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.

Structure-Potency Relationships for Epoxides in Allergic Contact Dermatitis.

Epoxides are known or proposed to be involved in skin sensitization in various ways. Some are encountered directly, and others have been shown to be formed abiotically and metabolically from various unsaturated chemicals. They can react as SN2 electrophiles. To date no quantitative mechanistic models (QMMs) are known for skin sensitization potency of this subcategory of SN2 electrophiles. Here we have considered the reaction mechanistic chemistry of epoxides and combined published experimental kinetic data (rate constants k for reaction with a cysteine-based peptide) together with calculated hydrophobicity data (logP) to derive a QMM correlating potency in the local lymph node assay (LLNA), expressed as EC3, with a relative alkylation index (RAI, calculated as logk + 0.4 logP). The QMM equation, pEC3 = 2.42(±0.26) RAI + 4.04 (±0.25), n = 9, R2 = 0.928, R2(adj) = 0.917, F = 90, s = 0.18, fits the data well, with one positive outlier. The outlier can be rationalized by its exhibiting an alert for oxidation of an amine moiety to give, in this case, the highly reactive glycidaldehyde. The epoxide QMM predicts the potency of a nonepoxide SN2 electrophile (predicted EC3, 0.48%; observed EC3, 0.5%), which suggests that it could form the basis for a more general H-polar SN2 QMM that could be a valuable tool in skin sensitization risk assessment for this quite extensive and structurally diverse reaction mechanistic domain.

Chemical applicability domain of the Local Lymph Node Assay (LLNA) for skin sensitisation potency. Part 2. The biological variability of the murine Local Lymph Node Assay (LLNA) for skin sensitisation.

The Local Lymph Node Assay (LLNA) is the most common in vivo regulatory toxicology test for skin sensitisation, quantifying potency as the EC3, the concentration of chemical giving a threefold increase in thymidine uptake in the local lymph node. Existing LLNA data can, along with clinical data, provide useful comparator information on the potency of sensitisers. Understanding of the biological variability of data from LLNA studies is important for those developing non-animal based risk assessment approaches for skin allergy. Here an existing set of 94 EC3 values for 12 chemicals, all tested at least three times in the same vehicle have been analysed by calculating standard deviations (SD) for logEC3 values. The SDs range from 0.08 to 0.22. The overall SD for the 94 logEC3 values is 0.147. Thus the 95% confidence limits (2xSD) for LLNA EC3 values are within a factor of 2, comparable to those for physico-chemical measurements such as partition coefficients and solubility. The residual SDs of Quantitative Mechanistic Models (QMMs) based on physical organic chemistry parameters are similar to the overall SD of the LLNA, indicating that QMMs of this type are unlikely to be bettered for predictive accuracy.

Kinetic-Based Reactivity for Michael Acceptors: Structural Activity Relationships and Its Relationship to Excess Acute Fish Toxicity.

Acute aquatic toxicity is divided into the "physical" mode governed by weak, non-covalent interactions and the "chemical" mode governed by covalent reactions. The potency of chemical interactions is typically expected to be greater than that for physical ones. This enhanced potency is called "excess" toxicity. As databases have become complex, substances thought to elicit a chemical mode reveal a lack of excess toxicity. One mechanism where the latter is prevalent is Michael-type addition. A series of α-ß-unsaturated substances were evaluated for reactivity. Second order rate constants (k') were calculated (M-1 s-1) and found to vary from >4000 to <0.0003. The electron-withdrawing capacity of the polar group impacts k' values; the sequence is nitro > carbonyl or sulfone â‰« sulfoxide, nitrile or amide. When the α-carbon and/or the ß-carbon of the π-system are substituted, the k' value is sharply reduced. Excess toxicity is associated with k' values >0.01 (M-1 s-1).

A practical guidance for Cramer class determination.

Expanded use of the Threshold of Toxicological Concern (TTC) methodology has brought into discussion the intent of the original questions used in the Cramer scheme or Cramer decision tree. We have analysed, both manually and by Toxtree software, a large dataset of fragrance ingredients and identified several issues with the original Cramer questions. Some relate to definitions and wording of questions; others relate to in silico interpretation of the questions. We have endeavoured to address all of these inconsistencies and misinterpretations without changing the basic structure and principles of the original decision tree. Based on the analysis of a large data set of over 2500 fragrance ingredients, we found that most of the 33 questions in the original Cramer scheme are straightforward. Through repeated examination each of the 33 questions, we found 14 where the logic underlying the development of the rule is unclear. These questions are well served by minor wording changes and/or further explanation designed to capture what we perceive to be the intent of the original decision tree. The findings reported here could be used as a guidance for conducting Cramer classification and provide advices for the improvement of the in silico tools.

