Identifying chemicals based on receptor binding/bioactivation/mechanistic explanation associated with potential to elicit hepatotoxicity and to support structure activity relationship-based read-across.

The liver is the most common target organ in toxicology studies. The development of chemical structural alerts for identifying hepatotoxicity will play an important role in in silico model prediction and help strengthen the identification of analogs used in structure activity relationship (SAR)- based read-across. The aim of the current study is development of an SAR-based expert-system decision tree for screening of hepatotoxicants across a wide range of chemistry space and proposed modes of action for clustering of chemicals using defined core chemical categories based on receptor-binding or bioactivation. The decision tree is based on âˆ¼ 1180 different chemicals that were reviewed for hepatotoxicity information. Knowledge of chemical receptor binding, metabolism and mechanistic information were used to group these chemicals into 16 different categories and 102 subcategories: four categories describe binders to 9 different receptors, 11 categories are associated with possible reactive metabolites (RMs) and there is one miscellaneous category. Each chemical subcategory has been associated with possible modes of action (MOAs) or similar key structural features. This decision tree can help to screen potential liver toxicants associated with core structural alerts of receptor binding and/or RMs and be used as a component of weight of evidence decisions based on SAR read-across, and to fill data gaps.

An interim internal Threshold of Toxicologic Concern (iTTC) for chemicals in consumer products, with support from an automated assessment of ToxCast™ dose response data.

Additional non-animal methods are urgently needed to meet regulatory and animal welfare goals. TTC is a broadly used risk assessment tool. TTC based on external dose has limited utility for multi-route exposure and some types of structure activity relationship assessments. An internal TTC (iTTC), where thresholds are based on blood concentration, would extend the applicability of TTC. While work is on-going to develop robust iTTC thresholds, we propose an interim conservative iTTC. Specifically, an interim iTTC of 1 µM, supported by the published experience of the pharmaceutical industry, a literature review of non-drug chemical/receptor interactions, and analysis of ToxCast™ data. ToxCast™ data were used to explore activity versus the 1 µM interim iTTC and recommendations for the analysis and interpretation of HTS data. Test concentration-based points of departure were classified to identify quality of fit to the Hill Model. We identified, for exclusion from the approach, estrogen receptor and androgen receptor targets as potent chemical/receptor interactions potentially associated with low dose exposure to non-pharmaceutical active ingredients in addition to the original TTC exclusions. With these exclusions, we conclude that a 1 µM plasma concentration is unlikely to be associated with significant biological effects from chemicals not intentionally designed for biological activity.

Intrinsic relative potency of a series of pyrrolizidine alkaloids characterized by rate and extent of metabolism.

1,2-Unsaturated pyrrolizidine alkaloids (PAs) are sometimes present in foods or herbal supplements/medicines as impurities and pose potential concerns for liver genotoxicity/carcinogenicity. PAs display a strong structure toxicity relationship, however, current regulatory approaches to risk assessment take the precautionary approach of assuming all PAs display the same potency as the most toxic congeners lasiocarpine (LAS) and riddelliine (RID). Here we explore the relative potencies of a series of structurally diverse PAs by measuring DNA adduct formation in vitro in a rat sandwich culture hepatocyte (SCH) cell system. The adducts generated are consistent with those identified in vivo as biomarkers of PA exposure and potential liver-tumor formation. DNA reactive PAs require metabolic activation to form intermediates that bind DNA, therefore, adduct formation is a direct reflection of reactive metabolite formation. Since the area under the concentration versus time curve (AUC) for the depletion of parent PA from the extracellular media is a measure of PA exposure, the ratio of adducts/AUC provides a measure of hepatocyte exposure to DNA-binding metabolites corresponding to an intrinsic potency for DNA adduct formation. Intrinsic potencies relative to potencies for LAS compare well with existing relative potency data further affirming that PA toxicity varies considerably with chemical structure.

Challenges in working towards an internal threshold of toxicological concern (iTTC) for use in the safety assessment of cosmetics: Discussions from the Cosmetics Europe iTTC Working Group workshop.

