OECD workshop consensus report: Ethical considerations with respect to human derived products, specifically human serum, in OECD test guidelines.

The ethical needs and concerns with use and sourcing of human materials, particularly serum, in OECD in vitro test guidelines were explored in a dedicated international workshop held in 2019. The health-related aspects of the donation procedure, including tissue screening, donor health, laboratory work health protection, permission from the donor for commercial use, payment of the donors and the potential for exploitation of low-income populations and data protection of the donors; supply, availability, and competition with clinical needs; traceability of the serum and auditability/GLP needs for the Test Guideline Programme, were examined. Here we provide the recommendations of the workshop with respect to the use of human serum, and potentially other human reagents, specifically with regard to test method development for OECD Test Guideline utility as part of the Mutual Acceptance of Data requirement across all OECD member countries. These include informed donor consent terminology, a checklist of human serum information requirements to be included with the Good Laboratory Practise report, and suitable sources for human serum to ensure waste supplies are used, that can no longer be used for medical purposes, ensuring no competition of supply for essential medical use.

Addressing Animal Welfare Issues in Fetal Blood Collection for Fetal Bovine Serum Production.

There is ethical debate over whether fetal calves suffer when their dam is slaughtered and fetal blood extracted by cardiac puncture for fetal bovine serum (FBS) production. Yet, the serum industry does not follow best practice, as recommended by the European Food Safety Authority (EFSA), to avoid fetal distress. We discuss the key elements of this debate, and recommend how the serum industry can alter its practices to improve animal welfare.

Addressing potential ethical issues regarding the supply of human-derived products or reagents in in vitro OECD Test Guidelines.

The number and scope of Organisation for Economic Cooperation and Development (OECD) in vitro test guidelines (TGs) are increasing, in an effort to both improve human relevance and replace in vivo animal testing.  In vitro test methods being developed for TG use are increasing the use of human based reagents, in combination with, or replacing animal derived reagents, and demand for human reagents is likely to grow in the near future.  There are a range of issues associated with the ethical use of human reagents, particularly human serum, in the adaptation and development of in vitro TGs, especially to ensure that there is no human exploitation, legal requirements are adhered to, and that the origin of the reagent is assured. To address these concerns, the OECD has instigated a workshop on ethics, sources, availability and traceability of human based reagents for TG purposes, to be held in March, 2019. The focus is to provide guidance on acceptable sources of human serum for use in in vitro TGs, in terms of donor ethics and informed consent regarding commercial use and Quality Control for safety and consistent performance, with a view to providing guidance to support the adaptation and/or development of in vitro TGs using human reagents, and to ensure that in reporting the test results to regulators, clearly defined ethical and traceability aspects are adequately addressed, for the Mutual Acceptance of Data principle to be accepted in all OECD member countries. This thought-starter provides a discussion basis to achieve those objectives.

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.

Non-animal Replacements for Acute Toxicity Testing.

Current approaches to predicting adverse effects in humans from acute toxic exposure to cosmetic ingredients still heavily necessitate the use of animals under EU legislation, particularly in the context of the REACH system, when cosmetic ingredients are also destined for use in other industries. These include the LD50 test, the Up-and-Down Procedure and the Fixed Dose Procedure, which are regarded as having notable scientific deficiencies and low transferability to humans. By expanding on previous in vitro tests, such as the animal cell-based 3T3 Neutral Red Uptake (NRU) assay, this project aims to develop a truly animal-free predictive test for the acute toxicity of cosmetic ingredients in humans, by using human-derived cells and a prediction model that does not rely on animal data. The project, funded by Innovate UK, will incorporate the NRU assay with human dermal fibroblasts in animal product-free culture, to generate an in vitro protocol that can be validated as an accepted replacement for the currently available in vivo tests. To date, the project has successfully completed an assessment of the robustness and reproducibility of the method, by using sodium lauryl sulphate (SLS) as a positive control, and displaying analogous results to those of the original studies with mouse 3T3 cells. Currently, the testing of five known ingredients from key groups (a surfactant, a preservative, a fragrance, a colour and an emulsifier) is under way. The testing consists of initial range-finding runs followed by three valid runs of a main experiment with the appropriate concentration ranges, to generate IC50 values. Expanded blind trials of 20 ingredients will follow. Early results indicate that this human cell-based test holds the potential to replace aspects of in vivo animal acute toxicity testing, particularly with reference to cosmetic ingredients.