Search

New Approach Methodologies (NAMs) are considered to include any in vitro, in silico or chemistry-based method, as well as the strategies to implement them, that may provide information that could inform chemical safety assessment. Current chemical legislation in the European Union is limited in its acceptance of the widespread use of NAMs. The European Partnership for Alternative Approaches to Animal Testing (EPAA) therefore convened a 'Deep Dive Workshop' to explore the use of NAMs in chemical safety assessment, the aim of which was to support regulatory decisions, whilst intending to protect human health. The workshop recognised that NAMs are currently used in many industrial sectors, with some considered as fit for regulatory purpose. Moreover, the workshop identified key discussion points that can be addressed to increase the use and regulatory acceptance of NAMs. These are based on the changes needed in frameworks for regulatory requirements and the essential needs in education, training and greater stakeholder engagement as well the gaps in the scientific basis of NAMs.

Skin sensitization following the covalent modification of proteins by low molecular weight chemicals (haptenation) is mediated by cytotoxic T lymphocyte (CTL) recognition of human leukocyte antigen (HLA) molecules presented on the surface of almost all nucleated cells. There exist 3 nonmutually exclusive hypotheses for how haptens mediate CTL recognition: direct stimulation by haptenated peptides, hapten modification of HLA leading to an altered HLA-peptide repertoire, or a hapten altered proteome leading to an altered HLA-peptide repertoire. To shed light on the mechanism underpinning skin sensitization, we set out to utilize proteomic analysis of keratinocyte presented antigens following exposure to 2,4-dinitrochlorobenzene (DNCB). We show that the following DNCB exposure, cultured keratinocytes present cysteine haptenated (dinitrophenylated) peptides in multiple HLA molecules. In addition, we find that one of the DNCB modified peptides derives from the active site of cytosolic glutathione-S transferase-ω. These results support the current view that a key mechanism of skin sensitization is stimulation of CTLs by haptenated peptides. Data are available via ProteomeXchange with identifier PXD021373.

Animal use for testing chemicals under REACH continues to increase, despite advances in non-animal safety science during the past 15 years. The application of modern science and technology, and the use of ‘next generation’ weight-of-evidence assessment approaches, are embedded in EU guidance for establishing the safety of cosmetics and foods – and of the ingredients used in these products. However, this is still not the case for the regulation of chemicals. Under the new Chemicals Strategy for Sustainability, thought leaders in human health and environmental protection are calling on the European Commission to quickly embrace the benefits of modern and innovative non-animal safety science, in place of outdated animal testing, if the EU is to be a leader in safe and sustainable innovation under the European Green Deal transformational change ambitions. The European Commission also needs to enable companies to meet their legal obligation to only conduct animal testing as a last resort, by providing a more flexible, science-based and consistent regulatory framework for assuring chemical safety, which supports the integration of data from different sources. We are at a tipping point for closing the gap between regulatory chemicals testing and modern safety science. It is time to join forces, across policy makers, scientists, regulators and lawyers, to lead the paradigm shift needed to deliver what EU citizens want – namely, chemicals and products that are safe and sustainable, without resorting to animal testing.

BACKGROUND: There is considerable interest in understanding the immunological variables that have the greatest influence on the effectiveness of sensitization by contact allergens, particularly in the context of developing new paradigms for risk assessment of novel compounds OBJECTIVES: To examine the relationship between patch test score for 3 different contact allergens and the characteristics of T-cell responses.PATIENTS/MATERIALS/METHODS: A total of 192 patients with confirmed nickel, p-phenylenediamine (PPD) or methylisothiazolinone allergy were recruited from the Contact Dermatitis Investigation Unit at Salford Royal Hospital. Severity of allergy was scored by patch testing and peripheral blood lymphocytes characterized for T cell phenotype by flow cytometry and proliferative activity by radiolabelled thymidine incorporation. Comparisons were drawn with buffy coat samples from healthy volunteers.RESULTS: Patch test positivity to nickel, PPD and MI was associated with changes in the phenotype of peripheral blood T-cells: increases in naïve cells, decreases in Treg cell frequency and the CD4+ :CD8hi ratio and increased expression of the skin homing marker CLA, particularly for those patients with a +++ patch test score.CONCLUSIONS: This increased understanding of the characteristics of the T cell responses to contact allergens may provide parameters to better measure health risks associated with skin sensitization. This article is protected by copyright. All rights reserved.

Our aim is to develop and apply next generation approaches to skin allergy risk assessment that do not require new animal test data and better quantify uncertainties. We introduce the concept of the population threshold – a chemical-specific exposure level at which no individual in a population will experience induction of contact allergy to a chemical. A Bayesian multilevel (hierarchical) regression model is developed to estimate this population threshold under the conditions of a human-repeat-insult-patch-test. The approach is built on historical human (HRIPT) and murine (LLNA) data but, importantly, enables prediction based on in vitro (DPRA, Keratinosens™, hCLAT and U-SENS™) data. The Bayesian probabilistic framework allows us to explicitly quantify the uncertainty in the population threshold. Our skin allergy risk assessment defined approach (SARA DA) is used to estimate population thresholds for 30 chemicals using a weight-of-evidence across publicly available human, murine and in vitro data. Additionally, estimates for a further 43 chemicals are presented using chemical-specific data from in vitro assays only. Comparisons are made with current risk assessment metrics and across data types. We demonstrate that the approach can be used to derive a point-of-departure for next generation risk assessment based on in vitro data only.