The Threshold of Toxicological Concern (TTC) is an important risk assessment tool which establishes acceptable low-level exposure values to be applied to chemicals with limited toxicological data. One of the logical next steps in the continued evolution of TTC is to develop this concept further so that it is representative of internal exposures (TTC based on plasma concentration). An internal TTC (iTTC) would provide threshold values that could be utilized in exposure-based safety assessments. As part of a Cosmetics Europe (CosEu) research program, CosEu has initiated a project that is working towards the development of iTTCs that can be used for the human safety assessment. Knowing that the development of an iTTC is an ambitious and broad-spanning topic, CosEu organized a Working Group comprised a balance of multiple stakeholders (cosmetics and chemical industries, the EPA and JRC and academia) with relevant experience and expertise and workshop to critically evaluate the requirements to establish an iTTC. Outcomes from the workshop included an evaluation on the current state of the science for iTTC, the overall iTTC strategy, selection of chemical databases, capture and curation of chemical information, ADME and repeat dose data, expected challenges, as well as next steps and ongoing work.

Structure activity relationship (SAR) toxicological assessments: The role of expert judgment.

Structure activity relationships (SAR) and read-across are widely used animal alternative approaches for filling toxicological data gaps. A framework describing the use of expert judgment in evaluating analogs for SAR has been published and widely cited, however, reliance on expert judgment can introduce inconsistent results across experts and hinder transparency. Here we explore the use of a quantitative similarity score between an analog and a Structure of Interest (SOI) to see if these scores correlate with the expert judgement-based suitability rankings. We find these global similarity scores representing a "whole-molecule" view of similarity to be insensitive to differences in local structure which may be important for toxicity, and, therefore, cannot be substituted for expert judgement-based similarity rankings. In this paper, we suggest that the next step in the progression of SAR approaches retains the insights from expert judgment, but facilitates consistency and transparency through the development of rating "rules". This report outlines and defines analog rating rules for several compound categories. While not comprehensive, the exercises demonstrate the development of rules for categories with a large spread in molecular weight and alkyl chain length and explains the advantages that we see in this approach compared to relying solely on a computational approach or an unstructured expert judgement approach. These rules may be incorporated into analog searching work flows to define boundaries for analogs "suitable" for read-across.

Toward Good Read-Across Practice (GRAP) guidance.

Grouping of substances and utilizing read-across of data within those groups represents an important data gap filling technique for chemical safety assessments. Categories/analogue groups are typically developed based on structural similarity and, increasingly often, also on mechanistic (biological) similarity. While read-across can play a key role in complying with legislations such as the European REACH regulation, the lack of consensus regarding the extent and type of evidence necessary to support it often hampers its successful application and acceptance by regulatory authorities. Despite a potentially broad user community, expertise is still concentrated across a handful of organizations and individuals. In order to facilitate the effective use of read-across, this document aims to summarize the state-of-the-art, summarizes insights learned from reviewing ECHA published decisions as far as the relative successes/pitfalls surrounding read-across under REACH and compile the relevant activities and guidance documents. Special emphasis is given to the available existing tools and approaches, an analysis of ECHA's published final decisions associated with all levels of compliance checks and testing proposals, the consideration and expression of uncertainty, the use of biological support data and the impact of the ECHA Read-Across Assessment Framework (RAAF) published in 2015.

Respiratory allergenic potential of plant-derived proteins: Understanding the relationship between exposure and potency for risk assessments.

Botanical ingredients (ingredients derived from plants) are finding increasing application in personal care products and the public perceives these ingredients to be safe. However, some proteins in botanicals have the potential to cause immediate-type (IgE-mediated) respiratory allergic reactions. Although reports of such reactions are uncommon, when they do occur, they can be severe. Experience with soap containing wheat proteins illustrated that under certain specific conditions, consumers may be affected. Establishing safe exposure levels for botanical proteins has been challenging. Industrial enzymes provide a rich reference dataset based on their historical association with allergic reactions among workers, which includes robust dose-response information. In the absence of similar data on the potency of plant proteins, a conservative default approach has historically been applied based on information derived from allergenic enzymes. In this article we review the historical default approach and dataset for setting limits for plant proteins in botanical ingredients based on analogy to industrial enzymes followed by a synthesis of literature data on allergic reactions following inhalation exposure to plant-derived proteins. The aim is to share relevant background information and display the relationship between exposure and potency as a first step in the development of a strategy for the development of an improved approach to support the risk assessment of plant-derived proteins.

