A botanical reference set illustrating a weight of evidence approach for skin sensitization risk assessment.

Recent years have seen an increase in the use of botanicals and natural substances (BNS) in consumer products such as cosmetics and household care products. Most work conducted to date to assess botanicals for human safety has focused their use as dietary supplements and thus on systemic toxicity. However, the induction of skin sensitization is a possible adverse effect of natural products in particular those that come into skin contact, especially for cosmetics that remain on the skin and are not rinsed off following use. Assessments of BNS ingredients are often challenging for a number of reasons: the BNS are complex mixtures that can be of mostly unknown composition; the composition can be highly variable even within the same plant species and dependent on how processed; the physical form of the BNS raw material can vary from a highly concentrated powdered extract to a liquid extract containing only a small percentage of the BNS; testing of the BNS raw materials in New Approach Methods (NAM) has uncertainty as these methods are often not developed or validated for complex mixtures. In this study, a reference set of 14 selected BNS which span the range of skin sensitization potential was complied. These data were used in a Weight of Evidence (WoE) approach to evaluate their skin sensitization potential with each of the data rich BNS being classified as either having strong evidence of inducing skin sensitization based on human topical use history, animal data, clinical data, composition data and NAM data, or having some but more limited (weak) evidence of inducing skin sensitization, or having strong evidence of no skin sensitization potential. When available data have sufficient potency related information, sensitization potency assessment is also provided based on WoE, classifying these BNS as either strong, moderate, or weak sensitizers, or non-sensitizers. An outline for a BNS skin sensitization risk assessment framework is proposed starting with exposure-based waiving and WoE assessment for higher exposures. In addition to demonstrating the application of the WoE approach, the reference set presented here provides a set of 'data rich' botanicals which cover a range of sensitization potencies that could be used for evaluating existing test methods or aid in the development of new predictive models for skin sensitization.

Development of a peptide reactivity assay for screening botanicals and natural substances: Proof of concept studies.

Consumer products containing botanicals or natural substances (BNS) are often preferred because there is a perception that 'natural' is safe. As with any product ingredient, a thorough safety assessment must be conducted, including a determination of skin sensitization potential. A modification of the Peroxidase Peptide Reactivity Assay (PPRA) was explored for screening BNS (B-PPRA) for their reactivity to a model cysteine peptide. The PPRA incorporates a horseradish peroxidase­hydrogen peroxide (+HRP/P) oxidation system for the activation of potential pre- and pro-haptens. BNS test materials contained <2% botanical constituent in either glycerin/water or propylene glycol/water. Stock solutions prepared in acetonitrile were diluted to 8 working concentrations. Direct reactivity was determined in reaction mixtures containing peptide and deferoxamine in potassium phosphate buffer. Enzyme-mediated reactivity determinations were performed with addition of +HRP/P. Initial studies demonstrated that results were reproducible and impact of carrier low. To determine the sensitivity of the assay, experiments were conducted with chamomile extract spiked with three sensitizers. Peptide depletion was observed in the +HRP/P reaction mixtures with isoeugenol spikes as low as 0.05%. The B-PPRA shows promise as a screening method for skin sensitization potential and could become part of a framework for the skin sensitization safety assessment of BNS.

Critical Evaluation of Low-Molecular Weight Respiratory Sensitizers and Their Protein Reactivity Potential Toward Lysine Residues.

Interest in the development of methods to evaluate the respiratory sensitization potential of low-molecular weight chemicals continues, but no method has yet been generally accepted or validated. A lack of chemical reference standards, together with uncertainty regarding relevant immunological mechanisms, has hampered method development. The first key event in the development of either skin or respiratory sensitization is the formation of stable adducts of the chemical with host proteins. This event is measured in the Direct Peptide Reactivity Assay using cysteine- and lysine-containing model peptides. It is hypothesized that protein reactivity and subsequent adduct formation may represent the earliest point of divergence in the pathways leading to either skin or respiratory sensitization. Direct Peptide Reactivity Assay data for 200 chemicals were compiled and grouped into respiratory, skin and nonsensitizers. Chemicals grouping was based on extensive literature research and expert judgment. To evaluate if chemical groups represent different peptide reactivity profiles, peptide reactivity data were clustered and compared with information on protein binding mechanisms and chemical categories available via the Organization for Economic Co-operation and Development. Toolbox. Respiratory sensitizers (n = 15) showed a significant (3-fold) higher lysine reactivity than skin sensitizers (n = 129). However, this difference was driven largely by the high representation of acid anhydrides among the respiratory sensitizers that showed clear lysine selectivity. Collectively, these data suggest that preferential reactivity for either cysteine or lysine is associated primarily with chemical structure, and that lysine preference is not a unifying characteristic of chemical respiratory allergens.