At the ESCD congress held in Manchester in 2016, a session was organized to encourage more dialogue between clinicians with expertise in skin sensitization and toxicologists seeking to provide effective risk assessment to prevent human health issues. That session focused on the remaining uncertainties regarding the induction and regulation of skin sensitization in humans, and the opportunities and challenges associated with the refinement and improvement of risk assessment methodologies. This short article, prompted by those discussions, debates what the authors regard as being among the most important and most intriguing uncertainties about skin sensitization and allergic contact dermatitis in humans, and the most significant opportunities for improving risk assessment. The aim has been to provide a basis for mapping out the areas that might benefit from a closer alignment between the relevant clinical community and toxicologists charged with the responsibility of ensuring that skin sensitization risks are understood and managed.

Diphenylcyclopropenone (DPC) is an organic chemical hapten which induces allergic contact dermatitis and is used in the treatment of warts, melanoma, and alopecia areata. This therapeutic setting therefore provided an opportunity to study T cell receptor (TCR) repertoire changes in response to hapten sensitization in humans. Repeated exposure to DPC induced highly dynamic transient expansions of a polyclonal diverse T cell population. The number of TCRs expanded early after sensitization varies between individuals and predicts the magnitude of the allergic reaction. The expanded TCRs show preferential TCR V and J gene usage and consist of clusters of TCRs with similar sequences, two characteristic features of antigen-driven responses. The expanded TCRs share subtle sequence motifs that can be captured using a dynamic Bayesian network. These observations suggest the response to DPC is mediated by a polyclonal population of T cells recognizing a small number of dominant antigens.

Next generation Risk Assessment (NGRA) is an exposure-led, hypothesis-driven approach which integrates new approach methodologies (NAMs) to assure safety without generating animal data. This hypothetical skin allergy risk assessment of two consumer products - face cream containing 0.1% coumarin and deodorant containing 1% coumarin - demonstrates the application of our skin allergy NGRA framework which incorporates our Skin Allergy Risk Assessment (SARA) Model. SARA uses Bayesian statistics to provide a human relevant point of departure and risk metric for a given chemical exposure based upon input data that can include both NAMs and historical in vivo studies. Regardless of whether NAM or in vivo inputs were used, the model predicted that the face cream and deodorant exposures were low and high risk respectively. Using only NAM data resulted in a minor underestimation of risk relative to in vivo. Coumarin is a predicted pro-hapten and consequently, when applying this mechanistic understanding to the selection of NAMs the discordance in relative risk could be minimized. This case study demonstrates how integrating a computational model and generating bespoke NAM data in a weight of evidence framework can build confidence in safety decision making.

The synthesis of a 72-membered macrocyclic tetraester bolaamphiphile is accomplished in six chemical steps from commercially available starting materials using copper-accelerated azide–alkyne coupling to close the macrocycle in high yield. Related diester amphiphiles and an acyclic tetraester bolaamphiphile were also prepared. The set of lipids bearing nitrophenyl phosphate head groups were incorporated into phospholipid vesicles but failed to undergo phosphate hydrolysis in basic conditions, undergoing efficient elimination in competition. The same lipid cores bearing phosphate-linked nitrobenzoxadiazole (NBD) head groups also incorporated into phospholipid vesicles and the NBD fluorescence was quenched with cobalt ions. The proportion of membrane-spanning bolaamphiphiles was determined from the ratio of cobalt quenching in the presence and in the absence of a detergent. The macrocyclic bolaamphiphile is incorporated into phospholipid vesicles such that 48 ± 4% of the NBD head groups are in the outer leaflet, consistent with a membrane-spanning orientation. The acyclic bolaamphiphile is incorporated with 75 ± 3% of the NBD head groups accessible to quencher in the absence of a detergent suggesting U-shaped incorporation in the outer leaflet of the bilayer membrane. In ring size and spanning ability, the macrocyclic bolaamphiphile mimics naturally occurring macrocyclic archaeal lipids.

Efforts are underway to transform regulatory toxicology and chemical safety assessment from a largely empirical science based on direct observation of apical toxicity outcomes in whole organism toxicity tests to a predictive one in which outcomes and risk are inferred from accumulated mechanistic understanding. The adverse outcome pathway (AOP) framework provides a systematic approach for organizing knowledge that may support such inference. Likewise, computational models of biological systems at various scales provide another means and platform to integrate current biological understanding to facilitate inference and extrapolation. We argue that the systematic organization of knowledge into AOP frameworks can inform and help direct the design and development of computational prediction models that can further enhance the utility of mechanistic and in silico data for chemical safety assessment. This concept was explored as part of a workshop on AOP-Informed Predictive Modeling Approaches for Regulatory Toxicology held September 24–25, 2015. Examples of AOP-informed model development and its application to the assessment of chemicals for skin sensitization and multiple modes of endocrine disruption are provided. The role of problem formulation, not only as a critical phase of risk assessment, but also as guide for both AOP and complementary model development is described. Finally, a proposal for actively engaging the modeling community in AOP-informed computational model development is made. The contents serve as a vision for how AOPs can be leveraged to facilitate development of computational prediction models needed to support the next generation of chemical safety assessment.

