A practice of expert review by read-across using QSAR Toolbox.

The International Council for Harmonisation of Technical Requirement for Pharmaceuticals for Human Use (ICH) M7 guideline on 'Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk' provides the application of two types of quantitative structure-activity relationship (QSAR) systems (rule- and statistics-based) as an alternative to the Ames test for evaluating the mutagenicity of impurities in pharmaceuticals. M7 guideline also states that the expert reviews can be applied when the outcomes of the two QSAR analyses show any conflicting or inconclusive prediction. However, the guideline does not provide any information of how to conduct expert reviews. Therefore, a conservative approach was chosen in this study, which is based on the intention to capture any mutagenic chemical substances. The 36 chemical substances, which are the model chemical substances in which positive mutagenicity was not observed according to the two types of QSAR analyses (i.e. the results are either conflicting or both negative), were selected from the list of chemical substances with strong mutagenicity known as the reported chemicals under the Industrial Safety and Health Act in Japan. The QSAR Toolbox was used in this study to rationally determine the positive mutagenicity of the 36 model chemical substances by applying a read-across method, a technique to evaluate the endpoint of the model chemical substances using the endpoint information of chemicals that are structurally similar to the model chemical substances. Resulting from the expert review by the read-across method, the 23 model chemical substances (63.8%) were rationally concluded as positive. In addition, 9 out of 11 model chemical substances that were assessed as negative for mutagenicity by both of the QSAR systems had positive analogues, supporting their mutagenicity. These results suggested that the read-across is a useful method, when conducting a conservative approach intended to capture any mutagenic chemical substances.

In silico approaches to genetic toxicology: progress and future.

Computational toxicology, also called 'in silico toxicology', is based on scientific knowledge gained from different scientific fields and on the premise that the toxicity of a chemical, depending on its intrinsic nature, can be predicted from its molecular structure and inferred from the properties of similar compounds whose activities are known. With the aim of providing faster, more economical, animal-free tools for predicting toxicity, the 'old' and well established science of Structure-Activity Relationships plays a crucial role, with increasing applications to the assessment of chemical genotoxicity and carcinogenicity. The development of the Structure-Activity Relationships algorithms is a continuous process, and new models, as well as newer versions of applications, are continuously becoming available. This Mutagenesis Special Issue presents a collection of papers on the recent advances in the field, and provides a precious snapshot in time with the most updated information available today.

Predicting Ames Mutagenicity Using Conformal Prediction in the Ames/QSAR International Challenge Project.

Valid and predictive models for classifying Ames mutagenicity have been developed using conformal prediction. The models are Random Forest models using signature molecular descriptors. The investigation indicates, on excluding not-strongly mutagenic compounds (class B), that the validity for mutagenic compounds is increased for the predictions based on both public and the Division of Genetics and Mutagenesis, National Institute of Health Sciences of Japan (DGM/NIHS) data while less so when using only the latter data source. The former models only result in valid predictions for the majority, non-mutagenic, class whereas the latter models are valid for both classes, i.e. mutagenic and non-mutagenic compounds. These results demonstrate the importance of data consistency manifested through the superior predictive quality and validity of the models based only on DGM/NIHS generated data compared to a combination of this data with public data sources.

Critical evaluation of 2-ethylhexyl acrylate dermal carcinogenicity studies using contemporary criteria.

Skin tumors have been observed in C3H/HeJ mice following treatment with high and strongly irritating concentrations of 2-ethylhexyl acrylate (2-EHA). Dermal carcinogenicity studies performed with 2-EHA are reviewed, contrasting the results in two mouse strains (C3H/HeJ and NMRI) under different dosing regimens. Application of contemporary evaluation criteria to the existing dermal carcinogenicity dataset demonstrates that 2-EHA induces skin tumors only at concentrations exceeding an maximum tolerated dose (MTD) and in the immune-dysregulated C3H/HeJ mouse model. Overall, the available chronic toxicity and genotoxicity data on 2-EHA support a non-genotoxic chemical irritant mechanism, whereby chronic irritation leads to inflammation, tissue injury, and wound repair, the latter of which is disrupted in C3H/HeJ mice and leads to tumor formation. Tumor response information in excess of an MTD should not be considered in a human hazard or risk assessment paradigm. For the purposes of an appropriate hazard assessment, 2-EHA did not cause or initiate dermal carcinogenesis in an immune competent (NMRI) mouse model, and, even in the immune compromised C3H/HeJ model, did not induce skin tumors at doses which did not exceed the MTD.

