Significance of melanin distribution in the epidermis for the protective effect against UV light.

Melanin, the most abundant skin chromophore, is produced by melanocytes and is one of the key components responsible for mediating the skin's response to ultraviolet radiation (UVR). Because of its antioxidant, radical scavenging, and broadband UV absorbing properties, melanin reduces the penetration of UVR into the nuclei of keratinocytes. Despite its long-established photoprotective role, there is evidence that melanin may also induce oxidative DNA damage in keratinocytes after UV exposure and therefore be involved in the development of melanoma. The present work aimed at evaluating the dependence of UV-induced DNA damage on melanin content and distribution, using reconstructed human epidermis (RHE) models. Tanned and light RHE were irradiated with a 233 nm UV-C LED source at 60 mJ/cm2 and a UV lamp at 3 mJ/cm2. Higher UV-mediated free radicals and DNA damage were detected in tanned RHE with significantly higher melanin content than in light RHE. The melanin distribution in the individual models can explain the lack of photoprotection. Fluorescence lifetime-based analysis and Fontana-Masson staining revealed a non-homogeneous distribution and absence of perinuclear melanin in the tanned RHE compared to the in vivo situation in humans. Extracellularly dispersed epidermal melanin interferes with photoprotection of the keratinocytes.

Investigating the surfactant antagonism with the Open Source Reconstructed Epidermis (OS-Rep) model.

Under the current EU chemicals legislation, in vitro test methods became the preferred methods to identify and classify the skin irritation potential of chemicals and mixtures. Among these, especially in vitro skin models are widely used. For surfactants, a well-known group of typically irritating chemicals, it is a long-standing experience that the irritation potential of a mixture of surfactants is typically lower than the irritation potential of the single surfactants, an effect usually described as surfactant antagonism. In order to evaluate if this effect can be observed in skin model systems as well, the irritation potential of the surfactants and of their mixtures was determined in the Open Source Reconstructed Epidermis (OS-REp) models. Combinations of sodium dodecyl sulfate or linear alkylbenzene sulfonate with cocoamidopropyl betain and alkyl polyglycosid, respectively, resulted in a clear decrease of the irritation potential compared to the irritation exerted by the single surfactants. The effect appeared to be primarily driven by the mixture's lower ability to damage the skin model's barrier, as shown by a reduced fluorescein permeation.

Extending the applicability domain of the human cell line activation test (h-CLAT).

Cosmetic ingredients must be toxicologically assessed to determine their skin sensitizing potential. The in vitro human cell line activation test (h-CLAT; OECD TG 442E) addresses the activation of dermal dendritic cells by analyzing specific protein expression after exposure of THP-1 cells to the test chemical. According to the protocol, FITC-labeled antibodies are used for protein detection. However, some chemicals show strong autofluorescence at FITC-specific wavelengths so that antibody-specific signals cannot be distinguished appropriately from autofluorescence background. This leads to inconclusive or false-negative predictions. Alternative fluorochromes can be used if their equivalence with the FITC-labeled antibodies is proven. In the current paper we describe the results of a proficiency exercise, based on the proficiency chemicals listed in the guideline, with FITC-labeled antibodies as the benchmark and APC-labeled anti­bodies as an alternative detection system. APC emits fluorescence at longer wavelengths, thus avoiding interference in the FITC spectrum. Irrespective of the employed fluorochrome, all chemicals were classified correctly, and the EC150 and 200 values were in the same order of magnitude. Hence, the equivalence in performance of FITC- and APC-labeled antibodies was demonstrated, and the respective demand of the guideline was fulfilled. In a case study, we then tested a proprietary oxidative hair dye using both fluorochromes. Using APC-labeled antibodies, the hair dye was unambiguously identified as a sensitizer, whereas no classification could be made with the FITC-labeled antibodies. With APC, fluorescence interference can be circumvented and the applicability domain of the h-CLAT extended to include autofluorescent chemicals.

In vitro eye irritation testing using the open source reconstructed hemicornea - a ring trial.

