three rs potential in the development and quality control of ... · nisations (e.g. who, o.i.e.)...

ALTEX 18, Suppl. /01 13

SummaryImmunobiologicals (vaccines, immunoglobulins and –sera) areconsidered to be the most cost-effective tools in the preventionof infectious diseases. Their importance will further increasedue to various eradication programmes of the WHO and EUand the emergence of new infectious diseases or the re-emer-gence of diseases as diphtheria and tuberculosis. The produc-tion and quality control of immunobiologicals are regulated bymonographs and guidelines, which are issued by internationalor national Pharmacopoeias (e.g. Ph. Eur.), international orga-nisations (e.g. WHO, O.I.E.) and international regulatory bodies (e.g. EMEA). Their purpose is to assure the quality of theproduct, i.e. its safety and potency. It is estimated that 10 millions of laboratory animals are world-wide used for theproduction and quality control of immunobiologicals, of which

80% are needed for the safety and potency testing of the finished product (batch control).In recent decades, the use of Three Rs principles has been re-cognised by the above mentioned organisations and various national competent authorities and had been incorporated intogeneral monographs and guidelines. Several tests with ques-tionable relevance have been deleted from Ph. Eur. monographs

(e.g. abnormal toxicity test, extraneous agents testing of viralvaccines for carnivores) or are now carried out during produc-tion. Reduction of the number of animals used could be achievedby introducing single-dilution tests. A large number of immuno-chemical tests have been developed, which could completely orpartly replace the use of animals for potency testing, however,only a few have been validated so far (e.g. ToBI and ELISA forpotency testing of human and veterinary tetanus vaccine; ELI-SA for potency testing of erysipelas vaccine). Regulatory acceptance of validated alternative methods is still a criticalstep. In particular, the period between successful validation andthe implementation appears to be far too long. Reasons for thiscould be the slow process of multinational agreement to revisepharmacopoeial monographs and guidelines, and the time-consuming and expensive production of sufficient reference material (antigen, sera etc) for the new test systems.The shift in the quality control concept from reliance on finalbatch testing to the concept of consistency of production offersthe opportunity to reduce the numbers of animals being usedand promote the use of alternative methods. Emphasis is put ona combination of in vitro tests, which could make it possible tomonitor batch-to-batch consistency. This new concept of quality

Three Rs Potential in the Development andQuality Control of Immunobiologicals

Marlies Halderon behalf of AGAATI, NL-Utrecht

Content1 Introduction (p. 15)2 Concepts of quality and safety control of Immuno-

biologicals (p. 15)2.1 History of quality control (p. 15)2.2 Standards and reference preparations (p. 15)2.3 Uniqueness of each vaccine batch versus consistency

of production (p. 16)3 Regulation and acceptance (p. 16)3.1 National and international control authorities (p. 16)3.2 National and international pharmacopoeias (p. 16)3.3 European guidelines and Council Directives (p. 17)3.4 International organisations (p. 17)3.4.1 WHO (p. 17)3.4.2 O.I.E (p. 17)3.5 International harmonisation (p. 18)3.6 Legal and ethical background to the Three Rs (p. 18)4 Development and validation of alternatives (p. 18)4.1 General aspects (p. 18)4.1.1 Who develops and validates? (p. 18)4.1.2 Who is sponsoring? (p. 18)4.2 Special aspects (p. 18)

4.2.1 Vaccines and immunosera for human use (p. 19)4.2.1.1 Bacterial vaccines (p. 19)4.2.1.2 Viral vaccines (p. 21)4.2.1.3 Immunosera/Antitoxins (p. 22)4.2.1.4 Tuberculins for human use (p. 23)4.2.1.5 Test for pyrogens (p. 23)4.2.2 Vaccine and immunosera for veterinary use (p. 24)4.2.2.1 The target animal safety test (p. 24)4.2.2.2 Bacterial vaccines (p. 24)4.2.2.3 Viral vaccines (p. 26)4.2.2.4 Immunosera and Antitoxins (p. 29)4.2.3 Humane endpoints (p. 29)5 Progress and criticism (p. 30)5.1 Progress (p. 30)5.1.1 General aspects (p. 30)5.1.2 Specific aspects (p. 30)5.2 Criticism (p. 31)6 Future aspects (p. 32)6.1 Consistency of production (p. 32)6.2 Antigen quantitation versus serological methods (p. 32)6.3 Novel vaccine production technologies and new

vaccines (p. 32)References (p. 33)

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ALTEX 18, Suppl. /0114

control is already in place for the new well-defined vaccines. In most cases, non-animal methods are used for monitoringconsistency at critical steps in the production and testing of avaccine. Whether the concept of consistency of productioncould be also applied to the conventional, less-defined products,should be investigated. Only little progress has been achieved with regard to interna-tional harmonisation. Most of the manufacturers produce for theworld market, so harmonisation of the requirements or mutualrecognition of tests would help to reduce the use of animals.There is agreement that for the time being animals will still beneeded for the development of vaccines in order to gain bestknowledge on the disease, the pathogen and the specific immuneresponse, including: pathogenesis, identification of the pro-tective antigens, the way the antigen is processed, the dynamicsof the immune response, the induction of memory, and the selection of the best adjuvant. With regard to routine batch release of conventional products, a number of Three Rs ap-proaches are already available and should further be developedand validated. Whereas routine batch release of new productsshould be based on in vitro methods already established duringtheir development.

Zusammenfassung: Das 3R Potenzial bei der Entwicklung undQualitätskontrolle von ImmunobiologikaImmunobiologika (Impfstoffe, Immunglobuline und –seren) sinddie kostengünstigste Möglichkeit, Infektionskrankheiten vorzu-beugen. Ihre Bedeutung wird noch weiter zunehmen, vor allemim Hinblick auf die verschiedenen Bekämpfungsprogramme derWHO und EU, dem Auftauchen von neuen Infektionskrankhei-ten oder der Zunahme von Krankheiten wie Diphtherie und Tuberkulose. Die Produktion und Qualitätskontrolle von Immunobiologikawerden durch Monographien und Richtlinien bestimmt, die in-ternationale oder nationale Arzneibücher (z.B. Ph. Eur.), inter-nationale Organisationen (z.B. WHO, O.I.E.) sowie interna-tionale Behörden (z.B. EMEA) erstellen und herausgeben. Siedienen der Qualitätssicherung der Produkte, d.h. der Über-prüfung der Unbedenklichkeit und Wirksamkeit. Man schätzt,dass pro Jahr 10 Millionen Tiere für die Produktion und Qualitätskontrolle von Immunobiologika verbraucht werden,davon 80% für die Überprüfung der Unbedenklichkeit undWirksamkeit des Endprodukts (Chargenprüfung).In den vergangenen Jahrzehnten haben die oben genannten Or-ganisationen und verschiedene nationale Behörden die Bedeu-tung der 3R erkannt und ihre Prinzipien in die allgemeinen Monographien und Richtlinien aufgenommen. Einige Tests mitfraglicher Relevanz wurden aus den Ph. Eur. Monographien gestrichen (z.B. Anomale Toxizität, Fremdvirusausschluss bei

viralen Lebendimpfstoffen für Carnivoren) oder werden jetztwährend der Produktion durchgeführt. Die Einführung von Ein-Punkt Tests führte ebenfalls zur Reduzierung der Tierzahlen. Eine Reihe von Alternativmethoden wurde bereits entwickelt,die die Wirksamkeitsprüfung am Tier ganz oder teilweise er-setzen könnte, aber nur wenige wurden bis jetzt validiert (z.B. der ToBI-Test und ein ELISA für die Wirksamkeitsprüfungvon Tetanusimpfstoffen für Mensch und Tier; ein ELISA für dieWirksamkeitsprüfung von Rotlaufimpfstoffen). Die behördlicheAkzeptanz von validierten Alternativmethoden erweist sich im-mer noch als kritischer Schritt. So erscheint die Zeitspanne vonder erfolgreich abgeschlossenen Validierung bis zum Vollzug ingesetzliche Vorschriften als viel zu lang. Gründe hierfür mögender langsame multinationale Einigungsprozess zur Revidierungvon Monographien und Richtlinien sowie die zeitraubende undkostenintensive Herstellung von Referenzmaterialien (Antigene,Seren, usw.) für die neuen Methoden sein.Die Änderung des Konzepts der Qualitätskontrolle von Im-munobiologika von der reinen Endproduktkontrolle hin zurKontrolle der Produktionskonsistenz eröffnet die Möglichkeit,die Tierzahlen weiter zu reduzieren und den Einsatz von Alter-nativmethoden zu fördern. Hierbei wird auf eine Kombinationvon in vitro Tests gesetzt, die es ermöglichen sollen, die Konsi-stenz zwischen Chargen zu überprüfen. Das neue Konzept derQualitätskontrolle wird bereits bei neuen, gut definierten Pro-dukten eingesetzt, und in den meisten Fällen werden tier-versuchsfreie Methoden zur Überwachung der Konsistenz besonders kritischer Schritte in der Produktion und Über-prüfung von Impfstoffen angewendet. Inwiefern sich diesesKonzept auch für konventionelle, weniger gut definierte Produkte eignet, sollte untersucht werden.Nur wenig Erfolg wurde bei der internationalen Harmonisie-rung erzielt. Nachdem jedoch die meisten Hersteller für denWeltmarkt produzieren, würde die Harmonisierung von Vor-schriften oder die gegenseitige Anerkennung von Tests dazu beitragen, die Tierzahlen zu reduzieren.Auch in Zukunft werden Tiere für die Entwicklung von Impf-stoffen gebraucht, vor allem um mehr über eine Krankheit zuerfahren, das pathogene Agens und die spezifische Immun-antwort zu erforschen, einschliesslich der Pathogenese, des protektiven Antigens, der Aufbereitung des Antigens, der Dyna-mik der Immunantwort, der Induktion des Immungedächtnissesund der Auswahl des besten Adjuvants.Was die Chargenprüfung der konventionellen Immunobiologikaanbelangt, sind bereits eine Reihe von Alternativmethoden vor-handen, die weiter entwickelt und validiert werden sollten. Zur Chargenprüfung von neuen Immunobiologika sollten be-reits während der Produktentwicklung in vitro Methoden ent-wickelt und etabliert werden.

Keywords: vaccines, immunosera, quality control, Three Rs methods, regulatory acceptance

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1 Introduction

It is estimated that more than 10 millionanimals a year are used worldwide forthe development, production and qualitycontrol of immunobiologicals. Figuresfrom the Netherlands show that about20% of the animals are used for the de-velopment of new or improved productsand about 80% in the routine qualitycontrol of batches of immunobiologicals(Hendriksen, 2000). The use of animalsfor the actual production of vaccines isrestricted to very few cases such as suck-ling mice rabies vaccines in developingcountries, monkey kidney cells for somepolio virus propagation, rabbits for rab-bit viral haemorrhagic disease vaccine,chickens for avian coccidiosis vaccineand cattle for lungworm disease.

Production and quality control is reg-ulated by monographs or guidelines,which are issued by international or na-tional Pharmacopoeias (e.g. EuropeanPharmacopoeia [Ph. Eur.]), interna-tional organisations (e.g. World HealthOrganization [WHO], Office Interna-tional des Epizooties [O.I.E.]) and inter-national regulatory bodies (e.g. EuropeanMedicines Evaluation Agency[EMEA]). These specify tests, whichcan be divided into two categories: safe-ty and efficacy tests during licensing,and safety and potency tests for batchquality control. Efficacy and potencytests ensure that the product inducesprotective immunity after administra-tion whilst safety tests ensure that theproduct does not induce abnormal ad-verse reactions.

Some batch safety tests (target animaltest for veterinary vaccines, abnormaltoxicity test or the extraneous agenttesting of poultry vaccines) are of ques-tionable relevance and other safety tests(e.g. neurovirulence testing of live poliovirus vaccines in monkeys) raisevery serious ethical concerns.

With regard to batch potency testing,most live vaccines are tested with invitro methods and do not require ani-mals. However, the testing of inac-tivated vaccines often requires largenumbers of animals: e.g. more than 100animals per batch are required for thepotency testing of diphtheria, tetanus,whole pertussis, erysipelas and rabiesvaccines. In addition to the large num-

ber of animals used, the potency testingof inactivated vaccines is often based ona vaccination and challenge test (e.g.clostridial, erysipelas, leptospiral vacci-nes) which involves considerable painand suffering for the animals since, onaverage, 50% of the animals will suc-cumb to the challenge and may die fromthe effects of toxicity or infection. In re-cent years, national control authorities,industry and regulatory bodies have made great efforts to develop, stand-ardise and validate alternatives to thesevaccination-challenge tests, and also torefine these tests and promote the use ofhumane endpoints.

There are two main approaches forthe replacement of challenge tests: a)antigen quantitation which completelyreplaces the animal test (e.g. ELISAtests for rabies, hepatitis B, leptospiral,Newcastle disease vaccines); and b) thereplacement of the challenge procedure

with an immunological technique whichallows the measurement of the appro-priate response to vaccination. In manycases this is a simple serological modelin which antibodies induced by the vac-cine in the animal are quantified usingimmunochemical techniques such asneutralisation tests in cell cultures (e.g.Diphtheria toxoid, Clostridium (C.) sep-ticum, C. novyi and C. perfringens vac-cines), ELISA procedures (e.g. pertus-sis, botulinum, tetanus, erysipelas,leptospiral, C. septicum, C. novyi and C.perfringens vaccines), modified ELISAmethods (ToBI test for tetanus vac-cines), or a host of other techniques. Insome cases, in vitro methods for theevaluation of cell-mediated responsesmay also be applied.

2 Concepts of quality and safetycontrol of immunobiologicals

2.1 History of quality controlInitially, when the first immunobiologi-cals such as Jenner’s smallpox vaccine,Pasteur’s vaccines or erysipelas antise-rum were invented more than a centuryago, they were not submitted to qualitycontrol. As a result the vaccines some-times contained an insufficiently inacti-vated virulent strain, were contaminatedwith other pathogens, or were of insuffi-cient potency. It soon became apparentthat large differences in quality betweenbatches of the same vaccine could occurand, in consequence, the first gov-ernmental regulations for batch qualitycontrol were introduced.

Historically, the way regulatory re-quirements for immunobiologicals havedeveloped, has been somewhat disaster-led (Tab. 1).

Without animal experiments the qualityof vaccines would not have been as goodas it is today, and their successful use onsuch a large scale would never have been possible.

2.2 Standards and referencepreparationsFor traditional vaccines such as DTP, rabies or erysipelas, the potency is ex-pressed as a relative value. This is achieved by comparing the potency ofthe test vaccine to a reference vaccine.The WHO, the Ph. Eur. or national control authorities provide such standardor reference preparations. Comparison ofthe test and the reference vaccine is based on multi-dilution tests, which arecarried out for licensing and as batch

Tab. 1: Immunobiological-related accidents

Year Cases DeathsToxin in vaccine Diphtheria Dallas 1929 96 10

Concord 1924 21 n.d.Bridgewater 1924 22 n.d.Baden 1924 28 7Kyoto 1948 600 68

Incomplete inactivation Polio Cutter incident 1955 260 5Contamination Tetanus in with toxin D-antiserum St. Louis 1901 20 14Wrong culture BCG Lübeck 1930 135 72

n.d. = no data availableSource: Hendriksen, 1996

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potency test. This approach uses largenumbers of animals; however, this hasbeen recognised and some of the mono-graphs now allow the use of single-dilution test (see Progress and Criticism).