Genotoxicity testing: progress and prospects for the next decade.

INTRODUCTION: Genotoxicity and mutagenicity analyses have a significant role in the identification of hazard effects of therapeutic drugs, cosmetics, agrochemicals, industrial compounds, food additives, natural toxins and nanomaterials for regulatory purposes. To evaluate mutagenicity or genotoxicity, different in vitro and in vivo methodologies exert various genotoxicological endpoints such as point mutations, changes in number and structure of chromosomes. Areas covered: This review covered the basics of genotoxicity and in vitro/in vivo methods for determining of genetic damages. The limitations that have arisen as a result of the common use of these methods were also discussed. Finally, the perspectives of further prospects on the use of genotoxicity testing and genotoxic mode of action were emphasized. Expert opinion: The solution of actual and practical problems of genetic toxicology is inarguably based on the understanding of DNA damage mechanisms at molecular, subcellular, cellular, organ, system and organism levels. Current strategies to investigate human health risks should be modified to increase their performance for more reliable results and also new techniques such as toxicogenomics, epigenomics and single cell approaches must be integrated into genetic safety evolutions. The explored new biomarkers by the omic techniques will provide forceful genotoxicity assessment to reduce the cancer risk.

NanoGenotoxicology: present and the future.

This Mutagenesis special issue is on the topic of nanogenotoxicology. It unites a collection of reports that provide insight into: (i) the properties of engineered nanomaterials (ENMs) that contribute to genotoxicity, (ii) the genotoxic mechanisms associated with DNA damage observed in both in vitro and in vivo tests and (iii) the future test systems that will provide more accurate prediction of ENM genotoxicity to support regulatory hazard assessment frameworks. The contributions within therefore provide collective oversight of our current understanding, coupled to future perspectives aimed at overcoming technical hurdles and describing novel analytical methods to further advance the field.

Advanced Approaches to Model Xenobiotic Metabolism in Bacterial Genotoxicology In Vitro.

During the past 30 years there has been considerable progress in the development of bacterial test systems for use in genotoxicity testing by the stable introduction of expression vectors (cDNAs) coding for xenobiotic-metabolizing enzymes into bacterial cells. The development not only provides insights into the mechanisms of bioactivation of xenobiotic compounds but also evaluates the roles of enzymes involved in metabolic activation or inactivation in chemical carcinogenesis. This review describes recent advances in bacterial genotoxicity assays and their future prospects, with a focus on the development and application of genetically engineering bacterial cells to incorporate some of the enzymatic activities involved in the bio-activation process of xenobiotics. Various genes have been introduced into bacterial umu tester strains encoding enzymes for genotoxic bioactivation, including bacterial nitroreductase and O-acetyltransferase, human cytochrome P450 monooxygenases, rat glutathione S-transferases, and human N-acetyltransferases and sulfotransferases. Their application has provided new tools for genotoxicity assays and for studying the role of biotransformation in chemical carcinogenesis in humans.

Next generation testing strategy for assessment of genomic damage: A conceptual framework and considerations.