The aim of the present ring trial was to test whether two new methodological approaches for the in vitro classification of eye irritating chemicals can be reliably transferred from the developers' laboratories to other sites. Both test methods are based on the well-established open source reconstructed 3D hemicornea models. In the first approach, the initial depth of injury after chemical treatment in the hemicornea model is derived from the quantitative analysis of histological sections. In the second approach, tissue viability, as a measure for corneal damage after chemical treatment, is analyzed separately for epithelium and stroma of the hemicornea model. The three independent laboratories that participated in the ring trial produced their own hemicornea models according to the test producer's instructions, thus supporting the open source concept. A total of 9 chemicals with different physicochemical and eye-irritating properties were tested to assess the between-laboratory reproducibility (BLR), the predictive performance, as well as possible limitations of the test systems. The BLR was 62.5% for the first and 100% for the second method. Both methods enabled to discriminate Cat. 1 chemicals from all non-Cat. 1 substances, which qualifies them to be used in a top-down approach. However, the selectivity between No Cat. and Cat. 2 chemicals still needs optimization.

Cosmetics Europe compilation of historical serious eye damage/eye irritation in vivo data analysed by drivers of classification to support the selection of chemicals for development and evaluation of alternative methods/strategies: the Draize eye test Reference Database (DRD).

A thorough understanding of which of the effects assessed in the in vivo Draize eye test are responsible for driving UN GHS/EU CLP classification is critical for an adequate selection of chemicals to be used in the development and/or evaluation of alternative methods/strategies and for properly assessing their predictive capacity and limitations. For this reason, Cosmetics Europe has compiled a database of Draize data (Draize eye test Reference Database, DRD) from external lists that were created to support past validation activities. This database contains 681 independent in vivo studies on 634 individual chemicals representing a wide range of chemical classes. A description of all the ocular effects observed in vivo, i.e. degree of severity and persistence of corneal opacity (CO), iritis, and/or conjunctiva effects, was added for each individual study in the database, and the studies were categorised according to their UN GHS/EU CLP classification and the main effect driving the classification. An evaluation of the various in vivo drivers of classification compiled in the database was performed to establish which of these are most important from a regulatory point of view. These analyses established that the most important drivers for Cat 1 Classification are (1) CO mean ≥ 3 (days 1-3) (severity) and (2) CO persistence on day 21 in the absence of severity, and those for Cat 2 classification are (3) CO mean ≥ 1 and (4) conjunctival redness mean ≥ 2. Moreover, it is shown that all classifiable effects (including persistence and CO = 4) should be present in ≥60 % of the animals to drive a classification. As a consequence, our analyses suggest the need for a critical revision of the UN GHS/EU CLP decision criteria for the Cat 1 classification of chemicals. Finally, a number of key criteria are identified that should be taken into consideration when selecting reference chemicals for the development, evaluation and/or validation of alternative methods and/or strategies for serious eye damage/eye irritation testing. Most important, the DRD is an invaluable tool for any future activity involving the selection of reference chemicals.

Determining the Depth of Injury in Bioengineered Tissue Models of Cornea and Conjunctiva for the Prediction of All Three Ocular GHS Categories.

The depth of injury (DOI) is a mechanistic correlate to the ocular irritation response. Attempts to quantitatively determine the DOI in alternative tests have been limited to ex vivo animal eyes by fluorescent staining for biomarkers of cell death and viability in histological cross sections. It was the purpose of this study to assess whether DOI could also be measured by means of cell viability detected by the MTT assay using 3-dimensional (3D) reconstructed models of cornea and conjunctiva. The formazan-free area of metabolically inactive cells in the tissue after topical substance application is used as the visible correlate of the DOI. Areas of metabolically active or inactive cells are quantitatively analyzed on cryosection images with ImageJ software analysis tools. By incorporating the total tissue thickness, the relative MTT-DOI (rMTT-DOI) was calculated. Using the rMTT-DOI and human reconstructed cornea equivalents, we developed a prediction model based on suitable viability cut-off values. We tested 25 chemicals that cover the whole range of eye irritation potential based on the globally harmonized system of classification and labelling of chemicals (GHS). Principally, the MTT-DOI test method allows distinguishing between the cytotoxic effects of the different chemicals in accordance with all 3 GHS categories for eye irritation. Although the prediction model is slightly over-predictive with respect to non-irritants, it promises to be highly valuable to discriminate between severe irritants (Cat. 1), and mild to moderate irritants (Cat. 2). We also tested 3D conjunctiva models with the aim to specifically address conjunctiva-damaging substances. Using the MTT-DOI method in this model delivers comparable results as the cornea model, but does not add additional information. However, the MTT-DOI method using reconstructed cornea models already provided good predictability that was superior to the already existing established in vitro/ex vivo methods.