Chemical Safety Assessment Using Read-Across: Assessing the Use of Novel Testing Methods to Strengthen the Evidence Base for Decision Making.

BACKGROUND: Safety assessment for repeated dose toxicity is one of the largest challenges in the process to replace animal testing. This is also one of the proof of concept ambitions of SEURAT-1, the largest ever European Union research initiative on alternative testing, co-funded by the European Commission and Cosmetics Europe. This review is based on the discussion and outcome of a workshop organized on initiative of the SEURAT-1 consortium joined by a group of international experts with complementary knowledge to further develop traditional read-across and include new approach data. OBJECTIVES: The aim of the suggested strategy for chemical read-across is to show how a traditional read-across based on structural similarities between source and target substance can be strengthened with additional evidence from new approach data--for example, information from in vitro molecular screening, "-omics" assays and computational models--to reach regulatory acceptance. METHODS: We identified four read-across scenarios that cover typical human health assessment situations. For each such decision context, we suggested several chemical groups as examples to prove when read-across between group members is possible, considering both chemical and biological similarities. CONCLUSIONS: We agreed to carry out the complete read-across exercise for at least one chemical category per read-across scenario in the context of SEURAT-1, and the results of this exercise will be completed and presented by the end of the research initiative in December 2015.

A strategy for safety assessment of chemicals with data gaps for developmental and/or reproductive toxicity.

Alternative methods for full replacement of in vivo tests for systemic endpoints are not yet available. Read across methods provide a means of maximizing utilization of existing data. A limitation for the use of read across methods is that they require analogs with test data. Repeat dose data are more frequently available than are developmental and/or reproductive toxicity (DART) studies. There is historical precedent for using repeat dose data in combination with a database uncertainty factor (UF) to account for missing DART data. We propose that use of the DART decision tree (Wu et al., 2013), in combination with a database UF, provides a path forward for DART data gap filling that better utilizes all of the data. Our hypothesis was that chemical structures identified by the DART tree as being related to structures with known DART toxicity would potentially have lower DART NOAELs compared to their respective repeat dose NOAELs than structures that lacked this association. Our analysis supports this hypothesis and as a result also supports that the DART decision tree can be used as part of weight of evidence in the selection of an appropriate DART database UF factor.

Use of read-across and computer-based predictive analysis for the safety assessment of PEG cocamines.

In the European Union animal testing has been eliminated for cosmetic ingredients while the US Cosmetic Ingredient Review Expert Panel may request data from animal studies. The use of read-across and predictive toxicology provides a path for filling data gaps without additional animal testing. The PEG cocamines are tertiary amines with an alkyl group derived from coconut fatty acids and two PEG chains of varying length. Toxicology data gaps for the PEG cocamines can be addressed by read-across based on structure-activity relationship using the framework described by Wu et al. (2010) for identifying suitable structural analogs. Data for structural analogs supports the conclusion that the PEG cocamines are non-genotoxic and not expected to exhibit systemic or developmental/reproductive toxicity with use in cosmetics. Due to lack of reliable dermal sensitization data for suitable analogs, this endpoint was addressed using predictive software (TIMES SS) as a first step (Laboratory of Mathematical Chemistry). The prediction for PEG cocamines was the same as that for PEGs, which have been concluded to not present a significant concern for dermal sensitization. This evaluation for PEG cocamines demonstrates the utility of read-across and predictive toxicology tools to assess the safety of cosmetic ingredients.

A framework to facilitate consistent characterization of read across uncertainty.

A process for evaluating analogues for use in structure activity relationship (SAR) assessments was previously published (Wu et al., 2010) and tested using a series of case studies (Blackburn et al., 2011). SAR-based "read across" approaches continue to be broadly used to address toxicological data gaps. The potential additional uncertainty introduced into risk assessments as a result of application of read across approaches to fill data gaps has been widely discussed (OECD, 2007; ECETOC, 2012; Patlewicz et al., 2013), but to date a systematic framework to guide the characterization of uncertainty in read across assessments has not been proposed. The current manuscript presents both a systematic framework to describe potential areas of additional uncertainty that may arise in read across (evaluated based on the number and suitability of analogues contributing data, severity of the critical effect, and effects and potency concordance), as well as a questionnaire for evaluating and documenting consideration of these potential additional sources of uncertainty by risk assessors. Application of this framework represents a next step in standardizing the read across process, both by providing a means to transparently assign a level of uncertainty to a SAR-based read across assessment and by facilitating consistency in read across conclusions drawn by different risk assessors.

