Integrated skin sensitization assessment based on OECD methods (III): Adding human data to the assessment_suppl1

Skin sensitization in silico protocol

We previously developed a test for detecting naturally occurring protein-induced skin sensitization based on the markers and criteria of the human cell-line activation test (h-CLAT) and showed that the h-CLAT was useful for assessing the allergenic potency of proteins. However, test proteins were contaminated with varying amounts of lipopolysaccharide (LPS), which might have contributed to the stimulation of CD86 and CD54 expression. In this study, we developed a method to exclude the effects of LPS in the assessment of skin sensitization by naturally occurring proteins. We tested two inhibitors [the caspase-1 inhibitor acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-cmk; hereafter referred to as YVAD), which can mitigate the LPS-induced increases in CD54 expression, and polymyxin B (PMB), which suppresses the effect of LPS by binding to its lipid moiety (i.e., the toxic component of LPS)]. After a 24 hr exposure, YVAD and PMB reduced LPS-induced CD86 and CD54 expression. In particular, the effect of PMB was dependent upon pre-incubation time and temperature, with the most potent effect observed following pre-incubation at 37°C for 24 hr. Moreover, only pre-incubation with cell-culture medium (CCM) at 37°C for 24 hr showed an inhibitory effect similar to that of PMB, with this result possibly caused by components of CCM binding to LPS. Similar effects were observed in the presence of ovalbumin (with 1070 EU/mg LPS) and ovomucoid, and lysozyme (with 2.82 and 0.234 EU/mg LPS, respectively) in CCM. These results indicated that PMB and CCM effectively eliminated the effects of LPS during assessment of protein allergenicity, thereby allowing a more accurate evaluation of the potential of proteins to induce skin sensitization.

Skin sensitization is a major environmental and occupational health hazard. Although many chemicals have been evaluated in humans, there have been no efforts to model these data to date. We have compiled, curated, analyzed, and compared the available human and LLNA data. Using these data, we have developed reliable computational models and applied them for virtual screening of chemical libraries to identify putative skin sensitizers. The overall concordance between murine LLNA and human skin sensitization responses for a set of 135 unique chemicals was low (R = 28-43%), although several chemical classes had high concordance. We have succeeded to develop predictive QSAR models of all available human data with the external correct classification rate of 71%. A consensus model integrating concordant QSAR predictions and LLNA results afforded a higher CCR of 82% but at the expense of the reduced external dataset coverage (52%). We used the developed QSAR models for virtual screening of CosIng database and identified 1061 putative skin sensitizers; for seventeen of these compounds, we found published evidence of their skin sensitization effects. Models reported herein provide more accurate alternative to LLNA testing for human skin sensitization assessment across diverse chemical data. In addition, they can also be used to guide the structural optimization of toxic compounds to reduce their skin sensitization potential.

Skin sensitisation is the regulatory endpoint that has been at the centre of concerted efforts to replace animal testing in recent years, as demonstrated by the Organisation for Economic Co-operation and Development (OECD) adoption of five non-animal methods addressing mechanisms under the first three key events of the skin sensitisation adverse outcome pathway. Nevertheless, the currently adopted methods, when used in isolation, are not sufficient to fulfil regulatory requirements on the skin sensitisation potential and potency of chemicals comparable to that provided by the regulatory animal tests. For this reason, a number of defined approaches integrating data from these methods with other relevant information have been proposed and documented by the OECD. With the aim to further enhance regulatory consideration and adoption of defined approaches, the European Union Reference Laboratory for Alternatives to Animal testing in collaboration with the International Cooperation on Alternative Test Methods hosted, on 4–5 October 2016, a workshop on the international regulatory applicability and acceptance of alternative non-animal approaches, i.e., defined approaches, to skin sensitisation assessment of chemicals used in a variety of sectors. The workshop convened representatives from more than 20 regulatory authorities from the European Union, United States, Canada, Japan, South Korea, Brazil and China. There was a general consensus among the workshop participants that to maximise global regulatory acceptance of data generated with defined approaches, international harmonisation and standardisation are needed. Potential assessment criteria were defined for a systematic evaluation of existing defined approaches that would facilitate their translation into international standards, e.g., into a performance-based Test Guideline. Informed by the discussions at the workshop, the ICATM members propose practical ways to further promote the regulatory use and facilitate adoption of defined approaches for skin sensitisation assessments.

