Current status and future challenges of genotoxicity OECD Test Guidelines for nanomaterials: a workshop report.

Genotoxicity testing for nanomaterials remains challenging as standard testing approaches require some adaptation, and further development of nano-specific OECD Test Guidelines (TGs) and Guidance Documents (GDs) are needed. However, the field of genotoxicology continues to progress and new approach methodologies (NAMs) are being developed that could provide relevant information on the range of mechanisms of genotoxic action that may be imparted by nanomaterials. There is a recognition of the need for implementation of new and/or adapted OECD TGs, new OECD GDs, and utilization of NAMs within a genotoxicity testing framework for nanomaterials. As such, the requirements to apply new experimental approaches and data for genotoxicity assessment of nanomaterials in a regulatory context is neither clear, nor used in practice. Thus, an international workshop with representatives from regulatory agencies, industry, government, and academic scientists was convened to discuss these issues. The expert discussion highlighted the current deficiencies that exist in standard testing approaches within exposure regimes, insufficient physicochemical characterization, lack of demonstration of cell or tissue uptake and internalization, and limitations in the coverage of genotoxic modes of action. Regarding the latter aspect, a consensus was reached on the importance of using NAMs to support the genotoxicity assessment of nanomaterials. Also highlighted was the need for close engagement between scientists and regulators to (i) provide clarity on the regulatory needs, (ii) improve the acceptance and use of NAM-generated data, and (iii) define how NAMs may be used as part of weight of evidence approaches for use in regulatory risk assessments.

Evaluation of the applicability of existing (Q)SAR models for predicting the genotoxicity of pesticides and similarity analysis related with genotoxicity of pesticides for facilitating of grouping and read across: An EFSA funded project.

To facilitate the practical implementation of the guidance on the residue definition for dietary risk assessment, EFSA has organized an evaluation of applicability of existing in silico models for predicting the genotoxicity of pesticides and their metabolites, including literature survey, application of QSARs and development of Read Across methodologies. This paper summarizes the main results. For the Ames test, all (Q)SAR models generated statistically significant predictions, comparable with the experimental variability of the test. The reliability of the models for other assays/endpoints appears to be still far from optimality. Two new Read Across approaches were evaluated: Read Across was largely successful for predicting the Ames test results, but less for in vitro Chromosomal Aberrations. The worse results for non-Ames endpoints may be attributable to the several revisions of experimental protocols and evaluation criteria of results, that have made the databases qualitatively non-homogeneous and poorly suitable for modeling. Last, Parent/Metabolite structural differences (besides known Structural Alerts) that may, or may not cause changes in the Ames mutagenicity were identified and catalogued. The findings from this work are suitable for being integrated into Weight-of-Evidence and Tiered evaluation schemes. Areas needing further developments are pointed out.

Data-based review of QSARs for predicting genotoxicity: the state of the art.

With the aim of providing faster, more economical, animal-free tools to predict toxicity, quantitative structure-activity relationships (QSAR) approaches are increasingly applied to the chemical risk assessment-in particular genotoxicity and carcinogenicity. The more recent period of time has witnessed refinements of the predictive systems, with the collection of larger training sets and continued fine-tuning, together with an increased interest for the use of QSAR by regulatory authorities. This literature review provides an updated snapshot of the present state of the art in the evaluation of QSAR methods as applied to genotoxicity. Overall, the abilities of software tools to predict Ames test mutagenicity were comparable with previously published evaluations, with sensitivity ranging 0.72-0.96, and specificity ranging 0.65-0.86 in applications to public data sets. These values compare quite fairly with the intrinsic variability of the Ames test. A preliminary analysis indicated a consistency with the results of the Japan Division of Genetics and Mutagenesis, National Institute of Health Sciences of Japan (DGM/NIHS) Ames/QSAR international collaborative project. Applications to a variety of external test sets pointed to the need of further improvements of the coverage/representation of the whole chemical space. Combinations of tools showed that sensitivity is usually increased at the expense of a decrease in specificity, whereas the supervision by expert judgement generated more equilibrated results. This points to the existence of a large area of context-dependent expert knowledge, which has not been formalised yet and has the potential to substantially improve the prediction systems. The overall evidence suggests that (Q)SARs for the Ames test have sufficient reliability for use in prioritisation processes as well as to support regulatory decisions in combination with other evidence. The use of highly sensitive genotoxicity QSARs in tiered integrations with other tools is suggested as a mean to shortlist chemicals for which no further testing is necessary.

