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Threshold of Toxicological Concern (TTC) aids assessment of human health risks from exposure to low levels of chemicals when toxicity data are limited. The objective here was to explore the potential refinement of exposure for applying the oral TTC to chemicals found in cosmetic products, for which there are limited dermal absorption data. A decision tree was constructed to estimate the dermally absorbed amount of chemical, based on typical skin exposure scenarios. Dermal absorption was calculated using an established predictive algorithm to derive the maximum skin flux adjusted to the actual 'dose' applied. The predicted systemic availability (assuming no local metabolism), can then be ranked against the oral TTC for the relevant structural class. The predictive approach has been evaluated by deriving the experimental/prediction ratio for systemic availability for 22 cosmetic chemical exposure scenarios. These emphasise that estimation of skin penetration may be challenging for penetration enhancing formulations, short application times with incomplete rinse-off, or significant metabolism. While there were a few exceptions, the experiment-to-prediction ratios mostly fell within a factor of 10 of the ideal value of 1. It can be concluded therefore, that the approach is fit-for-purpose when used as a screening and prioritisation tool., (Copyright © 2016 International Life Sciences Institute Europe. Published by Elsevier Inc. All rights reserved.)

Category formation, grouping and read across methods are broadly applicable in toxicological assessments and may be used to fill data gaps for chemical safety assessment and regulatory decisions. In order to facilitate a transparent and systematic approach to aid regulatory acceptance, a strategy to evaluate chemical category membership, to support the use of read-across predictions that may be used to fill data gaps for regulatory decisions is proposed. There are two major aspects of any read-across exercise, namely assessing similarity and uncertainty. While there can be an over-arching rationale for grouping organic substances based on molecular structure and chemical properties, these similarities alone are generally not sufficient to justify a read-across prediction. Further scientific justification is normally required to justify the chemical grouping, typically including considerations of bioavailability, metabolism and biological/mechanistic plausibility. Sources of uncertainty include a variety of elements which are typically divided into two main issues: the uncertainty associated firstly with the similarity justification and secondly the completeness of the read-across argument. This article focuses on chronic toxicity, whilst acknowledging the approaches are applicable to all endpoints. Templates, developed from work to prepare for the application of new toxicological data to read-across assessment, are presented. These templates act as proposals to assist in assessing similarity in the context of chemistry, toxicokinetics and toxicodynamics as well as to guide the systematic characterisation of uncertainty both in the context of the similarity rationale, the read across data and overall approach and conclusion. Lastly, a workflow for reporting a read-across prediction is suggested., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Chemical regulation is challenged by the large number of chemicals requiring assessment for potential human health and environmental impacts. Current approaches are too resource intensive in terms of time, money and animal use to evaluate all chemicals under development or already on the market. The need for timely and robust decision making demands that regulatory toxicity testing becomes more cost-effective and efficient. One way to realize this goal is by being more strategic in directing testing resources; focusing on chemicals of highest concern, limiting testing to the most probable hazards, or targeting the most vulnerable species. Hypothesis driven Integrated Approaches to Testing and Assessment (IATA) have been proposed as practical solutions to such strategic testing. In parallel, the development of the Adverse Outcome Pathway (AOP) framework, which provides information on the causal links between a molecular initiating event (MIE), intermediate key events (KEs) and an adverse outcome (AO) of regulatory concern, offers the biological context to facilitate development of IATA for regulatory decision making. This manuscript summarizes discussions at the Workshop entitled "Advancing AOPs for Integrated Toxicology and Regulatory Applications" with particular focus on the role AOPs play in informing the development of IATA for different regulatory purposes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The assessment of data quality is a crucial element in many disciplines such as predictive toxicology and risk assessment. Currently, the reliability of toxicity data is assessed on the basis of testing information alone (adherence to Good Laboratory Practice (GLP), detailed testing protocols, etc.). Common practice is to take one toxicity data point per compound - usually the one with the apparently highest reliability. All other toxicity data points (for the same experiment and compound) from other sources are neglected. To show the benefits of incorporating the "less reliable" data, a simple, independent, statistical approach to assess data quality and reliability on a mathematical basis was developed. A large data set of toxicity values to Aliivibrio fischeri was assessed. The data set contained 1813 data points for 1227 different compounds, including 203 identified as non-polar narcotic. Log KOW values were calculated and non-polar narcosis quantitative structure-activity relationship (QSAR) models were built. A statistical approach to data quality assessment, which is based on data outlier omission and confidence scoring, improved the linear QSARs. The results indicate that a beneficial method for using large data sets containing multiple data values per compound and highly variable study data has been developed. Furthermore this statistical approach can help to develop novel QSARs and support risk assessment by obtaining more reliable values for biological endpoints., (Copyright © 2014 Elsevier B.V. All rights reserved.)