It is also evident that with the develop-ment of new vaccines, the control au-thorities and manufacturers have to deal more and more with product-specific aspects. The use of one single universalworldwide standard, against which it isvalid to test each product against, mightno longer be possible. For newer vacci-nes, for example Haemophilus influenzaeb, recombinant Hepatitis B vaccine, acel-lular pertussis vaccine, and certainly thenew vaccine combinations the tendencyis for manufacturers to use clinical stand-ards to test consistency of production, i.e.a clinical standard is a batch of a vaccine, which has been used in clinicaltrials and shown to be efficacious (Dob-belaer, 2000).

2.3 Uniqueness of each vaccine batchversus consistency of productionBiologicals are derived from living organ-isms in a batch-wise procedure, whichmeans that their characteristics can varyfrom batch to batch. Therefore, eachbatch produced in one production run isconsidered as unique and undergoesstrict quality control with emphasis ontesting the finished product testing.

In recent years, it had been emphasisedthat consistency of production is essen-tial and quality control should monitor crit-ical steps during production and controlof a biological rather than rely on controlof the final batch (Griffiths, 1996). Thisconcept is mainly applied to new, well-defined biologicals; however, it could also be introduced into the production ofconventional, less-defined products(Hendriksen et al., 1998; Lucken, 1999;Leenaars et al., 2001). Consistency ofproduction means that each batch of aproduct is of the same quality and is within the same specifications as a batch,which has been shown to be safe and ef-ficacious in human trials or in the targetanimal species. Generally, alternativemethods such as physiochemical or im-munochemical methods are better able tomonitor consistency than in vivo (e.g.vaccination-challenge procedures) tests.This is because of the parameters meas-ured (e.g. antibody response versus

lethality) and the additional inherent variability of the classical challenge models (Hendriksen et al., 1998).

The shift in the quality control conceptfrom reliance on the final batch testing tothe concept of consistency of productionoffers the opportunity to reduce the num-bers of animals being used and promotethe use of alternative methods. Emphasisis put on a combination of in vitro testswhich could make it possible to monitorbatch-to-batch consistency even for traditional vaccines like tetanus anddiphtheria toxoids (Leenaars et al.,2001).

3 Regulation and acceptance

3.1 National and international controlauthoritiesIn Europe, competent control authoritiesregulate the authorisation and batch re-lease, based on the Ph. Eur. and nationalpharmacopoeiae. If there is no mono-graph for a particular product or the monographs do not specify exactly the method to be used, the competentcontrol authority sets the requirements to be fulfilled or requests a certain method to be used during the licensingprocedure.

Since the establishment of the EMEA(European Medicines Evaluation Agen-cy, London, UK, www.emea.eu.int/) in1995, a centralised authorisation pro-cedure is compulsory for biotech productsin the 15 Member States (MS) of the European Union. This centralised pro-cedure can also be used for new and innovative products. Other medicinalproducts can still undergo the decentralisedprocedure in one of the MS. The EMEArelies on two scientific committees, theCommittee for Proprietary MedicinalProducts (CPMP) and the Committee forVeterinary Medicinal Products (CVMP),each of which comprise 30 members nom-inated by the 15 MS. The CPMP and theCVMP set up guidelines for medicinalproducts, often in cooperation with spe-cific CPMP/CVMP working parties(WP). The Biotech-WP provides spe-cific expertise for the CPMP on humanimmunological products. The Immuno-logical Veterinary Medicinal WP (IWP)advises the CVMP for example on gen-eral policy issues such as the elaboration

and revision of guidelines on immuno-logical products. The guidelines for thetesting of medicinal products are includedin The Rules Governing Medicinal Products in the European Union (Euro-pean Union, 1999). In 1997, the Safety-WP adopted a position paper on the re-placement of animal studies by in vitromodels (EMEA, 1997), which addressesthe feasibility of replacing in vivo studieswith in vitro investigations in the preclinical development of medicinalproducts and gives advices on their validation and incorporation into CPMP Notes for guidance.

3.2 National and internationalpharmacopoeiasIn Europe, requirements for pharmaceu-tical products are laid down in the Ph.Eur. Since its elaboration in 1964, 28 Eu-ropean countries (including the European

Fig. 1: Organigramme EMEA ( EuropeanMedicines Evaluation Agency)

EXECUTIVE DIRECTORFinancial controller, a.i.

Directorate

PRE-AUTHORISATION EVALUATION OFMEDICINES FOR HUMAN USE

Scientific advice and orphan drugsQuality of medicines

Safety and efficacy of medicines

POST-AUTHORISATION EVALUATIONOF MEDICINES FOR HUMAN USERegulatory affairs and organisational

supportPharmacovigilance and post-

authorisation safety and efficacy ofmedicine

VETERINARY MEDICINES AND ITVeterinary marketing authorisation

proceduresSafety of veterinary medicines

Information technology

ADMINISTRATIONPersonnel, budget and facilities

Accounting

Commission services at the EMEA in London

ETOMEP

TECHNICAL COORDINATIONInspections

Document management and publishingConference services

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Union) have signed the Convention ofthe Ph. Eur. Another 18 European andNon-European countries (including theWHO) are observers.

The Ph. Eur. includes general notices,methods of analysis (e.g. biologicaltests), general texts (e.g. general texts onvaccines), general monographs (e.g. vaccines for veterinary use) and specificmonographs. A specific monograph is divided into the following sections: Definition, Production, Identification,Tests, Storage and Labelling. The Production section applies to the manu-facturer and stipulates usually extensivesafety and immunogenicity testing,which is, however, only performed oncein the lifetime of a product during the licensing procedure. The Tests sectionapplies to the manufacturers and to the control authorities, and the tests specified here have to be carried out oneach batch of a product by the manufac-turer but not necessarily by the controlauthority.

The Ph. Eur. Commission has 21Groups of Experts, of which Group ofExperts 15 (Sera and Vaccines) andGroup of Experts 15V (Veterinary Seraand Vaccines) are responsible for thedrafting of monographs on vaccines, anti-sera and antitoxins in collaboration withthe Ph. Eur. secretariat. Group of Ex-perts 6 (Biological Substances) andGroup of Experts 6B (Human Blood andBlood Products) may also draft mono-graphs on immunobiologicals such ashormones, immunoglobulins or otherblood-derived products. The draft mo-nographs are published in Pharmeuropafor public consultation. The expertgroup reviews the monographs in thelight of the comments received, and they are finally adopted by the Ph. Eur.Commission.

Countries, which have signed the Convention of the Ph. Eur. are legally obliged to implement the texts of the Ph. Eur. into national legislation.

Apart from the Ph. Eur. Secretariat, theEuropean Directorate for the Quality ofMedicines (EDQM) of the Ph. Eur. comprises the division for publications,the laboratory and Division IV, which is responsible for the biological stand-ardisation programme and the European network of OMCLs.

3.3 European guidelines and CouncilDirectivesWithin the European Union, the qualitycontrol of vaccines is regulated – in addi-tion to the Ph. Eur. - by the followingCouncil Directives and guidelines: • for human vaccines by Council Direc-

tive 89/342/EEC extending the scope ofCouncil Directives 65/65/EEC and75/319/EEC and laying down addi-tional provisions for immunologicalmedicinal products consisting of vac-cines, toxins or serums and allergens;

• for veterinary vaccines by Council Di-rective 90/677/EEC extending the scope of Council Directive 81/851/EEC on the approximation of the laws of theMember States relating veterinary medicinal products and laying downadditional provisions for immuno-logical veterinary medicinal products;

• “The Rules Governing Medicinal Products in the European Union” in-corporates testing guidelines issued bythe EU (EU, 1999). According to the Directives and guide-

lines, quality control of each batch of avaccine produced in one production runis mandatory in order to assure its safetyand immunogenicity.

3.4 International organisationsThere are two international organi-sations, which publish guidelines for thequality control of immunobiologicals:the WHO and the O.I.E.

3.4.1 WHOThe WHO was created in 1948 as a spe-cialised agency of the United Nations.The objective of the WHO is “the attain-ment by all peoples of the highest possiblelevel of health”. In a wide range of func-tions, two are specifically addressed tovaccines and biologicals: to stimulate andadvance work on the prevention and con-trol of epidemic, endemic and other dis-eases; and to establish and stimulate theestablishment of international standardsfor biological, pharmaceutical and similarproducts. It is essential that governmentalinstitutions and international organisa-tions co-operate with the WHO in devel-oping and promoting harmonisation ofvaccine standards (Vannier et al., 1997).

The International Biological Refer-ence Preparations (IBRP) are establishedby the WHO Expert Committee on Bio-

logical Standardization (ECBS), whichmeets annually and addresses medicinalproducts (among them vaccines) for human use but also veterinary vaccinesagainst diseases of zoonotic relevance.The use of IBRPs contributes to the re-duction of animal tests by using harmo-nised test requirements and thus avoidingretesting. The actual list of IBRPs wasrecently published (WHO, 2000).

The Vaccines & Biologicals Depart-ment of the WHO regularly publishesand updates guidelines for internationalvaccine standardisation in its TechnicalReports Series. Within the framework ofthe Global Training Network on VaccineQuality, it regularly organises trainingcourses on vaccine quality control for de-veloping countries, which also addressthe standardisation/optimisation of theuse and the reduction of laboratory ani-mals for the production and quality con-trol of vaccines.

3.4.2 O.I.EThe O.I.E. issues the Manual of Stan-dards for Diagnostic Tests and Vaccines,which is edited by its Standards Com-mission and distributed world-wide(O.I.E., 2000). It contains recommen-dations for (a) “prescribed tests” for di-agnosis, and (b) requirements for vac-cines for list A and B diseases. List A diseases are transmissible diseases thathave the potential for very serious and ra-pid spread, irrespective of national borders, that are of serious socio-eco-nomic or public health consequence andthat are of major importance in the inter-national trade of animals and animal pro-ducts. List B diseases are transmissiblediseases that are considered to be of socio-economic and/or public health importance within countries and that aresignificant in the international trade ofanimals and animal products.

The chapter on vaccines includes in-formation on recommended vaccines, data on seed management, characteristicsof the vaccine strains, culture conditions,validation of vaccines, manufacturing,in-process controls, sterility tests, safetytests, and potency tests. The O.I.E. dis-tributes this information and publishesthe annual reports of the O.I.E. StandardsCommission, which undoubtedly contri-bute to international harmonisation(Blancou and Truszczynski, 1997).

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In 1994, the O.I.E. set up an Ad hocGroup on the harmonisation of veter-inary medicines, which was the first steptowards the creation of the “VeterinaryInternational Cooperation on Harmonisa-tion” (VICH).

3.5 International harmonisationEuropeThe European Commission representsthe fifteen Member States of the EU. TheCommission is working, through harmo-nisation of technical requirements andprocedures, to achieve a single market inpharmaceuticals, which would allow freemovement of products throughout theEU. The CPMP and CVMP of the EMEAprovide technical and scientific supportfor International Conference on Harmo-nisation (ICH) and VICH activities.

The International Conference onHarmonisationThe ICH was established in 1990. It is ajoint activity of regulators and industryas equal partners in the scientific andtechnical discussions of the testing pro-cedures, which are required to ensure andassess the safety, quality and efficacy ofmedicines for human use. The focus ofICH is on the technical requirements formedicinal products containing newdrugs. The six founder members of theICH are the European Commission re-presenting the 15 EU MS, the EuropeanFederation of Pharmaceutical Industries'Associations (EFPIA), the Japanese Min-istry of Health and Welfare, the JapanPharmaceutical Manufacturers Associa-tion, the US Food and Drug Administra-tion, and the Pharmaceutical Researchand Manufacturers of America. There arethree observers, the WHO, the EuropeanFree Trade Area (represented at ICH bySwitzerland) and Canada. The ICHsecretariat is run by the International Federation of Pharmaceutical Manufac-turers Association (IFPMA), which is afederation of member associations representing the research-based pharma-ceutical industry and other manufac-turers of prescription medicines in 56countries throughout the world. IFPMAhas closely been associated with ICH,since its inception to ensure contact withthe research-based industry, outside theICH regions. IFPMA has two seats onthe ICH Steering Committee.

Veterinary International Cooperation onHarmonisationThe Veterinary International Cooperationon Harmonisation (VICH) was launchedin 1996. VICH focuses on harmonisingregistration requirements for veterinarymedicinal products in the EU, USA andJapan. Countries not involved in theVICH are kept informed on its progressthrough the O.I.E.

A Working Group on Target Animal Safety of veterinary medicines was recently established and had its first meeting in November 2000.

3.6 Legal and ethical background tothe Three RsThere is a legal and ethical obligation forthe countries, which have signed theConvention of the Council of Europe andin particular, for the MS of the EuropeanUnion. Both, the European Conventionfor the Protection of Animals Used forExperimental and other scientific pur-poses, ETS 123 (Council of Europe,1986) and Directive 86/609/EEC (EEC,1986) claim that• “an (animal) experiment shall not be

performed if another scientifically satisfactory method of obtaining the result sought, not entailing the use ofan animal, is reasonable and practi-cably available”; (replacement)

• “in a choice between experiments, those which use the minimum numberof animals … cause the least pain, suffering, distress, and lasting harmand which are most likely to provide satisfactory results shall be selected”;(reduction and refinement) and

• “all experiments shall be designed toavoid distress and unnecessary painand suffering to experimental ani-mals”; (refinement).In addition, Directive 86/609/EEC

states in Article 23 that the EuropeanCommission and the MS should initiateThree Rs studies.

4 Development and validation ofalternatives

4.1 General aspects

4.1.1 Who develops and validates?In the last fifteen years, a number of al-ternatives have been developed for the

quality and safety control of immunobio-logicals by national control authorities,academia, and manufacturers. In princi-ple, there are two different approaches:the development of product-specific methods, which are specifically designedand validated by manufacturers for theirproducts, and the development of referen-ce methods, which can be used for a prod-uct group e.g. tetanus or rabies vaccines.

Alternative reference methods, whichmight replace, for example, a pharma-copoeial test, are validated in an interna-tional collaborative study. Within Europe,the Council of Europe and the EuropeanCommission have initiated the Biolo-gical Standardisation Programme (BSP),which is dedicated to validate alternativemethods (Council of Europe, 1996a;Buchheit, 2001). In recent years, the Eu-ropean Centre for the Validation of Alter-native Methods (ECVAM, Institute ofHealth and Consumer Protection; JRC, I-Ispra) established by the European Com-mission also got involved in the valida-tion of alternative methods for the testingof biologicals. Several worldwide valida-tion studies have been run under the aus-pices of WHO, e.g. validation of a trans-genic mouse model and a molecularbiological test to replace the neuro-virulence testing of oral poliomyelitisusing monkeys.