Workshop: use of "read-across" for chemical safety assessment under REACH.

Read-across has generated much attention, since it may be used as an alternative approach for addressing the information requirements under REACH. Experience in the application of "read-across" has undoubtedly been gained within the context of the 2010 registrations (>1000 tonnes/annum). Industry, European Chemicals Agency (ECHA) and EU Member States all conceptually accept read-across approaches but difficulties still remain in applying them consistently in practice. A workshop on the 'Use of Read-Across for Chemical Safety Assessment under REACH', organised by ECHA with the active support of Cefic LRI was held on the 3rd October 2012 to gain insight on how ECHA evaluates read-across justifications, to share Industry experiences with read-across approaches and to discuss practical strategies to help develop scientifically valid read-across for future submissions.

Guiding principles for the implementation of non-animal safety assessment approaches for cosmetics: skin sensitisation.

Characterisation of skin sensitisation potential is a key endpoint for the safety assessment of cosmetic ingredients especially when significant dermal exposure to an ingredient is expected. At present the mouse local lymph node assay (LLNA) remains the 'gold standard' test method for this purpose however non-animal test methods are under development that aim to replace the need for new animal test data. COLIPA (the European Cosmetics Association) funds an extensive programme of skin sensitisation research, method development and method evaluation and helped coordinate the early evaluation of the three test methods currently undergoing pre-validation. In May 2010, a COLIPA scientific meeting was held to analyse to what extent skin sensitisation safety assessments for cosmetic ingredients can be made in the absence of animal data. In order to propose guiding principles for the application and further development of non-animal safety assessment strategies it was evaluated how and when non-animal test methods, predictions based on physico-chemical properties (including in silico tools), threshold concepts and weight-of-evidence based hazard characterisation could be used to enable safety decisions. Generation and assessment of potency information from alternative tools which at present is predominantly derived from the LLNA is considered the future key research area.

Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010.

The 7th amendment to the EU Cosmetics Directive prohibits to put animal-tested cosmetics on the market in Europe after 2013. In that context, the European Commission invited stakeholder bodies (industry, non-governmental organisations, EU Member States, and the Commission's Scientific Committee on Consumer Safety) to identify scientific experts in five toxicological areas, i.e. toxicokinetics, repeated dose toxicity, carcinogenicity, skin sensitisation, and reproductive toxicity for which the Directive foresees that the 2013 deadline could be further extended in case alternative and validated methods would not be available in time. The selected experts were asked to analyse the status and prospects of alternative methods and to provide a scientifically sound estimate of the time necessary to achieve full replacement of animal testing. In summary, the experts confirmed that it will take at least another 7-9 years for the replacement of the current in vivo animal tests used for the safety assessment of cosmetic ingredients for skin sensitisation. However, the experts were also of the opinion that alternative methods may be able to give hazard information, i.e. to differentiate between sensitisers and non-sensitisers, ahead of 2017. This would, however, not provide the complete picture of what is a safe exposure because the relative potency of a sensitiser would not be known. For toxicokinetics, the timeframe was 5-7 years to develop the models still lacking to predict lung absorption and renal/biliary excretion, and even longer to integrate the methods to fully replace the animal toxicokinetic models. For the systemic toxicological endpoints of repeated dose toxicity, carcinogenicity and reproductive toxicity, the time horizon for full replacement could not be estimated.

Local lymph node data for the evaluation of skin sensitization alternatives: a second compilation.

BACKGROUND: Development, evaluation and validation of alternatives to skin sensitisation testing require the availability of reliable databases with which comparative analyses can be conducted to establish performance characteristics. To facilitate this we have published previously a database comprising results from local lymph node assays (LLNAs) conducted with 211 chemicals. That database embraced a substantial range of chemistry, and of relative skin sensitising potency, and has found application in the assessment of new or refined methods. OBJECTIVE: In this paper we describe a second compilation to extend the LLNA database. METHODS: This second data compilation was derived from previously conducted LLNA studies involving an additional 108 chemicals. In addition, the first database contained a small number of inaccuracies, affecting results recorded with a few chemicals. In this paper these have been corrected. RESULTS: The inclusion of 108 new substances has served to extend and consolidate the areas of chemistry covered by the database. In addition, the entire dataset was evaluated for pre and prohaptens which will facilitate the choice of chemicals for alternative assay developments. CONCLUSIONS: It is anticipated that the new revised and extended database totalling over 300 chemicals will now serve as the primary resource to support the development and evaluation of new approaches to hazard identification and potency assessment.