Improvement of quantitative structure-activity relationship (QSAR) tools for predicting Ames mutagenicity: outcomes of the Ames/QSAR International Challenge Project.

The International Conference on Harmonization (ICH) M7 guideline allows the use of in silico approaches for predicting Ames mutagenicity for the initial assessment of impurities in pharmaceuticals. This is the first international guideline that addresses the use of quantitative structure-activity relationship (QSAR) models in lieu of actual toxicological studies for human health assessment. Therefore, QSAR models for Ames mutagenicity now require higher predictive power for identifying mutagenic chemicals. To increase the predictive power of QSAR models, larger experimental datasets from reliable sources are required. The Division of Genetics and Mutagenesis, National Institute of Health Sciences (DGM/NIHS) of Japan recently established a unique proprietary Ames mutagenicity database containing 12140 new chemicals that have not been previously used for developing QSAR models. The DGM/NIHS provided this Ames database to QSAR vendors to validate and improve their QSAR tools. The Ames/QSAR International Challenge Project was initiated in 2014 with 12 QSAR vendors testing 17 QSAR tools against these compounds in three phases. We now present the final results. All tools were considerably improved by participation in this project. Most tools achieved >50% sensitivity (positive prediction among all Ames positives) and predictive power (accuracy) was as high as 80%, almost equivalent to the inter-laboratory reproducibility of Ames tests. To further increase the predictive power of QSAR tools, accumulation of additional Ames test data is required as well as re-evaluation of some previous Ames test results. Indeed, some Ames-positive or Ames-negative chemicals may have previously been incorrectly classified because of methodological weakness, resulting in false-positive or false-negative predictions by QSAR tools. These incorrect data hamper prediction and are a source of noise in the development of QSAR models. It is thus essential to establish a large benchmark database consisting only of well-validated Ames test results to build more accurate QSAR models.

Endocrine Disruptors: Data-based survey of in vivo tests, predictive models and the Adverse Outcome Pathway.

The protection from endocrine disruptors is a high regulatory priority. Key issues are the characterization of in vivo assays, and the identification of reference chemicals to validate alternative methods. In this exploration, publicly available databases for in vivo assays for endocrine disruption were collected and compared: Rodent Uterotrophic, Rodent Repeated Dose 28-day Oral Toxicity, 21-Day Fish, and Daphnia magna reproduction assays. Only the Uterotrophic and 21-Day Fish assays results correlated with each other. The in vivo assays data were viewed in relation to the Adverse Outcome Pathway, using as a probe 18 ToxCast in vitro assays for the ER pathway. These are the same data at the basis of the EPA agonist ToxERscore model, whose good predictivity was confirmed. The multivariate comparison of the in vitro/in vivo assays suggests that the interaction with receptors is a major determinant of in vivo results, and is the critical basis for building predictive computational models. In agreement with the above, this work also shows that it is possible to build predictive models for the Uterotrophic and 21-Day Fish assays using a limited selection of Toxcast assays.

Formation and Repair of Mismatches Containing Ribonucleotides and Oxidized Bases at Repeated DNA Sequences.

The cellular pool of ribonucleotide triphosphates (rNTPs) is higher than that of deoxyribonucleotide triphosphates. To ensure genome stability, DNA polymerases must discriminate against rNTPs and incorporated ribonucleotides must be removed by ribonucleotide excision repair (RER). We investigated DNA polymerase ß (POL ß) capacity to incorporate ribonucleotides into trinucleotide repeated DNA sequences and the efficiency of base excision repair (BER) and RER enzymes (OGG1, MUTYH, and RNase H2) when presented with an incorrect sugar and an oxidized base. POL ß incorporated rAMP and rCMP opposite 7,8-dihydro-8-oxoguanine (8-oxodG) and extended both mispairs. In addition, POL ß was able to insert and elongate an oxidized rGMP when paired with dA. We show that RNase H2 always preserves the capacity to remove a single ribonucleotide when paired to an oxidized base or to incise an oxidized ribonucleotide in a DNA duplex. In contrast, BER activity is affected by the presence of a ribonucleotide opposite an 8-oxodG. In particular, MUTYH activity on 8-oxodG:rA mispairs is fully inhibited, although its binding capacity is retained. This results in the reduction of RNase H2 incision capability of this substrate. Thus complex mispairs formed by an oxidized base and a ribonucleotide can compromise BER and RER in repeated sequences.

