing herbs and spices were able to inhibit SULT activity. Flavonoids including quercetin, kaempferol, myricetin, apigenin, and nevadensin were the major constituents responsible for this inhibition with Ki values in the nano to micromolar range. In human HepG2 cells exposed to the proximate carcinogen 1ʹ-hydroxyestragole, the various flavonoids were able to inhibit estragole DNA adduct formation and shift metabolism in favor of glucuronidation which is a detoxification pathway for 1ʹ-hydroxyestragole. In a next step, the kinetics for SULT inhibition were incorporated in physiologically based biokinetic (PBBK) models for estragole in rat and human to predict the effect of co-exposure to estragole and (mixtures of) the different flavonoids on the bioactivation in vivo. The PBBK-model-based predictions indicate that the reduction of estragole bioactivation in rat and human by co-administration of the flavonoids is dependent on whether the intracellular liver concentrations of the flavonoids can reach their Ki values. It is expected that this is most easily achieved for nevadensin which has a Ki value in the nanomolar range and is, due to its methyl ation, more metabolically stable than the other flavonoid

Proceedings of a workshop to address animal methods bias in scientific publishing

Animal methods bias in scientific publishing is a newly defined type of publishing bias describing a preference for animal-based methods where they may not be necessary or where nonanimal-based methods may already be suitable, which impacts the likelihood or timeliness of a manuscript being accepted for publication. This article covers the output from a workshop between stakeholders in publishing, academia, industry, government, and non-governmental organizations. The intent of the workshop was to exchange perspectives on the prevalence, causes, and impact of animal methods bias in scientific publishing, as well as to explore mitigation strategies. Output from the workshop includes summaries of presentations, breakout group discussions, participant polling results, and a synthesis of recommendations for mitigation. Overall, participants felt that animal methods bias has a meaningful impact on scientific publishing, though more evidence is needed to demonstrate its prevalence. Significant consequences of this bias that were identified include the unnecessary use of animals in scientific procedures, the continued reliance on animals in research—even where suitable nonanimal methods exist, poor rates of clinical translation, delays in publication, and negative impacts on career trajectories in science. Workshop participants offered recommendations for journals, publishers, funders, governments, and other policy makers, as well as the scientific community at large, to reduce the prevalence and impacts of animal methods bias. The workshop resulted in the creation of working groups committed to addressing animal methods bias and activities are ongoing

Exposure modelling in Europe : how to pave the road for the future as part of the European Exposure Science Strategy 2020-2030

Exposure models are essential in almost all relevant contexts for exposure science. To address the numerous challenges and gaps that exist, exposure modelling is one of the priority areas of the European Exposure Science Strategy developed by the European Chapter of the International Society of Exposure Science (ISES Europe). A strategy was developed for the priority area of exposure modelling in Europe with four strategic objectives. These objectives are (1) improvement of models and tools, (2) development of new methodologies and support for understudied fields, (3) improvement of model use and (4) regulatory needs for modelling. In a bottom-up approach, exposure modellers from different European countries and institutions who are active in the fields of occupational, population and environmental exposure science pooled their expertise under the umbrella of the ISES Europe Working Group on exposure models. This working group assessed the state-of-the-art of exposure modelling in Europe by developing an inventory of exposure models used in Europe and reviewing the existing literature on pitfalls for exposure modelling, in order to identify crucial modelling-related strategy elements. Decisive actions were defined for ISES Europe stakeholders, including collecting available models and accompanying information in a living document curated and published by ISES Europe, as well as a long-term goal of developing a best-practices handbook. Alongside these actions, recommendations were developed and addressed to stakeholders outside of ISES Europe. Four strategic objectives were identified with an associated action plan and roadmap for the implementation of the European Exposure Science Strategy for exposure modelling. This strategic plan will foster a common understanding of modelling-related methodology, terminology and future research in Europe, and have a broader impact on strategic considerations globally.Peer reviewe

Alternative methods for regulatory toxicology – a state-of-the-art review

This state-of-the art review is based on the final report of a project carried out by the European Commission’s Joint Research Centre (JRC) for the European Chemicals Agency (ECHA). The aim of the project was to review the state of the science of non-standard methods that are available for assessing the toxicological and ecotoxicological properties of chemicals. Non-standard methods refer to alternatives to animal experiments, such as in vitro tests and computational models, as well as animal methods that are not covered by current regulatory guidelines. This report therefore reviews the current scientific status of non-standard methods for a range of human health and ecotoxicological endpoints, and provides a commentary on the mechanistic basis and regulatory applicability of these methods. For completeness, and to provide context, currently accepted (standard) methods are also summarised. In particular, the following human health endpoints are covered: a) skin irritation and corrosion; b) serious eye damage and eye irritation; c) skin sensitisation; d) acute systemic toxicity; e) repeat dose toxicity; f) genotoxicity and mutagenicity; g) carcinogenicity; h) reproductive toxicity (including effects on development and fertility); i) endocrine disruption relevant to human health; and j) toxicokinetics. In relation to ecotoxicological endpoints, the report focuses on non-standard methods for acute and chronic fish toxicity. While specific reference is made to the information needs of REACH, the Biocidal Products Regulation and the Classification, Labelling and Packaging Regulation, this review is also expected to be informative in relation to the possible use of alternative and non-standard methods in other sectors, such as cosmetics and plant protection products.JRC.I.5-Systems Toxicolog