There is demand for methodologies to establish levels of safety concern associated with dietary exposures to chemicals for which no toxicological data are available. In such situations, the application of in silico methods appears promising. To make safety statement requires quantitative predictions of toxicological reference points such as no observed adverse effect level and carcinogenic potency for DNA-reacting chemicals. A decision tree (DT) has been developed to aid integrating exposure information and predicted toxicological reference points obtained with quantitative structure activity relationship ((Q)SAR) software and read across techniques. The predicted toxicological values are compared with exposure to obtain margins of exposure (MoE). The size of the MoE defines the level of safety concern and should account for a number of uncertainties such as the classical interspecies and inter-individual variability as well as others determined on a case by case basis. An analysis of the uncertainties of in silico approaches together with results from case studies suggest that establishing safety concern based on application of the DT is unlikely to be significantly more uncertain than based on experimental data. The DT makes a full use of all data available, ensuring an adequate degree of conservatism. It can be used when fast decision making is required., (Copyright © 2013 ILSI Europe. Published by Elsevier Inc. All rights reserved.)

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance., (Copyright © 2013 Elsevier Inc. All rights reserved.)

For more than a decade, the integration of human and environmental risk assessment (RA) has become an attractive vision. At the same time, existing European regulations of chemical substances such as REACH (EC Regulation No. 1907/2006), the Plant Protection Products Regulation (EC regulation 1107/2009) and Biocide Regulation (EC Regulation 528/2012) continue to ask for sector-specific RAs, each of which have their individual information requirements regarding exposure and hazard data, and also use different methodologies for the ultimate risk quantification. In response to this difference between the vision for integration and the current scientific and regulatory practice, the present paper outlines five medium-term opportunities for integrating human and environmental RA, followed by detailed discussions of the associated major components and their state of the art. Current hazard assessment approaches are analyzed in terms of data availability and quality, and covering non-test tools, the integrated testing strategy (ITS) approach, the adverse outcome pathway (AOP) concept, methods for assessing uncertainty, and the issue of explicitly treating mixture toxicity. With respect to exposure, opportunities for integrating exposure assessment are discussed, taking into account the uncertainty, standardization and validation of exposure modeling as well as the availability of exposure data. A further focus is on ways to complement RA by a socio-economic assessment (SEA) in order to better inform about risk management options. In this way, the present analysis, developed as part of the EU FP7 project HEROIC, may contribute to paving the way for integrating, where useful and possible, human and environmental RA in a manner suitable for its coupling with SEA., (Copyright © 2013 Elsevier B.V. All rights reserved.)

To ascertain the genetic basis of pediatric Burkitt lymphoma (pBL), we performed clinical-grade next-generation sequencing of 182 cancer-related genes on 29 formalin-fixed, paraffin embedded primary pBL samples. Ninety percent of cases had at least one mutation or genetic alteration, most commonly involving MYC and TP53. EBV(-) cases were more likely than EBV(+) cases to have multiple mutations (P < .0001). Alterations in tumor-related genes not previously described in BL were identified. Truncating mutations in ARID1A, a member of the SWI/SNF nucleosome remodeling complex, were seen in 17% of cases. MCL1 pathway alterations were found in 22% of cases and confirmed in an expanded panel. Other clinically relevant genomic alterations were found in 20% of cases. Our data suggest the roles of MCL1 and ARID1A in BL pathogenesis and demonstrate that comprehensive genomic profiling may identify additional treatment options in refractory disease.