A data-based exploration of the adverse outcome pathway for skin sensitization points to the necessary requirements for its prediction with alternative methods.

This paper presents new data-based analyses on the ability of alternative methods to predict the skin sensitization potential of chemicals. It appears that skin sensitization, as shown in humans and rodents, can be predicted with good accuracy both with in vitro assays and QSAR approaches. The accuracy is about the same: 85-90%. Given that every biological measure has inherent uncertainty, this performance is quite remarkable. Overall, there is a good correlation between human data and experimental in vivo systems, except for sensitizers of intermediate potency. This uncertainty/variability is probably the reason why alternative methods are quite efficient in predicting both strong and non-sensitizers, but not the intermediate potency sensitizers. A detailed analysis of the predictivity of the individual approaches shows that the biological in vitro assays have limited added value in respect to the in chemico/QSAR ones, and suggests that the primary interaction with proteins is the rate-limiting step of the entire process. This confirms evidence from other fields (e.g., carcinogenicity, QSAR) indicating that successful predictive models are based on the parameterization of a few mechanistic features/events, whereas the consideration of all events supposedly involved in a toxicity pathway contributes to increase the uncertainty of the predictions.

Alternative Toxicity Testing: Analyses on Skin Sensitization, ToxCast Phases I and II, and Carcinogenicity Provide Indications on How to Model Mechanisms Linked to Adverse Outcome Pathways.

This article studies alternative toxicological approaches, with new (skin sensitization, ToxCast) and previous (carcinogenicity) analyses. Quantitative modeling of rate-limiting steps in skin sensitization and carcinogenicity predicts the majority of toxicants. Similarly, successful (Quantitative) Structure-Activity Relationships models exploit the quantification of only one, or few rate-limiting steps. High-throughput assays within ToxCast point to promising associations with endocrine disruption, whereas markers for pathways intermediate events have limited correlation with most endpoints. Since the pathways may be very different (often not simple linear chains of events), quantitative analysis is necessary to identify the type of mechanism and build the appropriate model.

A template wizard for the cocreation of machine-readable data-reporting to harmonize the evaluation of (nano)materials.

Making research data findable, accessible, interoperable and reusable (FAIR) is typically hampered by a lack of skills in technical aspects of data management by data generators and a lack of resources. We developed a Template Wizard for researchers to easily create templates suitable for consistently capturing data and metadata from their experiments. The templates are easy to use and enable the compilation of machine-readable metadata to accompany data generation and align them to existing community standards and databases, such as eNanoMapper, streamlining the adoption of the FAIR principles. These templates are citable objects and are available as online tools. The Template Wizard is designed to be user friendly and facilitates using and reusing existing templates for new projects or project extensions. The wizard is accompanied by an online template validator, which allows self-evaluation of the template (to ensure mapping to the data schema and machine readability of the captured data) and transformation by an open-source parser into machine-readable formats, compliant with the FAIR principles. The templates are based on extensive collective experience in nanosafety data collection and include over 60 harmonized data entry templates for physicochemical characterization and hazard assessment (cell viability, genotoxicity, environmental organism dose-response tests, omics), as well as exposure and release studies. The templates are generalizable across fields and have already been extended and adapted for microplastics and advanced materials research. The harmonized templates improve the reliability of interlaboratory comparisons, data reuse and meta-analyses and can facilitate the safety evaluation and regulation process for (nano) materials.

In vitro cell transformation assays for an integrated, alternative assessment of carcinogenicity: a data-based analysis.

