Assessment Of Skin Sensitization Potential Research Articles

Assessment of skin sensitization potential of zinc oxide, aluminum oxide, manganese oxide, and copper oxide nanoparticles through the local lymph node assay: 5-bromo-deoxyuridine flow cytometry method

ABSTRACT The advent of nanotechnology has significantly spurred the utilization of nanoparticles (NPs) across diverse sectors encompassing industry, agriculture, engineering, cosmetics, and medicine. Metallic oxides including zinc oxide (ZnO), copper oxide (CuO), manganese oxide (Mn2O3), and aluminum oxide (Al2O3), in their NP forms, have become prevalent in cosmetics and various dermal products. Despite the expanding consideration of these compounds for dermal applications, their potential for initiating skin sensitization (SS) has not been comprehensively examined. An in vivo assay, local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method (LLNA: BrdU-FCM) recognized as an alternative testing method for screening SS potential was used to address these issues. Following the OECD TG 442B guidelines, NPs suspensions smaller than 50 nm size were prepared for ZnO and Al2O3 at concentrations of 10, 25, and 50%, and Mn2O3 and CuO at concentrations of 5, 10, and 25%, and applied to the dorsum of each ear of female BALB/c mice on a daily basis for 3 consecutive days. Regarding the prediction of test substance to skin sensitizer if sensitization index (SI)≥2.7, all 4 NPs were classified as non-sensitizing. The SI values were below 2.06, 1.33, 1.42, and 0.99 for ZnO, Al2O3, Mn2O3, and CuO, respectively, at all test concentrations. Although data presented were negative with respect to adverse SS potential for these 4 NPs, further confirmatory tests addressing other key events associated with SS adverse outcome pathway need to be carried out to arrive at an acceptable conclusion on the skin safety for both cosmetic and dermal applications.

P17-34: Adoption of the OECD TG 442D for the in vitro assessment of skin sensitization potential of graphene-related materials

P17-34: Adoption of the OECD TG 442D for the in vitro assessment of skin sensitization potential of graphene-related materials

Strategy to Avoid Skin Sensitization: Application to Botanical Cosmetic Ingredients

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.

Expanding the applicability of the amino acid derivative reactivity assay: Determining a weight for preparation of test chemical solutions that yield a predictive capacity identical to the conventional method using molar concentration and demonstrating the capacity to detect sensitizers in liquid mixtures

IntroductionThe amino acid derivative reactivity assay (ADRA) is a novel in chemico alternative to animal testing for assessment of skin sensitization potential. The conventional ADRA protocol stipulates that test chemical solutions should be prepared to a specific molar concentration, allowing only for use of test chemicals with known molecular weights. Since many potential test substances are prepared by weight concentration or contain multiple unknown chemicals, this study was conducted to verify if it is possible to accurately assess the sensitization potential of test chemical solutions prepared at a specific weight concentration. Methods(1) Test chemical solutions for 82 chemicals were prepared at four different weight concentrations. Results were evaluated for agreement with in vivo results. (2) A liquid mixture comprising ten different non-sensitizers was prepared at 1 mg/mL. Ten different sensitizers of varying sensitization potencies were added individually to this mixture. The resulting pseudobinary mixtures were tested to confirm that the sensitizers could be detected. Results(1) The accuracies for test chemical solutions prepared at 0.5 and 0.2 mg/mL were 87.8% and 86.6%, respectively, which were roughly equivalent to the accuracy of 86.6% achieved with a solution prepared at the conventional molar concentration of 1 mM. In contrast, the accuracies for solutions prepared at 0.1 and 0.05 mg/mL were 82.9% and 74.4%, respectively, both of which were lower than that obtained with the conventional method. (2) Sensitizers added to the liquid mixture at 0.5 mg/mL were all correctly detected. DiscussionPreparing test chemical solutions at a weight concentration of 0.5 mg/mL decreased false negatives and increased false positives while improving prediction accuracy, which suggests that the sensitization potential of mixtures can also be assessed with this method.

