Joint positive control testing in guinea pig skin sensitization tests. A harmonized approach

A scheme for the performance of positive control studies within a coordinated group of laboratories was proposed (joint positive control testing). The procedure has been described, as well as the first results of the validation phase of this joint positive control testing project. Adoption of this proposal within the participating six laboratories would lead to a reduction in the number of guinea pigs required for reliability and sensitivity checks from current approximate 12 studies per year down to 2 studies, i.e., 150-300 fewer animals per year. Another benefit would be the use of a harmonized, and therefore more comparable, method to perform guinea pig tests and interpret the data. In the validation phase of joint reading of the positive control studies, the congruency of reading could clearly be demonstrated. From the experience gained up to now, it was possible to draw the conclusion that a coordinated interlaboratory approach for positive control testing was fully acceptable and an improvement with regard to animal welfare.     

Evaluation of in vitro predictive tests for irritation and allergic sensitization

Investigations of in vitro procedures to predict the potential of substances as skin irritants and as allergens inducing delayed hypersensitivity (contact dermatitis) are described, with indications of possible advances and known limitations. The examination of keratome slices of skin for release of enzymes, for changed histochemistry and for utilization of radioisotope-labelled amino acids will detect weak irritants but is of doubtful value for moderate irritants and will detect corrosive substances only through their inhibition of all cell activities. Fibroblast cultures, tested with Clostridium perfringens toxin and chemicals, show similar limitations in detecting moderate or severe irritants. Fibroblast cultures can be made more relevant to epidermal exposure by an overlying layer of agar containing keratin. In vitro tests to detect induction of sensitizing potential for delayed hypersensitivity have made little progress. The most promising approach is to treat antigen-presenting Langerhans cells with antigen and co-culture with lymphocytes. The lymphocytes may be examined for changes in receptor expression, for synthesis of interleukin 2, and possibly for responses to allergen if sufficient cells become specifically sensitized. There are several in vitro techniques to detect responses of in vivo- or in vitro-sensitized lymphocytes treated with antigen.     

Computational approaches for skin sensitization prediction

Abstract

Drugs, cosmetics, preservatives, fragrances, pesticides, metals, and other chemicals can cause skin sensitization. The ability to predict the skin sensitization potential and potency of substances is therefore of enormous importance to a host of different industries, to customers’ and workers’ safety. Animal experiments have been the preferred testing method for most risk assessment and regulatory purposes but considerable efforts to replace them with non-animal models and in silico models are ongoing. This review provides a comprehensive overview of the computational approaches and models that have been developed for skin sensitization prediction over the last 10 years. The scope and limitations of rule-based approaches, read-across, linear and nonlinear (quantitative) structure–activity relationship ((Q)SAR) modeling, hybrid or combined approaches, and models integrating computational methods with experimental results are discussed followed by examples of relevant models. Emphasis is placed on models that are accessible to the scientific community, and on model validation. A dedicated section reports on comparative performance assessments of various approaches and models. The review also provides a concise overview of relevant data sources on skin sensitization.     

Citral: identifying a threshold for induction of dermal sensitization

Citral [CAS# 5392-40-5; EINECS# 226-394-6; RIFM # 116; cis- and trans-3,7-dimethyl-2,6-Octadienal] is an important fragrance ingredient appreciated for its powerful lemon-aroma. It is widely used in fragrance formulations and incorporated into numerous consumer products. A comprehensive review of the dermal sensitization data available for citral was undertaken with the goal of identifying a threshold for the induction of dermal sensitization. In 2007, a complete literature search was conducted. On-line databases that were surveyed included Chemical Abstract Services and the National Library of Medicine. In addition, the toxicologic database of the Research Institute for Fragrance materials, Inc. (RIFM) was searched, which includes numerous unpublished reports. Based on a weight of evidence approach, the data from this survey demonstrate that the human NOEL (No Observed Effect Level) for induction of dermal sensitization to citral is 1400 microg/cm(2). The identification of this induction threshold will allow for risk assessments to focus on primary prevention of contact allergy to citral based on a new Quantitative Risk Assessment (QRA) paradigm. This subsequent assessment will form the basis of a risk management approach; specifically a new IFRA (International Fragrance Association) standard on the use of citral in consumer products.     