Experimental reactivity parameters for toxicity modeling: application to the acute aquatic toxicity of SN2 electrophiles to Tetrahymena pyriformis.

A diverse set of 60 haloaliphatic compounds were evaluated for reactivity with cysteine thiol groups in the previously described RC(50) assay using glutathione (GSH) as a model nucleophile. Reactivity was quantified by the RC(50) value, the concentration of test compound that produced 50% reaction of the GSH thiol groups in 120 min. Under standard conditions, RC(50) values are mathematically proportional to reciprocal rate constants. Quantitative structure-activity relationship (QSAR) analysis correlating acute aquatic toxicity (IGC(50)) to Tetrahymena pyriformis with RC(50) values was carried out. It was found that subdivision of the compounds into subdomains according to their reaction mechanism characteristics enabled toxicity-reactivity relationships to be identified. The largest subdomain consisting of 22 compounds in which a primary halogen is alpha to a carbonyl or other electronegative unsaturated group and which can be confidently assigned as S(N)2 electrophiles fits the equation pIGC(50) (mM) = 0.94 (+/-0.07) pRC(50) (mM) + 1.34 (+/-0.07), n = 22, r(2) = 0.889, r(2)(adj) = 0.884, s = 0.27, and F = 161. Compounds in which the halogen is not alpha to an unsaturated group are not reactive in the GSH assay and do not exhibit reactive toxicity to T. pyriformis. Compounds tested in which the halogen is alpha to an unsaturated nonelectronegative group were found to be less toxic in the assay than predicted by the above QSAR equation. Within a subdomain of 21 compounds having a halogen alpha to an electronegative unsaturated group that, in the absence of experimental evidence, could not be confidently assigned as S(N)2 electrophiles, 2-bromoalkanoates of general structure R(1)CHBrCO(2)R(2), 2-bromopropionamide, and 2-haloalkanoic acids of general formula R(1)CHXCO(2)H (nine compounds in total) are all well-predicted by the above equation. Of the other 12 compounds of this subdomain, eight are substantially less toxic than predicted by the above equation and are considered to react differently, whereas the alpha-halonitriles (four compounds) are more toxic than predicted and fit a correlation of their own: pIGC(50) = 1.01 (+/-0.05) pRC(50) + 2.04 (+/-0.05), n = 4, r(2) = 0.995, r(2)(adj) = 0.992, s = 0.08, and F = 381, with a similar slope but larger intercept. An explanation in terms of their physical chemistry and possible involvement of released cyanide ion is suggested.

Chemical mechanisms for skin sensitization by aromatic compounds with hydroxy and amino groups.

It is well-known that aromatic diamino-, dihydroxy-, and amino-hydroxy compounds, with NH(2) and OH groups in ortho- or para-positions relative to each other, are strong skin sensitizers. In this paper, we analyze published potency and cross-reactivity data, whereby animals sensitized to one of these compounds are challenged with other compounds. The data are consistent with two parallel chemical reaction mechanisms: oxidation to electrophilic (protein reactive) quinones or quinone imines or formation of protein-reactive free radicals such as the Wurster salt, which can be formed by para-phenylene diamine. Compounds with NH(2) and OH groups meta to each other have also been found to be skin sensitizers, in some cases quite strong sensitizers. For these compounds, direct formation of quinones or quinone imines is not possible, and free radicals of the Wurster salt type are not favored. Here, we present a molecular mechanism to rationalize the sensitization potential of such compounds and, using the results of quantum mechanics calculations, show how this mechanism can explain observed structure-potency trends.

Reactivity profiling: covalent modification of single nucleophile peptides for skin sensitization risk assessment.

The molecular basis of chemical allergy is rooted in the ability of an allergen (hapten) to modify endogenous proteins. This mechanistic understanding aided development of screening assays which generate reproducible quantitative and qualitative reactivity data. Such assays use model peptides with a limited number and type of protein nucleophiles, and the data does not reflect the specificity, variety, and complexity of hapten interactions with multiple nucleophiles. Building on these developments, we extended the standardized approach to maximize the type and the amount of information that can be derived from an in chemico assay. We used a panel of six single nucleophile peptides and individually optimized the incubation conditions to favor chemical modification. Employing liquid chromatography tandem mass spectrometry (LC-MS/MS) technique, we simultaneously obtained multiple quantitative and qualitative measurements (% peptide depletion, adducts formation, and peptide dimerization for Cys-containing peptide). Using these methods, we obtained reactivity data for 36 chemicals of known skin sensitizing potency. By optimizing incubation conditions, we ensured detection of all reactive chemicals. We explored the LC-MS/MS approach to generate kinetic data for 10 chemicals allowing further characterization of reactivity and a potentially more robust quantitative reactivity descriptor. Our ultimate aim is to integrate this dataset with available physicochemical data and outputs from other predictive assays, all addressing different key steps in the induction of sensitization, to help us make decisions about the safe use of chemicals without using animal tests. The epidermal protein target sites, modification of which may be immunogenic and lead to induction of skin sensitization, are currently unknown. Increasing the understanding of this process may help further refine in chemico reactivity assays as well as aid the interpretation of the reactivity data.

