Every year, thousands of rabbits suffer and die in so-called pyrogen tests. And this despite the fact that an animal-free test method has been available for over 10 years. This article gives the detailed background information which accompanied our successful campaign "Save 6,000 rabbits - stop pyrogen testing!"

Pyrogens

Pyrogens are toxins that can cause fever in humans or even result in septic shock (blood poisoning) with a potentially fatal outcome. Substances that are administered by bypassing the gastrointestinal tract are legally required to be tested for these fever-causing contaminants. These include substances that are injected into veins and muscles or under the skin, such as vaccines, infusion solutions, and injectable drugs, but also medical materials that are implanted in the human body, such as hip prostheses or artificial heart valves. The European Pharmacopoeia requires a quality control of each batch of these products to protect patients from harm.

Pyrogens can originate from different sources:

Bacterial pyrogens:

• Endotoxins (“internal toxins”) of gram-negative bacteria are lipopolysaccharides (LPS), which are released from the bacterial wall during its disintegration
• Components of gram-positive bacteria

Other pyrogens:

• Components of viruses
• Components of fungi
• Abrasion from rubber, plastic, or metal 

Animal experiment: Rabbit Pyrogen Test (RPT)

Rabbits are confined in small boxes for hours. ©One Voice

As in humans, pyrogens also cause an increase in body temperature in rabbits. In the Rabbit Pyrogen Test (RPT), also known as the "classical pyrogen test", the test substance is injected into rabbits’ ear veins. To do this, the animals are put into small boxes in which they cannot move, with only their head sticking out. To continuously measure the temperature of the animals, a thermometer is inserted into their rectum. The rabbits have to endure this for several hours. They are often not killed directly afterwards but are used in further pyrogen tests. The fixation in the box alone is associated with enormous stress for the animals. Due to the repeated injections, they suffer from painful thickenings of the veins. Depending on whether fever-inducing substances are contained in the sample or not, they might develop fever, respiratory distress, or shock. After a certain "usage period", the rabbits are killed. The rabbit pyrogen test degrades sentient animals to pure measuring devices that are killed and thrown away after “use”.

Animal consuming test: Limulus-Amebocyte-Lysate (LAL)-test

Horseshoe crabs are fished out of the sea so their blood can be extracted. ©SATIS

The Limulus-Amebocyte-Lysate (LAL)-test, also known as the Bacterial Endotoxin Test (BET), was developed in the 1950s. The blood of wild-caught horseshoe crabs (Limulus polyphemus) is used. These primeval animals, which are up to 85 cm long, have been living on Earth since before the time of the dinosaurs. All four species - three of them in Southeast Asia and one living off the North American Atlantic coast - are equally affected by the capture. The animals are caught and taken to specialized laboratories where up to one-third of their blue blood is drawn without anesthesia.

Many do not survive the long exposure to the air, the transport, and/or the process of blood withdrawal. Of the surviving animals, 13% are sold to local fishermen as bait; the rest are released back into the sea (1).

Every year, 500,000 horseshoe crabs are caught off the east coast of the USA alone. Around 130,000 of them die. (1) Various studies have shown that injuries and disorientation of released animals can lead to higher susceptibility to disease and to reduced reproduction rates. In addition, the ecosystem is disturbed by the massive catch (1).

The LAL-test has grown into a profitable market. Today, more than 90% of pyrogen testing is performed with this test, with worldwide sales of approximately $660 million (2). The company Charles River Laboratories in Charleston, South Carolina, USA operates a facility where blood is drawn from horseshoe crabs (1). Charles River is one of the world's largest breeders of so-called laboratory animals, and involved in the LAL-test as an "alternative" as well.

The horseshoe crab's blood cells, called amebocytes, clot when exposed to pyrogens. Apart from ethical concerns, the disadvantage of the LAL-test is that it can only detect endotoxins originating from gram-negative bacteria (LPS), while those from gram-positive bacteria cannot be detected. Obviously, the LAL-test should be rejected as an "alternative" to the rabbit test: it is associated with animal suffering, just like the rabbit test, and it is not able to detect all pyrogens.