Plant extracts, polymers and new approach methods: Practical experience with skin sensitization assessment

In modern animal husbandry, a large proportion of diseases of non-infectious etiology is occupied by animal injury, which leads to damage to the skin. We have developed a new antiseptic wound healing drug, which includes octenidine dihydrochloride, pantothenic acid and auxiliary components that potentiate the action of the main components of the drug. The study of sensitizing activity included two studies - the method of skin applications and a conjunctival test. It was conducted in accordance with the "Guidelines for conducting preclinical studies of medicines" edited by A.N. Mironov (2012), GOST 32375-2013 "Testing methods for the effects of chemical products on the human body. Skin sensitization assessment tests". The skin sensitization test included the determination of induction and provocative exposure, and was conducted on guinea pigs. Two groups of animals were formed – the study group and the control group. There were 20 animals in the study group and 10 in the control group. The second part of the study of the sensitizing activity of the drug is a conjunctival test. Guinea pigs were used for the study. Two groups of animals were formed – the study group and the control group, each with 10 animals. When assessing skin sensitization, the following results were obtained: redness, local increase in skin temperature, itching, thickening of the skin fold, peeling of the skin – were absent. There were no systemic changes, and the behavior of the animals did not change throughout the study. When examining a conjunctival sample, a gel with octenidine dihydrochloride caused redness in the area of the tear duct, cornea and sclera 15 minutes after administration of the drug under the upper eyelid. After 24 and 48 hours, there was no redness. There was no itching during each observation stage. There were no changes in the control group. No systemic changes were detected in both groups during the entire study period.

Incorporating integrated testing strategy (ITSv1) defined approach into read-across (RAx) in predicting skin sensitization potency: ITSv1-based RAx

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 physiologically- and 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.

Serious cases of allergic contact dermatitis (ACD) caused by exposure to 3,5,6-tetrachloro-4-(methylsulfonyl)pyridine (TCMSP; CAS no. 13108-52-6) used as an antimicrobial agent for desk mats have been reported in Japan. A quantitative risk assessment (QRA) of TCMSP contained in desk mats was performed retrospectively. A local lymph node assay (LLNA): BrdU-ELISA was conducted to determine a reliable EC1.6 value for TCMSP. The acceptable exposure level (AEL) for TCMSP was derived from the EC1.6 value by applying sensitization assessment factors (SAFs). The exposure level was estimated based on the assumption referring to the use conditions of thiabendazole in the same purpose. Then, the estimated exposure level was compared with the AEL to evaluate the skin sensitization risk. The AEL was calculated as 0.00458 μg/cm2 based on the EC1.6 value (0.011%, 2.75 μg/cm2 ) by applying a composite SAF of 600. The estimated TCMSP exposure level from the desk mat was 500 times greater than the AEL, indicating a serious skin sensitization risk. Assessments of skin sensitization potencies of chemicals to be used in consumer products are strongly recommended, and QRAs should be performed at the pre-marketing stage to avoid the skin sensitization risk in consumers.

In vitro RHE skin sensitisation assays: Applicability to challenging substances

Despite widespread use of silicon dioxide (SiO2) NPs in industry and in our daily lives, no studies so far have evaluated the potential of their skin phototoxicity and sensitization. This study was designed to investigate the potential of phototoxicity and sensitization of SiO2 NPs. Assessment of the potential of skin phototoxicity was carried out using the 3T3 neutral red uptake test, an HSEM, and an animal model. The potential of skin sensitization was evaluated by a non-radioisotope local lymph node assay (non-RI LLNA). Findings from the present study suggest that the HSEM may be a reasonable model system for evaluation of skin phototoxicity of NPs. In addition, our data demonstrate that non-RI LLNA may be a useful method for identification of skin sensitization of NPs. In this study, we showed that SiO2 NPs do not induce phototoxicity or skin sensitization.

The hazards and potency of skin sensitizers are traditionally determined using animal tests such as the local lymph node assay (LLNA); however, significant progress has been made in the development of non-animal test methods addressing the first three mechanistic key events of adverse outcome pathway in skin sensitization. We developed the epidermal sensitization assay (EpiSensA), which is a reconstructed human epidermis-based assay, by measuring four genes related to critical keratinocyte responses during skin sensitization. Four in vitro skin sensitization test methods (EpiSensA, direct peptide reactivity assay [DPRA], KeratinoSens™, and human cell line activation test [h-CLAT]) were systematically evaluated using 136 chemicals including lipophilic chemicals and pre/pro-haptens, which may be related to assay-specific limitations. The constructed database included existing and newly generated data. The EpiSensA showed a broader applicability domain and predicted the hazards with 82.4% and 78.8% accuracy than LLNA and human data. The EpiSensA could detect 76 out of 88 sensitizers at lower concentrations than the LLNA, indicating that the EpiSensA has higher sensitivity for the detection of minor sensitizing constituents. These results confirmed the potential use of the EpiSensA in evaluating a mixture of unknown compositions that can be evaluated by animal tests. To combine different information sources, the reconstructed human epidermis-based testing strategy (RTS) was developed based on weighted multiple information from the EpiSensA and TImes MEtabolism Simulator platform for predicting Skin Sensitization (TIMES-SS; RTSv1) or Organization for Economic Cooperation and Development (OECD) QSAR Toolbox automated workflow (RTSv2). The predictivities of the hazards and Globally Harmonized System (GHS) subcategories were equal to or better than the defined approaches (2 out of 3, integrated testing strategy [ITS]v1, and ITSv2) adopted as OECD Guideline 497.