Read-across to rank skin sensitization potential: subcategories for the Michael acceptor domain.

BACKGROUND: Eliminating animal testing for skin sensitization is a significant challenge in consumer safety risk assessment. To be able to perform resilient risk assessments in the future, one will need alternative approaches to fill the data gaps. OBJECTIVES: To this end, we propose a subcategory-based read-across approach to estimate and rank skin sensitization potential of chemicals. The example described here is for the mechanism of Michael-type nucleophilic addition with subcategories being limited to carbonyl-containing compounds. PATIENTS/METHODS: In this approach, in silico tools based on structural alerts were used to determine both the mechanism of protein binding and the relative subcategories within that mechanism. RESULTS: Fifty compounds previously evaluated in the in vivo mouse local lymph node assay (LLNA) were placed in 10 subcategories defined by their polarized alpha,beta-unsaturated substructure. To offset the limitations and skewness of the published in vivo data, in chemico glutathione (GSH) depletion data also were included. CONCLUSIONS: It was shown that the read-across approach can be successfully used to rank qualitatively skin sensitization potential of an untested carbonyl-containing Michael acceptor chemical by using subcategories. Moreover, the use of the more resilient in chemico GSH depletion data added further support to the read-across result.

Does the extreme skin sensitization potency of benzoquinone result from special chemistry?

BACKGROUND: Benzoquinone is known to be an extreme skin sensitizer. Until now there has been no consideration of whether it owes its extreme skin sensitization potency to unique aspects of its chemistry or simply to its high reactivity as a Michael acceptor. OBJECTIVES: To answer these questions, we have applied two mechanistic approaches to estimate a theoretical potency value for benzoquinone. PATIENTS/METHODS: A mechanistic read-across (MRA) approach, using known potency data for moderate sensitizers in the Michael acceptor domain, and a published quantitative mechanistic model (QMM) for skin sensitization potency of Michael acceptors, all in the moderate and weak range, were used. RESULTS: The read-across approach gives a theoretical local lymph node assay (LLNA) EC3 value of 0.04%, and the QMM approach gives a theoretical value of 0.013%, compared with an experimental value of 0.010%. CONCLUSIONS: The good agreement between the theoretical and experimental EC3 values supports the strength of the MRA and QMM approaches, and provides strong evidence that the extreme sensitization potency of benzoquinone can be attributed solely to its high reactivity as a Michael acceptor.

Assuring consumer safety without animal testing: a feasibility case study for skin sensitisation.

Allergic Contact Dermatitis (ACD; chemical-induced skin sensitisation) represents a key consumer safety endpoint for the cosmetics industry. At present, animal tests (predominantly the mouse Local Lymph Node Assay) are used to generate skin sensitisation hazard data for use in consumer safety risk assessments. An animal testing ban on chemicals to be used in cosmetics will come into effect in the European Union (EU) from March 2009. This animal testing ban is also linked to an EU marketing ban on products containing any ingredients that have been subsequently tested in animals, from March 2009 or March 2013, depending on the toxicological endpoint of concern. Consequently, the testing of cosmetic ingredients in animals for their potential to induce skin sensitisation will be subject to an EU marketing ban, from March 2013 onwards. Our conceptual framework and strategy to deliver a non-animal approach to consumer safety risk assessment can be summarised as an evaluation of new technologies (e.g. 'omics', informatics), leading to the development of new non-animal (in silico and in vitro) predictive models for the generation and interpretation of new forms of hazard characterisation data, followed by the development of new risk assessment approaches to integrate these new forms of data and information in the context of human exposure. Following the principles of the conceptual framework, we have been investigating existing and developing new technologies, models and approaches, in order to explore the feasibility of delivering consumer safety risk assessment decisions in the absence of new animal data. We present here our progress in implementing this conceptual framework, with the skin sensitisation endpoint used as a case study.

Chemical reactivity indices and mechanism-based read-across for non-animal based assessment of skin sensitisation potential.