Synthetic method: rFC-test

The LPS-induced agglutination of the horseshoe crab´s blood is based on a cascade of different enzymatic reactions that are sequentially activated by LPS. As early as 1997, scientists at the University of Singapore succeeded in synthetically producing the first molecule in this cascade: factor C. (1) As a result, various commercially available tests with recombinant factor C (rFC) were developed that detect LPS just as reliably as the horseshoe crab blood. However, there is still no comprehensive replacement for the LAL. The reason for this is the powerful LAL industry in the USA, which again succeeded in 2020 in preventing the rFC-test from being approved as an equivalent “alternative” for the LAL-test in the USA (3).

In January 2021, the rFC test was included into the European Pharmacopoeia in addition to the other approved tests (RPT, MAT, LAL). Starting in May 2025, the rFC test will also be approved by the United States Pharmacopeia (USP) (4).

Human-based method: Monocyte Activation Test (MAT)

The monocyte activation test (MAT) uses a completely different approach, based on human blood samples. The first steps in developing the MAT were made as early as 1984. In the 1990s, a test using human blood cells was developed at the University of Konstanz (5). Upon contact with pyrogens, cells of the immune system, in particular certain white blood cells (monocytes), release signaling substances (so-called interleukins), which mediate the fever reaction within the organism. The in-vitro test makes use of this reaction, and the substances to be tested are combined with a small amount of blood from a healthy, voluntary donor. If pyrogens are present, the white blood cells produce interleukin, and the amount of released interleukin can be determined. A blood donation of 500 ml is sufficient to perform up to 50,000 tests (2).

Antibodies - which are usually produced in animals - are used for quantification of the interleukins. Fetal calf serum (FCS) is also used as a supplement for the cell culture media. As a result, the MAT is unfortunately not animal-free. Today, however, it is possible and even recommended by ECVAM, the European Center for the Validation of Alternatives to Animal Testing, to use synthetically produced antibodies (6). Switching to a synthetic FCS-free cell culture medium also poses no problem (7).

In contrast to the two animal tests using rabbits or horseshoe crabs, not only solutions but also powders, gaseous substances, blood transfusions, cell therapies and solid materials such as implants, and nanomaterials can be tested with the MAT. In addition, a much wider range of pyrogens can be detected, including pyrogens from gram-positive bacteria, which are not covered by the LAL, and pyrogens from fungi (2,5). Moreover, the use of human cells means that the results can be transferred to humans. Furthermore, the test only costs a tenth of the rabbit pyrogen test. (2)

The MAT can be performed by using standardized and commercially available test kits. There are different variants that are equally approved in the European Pharmacopoeia: tests with fresh human whole blood or frozen blood from human voluntary donors are available, as well as a test based on a human cell line (Mono Mac 6 cells).

Table 1: Comparison of RPT, LAL, and MAT (2,8,9)

Legal regulations

The rabbit pyrogen test was included in the British Pharmacopoeia (BP) in 1912 and in the US Pharmacopoeia (USP) in 1942. (10) In the European Pharmacopoeia, too, the rabbit test had been the standard for testing for fever-inducing substances for decades. In the 1980s, the Limulus amebocyte lysate (LAL) test was added as an "alternative".

In 2005, the MAT was internationally validated. (11) In 2009 and 2010, the US FDA (Food and Drug Administration) accepted the MAT, and it was included in the European Pharmacopoeia (monograph 2.6.30) as the method of choice compared to older methods. (12) All three methods are listed in the 2017 pharmacopoeia. It is stated that the MAT should be given preference over the rabbit test. However, the MAT must be validated product-specifically before it can be considered suitable. (13) This means that each manufacturer or laboratory carrying out the test has to adapt the test for the specific product to be tested, thus making it harder to use this less cruel and more reliable method.

In Germany, the end of the rabbit test is finally approaching, as it will be removed from the European Pharmacopoeia in July 2025 (14). This will force manufacturers to replace the rabbit test with other methods. Another good news is that from May 2025, the rFC test will be recognized by the U.S. Pharmacopeia (4). 

