Since 1986, the European Pharmacopoeia (Ph. Eur.) Commission and its experts have worked continuously to ensure that the 3Rs principles of Replacement, Reduction and Refinement of animal testing are applied when revising or drafting the texts published in the Ph. Eur. As a result of the Ph. Eur. Commission’s efforts, no Ph. Eur. monographs on medicinal products derived from human blood and plasma describe any tests requiring the use of animals. As far as human and veterinary vaccines are concerned, an in vitro method has in many cases been introduced as an alternative to or replacement for in vivo testing.

Various strategies are also used to promote reduction and refinement with regard to in vivo assays, for example serology assays or single dilution assays for diphtheria, tetanus, acellular pertussis and rabies (veterinary/human) vaccines.

The major milestones that have marked the past several years are described below.

These decisions are fully in line with the EDQM’s mission to promote and protect human and animal health, and with its commitment to the 3Rs principles.

Share your proposals for replacement of in vivo methods

The Ph. Eur. Commission welcomes any data supporting the replacement of the remaining in vivo methods and proposals for replacement methods, both of which can be sent to the Commission Secretariat via the Helpdesk.

History of Replacement, Reduction and Refinement of animal testing (3Rs) at the EPC since 2012

2024

At its 179th Session in June 2024, the EPC bid “adieu” to the rabbit pyrogen test (RPT) in the Ph. Eur. monographs. The product of a broad exercise aiming to completely remove the RPT from the Ph. Eur., the EPC adopted 57 revised texts from which the RPT had been deleted, together with a new general chapter on Pyrogenicity (5.1.13), thus marking the end of the RPT era in the Ph. Eur. A list of these texts can be found here. This is a major achievement for animal welfare and for the advancement of modern in vitro approaches for pyrogenicity testing. As a result, the use of the RPT will no longer be required in any Ph. Eur. text and it will be the responsibility of medicine developers to select a suitable in vitro test (e.g. the monocyte-activation test) to control the pyrogenicity of their product, based on a risk assessment as described in the new general chapter.

At its 178th Session in March 2024, the EPC adopted two new texts: a general monograph on Gene therapy medicinal products for human use (3186) and a general chapter on Additional information on gene therapy medicinal products for human use (5.34). These texts include the option to test for bacterial endotoxins using recombinant factor C as an alternative to the limulus amoebocyte lysate test, which uses blood from horseshoe crabs. Together, these texts serve as a replacement for general chapter 5.14. Gene transfer medicinal products for human use, which was suppressed from the Ph. Eur. with the publication of Supplement 11.7.

2023

At its 177th Session in November 2023, the EPC decided to engage on a path that should ultimately lead to the suppression of the general chapters on Histamine (2.6.10) and Depressor substances (2.6.11) from the Ph. Eur. Both chapters describe tests that measure the effect of histamine and histamine-like substances, using guinea-pigs and cats respectively. Histamine (2.6.10) is referenced in the Production section of four monographs, while Depressor substances (2.6.11) is a standalone chapter. In addition, ten other monographs have requirements – manufacturing processes to eliminate or minimise substances lowering blood pressure – in their Production section that are remainders of these tests, although the chapters are not explicitly referenced.

To this end, the EPC approved a strategy for the suppression of these two chapters from the Ph. Eur. along with the corresponding statements in the Production section of the ten monographs mentioned previously. This strategy was set out in an article published in March 2024 in Pharmeuropa Bio & Scientific Notes. It is expected that this will be completed by the publication of the 12th Edition of the Ph. Eur.

At its 176th Session in June 2023, the EPC adopted the new general chapter Monocyte-activation test (MAT) for vaccines containing inherently pyrogenic components (2.6.40), which covers the use of the MAT to monitor the production consistency of inherently pyrogenic vaccines and will be of great help to stakeholders wishing to implement the MAT for such vaccines.  

At the same session, the EPC also adopted an updated version of the general chapter, Monocyte-activation test (2.6.30), that has been revised to streamline the test and bring it in line with current practices. This technical revision was part of the broader exercise to remove the rabbit pyrogen test (described in general chapter 2.6.8. Pyrogens) from the Ph. Eur. and the very first step towards it (see Strategy to completely replace the rabbit pyrogen test in the Ph. Eur. from 2022 below).

