New WHO guidelines on non-animal tests for quality control of biologics

We were commissioned by the World Health Organisation (WHO) – the gatekeeper for global standards for vaccine and biologics safety – in 2019 to audit its guidelines for batch release testing, an area that is estimated to use around 10 million animals a year in tests that can involve significant suffering. In direct response to our recommendations, the WHO has developed new guidelines that strongly encourage manufacturers and regulators to replace or remove animal-based quality control methods whenever scientifically justified.

Some currently used animal tests are expensive and have a high degree of variability which cause delays to the release of essential medicines. Non-animal technologies can offer more precise and robust assessment of safety and efficacy for biologics, but differences in testing requirements between countries and across product-specific guidelines has been a significant barrier to their routine adoption. This is compounded by a lag in updating guidance to reflect the latest replacement technologies.

WHO guidelines are adopted by national regulatory authorities and control laboratories around the world and in an unprecedented step towards international harmonisation of non-animal testing requirements, WHO have confirmed that the new guidance supersedes all previous recommendations for biologics. The guidelines align with ambitions in the UK government’s strategy to support the development, validation and uptake of alternative methods, including replacing both the rabbit pyrogen test (see case study below) and adventitious agent testing in animals. We are looking forward to continuing to work with WHO on their implementation plans.

Case study: Pyrogen testing

The new WHO guidance highlights that in vitro assays are superior or equivalent to traditional animal tests for pyrogenicity testing and make a strong recommendation for the replacement of the rabbit pyrogen test with the monocyte activation test (MAT).

Pyrogens are contaminants that activate the innate immune system and cause a fever. The rabbit pyrogen test, which involves intravenous administration of the substance to assess whether it raises the animals’ body temperature, has been widely reported to be prone to both false positive and false negative results, to have significant technical limitations and to be poorly predictive of responses in humans. The MAT is an in vitro alternative that detects activation of the immune system using human blood samples or cell lines, more closely reflecting human immune responses and providing a reliable, reproducible and sensitive measure of pyrogenicity. MAT testing kits in ready-to-use formats are commercially available and engineered cell lines for use in the assay continue to be developed, improving global access to the assay. As part of our ongoing project funded by the Gates Foundation, we recently ran a stakeholder survey on the use of the MAT as a replacement for the rabbit pyrogen test to better understand current practices and barriers to implementation.

About the review

Our project to embed 3Rs approaches in WHO guidance documents for biological product quality control was led by Dr Elliot Lilley with the support of an expert international working group, including 29 companies from 19 countries. We have:

• Identified almost 350 separate instances where animal tests are currently recommended.
• Developed 3Rs-focused text for each instance, including replacing animal tests with validated non-animal approaches, alongside opportunities to reduce the number of animals involved and refine procedures.
• Engaged extensively with the global biologicals manufacturer and regulator communities to understand the sector and regional perspectives that may impact the adoption of 3Rs approaches.
• Generated a searchable database as a resource for the community to access recommendations relevant to their work quickly and easily.
• Been involved throughout the drafting and consultation process of the new guidelines, including contributing text, providing feedback and responding to public consultations.
• Initiated a follow-on project to support delivery of the 3Rs impacts of our recommendations, funded by the Gates Foundation.

The new guidelines establish a clear international mandate to transition away from animal models for quality control testing and suggest specific non-animal test types and products in line with our report and in our database. They were accepted by the Expert Committee on Biological Standardization (ECBS) in October 2025 and will be formally published as a resource in the WHO Institutional Repository for Information Sharing (IRIS) and in the WHO Technical Report Series in Spring 2026.