The skin sensitisation potential of chemicals is currently assessed using in vivo methods where the murine local lymph node assay (LLNA) is typically the method of first choice. Current regulatory initiatives are driving the impetus for the use of in vitro/in silico alternative approaches to provide the relevant information needed for the effective assessment of skin sensitisation, for both hazard characterisation and risk assessment purposes. A chemical must undergo a number of steps for it to induce skin sensitisation but the main determining step is formation of a stable covalent association with carrier protein. The ability of a chemical to react covalently with carrier protein nucleophiles relates to both its electrophilic reactivity and its hydrophobicity. This paper focuses on quantitative indices of electrophilic reactivity with nucleophiles, in a chemical mechanism-of-action context, and compares and contrasts the experimental approaches available to generate reactivity data that are suitable for mathematical modelling and making predictions of skin sensitisation potential, using new chemistry data correlated against existing in vivo bioassay data. As such, the paper goes on to describe an illustrative example of how quantitative kinetic measures of reactivity can be usefully and simply applied to perform mechanism-based read-across that enables hazard characterisation of skin sensitisation potential. An illustration of the types of quantitative mechanistic models that could be built using databases of kinetic measures of reactivity, hydrophobicity and existing in vivo bioassay data is also given.

Determinants of skin sensitisation potential.

Skin sensitisation is an important toxicological endpoint. The possibility that chemicals used in the workplace and in consumer products might cause skin sensitisation is a major concern for individuals, for employers and for marketing. In European REACH (Registration, Evaluation, and Authorisation of Chemicals) legislation, the sensitising potential should therefore be assessed for chemicals below the 10 ton threshold. Development of methods for prediction of skin sensitisation potential without animal testing has been an active research area for some time, but has received further impetus with the advent of REACH and the EU Cosmetics Directive (EU 2003). This paper addresses the issue of non-animal based prediction of sensitisation by a mechanistic approach. It is known that the sequence of molecular, biomolecular and cellular events between exposure to a skin sensitiser and development of the sensitised state involves several stages, in particular penetration through the stratum corneum, covalent binding to carrier protein, migration of Langerhans cells, presentation of the antigen to naïve T-cells. In this paper each of these stages is considered with respect to the extent to which it is dependent on the chemical properties of the sensitiser. The evidence suggests that, although penetration of the stratum corneum, stimulation of migration and maturation of Langerhans cells, and antigen recognition are important events in the induction of sensitisation, except in certain specific circumstances they can be taken for granted. They are not important factors in determining whether a compound will be a sensitiser or not, nor are they important factors in determining how potent one sensitiser will be relative to another. The ability to bind covalently to carrier protein is the major structure-dependent determinant of skin sensitisation potential. A chemistry-based prediction strategy is proposed involving reaction mechanistic domain assignment, reactivity and hydrophobicity determination, and application of quantitative mechanistic modelling (QMM) or read-across.

Identifying the structural requirements for chromosomal aberration by incorporating molecular flexibility and metabolic activation of chemicals.

Modeling the potential of chemicals to induce chromosomal damage has been hampered by the diversity of mechanisms which condition this biological effect. The direct binding of a chemical to DNA is one of the underlying mechanisms that is also responsible for bacterial mutagenicity. Disturbance of DNA synthesis due to inhibition of topoisomerases and interaction of chemicals with nuclear proteins associated with DNA (e.g., histone proteins) were identified as additional mechanisms leading to chromosomal aberrations (CA). A comparative analysis of in vitro genotoxic data for a large number of chemicals revealed that more than 80% of chemicals that elicit bacterial mutagenicity (as indicated by the Ames test) also induce CA; alternatively, only 60% of chemicals that induce CA have been found to be active in the Ames test. In agreement with this relationship, a battery of models is developed for modeling CA. It combines the Ames model for bacterial mutagenicity, which has already been derived and integrated into the Optimized Approach Based on Structural Indices Set (OASIS) tissue metabolic simulator (TIMES) platform, and a newly derived model accounting for additional mechanisms leading to CA. Both models are based on the classical concept of reactive alerts. Some of the specified alerts interact directly with DNA or nuclear proteins, whereas others are applied in a combination of two- or three-dimensional quantitative structure-activity relationship models assessing the degree of activation of the alerts from the rest of the molecules. The use of each of the alerts has been justified by a mechanistic interpretation of the interaction. In combination with a rat liver S9 metabolism simulator, the model explained the CA induced by metabolically activated chemicals that do not elicit activity in the parent form. The model can be applied in two ways: with and without metabolic activation of chemicals.