Framework for identifying chemicals with structural features associated with the potential to act as developmental or reproductive toxicants.

Developmental and reproductive toxicity (DART) end points are important hazard end points that need to be addressed in the risk assessment of chemicals to determine whether or not they are the critical effects in the overall risk assessment. These hazard end points are difficult to predict using current in silico tools because of the diversity of mechanisms of action that elicit DART effects and the potential for narrow windows of vulnerability. DART end points have been projected to consume the majority of animals used for compliance with REACH; thus, additional nonanimal predictive tools are urgently needed. This article presents an empirically based decision tree for determining whether or not a chemical has receptor-binding properties and structural features that are consistent with chemical structures known to have toxicity for DART end points. The decision tree is based on a detailed review of 716 chemicals (664 positive, 16 negative, and 36 with insufficient data) that have DART end-point data and are grouped into defined receptor binding and chemical domains. When tested against a group of chemicals not included in the training set, the decision tree is shown to identify a high percentage of chemicals with known DART effects. It is proposed that this decision tree could be used both as a component of a screening system to identify chemicals of potential concern and as a component of weight-of-evidence decisions based on structure-activity relationships (SAR) to fill data gaps without generating additional test data. In addition, the chemical groupings generated could be used as a starting point for the development of hypotheses for in vitro testing to elucidate mode of action and ultimately in the development of refined SAR principles for DART that incorporate mode of action (adverse outcome pathways).

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.

Correlation of chemical structure with reproductive and developmental toxicity as it relates to the use of the threshold of toxicological concern.

In the absence of toxicological data on a chemical, the threshold of toxicological concern (TTC) approach provides a system to estimate a conservative exposure below which there is a low probability of risk for adverse health effects. The original toxicology dataset underlying the TTC was based on NOELs from repeat dose studies. Subsequently there have been several efforts to assess whether or not these limits are also protective for reproductive/developmental effects. This work expands the database of chemicals with reproductive and developmental data, presents these data in a comprehensive and transparent format and groups the chemicals according to the TTC "Cramer Class" rules. Distributions of NOAELs from each of these classes were used to assess whether the previously proposed TTC values based on repeat dose data are protective for reproductive/developmental toxicity endpoints as well. The present analysis indicates that, for each Cramer Class, the reproductive and developmental endpoints would be protected at the corresponding general TTC tiers derived by Munro et al. (1996).

Case studies to test: A framework for using structural, reactivity, metabolic and physicochemical similarity to evaluate the suitability of analogs for SAR-based toxicological assessments.

A process for evaluating analogs for use in SAR (Structure-Activity Relationship) assessments was previously published (Wu et al. 2010). Subsequently, this process has been updated to include a decision tree for estrogen binding (from US EPA) and flags for developmental and reproductive toxicity (DART). This paper presents the results of blinded case studies designed to test this updated framework. The results of these case studies support the conclusion that the process outlined by Wu et al. (2010) can be successfully applied to develop surrogate values for risk assessment. The read across results generated by the process were shown to be protective when compared to the actual toxicity data. Successful application of the approach requires significant expertise as well as discipline to not overstep the boundaries of the defined analogs and the rating system. The end result of this rigor can be the inability to read across all endpoints for all chemicals resulting in data gaps that cannot be filled using read across, however, this reflects the current state of the science and is preferable to making non-protective decisions. Future work will be targeted towards expanding read across capabilities. Two examples of a broader category approach are also shown.

A framework for using structural, reactivity, metabolic and physicochemical similarity to evaluate the suitability of analogs for SAR-based toxicological assessments.