The study of the chemical carcinogenesis mechanisms and the design of efficient prevention strategies and measures are of crucial importance to protect human health. The long-term carcinogenesis bioassays have played a central role in protecting human health, but for ethical and practical reasons their use is dramatically diminishing, and the genotoxicity short-term tests have taken the pivotal role in the pre-screening of carcinogenicity. However, there is evidence that this strategy is not sensitive enough to detect all genotoxic carcinogens and it cannot detect nongenotoxic carcinogens. In a previous article, we have shown that an integrated strategy consisting of the in vitro Ames and Syrian Hamster Embryo cells transformation assays, combined with structure-activity relationships, is a valid alternative to the present pre-screening strategies. Here, we expand the previous investigation by (i) including results of cell transformation assays on inorganics, together with an additional assay (Bhas 42), and (ii) considering new structural alerts for nongenotoxic carcinogenicity. We also present a new analysis on global relationships between toxicological endpoints. The new results confirm that the previously proposed integrated, alternative strategy is an efficient tool to identify both genotoxic and nongenotoxic carcinogens, with an estimated 90-95% sensitivity.

New perspectives in toxicological information management, and the role of ISSTOX databases in assessing chemical mutagenicity and carcinogenicity.

Currently, the public has access to a variety of databases containing mutagenicity and carcinogenicity data. These resources are crucial for the toxicologists and regulators involved in the risk assessment of chemicals, which necessitates access to all the relevant literature, and the capability to search across toxicity databases using both biological and chemical criteria. Towards the larger goal of screening chemicals for a wide range of toxicity end points of potential interest, publicly available resources across a large spectrum of biological and chemical data space must be effectively harnessed with current and evolving information technologies (i.e. systematised, integrated and mined), if long-term screening and prediction objectives are to be achieved. A key to rapid progress in the field of chemical toxicity databases is that of combining information technology with the chemical structure as identifier of the molecules. This permits an enormous range of operations (e.g. retrieving chemicals or chemical classes, describing the content of databases, finding similar chemicals, crossing biological and chemical interrogations, etc.) that other more classical databases cannot allow. This article describes the progress in the technology of toxicity databases, including the concepts of Chemical Relational Database and Toxicological Standardized Controlled Vocabularies (Ontology). Then it describes the ISSTOX cluster of toxicological databases at the Istituto Superiore di Sanitá. It consists of freely available databases characterised by the use of modern information technologies and by curation of the quality of the biological data. Finally, this article provides examples of analyses and results made possible by ISSTOX.

IARC classes 1 and 2 carcinogens are successfully identified by an alternative strategy that detects DNA-reactivity and cell transformation ability of chemicals.

For decades, traditional toxicology has been the ultimate source of information on the carcinogenic potential of chemicals; however with increasing demand on regulation of chemicals and decreasing resources for testing, opportunities to accept "alternative" approaches have dramatically expanded. The need for tools able to identify carcinogens in shorter times and at a lower cost in terms of animal lives and money is still an open issue, and the present strategies and regulations for carcinogenicity pre-screening do not adequately protect human health. In previous papers, we have proposed an integrated in vitro/in silico strategy that detects DNA-reactivity and tissue disorganization/disruption by chemicals, and we have shown that the combination of Salmonella and Structural Alerts for the DNA-reactive carcinogens, and in vitro cell transformation assays for nongenotoxic carcinogens permits the identification of a very large proportion (up to 95%) of rodent carcinogens, while having a considerable specificity with the rodent noncarcinogens. In the present paper we expand the previous investigation and show that this alternative strategy identifies correctly IARC Classes 1 and 2 carcinogens. If implemented, this alternative strategy can contribute to improve the protection of human health while decreasing the use of animals.

A comparative survey of chemistry-driven in silico methods to identify hazardous substances under REACH.