Summary Background Methylisothiazolinone (MI), a preservative that is commonly used in personal care products, is now recognized as an important contact allergen in both cosmetic and occupational settings. Objectives To analyse T lymphocyte responses to MI, in order to provide important information regarding the relationship between the nature of such responses and skin sensitization potency. Methods Proliferative responses to free MI and to an MI–human serum albumin (HSA) conjugate were measured according to [3H]thymidine incorporation (n = 56 donors; patch test scores of + in 20, ++ in 29, and +++ in 7). Peripheral blood mononuclear cells were cultured in the presence of MI (0.001–1 µg/ml) or MI–HSA (0.001–100 µg/ml). Proliferating CD4+ and CD8+ T lymphocytes were identified by flow cytometry with the intracellular marker Ki-67. Results For free MI, modest positive responses were recorded for 7 of 31 donors. In contrast, MI–HSA stimulated more marked responses in 17 of 31 donors. Characterization of positive proliferative responses showed variable patterns of proliferating CD4+ and CD8+ T lymphocytes from donors with the same patch test scores and similar maximal values. Conclusions MI–HSA is able to induce secondary responses in lymphocytes drawn from sensitized subjects, and provides a more effective source of antigen than free MI. Furthermore, individual donors show differential activity profiles with respect to T lymphocyte subsets.

Whereas T lymphocyte (T cell) activation is the key event in the acquisition of skin sensitization and subsequent elicitation of allergic contact dermatitis, the humoral component of immune responses to organic contact allergens has received little consideration. There is evidence that, in experimental animals, topical exposure to potent contact allergens is associated with B cell activation and proliferation, and hapten-specific antibody production. However, there is very limited evidence available for anti-hapten antibody responses being induced following topical exposure of humans to contact allergens. Nevertheless, it is important to appreciate that there are almost no negative studies in which evidence for antibody production as the result of skin sensitization has been sought and not found. That is, there is absence of evidence rather than evidence of absence. Furthermore, exposure to chemical respiratory allergens, in which the skin has been implicated as a potential route of sensitization, results in anti-hapten antibody responses. It is proposed that skin sensitization to contact allergens will normally be accompanied by antibody production. The phenomenon is worthy of investigation, as anti-hapten antibodies could potentially influence and/or regulate the induction of skin sensitization. Moreover, such antibodies may provide an informative correlate of the extent to which sensitization has been acquired.

Allergic contact dermatitis (ACD) is driven by the activation and proliferation of allergen-specific memory T-lymphocytes and is currently diagnosed by patch testing with a selected panel of chemical allergens. The lymphocyte transformation test (LTT) can be used to monitor ex vivo T-lymphocyte responses to antigens, including contact allergens. The LTT is not viewed as being an alternative to patch testing, but it does seek to reflect experimentally skin sensitization to specific chemicals. The LTT is based on stimulation in vitro of antigen-driven T-lymphocyte proliferation. That is, exposure in culture of primed memory T-lymphocytes to the relevant antigen delivered in an appropriate configuration will provoke a secondary response that reflects the acquisition of skin sensitization. The technical aspects of this test and the utility of the approach for investigation of immune responses to contact allergens in humans are reviewed here, with particular emphasis on further development and refinement of the protocol. An important potential application is that it may provide a basis for characterizing those aspects of T-lymphocyte responses to contact allergens that have the greatest influence on skin sensitizing potency and this will be considered in some detail.

Although adoption of skin sensitization in vivo assays for hazard identification is likely to be successful in the next few years, this does not replace their use in potency prediction. Notably, measurement of potency of skin sensitizers in the local lymph node assay has been important. However, this local lymph node assay potency measure has not been formally assessed against a range of substances of known human sensitizing potential, because the latter is lacking. Accordingly, criteria for human data have been established that characterize 6 categories of human sensitizing potency, with 1 the most potent and 5 the least potent; category 6 represents true nonsensitizers. The literature has been searched, and 131 chemicals assigned into these categories according to their intrinsic potency judged only by the available human information. The criteria and data set generated provide a basis for examination of the capacity of nonanimal approaches for the determination of human sensitization potency.

In a previous EPAA-Cefic LRI workshop in 2011, issues surrounding the use and interpretation of results from the local lymph node assay were addressed. At the beginning of 2013 a second joint workshop focused greater attention on the opportunities to make use of non-animal test data, not least since a number of in vitro assays have progressed to an advanced position in terms of their formal validation. It is already recognised that information produced from non-animal assays can be used in regulatory decision-making, notably in terms of classifying a substance as a skin sensitiser. The evolution into a full replacement for hazard identification, where the decision is not to classify, requires the generation of confidence in the in vitro alternative, e.g. via formal validation, the existence of peer reviewed publications and the knowledge that the assay(s) are founded on key elements of the Adverse Outcome Pathway for skin sensitisation. It is foreseen that the validated in vitro assays and relevant QSAR models can be organised into formal testing strategies to be applied for regulatory purposes by the industry. To facilitate progress, the European Partnership for Alternative Approaches to animal testing (EPAA) provided the platform for cross-industry and regulatory dialogue, enabling an essential and open debate on the acceptability of an in vitro based integrated strategy. Based on these considerations, a follow up activity was agreed upon to explore an example of an Integrated Testing Strategy for skin sensitisation hazard identification purposes in the context of REACH submissions.