Refinement of the Peroxidase Peptide Reactivity Assay and Prediction Model for Assessing Skin Sensitization Potential.

A peptide reactivity assay with an activation component was developed for use in screening chemicals for skin sensitization potential. A horseradish peroxidase-hydrogen peroxide (HRP/P) oxidation system was incorporated into the assay for characterizing reactivity of hapten and pre-/prohapten sensitizers. The assay, named the Peroxidase Peptide Reactivity Assay (PPRA) had a predictive accuracy of 83% (relative to the local lymph node assay) with the original protocol and prediction model. However, apparent false positives attributed to cysteine depletion at relatively high chemical concentrations and, for some chemicals expected to react with the -NH2 group of lysine, little to no depletion of the lysine peptide were observed. To improve the PPRA, cysteine peptide reactions with and without HRP/P were modified by increasing the number of test concentrations and refining their range. In addition, removal of DL-dithiothreitol from the reaction without HRP/P increased cysteine depletion and improved detection of reactive aldehydes and thiazolines without compromising the assay's ability to detect prohaptens. Modification of the lysine reaction mixture by changing the buffer from 0.1 M ammonium acetate buffer (pH 10.2) to 0.1 M phosphate buffer (pH 7.4) and increasing the level of organic solvent from 1% to 25% resulted in increased lysine depletion for known lysine reactive chemicals. Refinement of the prediction model improved the sensitivity, specificity, and accuracy for hazard identification. These changes resulted in significant improvement of the PPRA making it is a reliable method for predicting the skin sensitization potential of all chemicals, including pre-/prohaptens and directly reactive haptens.

The kinetic direct peptide reactivity assay (kDPRA): Intra- and inter-laboratory reproducibility in a seven-laboratory ring trial

While the skin sensitization hazard of substances can be identified using non-animal methods, the classification of potency into UN GHS sub-categories 1A and 1B remains challenging. The kinetic direct peptide reactivity assay (kDPRA) is a modification of the DPRA wherein the reaction kinetics of a test substance towards a synthetic cysteine-containing peptide are evaluated. For this purpose, several concentrations of the test substance are incubated with the synthetic peptide for several incubation times. The reaction is stopped by addition of monobromobimane, which forms a fluorescent complex with the free cysteine of the model peptide. The relative remaining non-depleted amount of peptide is determined. Kinetic rate constants are derived from the depletion vs concentration and time matrix and used to distinguish between UN GHS sub-category 1A sensitizers and test substances in sub-category 1B/not classified test substances. In this study, we present a ring trial of the kDPRA with 24 blind-coded test substances in seven laboratories. The intra- and inter-laboratory reproducibility were 96% and 88%, respectively (both for differentiating GHS Cat 1A sensitizers from GHS Cat 1B/not classified). Following an independent peer review, the kDPRA was considered to be acceptable for the identification of GHS Cat 1A skin sensitizers. Besides GHS Cat 1A identification, the kDPRA can be used as part of a defined approach(es) with a quantitative data integration procedure for skin sensitization potency assessment. For this aim, next to reproducibility of classification, the quantitative reproducibility and variability of the rate constants were quantified in this study.

Proteomic and Bioinformatic Analyses for the Identification of Proteins With Low Allergenic Potential for Hazard Assessment.