4.1.2 Who is sponsoring?Financial support to the development wasmainly given by national governments(e.g. BMBF, Swiss National Science Foundation), semi-governmental institu-tions (e.g. ZON), national institutes (e.g.RIVM), European authorities (e.g. Euro-pean Commission research programmes)or international organisation (WHO,Council of Europe), national alternativecentres, foundations (e.g. set, Stiftung3R, DZ, FFVFF) and industry (e.g. In-VITRO). International validation studiesare mostly sponsored on a European leveland international level, e.g. by the BSP ofEDQM, the European Comission (e.g.ECVAM) and the WHO.

4.2 Special aspectsThe following two sections cover animaltests and possible alternatives to thebatch potency and batch safety testing ofvaccines and immunosera. In some cases, certain classes of vaccines (e.g.

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clostridial vaccines) or vaccines for a certain species (e.g. avian vaccines) arereviewed together. Annex 1 and Annex 2include tables, which summarise the numbers of animals needed for batch safety and batch potency testing, list dis-tress categories and refer to the chapters.

4.2.1 Vaccines and immunosera forhuman use

4.2.1.1 Bacterial vaccinesBCG vaccineSafetyThere are two safety tests stipulated,which involve the use of animals: the testfor virulent mycobacteria (subcutaneousor intramuscularly injection of 6 guineapigs) and the test for excessive dermalreactivity (intradermal injection of testand reference vaccine into two groups of6 guinea pigs). Suitable alternatives tothese animal tests are not available.

Cholera vaccinePotencyThe Ph. Eur. monograph on cholera vaccine includes the test for antibodyproduction, which is a potency test basedon immunisation of 6 animals (guineapigs, rabbits, mice) and estimation of serum antibodies with suitable methods.Alternatives, which could replace this serological model, e.g. antigen quantifi-cation, are not available.

Diphtheria vaccineSafetyBoth, the WHO and the Ph. Eur. stipu-late tests for diphtheria toxin to be carriedout on the final bulk (absence of toxin; irreversibility of toxoid) and the final lot(specific toxicity). Currently, there arethree methods used, which are two ani-mal tests involving subcutaneous or intra-dermal inoculation of five respectivelyone guinea pig, and an in vitro methodusing cell cultures.

In 2000, the monograph on diphtheriavaccine and vaccines containing a diph-theria component was revised (Councilof Europe, 2000a). It is intended to com-bine the test for absence of toxin and irreversibility of toxin and to use cellcultures for the detection of diphtheriatoxin. The test for specific toxicity onthe final bulk and the final lot will be deleted.

The use of Vero cells for the detectionof diphtheria toxin was first described byAbreo and Stainer (1985); and furtheroptimised and standardised by van derGun et al. (1999); however, a validationstudy is still required.

A rapid enzyme immunoassay has beendeveloped for diagnostic purposes,which might also be used for diphtheriavaccines (Engler and Efstratiou, 1999).

PotencyThe Ph. Eur. includes two methods forthe potency testing of diphtheria vac-cines, both are classical multi-dilutionchallenge assays. The first method is carried out with least 6 groups of guineapigs (exact number not stated in the monograph, in practice 8-12 animals) areimmunised and subcutaneously (lethally)challenged. The second test is based onintradermal challenge of 5 immunisedguinea pigs and evaluation of the dermalreaction. The WHO requires either theintradermal challenge or a serologicalmethod used for the estimation of diph-theria antibodies in the serum of immu-nised mice or guinea pigs. Until today,only the WHO permits the use of a single-dilution assay once the consistency ofproduction and testing has been estab-lished; the revised Ph. Eur. text 2.7.6. Assay of Diphtheria Vaccine (absorbed)(Council of Europe, 2000b) now includesthe option to use a single-dilution assay.

The most promising serological meth-od is the Vero cell test, which is alreadyallowed by WHO (WHO, 1995). Valida-tion of this method is foreseen in the BSPof EDQM. Other possibilities for the esti-mation of serum antibodies are the ToBItest (Hendriksen et al., 1989), an ELISAprocedure (Moon et al., 1999) and two types of double antigen immunoassays,of which one could also detect tetanus antibodies (Aggerbeck et al., 1996; Kri-stiansen et al., 1997; Azhari et al., 1999).

Haemophilus type B conjugate vaccineSafetyA pyrogen test is carried out on the finalbulk for safety control purposes (see4.2.1.5 Test for pyrogens).

PotencyThe potency testing of haemophilus vac-cines is based on a serological animalmodel: a group of eight mice is immu-

nised with the test vaccine and after a given period the serum antibodies areestimated and compared to those of agroup of eight non-immunised mice. Forliquid products, this test is not carried outon the final lot but only on the final bulk.

There is a need to evaluate whether thenumber of animals required could be reduced, in particular, the number of control animals appears rather high.

Meningococcal polysaccharide vaccineSafetyA pyrogen test is carried out for safetycontrol purposes (see 4.2.1.5 Test for pyrogens).

Acellular pertussis vaccines (ACPVs)SafetyThe Ph. Eur. monograph on acellular per-tussis vaccine stipulates two safety teststo be carried out on the final lot, the testfor absence of residual pertussis toxin andthe test for reversibility of toxoid. The so-called histamine-sensitisation (HS) test iscarried out in both cases: five mice areimmunised with ACPV, five control ani-mals are injected with diluent, and after agiven period intraperitoneally challengedwith histamine. An in vivo alternative tothe HS test is the leukocytosis promotion(LP) test, which is considered not to beconsistently reliable (Corbel et al., 1999).The in vitro alternative, the Chinese ham-ster ovary (CHO) clustering test, can beused for the testing of bulk componentsbut not for absorbed vaccines, since theadjuvant might interfere with the cells.

PotencyPotency testing of ACPV is based on a serological multi-dilution test: 6 groups ofmice are immunised with dilutions of thetest vaccine and reference vaccine and after a given period the animals are bledand the serum antibodies are estimatedwith an immunochemical method. The tester can choose the number of animalsper group, which should be suitable to meetthe requirements for a valid test. The mo-nograph allows the use of a single-dilutiontest provided that the tester has gained sufficient experience with the method.

Whole cell pertussis vaccine (WCPVs)Specific toxicity testingAccording to the current Ph. Eur. require-ments, the specific toxicity of WCPV is

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tested with the mouse weight gain(MWG) test, which can be regarded as anon-specific test estimating overall tox-icity. At least 10 mice are injected withthe vaccine to be tested; their weight is re-corded on day 3 and day 7 and comparedto the weight of a control group. The re-cently revised Ph. Eur. monograph stipu-lates the use of only 5 guinea pigs ac-cording to the proposal of Weisser andHechler (1997) (Council of Europe, 2000c).

Bordetella pertussis is known to pro-duce at least five toxins; however, it is notclear to what extent they cause reaction tothe vaccine after injection into humans. Anumber of animal and non-animal test systems have been developed for the de-tection of specific toxins (Tab. 2) and fourof them were recently compared in a large-scale collaborative study (van Straaten-van de Kappelle et al., 1997). From thisstudy it was concluded that the MWG testis not the most suitable test to detect per-tussis toxin activity and that the HS testand the LP test might be more suitable forthis purpose. However, the authors under-line the importance of further opti-misation and standardisation of the test sy-stems. They emphasised that the Limulusamebocyte lysate (LAL) test should be used to measure endotoxin levels,whereas the CHO clustering test couldonly be used for adjuvant-free vaccines.

Since the HS test is based on a chal-lenge procedure with histamine and subsequent death of the animals in thepresence of pertussis toxin, it is a moresevere procedure than the LP test.

PotencyThe potency testing of WCPVs is a clas-sic multiple-dilution challenge test (Ken-

drick test): groups of at least 16 mice areimmunised with serial dilutions of thetest vaccine and a reference preparation.14-17 days after immunisation, the ani-mals are intracerebrally challenged withlive pertussis bacteria, observed for 14days and the survival rates are evaluated.This intracerebral mouse protection testis stipulated (with minor differences) byall international requirements. It uses largenumbers of animals and inflicts severedistress on the mice. The precision andreproducibility of the test is poor (re-viewed in Weisser and Hechler, 1997).

Modification of the animal modelseems to be possible, thus the intrace-rebral challenge could be replaced withaerosol challenge, which is less distressingfor the animals and is not based on thelethal endpoint (Canthaboo et al., 1999a).

The most promising alternative meth-od developed is a whole cell ELISA,which estimates pertussis antibodies inthe serum of immunised mice and avoidsintracerebral challenge. A collaborativestudy with five participating laboratoriesrevealed that the whole cell ELISA is asuitable method for the potency testingof WCPVs (van der Ark et al., 2000).

Canthaboo et al. (1999b) report an al-ternative method, which is based on theestimation of nitric oxide induction inmacrophages of mice immunised withWCPV. This method aims to replace theintracerebral challenge, however, it isstill under development.

Pneumococcal polysaccharide vaccineSafetyA pyrogen test is carried out for safetycontrol purposes (see 4.2.1.5 Test for pyrogens).

Tetanus vaccineSafetyThe current Ph.Eur. monographs stipu-lates three animal tests to be carried outfor the detection of tetanus toxin, whichare carried out on the toxoid bulk (ab-sence of toxin, irreversibility of toxoid)and on the final bulk (specific toxicity).The freedom from residual and rever-sible tetanus toxicity of the toxoid bulkis tested in guinea pigs or mice. Weisserand Hechler (1997) have outlined possi-bilities for refinement and reduction ofthese animal tests. The revised Ph. Eur.monograph includes significant changeswhich are the combination of the twotests on the toxoid bulk carried out inguinea pigs and the deletion of the specific toxicity test on the final bulk(Council of Europe, 2000d).

A promising in vitro approach for thedetection of tetanus toxin, an endopepti-dase test, has recently be developed(Ekong and Sesardic, 1999; Sesardic etal., 2000a); however, further standardi-sation and validation are still required.

PotencyAccording to the requirements of the Ph.Eur. monograph, a classic multi-dilutionvaccination challenge test has to be per-formed for the potency testing of humantetanus vaccines. Guinea pigs or micecan be used for this purpose. A series ofat least three dilutions of the vaccine andthe reference preparation are adminis-tered subcutaneously. The exact numberof animals to be used per group is notstated, but the monograph prescribes thatit must be sufficient to meet the statisticalrequirements. Four weeks after immuni-sation, the animals are challenged with

either a lethal or a paralytic dose of tetanus toxin, and afterone week of observation, thesurvival rate is analysed. Thechallenge with the tetanus toxincauses severe suffering to theanimals, as at least 50% of theanimals die of tetanus or developparalysis. The recently revisedPh. Eur. text 2.7.8. Assay of tetanus vaccine (absorbed) for-sees the use of a single-dilutionassay provided that the testerhas sufficient experience withthe method for a given product(Council of Europe, 2000e).

Tab. 2: Alternatives to the mouse weight gain test

Method Toxin Animals Status Reference

Histamine-Sensitisation Pertussis Multi-dilution Allowed by WHO van Straaten-van de

(HS) test test in mice* Kappelle et al., 1997

Leukocytosis Promotion Pertussis Multi-dilution Allowed by WHO van Straaten-van de

(LP) test test in mice Kappelle et al., 1992

Limulus Amebocyte Endotoxin - Allowed by WHO van Straaten-van de

Lysate (LAL) test (LPS) Kappelle et al., 1997

Chinese Hamster Ovary Pertussis - Allowed by WHO; Fujiwara and Iwasa,

(CHO) clustering test** Used for PT 1989

detection in acellular

pertussis vaccines

* = HS test is used as a single-dilution test for the quality control of acellular pertussis vaccines** = only for adjuvant-free vaccines

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This will significantly reduce the num-ber of animals required for the potencytesting.

During the last decade, two serologicaltest systems, an ELISA procedure andthe toxin binding inhibition test (ToBI)were developed, which measure the levelof tetanus antibodies in the sera of theimmunised animals (Hendriksen et al.,1991; Hendriksen et al., 1994).

A validation study was initiated in1996 with the financial support ofECVAM and EDQM. The main objec-tives of the validation study were to re-place the toxin challenge with in vitroestimation of tetanus antibodies; to re-place the multi-dilution (quantitative) testwith a single-dilution (qualitative) test;and to use guinea pigs instead of mice forthe immunisation with tetanus vaccine(Winsnes et al., 1999). The results in-dicate a very good correlation between theantibody concentration assessed by thetwo serological methods and death/surviv-al of the guinea pigs after the challenge:the predictive value for the ToBI test is94% (range 92-97%, for six laboratories)and 92% for the ELISA method (range91-95%, for six laboratories). Antibodyconcentrations determined by ELISAand ToBI were generally in the same range(Council of Europe, 2000f). It is hopedthat the ELISA and the ToBI test will beallowed for batch potency testing soon. WHO already permits the use ofserological tests for batch potency testingof human tetanus vaccines provided thatit has been validated for vaccines of thesame type (WHO, 1995).

Typhoid vaccinesThe Ph. Eur. contains three monographson typhoid vaccines, which are typhoidpolysaccharide vaccines, oral live typhoid vaccines (strain TY 21 A) andtyphoid vaccines. Only the latter stipulatestests in animals; however, it is no longerrelevant.

4.2.1.2 Viral vaccinesHepatitis vaccinesPotencyThe three Ph. Eur. monographs on hepa-titis vaccine A, hepatitis B and the com-bined product stipulate that potencytesting should be carried out in vivo or invitro. The in vivo potency test is a sero-logical test which is carried out in mice

or guinea pigs. The in vitro test is basedon immunochemical determination of theantigen content and has to be approvedby the National Control Authority. Des-camps et al. (1999) report that the use ofantigen quantification for batch releasereduced the number of mice by 60% atone of the main hepatitis vaccine manu-facturer. However, not all competent authorities accept the in vitro method andtherefore, animals are still used. In themeantime, the WHO has also modifiedits requirements on hepatitis B vaccinesand allows the use of antigen quantifi-cation for batch release (WHO, 1999a).

Influenza vaccinesThe Ph. Eur. includes three monographson influenza vaccines, which are inac-tivated split virion, surface antigen andwhole virion influenza vaccines. The mo-nographs stipulate a test for inactivationto be carried out in fertilised chickeneggs. There is a need to evaluate whethercell cultures would be suitable for thispurpose, since at least influenza virusstrain B grows on MDCK cells.

Poliomyelitis vaccinesProductionPoliomyelitis vaccines deserve a specialconsideration since monkeys might beused for their production and qualitycontrol (neurovirulence testing of oralpoliomyelitis vaccine). Some vaccinemanufacturers use primary and subcul-tured monkey kidney cells for the propa-gation of the vaccine virus, other use human diploid cell lines or Vero cells. Ithas been discussed whether the use ofprimary monkey kidney cells should cease. However, in the light of the WHOpoliomyelitis eradication campaign andthe decreasing need of poliomyelitis vac-cine, it was been claimed that far moremonkeys would be needed to re-establishconsistency of production than would besaved by change of cell substrate.

Inactivated poliovirus vaccines (IPV)PotencyThe Ph. Eur. monograph includes threemulti-dilution serological animal modelsfor the potency testing of IPV. Since atest with the reference vaccine is carriedout in parallel, at least 60 chicks, guineapigs or rats are required per test. The ratpotency test, which has been evaluated in

a collaborative study, is now recommen-ded by Ph. Eur. (Wood and Heath, 2000).

The potency test can be omitted on thefinal lot provided that it had been per-formed with satisfactory results on the final bulk.