Summary Consumer safety risk assessment of skin sensitization requires information on both consumer exposure to the ingredient through product use and the hazardous properties of the ingredient. Significant progress has been made in determining the hazard potential of ingredients without animal testing. However, hazard identification is insufficient for risk assessment, and an understanding of the dose-response is needed. Obtaining such knowledge without animal testing is challenging and requires applying available mechanistic knowledge to both assay development and the integration of these data. The recent OECD report “The Adverse Outcome Pathway for Skin Sensitization Initiated by Covalent Binding to Proteins” presents the available mechanistic knowledge of the sensitization response within an adverse outcome pathway (AOP). We propose to use this AOP as the mechanistic basis for physiologicallyand mechanistically-based toxicokinetic-toxicodynamic models of the sensitization response. The approach would be informed by non-animal data, provide predictions of the dose-response required for risk assessment, and would be evaluated against human clinical data.

Methylisothiazolinone (MI), a preservative that is commonly used in personal care products, is now recognized as an important contact allergen in both cosmetic and occupational settings.To analyse T lymphocyte responses to MI, in order to provide important information regarding the relationship between the nature of such responses and skin sensitization potency.Proliferative responses to free MI and to an MI-human serum albumin (HSA) conjugate were measured according to [(3) H]thymidine incorporation (n = 56 donors; patch test scores of + in 20, ++ in 29, and +++ in 7). Peripheral blood mononuclear cells were cultured in the presence of MI (0.001-1 µg/ml) or MI-HSA (0.001-100 µg/ml). Proliferating CD4(+) and CD8(+) T lymphocytes were identified by flow cytometry with the intracellular marker Ki-67.For free MI, modest positive responses were recorded for 7 of 31 donors. In contrast, MI-HSA stimulated more marked responses in 17 of 31 donors. Characterization of positive proliferative responses showed variable patterns of proliferating CD4(+) and CD8(+) T lymphocytes from donors with the same patch test scores and similar maximal values.MI-HSA is able to induce secondary responses in lymphocytes drawn from sensitized subjects, and provides a more effective source of antigen than free MI. Furthermore, individual donors show differential activity profiles with respect to T lymphocyte subsets.

The use of Integrated Testing Strategies (ITS) permits the combination of diverse types of chemical and toxicological data for the purposes of hazard identification and characterisation. In November 2008, the European Partnership for Alternative Approaches to Animal Testing (EPAA), together with the European Centre for the Validation of Alternative Methods (ECVAM), held a workshop on Overcoming Barriers to Validation of Non-animal Partial Replacement Methods/Integrated Testing Strategies, in Ispra, Italy, to discuss the extent to which current ECVAM approaches to validation can be used to evaluate partial replacement in vitro test methods (i.e. as potential ITS components) and ITS themselves. The main conclusions of these discussions were that formal validation was only considered necessary for regulatory purposes (e.g. the replacement of a test guideline), and that current ECVAM approaches to validation should be adapted to accommodate such test methods (1). With these conclusions in mind, a follow-up EPAA–ECVAM workshop was held in October 2009, to discuss the extent to which existing validation principles are applicable to the validation of ITS test methods, and to develop a draft approach for the validation of such test methods and/or overall ITS for regulatory purposes. This report summarises the workshop discussions that started with a review of the current validation methodologies and the presentation of two case studies (skin sensitisation and acute toxicity), before covering the definition of ITS and their components, including their validation and regulatory acceptance. The following main conclusions/recommendations were made: that the validation of a partial replacement test method (for application as part of a testing strategy) should be differentiated from the validation of an in vitro test method for application as a stand-alone replacement, especially with regard to its predictive capacity; that, in the former case, the predictive capacity of the whole testing strategy (rather than of the individual test methods) would be more important, especially if the individual test methods had a high biological relevance; that ITS allowing for flexible and ad hoc approaches cannot be validated, whereas the validation of clearly defined ITS would be feasible, although practically quite difficult; and that test method developers should be encouraged to develop and submit to ECVAM not only full replacement test methods, but also partial replacement methods to be placed as parts of testing strategies. The added value from the formal validation of testing strategies, and the requirements needed in view of regulatory acceptance of the data, require further informed discussion within the EPAA forum on the basis of case studies provided by industry.

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.

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 Commissions 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 79 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 57 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.

The sensitizing potential of chemicals is usually identified and characterized using one of the available animal test methods, such as the mouse local lymph node assay. Due to the increasing public and political concerns regarding the use of animals for the screening of new chemicals, the Colipa Skin Tolerance Task Force collaborates with and/or funds research groups to increase and apply our understanding of the events occurring during the acquisition of skin sensitization. Knowledge gained from this research is used to support the development and evaluation of novel alternative approaches for the identification and characterization of skin sensitizing chemicals. At present one in chemico (direct peptide reactivity assay (DPRA)) and two in vitro test methods (cell based assays (MUSST and h-CLAT)) have been evaluated within Colipa inter-laboratory ring trials and accepted by the European Centre for the Validation of Alternative Methods (ECVAM) for pre-validation. Data from all three test methods will be used to support the development of testing strategy approaches for skin sensitizer potency prediction. The replacement of the need for animal testing for skin sensitization risk assessment is viewed as ultimately achievable and the next couple of years should set the timeline for this milestone.

