Principles and Procedures for Assessment of Acute Toxicity Incorporating In Silico Methods.

Acute toxicity in silico models are being used to support an increasing number of application areas including (1) product research and development, (2) product approval and registration as well as (3) the transport, storage and handling of chemicals. The adoption of such models is being hindered, in part, because of a lack of guidance describing how to perform and document an in silico analysis. To address this issue, a framework for an acute toxicity hazard assessment is proposed. This framework combines results from different sources including in silico methods and in vitro or in vivo experiments. In silico methods that can assist the prediction of in vivo outcomes (i.e., LD50) are analyzed concluding that predictions obtained using in silico approaches are now well-suited for reliably supporting assessment of LD50-based acute toxicity for the purpose of GHS classification. A general overview is provided of the endpoints from in vitro studies commonly evaluated for predicting acute toxicity (e.g., cytotoxicity/cytolethality as well as assays targeting specific mechanisms). The increased understanding of pathways and key triggering mechanisms underlying toxicity and the increased availability of in vitro data allow for a shift away from assessments solely based on endpoints such as LD50, to mechanism-based endpoints that can be accurately assessed in vitro or by using in silico prediction models. This paper also highlights the importance of an expert review of all available information using weight-of-evidence considerations and illustrates, using a series of diverse practical use cases, how in silico approaches support the assessment of acute toxicity.

A cross-industry collaboration to assess if acute oral toxicity (Q)SAR models are fit-for-purpose for GHS classification and labelling.

This study assesses whether currently available acute oral toxicity (AOT) in silico models, provided by the widely employed Leadscope software, are fit-for-purpose for categorization and labelling of chemicals. As part of this study, a large data set of proprietary and marketed compounds from multiple companies (pharmaceutical, plant protection products, and other chemical industries) was assembled to assess the models' performance. The absolute percentage of correct or more conservative predictions, based on a comparison of experimental and predicted GHS categories, was approximately 95%, after excluding a small percentage of inconclusive (indeterminate or out of domain) predictions. Since the frequency distribution across the experimental categories is skewed towards low toxicity chemicals, a balanced assessment was also performed. Across all compounds which could be assigned to a well-defined experimental category, the average percentage of correct or more conservative predictions was around 80%. These results indicate the potential for reliable and broad application of these models across different industrial sectors. This manuscript describes the evaluation of these models, highlights the importance of an expert review, and provides guidance on the use of AOT models to fulfill testing requirements, GHS classification/labelling, and transportation needs.

In Silico Approaches In Carcinogenicity Hazard Assessment: Current Status and Future Needs.

Historically, identifying carcinogens has relied primarily on tumor studies in rodents, which require enormous resources in both money and time. In silico models have been developed for predicting rodent carcinogens but have not yet found general regulatory acceptance, in part due to the lack of a generally accepted protocol for performing such an assessment as well as limitations in predictive performance and scope. There remains a need for additional, improved in silico carcinogenicity models, especially ones that are more human-relevant, for use in research and regulatory decision-making. As part of an international effort to develop in silico toxicological protocols, a consortium of toxicologists, computational scientists, and regulatory scientists across several industries and governmental agencies evaluated the extent to which in silico models exist for each of the recently defined 10 key characteristics (KCs) of carcinogens. This position paper summarizes the current status of in silico tools for the assessment of each KC and identifies the data gaps that need to be addressed before a comprehensive in silico carcinogenicity protocol can be developed for regulatory use.

Dietary intake of phloridzin from natural occurrence in foods.

Type 2 diabetes mellitus (T2DM) is one of the major diseases of our times. Besides being a considerable inconvenience for the patient, the associated healthcare expenses are tremendous. One of the cornerstones of T2DM prevention is a healthy diet, including a variety of fruits and vegetables. Apples are touted to have health benefits, and the apple polyphenol, phloridzin, has gained interest in recent years as it can reduce intestinal sugar uptake by inhibition of the Na/glucose cotransporter 1. By researching the amount of phloridzin in different food sources and linking them to their consumption data, we could estimate the average and high-level phloridzin consumption in Europe. On average, European people consume 0·7-7·5 mg/d phloridzin, the main contributors being apples and apple juice. High-level consumers may get up to 52 mg/d of phloridzin. Older people are more at risk of developing T2DM, yet they consume less phloridzin than adolescents and adults, as determined by our survey. Management of blood glucose levels might be improved by the consumption of phloridzin, as has been shown in recent clinical trials; these trials used phloridzin-enriched apple extract at doses exceeding those from normal food consumption. There are, however, indications that consumption of average to high levels of phloridzin via food might also contribute to reduced sugar load and a reduction in T2DM risk.

Genetic toxicology in silico protocol.