There are a large number of chemicals in current use for which adequate toxicity data are not available. Whilst there are clear ethical and legal obligations to obtain data from sources other than in vivo experiments wherever possible, in certain cases in vivo assays may be deemed necessary. In such circumstances, it is essential to ensure that the maximum amount of high quality data is obtained from the minimum number of animals, using the most humane procedures, in accordance with the philosophy of reduction, refinement and replacement (3Rs). The aim of this report is to provide a strategy for anyone involved in animal experimentation, for either toxicological or pharmacological purposes, as to how in vivo experiments may be optimised. The impact of generic and endpoint specific sources of variability has been highlighted in a proof-of-principle analysis considering the variation in protocols for assays for four human health endpoints (skin sensitisation, reproductive/developmental toxicity, mutagenicity and carcinogenicity). Other factors such as operator training, experimental/statistical design, use of lower species and use of combined assays are also discussed. Recommendations for optimisation of in vivo assays, in terms of the 3Rs philosophy, applied to performing tests, harvesting data and appropriate reporting are summarised as a checklist of issues to be addressed prior to undertaking such assays., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The need to assess the ability of a chemical to act as a mutagen is one of the primary requirements in regulatory toxicology. Several pieces of legislation have led to an increased interest in the use of in silico methods, specifically the formation of chemical categories and read-across for the assessment of toxicological endpoints. One of the key steps in the development of chemical categories for mutagenicity is defining the mechanistic organic chemistry associated with the formation of a covalent bond between DNA and an exogenous chemical. To this end this study has analysed, by use of a large set of mutagenicity data (Ames test), the mechanistic coverage of a recently published set of in silico structural alerts developed for category formation. The results show that the majority of chemicals with a positive result in the Ames test were assigned at least one covalent binding mechanism related to the formation of a DNA adduct. The remaining chemicals with positive data in the Ames assay were subjected to a detailed mechanistic analysis from which 26 new structural alerts relating to covalent binding mechanisms were developed. In addition, structural alerts for radical and non-covalent intercalation mechanisms were also defined. The structural alerts outlined in this study are not intended to predict mutagenicity but rather to identify mechanisms associated with covalent and non-covalent DNA binding. This mechanistic profiling information can then be used to form chemical categories suitable for filling data gaps via read-across. A strategy for chemical category formation for mutagenicity is also presented., (© 2012 Elsevier B.V. All rights reserved.)

Anxiety confers increased risk for inflammatory diseases, and elevated inflammatory activity in anxious individuals may contribute to this increased risk. One complication, however, is that anxiety could be associated with inflammatory activity either through a specific anxiety pathway or through a more general negative emotionality pathway. To investigate, we measured levels of the stress hormone cortisol, the pro-inflammatory cytokine interleukin-6 (IL-6), and the systemic inflammatory marker C-reactive protein (CRP), as well as depression and neuroticism, in clinically anxious and non-anxious adults. Compared with non-anxious participants, clinically anxious participants exhibited significantly lower levels of morning cortisol and significantly higher levels of IL-6, independent of age, sex, and depressive symptoms. These group differences were robust when controlling for neuroticism. Conversely, the groups had equivalent levels of CRP in all analyses. Results are indicative of anxiety-specific effects on inflammatory activity, and highlight a pathway by which anxiety may increase risk for inflammatory diseases., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Improvements in analytical techniques have led to an increased awareness of the presence of pharmaceuticals in the environment. Concern is now raised as to the potential adverse effects these compounds may have on non-target organisms, particularly under conditions of chronic exposure. There is a paucity of experimental ecotoxicity data available for pharmaceuticals, hence the use of in silico tools to predict toxicity is a pragmatic option. Previous studies have used the ECOSAR program to predict environmental toxicity of pharmaceuticals, however, these models were developed using industrial chemicals and the applicability of the models to predict effects of pharmaceuticals should be carefully considered. In this study ECOSAR was used to assign 364 diverse pharmaceuticals to recognised chemical classes and hence predict their aquatic toxicity. Confidence in the predictions was assessed in terms of whether the assigned class was realistically representative of the pharmaceutical in question. The correlation between experimentally determined toxicity values (where these were available) and those predicted by ECOSAR was investigated in terms of confidence in the prediction. ECOSAR was shown to make reasonable predictions for certain pharmaceuticals considered to be within the applicability domain of the models, but predictions were less reliable for compounds judged to fall outwith the domain of the models. This study is not critical of ECOSAR or the class based approach to predicting toxicity, but demonstrates the importance of using expert judgement to ascertain whether or not use of a particular model is appropriate when the specific chemistry of a query compound is considered.