This paper presents an inventory of in silico screening tools to identify substance properties of concern under the European chemicals' legislation REACH. The objective is to support the selection and implementation of appropriate tools as building blocks within integrated testing strategies (ITS). The relevant concerns addressed are persistence, bioaccumulation potential, acute and long-term aquatic toxicity, PBT/vPvB properties ((very) persistent, (very) bioaccumulative, toxic), CMR (carcinogenicity, mutagenicity, reproductive toxicity), endocrine disruption and skin sensitisation. The inventory offers a comparative evaluation of methods with respect to the underlying algorithms (how does the method work?) and the applicability domains (when does the method work?) as well as their limitations (when does the method not work?). The inventory explicitly addresses the reliability of predictions of different in silico models for diverse chemicals by applicability domain considerations. The confidence in predictions can be greatly improved by consensus modelling that allows for taking conflicting results into account. The inventory is complemented by a brief discussion of socio-economic tools for assessing the potential efficiency gains of using in silico methods compared to traditional in vivo testing of chemical hazards.

The new ISSMIC database on in vivo micronucleus and its role in assessing genotoxicity testing strategies.

This paper presents a new curated database on in vivo micronucleus mutagenicity results, called ISSMIC. It is freely available at: http://www.iss.it/ampp/dati/cont.php?id=233&lang=1&tipo=7. The experimental results were critically reviewed, and evidence on target cell exposure was considered as well. The inspection of ISSMIC demonstrates that a large proportion of reported negative results in the literature (231 out 566 ISSMIC chemicals) lack a clear-cut, direct demonstration of toxicity at the target cells. Using this updated database, the predictive value of a compilation of Structural Alerts (SA) for in vivo micronucleus recently implemented in the expert system Toxtree was investigated. Individually, most of the SA showed a high Positive Predictivity (∼80%), but the need for further expanding the list of alerts was pointed out as well. The role of in vivo micronucleus in strategies for carcinogenicity prediction was re-evaluated. In agreement with previous analyses, the data point to a low overall correlation with carcinogenicity. In addition, given the cost in animal lives and the time required for the experimentation, in many programs, the in vivo tests are used only to assess in vitro positive results. The ability of in vivo micronucleus to identify real positives (i.e. carcinogens) among chemicals positive in Salmonella or among chemicals inducing in vitro chromosomal aberrations was studied. It appears that the in vivo micronucleus test does not have added value and rather impairs the prediction ability of the in vitro tests alone. The overall evidence indicates that in vivo micronucleus--in its present form--cannot be considered an useful tool for routine genotoxicity testing but should be used in targeted mechanistic studies.

Assessment and validation of US EPA's OncoLogic® expert system and analysis of its modulating factors for structural alerts.

OncoLogic® is a software program able to screen chemical compounds for toxicological effects. The software predicts the potential carcinogenicity of chemicals by applying rules of structure activity relationship (SAR) analysis. To validate the predictivity of OncoLogic® (Version 7.0), 123 compounds tested with the long-term carcinogenicity bioassay on rodents were extracted from the ISSCAN database and were analyzed. The concordance between the OncoLogic® SAR analysis and the bioassay results was high. To better understand the strength of the SAR science in OncoLogic®, we investigated the influence of a select group of modulating factors on the predictions by the structural alerts.

Alternative strategies for carcinogenicity assessment: an efficient and simplified approach based on in vitro mutagenicity and cell transformation assays.

The need for tools able to predict chemical carcinogens in less time and at a lower cost in terms of animal lives and money is still a research priority, even after several decades of effort in that direction. Now, new regulatory requirements (e.g. the Registration, Evaluation, Authorisation and Restriction of Chemical substances recently implemented in Europe) have even increased the pressure to develop new tools in this field. Drawbacks of the present testing strategies have come to light again recently especially in view of new requirements in worldwide regulations. Among these are (i) the lack of assays able to identify non-genotoxic carcinogens, (ii) the exaggerated rate of misleading (false) positive results of the in vitro mammalian cell-based short-term mutagenicity tests and (iii) the extremely low sensitivity of in vivo short-term mutagenicity tests. Within this perspective, we analyse the contribution of cell transformation assays (CTAs), and we show that they are a valid complement to tools able to detect DNA-reactive carcinogens. We also show that a tiered strategy, with inexpensive and fast tests in Tier 1 (e.g. the Ames test or structural alerts) and the Syrian hamster embryo CTA in Tier 2, is able to identify up to 90% of carcinogens.

FAIRification of nanosafety data to improve applicability of (Q)SAR approaches: A case study on in vitro Comet assay genotoxicity data.