Several of the texts adopted at the 175th Session of the Ph. Eur. Commission (EPC) in March 2023 were revised in keeping with the 3Rs principles.

Revision of veterinary vaccine monographs

The following monographs were revised to delete the in vivo residual live virus test at final product stage when there is no reversion to virulence, relying instead on the in-process test performed, preferably using cell cultures. All remaining residual live virus tests are performed in vitro.

• Equine influenza vaccine (inactivated) (0249);
• Aujeszky’s disease vaccine (inactivated) for pigs (0744);
• Newcastle disease vaccine (inactivated) (0870);
• Avian infectious bronchitis vaccine (inactivated) (0959);
• Avian infectious bursal disease vaccine (inactivated) (0960);
• Porcine influenza vaccine (inactivated) (0963);
• Egg drop syndrome ’76 vaccine (inactivated) (1202);
• Avian paramyxovirus 3 vaccine (inactivated) for turkeys (1392); and
• Rabbit haemorrhagic disease vaccine (inactivated) (2325).

The following monographs were revised to reduce the number of controls for in vivo routine batch potency tests:

• Newcastle disease vaccine (inactivated) (0870);
• Avian infectious bronchitis vaccine (inactivated) (0959);
• Avian infectious bursal disease vaccine (inactivated) (0960);
• Egg drop syndrome ’76 vaccine (inactivated) (1202);
• Mycoplasma gallisepticum vaccine (inactivated) (1942);
• Fowl cholera vaccine (inactivated) (1945);
• Salmonella Enteritidis vaccine (inactivated) for chickens (1947); and
• Salmonella Typhimurium vaccine (inactivated) for chickens (2361).

The following monographs were revised to add humane endpoints (immunogenicity):

• Avian infectious encephalomyelitis vaccine (live) (0588);
• Fowl cholera vaccine (inactivated) (1945); and
• Rabbit haemorrhagic disease vaccine (inactivated) (2325).

2022

Strategy to completely replace the rabbit pyrogen test in the Ph. Eur.

In June 2021, the EPC took the decision to completely replace the rabbit pyrogen test (RPT) in the Ph. Eur. within approximately five years. The pyrogenicity strategy paper, published in Pharmeuropa Technical Information in September 2022, provides more information on how this will be tackled.

Revision of the rabies vaccine monograph to ban the NIH test for routine testing

At its 172nd Session in March 2022, the EPC adopted the revised monograph on Rabies vaccine (inactivated) for veterinary use (0451). The revised text reserves the NIH test for the development and qualification of standards/reference preparations only and it can no longer be used as a routine batch potency test.

Revision of veterinary vaccine monographs to delete the in vivo residual live virus test at final product stage

The monograph on Rabies vaccine (inactivated) for veterinary use (0451) was the first monograph revised to delete the in vivo residual live virus test at final product stage, relying instead on the in-process test performed, preferably using cell cultures, since there is no reversion to virulence. Should a residual live virus test still need to be conducted, this must be performed in vitro.

Revision of the monograph on Clostridium botulinum vaccine for veterinary use (0360)

Following the adoption of the revised monograph on Botulinum toxin type A for injection (2113), a section on the application of humane endpoints in the LD50 assay was also introduced in the vaccine monograph to reduce animal suffering. This provided an opportunity to specifically mention that cell-based assays were good candidates for a validated in vitro procedure as a possible alternative to the routine batch potency test.

Revision of the monograph on Radiopharmaceutical preparations (0125)

The section on the performance of the physiological distribution test was removed.

2021

Several of the texts adopted at the 170th Session of the Ph. Eur. Commission in June 2021 had been revised in keeping with the 3Rs principles.

Review of toxicity testing requirements for diphtheria vaccines

These monographs were revised to delete the test for specific toxicity (test in guinea pigs) that is performed on the final lot as part of the validation of the production process.

The decision to remove the test was taken following a thorough re-assessment of toxicity testing requirements for these vaccines, and follows recent similar exercises for other toxoid vaccines.

Review of veterinary vaccine monographs

Three monographs on clostridial vaccines for veterinary use were also revised, submitted to the Commission and adopted:

• Clostridium septicum vaccine for veterinary use (0364);
• Clostridium perfringens vaccine for veterinary use (0363); and
• Clostridium novyi (type B) vaccine for veterinary use (0362).