A previously developed function, the Modelling Power Plot, has been applied to QSARs developed using partial least squares (PLS) following variable selection from a genetic algorithm (GA). Modelling power (Mp) integrates the predictive and descriptive capabilities of a QSAR. With regard to QSARs for narcotic toxic potency, Mp was able to guide the optimal selection of variables using a GA. The results emphasise the importance of Mp to assess the success of the variable selection and that techniques such as PLS are more robust following variable selection.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS, e.g. nitric oxide, NO(*)) are well recognised for playing a dual role as both deleterious and beneficial species. ROS and RNS are normally generated by tightly regulated enzymes, such as NO synthase (NOS) and NAD(P)H oxidase isoforms, respectively. Overproduction of ROS (arising either from mitochondrial electron-transport chain or excessive stimulation of NAD(P)H) results in oxidative stress, a deleterious process that can be an important mediator of damage to cell structures, including lipids and membranes, proteins, and DNA. In contrast, beneficial effects of ROS/RNS (e.g. superoxide radical and nitric oxide) occur at low/moderate concentrations and involve physiological roles in cellular responses to noxia, as for example in defence against infectious agents, in the function of a number of cellular signalling pathways, and the induction of a mitogenic response. Ironically, various ROS-mediated actions in fact protect cells against ROS-induced oxidative stress and re-establish or maintain "redox balance" termed also "redox homeostasis". The "two-faced" character of ROS is clearly substantiated. For example, a growing body of evidence shows that ROS within cells act as secondary messengers in intracellular signalling cascades which induce and maintain the oncogenic phenotype of cancer cells, however, ROS can also induce cellular senescence and apoptosis and can therefore function as anti-tumourigenic species. This review will describe the: (i) chemistry and biochemistry of ROS/RNS and sources of free radical generation; (ii) damage to DNA, to proteins, and to lipids by free radicals; (iii) role of antioxidants (e.g. glutathione) in the maintenance of cellular "redox homeostasis"; (iv) overview of ROS-induced signaling pathways; (v) role of ROS in redox regulation of normal physiological functions, as well as (vi) role of ROS in pathophysiological implications of altered redox regulation (human diseases and ageing). Attention is focussed on the ROS/RNS-linked pathogenesis of cancer, cardiovascular disease, atherosclerosis, hypertension, ischemia/reperfusion injury, diabetes mellitus, neurodegenerative diseases (Alzheimer's disease and Parkinson's disease), rheumatoid arthritis, and ageing. Topics of current debate are also reviewed such as the question whether excessive formation of free radicals is a primary cause or a downstream consequence of tissue injury.

Regulatory guidelines for the assessment of acute dermal and ocular toxicity refer to the need to take the pH values of chemicals into consideration, since the acidic and basic properties of chemicals are known to play a role in the generation of acute dermal and ocular lesions. However, not all test guidelines provide an objective interpreting pH measurements in terms of acute skin or eye toxicity. The aim of this study was to develop classification models based on pH data for predicting the potential of chemicals to cause skin corrosion, skin irritation and eye irritation. The possible application of these models in the context of tiered testing strategies is discussed.

The use of computer-aided methods to predict the toxicity of drugs is described. These methods can assist in the identification of toxic compounds early in the drug development process. Thus, there is potential for these methods to be combined with combinatorial synthesis and library design. Quantitative structure-activity relationships allow for the prediction of individual endpoints, usually for restricted groups of compounds. Expert systems for toxicity prediction are based on a number of methodologies, each with its own strengths and weaknesses. The relative merit of each individual technique and methodology is described. However, more toxicity data are required, both to produce and to validate expert systems. Potential sources of new data include the use of high-throughput screening and microarrays for toxicology.

The toxicities of benzoic acids to Vibrio fischeri, Daphnia magna and carp were measured. The results showed that the toxicity to V. fischeri and Daphnia decreased in the order of bromo > chloro > fluoro approximately equal to aminobenzoic acids. The toxicity of substituted benzoic acids to carp and Daphnia was much lower that to V. fischeri. The results also showed that the toxicity of benzoic acids to Daphnia decreased as the pH increased. It is suggested that ionized and non-ionized forms have different toxic responses. The non-ionized form may play an important role in toxicity because the toxicity of benzoic acids to Daphnia greatly decreases as the pH increases. The toxicity of benzoic acids to Daphnia may operate through non-polar narcosis, based on the regression results between the toxicities and partition coefficients (log P) and apparent partition coefficients (log D). However, toxicity cannot be predicted from non-polar baseline models because the ionized and non-ionized form of benzoic acids have different contributions to toxicity. Compared with the single descriptors, the prediction of toxicity of the benzoic acids was improved remarkably by using log P with pKa and log P with ELUMO. For the toxicity of benzoic acids to V. fischeri, it is suggested that the toxic mechanism may be different from the mechanism in Daphnia and carp. A probable reason is that V. fischeri is a unicellular organism with low lipid content, and hence both ionized and non-ionized forms of benzoic acids can easily cross the cell membrane and contribute to toxicity.