For potency testing, it is then sufficientto quantify the antigen content.

Live oral poliomyelitis vaccines (OPV)Neurovirulence testLive attenuated virus vaccines are testedfor neurovirulence in monkeys. The aimof the test is to confirm that the attenuatedvaccine virus strain has not reverted toneurovirulence. In the case of OPVs, thistest is carried out on each master seed lot,working seed lot and on each monovalentbulk. Monkeys (for example, Macaca fascicularis, Cercopithecus aethiops) areintraspinally injected with the test or re-ference preparation and killed after a given observation period and the centralnervous system is checked for the specificneuronal lesions of poliovirus. Accordingto the Ph. Eur. and WHO requirements, atleast 80 monkeys are needed for the quality control of a trivalent bulk.

Alternative tests have been developedand are, at least for poliovirus type 3, close to regulatory acceptance (Wood,1999). The alternatives are the MAPRECtest (molecular analysis by PCR and re-striction enzyme cleavage; Chumakov etal., 1991), which detects neurovirulencespecific mutations, and a neurovirulencetest in transgenic mice (TgPVR21 mice),which express the human cellular re-ceptor for polioviruses (Koike, 1991).Wood and Macadam (1997) and Dragunsky et al. (1996) have reviewedthe specifications of these test systems.

The MAPREC test for poliovirus type3 is already an option in the WHO re-quirements, which suggest that only pre-parations, which pass the MAPREC test,should be tested in monkeys in order todetect other mutations. WHO Interna-tional Standard and Reference Preparationand a SOP are now available for polio-virus type 3 but not yet for type 1 and 2(Wood, 1999). The WHO has endorsedthe MAPREC test as the in vitro test ofpreference for the quality control of poliovirus type 3 (Wood et al., 2000).

The WHO has organised a collabora-tive study to validate the transgenic mouse model for neurovirulence testing

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of poliovirus type 3 vaccines (Wood,1999) and the TgPVR21 mouse model isnow recommended as an alternative tothe test in monkeys (Wood et al., 2000).Studies on the suitability of TgPVR21mice for neurovirulence testing of polio-virus type 1 and 2 are in progress.

Horie et al. (1998) modified the MA-PREC test and developed the NON-RIMAPREC, which does not involve theuse of radioisotopes. A further moleculartest has been developed by Kaul et al.(1998), who used Taq Man PCR for thequantification of neurovirulence specificmutations. Proudnikov et al. (2000) useda new technology of hybridisation for thedetection and quantification of neuro-virulent mutants in OPVs, which can beused for screening samples for knownmutants as well as for new mutations.

Rabies vaccinePotencyAccording to the WHO requirements andthe Ph. Eur. monograph, the potency ofinactivated rabies vaccines for humanuse is estimated with the NIH test. TheNIH test is a multi-dilution challengetest: at least 6 groups of mice are immu-nised with serial dilutions of test and re-ference preparation. The animals are in-tracerebrally challenged with virulentrabies virus and observed for signs of ra-bies for 14 days. The NIH test requires ahigh number of animals (up to 170 miceper batch, Weisser and Hechler, 1997)and causes severe distress to them. Theuse of non-lethal endpoints should beconsidered (see 4.2.3 Humane end-points). Various alternative methods havebeen developed which could replace theNIH test. These methods are either basedon serology or antigen quantification andsome are listed in Table 3.

Recently, a validation study has beenperformed involving seven laboratories:the potency of five commercially availa-ble human and veterinary rabies vaccinesand one reference preparation was testedwith an ELISA procedure (Rooijakkerset al., 1996), which estimates rabies virusantigens, glycoprotein (G) and nucleo-protein (N). All the participating labora-tories carried out the requested assaysand generated valid data. The resultsclearly show that the reproducibility ofthe two ELISA kits is by far superior tothat of the NIH test (unpublished data).

4.2.1.3 Immunosera/AntitoxinsC. botulinum antitoxin for human usePotencyThe Ph. Eur. stipulates a classical toxinneutralisation test in mice for the poten-cy testing of botulinum antitoxin, whichrequires large numbers of animals (atleast, 150 mice per batch). Possibilitiesfor reduction and refinement have beenreviewed by Weisser and Hechler (1997).An in vitro method for the detection ofneutralising antibodies against botulinumtoxin type A has recently be reported(Martin and Sesardic, 1999; Sesardic etal., 2000b), which is dependent on theactivity of a botulinum type A proteaseon a synthetic substrate.

Diphtheria antitoxinPotencyThe potency testing of diphtheria anti-toxin is based on an in vivo intradermaltoxin neutralisation test in guinea pigs orrabbits. Serial dilutions of the test anti-toxin are mixed with a given dose ofdiphtheria toxin and each dilution is intradermally injected into two animals.Weisser and Hechler (1997) suggestedthat the total number of animals could bereduced by using a number of injectionsites on each animal. Thus, one animalwould be sufficient for the complete titration of the test antitoxin and the re-ference preparation. With regard to re-placement, there is a need to evaluatewhether any of the in vitro methods developed for the potency testing ofdiphtheria vaccines could also be usedfor the potency testing of diphtheria an-titoxins (e.g. Vero cell test).

European viper venom antiserumPotencyEuropean viper venom antiserum is pro-tective against the venom of five viperspecies and the potency of each test anti-serum has to be tested against the five ven-oms. The PD50 value for each venom isdetermined with an in vivo toxin neutra-lisation test in mice. Weisser and Hechler(1997) report that around 400 mice areneeded per batch. In addition, about 50%of the mice are not protected against thevenom and suffer extremely.

A number of alternative methods havebeen developed, however, all of them forthe potency testing of non-Europeansnake venom. Most of the alternativesare based on in vitro neutralisation ofspecific snake venom effects, e.g. anti-procoagulating, myonecrotic, haemato-lytic or proteolytic effects, on cell cul-tures (da Silva et al., 1982; Warrell et al.,1986; Guitterrez et al., 1988; Laing etal., 1992; Gowda and Middlebrook,1993; de Araújo et al., 1999). Variousimmunochemical procedures have beenassessed for the detection of snake ven-om antibodies in the sera of patients andin antisera (Theakston, 1983); however,no studies are reported on the suitabilityof these methods for the potency testingof European snake venom antiserum. Inthe light of the large number of animalsrequired per batch and the severe distress inflicted on the animals, effortsshould be made to investigate whetherimmunochemical methods or in vitroneutralisation could be used for the potency testing of European snake venom.

Tab. 3: Alternative methods for the poteny testing of rabies vaccines

Method Animals Reference Status

Antibody estimation

Rapid fluorescent focus Yes (5 mice) Smith et al., 1973 WHO method;

inhibition test Council of Europe, 1998a Ph. Eur. method

ELISA Yes Joffret et al, 1991 Commercially available

Antigen quantification

Single radial diffusion No Ferguson et al., 1984 WHO method

Vogel et al., 1989 Accepted in Austria

Antibody binding test Depending on Arko et al., 1973 WHO method

the method used

ELISA (G antigen) No Gamoh et al., 1996

ELISA No Rooijakkers et al., 1996 Validated (unpublished

(G and N protein) data)

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Coded samples of tetanus antisera andimmunoglobulins including samples ofinferior quality were tested in six labora-tories. Comparison of the ToBI data withthe in vivo data (provided by the manu-facturers) showed a high agreement inthe ranking of the potencies of the sam-ples. The best results were obtained forthe human tetanus immunoglobulins: in-terlaboratory variation was <15% andthe ranking of the samples was identicalin all laboratories. It is therefore conclu-ded that a formal validation study of theToBI test for the potency testing of hu-man tetanus immunoglobulins should beinitiated. With regard to the tetanus anti-sera for human and veterinary use, itshould be considered whether the mono-graph on tetanus antiserum for humanuse should be deleted since tetanus anti-serum of equine origin is no longer usedfor humans or whether the two mono-graphs should be harmonised and the re-quirements on the quality and safety ofthe products be updated to reflect thepresent state-of-the-art. Nevertheless,validation of the ToBI test for the poten-cy testing of tetanus antisera is recom-mended. Provided that the ToBI testcould be incorporated into the Ph. Eur.monographs, at least 2.000 animals peryear could be saved in Germany (Weis-ser and Hechler, 1997).

Kolbe and Clough (1999) developed acompetitive ELISA for the estimation oftetanus antibodies in equine tetanus antise-rum for veterinary use. They compared theELISA results of nine batches of tetanusantiserum with the in vivo results obtainedwith the toxin neutralisation test in guineapigs (US requirements) and found a goodcorrelation. However, data on the repeat-ability and the reproducibility of resultsfrom other laboratories were not provided.

4.2.1.4 Tuberculins for human useThere are two monographs on human tu-berculins in the Ph. Eur., which stipulatethe use of animals for toxicity (two guinea pigs), sensitisation (3 guineapigs) and potency (6 guinea pigs) testing.The test for identification is also carriedout in animals but can be combined withthe potency testing.

The test for live mycobacteria was re-cently deleted and replaced by new andmore sensitive culture methods (Councilof Europe, 2000g).

Weisser and Hechler (1997) questionthe relevance of the toxicity test since itdoes not provide any further information.Alternatives to the test for sensitisation arenot available, however, the number ofcontrol animals could be reduced fromthree to one. There have been several attempts to develop alternatives for thepotency testing of tuberculins, however,none has been validated. The most promising approach is the lymphocyte stim-ulation test (Hasloev et al., 1986), whichmeasures in vitro the cytokine release ofT-cells in response to mycobacteria. Weis-ser and Hechler (1997) suggest that poten-cy could be tested in human volunteers,since in house reference preparations arealso calibrated in humans against the in-ternational reference. Further possibilitiescould be the use of in vitro skin models.

For the time being, the design of the cur-rent potency test in animals could be re-fined (e.g. individual evaluation of eachanimal) and potency testing should onlybe carried out during production and onthe final bulk, which would significantlyreduce the total number of animals needed.

4.2.1.5 Test for pyrogensThe Ph. Eur. stipulates the test for pyro-gens for a number of immunobiologicals(Tab. 4). It assesses the fever reaction inthree rabbits induced by pyrogens.

A well-established alternative to thepyrogen test in rabbits is the in vitro Li-mulus amebocyte lysate (LAL) assay,which has already replaced the rabbit testin a number of Ph. Eur. monographs (testfor bacterial endotoxins). However, it canonly detect cell wall components ofgram-negative bacteria but not pyrogeni-city caused by gram-positive bacteria,fungi or other possible pyrogens. Withregard to the products listed in Table 4, itshould be possible to use the test for bac-terial endotoxins for pyrogenicity testingof meningococcal vaccines, since the pyrogens to be detected are endotoxins.The monograph on hepatitis B vaccinesexplicitly states that a validated test forbacterial endotoxins could replace therabbit pyrogen test.

The fact that the Ph. Eur. has estab-lished a new Expert Group for AlternativePyrogen Testing reflects the importanceof these issues.

A number of in vitro tests based on thehuman fever reaction have been devel-

Gas-gangrene antitoxinsPotencyThe Ph. Eur. includes four monographson gas-gangrene antitoxins, which areGas-gangrene Antitoxin (C. perfringens),Gas-gangrene Antitoxin (C. septicum),Gas-gangrene Antitoxin (C. novyi) andMixed Gas-gangrene Antitoxin. The potency testing of gas-gangrene antitoxinsis based on in vivo toxin neutralisation inmice or “other suitable animals”. Serialdilutions of the test antitoxin are mixedwith a given dose of toxin and each di-lution is intramuscularly (C. novyi) or in-travenously (C. perfringens, C. septicum)injected into a group of 6 animals. Weis-ser and Hechler (1997) reported that thetotal number of animals needed for thepotency testing of a batch of mixed gas-gangrene antitoxin is about 150.They recommend that the number of animals per group should be reducedfrom six to between one to three.

Alternative methods could either bebased on in vitro neutralisation of the cyto-pathic effects caused by clostridial toxins(Knight et al., 1986 and 1990; Bette etal., 1989; and Tab. 6) or modification ofimmunochemical methods, which havebeen developed for the potency testing ofclostridial vaccines (Tab. 6).

Tetanus immunoglobulins and antiserafor human usePotencyThe Ph. Eur. monographs Human TetanusImmunoglobulin and Tetanus Antiserumfor Human Use (equine origin) and Teta-nus Antiserum for Veterinary Use (equineorigin) stipulate a toxin neutralisation testin mice for the potency testing of theseproducts. The monograph Human TetanusImmunoglobulin has recently been revisedand the use of validated serological invitro methods is now allowed; however,no reference method is proposed or described, and the in vivo test is still used.

Studies performed at the PEI had shownthat the EIA and the RIE are promisingmethods (Mainka and Haase, 1995; Zott,1996) for replacement of the in vivo test.With the financial support of ECVAM,the PEI initiated the standardisation ofthese two methods and modified the ToBItest for the potency testing of tetanus immunoglobulins and antisera, whichproved to be the most suitable method forprevalidation (Ebert et al., 1998).

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oped (Tab. 5). The in vitro approachesuse leukocyte cell lines, isolated primaryblood cells or whole blood and measurethe release of fever mediators in res-ponse to pyrogens. A collaborative studyis in progress, which aims to validate themost suitable model(s) for pyrogenicitytesting of immunobiologicals, pharma-ceuticals, medical devices etc. (Hartunget al., 2001).

4.2.2 Vaccine and immunosera forveterinary use

4.2.2.1 The target animal safety testThe target animal safety test (TAST) is required by Ph. Eur. monographs and various EU guidelines on veterinary vac-cines. It is carried out on the finished product and should detect non-specificcontamination. At least two animals of thetarget species are injected with the 2-fold(inactivated vaccines) or 10-fold (live vac-cines) recommended dose of the vaccineto be tested. None of the animals shouldshow abnormal or systemic reactions during a given observation period. Signi-ficant differences in the numbers of animals

required (mammals: 2 animals; poultry: atleast 10 animals), the administration scheme and the period of observation areevident between the monographs.

During the last few years, the rele-vance of the TAST has more and morebeen questioned, because the introductionof Good Laboratory Practice (GLP) andGood Manufacturing Practice (GMP) intothe manufacturing of vaccines has significantly increased the safety andquality of the products, and some of theanimal tests carried out for purity and safety purposes appear now to be super-fluous (Roberts and Lucken, 1996; Zee-gers et al., 1997; Pastoret et al., 1997;Cussler et al., 2000a).

Two studies have been carried out bythe PEI and AGAATI, which aimed toevaluate the relevance of TAST by retro-spective data analysis. The results ob-tained to date confirm its questionable relevance. The test design, the evaluationcriteria and the requirement to be per-formed for batch release can no longer bejustified. For several vaccines, it was sho-wn that the TAST could be omitted, atleast for routine batch control purposes

(Bruckner et al., 2000; Pössnecker andCussler, 1998; Cussler and Pössnecker,2000). The final reports of the studies willbe available shortly. At that time, consider-ation should be given to contacting theEuropean regulatory authorities, askingfor modification of their guidelines andmonographs and specifying the deletionof the TAST on grounds that it is no longer relevant. However, if the TAST isstill necessary for specific products orgroups of products, the test design and re-quirements should be revised and clearlydefined in order to obtain reliable results.