Use of botanicals and natural substances in consumer products has increased in recent years. Such extracts can contain protein that may theoretically represent a potential risk of IgE-mediated allergy. No method has yet been generally accepted or validated for assessment of the allergenic potential of proteins. For development of suitable methods datasets of allergenic and nonallergenic (or low allergenic) proteins are required that can serve, respectively, as positive and negative controls. However, data are unavailable on proteins that lack or have low allergenic potential. Here, low allergenic potential proteins are identified based on the assumption that proteins with established human exposure, but with a lack of an association with allergy, possess low allergenic potential. Proteins were extracted from sources considered to have less allergenic potential (corn, potato, spinach, rice, and tomato) as well as higher allergenic potential (wheat) regarding common allergenic foods. Proteins were identified and semi-quantified by label-free proteomic analysis conducted using mass spectrometry. Predicted allergenicity was determined using AllerCatPro (https://allercatpro.bii.a-star.edu.sg/). In summary, 9077 proteins were identified and semi-quantified from 6 protein sources. Within the top 10% of the most abundant proteins identified, 178 characterized proteins were found to have no evidence for allergenicity predicted by AllerCatPro and were considered to have low allergenic potential. This panel of low allergenic potential proteins provides a pragmatic approach to aid the development of alternative methods for robust testing strategies to distinguish between proteins of high and low allergenic potential to assess the risk of proteins from natural or botanical sources.

Application of in vitro skin penetration measurements to confirm and refine the quantitative skin sensitization risk assessment of methylisothiazolinone.

Use of quantitative risk assessment (QRA) for assessing the skin sensitization potential of chemicals present in consumer products requires an understanding of hazard and product exposure. In the absence of data, consumer exposure is based on relevant habits and practices and assumes 100% skin uptake of the applied dose. To confirm and refine the exposure, a novel design for in vitro skin exposure measurements was conducted with the preservative, methylisothiazolinone (MI), in beauty care (BC) and household care (HHC) products using realistic consumer exposure conditions. A difference between measured exposure levels (MELs) for MI in leave-on versus rinse-off BC products, and lower MELs for MI in HHC rinse-off compared to BC products was demonstrated. For repeated product applications, the measured exposure was lower than estimations based on summation of applied amounts. Compared to rinse-off products, leave-on applications resulted in higher MELs, correlating with the higher incidences of allergic contact dermatitis associated with those product types. Lower MELs for MI in rinse-off products indicate a lower likelihood to induce skin sensitization, also after multiple daily applications. These in vitro skin exposure measurements indicate conservatism of default exposure estimates applied in skin sensitization QRA and might be helpful in future risk assessments.

A dataset on 145 chemicals tested in alternative assays for skin sensitization undergoing prevalidation.

Skin sensitization is a key endpoint for cosmetic ingredients, with a forthcoming ban for animal testing in Europe. Four alternative tests have so far been submitted to ECVAM prevalidation: (i) MUSST and (ii) h-Clat assess surface markers on dendritic cell lines, (iii) the direct peptide reactivity assay (DPRA) measures reactivity with model peptides and (iv) the KeratinoSens(TM) assay which is based on detection of Nrf2-induced luciferase. It is anticipated that only an integrated testing strategy (ITS) based on a battery of tests might give a full replacement providing also a sensitization potency assessment, but this concept should be tested with a data-driven analysis. Here we report a database on 145 chemicals reporting the quantitative endpoints measured in a U937- test, the DPRA and KeratinoSens(TM) . It can serve to develop data-driven ITS approaches as we show in a parallel paper and provides a view as to the current ability to predict with in vitro tests as we are entering 2013. It may also serve as reference database when benchmarking new molecules with in vitro based read-across and find use as a reference database when evaluating new tests. The tests and combinations thereof were evaluated for predictivity, and overall a similar predictivity was found as before on three-fold smaller datasets. Analysis of the dose-response parameters of the individual tests indicates a correlation to sensitization potency. Detailed analysis of chemicals false-negative and false-positive in two tests helped to define limitations in the tests but also in the database derived from animal studies.

Chemical allergy: translating biology into hazard characterization.