There is considerable interest in the development of in vitro methods for the identification of contact sensitizers, including those that use cultured dendritic cells (DC), key players in cutaneous immune responses. Chemical allergens, such as dinitrobenzene sulfonic acid (DNBS), or skin irritants, such as benzene sulfonic acid (BS), induce modest changes in DC phenotype. In an attempt to increase the sensitivity of DC responses, DC have been co-cultured with chemical and DC activators (toll-like receptor [TLR] ligands). Cells were cultured with DNBS or BS at doses of equivalent cytotoxicity, together with sub-optimal doses of selected TLR ligands (Pam(3)Cys-Ser-(Lys)(4) [PAM], TLR1-2; macrophage-activating lipopeptide-2 [MALP-2], TLR6-2; or flagellin; TLR5). Both chemicals caused a decline in cell viability. DNBS induced a higher proportion of late apoptotic/necrotic cells whereas BS was associated with early apoptotic cells, suggesting different mechanisms of cell death. Some synergy was observed for interleukin (IL)-6 and tumor necrosis factor alpha production for DC co-cultured with BS and MALP-2/PAM. In contrast, there were marked synergistic effects on IL-6 secretion when DC were cultured with DNBS and flagellin. It may be possible to exploit this enhanced sensitivity of flagellin-activated DC for chemical allergen for the development of in vitro skin sensitization assays.

We have developed an in silico model of the induction of skin sensitisation, in order to characterise and quantify the contribution of each pathway to the overall biological process. This analysis has been used to guide our research on skin sensitisation and in vitro test development programmes, and provides a theoretical rationale for the interpretation and integration of non-animal predictive data for risk assessment (RA) purposes. The in vivo mouse Local Lymph Node Assay (LLNA) is now in widespread use for the evaluation of skin sensitisation potential and potency. Recent changes in European Union (EU) legislation (i.e. the 7th Amendment to the EU Cosmetics Directive) have made the development of non-animal approaches to provide the data for skin sensitisation RA a key business need. Several in vitro predictive assays have already been developed for the prediction of skin sensitisation. However, these are based on the determination of a small number of pathways within the overall biological process, and our understanding of the relative contribution of these individual pathways to skin sensitisation induction is limited. To address this knowledge gap, a “systems biology” approach has been used to construct a computer-based mathematical model of the induction of skin sensitisation, in collaboration with Entelos, Inc. The biological mechanisms underlying the induction phase of skin sensitisation are represented by nonlinear ordinary differential equations and defined by using information from over 500 published papers. By using the model, we have identified knowledge gaps for future investigative research, and key factors that have a major influence on the induction of skin sensitisation (e.g. TNF-α production in the epidermis). The relative contribution of each of these key pathways has been assessed by determining their contributions to the overall process (e.g. sensitiser-specific T-cell proliferation in the draining lymph node). This information provides a biologically-relevant rationale for the interpretation and potential integration of diverse types of non-animal predictive data. Consequently, the Skin Sensitisation Physiolab® (SSP) platform represents one approach to integration that is likely to prove an invaluable tool for hazard evaluation in a new framework for consumer safety RA.

Allergic contact dermatitis is, to a considerable extent, a preventable disease. Limitation of allergic contact dermatitis can be achieved by correct detection of skin sensitizers, characterization of potency, understanding of human skin exposure and the application of adequate risk assessment and management strategies. A range of methods exist currently that have been proven to be highly accurate in terms of the predictive identification of chemicals that possess skin sensitizing properties. In addition, certain methods, notably the local lymph node assay, also deliver valuable information regarding the relative potency of identified sensitizers. Great use can be made of this potency information in the application of quantitative risk assessments (although, of course, such assessments also depend on the availability of accurate data on human skin exposure). However, the challenge now to be faced is how to obtain the same quality of information on the potency of skin sensitizing chemicals using solely in ...

Allergic contact dermatitis (ACD) is to a considerable extent a preventable disease. Limitation of ACD can be achieved by correct detection of skin sensitizers, characterization of potency, understanding of human skin exposure, and the application of adequate risk assessment and management strategies. A range of methods now exist that have been proven to be very accurate in terms of the predictive identification of chemicals that possess skin sensitizing properties. In addition, certain methods, notably the local lymph node assay (LLNA), also deliver valuable information of the relative potency of identified sensitizers. Great use can be made of this potency information in the application of quantitative risk assessments (although of course such assessments depend also on the availability of accurate data on human skin exposure). However, the challenge now to be faced is how to obtain the same quality of information on the potency of skin sensitizing chemicals using solely in vitro and in silico methods. With the forthcoming elimination of in vivo tests, the opportunities being exploited for in vitro test development focus on key elements of the sensitization process, such as peptide binding and dendritic cell activation. What has to then be addressed is how information from such in vitro assays is integrated, together with data on epidermal bioavailability, to deliver an assessment of the allergen potency.

