SkinEthic HCE Time-to-Toxicity on solids: A test method for distinguishing chemicals inducing serious eye damage, eye irritation and not requiring classification and labelling.

This study describes the development of a Time-to-Toxicity approach for solids (TTS) based on the SkinEthic™ HCE tissue construct, capable to distinguish chemicals that do not require classification for serious eye damage/eye irritation (No Cat.) from chemicals that require classification for eye irritation (Cat. 2), and serious eye damage (Cat. 1). Briefly, the time-to-toxicity of 69 solids was evaluated by exposing SkinEthic™ HCE tissue constructs to the test chemical for two different time periods (30-min, and 120-min). Based on the viability observed for the different exposure periods, a classification was assigned. The within laboratory reproducibility in terms of concordance in classifications (3 UN GHS categories), based on a set of 48 solids, was 93.7%. Furthermore, 73.6% Cat. 1 (N = 24), 55.6% Cat. 2 (N = 15) and 72.2% No Cat. (N = 30) were correctly identified with the SkinEthic™ HCE TTS test method. This study provides evidence that the SkinEthic™ HCE Time-to-Toxicity method (multiple exposure times) can distinguish Cat. 2 solids from Cat. 1 solids. This is an added value compared to the SkinEthic™ HCE EITS method (single exposure time) that can distinguish No Cat. chemicals from chemicals that do require classification and labelling for eye irritation/serious eye damage (Cat. 2/Cat. 1).

Development of the SkinEthic HCE Time-to-Toxicity test method for identifying liquid chemicals not requiring classification and labelling and liquids inducing serious eye damage and eye irritation.

This study describes the development of a Time-to-Toxicity approach for liquids (TTL) based on the SkinEthic™ HCE tissue construct, capable to distinguish chemicals that do not require classification for serious eye damage/eye irritation (No Cat.) from chemicals that require classification for eye irritation (Cat. 2), and serious eye damage (Cat. 1). Briefly, the Time-to-Toxicity of 56 liquids was evaluated by exposing SkinEthic™ HCE tissue constructs to the test chemical for three different time periods (5-min, 16-min, and 120-min). Based on the viability observed for the different exposure periods, a classification was assigned. The within laboratory reproducibility in terms of concordance in classifications (3 UN GHS categories), based on a set of 50 liquids, was 80.0%. Furthermore, 84.3% Cat. 1 (N = 17), 79.4% Cat. 2 (N = 21) and 72.2% No Cat. (N = 18) were correctly identified with the SkinEthic™ HCE TTL test method. This study provides evidence that the SkinEthic™ HCE Time-to-Toxicity method (multiple exposure times) is capable of distinguishing Cat. 2 liquids from Cat. 1 liquids. This is an advantage compared to the SkinEthic™ HCE EITL method (single exposure time) that can distinguish No Cat. chemicals from chemicals that do require classification and labelling for eye irritation/serious eye damage (Cat. 2/Cat. 1).

Usefulness of the EpiSkin™ reconstructed human epidermis model within Integrated Approaches on Testing and Assessment (IATA) for skin corrosion and irritation.

Predictive capacity of the EpiSkin™ model was evaluated on 87 chemicals using the Bottom-Up and the Top-Down testing approaches recommended within Integrated Approach on Testing and Assessment for the identification of both skin irritation and corrosion hazards. Classified (UN GHS Cat. 1 and Cat. 2) chemicals were identified with a very high sensitivity (≥94%) and the non-classified (UN GHS Cat. 3 and No Cat.) chemicals with an appropriate specificity (70%). Very high sensitivities were obtained for the identification of Cat. 1 chemicals (≥98%), very high specificities for non-Cat. 1 chemicals (93%), and accuracies of -95% for the identification of skin corrosives vs. non-corrosives by both approaches. Overall accuracies of 72% were found for predicting the single (sub)categories: non-classified, Cat. 2, Subcat. 1B/1C and Subcat. 1A. Results indicated the testing strategies to be more predictive than the individual assays on a conservative safety approach. Finally, no extreme misclassifications (no under-prediction of in vivo Subcat. 1A as non-Cat. 1, and no over-prediction of non-classified chemical as Subcat. 1A) occur. These findings, independently of the approach used, confirm the usefulness of the EpiSkin™ in vitro model for a safe prediction of the skin irritant and corrosive hazards of chemicals.

SkinEthic™ HCE Eye Irritation Test: Similar performance demonstrated after long distance shipment and extended storage conditions.

Assessment of ocular irritation risk is an international regulatory requirement in the safety evaluation of products. In response to this need, L'Oréal developed the SkinEthic™ Human Corneal Epithelium (HCE) Eye Irritation Test (EIT) that has been included in OECD Test Guideline 492. SkinEthic™ HCE EIT is able to correctly and reliably identify chemicals not requiring classification versus labelling for eye irritation or serious eye damage according to UN GHS. In an effort to promote its global use, the performance of the method was evaluated after long-distance shipment and compared to European shipment conditions. Results obtained by Cosmos Technical Center (Japan) after extended tissues transit were compared to results obtained in L'Oréal (France). Thirty-nine out of 40 blinded chemicals, representing different functional chemical classes, were consistently classified in both laboratories. The SkinEthic™ HCE EIT test method was also evaluated for its performance after extended storage of the tissues. The performance was in agreement with the values reported in OECD TG 492, with an overall accuracy of 87.1% (based on 119 chemicals), sensitivity of 95.5% and specificity of 73.5%. The reliability and relevance of SkinEthic™ HCE EIT test method after long-distance shipment and extended storage remain in agreement with regulatory validation criteria.