The monographs had been modified:

• to replace the use of mice as indicator of toxicity with a suitable in vitro method – the minimum lethal dose (MLD) and total combining power (TCP) assays – for in-process quality control tests which traditionally use many animals;
• to favour the use of in vitro methods for the routine batch potency test (several in vitro methods are available);
• to allow manufacturers to switch from an animal test for vaccine identification to an in vitro test;
• to delete the test for residual toxicity on the final product because no reversion to toxicity has ever been reported and this is in line with the general monograph Vaccines for veterinary use (0062); the conditions under which the residual toxicity test may be omitted on the final bulk and the final batch are stated.

Review of a radiopharmaceutical monograph

The monograph on Technetium (99mTc) macrosalb injection (0296) was revised to transfer the (in vivo) physiological distribution test from the Tests section to the Production section, since the (in vitro) particle size test is also considered to be an indicator of production consistency and thus of physiological distribution. With the modified text adopted by the Commission, the physiological distribution test is now no longer to be performed routinely, resulting in a considerable reduction in the number of animals used in testing.

Revision of Botulinum toxin type A monograph

The commitment to the 3Rs principles continued with a revised version of the Botulinum toxin type A monograph, also adopted at the session. The modified text provides up-to-date information on alternative in vitro methods for potency and now includes a section on the application of humane end-points in the LD50 assay to reduce animal suffering.

2020

Review of veterinary vaccine monographs to promote the 3R principles

At its 167th Session in June 2020, the Ph. Eur. Commission adopted revised versions of three monographs on vaccines for veterinary use which had been modified to promote the 3R principles:

• Canine parvovirosis vaccine (live) (0964) to reduce to a minimum the number of dogs used in the safety tests;
• Equine herpesvirus vaccine (inactivated) (1613) to stress that an in vitro alternative method should be preferred for the routine batch potency test;
• and Avian infectious bronchitis vaccine (live) (0442) to encourage the development of alternative methods, such as Polymerase Chain Reaction (PCR), for the recovery of the virus from tracheal swabs.

2019

Review of toxicity testing requirements for tetanus vaccines

At its 165th Session in November 2019, the Ph. Eur. Commission adopted revised versions of its monographs on tetanus vaccines from which three animal tests have been suppressed. The Test for specific toxicity and the Test for residual toxicity – performed on the final lot for human and veterinary vaccines, respectively – and the Test for irreversibility of tetanus toxoid carried out on the bulk purified toxoid (all tests in guinea pigs) have been deleted.

The decision to remove the tests was taken following a re-assessment of toxicity testing requirements for these vaccines.

2018

Replacement of the Histamine sensitisation test (HIST) for residual pertussis toxin testing

At its 162nd Session in November 2018, the European Pharmacopoeia Commission adopted the replacement of the Histamine sensitisation test (HIST) in mice with a standardised CHO cell clustering assay for residual pertussis toxin testing, in general chapter 2.6.33 Residual pertussis toxin and in ten individual monographs on acellular pertussis vaccines.

As part of the same revision exercise, the test for irreversibility of pertussis toxoid and the requirement to test the final lot for residual pertussis toxin have been removed from individual monographs on acellular pertussis vaccines.

2017

Suppression of the Test for Abnormal Toxicity from the European Pharmacopoeia

In November 2017 at its 159th Session, the Ph. Eur. Commission adopted 49 monographs, 36 of which were on vaccines for human use, which had been revised to remove the reference to the test for abnormal toxicity. General chapter Abnormal toxicity (2.6.9) will therefore no longer be cited in any monograph and will also be deleted. As a result, the test for abnormal toxicity will be completely suppressed from the Ph. Eur. as of Supplement 9.6 (implementation date 1 January 2019).

2016

New chapter in Ph. Eur: Substitution of in vivo method(s) by in vitro method(s) for the quality control of vaccines (5.2.14)

Again encouraging the transition from in vivo to in vitro methods, the Ph. Eur. groups of experts on vaccines have developed a new general chapter entitled Substitution of in vivo method(s) by in vitro method(s) for the quality control of vaccines (5.2.14). The text was adopted at the 156th Session and provides guidelines on validation of substitute methods where a direct head-to-head comparison with an existing in vivo method is not possible

Revised Monocyte-activation test (2.6.30): an alternative to pyrogen testing

Extensively overhauled with input from industry representatives, academics, regulatory authorities and Official Medicines Control Laboratories, the revised version of the general chapter Monocyte-activation test (2.6.30) was adopted by the Commission at its 155th Session. Revision of this chapter (first published in the Ph. Eur. in 2010) was intended to promote use of the test by stakeholders particularly as a replacement for the pyrogens test which is conducted on laboratory animals.