Whereas T lymphocyte (T cell) activation is the key event in the acquisition of skin sensitization and subsequent elicitation of allergic contact dermatitis, the humoral component of immune responses to organic contact allergens has received little consideration. There is evidence that, in experimental animals, topical exposure to potent contact allergens is associated with B cell activation and proliferation, and hapten-specific antibody production. However, there is very limited evidence available for anti-hapten antibody responses being induced following topical exposure of humans to contact allergens. Nevertheless, it is important to appreciate that there are almost no negative studies in which evidence for antibody production as the result of skin sensitization has been sought and not found. That is, there is absence of evidence rather than evidence of absence. Furthermore, exposure to chemical respiratory allergens, in which the skin has been implicated as a potential route of sensitization, results in anti-hapten antibody responses. It is proposed that skin sensitization to contact allergens will normally be accompanied by antibody production. The phenomenon is worthy of investigation, as anti-hapten antibodies could potentially influence and/or regulate the induction of skin sensitization. Moreover, such antibodies may provide an informative correlate of the extent to which sensitization has been acquired.

As documented in the recent OECD report 'the adverse outcome pathway for skin sensitisation initiated by covalent binding to proteins' (OECD, 2012), the chemical and biological events driving the induction of human skin sensitisation have been investigated for many years and are now well understood. Several non-animal test methods have been developed to predict sensitiser potential by measuring the impact of chemical sensitisers on these key events (Adler et al., 2011; Maxwell et al., 2011); however our ability to use these non-animal datasets for risk assessment decision-making (i.e. to establish a safe level of human exposure for a sensitising chemical) remains limited and a more mechanistic approach to data integration is required to address this challenge. Informed by our previous efforts to model the induction of skin sensitisation (Maxwell and MacKay, 2008) we are now developing two mathematical models ('total haptenated protein' model and 'CD8(+) T cell response' model) that will be linked to provide predictions of the human CD8(+) T cell response for a defined skin exposure to a sensitising chemical. Mathematical model development is underpinned by focussed clinical or human-relevant research activities designed to inform/challenge model predictions whilst also increasing our fundamental understanding of human skin sensitisation. With this approach, we aim to quantify the relationship between the dose of sensitiser applied to the skin and the extent of the hapten-specific T cell response that would result. Furthermore, by benchmarking our mathematical model predictions against clinical datasets (e.g. human diagnostic patch test data), instead of animal test data, we propose that this approach could represent a new paradigm for mechanistic toxicology.

Systemic toxicity testing forms the cornerstone for the safety evaluation of substances. Pressures to move from traditional animal models to novel technologies arise from various concerns, including: the need to evaluate large numbers of previously untested chemicals and new products (such as nanoparticles or cell therapies), the limited predictivity of traditional tests for human health effects, duration and costs of current approaches, and animal welfare considerations. The latter holds especially true in the context of the scheduled 2013 marketing ban on cosmetic ingredients tested for systemic toxicity. Based on a major analysis of the status of alternative methods (Adler et al., 2011) and its independent review (Hartung et al., 2011), the present report proposes a roadmap for how to overcome the acknowledged scientific gaps for the full replacement of systemic toxicity testing using animals. Five whitepapers were commissioned addressing toxicokinetics, skin sensitization, repeated-dose toxicity, carcinogenicity, and reproductive toxicity testing. An expert workshop of 35 participants from Europe and the US discussed and refined these whitepapers, which were subsequently compiled to form the present report. By prioritizing the many options to move the field forward, the expert group hopes to advance regulatory science.

T lymphocytes mediate skin sensitisation and allergic contact dermatitis. Not unexpectedly, therefore, there is considerable interest in the use of T lymphocyte-based assays as alternative strategies for the identification of skin sensitising chemicals. However, in addition to accurate identification of hazards the development of effective risk assessments requires that information is available about the relative skin sensitising potency of contact allergens. The purpose of this article is to consider the relationships that exist between the characteristics of T lymphocyte responses to contact allergens and the effectiveness/potency of sensitisation. We propose that there are 3 aspects of T lymphocyte responses that have the potential to impact on the potency of sensitisation. These are: (a) the magnitude of response, and in particular the vigour and duration of proliferation and the clonal expansion of allergen-reactive T lymphocytes, (b) the quality of response, including the balance achieved between effector and regulatory cells, and (c) the breadth of response and the clonal diversity of T lymphocyte responses. A case is made that there may be opportunities to exploit an understanding of T lymphocyte responses to contact allergens to develop novel paradigms for predicting skin sensitising potency and new approaches to risk assessment.

Allergic contact dermatitis is a delayed-type hypersensitivity reaction induced by small reactive chemicals (haptens). Currently, the sensitising potential and potency of new chemicals is usually characterised using data generated via animal studies, such as the local lymph node assay (LLNA). There are, however, increasing public and political concerns regarding the use of animals for the testing of new chemicals. Consequently, the development of in vitro, in chemico or in silico models for predicting the sensitising potential and/or potency of new chemicals is receiving widespread interest. The Colipa Skin Tolerance task force currently collaborates with and/or funds several academic research groups to expand our understanding of the molecular and cellular events occurring during the acquisition of skin sensitisation. Knowledge gained from this research is being used to support the development and evaluation of novel alternative approaches for the identification and characterisation of skin sensitizing chemicals. At present three non-animal test methods (Direct Peptide Reactivity Assay (DPRA), Myeloid U937 Skin Sensitisation Test (MUSST) and human Cell Line Activation Test (hCLAT)) have been evaluated in Colipa interlaboratory ring trials for their potential to predict skin sensitisation potential and were recently submitted to ECVAM for formal pre-validation. Data from all three test methods will now be used to support the study and development of testing strategy approaches for skin sensitiser potency prediction. This publication represents the current viewpoint of the cosmetics industry on the feasibility of replacing the need for animal test data for informing skin sensitisation risk assessment decisions.