For several decades, regulatory testing schemes for genetic damage have been standardized where the tests being utilized examined mutations and structural and numerical chromosomal damage. This has served the genetic toxicity community well when most of the substances being tested were amenable to such assays. The outcome from this testing is usually a dichotomous (yes/no) evaluation of test results, and in many instances, the information is only used to determine whether a substance has carcinogenic potential or not. Over the same time period, mechanisms and modes of action (MOAs) that elucidate a wider range of genomic damage involved in many adverse health outcomes have been recognized. In addition, a paradigm shift in applied genetic toxicology is moving the field toward a more quantitative dose-response analysis and point-of-departure (PoD) determination with a focus on risks to exposed humans. This is directing emphasis on genomic damage that is likely to induce changes associated with a variety of adverse health outcomes. This paradigm shift is moving the testing emphasis for genetic damage from a hazard identification only evaluation to a more comprehensive risk assessment approach that provides more insightful information for decision makers regarding the potential risk of genetic damage to exposed humans. To enable this broader context for examining genetic damage, a next generation testing strategy needs to take into account a broader, more flexible approach to testing, and ultimately modeling, of genomic damage as it relates to human exposure. This is consistent with the larger risk assessment context being used in regulatory decision making. As presented here, this flexible approach for examining genomic damage focuses on testing for relevant genomic effects that can be, as best as possible, associated with an adverse health effect. The most desired linkage for risk to humans would be changes in loci associated with human diseases, whether in somatic or germ cells. The outline of a flexible approach and associated considerations are presented in a series of nine steps, some of which can occur in parallel, which was developed through a collaborative effort by leading genetic toxicologists from academia, government, and industry through the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Genetic Toxicology Technical Committee (GTTC). The ultimate goal is to provide quantitative data to model the potential risk levels of substances, which induce genomic damage contributing to human adverse health outcomes. Any good risk assessment begins with asking the appropriate risk management questions in a planning and scoping effort. This step sets up the problem to be addressed (e.g., broadly, does genomic damage need to be addressed, and if so, how to proceed). The next two steps assemble what is known about the problem by building a knowledge base about the substance of concern and developing a rational biological argument for why testing for genomic damage is needed or not. By focusing on the risk management problem and potential genomic damage of concern, the next step of assay(s) selection takes place. The work-up of the problem during the earlier steps provides the insight to which assays would most likely produce the most meaningful data. This discussion does not detail the wide range of genomic damage tests available, but points to types of testing systems that can be very useful. Once the assays are performed and analyzed, the relevant data sets are selected for modeling potential risk. From this point on, the data are evaluated and modeled as they are for any other toxicology endpoint. Any observed genomic damage/effects (or genetic event(s)) can be modeled via a dose-response analysis and determination of an estimated PoD. When a quantitative risk analysis is needed for decision making, a parallel exposure assessment effort is performed (exposure assessment is not detailed here as this is not the focus of this discussion; guidelines for this assessment exist elsewhere). Then the PoD for genomic damage is used with the exposure information to develop risk estimations (e.g., using reference dose (RfD), margin of exposure (MOE) approaches) in a risk characterization and presented to risk managers for informing decision making. This approach is applicable now for incorporating genomic damage results into the decision-making process for assessing potential adverse outcomes in chemically exposed humans and is consistent with the ILSI HESI Risk Assessment in the 21st Century (RISK21) roadmap. This applies to any substance to which humans are exposed, including pharmaceuticals, agricultural products, food additives, and other chemicals. It is time for regulatory bodies to incorporate the broader knowledge and insights provided by genomic damage results into the assessments of risk to more fully understand the potential of adverse outcomes in chemically exposed humans, thus improving the assessment of risk due to genomic damage. The historical use of genomic damage data as a yes/no gateway for possible cancer risk has been too narrowly focused in risk assessment. The recent advances in assaying for and understanding genomic damage, including eventually epigenetic alterations, obviously add a greater wealth of information for determining potential risk to humans. Regulatory bodies need to embrace this paradigm shift from hazard identification to quantitative analysis and to incorporate the wider range of genomic damage in their assessments of risk to humans. The quantitative analyses and methodologies discussed here can be readily applied to genomic damage testing results now. Indeed, with the passage of the recent update to the Toxic Substances Control Act (TSCA) in the US, the new generation testing strategy for genomic damage described here provides a regulatory agency (here the US Environmental Protection Agency (EPA), but suitable for others) a golden opportunity to reexamine the way it addresses risk-based genomic damage testing (including hazard identification and exposure). Environ. Mol. Mutagen. 58:264-283, 2017. © 2016 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.