All cosmetics products must be safe under foreseeable conditions of use. Allergenic responses are one of the most frequent adverse reactions noted for cosmetics. Thus, the EU cosmetics legislation requires skin sensitisation assessment for all cosmetics ingredients, including the regulated ones (for which the full toxicological dossier needs to be analysed by the Scientific Committee on Consumer Safety (SCCS)) and those (perceived as less toxic) which are assessed by industrial safety assessors. Regardless of who performs the risk assessment, it should be carried out using scientifically and regulatory body-accepted methods. In the EU, reference methods for chemical toxicity testing are defined in the relevant Annexes (VII–X) of the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) Regulation. Recommendations for Skin Sensitization (Skin Sens) testing are provided in Annex VII, and this particular endpoint information is required for all EU-registered chemicals. Historically, in vivo animal and human methods have been used. Both raise ethical doubts, and some of them cause practical problems in the objective analysis of skin sensitising potency. Previous decades of huge effort have resulted in the regulatory acceptance of the alternative Skin Sens IATA (Integrated Approaches to Testing and Assessment) and NGRA (Next Generation Risk Assessment). Regardless of the testing issues, a serious sociological problem are observed within the market: the consumer assumes the presence of strong sensitisers in cosmetics formulations and insufficient risk management tools used by the industry. The present review aims to provide an overview of methods for assessing skin sensitisation. Additionally, it aims to answer the following question: what are the most potent skin sensitisers used in cosmetics? The answer considers the mechanistic background along with the actual regulatory status of ingredients and practical examples of responsible industry solutions in the area of risk management.

Assays to predict the skin sensitisation potential of substances commonly address the molecular initiating event (MIE) of the adverse outcome pathway (AOP), which is the covalent binding of chemicals to proteins. This represents the first key event in the pathway. In chemico approaches have emerged as validated alternatives to conventional animal testing methods to determine this process. This review explores the integration of biosensor-based approaches to supplement other in chemico methods, for use as alternatives to the 'gold standard' Local Lymph Node Assay (LLNA) in skin sensitisation testing. While surface plasmon resonance-based biosensors have shown promise, they exhibit limitations such as poor sensitivity. To overcome such limitations, this review highlights the use of impedance-based biosensors in the study of hapten-skin protein interactions, which are associated with the MIE in skin sensitisation. Impedance-based biosensors have exhibited impressive performance, with a specificity of 97%, a sensitivity of 83% and an accuracy of 92% being obtained in one study (N = 49 substances). The advantages of impedance-based biosensors include label-free methodologies, high sensitivity, low cost and operational simplicity. This review also covers recent advancements in the use of biosensors in cosmetic studies and skin research, as well as comparisons of the limitations of the various in chemico methods and future perspectives for skin sensitisation assessment. The use of impedance-based biosensors as part of integrated testing approaches alongside other in chemico testing methods, can represent a reliable approach for skin sensitisation assessment, while supporting the reduction and replacement of animal use in toxicity testing.

Assessment of skin sensitization potential is mandatory for ingredients dedicated to topical applications. A battery of in vitro tests covering the key steps of the Adverse Outcome Pathway (AOP) for skin sensitization has been recommended to replace animal testing. However, despite international guidelines on in vitro methods, there is no universal approach defining their combination. The purpose of this work was to assess skin sensitization of botanical ingredients relying on a previously developed in vitro testing strategy. This tool focused on complex and poorly water-soluble substances, which were not already covered. Sixteen botanical extracts were tested in a sequential approach, starting with Sens-Is, supplemented by Organization for Economic Cooperation and Development (OECD) in vitro methods when necessary. The results on the selected well-known botanical sensitizers complemented the relevance of the strategy. Testing on experimental botanicals could detect skin sensitizers. In addition, phytochemistry was a determining support to identify and remove the components at the origin of the effect. Altogether, these results enlarged the scope of the methodology to various ingredient categories and chemical natures, contributing to place on the market new ingredients, safe for workers and end-users.