Adaptation of the human Cell Line Activation Test (h-CLAT) to animal-product-free conditions

Abstract

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.

The Overt and Hidden Use of Animal-Derived Products in Alternative Methods for Skin Sensitisation: A Systematic Review

“… DC: dendritic cells; PB: peripheral blood; IL: interleukin; RT-PCR: reverse transcriptase polymerase chain reaction; FCS: fetal calf serum; BSA: bovine serum albumin; HLA-DR: human leukocyte antigen DR isotype; LC: Langerhans cells; ELISA: enzyme-linked immunosorbent assay; qRT-PCR: quantitative real-time polymerase chain reaction; FBS: fetal bovine serum; CREM: cyclic adenosine monophosphate-responsive element modulator; CCR2: monocyte chemotactic protein-1 receptor; MHC: major histocompatibility complex; MIP: macrophage inflammatory protein. a Animal-derived products that were clearly mentioned in the paper. b Potentially animal-derived products that were mentioned in the paper — that is, when the products that were mentioned are usually animal-derived, but no specific information was provided regarding their source in this particular case. c Animal-derived products that were probably used, according to the technique, but were not actually mentioned in the paper. d Not used in the h-CLAT adapted to animal product-free conditions (Edwards et al). 82 e Not used in the h-CLAT adapted to chemically defined culture (Marigliani et al). 83 …”

“…Human platelet lysate is a promising alternative, 159 as is human serum, which has already been used in validated alternative methods for skin sensitisation. 38,82 These two methods 38,82 use commercial human plasma (pooled human male AB plasma) as the source, which is more defined than animal serum but is still ‘ill-defined’. As such supplements are human-derived and contain unidentified trace elements and unknown factors, their availability is limited and they pose the risk of human pathogen transmission.…”

“…161 However, there are ‘animal-free’ anticoagulants that could be used, such as citrate. 38,82 Concerns regarding animal-derived heparin also apply to all in vitro methods that use blood-derived cells.…”

“…However, with regard to the h-CLAT, it is a promising development that THP-1 cells are used in this test, and also that the method has recently been adapted to animal product-free conditions by using human serum, human albumin and synthetic antibodies. 82…”

“…Despite the extensive use of BSA in immunological methods, several non-animal alternative products exist, including detergents, polymers, 171 plant proteins (such as lectins) 172 and human serum albumin. 82,117…”