LB756 Determination of contact sensitization potential of chemicals using in vitro reconstructed normal human epidermal model EpiDerm: Impact of the modality of application

Assessment of skin sensitization potential has traditionally been conducted in animal models, such as the Mouse Local Lymph Node Assay (LLNA) and the Guinea Pig Maximisation Test (GPMT). However, a growing focus and consensus for minimizing animal use have stimulated the development of in vitro methods to assess skin sensitization. Interleukin-18 (IL-18) release in reconstructed human epidermal models has been identified as a potentially useful endpoint for the identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability. The purpose of this study was to investigate the impact of the modality of chemical exposure on the predictive capacity of the assay. EpiDerm tissue viability assessed by MTT assay and IL-18 release assessed by ELISA were evaluated after 24 h topical exposure to test chemicals either impregnated in 8 mm diameter paper filters or directly applied to the surface of EpiDerm. Acetone: olive oil (4:1) was used as vehicle in all cases. A total of five chemicals from 3 different sources were tested. The testing set included 3 senzitizers, namely 2,4-dinitrochlorobenzene, cinnamaldehyde and isoeugenol/eugenol, and 2 non senzitizers, lactic acid and salicylic acid. Four independent dose – response experiments were conducted in 3 laboratories, resulting in correct prediction of the sensitizing potency of test chemicals. The assessment of IL-18 release using in vitro reconstructed normal human epidermal model EpiDerm appears to be a promising tool for in vitro determination of contact sensitization potential.

Application of the KeratinoSens™ assay for assessing the skin sensitization potential of agrochemical active ingredients and formulations

Assessment of skin sensitization potential is an important component of the safety evaluation process for agrochemical products. Recently, non-animal approaches including the KeratinoSens™ assay have been developed for predicting skin sensitization potential. Assessing the utility of the KeratinoSens™ assay for use with multi-component mixtures such as agrochemical formulations has not been previously evaluated and is a significant need. This study was undertaken to evaluate the KeratinoSens™ assay prediction potential for agrochemical formulations. The assay was conducted for 8 agrochemical active ingredients (AIs) including 3 sensitizers (acetochlor, meptyldinocap, triclopyr), 5 non-sensitizers (aminopyralid, clopyralid, florasulam, methoxyfenozide, oxyfluorfen) and 10 formulations for which in vivo sensitization data were available. The KeratinoSens™ correctly predicted the sensitization potential of all the AIs. For agrochemical formulations it was necessary to modify the standard assay procedure whereby the formulation was assumed to have a common molecular weight. The resultant approach correctly predicted the sensitization potential for 3 of 4 sensitizing formulations and all 6 non-sensitizing formulations when compared to in vivo data. Only the meptyldinocap-containing formulation was misclassified, as a result of high cytotoxicity. These results demonstrate the promising utility of the KeratinoSens™ assay for evaluating the skin sensitization potential of agrochemical AIs and formulations.

In silico risk assessment for skin sensitization using artificial neural network analysis.

The sensitizing potential of chemicals is usually identified and characterized using in vivo methods such as the murine local lymph node assay (LLNA). Due to regulatory constraints and ethical concerns, alternatives to animal testing are needed to predict the skin sensitization potential of chemicals. For this purpose, an integrated evaluation system employing multiple in vitro and in silico parameters that reflect different aspects of the sensitization process seems promising. We previously reported that LLNA thresholds could be well predicted by using an artificial neural network (ANN) model, designated iSENS ver. 2 (integrating in vitro sensitization tests version 2), to analyze data obtained from in vitro tests focused on different aspects of skin sensitization. Here, we examined whether LLNA thresholds could be predicted by ANN using in silico-calculated descriptors of the three-dimensional structures of chemicals. We obtained a good correlation between predicted LLNA thresholds and reported values. Furthermore, combining the results of the in vitro (iSENS ver. 2) and in silico models reduced the number of chemicals for which the potency category was under-estimated. In conclusion, the ANN model using in silico parameters was shown to be have useful predictive performance. Further, our results indicate that the combination of this model with a predictive model using in vitro data represents a promising approach for integrated risk assessment of skin sensitization potential of chemicals.

Towards AOP application – Implementation of an integrated approach to testing and assessment (IATA) into a pipeline tool for skin sensitization

Since the OECD published the Adverse Outcome Pathway (AOP) for skin sensitization, many efforts have focused on how to integrate and interpret nonstandard information generated for key events in a manner that can be practically useful for decision making. These types of frameworks are known as Integrated Approaches to Testing and Assessment (IATA). Here we have outlined an IATA for skin sensitization which focuses on existing information including non testing approaches such as QSAR and read-across. The IATA was implemented into a pipeline tool using OASIS technology to provide a means of systematically collating and compiling relevant information which could be used in an assessment of skin sensitization potential. A test set of 100 substances with available skin sensitization information was profiled using the pipeline IATA. In silico and in chemico profiling information alone was able to correctly predict skin sensitization potential, with a preliminary accuracy of 73.85%. Information from other relevant endpoints (e.g., Ames mutagenicity) was found to improve the accuracy (to 87.6%) when coupled with a reaction chemistry mechanistic understanding. This pipeline platform could be useful in the assessment of skin sensitization potential and marks a step change in how non testing approaches can be practically applied.

