Amending the U-SENS™ skin sensitization test method for interfering auto-fluorescent chemicals.

Limitations of the applicability domain of new approach methodologies (NAM) present a major challenge for the testing of cosmetic ingredients in Europe, as the regulation does not allow to resort to in vivo test method. Therefore, research focused on overcoming such limitations of established in vitro test methods is frequently conducted. Here, we address a limitation of the U-SENS™, an in vitro skin sensitization test method that addresses the key event 3 on activation of dendritic cells of the adverse outcome pathway (AOP) for skin sensitization. The applicability domain of the U-SENS™ excludes autofluorescent substances that can interfere with the measurement of the expression of CD86, i.e., the primary readout. An evaluation of several fluorochromes identified APC as most suitable for testing auto-fluorescent chemicals. Acceptance criteria were reproducibly met when using the APC-labelled antibody. Equivalent performance in terms of reproducibility and skin sensitisation hazard assessment of the standard FITC-labelled antibodies and the APC-labelled antibodies was demonstrated by testing 40 substances. Finally, the value of the expanded technical applicability domain was highlighted with a case study using sulfuretin. In conclusion, we successfully demonstrated the expansion of the U-SENS™ applicability domain to interfering auto-fluorescent chemicals by using APC-labelled antibodies.

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).

The reconstructed skin model as a new tool for investigating in vitro dermal fillers: increased fibroblast activity by hyaluronic acid.

BACKGROUND: Clinical studies on dermal fillers have essentially focused upon visible improvement of skin quality and any eventual side effects, whereas very little is known about their detailed biological effects. OBJECTIVES: New skin equivalent models were created to investigate the biological impact of hyaluronic acid (HA) fillers on the dermal compartment in vitro. MATERIALS AND METHODS: Two different reconstructed skin models were developed to incorporate HA within the collagen fibers. In the mixed model, HA was distributed throughout the whole collagen gel whereas the HA was concentrated in the center of collagen gel in the inclusion model. RESULTS: A comparison of the addition of fillers in two models of reconstructed skin has permitted a better understanding of the biological impact of HA fillers. Protein profiling of supernatants from both models suggested a regulation of MMP-1 secretion by fibroblasts as a function of HA volume, distribution in the dermis and degree of cross-linking. Immunostaining of the inclusion model revealed increased production of type I and III procollagens close to the cross-linked HA. Fibroblasts located in this area showed a fusiform morphology as well as an increase in -smooth actin expression. The observed increase in collagen production may thus result in part from tension in fibroblasts surrounding the cross-linked HA. CONCLUSION: The inclusion reconstructed skin model, as compared to the mixed model, presented here, appears to be a useful tool for investigating the properties of various fillers in vitro and closer to the in vivo situation; our results show that HA fillers promote in vitro remodeling of the dermis by fibroblasts.