10 animal tests that should be fully replaced
It may be commonly assumed that tests on animals that have non-animal replacements are no longer conducted, or only vary rarely. The reality is that such tests can persist, and even increase, long after suitable non-animal methods become available.
To draw attention to this issue we created the Replace Animal Tests (RAT) List, describing ten tests typically performed to satisfy legal requirements which are still conducted using animals, despite there being non-animal replacements known to give reliable results.
While testing happens globally, the EU is currently the only major region that publishes annual reports detailing the number of tests conducted on animals. So, to illustrate the potential scale of animal use in these ten tests, we have included figures from the EU as an example for each.
The total number of animals still being used in these ten tests could be 1.4 million every year in Europe alone, based on statistics for the year 2020.
Over the last 30 years, there have been significant developments in the replacement of tests on animals used to meet legal information requirements. Non-animal methods have been developed that can now replace, wholly or in part, a number of animal tests across several product sectors.
However, our experience has been that actually replacing tests on animals takes much longer than it should. We have seen how valid non-animal methods for assessing skin irritation, skin sensitisation and batch safety have taken years to be accepted and we know that tests on animals are still being conducted to provide this type of information. This is unacceptable. In many cases, the problem lies with lack of clarity from, and enforcement by, regulatory authorities, as well as the absence of a joined-up approach for acceptance of non-animal methods around the world. Since non-animal tests can be cheaper, faster and more accurate than the tests on animals they replace, it is in the interests of animal welfare and good science to speed up their acceptance and to expand their use.
Overcoming the remaining hurdles so that these tests on animals can be fully replaced is a matter of urgency.
Species: Mostly rabbits
Number of tests in the EU: 4,070 per year
Product test is used for: Chemicals, medicines, medical devices
Animal method: A test substance is rubbed on to a 6 cm area of a rabbit’s shaved skin and held in place with a bandage for four hours. The rabbit is then examined for signs of skin damage for 14 days. If there are no signs of irritation, two more rabbits are used in a confirmatory test. Rabbits are singly housed and can suffer from painful skin reactions and rashes.
Replacement method: Reconstituted human skin models can be used in most cases. They are more predictive than the rabbit test.
Species: Rabbits
Number of tests in the EU: 491 per year
Product test is used for: Chemicals, medicines, medical devices, biocides
Animal method: A test substance is placed in the eyes of live rabbits and left for at least one hour before being washed out. Their eyes are then examined for signs of irritation and damage over three days. If there are no signs of severe irritation in the initial test, two more rabbits are used in a confirmatory test. Rabbits are singly housed and can experience painful damage in their eyes that can lead to blindness.
Replacement method: Reconstituted human eye models can be used to detect non-irritants. Eyes from hens and cows killed for food can also be used to identify severe irritants and non-irritants. Work is underway to develop a "defined approach” – a combination of chemical-, computer- and cell-based tests – that will enable the non-animal methods to provide additional information about the severity of irritation.
Species: Mice, guinea pigs
Number of tests in the EU: 38,024 per year
Product test is used for: Chemicals, medicines, medical devices
Animal method: A test substance is rubbed on to the shaved skin of guinea pigs who are then observed daily for signs of an allergic reaction for almost a month. They may suffer from painful skin reactions and rashes. In the mouse test, a substance is painted on to the ears of mice every day for three days. The mice are then killed three days later, and their ears are dissected.
Replacement method: Several chemical-, computer- and cell-based tests can be used in combination formalised in a ‘defined approach’ guideline in 2021. These tests include the direct peptide reactivity assay (DPRA), which measures the binding of a substance to proteins, the keratinocyte assay and the human cell line activation test (h-CLAT) based on human immune cells.
Species: Rabbits
Number of tests in the EU: 24,139 per year
Product test is used for: Medicines (human), medical devices
Animal method: Rabbits are restrained in boxes for up to eight hours per test, with food and water restriction prior to this. They are injected with a test substance into a vein in their ears which often causes damage, and their temperature is monitored through probes inserted deep into their rectums.