The induction by chemicals of allergic sensitization and allergic disease is an important and challenging branch of toxicology. Skin sensitization resulting in allergic contact dermatitis represents the most common manifestation of immunotoxicity in humans, and many hundreds of chemicals have been implicated as skin sensitizers. There are far fewer chemicals that have been shown to cause sensitization of the respiratory tract and asthma, but the issue is no less important because hazard identification remains a significant challenge, and occupational asthma can be fatal. In all areas of chemical allergy, there have been, and remain still, intriguing challenges where progress has required a close and productive alignment between immunology, toxicology, and clinical medicine. What the authors have sought to do here is to exemplify, within the framework of chemical allergy, how an investment in fundamental research and an improved understanding of relevant biological and biochemical mechanisms can pay important dividends in driving new innovations in hazard identification, hazard characterization, and risk assessment. Here we will consider in turn three specific areas of research in chemical allergy: (1) the role of epidermal Langerhans cells in the development of skin sensitization, (2) T lymphocytes and skin sensitization, and (3) sensitization of the respiratory tract. In each area, the aim is to identify what has been achieved and how that progress has impacted on the development of new approaches to toxicological evaluation. Success has been patchy, and there is still much to be achieved, but the journey has been fascinating and there have been some very important developments. The conclusion drawn is that continued investment in research, if coupled with an appetite for translating the fruits of that research into imaginative new tools for toxicology, should continue to better equip us for tackling the important challenges that remain to be addressed.

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

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

The impact of LLNA group size on the identification and potency classification of skin sensitizers: a review of published data.

The Organisation for Economic Co-operation and Development (OECD) Test Guideline 429 for the local lymph node assay (LLNA) indicates a minimum of 4 mice per dose group, or of 5 mice if statistics are required. Recent discussions at the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) have led to suggestions that there should be a change to LLNA protocol requirements to mandate a minimum of 5 mice per group. Although it is not certain that any such proposal will be made, the debate is an important one and prompts reconsideration of animal requirements in the LLNA. In this paper we have conducted an analysis of published data from our own laboratories to determine whether the use of 4 or of 5 mice has had any practical impact on the outcome of the assay. Of the data sets for 17 chemicals in the 4-animal assay (14 positive, 1 uncertain, and 2 negative), 16 results were identical in the 5-animal assay. A marginally positive result in the 4-animal assay was negative in the 5-animal assay. Where potency determinations were made, the outcomes were essentially identical in the 2 forms of the LLNA. Consequently, it is concluded that there is no scientific justification for removing the option to use a 4-animal version of the LLNA.

Determination of in vivo dose response and allergen-specific T cells in subjects contact-sensitized to squaric acid dibutyl ester.

BACKGROUND: Squaric acid dibutyl ester (SADBE) is a known contact sensitizer, but dose-response data are not defined. OBJECTIVE: To determine the relationship between sensitization dose and contact hypersensitivity (CHS) response to SADBE in human volunteers. The study also aimed to investigate whether SADBE-reactive blood T cells could be detected using ex vivo mature dendritic cells (DCs) as antigen-presenting cells. METHOD: Forty healthy volunteers were sensitized to either 12.5, 25, 50, or 250 microg of SADBE in a 48 microL volume. This was followed by elicitation 2 weeks later with five doses (0, 0.2, 2, 20, and 200 microg in 20 microL). An additional 10 subjects received the elicitation doses without prior sensitization. Blood samples obtained after sensitization were purified into T cells and mature DCs. RESULTS: A direct relationship between sensitization dose and in vivo CHS response was observed. The SADBE dose that effectively sensitized 50% of the population (ED50) was 22 microg/cm2. Significant SADBE-specific T-cell proliferation in vitro was not observed 2 weeks after sensitization but became evident after elicitation. CONCLUSION: This study establishes the in vivo dose-response characteristics of immune reactivity to SADBE and antigen-specific T-cell reactivity.

Dose metrics in the acquisition of skin sensitization: thresholds and importance of dose per unit area.