Toxicogenomics in Environmental Science.

This chapter reviews the current knowledge and recent progress in the field of environmental, aquatic ecotoxicogenomics with a focus on transcriptomic methods. In ecotoxicogenomics the omics technologies are applied for the detection and assessment of adverse effects in the environment, and thus are to be distinguished from omics used in human toxicology [Snape et al., Aquat Toxicol 67:143-154, 2004]. Transcriptomic methods in ecotoxicology are applied to gain a mechanistic understanding of toxic effects on organisms or populations, and thus aim to bridge the gap between cause and effect. A worthwhile effect-based interpretation of stressor induced changes on the transcriptome is based on the principle of phenotypic-anchoring [Paules, Environ Health Perspect 111:A338-A339, 2003]. Thereby, changes on the transcriptomic level can only be identified as effects if they are clearly linked to a specific stressor-induced effect on the macroscopic level. By integrating those macroscopic and transcriptomic effects, conclusions on the effect-inducing type of the stressor can be drawn. Stressor-specific effects on the transcriptomic level can be identified as stressor-specific induced pathways, transcriptomic patterns, or stressors-specific genetic biomarkers. In this chapter, examples of the combined application of macroscopic and transcriptional effects for the identification of environmental stressors, such as aquatic pollutants, are given and discussed. By means of these examples, challenges on the way to a standardized application of transcriptomics in ecotoxicology are discussed. This is also done against the background of the application of transcriptomic methods in environmental regulation such as the EU regulation Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Aplicación de un sistema in silico múltiple para la priorización en la selección de compuestos antimaláricos / Multiple in silico genetic toxicity alerting tools for antimalarial compounds prioritization

Introducción: Como parte del compromiso de GSK en la reducción del fracaso en las fases de desarrollo clínico y preclínico, se ha implementado en las fases más tempranas de desarrollo una estrategia para evitar los problemas de genotoxicidad que son los que, en mayor manera, pueden obstaculizar la progresión a fases más avanzadas. El objetivo de este trabajo es aplicar la nueva estrategia de GSK para la priorización de compuestos que permita seleccionar aquellas estructuras con menor riesgo de genotoxicidad utilizando una combinación de herramientas computacionales que predice el resultado del test de Ames. Materiales y Métodos: Compuestos de la colección de GSK, activos en el screening fenotípico frente a P. falciparum, fueron utilizados en este estudio. Tres modelos, Derek Nexus (Lhasa Limited, Leeds, UK), Leadscope y un método de mecánica cuántica desarrollado internamente se utilizaron para las predicciones in silico. Resultados: la combinación de los tres modelos de predicción tuvo un porcentaje de éxito del 75% con sólo 1 falso positivo. Conclusiones: Moléculas con 2 o más alertas de genotoxicidad generadas por este sistema múltiple deberían ser despriorizadas o ensayadas experimentalmente cuanto antes para descartar su riesgo de genotoxicidad (AU)

Introduction: As part of the commitment of GSK in reducing attrition rate in clinical and preclinical stages, it has been set up in early stages of development (H2L, Lead Op) a candidate quality strategy to avoid genotoxicity liabilities that mainly can stop the progression of compound towards advanced stages. The aim of the study is to apply the new strategy in order to triage structures with less genotoxicity risk by means an in silico multiple system that predicts the outcome of Ames test. Material & Methods: Active compounds against P. falciparum phenotypic screening from GSK collection were used. Three different models: Derek Nexus (Lhasa Limited, Leeds, UK), Leadscope and a quantum mechanics method developed internally were used for in silico predictions. Results: The use of three models have an accurately success rate, greater than 75% with only 1 false positives. Conclusions: Those molecules that fire 2 or more genotoxicity alerts should be deprioritised or tested experimentally in Ames test to confirm or discharge the genotoxicity risk (AU)

Where will genetic toxicology testing be in 30 years' time? Summary report of the 25th Industrial Genotoxicity Group Meeting, Royal Society of Medicine, London, November 9, 2011.