Two recent cases show that a passedTAST does not guarantee a safe vaccine.As Falcone et al. (1999) report, hundredsof cattle died after vaccination becausethe infectious bovine rhinotracheitis(IBR) vaccine used had been contamina-ted with bovine viral diarrhoea (BVD)virus. The IBR vaccine had been testedaccording the Ph. Eur. monograph andhad been released. Since the serologicalstatus of the two calves used in the TASThad not been BVD negative, the contami-nation with BVD could not be detected.In France, the marketing authorisationfor a dog vaccine was withdrawn, whenseveral puppies died of distemper aftervaccination (Anon., 2000).

4.2.2.2 Bacterial vaccinesAnthrax spore vaccinePotencyThe Ph. Eur. monograph on anthrax spore vaccine stipulates a vaccination-challenge test in 13 guinea pigs or rabbitsor 8 sheep for the potency testing. Weisserand Hechler (1997) suggested the re-vision of the monograph and proposed

Tab. 4: Ph. Eur. monographs on immunobiologicals stipulating the test for pyrogens in rabbits

Haemophilus type B conjugate vaccineMeningococcal polysaccharide vaccinePneumococcal polysaccharide vaccineHepatitis B vaccine (rDNA)Rabies vaccine for human use prepared in cell culturesTick-borne encephalitis vaccine Human normal immunoglobulin (applies also to all "special” human immunoglobulins, e.g.Human tetanus immunoglobulinHuman normal immunoglobulin for intravenous use

Tab. 5: Alternatives to the test for pyrogens in rabbits

In vitro models Parameter Reference

Leukocyte cell lines

MonoMac cell line IL-6, TNF-α Taktak et al., 1991

THP-1; RAW 264.7 Neopterin, NO release Peterbauer et al., 1999; 2000

Sub clones of THP-1 TNF-α Eperon and Jungi, 1996; Eperon et al., 1997

and MonoMAC cells

Isolated primary leukocytes IL-1, IL-6, TNF-α Duff and Atkins, 1982; Dinarello et al., 1984; Poole et al., 1988a&b;

Chia and McManua, 1990; Wind Hansen and Dencker Christensen, 1990;

Morin et al., 1991

Whole blood models IL-1, IL-6 Hartung and Wendel, 1995, 1996; Pool et al., 1998; Hartung et al., 2000

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that the potency should be determined invivo only during licensing. After estab-lishment of a correlation between thenumber of live spores and the protectionachieved in the target species, it wouldsuffice for batch potency testing to de-termine the number of live spores invitro, which is state-of-the-art for otherlive bacterial vaccines.

Brucellosis vaccineFifty percent persistence timeThe test should show that the vaccinestrain persists for a certain period in theorganism. The bacteria localise intra-cellularly and induce the cellular immuneresponse. 32 mice are subcutaneously in-jected with the vaccine to be tested andtheir spleens are examined for brucel-losis bacteria after given periods. Alterna-tives to this test have not been developed.Weisser and Hechler (1997) recommendthat it should only be performed duringlicensing and not on the finished product.

Fish vaccinesThe Ph. Eur. includes three monographson fish vaccines, which are furunculosisvaccine, vibriosis (cold-water) vaccineand vibriosis vaccine. It is striking thatthese monographs stipulate the use of farmore animals, i.e. 10 fish for the safetytesting and 60 for the vaccination-chal-lenge or 35 fish for vaccination-serology,than monographs on mammalian vac-cines. The EU guideline on Specific Requirements for the Production andControl of Live and Inactivated VaccinesIntended for Fish stipulates even 30 fishfor the safety testing (EU, 1999). Itshould be investigated whether thesenumbers could be reduced.

Alternatives to the lethal vaccination-challenge test for potency testing of furunculosis vaccines are reported byWagner et al. (1998) who used a sand-wich-ELISA for the detection of potenti-ally protective antibodies, and Pund et al.(1998), who compared the reaction ofisolated lymphocytes of vaccinated andnon-vaccinated fish to Aeromonas salmo-nicida isolates.

Tetanus vaccine for veterinary usePotencyAn inter-laboratory validation study wascarried out with financial support of theEuropean Commission (allocated ECVAM

budget; Contract No B4-3081/91/7945).Based on the results, the Group of Ex-perts 15V of the European Pharma-copoeia Commission agreed in 1996 tomodify the monograph (Council of Euro-pe, 1996b) on tetanus toxoid vaccines forveterinary use according to the recom-mendations of the study (Hendriksen etal., 1994). The currently stipulated TNTwith mice will be replaced with an invitro immunoassay (for example, the ToBI test). However, new homologousreference sera of rabbit and guinea pigorigin were necessary to establish themethods for routine batch control. Aftercalibration of the reference sera in an in-ternational collaborative study organisedby EDQM (Lensing et al., 2000), biological reference preparations arenow available and the monograph willprobably come into force shortly.

Other clostridial vaccinesSafety - Residual toxicityThe Ph. Eur. monographs on C. botuli-num, C. novyi, C. perfringens, and C.septicum vaccines require the test for re-sidual toxicity, which involves subcuta-neous injection of a given vaccine doseinto 5 mice. In principle, cell culture meth-ods can be used for the detection of resi-dual toxicity of several toxoids, e.g. C.novyi and C. perfringens (Borrmann etal., 2001). However, in practice their useis mostly hindered by excipients such asformaldehyde.

PotencyFor a long time, potency testing of clos-tridial vaccines and immunosera in-volved large numbers of animals and in-flicted severe suffering on the animals. In1997, a symposium on clostridial vac-cines and immunosera organised by theEDQM, the PEI and the RIVM was heldin Strasbourg (Council of Europe,1997b). In the same year, the revision offour monographs on clostridial vaccineswas initiated which already incorporatedthe proposals of the symposium, for ex-ample the permission to use validatedimmunochemical methods or tests basedon toxin neutralisation in cell cultures forthe potency testing of C. novyi (Type B),C. perfringens, and C. septicum vac-cines, which would replace the mouse neu-tralisation test. The revised monographscame in force with the Ph. Eur. Supple-

ment 2001. A number of alternative meth-ods have been developed for this purpose(examples of which are listed in Table 6);however, none of them has been validated. The International VeterinaryIndustry Test Replacement Organisation(In-VITRO) and the EDQM initiated alarge-scale collaborative study for esta-blishing a multicomponent reference se-rum, which would facilitate to stand-ardise and validate these methods(Redhead et al., 1999). This new Europe-an Reference Preparation has recentlybecome available (Lucken et al., 2000).

With regard to C. chauvoei, the strin-gent pharmacopoeial requirements forthe vaccination-challenge test involvingguinea pigs and stipulating a 100% pro-tection rate were changed in 1998 andcame into force with Ph. Eur. Supple-ment 2001. In practice, potency testingnow requires significantly fewer animals(Redhead et al., 1999). Efforts have beenundertaken to identify the protective anti-gens for C. chauvoei and develop alter-native methods for the potency testing ofthese vaccines (Roth and Seifert, 1997;Hauer, 1997; Kijima-Tanaka, 1998; Hauer and Clough, 1999).

US control authorities allow the use ofantigen quantification for the potencytesting of inactivated vaccines; however,the regulations have not yet been changedfor clostridial vaccines. Hauer andClough (1999) report the development ofhybridoma cell lines which produce mo-noclonal antibodies directed against C.perfringens alpha, beta and epsilon toxin,C. sordelli lethal toxin and the flagella ofC. chauvoei. The antibodies are interna-tionally available and can be used in antigen quantification assays or other alternative methods.

Erysipelas vaccinePotencyThe Ph. Eur. monograph InactivatedSwine Erysipelas Vaccines has recentlybeen changed and came into force withPh. Eur. Supplement 2001. It currentlystipulates a single-dilution vaccination-challenge test in 10 mice instead of amulti-dilution-assay, which needed over100 mice for batch potency. Several prom-ising serological models have been de-veloped in recent years (Beckmann andCussler, 1994; Rosskopf-Streicher et al.,1998, 1999; Redhead, 1998), which

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could replace the challenge. The PEI hassuccessfully validated a direct ELISAprocedure (Rosskopf-Streicher et al.,2001): 10 mice are vaccinated with thetest vaccine and after a given immuni-sation period, the mice are bled and theantibodies to erysipelas are estimatedwith the ELISA. EDQM has asked thePEI to produce a reference coating anti-gen. It is hoped that the ELISA methodwill be accepted soon and incorporatedinto the monograph.

In the USA, work is in progress on thedevelopment of a sandwich ELISA forthe antigen quantification of erysipelasbacterins, which does not involve the useof animals (Hauer, 1998; Coe-Clough,1998).

Leptospira vaccines The current Ph. Eur. monograph Lepto-spiral vaccines for veterinary use coversvaccines containing Leptospira (L.) in-terrogans serovar canicola and serovaricterohaemorrhagiae. A hamster chal-lenge test is prescribed for the batch potency testing: a group of five hamsters

is immunised with the test vaccine. Aftera given immunisation period, the immu-nised hamster and a non-immunisedgroup of five hamsters are challengedwith the corresponding challenge strain.At least 80% of the non-protected hamsters die due to the infection, whichcauses severe distress and suffering.

The hamster challenge model is alsoused for the potency testing of L. hardjovaccines for cattle; however, the chal-lenge procedure is regarded as less severe, since the animals often survivethe infection. In this case, the re-isolationrate of bacteria from the kidneys is usedto determine the degree of protection.

Some major disadvantages of this mod-el are evident: large numbers of animalsare used for the potency test itself, but also for the maintenance of the virulenceof the challenge strains. Since humansare susceptible to infection with lepto-spiral bacteria, the test involves a high riskfor infection of the laboratory personnel(Weisser and Hechler, 1997; Marbehant,1999). At the workshop on Alternativesto Animal Challenge Tests in the Batch

Control of Leptospiral Vaccines for Ve-terinary Use (Council of Europe, 1999a) anumber of promising alternative methodswere presented, which are based eitheron serology (involving the immunisationof animals) or antigen quantification(animals are not involved) (Tab. 7). However, none of these methods has yetbeen validated in an international collab-orative study. Recently, USDA pre-sented antigen quantification assays for several leptospira vaccines (includingserovars canicola and icterohaemorrha-giae) suitable for in vitro potency testing.Those approved but pending StandardRequirements are listed underhttp://www.aphis.usda.gov/vs/cvb/lab/newsams.html.

4.2.2.3 Viral vaccinesAvian viral vaccinesThe Ph. Eur. contains two general textsand 13 monographs on poultry vaccines(Tab. 8). Tests in animals are prescribedfor extraneous agents, safety and potencytesting. In particular, large numbers ofanimals are needed for batch control. Re-

Tab. 6: Alternative methods for the potency testing of clostridial vaccines (excl. Tetanus)

Vaccine Method Status Reference

C. perfringens Type D Cell cultures (serology) Available Knight et al., 1990a; Payne et al., 1994;epsilon and beta Borrmann and Schulze, 1995, 1997

ELISA (serology) Used by industry for Wood, 1991batch release

Validation in progress Lucken, 1997

Prevalidated Ebert et al., 1999a

(and alpha toxin) ELISA (antigen quantification) Available Hauer and Clough, 1999

C. novyi Type B Cell cultures (serology) Borrmann et al., 1999

ELISA Used by industry for Wood, 1991batch release


C. septicum Cell cultures (serology) Available Knight et al., 1990b; Roth et al., 1999

ELISA (serology) Used by industry for Wood, 1991batch release


C. chauvoei ELISA (cellular antigens) Roth and Seifert, 1997

ELISA (flagella antigen; Validation in progress Hauer, 1997; Lucken, 1997; immunochemical and antigen Kijima-Tanaka, 1998; quantification) Hauer and Clough, 1999

C. sordelli ELISA (antigen quantification) Available Hauer and Clough, 1999

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presentatives of vaccine manufacturers,which participated in the joint AGAATI/ECVAM workshop on Three Rs Ap-proaches in the Production and QualityControl of Avian Vaccines, estimated thatabout 51.000 chickens and birds are usedeach year for the quality control of 1.800batches of avian vaccines (Bruckner etal., 2000).

The following evaluation will focus onthe tests stipulated for batch quality control since they offer more possibilities

for replacement and reduction. However,the report of the above workshop in-cludes a number of recommendations forthe implementation of the Three Rs in theproduction of avian vaccines.

Batch testing for extraneous agentsThe Ph. Eur. text 2.6.4 Avian Live VirusVaccines: Tests for Extraneous Agents inBatches of Finished Products stipulatestests in eggs, cell cultures and chicks inorder to detect a given list of possible

contaminants in avian live vaccines. Thetest in chicks (ten two-week-oldchicks/test) is regarded as less sensitivethan the tests in eggs and cell cultures(Bruckner et al., 2000). In addition, PCRmethods that are at least as sensitive asthe animal test are now available (Tab. 9).Some of them have already be validatedand accepted, others have recently beendeveloped and could be validated withinthe short-term.

Batch safety testingThe arguments against the TAST given inthe general section on the TAST also apply to avian vaccines. However, atleast ten chickens are required for avianvaccines in contrary to two for mammalianvaccines, without any scientific or statistical rationale for this difference.

Two studies are currently being per-formed by a working group at the PEI andby AGAATI (with the financial supportof the European Commission viaECVAM). Data on the target animal safety test are being collected from vac-cine manufacturers and control author-ities in Europe and analysed retrospec-tively. The results on avian vaccinesshow that all batches tested in the lastfew years passed the test (personal com-munication). Passing the test had no in-fluence on the number of the reports onvaccinovigilance collected from the field.The purpose and relevance of this test istherefore questionable, also since recentexperience showed that the test is notcapable of detecting major problemswith vaccine batches. Deletion of theTAST from the individual monographson avian vaccines is therefore recom-mended. It should be considered whetherthe TAST should be carried out on a limited number of batches for demonstra-tion of consistency of production or afterrelevant changes in the production pro-cess (Bruckner et al., 2000).

Batch potency testingIn contrast to live avian vaccines, wherepotency testing of a representative batchis sufficient, the potency of inactivatedavian vaccines has to be tested on eachvaccine batch. This requires a large num-ber of animals and involves suffering tothe animals if, under certain circum-stances, challenge with the infectiousagent is required. In most cases, sero-

Tab. 7: Current alternative methods for the potency testing of leptospira vaccines

Method Serovars Status Reference

a) Serology

MAT* L. hardjo Standardised Ebert et al., 1999b

ELISA L. hardjo Under development Ebert et al., 1999b

ELISA L. icterohaemorrhagiae Under development Schalling, 1999

and canicola

ELISA L. icterohaemorrhagiae Under development Varney, 1999

and canicola

b) Antigen quantification

ELISA L. copenhagi Standardised Hartskeel, 1999

and canicola

ELISA L. icterohaemorrhagiae Standardised Hickey, 1999

and canicola

ELISA L. icterohaemorrhagiae Standardised Martinon, 1999

and canicola

ELISA L. icterohaemorrhagiae, Standardised Ruby, 1999

canicola, pomona,

grippotyphosa

* modified O.I.E. method (2nd Edition of O.I.E. Manual; Goddard et al., 1986, 1991)

Tab. 8: European pharmacopoeia texts and monographs on avian vaccines

General texts

2.6.3. Avian Viral Vaccines: Tests for Extraneous Agents Seed Lots

2.6.4. Avian Live Virus Vaccines: Tests for Extraneous Agents in Batches of Finished Product

Individual monographs

Avian Infectious Bronchitis Vaccine (live)

Avian Infectious Bronchitis Vaccine (inactivated)

Avian Infectious Bursal Disease Vaccine (live)

Avian Infectious Bursal Disease Vaccine (inactivated)

Avian Infectious Encephalomyelitis Vaccine (live)

Avian Infectious Laryngotracheitis Vaccine (live)

Avian Viral Tenosynovitis Vaccine (live)

Duck Viral Hepatitis Vaccine (live)

Egg Drop Syndrome '76 Vaccine (inactivated)

Fowl-pox Vaccine (live)

Newcastle Disease Vaccine (live)

Newcastle Disease Vaccine (inactivated)

Marek's Disease Vaccine (live)

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logical methods are used for antibodyestimation after immunisation.