In silico toxicology (IST) approaches to rapidly assess chemical hazard, and usage of such methods is increasing in all applications but especially for regulatory submissions, such as for assessing chemicals under REACH as well as the ICH M7 guideline for drug impurities. There are a number of obstacles to performing an IST assessment, including uncertainty in how such an assessment and associated expert review should be performed or what is fit for purpose, as well as a lack of confidence that the results will be accepted by colleagues, collaborators and regulatory authorities. To address this, a project to develop a series of IST protocols for different hazard endpoints has been initiated and this paper describes the genetic toxicity in silico (GIST) protocol. The protocol outlines a hazard assessment framework including key effects/mechanisms and their relationships to endpoints such as gene mutation and clastogenicity. IST models and data are reviewed that support the assessment of these effects/mechanisms along with defined approaches for combining the information and evaluating the confidence in the assessment. This protocol has been developed through a consortium of toxicologists, computational scientists, and regulatory scientists across several industries to support the implementation and acceptance of in silico approaches.

In silico toxicology protocols.

The present publication surveys several applications of in silico (i.e., computational) toxicology approaches across different industries and institutions. It highlights the need to develop standardized protocols when conducting toxicity-related predictions. This contribution articulates the information needed for protocols to support in silico predictions for major toxicological endpoints of concern (e.g., genetic toxicity, carcinogenicity, acute toxicity, reproductive toxicity, developmental toxicity) across several industries and regulatory bodies. Such novel in silico toxicology (IST) protocols, when fully developed and implemented, will ensure in silico toxicological assessments are performed and evaluated in a consistent, reproducible, and well-documented manner across industries and regulatory bodies to support wider uptake and acceptance of the approaches. The development of IST protocols is an initiative developed through a collaboration among an international consortium to reflect the state-of-the-art in in silico toxicology for hazard identification and characterization. A general outline for describing the development of such protocols is included and it is based on in silico predictions and/or available experimental data for a defined series of relevant toxicological effects or mechanisms. The publication presents a novel approach for determining the reliability of in silico predictions alongside experimental data. In addition, we discuss how to determine the level of confidence in the assessment based on the relevance and reliability of the information.

Making reliable negative predictions of human skin sensitisation using an in silico fragmentation approach.

A previously published fragmentation method for making reliable negative in silico predictions has been applied to the problem of predicting skin sensitisation in humans, making use of a dataset of over 2750 chemicals with publicly available skin sensitisation data from 18 in vivo assays. An assay hierarchy was designed to enable the classification of chemicals within this dataset as either sensitisers or non-sensitisers where data from more than one in vivo test was available. The negative prediction approach was validated internally, using a 5-fold cross-validation, and externally, against a proprietary dataset of approximately 1000 chemicals with in vivo reference data shared by members of the pharmaceutical, nutritional, and personal care industries. The negative predictivity for this proprietary dataset was high in all cases (>75%), and the model was also able to identify structural features that resulted in a lower accuracy or a higher uncertainty in the negative prediction, termed misclassified and unclassified features respectively. These features could serve as an aid for further expert assessment of the negative in silico prediction.

Applicability of in silico genotoxicity models on food and feed ingredients.

Evaluation of the genotoxic potential of food and feed ingredients is required in the development of new substances and for their registration. In addition to in vitro and in vivo assays, in silico tools such as expert alert-based and statistical models can be used for data generation. These in silico models are commonly used among the pharmaceutical industry, whereas the food industry has not widely adopted them. In this study, the applicability of in silico tools for predicting genotoxicity was evaluated, with a focus on bacterial mutagenicity, in vitro and in vivo chromosome damage assays. For this purpose, a test set of 27 food and feed ingredients including vitamins, carotenoids, and nutraceuticals with experimental genotoxicity data was constructed from proprietary data. This dataset was run through multiple models and the model applicability was analyzed. The compounds were generally within the applicability domain of the models and the models predicted the compounds correctly in most of the cases. Although the regulatory acceptance of in silico tools as single data source is still limited, the models are applicable and can be used in the safety evaluation of food and feed ingredients.

Review of genotoxicity and rat carcinogenicity investigations with astaxanthin.