(Quantitative) structure-activity relationship ([Q]SAR) methodologies are widely applied to predict the (eco)toxicological effects of chemicals, and their use is envisaged in different regulatory frameworks for filling data gaps of untested substances. However, their application to the risk assessment of nanomaterials is still limited, also due to the scarcity of large and curated experimental datasets. Despite a great amount of nanosafety data having been produced over the last decade in international collaborative initiatives, their interpretation, integration and reuse has been hampered by several obstacles, such as poorly described (meta)data, non-standard terminology, lack of harmonized reporting formats and criteria. Recently, the FAIR (Findable, Accessible, Interoperable, and Reusable) principles have been established to guide the scientific community in good data management and stewardship. The EU H2020 Gov4Nano project, together with other international projects and initiatives, is addressing the challenge of improving nanosafety data FAIRness, for maximizing their availability, understanding, exchange and ultimately their reuse. These efforts are largely supported by the creation of a common Nanosafety Data Interface, which connects a row of project-specific databases applying the eNanoMapper data model. A wide variety of experimental data relating to characterization and effects of nanomaterials are stored in the database; however, the methods, protocols and parameters driving their generation are not fully mature. This article reports the progress of an ongoing case study in the Gov4nano project on the reuse of in vitro Comet genotoxicity data, focusing on the issues and challenges encountered in their FAIRification through the eNanoMapper data model. The case study is part of an iterative process in which the FAIRification of data supports the understanding of the phenomena underlying their generation and, ultimately, improves their reusability.

Towards FAIR nanosafety data.

Nanotechnology is a key enabling technology with billions of euros in global investment from public funding, which include large collaborative projects that have investigated environmental and health safety aspects of nanomaterials, but the reuse of accumulated data is clearly lagging behind. Here we summarize challenges and provide recommendations for the efficient reuse of nanosafety data, in line with the recently established FAIR (findable, accessible, interoperable and reusable) guiding principles. We describe the FAIR-aligned Nanosafety Data Interface, with an aggregated findability, accessibility and interoperability across physicochemical, bio-nano interaction, human toxicity, omics, ecotoxicological and exposure data. Overall, we illustrate a much-needed path towards standards for the optimized use of existing data, which avoids duplication of efforts, and provides a multitude of options to promote safe and sustainable nanotechnology.

Structural analysis and predictive value of the rodent in vivo micronucleus assay results.

In vivo genotoxicity studies-shortly followed by carcinogenicity-are posing high demand for test-related recourses in terms of animal lives and resources. Among those, the micronucleus test in rodents is the most widely used as a follow-up to positive in vitro mutagenicity results; therefore, the development and extensive use of estimation techniques based on the concept of Structure-Activity Relationships-such as (Quantitative) Structure-Activity Relationships, read-across and grouping of chemicals-might have a huge saving potential for this end point. In this paper, we present a newly derived compilation of Structural Alerts for the rodent in vivo micronucleus assay, thus providing a coarse-grain filter for preliminary screening of potentially in vivo mutagens. The compilation has been implemented as computerized rule of the expert system Toxtree and is freely available: http://ecb.jrc.ec.europa.eu/qsar/qsar-tools/index.php?c=TOXTREE. In addition, analyses on the performance of the micronucleus assay as pre-screening tool for carcinogenesis indicate that this assay is prone to give false-negative predictions and point to the need of improving the in vivo component of the present testing schemes.

Exploring in vitro/in vivo correlation: lessons learned from analyzing phase I results of the US EPA's ToxCast Project.

The research on alternative toxicological methods provides, among other things, a privileged viewpoint on one of the central issues of modern biomedical research--the relationship between (a) biological phenomena observed at the level of tissues and organisms and (b) their cellular and molecular bases as studied in isolated systems in vitro. The newly released ToxCast Phase 1 results, subject to initial analysis, converge with evidence from other fields (e.g., research on drug design with intensive use of omics technologies, traditional research on alternative tests) in indicating a low degree of the in vitro/in vivo correlation overall. In addition, this and other approaches point to the need for combining biological and chemical information in exploring the in vitro to in vivo connection.