A number of influences including legislation, industry and academia have encouraged advances in computational toxicology and high-throughput testing to probe more broadly putative toxicity pathways. The aim of the 25th United Kingdom Mutagen Society (UKEMS) Industrial Genotoxicity Group Annual Meeting 2011 was to explore current and upcoming research tools that may provide new cancer risk estimation approaches and discuss the genotoxicity testing paradigm of the future. The meeting considered whether computer modelling, molecular biology systems and/or adverse outcome pathway approaches can provide more accurate toxicity predictions and whether high-content study data, pluripotent stem cells or new scientific disciplines, such as epigenetics and adductomics, could be integrated into the risk assessment process. With close collaboration between industry, academia and regulators next generation predictive models and high-content tools have the potential to transform genetic toxicology testing in the 21st century.

Incorporating new technologies into toxicity testing and risk assessment: moving from 21st century vision to a data-driven framework.

Based on existing data and previous work, a series of studies is proposed as a basis toward a pragmatic early step in transforming toxicity testing. These studies were assembled into a data-driven framework that invokes successive tiers of testing with margin of exposure (MOE) as the primary metric. The first tier of the framework integrates data from high-throughput in vitro assays, in vitro-to-in vivo extrapolation (IVIVE) pharmacokinetic modeling, and exposure modeling. The in vitro assays are used to separate chemicals based on their relative selectivity in interacting with biological targets and identify the concentration at which these interactions occur. The IVIVE modeling converts in vitro concentrations into external dose for calculation of the point of departure (POD) and comparisons to human exposure estimates to yield a MOE. The second tier involves short-term in vivo studies, expanded pharmacokinetic evaluations, and refined human exposure estimates. The results from the second tier studies provide more accurate estimates of the POD and the MOE. The third tier contains the traditional animal studies currently used to assess chemical safety. In each tier, the POD for selective chemicals is based primarily on endpoints associated with a proposed mode of action, whereas the POD for nonselective chemicals is based on potential biological perturbation. Based on the MOE, a significant percentage of chemicals evaluated in the first 2 tiers could be eliminated from further testing. The framework provides a risk-based and animal-sparing approach to evaluate chemical safety, drawing broadly from previous experience but incorporating technological advances to increase efficiency.

Genotoxicity investigations on nanomaterials.

This review is based on the lecture presented at the April 2010 nanomaterials safety assessment Postsatellite to the 2009 EUROTOX Meeting and summarizes genotoxicity investigations on nanomaterials published in the open scientific literature (up to 2008). Special attention is paid to the relationship between particle size and positive versus negative outcome, as well as the dependence of the outcome on the test used. Salient conclusions and outstanding recommendations emerging from the information summarized in this review are as follows: recognize that nanomaterials are not all the same; therefore know and document what nanomaterial has been tested and in what form; take nanomaterials specific properties into account; in order to make your results comparable with those of others and on other nanomaterials: use or at least include in your studies standardized methods; use in vivo studies to put in vitro results into perspective; take uptake and distribution of the nanomaterial into account; and in order to become able to make extrapolations to risk for human: learn about the mechanism of nanomaterials genotoxic effects. Past experience with standard non-nanosubstances already had shown that mechanisms of genotoxic effects can be complex and their elucidation can be demanding, while there often is an immediate need to assess the genotoxic hazard. Thus, a practical and pragmatic approach to genotoxicity investigations of novel nanomaterials is the use of a battery of standard genotoxicity testing methods covering a wide range of mechanisms. Application of these standard methods to nanomaterials demands, however, adaptations, and the interpretation of results from the genotoxicity testing of nanomaterials needs additional considerations exceeding those used for standard size materials.

Need and potential value of the Pig-ain vivo mutation assay-a HESI perspective.