Evaluation of Sex Sensitivity in Local Lymph Node Assay Using Acephate and α- Hexylcinnamaldehyde

The current OECD Test Guideline for the conduct of the Local Lymph Node Assay (LLNA) recommends the use of only female mice for the assessment of skin sensitization potential for a given chemical. The NIH publication N° 99-4494 recommends that only female CBA mice be used, as they reportedly develop a stronger contact dermatitis response when compared to males however if male to be used, systematic studies evaluating potential sex differences should be conducted. Moreover, males were reported to display a larger variation in response due to a greater tendency to fight and to be involved in 'social ranking' behavior when group housed. However, there are several advantages to consider with the inclusion of male mice in LLNA testing including a more refined and responsible use of animals. Therefore, to begin to systematically assess the appropriateness of using male mice in the LLNA a comparative guideline study was conducted with individual housing of mice using a non-sensitizer and a sensitizer viz., Acephate 97 DF and α-hexylcinnamaldehyde (HCA) respectively. We have conducted an in-house study using a vehicle (dimethyl formamide) that is recommended by OECD 429 for the testing of mixtures in the LLNA for assessing skin sensitisation. We conducted the test according to the methods described by OECD 429 (Skin Sensitization: Local Lymph Node Assay). One common pesticide formulation, Acephate 97 DF and one known skin sensitizer - HCA, were each applied to the dorsum of both ears (25 µL per ear) of groups of 5 female CBA/J mice (male and female). Acephate 97 DF was applied at 3 separate concentrations and one concentration for HCA for 3 consecutive days (days 0, 1 and 2). A further group was given the vehicle (dimethyl formamide), alone. On day 5, all mice were injected intravenously (tail vein) with approximately 20 (±1) µCi of tritiated methyl thymidine. Five hours post-administration the uptake of 3H-thymidine into the auricular (local) lymph nodes draining the site of chemical application was measured in order to assess the proliferative response of the lymph node. The DPM values were measured individually for each mouse. Stimulation Index (SI Value) was calculated and are given below: - Acephate 97 DF (Male) (10%) 1.16 (25%) 1.58 (50%) 1.59 - Acephate 97 DF (Female (10%) 1.39 (25%) 1.55 (50%) 1.56 -

Artificial neural network analysis of data from multiple in vitro assays for prediction of skin sensitization potency of chemicals

In order to develop in vitro risk assessment systems for skin sensitization, it is important to predict a threshold from the murine local lymph node assay (LLNA). We first confirmed that the combination of the human Cell Line Activation Test (h-CLAT) and the SH test improved the accuracy and sensitivity of prediction of LLNA data compared with each individual test. Next, we assessed the mutual correlations among maximum amount of change of cell-surface thiols (MAC value) in the SH test, CV75 value (concentration giving 75% cell viability) in a cytotoxicity assay, EC150 and EC200 values (thresholds concentrations of CD86 and CD54 expression, respectively) in h-CLAT and published LLNA thresholds of 64 chemicals. Based on the results, we selected MAC value and the minimum of CV75, EC150 (CD86) and EC200 (CD54) as descriptors for the input layer of an artificial neural network (ANN) system. The ANN-predicted values were well correlated with reported LLNA thresholds. We also found a correlation between the SH test and the peptide-binding assay used to evaluate hapten-protein complex formation. Thus, this model, which we designate as the “iSENS ver. 1”, may be useful for risk assessment of skin sensitization potential of chemicals from in vitro test data.

Investigation of surface plasmon resonance biosensor for skin sensitizers studies

Non-animal testing methods are a current challenge in terms of the assessment of skin sensitization potential for new chemicals. Our objective was to investigate a surface plasmon resonance (SPR) biosensor to screen allergens against nucleophilic amino acids (cysteine, lysine and histidine) in a direct binding assay. Amino acids were immobilized on the sensor surface and exposed to different skin allergens (chemicals and fragrances) with varying sensitizing potential. Cysteine was found to be more reactive than lysine while histidine showed the lowest reactivity. The interactions observed were different depending on the allergen/amino acids involved. It appeared that weak allergens could quickly dissociate from the ligand, whereas strong and extreme allergens remained bound to the amino acids. The SPR report points allowed a good discrimination of the tested allergens. With this technology, we can observe low energy bindings and get information on the stability of the hapten/amino acid complex which seem relevant for the determination of skin sensitization potential. This prospective experiment showed the potential of real-time SPR to generate specific report points to refine the skin sensitization allergen assessment.