An integrated testing strategy for in vitro skin corrosion and irritation assessment using SkinEthic™ Reconstructed Human Epidermis.

The SkinEthic™ Reconstructed Human Epidermis (RHE) method has been formally adopted for the regulatory assessment of skin irritation (OECD TG 439) and corrosion (OECD TG 431). Recently, the OECD adopted an Integrated Approach on Testing and Assessment (IATA) for skin corrosion and skin irritation (OECD GD 203), which provides guidance on the integration of existing and new information in a modular approach for classification and labelling. The present study aimed to evaluate the use of the SkinEthic™ RHE model within the proposed OECD IATA. Data on 86 substances were integrated in a bottom-up and top-down testing strategy to assess their capacity for EU CLP and UN GHS classifications. For EU CLP, strategies showed an accuracy of 84.8% to discriminate non-classified from classified substances, 94.4% to discriminate corrosive from non-corrosive substances, and 68.5% to discriminate the four (sub)-categories. For UN GHS, strategies showed an accuracy of 89.5% to discriminate non-classified from classified substances, 93.4% to discriminate corrosive from non-corrosive substances, and 74.2% to discriminate four GHS (sub)-categories (excluding Category 3). In conclusion, the integration of SkinEthic™ RHE irritation and corrosion data in a bottom-up and top-down testing strategy allows the classification of substances according to EU CLP and UN GHS.

In vitro assessment of eye irritancy using the Reconstructed Human Corneal Epithelial SkinEthic HCE model: application to 435 substances from consumer products industry.

The 7th amendment of the EU Cosmetics Directive led to the ban of eye irritation testing for cosmetic ingredients in animals, effective from March 11th 2009. Over the last 20years, many efforts have been made to find reliable and relevant alternative methods. The SkinEthic HCE model was used to evaluate the in vitro eye irritancy potential of substances from a cosmetic industry portfolio. An optimized protocol based on a specific 1-h treatment and a 16-h post-treatment incubation period was first assessed on a set of 102 substances. The prediction model (PM) based on a 50% viability cut-off, allowed to draw up two classes (Irritants and Non-Irritants), with good associated sensitivity (86.2%) and specificity (83.5%). To check the robustness of the method, the evaluated set was expanded up to 435 substances. Final performances maintained a high level and were characterized by an overall accuracy value > 82% when using EU or GHS classification rules. Results showed that the SkinEthic HCE test method is a promising in vitro tool for the prediction of eye irritancy. Optimization datasets were shared with the COLIPA Eye Irritation Project Team and ECVAM experts, and reviewed as part of an ongoing progression to enter an ECVAM prospective validation study for eye irritation.

The in vitro skin irritation of chemicals: optimisation of the EPISKIN prediction model within the framework of the ECVAM validation process.

In view of the increasing need to identify non-animal tests able to predict acute skin irritation of chemicals, the European Centre for the Validation of Alternative Methods (ECVAM) focused on the evaluation of appropriate in vitro models. In vitro tests should be capable of discriminating between irritant (I) chemicals (EU risk: R38) and non-irritant (NI) chemicals (EU risk: "no classification"). Since major in vivo skin irritation assays rely on visual scoring, it is still a challenge to correlate in vivo clinical signs with in vitro biochemical measurements. Being particularly suited to test raw materials or chemicals with a wide variety of physical properties, in vitro skin models resembling in vivo human skin were involved in prevalidation processes. Among many other factors, cytotoxicity is known to trigger irritation processes, and can therefore be a first common event for irritants. A refined protocol (protocol 15min-18hours) for the EPISKIN model had been proposed for inclusion in the ECVAM formal validation study. A further improvement on this protocol, mainly based on a post-treatment incubation period of 42 hours (protocol 15min-42hours), the optimised protocol, was applied to a set of 48 chemicals. The sensitivity, specificity and accuracy with the MTT assay-based prediction model (PM) were 85%, 78.6% and 81.3% respectively, with a low rate of false negatives (12%). The improved performance of this optimised protocol was confirmed by a higher robustness (homogeneity of individual responses) and a better discrimination between the I and NI classes. To improve the MTT viability-based PM, the release of a membrane damage marker, adenylate kinase (AK), and of cytokines IL-1alpha and IL-8 were also investigated. Combining these endpoints, a simple two-tiered strategy (TTS) was developed, with the MTT assay as the first, sort-out, stage. This resulted in a clear increase in sensitivity to 95%, and a fall in the false-positive rate (to 4.3%), thus demonstrating its usefulness as a "decision-making" tool. The optimised protocol proved, both by its higher performances and by its robustness, to be a good candidate for the validation process, as well as a potential alternative method for assessing acute skin irritation.