2015

Revision of testing strategy for extraneous agents

In November 2015, the Ph. Eur. Commission's attention turned to the current requirements for extraneous agent testing. The aim was to rationalise these requirements without in any way compromising safety. To this end, a reference to the new general chapter 5.2.13, which lays down the requirements for healthy chicken flocks used for the production of inactivated vaccines for veterinary use, was introduced into 8 veterinary vaccine monographs. This rendered the test for specified extraneous agents previously performed on each final product obsolete. As part of the same initiative, the revised texts on Tests for extraneous agents in viral vaccines for human use (2.6.16) and Cell substrates for the production of vaccines for human use (5.2.3) were adopted at the 156th Session of the Ph. Eur. Commission. The changes include deletion of the tests on adult mice and guinea pigs and restricting the use of the test on suckling mice and control eggs to cases in which the tests provide risk mitigation.

Revision of general monograph Vaccines for veterinary use (0062): reduction of animal testing for veterinary vaccines

At its 153rd Session in November 2015, the Ph. Eur. Commission also adopted the revised general monograph Vaccines for veterinary use (0062) together with around 40 veterinary vaccine monographs in order to allow manufacturers to switch from an animal test for vaccine identification to an in vitro test, when appropriate. Not only was the reference to the antibody induction test for identification of all inactivated vaccines deleted from these monographs, but inactivation testing of all inactivated veterinary vaccines was revised to allow manufacturers to omit the second inactivation test.

Provision for additional systems for monitoring of production consistency and in vitro alternatives

The Ph. Eur.'s commitment to the 3Rs continued with the inclusion of provisions for additional systems to monitor the consistency of production, firstly in the General Notices (146th Ph. Eur. Commission Session), and then in the general monograph Vaccines for veterinary use (0062) and in three individual veterinary vaccine monographs, Canine leptospirosis vaccine (inactivated) (0447), Bovine leptospirosis vaccine (inactivated) (1939) and Infectious bovine rhinotracheitis vaccine (inactivated) (2674) (153rd Session). This was done in conjunction with implementation of the revised Directive 2010/63/EU on the protection of animals used for scientific purposes, and both recognised manufacturers’ efforts to develop new test methods and encouraged them to develop in vitro alternatives. It was also a reflection of the growing control over vaccine production in general.

2014

ELISA alternative introduced in Assay of hepatitis A vaccine (2.7.14

In June 2014 at its 149th Session, the Ph. Eur. Commission  introduced a validated ELISA method for determination of antigen content in Assay of hepatitis A vaccine (2.7.14) and thus providing  an alternative to the serology assay in mice.

2012

Reduction of unnecessary use of animals in pertussis vaccines

In June 2012, following adoption of the general chapter Residual pertussis toxin and irreversibility of pertussis toxoid (2.6.33), 9 revised monographs on human vaccines to reference this new general chapter were also adopted. The protocol described in the chapter, which is based on the outcome of a study from the Biological Standardisation Programme (BSP), facilitated standardisation of the method and therefore reduces the unnecessary use of animals.

Adoption of 80 veterinary vaccine monographs reducing the number of animals used in testing

During its 142nd Session (April 2012) and several years into an extensive and ambitious programme of works launched as part of harmonisation with VICH Guidelines 41 and 44, the Ph. Eur. Commission adopted some 80 veterinary vaccine monographs in addition to the general monograph on Vaccines for veterinary use and two general chapters (5.2.6 and 5.2.9), revised for greater consistency with European regulations.

The work programme also included monographs on vaccines for other species that were outside the scope of the VICH Guidelines, targeting harmonisation of the tests for increased virulence performed during vaccine development as a means of substantially reducing the number of animals used for testing.

During the same session, the Ph. Eur. Commission also voted to delete the target animal batch safety test (TABST) waiver, an option available for established vaccines since 2004, from all Ph. Eur. veterinary vaccines. This became effective on 1 April 2013.