The Health and Environmental Sciences Institute (HESI), a global branch of the International Life Sciences Institute (ILSI), initiated a project committee entitled "Relevance and Follow-up of Positive Results from In Vitro Genetic Toxicity Testing (IVGT)" with the overall objective of improving the scientific basis for the interpretation of results from genetic toxicology testing. The IVGT committee has also recognized the need to develop follow-up strategies for determining the relevance of in vitro test results to human health, and moving genetic toxicology testing from the sole purpose of hazard identification toward a more quantitative risk assessment approach. In this context, a group of experts evaluated the potential utility of the emerging in vivo mutational assessment model commonly known as the Pig-a gene mutation assay to follow-up positive in vitro genetic toxicology findings and to generate robust dose-response data for quantitative assessment of the in vivo mutagenicity. The IVGT experts participating in this effort represented academia, industry, and government agencies from across the globe and addressed such issues as the optimal sample size and experimental design for generating robust dose-response data. This expert group concluded that the emerging Pig-a gene mutation assay holds great promise as an in vivo mutagenicity assay, either as a stand-alone study or integrated into repeat-dose toxicology studies, and therefore supports further validation of the model.

XIX Congreso Español de Toxicología / No disponible

No disponible

In vitro genotoxicity test approaches with better predictivity: summary of an IWGT workshop.

Improving current in vitro genotoxicity tests is an ongoing task for genetic toxicologists. Further, the question on how to deal with positive in vitro results that are demonstrated to not predict genotoxicity or carcinogenicity potential in rodents or humans is a challenge. These two aspects were addressed at the 5th International Workshop on Genotoxicity Testing (IWGT) held in Basel, Switzerland, on August 17-19, 2009. The objectives of the working group (WG) were to make recommendations on the use of cell types or lines, if possible, and to provide evaluations of promising new approaches. Results obtained in rodent cell lines with impaired p53 function (L5178Y, V79, CHL and CHO cells) and human p53-competent cells (peripheral blood lymphocytes, TK6 and HepG2 cells) suggest that a reduction in the percentage of non-relevant positive results for carcinogenicity prediction can be achieved by careful selection of cells used without decreasing the sensitivity of the assays. Therefore, the WG suggested using p53- competent - preferably human - cells in in vitro micronucleus or chromosomal aberration tests. The use of the hepatoma cell line HepaRG for genotoxicity testing was considered promising since these cells possess better phase I and II metabolizing potential compared to cell lines commonly used in this area and may overcome the need for the addition of S9. For dermally applied compounds, the WG agreed that in vitro reconstructed skin models, once validated, will be useful to follow up on positive results from standard in vitro assays as they resemble the properties of human skin (barrier function, metabolism). While the reconstructed skin micronucleus assay has been shown to be further advanced, there was also consensus that the Comet assay should be further evaluated due to its independence from cell proliferation and coverage of a wider spectrum of DNA damage.

Follow-up actions from positive results of in vitro genetic toxicity testing.

Appropriate follow-up actions and decisions are needed when evaluating and interpreting clear positive results obtained in the in vitro assays used in the initial genotoxicity screening battery (i.e., the battery of tests generally required by regulatory authorities) to assist in overall risk-based decision making concerning the potential effects of human exposure to the agent under test. Over the past few years, the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing developed a decision process flow chart to be applied in case of clear positive results in vitro. It provides for a variety of different possibilities and allows flexibility in choosing follow-up action(s), depending on the results obtained in the initial battery of assays and available information. The intent of the Review Subgroup was not to provide a prescriptive testing strategy, but rather to reinforce the concept of weighing the totality of the evidence. The Review Subgroup of the IVGT committee highlighted the importance of properly analyzing the existing data, and considering potential confounding factors (e.g., possible interactions with the test systems, presence of impurities, irrelevant metabolism), and chemical modes of action when analyzing and interpreting positive results in the in vitro genotoxicity assays and determining appropriate follow-up testing. The Review Subgroup also examined the characteristics, strengths, and limitations of each of the existing in vitro and in vivo genotoxicity assays to determine their usefulness in any follow-up testing.