Synthetic astaxanthin has been extensively tested for safety. Genotoxicity studies including Ames and in vitro Micronucleus Tests show absence of genotoxic potential. Although a long-term mouse study showed no carcinogenicity potential, the rat carcinogenicity study with dietary dosages of 0 (control), 0 (placebo beadlet), 40, 200 and 1000 mg astaxanthin/kg bw/day showed an increased incidence of benign, hepatocellular adenoma in females only, at 200 mg/kg bw/day and above. There was no clear evidence of toxicity during the in-life phase. Discoloration of feces was observed and a reduction in body weight gain in all groups receiving beadlets, probably reflecting a nutritional influence. Blood sampling confirmed systemic exposure and some minor clinical chemistry differences in females at 200 and 1000 mg/kg bw/day. There was no effect on adjusted liver weight. Histopathological examination showed hepatic changes indicative of slight hepatotoxicity and hepatocyte regeneration in females at 200 and 1000 mg/kg bw/day, in addition to the adenoma. Taking into account this pathological background in the female rat, and a wide variety of other supporting information, it is concluded that the hepatocellular adenoma in female rats was secondary to hepatotoxicity and regeneration, and is most probably a species-specific phenomenon of doubtful human relevance.

Polyphenolic apple extracts: effects of raw material and production method on antioxidant effectiveness and reduction of DNA damage in Caco-2 cells.

A diet rich in fruits and vegetables is commonly perceived to be associated with reduced cancer risk, attributed to its high content of polyphenols. As apples represent a major polyphenol source in Western countries, we studied differentially produced extracts (1-100 microg/mL): two from different apple juices (AEs), one from pomace (APE), and one peel extract (PE) on their potential to reduce DNA oxidation damage and induce antioxidant defense in Caco-2 cells. Additionally, we measured direct antioxidant capacity (TEAC/ORAC) of the extracts. Quercetin-rich PE and APE most effectively diminished DNA damage and ROS level after 24 h incubation (PE > APE), whereas the AEs were only moderately effective. GPx activity was diminished for all extracts, with AEs > APE > PE. Direct antioxidant activity decreased in the order AEs > PE > APE, displaying no significant correlation with cellular markers. In conclusion, apple phenolics at low, nutritionally relevant concentrations may protect intestinal cells from ROS-induced DNA damage, mediated by cellular defense mechanisms rather than by antioxidant activity.

Formation of hydrogen peroxide in cell culture media by apple polyphenols and its effect on antioxidant biomarkers in the colon cell line HT-29.

Beneficial health effects of diets containing fruits have partly been attributed to polyphenols which display a spectrum of bioactive effects, including antioxidant activity. However, polyphenols can also exert prooxidative effects in vitro. In this study, polyphenol-mediated hydrogen peroxide (H(2)O(2)) formation was determined after incubation of apple juice extracts (AEs) and polyphenols in cell culture media. Effects of extracellular H(2)O(2 )on total glutathione (tGSH; =GSH + GSSG) and cellular reactive oxygen species (ROS) level of HT-29 cells were studied by coincubation +/- catalase (CAT). AEs ( > or =30 microg/mL) significantly generated H(2)O(2) in DMEM, depending on their composition. Similarly, H(2)O(2) was measured for individual apple polyphenols/degradation products (phenolic acids > epicatechin, flavonols > dihydrochalcones). Highest concentrations were generated by compounds bearing the o-catechol moiety. H(2)O(2) formation was found to be pH dependent; addition of CAT caused a complete decomposition of H(2)O(2) whereas superoxide dismutase was less/not effective. At incubation of HT-29 cells with quercetin (1-100 microM), generated H(2)O(2) slightly contributed to antioxidant cell protection by modulation of tGSH- and ROS-level. In conclusion, H(2)O(2) generation in vitro by polyphenols has to be taken into consideration when interpreting results of such cell culture experiments. Unphysiologically high polyphenol concentrations, favoring substantial H(2)O(2 )formation, are not expected to be met in vivo, even under conditions of high end nutritional uptake.

Antioxidant effectiveness of phenolic apple juice extracts and their gut fermentation products in the human colon carcinoma cell line caco-2.

Apples represent a major dietary source of antioxidative polyphenols. Their metabolic conversion by the gut microflora might generate products that protect the intestine against oxidative damage. We studied the antioxidant effectiveness of supernatants of fermented apple juice extracts (F-AEs, 6 and 24 h fermentation) and of selected phenolic degradation products, identified by HPLC-DAD-ESI-MS. Cell free antioxidant capacity of unfermented apple juice extracts (AEs) was decreased after fermentation by 30-50%. In the human colon carcinoma cell line Caco-2, F-AEs (containing <0.5% of original AE-phenolics) decreased the reactive oxygen species (ROS) level more efficiently than the F-blank (fermented without AE) but were less effective than the respective AEs. Similarly, antioxidant effectiveness of individual degradation products was lower compared to respective AE constituents. Glutathione level was slightly increased and oxidative DNA damage slightly decreased by fermented AE03, rich in quercetin glycosides. In conclusion, F-AEs/degradation products exhibit antioxidant activity in colon cells but to a lesser extent than the respective unfermented AEs/constituents.