Adaptation of the human Cell Line Activation Test (h-CLAT) to Animal-Product-Free Conditions.

Skin sensitisers are substances that can elicit allergic responses following skin contact and the process by which this occurs is described as skin sensitisation. Skin sensitisation is defined as a series of key events, that form an adverse outcome pathway (AOP). Key event three in the AOP is dendritic cell activation that can be modelled by the human Cell Line Activation Test (h-CLAT) and is typified by changes in cell surface markers CD54 and CD86 in dendritic cells. The h-CLAT is accepted at a regulatory level (OECD Test-Guideline (TG)442E) and can be used to assess skin sensitisation potential as part of an integrated approach to testing and assessment (IATA). Stakeholders in the cosmetics and chemical industries have scientific and ethical concerns relating to use of animal derived material and have communicated a strong preference for fully human based in vitro methods. Therefore, we adapted the h-CLAT to animal-product-free conditions and validated the adapted method with the proficiency panel substances in Annex II of TG442E, using 3 independent batches of pooled human serum. The modified method showed equivalence to the validated reference method (VRM), as all proficiency substances were correctly classified. Comparable values for CV75 (concentration yielding 75% cell viability), EC150 and EC200 (concentration yielding RFI of ≥150 for CD86 and ≥200 for CD54) were obtained. Data generated using the adapted method may be used in European REACH submissions, provided the proficiency data is included. We are seeking formal inclusion of the adaptation into TG442E, enabling compliance with global regulations.

Adaptation of the KeratinoSens™ skin sensitization test to animal-product-free cell culture

Skin sensitization is the process by which a substance induces an allergic reaction following skin contact. The process has been described as an adverse outcome pathway (AOP), including several key events, from skin penetration and covalent protein binding, to keratinocyte activation, dendritic cell activation and T-lymphocyte proliferation. The in vitro assay KeratinoSens™ measures the activation of keratinocytes. It is fully accepted at a regulatory level (OECD TG 442d) and complies with a range of legislation including the EU Cosmetics Regulation, REACH, and the CLP Regulation. Currently, many in vitro methods use animal-derived components in their cell culture systems. Many stakeholders in the cosmetics industry have both scientific and ethical concerns relating to this issue and have stated a strong preference for fully human in vitro test systems. We have adapted the KeratinoSens™ method to animal product-free condi­tions, and carried out an in-house validation with 21 reference substances, including those listed in the Performance Standards associated with OECD TG442d. The modified method was shown to be equivalent to the Validated Ref­erence Method (VRM), with comparable values for accuracy (85.7%), sensitivity (84.6%) and specificity (87.5%), and all acceptance criteria being met. In Europe, data generated by the adapted method may be used in REACH submissions, and we are now seeking approval to list the adaptation in OECD TG 442d, enabling formal compliance with a range of global regulations.

Fibronectin interactions with osteoblasts: identification of a non-integrin-mediated binding mechanism using a real-time fluorescence binding assay.

Fibronectin (Fn) is an extracellular matrix protein that interacts with specific integrins on the cell surface, initiating signal transduction processes that lead to a reorganization of the cytoskeleton and the assembly of focal adhesions. Cell surface proteoglycans or glycosaminoglycans (GAGs) such as heparan sulfate are also known to participate in the interaction of Fn with the cell surface by binding to two different heparin-binding domains. The influence of Fn and GAGs on the spreading and differentiation of human osteoblasts was also previously described. In the current work, a method developed in our laboratory is established to evaluate the interaction between Fn and human osteoblasts and the influence of GAGs on such interactions. This technique makes use of fluoresceinphosphatidylethanolamine (FPE) such that when inserted into the lipidic bilayer, it acts as a fluorescent indicator of membrane interactions. The results indicate that the binding profile of Fn with the osteoblast cell surface is best represented by a hyperbolic single binding site model with a membrane affinity of 120 nM. Removal of cell surface heparan sulfate by treatment with heparitinase indicates that the cell surface moiety is directly involved in the binding process. Studies directed to assess the influence of heparin on the interaction of Fn with osteoblasts reveal that although it does not hamper Fn binding to the cell surface, it blocks the initial attachment to Fn-coated surfaces, indicating that binding to the integrin receptor alone is not enough to promote cell attachment but that the participation of the cell-surface GAGs is also a necessary condition.