A systematic expert-driven process is presented for evaluating analogs for read across in SAR (structure activity relationship) toxicological assessments. The approach involves categorizing potential analogs based upon their degree of structural, reactivity, metabolic and physicochemical similarity to the chemical with missing toxicological data (target chemical). It extends beyond structural similarity, and includes differentiation based upon chemical reactivity and addresses the potential that an analog and target could show toxicologically significant metabolic convergence or divergence. In addition, it identifies differences in physicochemical properties, which could affect bioavailability and consequently biological responses observed in vitro or in vivo. The approach provides a stepwise decision tree for categorizing the suitability of analogs, which qualitatively characterizes the strength of the evidence supporting the hypothesis of similarity and level of uncertainty associated with their use for read across. The result is a comprehensive framework to apply chemical, biochemical and toxicological principles in a systematic manner to identify and evaluate factors that can introduce uncertainty into SAR assessments, while maximizing the appropriate use of all available data.

Tissue distribution of 20 nm, 100 nm and 1000 nm fluorescent polystyrene latex nanospheres following acute systemic or acute and repeat airway exposure in the rat.

Understanding tissue distribution and clearance of nanomaterials following different routes of exposure is needed for risk assessment. F344 female rats received single or multiple exposures to 20 nm, 100 nm or 1000 nm latex fluorospheres by intravenous (i.v.) injection or oral pharyngeal aspiration into the airways. The presence of fluorospheres in tissues was assessed up to 90-120 days after the final dose. Blood, perfusion fluid, bone marrow, brain, eyes, feces, gut, heart, kidney, liver, lung, muscle, skin, spleen, thymus, tongue, urine and uterus plus ovaries were collected for analysis. Liver, spleen and lung were the greatest tissue depots for all particles following i.v. injection. The proportion of 100 nm and 1000 nm but not 20 nm spheres significantly increased in the spleen over time. Lung was the greatest tissue depot for all particles following single or repeat airway exposure. Greater than 95% of 1000 nm spheres that were recovered were in the lung in contrast to 70-80% of 20 nm spheres or 89-95% of 100 nm spheres. All 3 sizes were found in gut or gut+feces 1-7 days after lung exposure. The thymus was the largest extra-pulmonary depot for the particles; up to 25% of recovered 20 nm particles were in the thymus up to 4 months after exposure compared to 6% of 100 nm particles and 1-3% of 1000 nm particles. A small proportion of 20 nm particles were detected in kidney following both acute and repeat airway exposure. Low numbers of particles were found in the circulation (blood, perfusion), bone marrow, brain, heart, liver and spleen but not in eye, muscle, skin, tongue, ovaries, uterus or urine. These data show that the tissue targets of nano- and micron-sized spheres are very similar whether exposure occurs systemically or via the airways while the proportion of particles in some tissues and tissue clearance varies based on particle size.

Application of the threshold of toxicological concern approach to ingredients in personal and household care products.

In the absence of chemical-specific data, the threshold of toxicological concern (TTC) provides a method to determine a conservative estimate of a chronic oral exposure below which there is a very low probability of risk. The TTC approach was originally developed to support exposures to indirect food additives and was based on linear low-dose risk estimates to assure protection in the event that the chemical was later determined to be a carcinogen. Subsequently, TTC values based on noncancer endpoints were proposed for chemicals without structural alerts for genotoxicity. The original database supporting the TTC values for noncancer endpoints includes >600 structurally diverse chemicals. The objectives of this work were to evaluate the applicability of the TTC database to ingredients used in consumer products based on a comparison of the diversity of chemical structures with those in the original TTC database and to confirm that the range of NOELs for these ingredients is consistent with the range of NOELs in the original database. The results show good coverage of the product ingredient structures and confirm that the NOELs for the ingredient chemicals are similar in range to the original dataset, supporting the use of the TTC for ingredients in consumer products.

Experimental synchronization of chaos in diode lasers with polarization-rotated feedback and injection.

We demonstrate experimental chaos synchronization between two chaotic semiconductor lasers subjected to polarization-rotated optical feedback and unidirectional injection. This system allows high-quality synchronization to be obtained between dissimilar lasers in a wide range of chaotic operating regimes. Another feature of this system is its operation at high characteristic frequencies, taking advantage of all-optical implementation. Time series and RF spectra showing synchronization are confirmed by high correlation coefficients in excess of 0.85.