Quantitative structure-activity relationships (QSARs) have been developed with the logarithm of the inverse of 15-min toxicity (pT15) to Vibrio fischeri (the acute Microtox test). Statistically, robust QSARs were found for alkanones acting by the nonreactive, baseline narcosis mechanism of action [pT15 = 0.99 (log Kow)-2.08, n = 6, s = 0.24, r2 = 0.988, F = 405]; aldehydes acting by the Schiff base-forming mechanism of electrophilicity [pT15 = 0.55(log Kow)-0.58, n = 6, s = 0.07, r2 = 0.994, F = 782]; and alkenals acting by the Michael-type acceptor mechanism of electrophilicity [pT15 = 0.52(log Kow) + 0.35, n = 6, s = 0.19, r2 = 0.914, F = 54.5]. Efforts to model toxicity across mechanism of action resulted in the development of a response surface [pT15 = 0.79(log Kow)-1.17 (ELUMO)-0.41, n = 19, s = 0.46, r2 = 0.892, F = 75.3]. In addition, an excellent correlation was found between Tetrahymena pyriformis [log(1/IGC50)] and V. fischeri. [log(1/IGC50) = 0.86(pT15)-0.25, n = 19, s = 0.25, r2 = 0.957, F = 405] toxicity.

Quantitative structure-activity relationships (QSARs) have been utilized to validate toxicity data to Vibrio fisheri (the acute Microtox test) at each of the 5-, 15- and 30-min endpoints. Statistically robust QSARs were found for chemicals acting by a non-polar narcosis mechanism of action (MOA). The baseline, non-polar narcosis QSARs were similar to those found in other aquatic organisms, thus indicating that for this MOA, the acute V. fisheri assay makes a suitable surrogate for higher test species. For the toxicity of phenols, previously modeled in other species with parameters describing membrane transport (i.e. hydrophobicity) and soft electrophilicity or ionization, no significant relationships were obtained. The use of acute V. fisheri toxicity data is not recommended for extrapolation to higher species for these compounds. Despite this recommendation, it was noted, however, that a 'minimal' phenolic toxicity was apparent that was analogous to polar narcosis in other species. This confirmed that a polar narcosis MOA does operate in the acute V. fisheri system analogous to other species. For all the compounds considered, there were highly significant correlations between the toxicities at each endpoint, indicating that data from different time endpoints were effectively interchangeable.

The structural basis of dapsone (4,4'-diaminodiphenyl sulphone) haemotoxicity has been determined by investigation of the in vitro bioactivation of a series of 4-substituted arylamines. In the presence of rat liver microsomes, dapsone (100 microM) was the most potent former of methaemoglobin in human erythrocytes (44.8 +/- 6.7%). Substitution of the sulphone group with sulphur (11.6 +/- 1.4% methaemoglobin), oxygen (4.5 +/- 1.1%), nitrogen (0.0 +/- 3.2%), carbon (13.6 +/- 0.8%) or a keto group (34.0 +/- 6.1%) resulted in a decrease in methaemoglobin formation. Only one compound, 4,4'-diaminodiphenylamine, generated significant (P < 0.001) amounts of methaemoglobin (25.6 +/- 2.5%) in the absence of NADPH. To assess further the role of the 4-substituent in methaemoglobinaemia, the toxicity of a series of 4-substituted aniline derivatives was also studied. Of the anilines studied, 4-nitroaniline caused the most methaemoglobin (36.5 +/- 8.0%), whilst aniline caused the least (0.3 +/- 0.5%). Overall, there was a significant correlation (r2 = 0.83) between the haemotoxicity and the Hammett constant, sigma(p), suggesting that it is the electron-withdrawing properties of the substituent that influence the methaemoglobin formation. In the presence of microsomes prepared from two human livers, dapsone was the most haemotoxic bis arylamine, whereas 4-iodoaniline was the most potent methaemoglobin former (60.6 and 73.6%) and aniline the least potent (1.1 and 2.4%). As a whole, these results indicate that the sulphonyl group, which is essential for the pharmacological activity of dapsone, is also largely responsible for the haemotoxicity seen with this drug.

This study investigated the relationships between the toxicities of common organic pollutants to the fathead minnow (Pimephales promelas), to Daphnia magna, to Tetrahymena pyriformis and in the Microtox test, which uses the luminescent bacterium Photobacterium phosphoreum. The toxicity data were compiled from the literature, with the exception of 40 experimentally determined Microtox data. Encouraging correlations are seen, indicating significant relationships between fish toxicities and those to lower organisms. When the toxicities of individual chemical classes are studied, further improvement is often seen in the correlations. Analysis of significant outliers from the inter-species relationships has led to the suggestion that the fathead minnow may be more susceptible to chemicals that are metabolised to reactive intermediates (such as the aldehydes). The fish may, however, be less susceptible to other chemical classes such as ketones and alcohols.