Use of rabbits for pyrogen testing

Until a few years ago, there were only rough estimates of how many rabbits were subjected to pyrogen testing each year. In 2000, for example, it was assumed that 80,000 rabbits were used for pyrogen testing in Germany (15), and for the years 2003 and 2010 figures of 200,000 rabbits per year in Europe were given. (16) In 2015, an article estimated the use of 170,000 rabbits in Europe and 400,000 worldwide. (2)

Since 2014, rabbits used for pyrogen testing have been recorded separately in the annual animal experimentation report of the Federal Ministry of Food and Agriculture (BMEL), (17) a task that is now being fulfilled by the German Federal Institute for Risk Assessment (BfR). According to the available reports, more than thousand rabbits are still used in Germany for the pyrogen test.

Rabbits used in pyrogen testing
Country	2014	2015	2016	2017	2018	2019	2020	2021	2022
Belgium	804	0	0	0	0	0	?	0	0
Bulgaria	?	?	?	?	?	?	?	300	270
Germany	7.259	6.992	347	5.591	6.291	6.457	3.223	1.371	1.078
France	?	?	?	6.191	?	?	10.325	9.711	7.145
Greece	?	?	?	?	?	?	?	0	12
United Kingdom	3.167	2.609	2.472	1.125	0	?	?	0	0
Ireland	?	570	506	312	145	525	943	657	966
Italy	?	?	?	?	?	?	?	1.531	1.518
Croatia	75	63	67	0	0	?	?	0	0
Austria	6.897	14.794	13.157	9.125	1.923	704	?	0	0
Poland	?	?	?	?	?	?	?	177	147
Rumania	?	?	?	?	?	?	?	25	0
Slovenia	?	111	40	59	64	?	68	68	73
Spain	9.595	9.960	9.878	9.472	9.472	8.802	7.005	7.005	6.429
Hungary	1.416	952	646	29	?	?	14	0	0
Total	29.213	36.051	27.113	31.904	17.895	16.488	21.578	20.895	17.638
EU*	-	46.543	38.384	35.172	30.453	30.277	23.855	20.895	17.638

Table 2: Number of rabbits used in pyrogen testing in some European countries. Any countries not listed here either did not list the pyrogen test separately in their statistics, or the information could not be obtained for other reasons (18,19,20)

* The first EU statistics according to the new method of counting based on the EU Animal Testing Directive 2010/63/EU were published in 2019, and cover the years 2015-2017. During this period, between 46,543 and 35,172 rabbits were used for pyrogen testing in the EU. The above list of countries therefore contains large gaps. France in particular should be mentioned here, where pyrogen tests are not listed separately, contrary to EU law.

Rabbit pyrogen tests in Germany

Pyrogen tests on rabbits are only carried out in 4 federal states of Germany. The vast majority (82% in 2018) is attributable to Bavaria.

Table 3: Number of rabbits used for pyrogen testing by federal states of Germany. No such tests have been registered in federal states that are not listed (21,22). We do not have any figures from the individual federal states for 2019 and 2020.

In Bavaria, the company Labor LS in Bad Bocklet, north of Bad Kissingen, is the main reason for the high numbers of animals used for pyrogen testing. When asked in April of 2021, the company stated that it would no longer offer the rabbit test, but that some pyrogen tests would still be carried out until the "changes have been approved by the supervisory authorities". The positive development in this laboratory is likely the reason for reduced animal numbers in 2020.

In Schleswig-Holstein, the contract laboratory LPT (later named Provivo Bioscience) had a facility at Gut Löhndorf in the district of Plön. After scandalous conditions were revealed by an undercover investigation, the LPT facility in Neu-Wulmstorf-Mienenbüttel in the district of Harburg, Lower Saxony, was closed by the authorities. The facility at Gut Löhndorf was closed in 2022.

The global "laboratory animal" breeding company Charles River also offers rabbit pyrogen testing on its website. However, these are probably not carried out at the German site in Sulzfeld, since according to the BMEL no rabbit pyrogen tests are carried out in Baden-Württemberg.