GAVIN MAXWELL, CATHERINE CLAPP, RICHARD CUBBERLEY, SERAYA DHADRA, NIKKI GELLATLY, STEPHEN GLAVIN, SARAH HADFIELD, SANDRINE JACQUOILLEOT, AMELIA JARMAN, IAN JOWSEY, SUE LOVELL, JACK MAYNE, CRAIG MOORE, RUTH PENDLINGTON, JULIETTE PICKLES, JOE REYNOLDS. OUARDA SAIB, DAVID SHEFFIELD, RICHARD STARK, WENDY SIMPSON, VICKI SUMMERFIELD. DAWEI TANG, SAM WINDEBANK & CAMERON MACKAY SAFETY & ENVIRONMENTAL ASSURANCE CENTRE (SEAC), UNILEVER R&D

Non-animal based approaches to risk assessment are now routinely used for assuring consumer safety for some endpoints (such as skin irritation) following considerable investment in developing and applying new methods over the past 20 years. Unilever's research programme into non-animal approaches for safety assessment is currently focused on the application of new technologies to risk assessments in the areas of skin allergy, cancer and general toxicity (including inhalation toxicity). In all of these areas, a long-term investment is essential to increase the scientific understanding of the underlying biological and chemical processes that we believe will ultimately form a sound basis for novel risk assessment approaches. Our research programme in these priority areas consists of in-house research as well as Unilever-sponsored academic research, involvement with EU-funded projects (e.g. Sens-it-iv, carcino-GENOMICS), participation in cross-industry collaborative research (e.g. COLIPA, EPAA) and ongoing involvement with other scientific initiatives on non-animal approaches to risk assessment (e.g. UK NC3Rs, US ‘Human Toxicology Project’ consortium).

Advances in the understanding of the biological mechanisms underlying skin sensitisation, and the need to comply with recent regulatory requirements, have favoured the development of alternative approaches, some of which may provide promising screening or partial replacement methods. However, efforts are still required to identify more-robust predictive endpoints, and to further optimise existing methods to be integrated into a testing strategy, with a view to achieving the full replacement of the current animal tests. The European Centre for the Validation of Alternative Methods (ECVAM) and the European Cosmetics Association (Colipa) have been collaborating with the aim of identifying a core set of reference chemicals for test method development and/or optimisation. The use of a common set of reference chemicals in the method development phase, would facilitate an early assessment of the performance of a method with respect to existing tests, and of its possible contribution to a testing strategy. By applying pre-defined criteria, existing databases were mined, and a list of 16 chemicals, including 12 positive controls, of which four require metabolic activation to act as sensitisers, and four negative controls, was collated. The chemicals and the criteria used for their selection are presented.

Dendritic cells (DCs) are required for the initiation of primary immune responses. The pattern of Toll-like receptor (TLR) expression on various subsets of these cells has been shown to differ, suggestive of distinct roles in influencing immune responses. We have examined here the responses of immature DCs derived from murine bone marrow (BMDCs) to a range of TLR ligands. BMDCs cultured for 6 days in the presence of granulocyte-macrophage colony-stimulating factor were stimulated for 24 hr with ligands to TLR1-2 [Pam(3)Cys-Ser-(Lys)(4) (PAM)], TLR2-6 (macrophage-activating lipopeptide-2 (MALP-2); zymosan or peptidoglycan (PG)], TLR3 (polyinosinic-polycytidylic acid), TLR4 [lipopolysaccharide R515 (LPS)], TLR5 (flagellin), TLR7 (polyuridylic acid) and TLR9 [CpG ODN2395 (CpG)]. DC activation was monitored using membrane marker expression and analysis of culture supernatants for cytokine/chemokine release. Ligands to TLR3 and TLR7 failed to activate BMDCs. All other TLR ligands caused elevated expression of membrane markers. PAM, MALP-2 and LPS induced high-level expression of proinflammatory cytokines and chemokines. Treatment with CpG was associated with a preferential type 1 cytokine and chemokine profile. Zymosan and PG were proinflammatory but also skewed towards a type 2 pattern of cytokines and chemokines. In contrast, flagellin did not cause marked secretion by BMDCs of cytokines or chemokines. These data for BMDCs are largely consistent with the reported TLR repertoire of freshly isolated murine Langerhans cells. In addition, murine BMDCs show selective responses to TLR ligands with respect to general activation, with differentiated cytokine patterns suggestive of potential priming for divergent immune responses.