In recent years, efforts have been un-dertaken to develop antigen-estimationbased in vitro methods, which do not re-quire animals. Most progress has beenachieved with models for the potencytesting of inactivated Newcastle disease(ND) vaccines (Maas et al., 1998; Maaset al., 2000). The approach used is basedon the quantification of the NDV anti-

gens, the HN-protein and F-protein,which induce the production of neu-tralising antibodies after vaccination.Two ELISA procedures have been devel-oped for this purpose. Preliminary resultsshow that the method is sensitive, spe-cific and reproducible. There is a goodcorrelation between the amount of virusestimated in the vaccine and the anti-bodies detected in the serum after immu-nisation (Maas et al., 2000).

Whether antigen quantification can be used for the potency testing of in-activated bursal disease vaccine is alsonow under investigation.

Foot-and-mouth disease vaccine (FMD)SafetyThe safety test in the Ph. Eur. mono-graph can be regarded as a test for suf-ficient inactivation of the virus strain.Cell cultures are more suitable for this

Tab. 9: Availability and status of non-animal methods for extraneous agents testing of batches of the finished product according to Ph. Eur. text 2.6.4

Agents Alternative methods Status References

Infectious avian Eggs (intravitelline injection) Accepted Paragraph 2.6.3.1 in Council of Europe, 1999bencephalomyelitis

Avian leucosis viruses Cell culture Accepted Covered by paragraph 2.6.3.3 in Council of Europe, 1999b;

PCR Accepted Häuptli et al., 1997

Avian nephritis virus Kidney cell culture Accepted Paragraph 2.6.3.2 of Council of Europe, 1999b

Avian reticuloendotheliosis Chick (or duck) embryo Accepted Paragraph 2.6.3.4 of Council of Europe, 1999bvirus fibroblasts +

immunofluorescence;PCR Available Tagaki et al., 1996

Egg drop syndrome virus Production substrate Available for substrates Paragraph 2.6.3.1 and 2.6.3.2 in Council of (fibroblasts, duck eggs) Europe, 1999bor other sensitive cells

Chicken anaemia virus Cells; Accepted Paragraph 2.6.3.5 in Council of Europe, 1999b;PCR Available Falcone et al., 2000

Marek`s disease virus Chick embryo fibroblasts Accepted Paragraph 2.6.3.2 in Council of Europe, 1999b

Newcastle disease virus Eggs (intra-allantoic Accepted Paragraph 2.6.3.1 in Council of Europe, 1999binoculation)PCR Available Stäuber et al., 1995

Infectious bursal disease virus Eggs (chorio-allantoic membrane Accepted Paragraph 2.6.3.1 in Council of Europe, 1999band intra-allantoic inoculation)PCR Under development

Infectious bronchitis virus Eggs (intra-allantoic inoculation) Accepted Paragraph 2.6.3.1 in Council of Europe, 1999bPCR Available Falcone et al., 1997

Infectious laryngotracheitis Eggs (chorio-allantoic Accepted Paragraph 2.6.3.1 in Council of Europe, 1999virus membrane inoculation)

PCR Available Vögtlin et al., 1999

Salmonella pullorum Culture Accepted Covered by the sterility test stipulated in (Council of Europe, 1997a)

Turkey rhinotracheitis virus Vero cells, chick embryo Accepted Paragraph 2.6.3.2 in Council of Europe, 1999bfibroblasts

Chlamydia spp Eggs (intravitelline injection) Accepted Paragraph 2.6.3.1 in Council of Europe, 1999b

Avian infectious haemorrhagic PCR Available Hess et al., 1999enteritis virus

Avian paramyxovirus-3 Eggs Accepted Paragraph 2.6.3.1 in Council of Europe, 1999b

Duck/goose parvoviruses Duck eggs/ duck embryo Accepted Paragraphs 2.6.3.1 and 2.6.3.2 in Council of fibroblasts Europe, 1999b

Duck enteritis virus Duck embryo fibroblasts Accepted Paragraphs 2.6.3.1 and 2.6.3.2 in Council of Europe, 1999b

Duck hepatitis viruses I and II Eggs Accepted Paragraph 2.6.3.1 in Council of Europe, 1999b

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purpose since they are more sensitive toinfectious virus and allow the testing oflarge volumes of test vaccine (Andersonet al., 1970; Barteling and Vreeswijk,1991).

PotencyPotency testing of foot-and-mouth disease vaccines is carried out as a multi-dilution vaccination-challenge test in 17cattle. In order to reduce the numbers ofanimals, a single-dilution vaccination-challenge could be used. Alternatives tothis highly distressing test have been developed and have been reviewed byWeisser and Hechler (1997); however,they have not yet been validated. Themost suitable alternatives are based oneither antigen quantification (Rweye-mamu et al., 1984; Black et al., 1984;Pay and Hingley; 1987) or estimation ofFMD antibodies in the serum of vac-cinated animals with a sandwich ELISAprocedure (Hamblin et al., 1986) or theplaque reduction test in BHK21-CTcells (Ahl et al., 1987). The latter method has been accepted for batch potency testing in the former GDR(Thalmann et al., 1987).

Rabies vaccine for veterinary useInactivated rabies vaccine According to the Ph. Eur. monographon rabies vaccine for veterinary use, thecomplete inactivation of rabies virus istested by intracerebral injection of 10mice. The sensitivity and relevance ofthis test were questioned (Hendriksen,1988; Weisser and Hechler, 1997). Themanufacturers are now allowed to carryout the test for inactivation in processimmediately before addition of the ad-juvant. Methods for in vitro detection ofactive rabies virus should then be used(for example, methods described by Ullrich, 1993; Blum, 1999) and the teston the finished product can be omitted.

PotencyThe Ph. Eur. monograph on rabies vac-cine for veterinary use allows estimationof rabies antibodies (for example RFFIT)and in vitro antigen quantification (forexample, with an ELISA procedure); however, it is required that the NIH test is performed in parallel (see also 4.2.3Humane endpoints).

Live rabies vaccinesPotencyLive rabies vaccines are used in foxes.For potency testing, a group of at least 25foxes is immunised with the test vaccine,after a given period the vaccines and atleast 10 non-vaccinated control animalsare challenged with the rabies virus. Al-ternatives to this challenge test have notbeen developed. There is a need to de-termine whether the above-mentionedmethods would be suitable to replace thechallenge.

Based on statistical methods, Weisserand Hechler (1997) showed that the num-ber of animals required could be reducedto 15 vaccines and 5 control animals.

The Ph. Eur. monograph allows skip-ping of the batch potency test providedthat consistency of production has beenproven. Since the TAST is not stipulatedfor this vaccine, it is the first veterinaryvaccine, which does not require tests inanimals for routine batch release purposes.

Classical swine fever vaccinePotencyAccording to the Ph. Eur. monograph,the potency of classical swine fever vac-cines is demonstrated with a vaccination-challenge test, which involves 12 piglets.Alternatives based on serum antibodyestimation (Wensvoort et al., 1988) orantigen quantification (Popa et al., 1987)are available; however, they have not yetbeen validated.

Other viral vaccinesFor a number of vaccines (Tab. 13 in Annex 2), the Ph. Eur. prescribes a sero-logical test for potency testing: animalsare immunised and the response to thevaccine is estimated with immunochemi-cal methods. Efforts should be made todevelop and validate in vitro methods(e.g. based on antigen quantification),which do not require animals.

4.2.2.4 Immunosera and antitoxinsTetanus antiserum for veterinary useWith regard to Tetanus Antiserum for Veterinary Use (equine origin) see Tetanus immunoglobulins and antiserafor human use.

Other clostridial antitoxins The Ph. Eur. includes three monographson clostridial antitoxins (C. novyi alpha

antitoxin, C. perfringens beta antitoxinand C. perfringens epsilon antitoxin) forveterinary use. At the symposium on Veterinary Clostridial Vaccines, it wasstated that “EDQM will enquire which seraare still manufactured and if appropriatereconsider the need for the present monographs” (Council of Europe,1997b). In principle, serological assays(e.g. EIAs) being suitable for the potencytesting of clostridial vaccines could beadapted for the testing of clostridial anti-toxins (Ebert et al., 1999c).

Erysipelas immunoserumPotencyThe Ph. Eur. monograph Swine Erysi-pelas Immunoserum also stipulates a multi-ple-dilution vaccination-challenge test inmice for the estimation of potency. ELI-SA procedures have been developed,which can be used for in vitro estimationof protective erysipelas antibodies in im-munosera (Dahms et al., 1991; Beck-mann and Cussler, 1993; Cussler et al.,1995), but have not yet been validated.

The TAST is stipulated in this mono-graph, which is in contrast to the othermonographs for immunosera. Retro-spective analysis of TAST data releasedon the German market show no irregu-larities (Pössnecker and Cussler, 1998).Deletion of the TAST from these mono-graphs is therefore recommended.

4.2.3 Humane endpointsThe potency testing of a number of human (whole-cell pertussis, diphtheria,tetanus, inactivated rabies) and veterinaryvaccines (clostridial, leptospirosis, ery-sipelas) is based on a vaccination-chal-lenge test, i.e. the capacity of the vaccine toprotect against infection or against toxinsis measured by challenging immunisedanimals with the relevant agent. Insuf-ficiently protected animals develop signsof the disease or eventually die of the disease (up to 50%). Most of the mono-graphs name death of the animal or severeclinical signs as endpoints, which usuallyinvolves considerable suffering and severe distress to the animals. Recently, astudy has been carried out which aimedto evaluate and validate the use of hu-mane endpoints as an alternative to severe clinical endpoints in potency testson immunobiologicals and to promotethe implementation and use of humane

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endpoints in guidelines and in the animallaboratory setting.

The working group established for thisstudy (Coenraad Hendriksen, RIVM,Bilthoven, The Netherlands; Klaus Cussler, PEI, Langen, Germany; DavidMorton, University of Birmingham, UK)decided to restrict the study to three vac-cines: erysipelas, rabies, and pertussisvaccines, and set up a list of the para-meters to be evaluated (body weight, bodytemperature and clinical signs, for ex-ample, ruffled fur, hunched back, changesin the movements, paresis, paralysis, pro-stration, agony) with animals monitoredtwice a day. The data obtained for pertussis showed that decrease in bodytemperature to 34.5°C and loss of muscularco-ordination are suitable humane end-points, which could be used for the potency testing of whole pertussis vac-cines. The duration of severe suffering ofthe animals could be shortened by 1-3days (Hendriksen et al., 1999).

Rabies-infected mice did not show anyincrease or decrease in body tempera-ture. Clinical signs and (loss of bodyweight) were more specific, and it wasconcluded that a 15% decrease of bodyweight and clinical signs of neurologicaldisorder used as a combined humaneendpoint would shorten the test period by3 days (Cussler et al., 1998). Guidanceon the use of humane endpoints in batchpotency testing of rabies vaccines is available on video (HELP, 2000).

For the potency testing of erysipelasvaccines, it was not possible to identifyearly predictors of lethality in mice. How-ever, to minimise pain and distress, it wasrecommended that terminally ill ani-mals characterised by lethargy and hypothermia (<34°C) should be killedhumanely.

The results of this study have been pre-sented at the International Conference onThe use of Humane Endpoints in AnimalExperiments for Biomedical Research(22 - 25 November, 1998, Zeist, TheNetherlands; Hendriksen and Morton,1999) and at the 3rd World Congress onAlternatives and Animal Use in the LifeSciences (29 August - 2 September, 1999,Bologna, Italy; Cussler et al., 2000b).

An example for the use of humaneendpoints in vaccination-challenge studies in large animals was recentlypresented by Johannes et al. (1999)

for the testing of swine erysipelas vac-cine.

The general idea of the study, the ap-plication of humane endpoints, has beenconsidered by the European Pharma-copoeia. The draft revision of the generalmonograph for veterinary vaccines nowreads: Animal tests.

In accordance with the provisions ofthe European Convention for the Pro-tection of Vertebrate Animals Used forExperimental and Other Scientific Purposes, tests must be carried out insuch a way as to use the minimum num-ber of animals and to cause the leastpain, suffering, distress or lasting harm.The criteria for judging tests in mono-graphs must be applied in the light ofthis. For example, if it is indicated thatan animal is considered to show positive,infected etc. when typical signs or deathoccur then as soon as sufficient indi-cation of a positive result is obtained the animal in question shall be either humanely killed or given suitable treatment to prevent unnecessary suf-fering (Council of Europe, 2001).

5 Progress and criticism

5.1 Progress

5.1.1 General aspectsThe Three Rs principle has become animportant issue in the development andquality control of immunobiologicals. Inrecent years a number of workshops, sem-inars and conferences have been de-dicated to general and special aspects ofthis issue. The Three Rs concept has been implemented into general guide-lines/monographs. Thus, the general notices in the 3rd Edition of Ph. Eur. allowthe replacement of any test method men-tioned in a monograph: The tests and as-say described are the official methodsupon which the standards of the Pharma-copoeia are based. With the agreementof the competent authority, alternativemethods of analysis may be used forcontrol purposes, provided that the methods used enable an unequivocal de-cision to be made as to whether compli-ance with the standards of the monogra-phs would be achieved if the officialmethods were used. In the event of doubtor dispute, the methods of analysis of the

Pharmacopoeia are alone authoritative(Council of Europe, 1997a).

Already in 1995, the WHO has pub-lished the Manual of Laboratory Methodsfor Potency Testing of Vaccines Used inthe WHO Expanded Programme on Im-munisation (WHO, 1995), which in-cludes in vivo and alternative methodsfor the potency testing of various humanvaccines (e.g. ToBI test for tetanus vac-cines, VERO cell test for diphtheria vac-cines), which can be used provided thatcorrelation can be demonstrated betweenantibody titres and protection.