Integrated decision strategies for skin sensitization hazard

One of the top priorities of the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) is the identification and evaluation of non‐animal alternatives for skin sensitization testing. Although skin sensitization is a complex process, the key biological events of the process have been well characterized in an adverse outcome pathway (AOP) proposed by the Organisation for Economic Co‐operation and Development (OECD). Accordingly, ICCVAM is working to develop integrated decision strategies based on the AOP using in vitro, in chemico and in silico information. Data were compiled for 120 substances tested in the murine local lymph node assay (LLNA), direct peptide reactivity assay (DPRA), human cell line activation test (h‐CLAT) and KeratinoSens assay. Data for six physicochemical properties, which may affect skin penetration, were also collected, and skin sensitization read‐across predictions were performed using OECD QSAR Toolbox. All data were combined into a variety of potential integrated decision strategies to predict LLNA outcomes using a training set of 94 substances and an external test set of 26 substances. Fifty‐four models were built using multiple combinations of machine learning approaches and predictor variables. The seven models with the highest accuracy (89–96% for the test set and 96–99% for the training set) for predicting LLNA outcomes used a support vector machine (SVM) approach with different combinations of predictor variables. The performance statistics of the SVM models were higher than any of the non‐animal tests alone and higher than simple test battery approaches using these methods. These data suggest that computational approaches are promising tools to effectively integrate data sources to identify potential skin sensitizers without animal testing. Published 2016. This article has been contributed to by US Government employees and their work is in the public domain in the USA.     

Is skin penetration a determining factor in skin sensitization potential and potency? Refuting the notion of a LogKow threshold for skin sensitization

It is widely accepted that substances that cannot penetrate through the skin will not be sensitizers. LogKow and molecular weight (MW) have been used to set thresholds for sensitization potential. Highly hydrophilic substances e.g. LogKow ≤ 1 are expected not to penetrate effectively to induce sensitization. To investigate whether LogKow >1 is a true requirement for sensitization, a large dataset of substances that had been evaluated for their skin sensitization potential under Registration, Evaluation, Authorisation and restriction of CHemicals (REACH), together with available measured LogKow values was compiled using the OECD eChemPortal. The incidence of sensitizers relative to non‐sensitizers above and below a LogKow of 1 was explored. Reaction chemistry principles were used to explain the sensitization observed for the subset of substances with a LogKow ≤0. 1482 substances were identified with skin sensitization data and measured LogKow values. 525 substances had a measured LogKow ≤ 1, 100 of those were sensitizers. There was no significant difference in the incidence of sensitizers above and below a LogKow of 1. Reaction chemistry principles that had been established for lower MW and more hydrophobic substances were found to be still valid in rationalizing the skin sensitizers with a LogKow ≤ 0. The LogKow threshold arises from the widespread misconception that the ability to efficiently penetrate the stratum corneum is a key determinant of sensitization potential and potency. Copyright © 2016 John Wiley & Sons, Ltd.     

Proposal for a risk assessment methodology for skin sensitization based on sensitization potency data

In this paper, we propose a quantitative risk assessment methodology for skin sensitization aiming at the derivation of 'safe' exposure levels for sensitizing chemicals, used e.g., as ingredients in consumer products. Given the limited number of sensitizers tested in human sensitization tests, such as the human repeat-insult patch test (HRIPT) or the human maximization test (HMT), we used EC3 values from the local lymph node assay (LLNA) in mice because they provide the best quantitative measure of the skin sensitizing potency of a chemical. A comparison of LLNA EC3 values with HRIPT and HMT LOEL, and NOEL values was carried out and revealed that the EC3, expressed as area dose, can be used as a surrogate value for the human NOEL in risk assessment. The uncertainty/extrapolation factor approach was used to derive (a) an 'acceptable non-sensitizing area dose' (ANSAD) to protect non-allergic individuals against skin sensitization and (b) an 'acceptable non-eliciting area dose' (ANEAD) to protect allergic individuals against elicitation of allergic contact dermatitis. For ANSAD derivation, interspecies, intraspecies and time extrapolation factors are applied to the LLNA EC3. For ANEAD derivation, additional application of a variable sensitization-elicitation extrapolation factor is proposed. Values for extrapolation factors are derived and discussed, the proposed methodology is applied to the sensitizers methylchloroisothiazolinone/methylisothiazolinone, cinnamic aldehyde and nickel and results are compared to published risk assessments.     