The local lymph node assay in practice: a current regulatory perspective

Following the formal acceptance of the local lymph node assay (LLNA) as an Organization for Economic Cooperation and Development (OECD) guideline in April 2002, the UK Health and Safety Executive (HSE) informed notifiers that this was now the method of choice for the assessment of skin sensitization potential under the EU notification scheme for new industrial chemicals (NONS). This paper summarizes the experience of the HSE for the 2-year period immediately following the issuing of this statement, during which 48 LLNA study reports were assessed for notification purposes. The issues discussed here include adherence to the OECD guideline, interpretation of results, and classification outcomes. Generally, notifying laboratories followed the OECD guideline successfully, with regard to the sex/ strain/numbers of mice used, the precise process used for measurement of cell proliferation, and the use of recommended vehicles and positive controls. Initially, use of the individual animal approach (measuring the cell proliferation in each animal rather than for a pooled dose group) highlighted problems caused by technical inexperience, but these were overcome by practice. Toxicity or irritation were found to be minor factors in dose selection; more important was the choice of vehicle to correctly maximize the test substance concentration, while maintaining appropriate application properties. Contrary to concerns that the LLNA would prove to be less sensitive or more sensitive than the traditionally used Guinea Pig Maximization Test (GPMT), the proportion of new substances classified as skin sensitizers was within the range observed in previous years. Although the sample size is relatively small, the experience of the HSE indicates that the LLNA is satisfactory for routine regulatory use.

A comparative study of the sensitivity of the 3-induction and 9-induction Buehler test procedures for assessing skin sensitisation potential

Assessment of skin sensitization potential is a mandatory requirement for the registration or notification of most types of chemicals and products. Until recently, two methods using the guinea pig as test model were the most widely accepted; the guinea pig maximisation test and the Buehler test. In the case of agrochemical formulations, which constitute the final end use product in contact with operators, industry and also some regulatory authorities consider the Buehler method more appropriate as the methodology is more relevant to likely exposure in the field. However, certain European regulatory authorities have become concerned about the sensitivity of the Buehler test for this purpose and have requested that a modified method is used in which additional applications of test materials are used during the induction phase of the protocol (a total of 9 rather than the normal 3). This study was designed to assess whether this modification was justified. Six reference substances (formaldehyde, alpha-hexylcinnamaldehyde, fragrance mix, thimerosal, mercaptobenzothiazole and phthalic anhydride); all mild to moderate skin sensitizing chemicals, were assessed in a study, which compared the use of 3 and 9 induction applications. The results of this study demonstrated that, although most of these sensitisers were detected by both protocols, the modified method (9 induction applications) was no more sensitive than the standard method (3 induction applications). As the modified protocol is also potentially more stressful to the animals, it is concluded that the use of additional induction applications in the Buehler test cannot be justified from either a scientific or an animal welfare perspective.

Risk assessment of sensitizing agents.

This review describes an approach that has been used to assess the skin sensitization risk of new product ingredients prior to and after marketing. The risk assessment process utilizes a comparative toxicological approach in which data on the inherent toxicity of a material and the exposure to it through manufacturing or consumer use or foreseeable misuse are integrated and compared with data generated by 'benchmark' materials of similar chemistry or product application, or both. This approach has been valuable in providing an accurate assessment of skin sensitization potential and the basis for eventual safe marketing of a wide range of consumer household and personal care products and topical pharmaceuticals.

The value of the local lymph node assay in quantitative structure-activity investigations.

The development of quantitative correlations between the physicochemical properties of a compound and its ability to act as a skin sensitizer is complicated by the number of variables associated with the current sensitization test data, combined with the absence of a truly objective end point. Recently, however, a novel approach to the assessment of skin sensitization potential, the local lymph node assay (LLNA), has been described, which determines the skin sensitization by measuring lymphocyte proliferation in lymph nodes draining the site of chemical exposure. The assay offers several advantages over traditional methods in the context of quantitative structure-activity relationship studies. In the present work, a range of bromoalkanes has been employed which demonstrate the robustness and reproducibility of the LLNA. Sensitizing activity increased with chain length up to a maximum at C15/C16, whereafter the response declined. The data were modelled against hydrophobicity, expressed as Clog P and (ClogP)2 to fit the biphasic nature of the results. The results demonstrate the utility of LLNA data for interpretation in the context of quantitative structure-activity relationships, the limited number of variables, inter-test reproducibility and quantitative end point, lending themselves to mathematical interpretations.