5.1.2 Specific aspectsAbnormal toxicity test (ATT)Based on the outcome of an enquiry ofthe Ph. Eur. Secretariat (Council of Euro-pe, 1994) and the outcome of a study ofthe PEI (Duchow et al., 1995), the Ph.Eur. Commission decided to delete theATT from all Ph. Eur. monographs forveterinary vaccines, veterinary and hu-man immunosera and immunoglobulinsand from most of the monographs for hu-man vaccines. Due to the introduction ofthe principles of GMP and GLP, the rele-vance of the ATT had been questioned fora number of years. The ATT is still re-quired by some Ph. Eur. monographs on human vaccines, however, it has beenshifted upstream, i.e. it is no longer per-formed on the finished product but inprocess. The ATT can also be omitted forthese vaccines, provided that the manu-facturer could demonstrate that a suf-ficient number of batches gave negativeresults (Schwanig et al., 1997). After deletion of the ATT, there are now severalPh. Eur. monographs on human vaccines,which do not stipulate tests in animals forroutine batch release (Tab. 10). The PEIestimates that these modifications of Ph.Eur. monographs will reduce the totalnumber of animals needed in Germanyfor vaccine quality control by 10 - 20.000mice and guinea pigs per year. Never-theless, the FDA and WHO still stipulatethe ATT; however, the WHO ExpertCommittee on Biological Standardizationrecently stated that the collection of global data on the value of the ATT wouldbe initiated (WHO, 1999b).

Upstream testingAs already mentioned above, severaltests, which used to be carried out on the

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finished product have been shifted up-stream in the production process and arecarried out on the bulk stage or even ear-lier. This principle has been introducedfor safety testing, for example, ATT, re-sidual toxicity testing of clostridial vac-cines for veterinary use, toxicity testingof tetanus and diphtheria vaccines for hu-man use, and for the potency testing ofseveral human vaccines (for example,Haemophilus influenza b, Hepatitis Aand B; see also Tab. 11 Annex 1).

The Ph. Eur. generally accepts the principle of upstream testing for residuallive virus in inactivated vaccines for veterinary use. This normally allows theuse of in vitro methods instead of tests(with questionable sensitivity) in animals.

Introduction of single-dilution testsMany potency tests have been or areclassical multi-dilution tests, which re-quire large number of animals. In recentyears, more and more guidelines/mono-graphs allow the manufacturer to replacethe multi-dilution test with single-dilution test (e.g. WHO for diphtheriaand tetanus, Ph. Eur. diphtheria, tetanus,acellular pertussis, erysipelas).

The potency test for swine erysipelasvaccine had to be changed due to the de-toriation of the International Standard.This included the introduction of a single-dilution assay, which reduced the numberof animals used by more than 70%.

Replacement of animal tests withalternative methodsIntroduction of in vitro methods (basedon antigen quantification) to replace ananimal based model (immunisation-chal-lenge or serological model) has beenachieved for several vaccines, e.g. hepa-titis A and B, inactivated rabies vaccine

for veterinary use. Replacement of thechallenge procedure during potencytesting of inactivated vaccines with invitro methods (serum antibody titrationon cell cultures, immunochemical meth-ods, etc) looks promising for a number ofvaccines; in particular, for tetanus vacci-ne for human and veterinary use (ToBItest and ELISA procedure) and erysipe-las vaccines (ELISA procedure).

With regard to live poliomyelitis vac-cine, the WHO has revised its recom-mendations for quality control and intro-duced the MAPREC test and a transgenicmouse model (TgPVR21 mice) for neurovirulence testing of poliovirus type3 to replace the test in monkeys (Wood etal., 2000).

5.2 CriticismAcceptance and implementation of alternative methodsThe participants of the ECVAM Work-shop 31 (Hendriksen et al., 1998) esti-mated that 9.5-11.5 years are neededfrom the development of a test to its im-plementation in monographs and guide-lines. In particular, the period betweensuccessful validation and the implemen-tation appears to be far too long (3.5years). Reasons for this could be theslow process of multinational agreementto revise pharmacopoeial monographsand guidelines, and the time-consumingand expensive production of sufficientreference material (antigen, sera etc) forthe new test systems. In the light of this,it is hardly understandable why the re-vised monograph on erysipelas vaccineand the currently revised monograph onhuman tetanus vaccine do not include astatement that validated alternative methods could be used for batch potencytesting. By the time of the revision, vali-dation of alternative methods for the po-tency testing of human tetanus vaccineand erysipelas vaccine were well on theirway and Ph. Eur. had been kept informed on the progress. In the case ofseveral clostridial vaccines, this approachof speeding up the implementation andpromoting the use of alternative methodshas been taken although none of the alter-natives had been validated by that time.

There should be an (better) informa-tion system to raise the awareness for ac-cepted alternatives and to ensure that the

legal obligation of implementing them isfollowed. There is evidence that someOMCLs are rather reluctant in acceptingthe use of Three Rs methods or the deletion of animal tests (for example, oneOMCL was still requiring the ATT although it had been deleted). If there isa need for training, then measures shouldbe taken to offer this to OMCLs and manufacturers.

Variation processChanges in the production of a vaccineand the quality control have to be li-censed by the control authorities. If a prod-uct is not centrally licensed, the manu-facturer has to apply for variation to eachnational control authority. This appliesalso to the introduction of an alternativemethod. There should be quick and ef-fective mechanisms for manufacturers toreach a harmonised decision after initiatinga variation to introduce an alternative fora product, which has not undergone acentralised procedure. Until now, this hasnot been achieved and it is most likelythat a manufacturer would not receive aharmonised reply from the competentauthorities (Communication of Dow inCouncil of Europe, 2000h; p. 281).

In addition, the EMEA and the na-tional control authorities should considerthe possibility of waiving or at least reducing the fees for variations, whichare in accordance with the requirementof Directive 86/609/EEC and replace, reduce or refine animal tests.

Retesting of vaccine for batch releaseOMCLs have the possibility to retest vac-cine batches after the manufacturer hassubmitted the dossier for batch release.However, different policies are used inOMCLs in Europe: some repeat all tests,others do not retest. It is clear that retesting uses a substantial number of ani-mals. Since the manufacturer has alreadytested these batches it is questionablewhether this approach can still be justifiedin the age of GMP and quality assurance.

Monographs/guidelines for newimmunobiologicalsAnimal tests are still easily introducedinto monographs without a rationale onnecessity, animal numbers or distress – ajustification could be given e.g. in the

Tab. 10: Human vaccines, which do notrequire animal testing for batch release

Influenza vaccine (split virion)

Influenza vaccine (surface antigen)

Influenza vaccine (whole virion)

Measles vaccine

Mumps vaccine

Rubella vaccine

Typhoid polysaccharide vaccine

Typhoid vaccine (live), (oral, strain Ty 21a)

Varicella vaccine

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preface, when monographs are publishedfor comments in Pharmeuropa.

International harmonisationHarmonisation at a European level will beor has already been overtaken by globali-sation. Most of the manufacturers pro-duce for the world market, so harmonisationof the requirements or mutual recognitionof tests would help to reduce the use ofanimals. However, little progress has been achieved. For example, there is nocommon international approach for po-tency testing of diphtheria and tetanusvaccines, which are amongst the mostused vaccines worldwide. Vaccine batches, which have already been testedaccording to the e.g. WHO requirements,have to be re-tested according to the e.g.Ph. Eur. requirements, due to the lack ofa universally accepted potency test.

European industry reports that the ad-ditional testing in the EU for those manu-facturers who bring in their products fromoffshore increases animal testing in suchsituations (communication of Vose inCouncil of Europe, 2000h, p. 270-272).

Another example is the ATT, whichhas been deleted in Ph. Eur. monographs(see above) but is still required by FDA,USDA and WHO. This problem has re-cently been highlighted at an interna-tional conference where representativesof the two organisations stressed the needto remove this test (Communication ofEgan in Council of Europe, 2000h, p. 282; Communication of Griffith inCouncil of Europe, 2000h, p. 283; WHO,1999b).

With regard to veterinary vaccines, dif-ferent approaches are evident in replacingbatch potency testing of inactivated vaccines. In Europe, the focus is on sero-logical models, which replace the chal-lenge procedure but still require animalswhereas the USA rely on antigen quanti-fication, which does not require animals(Hauer and Clough, 1999).

The ICH and the VICH should under-take efforts to harmonise requirementsand promote mutual recognition.

Target animal safety testAs already outlined in the paragraph onthe TAST, the relevance of this test hasbeen discussed for years without any evident progress. There is hope that the

new initiative of the VICH will be moresuccessful.

6 Future aspects

6.1 Consistency of productionAs mentioned at the beginning of this re-port, a new concept of quality control is al-ready in place for the new well-definedvaccines. In most cases, non-animal meth-ods are used for monitoring consistency at critical steps in the production andtesting of a vaccine. These methods can already be developed during the develop-ment of the product. Therefore, manufac-turers should be encouraged to developsuch methods and control authorities shouldbe encouraged to release batches, whichhave been tested with these methods.

Whether the concept of consistency ofproduction could be also applied to theconventional, less-defined products,should be investigated. In recent years,not only has the knowledge on these con-ventional products increased but alsohighly sophisticated methods such as immunochemical, physico-chemical, invitro functional methods became avail-able which could be used for an accuratefingerprinting of a vaccine (Leenaars etal., 2001). A battery of in vitro tests couldbe used to monitor the stability, confor-mation and integrity of antigens and todefine parameters, which correlate anti-genicity and immunogenicity (Hendrik-sen and Gupta, 2000).

Until now, the concept of consistencyof production is mainly applied to humanvaccines but not for veterinary vaccines.

6.2 Antigen quantitation versus serological methodsIn the introduction, it is stated that thereare two main approaches to replace in vi-vo potency testing of inactivated vac-cines: antigen quantitation and replace-ment of the challenge procedure with a invitro serological method. The latter ap-proach should be considered as an in-terim step, which has a high impact onthe reduction of animal numbers, andeven more important, the suffering due toinfection or intoxication is avoided. Thegoal, however, is the complete replace-ment of the immunisation-challenge model for batch testing purposes.

6.3 Novel vaccine productiontechnologies and new vaccinesIn the last decade, a number of develop-ments have taken place that might havean impact on the use of laboratory ani-mals for the production and quality con-trol of immunobiologicals. GMP andquality assurance systems have beenestablished and the concept of consist-ency of production has been introduced.Conventional vaccines are more definedsince the production technology has con-tinuously been improved. Biotechnolo-gical methods became available whichallow a better understanding of diseasesand mechanism of immune response andthus the improvement of existing immuno-biologicals and the development and production of new, well-defined products.

Regarding the vaccines listed in Annex 1& 2, it is evident that the future has al-ready started: there are several vaccines, so-called subunit (e.g. influenza, meningo-coccal, pneumococcal, HIB, Escherichiacoli, FeLV vaccines) or synthetic (e.g. he-patitis B) vaccines, which are well-definedand released without using animals forbatch potency testing. In contrast to at-tenuated live vaccines, whole cell bacterialvaccines or inactivated vaccines, subunitvaccines contain only fragments of the in-fectious agent; whereas synthetic vaccinesare produced chemically or with recom-binant methods (review article Liljeqvistand Ståhl, 1999). The so-called “Third Re-volution on Vaccines” started about 10years ago with the use of genetic immuni-sation or DNA vaccination. Instead of in-jecting the antigen into the target or-ganism, bacterial plasmid DNA encodingbacterial, viral or parasitic protein antigensare inoculated by various routes (intra-muscular, intradermal, intranasal, oral) andtechniques (needle inoculation, gene gun,air gun, food uptake). Depending on theadministration route, the DNA is taken upby e.g. muscle cells or skin Langerhanscells. This in vivo transfection of the targetcells results in the expression of antigens.The organism recognises the antigen(s)and generates humoral (antibody)- and/orcell-mediated immune response. The cor-relates of immunity can be manipulatede.g. by the method of vaccine delivery, pre-sence of genetic adjuvants or vaccine re-gimen (reviewed articles: Cichutek, 2000;Shedlock and Weiner, 2000).

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DNA vaccines have been used to stim-ulate protective immunity against manyinfectious pathogens, malignancies andautoimmune disorders in animal models(review articles: Donnelly et al., 1997;Donnelly and Ulmer, 1999; Cichutek,2000; Shedlock and Weiner, 2000; Heppell and Davis, 2000; Krishan, 2000;Liu and Ulmer, 2000) and entered intothe first clinical trials about five yearsago. There are ongoing clinical trials in Bcell lymphoma, HIV, influenza, malaria,herpes simplex, hepatitis B, FIV, etc(more information see www.dnavac-cine.com).

The ease of production, their stabilityand possibilities of transport are con-sidered to be the main advantages ofDNA vaccines over conventional vaccines.In addition, cultivation of dangerous in-fectious agents is no longer necessaryand they provide the possibility to inducelong-term immunity against one or multiple pathogens after a single shot.

Numerous articles on DNA vaccinesindicate that large numbers of animals(rodents, cats, primates) are used for thedevelopment of such vaccines. Since newpathogens are emerging, or well-knownpathogens re-emerging, even more ani-mals might be used for the developmentof new vaccines.

Concerns about the safety and efficacyof DNA vaccines have been consideredby EMEA, the WHO and FDA, all ofwhom issued guidelines on their produc-tion and quality control (FDA, 1996;EMEA, 1998; EMEA, 1999 (draft);WHO; 1998). According to the CVMPand CPMP, animals will still be neededduring production in order to demon-strate safety and efficacy; however, fullyvalidated in vitro expression assay wouldbe considered sufficient for establishingbatch potency; whereas the TAST is stillrequired for batch safety testing of veterinary DNA vaccines.

There is agreement that for the timebeing animals will still be needed for thedevelopment of vaccines in order to gainbest knowledge on the disease, the pathogen and the specific immune re-sponse, including: pathogenesis, identi-fication of the protective antigens, the waythe antigen is processed, the dynamics ofthe immune response, the induction ofmemory, and the selection of the best adjuvant (Hendriksen and Gupta, 2000).

With regard to routine batch release ofconventional products, a number ofThree Rs approaches are already availableand should further be developed and validated. Whereas routine batch releaseof new products should be based on invitro methods already established duringtheir development.