Allergic contact dermatitis is a common occupational and environmental health problem and many hundreds of chemicals have been implicated as skin sensitizers. Sensitization is acquired following topical exposure to a contact allergen and induction of a cutaneous immune response of an appropriate magnitude. For effective assessment and management of human health risks there is a need to appreciate the dose metrics that drive the induction of skin sensitization. The available evidence suggests that under most normal conditions of exposure it is the dose per unit area of chemical that has over-riding impact on the effectiveness of sensitization. The exception to this rule is when the area of the application site drops below a certain critical level. Here we review in detail the evidence which supports dose per unit area as being the critical exposure metric in the induction of skin sensitization, and the mechanistic bases for this relationship.

Dermal sensitization quantitative risk assessment (QRA) for fragrance ingredients.

Based on chemical, cellular, and molecular understanding of dermal sensitization, an exposure-based quantitative risk assessment (QRA) can be conducted to determine safe use levels of fragrance ingredients in different consumer product types. The key steps are: (1) determination of benchmarks (no expected sensitization induction level (NESIL)); (2) application of sensitization assessment factors (SAF); and (3) consumer exposure (CEL) calculation through product use. Using these parameters, an acceptable exposure level (AEL) can be calculated and compared with the CEL. The ratio of AEL to CEL must be favorable to support safe use of the potential skin sensitizer. This ratio must be calculated for the fragrance ingredient in each product type. Based on the Research Institute for Fragrance Materials, Inc. (RIFM) Expert Panel's recommendation, RIFM and the International Fragrance Association (IFRA) have adopted the dermal sensitization QRA approach described in this review for fragrance ingredients identified as potential dermal sensitizers. This now forms the fragrance industry's core strategy for primary prevention of dermal sensitization to these materials in consumer products. This methodology is used to determine global fragrance industry product management practices (IFRA Standards) for fragrance ingredients that are potential dermal sensitizers. This paper describes the principles of the recommended approach, provides detailed review of all the information used in the dermal sensitization QRA approach for fragrance ingredients and presents key conclusions for its use now and refinement in the future.

Preservatives and skin sensitization quantitative risk assessment.

BACKGROUND: Preservatives are an unfortunately common cause of allergic contact dermatitis (ACD). Often, this is in association with exposure to cosmetics or medicaments. Recently, a quantitative risk assessment (QRA) approach to the quantitation of safe exposure levels for sensitizers has been promulgated as a more effective tool for the identification of acceptable levels of potential sensitizers in consumer products. OBJECTIVE: To assess this QRA approach, which facilitates the prediction of acceptable exposure levels to skin sensitizers in consumer products, levels that are normally below the threshold for the induction of skin sensitization. METHODS: Retrospective QRA analysis on four preservatives in five consumer product types. RESULTS: The analysis shows that functional levels of preservatives may be somewhat above an ideal exposure level for some product types, an outcome that is consistent with the clinical picture. CONCLUSION: QRA represents a new tool that in the future should be used in combination with the assessment of microbiologic protection needs of specific product types to limit the problem of preservative ACD.

The reduced local lymph node assay: the impact of group size.

The local lymph node assay (LLNA) is a skin sensitization test that provides animal welfare benefits. To reduce animal usage further, a modified version (rLLNA) was proposed. Conducting the rLLNA as a screening test with a single high dose group and vehicle control differentiated accurately between skin sensitizers and non-sensitizers. This study examined whether a reduction in animal number/group is feasible. Historical data were utilized to examine the impact of conducting the rLLNA with two mice/group. To assess the effect on the stimulation index (SI) 41 datasets with individual animal data derived using five mice/group were analysed. SIs were calculated on all possible combinations of two control and two high dose group disintegrations per minute (dpm) values. For 25 of 33 sensitizer datasets, > 96% of possible dpm combinations resulted in a calculated SI > 3. The lowest percentages of positive SIs were observed with weak allergens when, in the standard LLNA, the mean SIs would have been nearer to the threshold value of 3. The results indicate that moderate, strong and extreme allergens are more likely than weak allergens to be identified as sensitizers when group sizes of two mice are used within the rLLNA. It is concluded that a rLLNA with two mice/group would display decreased sensitivity and is inappropriate for use in hazard identification.

Extrapolating local lymph node assay EC3 values to estimate relative sensitizing potency.