Replacement method: A test that uses human cells – the monocyte activation test (MAT) – was developed in the 2000s. The MAT works by measuring the immune response of human cryo-preserved blood to a test substance. A test using blood from horseshoe crabs (limulus amebocyte lysate; LAL) is another alternative to the live rabbit test, but the crabs are harmed and sometimes killed in the process. The rabbit test is promised to be deleted from EU guidelines by 2026.
Species: Mice
Number of tests in the EU: 273,955 per year
Product test is used for: Medicines (human)
Animal method: Mice have an injection into their abdomens with the botulinum toxin and, over the following three days, become increasingly paralysed. If left, mice in the higher dose groups will stop being able to breathe and suffocate to death. Approximately 50% of the mice die per test, and survivors are killed at the end of the test.
Replacement method: The major toxin manufacturers have now developed a cell-based test to replace the batch test for their botox products.
Species: Various including mice, rabbits, sheep, goats
Number of tests in the EU: 1 million* per year
Product test is used for: Many areas of research and testing
Animal method: Animals, including mice, rabbits, sheep and goats are used as “factories” to generate large numbers of antibodies. The process involves injecting animals with a substance that stimulates the immune system to produce specific antibodies. The animals often suffer from harmful side effects before their blood is taken to harvest the desired antibodies. Animals are killed when they are no longer useful.
Replacement method: Phage-display technology – endorsed in 2020 by the EU Reference Laboratory for alternatives to animal testing – can be used to produce a wide range of antibodies that are of higher quality, more stable, more relevant and more reproducible than the antibodies produced in animals. They are also much faster and cheaper to produce.
Species: Hamsters
Number of tests in the EU: 3,642 per year
Product test is used for: Medicines (vet)
Animal method: Hamsters are first injected with the vaccine and then, up to 30 days later, Leptospira bacteria are injected into their abdomens. For the batch to pass the test, 80% of the vaccinated animals must survive while 80% of the unvaccinated animals must die or show symptoms of infection, which includes bleeding internally and from the nose.
Replacement method: Enzyme-linked immunosorbent assays (ELISAs) are rapid immunological tests that can be used to detect specific antigens or antibodies. ELISAs have been developed and validated to replace the potency test in hamsters. These tests are more reliable and predictive.
Species: Can include mice, farmed animals, dogs, cats
Number of tests in the EU: 720* per year
Product test is used for: Medicines (vet)
Animal method: In the target and laboratory animal batch safety test animals are injected with two doses of an inactivated vaccine or ten doses of a live vaccine and then observed for at least two weeks. Animals can suffer painful reactions in the rare occasion the batch is contaminated and as a result of large injection volumes relative to the animals’ size (young animals are often used).
Replacement method: Improvements in the manufacturing process of vaccines have made animal batch tests redundant. Cell-based tests are also used as part of a “consistency approach” to ensure that each vaccine batch is consistent with previously tested batches.
Species: Mice, guinea pigs
Number of tests in the EU: 2,810* per year
Product test is used for: Medicines (human)
Animal method: Animals, typically mice and guinea pigs, are injected with a biological product (e.g. vaccines, sera, antibodies) and observed for signs of local and systemic reactions for at least seven days. If one of the animals dies or becomes ill, the test will be repeated a second time.
Replacement method: Improvements in the manufacturing process of biological products and post-market surveillance have made animal batch tests redundant. Cell-based tests are used as part of a “consistency approach” to ensure that each vaccine batch is consistent with previously tested batches.
Species: Mice
Number of tests in the EU: 25,884 per year
Product test is used for: Food
Animal method: Extracts from shellfish are injected into the abdomens of mice and the time until they die is recorded. If two thirds of the mice die within 24 hours, the shellfish is deemed unsafe for human consumption.
Replacement method: Analytical chemistry methods (high performance liquid chromatography and liquid chromatography-mass spectrometry methods) can be used to check for the presence of paralytic shellfish toxins, amnesic toxins and lipophilic toxins.
Notes: “Number of tests” means the number of tests performed on animals, which may not always be the same as the number of animals used in tests. The numbers of tests on animals have been taken directly from the official statistics for the EU and Norway for the year 2020, except where indicated by an asterisk (*) – for those numbers, details of the calculations used are available upon request. The total number of tests on animals = 1,373,735.