Conclusions and demands

An animal-free test using human blood - which offers not only ethical, but also scientific advantages over the traditional rabbit pyrogen test - was developed more than 30 years ago, validated 19 years ago, and introduced into European legislation 14 years ago. Nevertheless, thousands of rabbits still suffer and die for pyrogen testing. In 2022, over 17,000 rabbits were used in pyrogen tests in Europe and Norway, with more than 1,000 of them in Germany.

The removal of the rabbit test from the European Pharmacopoeia is an important step and a success of our campaign. However, this should not obscure the fact that animals continue to suffer and die in pyrogen testing due to the widespread use of the LAL test. In our campaign, "Agony in the blood factory - Stop the suffering of horseshoe crabs!", we therefore demand that the LAL test, for which the rFC test is a scientifically recognized replacement (23), is also removed from the European Pharmacopoeia. Pyrogen testing as a whole must be converted to exclusively animal-free methods.

Dr.  med. vet. Corina Gericke
30/07/2024

References

1. Maloney T et al. Saving the horseshoe crab: A synthetic alternative to horseshoe crab blood for endotoxin detection. PLOS Biology2018; 16(10): e2006607
2. Hartung T. The human whole blood pyrogen test – lessons learned in twenty years. ALTEX 2015; 32(2); 79-100
3. Horseshoe crab blood to remain big pharma’s standard as industry group rejects substitute. The Guardian, 31.05.2020
4. Years of advocacy pay off with policy change that improves science and protects horseshoe crabs, Physicians Committee for Responsible Medicine, 26.07.2024 
5. Hartung T et al. Entwicklung und Evaluierung eines Pyrogentests mit menschlichem Blut. ALTEX 1998; Suppl.: 9-10
6. Barroso V et al. EURL EVCAM Recommendation on Non-Animal Derived Antibodies. 2020 (accessed on 7.7.2020)
7. FCS-free – cell culture media without fetal calf serum. Doctors Against Animal Experiments, 09.03.2023 (accessed on 7.7.2020)
8. Fennrich et al. New applications of the human whole blood pyrogen assay (PyroCheck). ALTEX 1999; 16(3): 146-149
9. The human(e) alternative in pyrogen testing – PyroDetect system. Broschüre von Merck, 2018, S. 2
10. Wikipedia. Pyrogen. https://de.wikipedia.org/wiki/Pyrogen (accessed on 7.7.2020)
11. Hoffmann S et al. International validation of novel pyrogen tests based on human monocytoid cells. J Immunol 2005; 298(1-2): 161-173
12. Wunderlich CM. Adaption und Etablierung des Monozyten-Aktivierungstests (MAT) für die Durchführung mit bovinem Vollblut. Veterinärmedizinische Dissertation TiHo 2014; S. 22-23
13. EDQM. Europäisches Arzneibuch 2017, Ausgabe 9, Monographie 2.6.30
14. Finally: end of rabbit pyrogen test in sight, press release, Doctors Against Animal Experiments, 16.07.2024 (accessed on 14.08.2024) 
15. Gute Nachrichten für Versuchskaninchen. Deutschlandfunk, Sendung „Forschung Aktuell“, Januar 2000
16. Universitätsklinikum Tübingen. Ein Erfolg für die Kaninchen. Pressemitteilung vom 6.5.2010
17. Bundesministerium für Ernährung und Landwirtschaft. Tierversuchsstatistik 2014, 2015, 2016
18. European Commission: Animals used for Scientific Purposes (accessed on 7.7.2020)
19. ALURES – Animal Use Reporting - EU System 
20. Summary Report on the statistics on the use of animals for scientific purposes in the Member States of the European Union and Norway in 2020, 31.03.2023
21. Antwort des Bundesministeriums für Ernährung und Landwirtschaft (BMEL) auf die Kleine Anfrage mehrere Abgeordneter der Bundestagsfraktion DIE LINKE, 19.06.2020, Drucksache 19/19838
22. BfR: Zahlen zu den im Jahr 2020 verwendeten Versuchstieren, Bericht vom 16. Dezember 2021
23. LAL-Test - Horseshoe crabs suffer despite existing non-animal test methods, 04.04.2023