The assessment of the potency of a skin sensitizing chemical is a key starting point for its subsequent risk assessment/management. The Local Lymph Node Assay can provide information on the relative skin sensitizing potency of contact allergens by interpolation from the dose response curve the concentration of a chemical required to elicit a threshold positive response (EC3 value). However, interpolation requires that the dose response curve have at least one stimulation index (SI) value above and one SI value below the threshold value of 3. For instances where all test concentrations result in SI values above 3, there was a need to develop a method that would permit estimation of EC3 values. This has been achieved by log-linear extrapolation using the two lowest test concentrations from the dose response curve. Before applying this approach, it is important that data quality is assessed. The dose response must include concentrations on the linear portion of the curve and, ideally, the SI induced by the lowest dose should approach 3. Judicious use of this approach for extrapolating EC3 values can provide information on a likely potency classification for use in risk assessment and may avoid the need for repeat animal testing.

B220 analysis with the local lymph node assay: proposal for a more flexible prediction model.

The mouse local lymph node assay (LLNA) has been developed and validated for the identification of chemicals that have the potential to induce skin sensitisation. In common with other predictive test methods the accuracy of the LLNA is not absolute and experience has revealed that a few chemicals, including for instance a minority of skin irritants, may elicit false-positive reactions in the assay. To improve further the performance of the LLNA, and to eliminate or reduce false-positives, there has been interest in an adjunct method in which the ability of chemicals to cause increases in the frequency of B220(+) lymphocytes in skin-draining lymph nodes is measured. Previous studies suggest that the use of B220 analyses aligned with the standard LLNA may serve to distinguish further between contact allergens and skin irritants. In the original predictive model, chemicals were regarded as being skin sensitisers if they were able to induce a 1.25-fold or greater increase in the percentage of B220(+) cells within lymph nodes compared with concurrent vehicle controls. Although this first prediction model has proven useful, in the light of more recent experience, and specifically as a consequence of some variability observed in the frequency of B220(+) lymphocytes in nodes taken from vehicle control-treated animals, it is timely now to reconsider and refine the model. As a result a new prediction model is proposed in which reliance on the use of absolute thresholds is reduced, and in which small changes in control values can be better accommodated.

Mechanistic applicability domain classification of a local lymph node assay dataset for skin sensitization.

The goal of eliminating animal testing in the predictive identification of chemicals with the intrinsic ability to cause skin sensitization is an important target, the attainment of which has recently been brought into even sharper relief by the EU Cosmetics Directive and the requirements of the REACH legislation. Development of alternative methods requires that the chemicals used to evaluate and validate novel approaches comprise not only confirmed skin sensitizers and non-sensitizers but also substances that span the full chemical mechanistic spectrum associated with skin sensitization. To this end, a recently published database of more than 200 chemicals tested in the mouse local lymph node assay (LLNA) has been examined in relation to various chemical reaction mechanistic domains known to be associated with sensitization. It is demonstrated here that the dataset does cover the main reaction mechanistic domains. In addition, it is shown that assignment to a reaction mechanistic domain is a critical first step in a strategic approach to understanding, ultimately on a quantitative basis, how chemical properties influence the potency of skin sensitizing chemicals. This understanding is necessary if reliable non-animal approaches, including (quantitative) structure-activity relationships (Q)SARs, read-across, and experimental chemistry based models, are to be developed.

Dendritic cells and skin sensitization: biological roles and uses in hazard identification.

Recent advances have been made in our understanding of the roles played by cutaneous dendritic cells (DCs) in the induction of contact allergy. A number of associated changes in epidermal Langerhans cell phenotype and function required for effective skin sensitization are providing the foundations for the development of cellular assays (using DC and DC-like cells) for skin sensitization hazard identification. These alternative approaches to the identification and characterization of skin sensitizing chemicals were the focus of a Workshop entitled "Dendritic Cells and Skin Sensitization: Biological Roles and Uses in Hazard Identification" that was given at the annual Society of Toxicology meeting held March 6-9, 2006 in San Diego, California. This paper reports information that was presented during the Workshop.