Strategies for identifying and developing new anticonvulsant drugs

The identification of new anticonvulsant drugs depends on the use of different animal models of epilepsy. The models should be mechanism-independent, able to screen a large number of compounds, at limited cost and technical expertise. Primary screening models include genetic or reflex models of epilepsy and electrically and chemically induced seizures. Once active compounds have been identified, more advanced mechanistic and seizure-specific models are needed to refine the choice of a lead compound. These can be eitherin vivo orin vitro models. Models known to interact with specific receptors or the production of the putative neurotransmitters of neural excitability or inhibition are valuable in assessing possible mechanisms of action.In vitro models have evolved as important tools in correlating changes in electrical phenomena and therapeutic spectrum. The use of the hippocampal slice and the cultured neuron permits classification of anticonvulsant activity based on cellular actions of the drug. Interactions by the experimental drugs with specific subcellular fractions of the central nervous system augment information on possible mechanisms of action. The final choice of compounds for development requires synthesizing and comparing all of the pharmacodynamic information with the pharmacokinetic and toxicologic data. In the final analysis, no single animal model of epilepsy known today can assure the development of better drugs for all treatment of the epilepsies.     

Non-animal test methods for predicting skin sensitization potentials

Contact allergies are complex diseases, and it is estimated that 15-20 % of the general population suffers from contact allergy, with increasing prevalence. Evaluation of the sensitization potential of a substance is usually carried out in animal models. Nowadays, there is much interest in reducing and ultimately replacing current animal tests. Furthermore, as of 2013, the EU has posed a ban on animal testing of cosmetic ingredients that includes skin sensitization. Therefore, predictive and robust in vitro tests are urgently needed. In order to establish alternatives to animal testing, the in vitro tests must mimic the very complex interactions between the sensitizing chemical and the different parts of the immune system. This review article summarizes recent efforts to develop in vitro tests for predicting skin sensitizers. Cell-based assays, in chemico methods and, to a lesser extent, in silico methods are presented together with a discussion of their current status. With considerable progress having been achieved during the last years, the rationale today is that data from different non-animal test methods will have to be combined in order to obtain reliable hazard and potency information on potential skin sensitizers.     

Drug hypersensitivity reactions in skin: understanding mechanisms and the development of diagnostic and predictive tests

Cutaneous manifestations of drug hypersensitivity can be serious and potentially life threatening and may prevent effective drug therapy. T cells play an important role in the pathology of drug hypersensitivity reactions. Classical studies suggest that T-cell activation requires drug bioactivation, covalent binding to protein and antigen processing to stimulate an immune response. Recent studies have shown that drugs can also be presented to T cells in the absence of antigen processing and drug metabolism. In this article, sulfamethoxazole is used as a paradigm to describe the chemical mechanisms involved in the initiation and maintenance of an aberrant drug antigen specific T-cell response. Presentation of the same drug to different individuals can cause a variety of skin diseases. Such reactions have been classified according to the phenotype and functionality of the T-cell response. This review summarises the different forms of cutaneous hypersensitivity reactions and describes how T-cell clones generated from hypersensitive patients have been used to study the cellular mechanisms of anticonvulsant hypersensitivity. Potential uses of in vitro cell culture assays for patient diagnosis and drug evaluation are also discussed.     

An in vitro human skin test for assessing sensitization potential

Sensitization to chemicals resulting in an allergy is an important health issue. The current gold‐standard method for identification and characterization of skin‐sensitizing chemicals was the mouse local lymph node assay (LLNA). However, for a number of reasons there has been an increasing imperative to develop alternative approaches to hazard identification that do not require the use of animals. Here we describe a human in‐vitro skin explant test for identification of sensitization hazards and the assessment of relative skin sensitizing potency. This method measures histological damage in human skin as a readout of the immune response induced by the test material. Using this approach we have measured responses to 44 chemicals including skin sensitizers, pre/pro‐haptens, respiratory sensitizers, non‐sensitizing chemicals (including skin‐irritants) and previously misclassified compounds. Based on comparisons with the LLNA, the skin explant test gave 95% specificity, 95% sensitivity, 95% concordance with a correlation coefficient of 0.9. The same specificity and sensitivity were achieved for comparison of results with published human sensitization data with a correlation coefficient of 0.91. The test also successfully identified nickel sulphate as a human skin sensitizer, which was misclassified as negative in the LLNA. In addition, sensitizers and non‐sensitizers identified as positive or negative by the skin explant test have induced high/low T cell proliferation and IFNγ production, respectively. Collectively, the data suggests the human in‐vitro skin explant test could provide the basis for a novel approach for characterization of the sensitizing activity as a first step in the risk assessment process. Copyright © 2015 John Wiley & Sons, Ltd.