The use of Integrated Testing Strategies (ITS) in toxicological hazard identification and characterisation is becoming increasingly common as a method for enabling the integration of diverse types of toxicology data. At present, there are no existing procedures and guidelines for the construction and validation of ITS, so a joint EPAA WG5–ECVAM workshop was held with the following objectives: a) to investigate the role of ITS and the need for validation of ITS in the different industry sectors (pharmaceuticals, cosmetics, chemicals); b) to formulate a common definition of ITS applicable across different sectors; c) to explore how and when Three Rs methods are used within ITS; and d) to propose a validation rationale for ITS and for alternative methods that are foreseen to be used within ITS. The EPAA provided a platform for comparing experiences with ITS across different industry sectors. It became clear that every ITS has to be adapted to the product type, R&D stage, and regulatory context. However, common features of ITS were also identified, and this permitted the formulation of a general definition of ITS in a regulatory context. The definition served as a basis for discussing the needs, rationale and process of formal ITS validation. One of the main conclusions was that a formal validation should not be required, unless the strategy will serve as full replacement of an in vivo study used for regulatory purposes. Finally, several challenges and bottlenecks to the ITS validation were identified, and it was agreed that a roadmap on how to address these barriers would be established by the EPAA partners.

The surrogate light chain (SLC) consists of the polypeptides lambda5 and, in the mouse, either VpreB1 or VpreB2. SLC associates with BILL-Cadherin and other glycoproteins to form the pro-B cell receptor (pro-BCR) at the pre-BI cell stage, and with the immunoglobulin mu heavy chain to form the pre-BCR at the pre-BII cell stage. The function of the pro-BCR, if any, is unknown, whereas the pre-BCR is crucial for proliferative expansion of pre-BII cells. To shed light on the functional properties of VpreB1 and VpreB2 in vivo, mice with either one or two VpreB1, or one or two VpreB2, alleles have been investigated. We show that B cell development in mice with two VpreB1 alleles is indistinguishable from that of normal mice. In contrast, mice with two VpreB2 alleles show an approximately 1.6-fold increase in pre-BI and a 35% decrease in pre-BII cell numbers, while mice with only one VpreB2 allele show a reduction in B cell development manifested in a 2-fold enrichment in pre-BI cells and a 75% reduction in pre-BII cells. However, such a gene dosage effect is not observed for VpreB1. Our results suggest that the difference between VpreB1- and VpreB2-deficient mice is due to lower VpreB2 protein expression, thus limiting the formation of pre-BCRs and thereby the number of large, cycling pre-BII cells.

CD4+ Th1 cells play a critical role in the induction of cell-mediated immune responses that are important for the eradication of intracellular pathogens. Peptide-25 is the major Th1 epitope for Ag85B of Mycobacterium tuberculosis and is immunogenic in I-Ab mice. To elucidate the role of the TCR and IFN-gamma/IL-12 signals in Th1 induction, we generated TCR transgenic mice (P25 TCR-Tg) expressing TCR alpha- and beta-chains of Peptide-25-reactive cloned T cells and analyzed Th1 development of CD4+ T cells from P25 TCR-Tg. Naive CD4+ T cells from P25 TCR-Tg differentiate into both Th1 and Th2 cells upon stimulation with anti-CD3. Naive CD4+ T cells from P25 TCR-Tg preferentially develop Th1 cells upon Peptide-25 stimulation in the presence of I-Ab splenic antigen-presenting cells under neutral conditions. In contrast, a mutant of Peptide-25 can induce solely Th2 differentiation. Peptide-25-induced Th1 differentiation is observed even in the presence of anti-IFN-gamma and anti-IL-12. Furthermore, naive CD4+ T cells from STAT1 deficient P25 TCR-Tg also differentiate into Th1 cells upon Peptide-25 stimulation. Moreover, Peptide-25-loaded I-Ab-transfected Chinese hamster ovary cells induce Th1 differentiation of naive CD4+ T cells from P25 TCR-Tg in the absence of IFN-gamma or IL-12. These results imply that interaction between Peptide-25/I-Ab and TCR may primarily influence determination of the fate of naive CD4+ T cells in their differentiation towards the Th1 subset.

This article opens for debate and discussion the topic of how health-care professionals in the UK can contribute to the improvement of health in developing countries and learn from the process. In 1994, the 50th anniversary of the Bretton Woods agreement which established the World Bank, British and European government aid agencies had substantial funds to support projects aimed at developing countries. Today, the challenges of working in developing countries to create a modern health infrastructure are being mimicked in the newly-independent countries of eastern Europe. Thus, lessons learned in developing countries can be applied to Europe, although huge challenges remain as do questions about the direction of aid programs, particularly in sub-Saharan Africa. The Western perception is that the provision of aid also provides ways for individuals to supplement their income through schemes that would be considered corrupt in the West. In order to draw useful conclusions from the African experience, it must first be recognized that the situation has improved recently and also that practices viewed as corrupt in the West may simply be the way of doing business in Third World countries. Justification for replacing commission-based systems of local aid with modern management techniques rests in the efficiency of the modern systems. It may be a form of neocolonialism simply to impose Western values on other cultures. The upcoming efforts in eastern Europe will also challenge heretofore unquestioned assumptions about the way we do things. The most pressing challenges will arise from the sheer magnitude of the need to provide health care in an atmosphere which engenders apathy towards inadequate services. We must learn from our successes and recognize that without our aid efforts, millions of people, particularly women and children, would have suffered and died.