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Correspondence toOffice AGAATIYalelaan 17NC-3584 CL UtrechtE-mail: [email protected]

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Terms related to immunobiologicals/methods/production etc.ACPV Acellular pertussis vaccineATT Abnormal toxicity testBCG Bacillus of Calmette and Guérin (Mycobacterium

bovis BCG)BVD Bovine viral diarrhoeaBSP Biological Standardisation ProgrammeCHO Chinese hamster ovary cellsDPT Diphtheria-Pertussis-Tetanus vaccineELISA Enzyme linked immunosorbent assayFeLV Feline leucosis virusFMD Foot and mouth diseaseGLP Good Laboratory PracticeGMP Good Manufacturing PracticeHS Histamine sensitisation testIBR Infectious bovine rhinotracheitisIBRP International Biological Reference PreparationIL-1 Interleukin 1IL-6 Interleukin 6IPV Inactivated poliovirus vaccineLAL Limulus amebocyte lysateLP Leukocytosis promotion testLPS LipopolysaccharidesMAT Micro-agglutination testMAPREC Molecular analysis by PCR and restriction enzyme

cleavageMDCK Madin-Darby-Canine-Kidney cell lineMNVT Monkey neurovirulence testMWG Mouse weight gain testND Newcastle diseaseOPV Oral poliovirus vaccinePCR Polymerase chain reactionRFFIT Rapid fluorescent focus inhibition testSOP Standard operating procedureTAST Target animal safety testTNF-α Tumour necrosis factor αTNT Toxin neutralisation testToBI Toxin binding inhibition testWCPV Whole cell pertussis vaccine

List of abbreviations

Organisations/Institutions/etc.AGAATI Advisory Group on Alternatives to Animal Testing in

ImmunobiologicalsBMBF German Federal Ministry of Education and Research CPMP Committee for Proprietary Medicinal Products (EMEA)CVMP Committee for Veterinary Medicinal Products (EMEA)DZ Doerenkamp-Zbinden FoundationEC European CommissionECBS WHO Expert Committee on Biological StandardizationECVAM European Centre for the Validation of Alternative

Methods, Ispra, IEDQM European Directorate for the Quality of Medicine,

Strasbourg, FEFPIA European Federation of Pharmaceutical Industries'

AssociationsEMEA European Medicines Evaluation Agency, London, UKEPC European Pharmacopoeia CommissionEU European UnionFDA Food and Drug Administration, FFVFF Fonds für Versuchstierfreie Forschung, Zürich, CHIABS International Association for Biologicals, Geneva, CHICH International conference of HarmonisationIWP Immunological Veterinary Medicinal Working Party

(EMEA)IFMPA International Federation of Pharmaceutical

Manufacturers AssociationIn-VITRO The International Veterinary Industry Test Replacement

OrganisationJRC Joint Research Centre (European Commission) Ispra, IMS Member States of the European UnionNIH US National Institute of HealthO.I.E. Offices internationales des épizooties, Paris, FPEI Paul-Ehrlich-Institut, Langen, DPh. Eur. Pharmacopoea Europaea (European Pharmacopoeia),

Strasbourg, FRIVM National Institute of Public Health and the

Environment, Bilthoven, NLset Foundation for the Promotion of Research on

Replacement and Complementary Methods to ReduceAnimal Testing

USDA United States Department of Agriculture, VICH Veterinary International Cooperation on HarmonisationWHO World Health Organization, Geneva, CHZON Zorg Onderzoek Nederland,

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ANNEX 1Tab. 11: Animal tests stipulated for the batch testing of human vaccines and immunosera (based on Ph. Eur. Monographs, Council of Europe, 2000g)

Immunobiological Tests in Animals Carried Numbers/Species Distress* Commentsout on the Final Bulk/Lot

Bacterial vaccines

BCG vaccine Virulent mycobacteria 6 guinea pigs 1 May be omitted on the final lotExcessive dermal reactivity 6 guinea pigs 4 May be omitted on the final lot

Cholera vaccine Antibody production 6 rabbits or guinea pigs or mice 1Cholera vaccine, freeze-dried Antibody production 6 rabbits or guinea pigs or mice

Diphtheria vaccine Specific toxicity (only final lot) 5 guinea pigs/lot 1 Deletion foreseen(and combined vaccines cont. Absence of toxin 5 guinea pigs/bulk 1diphtheria component) Irreversibility of the toxoid in guinea pigs 1 In vitro should be preferred

guinea pigs or cell culturesPotency (challenge) guinea pigs, number not 4 Validation of serological test

defined; 20 control animals with Vero cells foreseen might be required

Haemophilus type B Pyrogens 3 rabbits 1 Table 5conjugate vaccine Potency (serology; final bulk) 16 mice 1

Meningococcal Pyrogens 3 rabbits 1 Table 5polysaccharide vaccine

Pertussis vaccine Specific toxicity 10 mice 1 Table 2(and combined vaccines cont. Potency (challenge) 136 mice 4 Serological model availablepertussis component)

Pertussis acellular vaccine Absence of pertussis toxin 5 mice 1Irreversibility of toxoid 5 mice 1Potency (serology; multi- mice (6 groups of 1dilution) appropriate number)

Pneumococcal poly- Pyrogens 3 rabbits 1 Table 5saccharide vaccine

Tetanus vaccine Specific toxicity (final lot) 5 guinea pigs 1 Deletion foreseen Absence of toxin 5 guinea pigs 1 Deletion as final bulk test forseenIrreversibility of the toxoid mice or guinea pigs 1 Deletion as final bulk test forseenPotency (challenge) guinea pigs or mice; 4 Single-dilution serological test

number not defined foreseen

Typhoid vaccine Antigenic power Susceptible laboratory animals 1 No longer relevantTyphoid vaccine, freeze-dried (estimation of antibodies)

Viral vaccines

Hepatitis A vaccine Potency Mice; number suitable to 1 In vitro method recommended(inactivated) (serology or meet statistical requirements

antigen content) -

Hepatitis B vaccine (rDNA) Pyrogens 3 rabbits 1Potency Mice or guinea pigs; number 1 In vitro method recommended(serology suitable to meet theor requirements for a valid testantigen content) -

Poliomyelitis vaccine Potency (serology 60 chickens/guinea pigs 1 Test in rats recommended in(inactivated) and or rats Ph. Eur. monograph

D-antigen content) -

Poliomyelitis vaccine (oral) Neurovirulence (monovalent > 80 monkeys 5 Alternatives available for harvest) polivirus type 3; under

investigation for type 1 and 2

Rabies vaccine for human Pyrogens 3 rabbits 1 Table 5use prepared in cell cultures Potency (challenge) 6 groups of mice of a suitable 4 Single-dilution assay allowed

size to meet the requirements Table 3for a valid test

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Immunobiological Tests in Animals Carried Numbers/Species Distress* Commentsout on the Final Bulk/Lot

Tick-borne Pyrogens 3 rabbits 1 Table 5encephalitis vaccine Potency (challenge) 6 groups of mice of a suitable 4

size to meet the requirements for a valid test

Yellow fever vaccine Potency (determination of 6 mice per dilution 4mouse LD50; challenge)

Immunosera/Antitoxin/Immunoglobulins

Botulinum antitoxin Potency 4 mice per dilution 4 Serological model available(in vivo toxin neutralisation)

Diphtheria antitoxin Potency 2 guinea pigs or rabbits 4 Vero cell test(intradermal toxin neutralisation) per dilution

European viper venom Potency 6 mice per dilution 4 No alternatives availableantiserum (for each venom, in vivo

toxin neutralisation)

Mixed gas-gangrene antitoxin Potency 6 mice per dilution 4 Table 6(for each toxin, in vivotoxin neutralisation)

Human tetanus Ig Potency 6 mice per dilution 4 Serological methods (in vivo toxin neutralisation (ToBI test) prevalidatedor serology)

Tetanus antitoxin Potency 6 mice per dilution 4 Serological methods (in vivo toxin neutralisation) (ToBI test) prevalidated

Human normal immunoglobulin Pyrogens 3 rabbits 1 Table 5

Human normal immuno- Pyrogens 3 rabbits 1 Table 5globulin for intravenous use

Tuberculins

Tuberculin purified protein Identification (combined with derivative for human use potency) (and Old tuberculin) Toxicity 2 guinea pigs 1 Deletion recommended

Sensitisation 6 guinea pigs 4 Alternative availablePotency 6 guinea pigs 1

* Distress categories: 1 – slight; 2 – moderate; 3 – severe, duration <1 day; 4 – severe, duration 1-7 days; 5 – severe, duration 7-30 days; 6 – severe, duration > 30 days

ANNEX 2Animal tests stipulated for the batch testing of veterinary vaccines and immunosera (based on Ph. Eur. Monographs, Council of Europe, 2000g)

Tab. 12: Batch potency testing with immunisation-challenge model

Immunobiological Tests in Animals Numbers/Species Distress* Comments

Bacterial vaccines

Anthrax spore vaccine Safety 2 susceptible animals 1Potency (challenge) 13 guinea pigs (or rabbits) 4 In vitro quantification of live

or 8 sheep anthrax spores

Brucellosis vaccine Safety 2 sheep 150% persistence time 64 mice 2

Clostridium botulinum vaccine Safety 2 susceptible animals 1Residual toxicity 5 mice 1Potency (challenge) 30 mice 4

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Clostridium chauvoei vaccine Safety 2 susceptible animals 1Potency (challenge) 15 guinea pigs 4 Table 6

Clostridium novyi Safety 2 sheep 1(type B) vaccine Residual toxicity 5 mice 1 May be omitted by manufacturer

Potency Table 6(in vivo neutralisation 10 rabbits 4

at least 2 animals/dilution Validated immunochemical at least one repetition methods are allowed for routine

batch controlor serology) 10 rabbits 1

Clostridium perfringens Safety 2 susceptible animals 1vaccine Residual toxicity 5 mice 1 May be omitted by manufacturer

Potency Table 6(in vivo neutralisation 10 rabbits 4

at least 2 animals/dilution/toxin Validated immunochemical at least one repetition methods are allowed for routine

batch controlor serology) 10 rabbits 1

Clostridium septicum vaccine Safety 2 susceptible animals 1Residual toxicity 5 mice 1 May be omitted by manufacturerPotency Table 6(in vivo neutralisation 10 rabbits 4

at least 2 animals/dilution/toxin Validated immunochemicalor at least one repetition methods are allowed for routine

batch controlserology) 10 rabbits 1

Furunculosis vaccine Safety 10 fish 1Potency Reduction of animal numbers (challenge 60 fish 4 should be investigatedor serology) 35 fish 1

Leptospira vaccine Safety 2 target animals 1Potency (challenge) 10 hamsters 4 Table 8

Swine erysipelas vaccine Safety 2 piglets 1Potency (challenge) 30 mice 4 Validated alternative available

Tetanus vaccine Toxicity 5 guinea pigs 1Potency (vaccination and at least 2 mice per dilution 4 Replacement of animal test with in vivo neutralisation) at least one repetition ELISA or ToBI is expected soon

Vibriosis (cold-water) vaccine Safety 10 fish 1 Reduction of animal numbers Potency should be investigated(challenge 60 fish 4or serology) 35 fish 1

Vibriosis vaccine Safety 10 fish 1 Reduction of animal numbersPotency should be investigated(challenge 60 fish 4or serology) 35 fish 1

Viral vaccines

Aujeszky’s disease vaccine Safety 3 piglets 1(inactivated) Potency (challenge 10 pigs 4

or suitable validated method)

Avian infectious bronchitis Safety combined; 10 chickens 1vaccine (inactivated) Extraneous agents Table 9

Potency (suitable validated method)

Avian infectious bronchitis Safety 10 chickens 1vaccine (live) Extraneous agents 10 chickens 1 Table 9

}

Tab. 12: continued

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Avian infectious bursal Safety combined; 10 chickens 1disease vaccine (inactivated) Extraneous agents 1 Table 9

Inactivation (only for certain 10 chickens 1strains)Potency (serology) 20 chickens 1

Avian infectious Safety 10 chickens 1encephalomyelitis vaccine (live) Extraneous agents 10 chickens 1 Table 9

Avian infectious Safety 10 chickens 1laryngotracheitis vaccine (live) Extraneous agents 10 chickens 1 Table 9

Avian paramyxovirus 3 Safety 10 turkeys 1(inactivated) Extraneous agents 10 chickens 1 Table 9

Potency (suitable validated method)

Duck viral hepatitis vaccine Safety 10 ducklings 1Extraneous agents 10 ducklings 1 Table 9

Foot-and-mouth disease Safety 3 cattle 1(ruminants) vaccine Potency (challenge) 17 cattle 3(inactivated)

Fowl-pox vaccine (live) Safety 10 chickens 1Extraneous agents 10 chickens 1

Newcastle disease Safety 10 chickens (or 10 other birds) 1vaccine (inactivated) Extraneous agents 10 chickens (or 10 other birds) 1 Table 9

Potency (serology 20 chickens or 1or challenge) 70 chickens (or 30 other birds) 4

Newcastle disease vaccine Safety 10 chickens 1(live) Extraneous agents 10 chickens 1 Table 9

Rabies vaccine (inactivated) Safety 2 dogs 1for veterinary use Inactivation (adjuvanted 10 mice 1

vaccine)Potency (serology) 5 mice 1

Swine fever vaccine (live), Safety 3 piglets 1classical Extraneous agents 10 mice 1

Potency (challenge) 12 piglets 4

Immunosera/Antitoxins

Clostridium novyi alpha Potency At least two mice per dilution 4antitoxin (in vivo neutralisation) step; At least one repetition

Clostridium perfringens Potency At least two mice per dilution 4beta antitoxin (in vivo neutralisation) step; At least one repetition

Clostridium perfringens Potency At least two mice per dilution 4epsilon antitoxin (in vivo neutralisation)

Swine erysipelas Safety 2 pigs 1immunoserum Potency (challenge) 70 mice 4

Tetanus antitoxin for Potency At least two mice per dilution 4veterinary use (in vivo neutralisation) step; At least one repetition


}

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Tab. 13: Batch potency testing is based on serological model

Immunobiological Batch tests involving animals Number of Animals/Species Distress*

Neonatal piglet colibacillosis vaccine (inactivated) Safety 2 piglets 1Potency (serology) 7 pigs or 1

7 rabbits, guinea pigs or mice 1

Neonatal ruminant colibacillosis vaccine (inactivated) Safety 2 calves 1Potency (serology) 7 rabbits, guinea pigs or mice 1

Porcine actinobacillosis vaccine Safety 2 pigs 1Potency (serology) 5 mice 1

Porcine progressive atrophic rhinitis vaccine Safety 2 pigs 1Potency (serology) 7 pigs or 7 laboratory animals

Canine parvovirus vaccine (inactivated) Safety 2 dogs 1Potency (serology) 2 dogs or 5 guinea pigs 1

Egg drop syndrome ’76 vaccine (inactivated) Safety 10 chickens 1Potency (serology) 10 chickens 1

Equine influenza vaccine (inactivated) Safety 2 horses 1Potency (serology) 5 guinea pigs

Feline calicivirosis vaccine (inactivated) Safety 2 cats 1Potency (serology) 15 mice

Feline infectious enteritis vaccine (inactivated) Safety 2 cats 1Potency (serology) 2 or 4 cats 1

or guinea pigs for routine control

Feline viral rhinotracheitis vaccine (inactivated) Safety 2 cats 1Potency (serology) 15 mice 1

Porcine influenza vaccine (inactivated) Safety 2 pigs 1Potency (serology) 5 guinea pigs

Porcine parvovirus vaccine (inactivated) Safety 2 pigs 1Potency (serology) 5 guinea pigs


Tab. 14: The only routine batch tests involving animals is the target animal safety test

Immunobiological Batch tests involving animals Numbers of Animals/Species Distress*

Aujeszky’s disease vaccine (live) Safety 3 piglets 1

Bovine parainfluenza virus vaccine (live) Safety 2 calves 1

Bovine respiratory syncytical virus vaccine (live) Safety 2 calves 1

Canine adenovirus vaccine (inactivated) Safety 2 dogs 1

Canine contagious hepatitis vaccine (live) Safety 2 dogs 1

Canine distemper vaccine (live) Safety 2 dogs 1

Canine parvovirus vaccine (live) Safety 2 dogs 1

Distemper vaccine (live) for mustelids Safety 2 ferrets 1

Feline calicivirosis vaccine (live) Safety 2 cats 1

Feline infectious enteritis vaccine (live) Safety 2 cats 1

Feline leukaemia vaccine (inactivated) Safety 2 cats 1

Feline viral rhinotracheitis vaccine (live) Safety 2 cats 1

Infectious bovine rhinotracheitis vaccine (live) Safety 2 calves 1


three rs potential in the development and quality control of ... · nisations (e.g. who, o.i.e.)...

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