Veterinary vaccine post-licensing safety testing: overview of current regulatory requirements and accepted alternatives

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Procedia in Vaccinology 5 (2011) 236 – 247 Procedia in Vaccinology 5 (2011) 236 – 247

NICEATM-ICCVAM # International Workshop on Alternative Methods to Reduce, Refine, and Replace the Use of Animals in Vaccine Potency and Safety Testing: State of the Science and Future Directions Bethesda, Maryland, USA, 14-16 September 2010

Veterinary vaccine post-licensing safety testing: overview of current regulatory requirements and accepted alternatives Glen Gifford , Pawan Agrawal, Donna Hutchings, and Oksana Yarosh Canadian Centre for Veterinary Biologics, Terrestrial Animal Health Division, Animal Health Directorate, Canadian Food Inspection Agency, 59 Camelot Drive, Ottawa, Ontario, Canada, K1A 0Y9

Abstract Manufacturers of veterinary vaccines frequently incorporate animal-based batch release safety tests into their quality assurance monitoring protocols to meet their internal quality standards and to conform to government regulatory requirements. These tests are conducted by vaccinating target species animals or laboratory animals with a single dose or multiple doses of the test batch, and observing the vaccinated animals for signs of local or systemic adverse reactions. Manufacturers, standard-setting bodies, animal welfare advocacy groups, and regulatory agencies are actively investigating alternative methods to reduce their reliance on animal-based methods for batch release safety testing. Approaches which have been implemented or proposed include harmonizing technical requirements, developing in vitro tests, refining the existing animal tests, improving adverse reaction monitoring (vaccinovigilance), and improving manufacturing methods and quality controls to reduce batch-to-batch variability. An approach, known as the consistency approach, is increasingly being acknowledged as a potentially viable alternative to animal-based batch release tests for vaccines. This paper will provide an overview of currently utilized batch release safety tests for veterinary vaccines, the associated regulatory requirements, and some potentially acceptable alternative approaches for reducing, refining, and replacing the use of animals in these tests.

© 2011 Published by Elsevier Ltd. Selection and/or peer-review under responsibility of the National Toxicology © 2011 Interagency Published by Elsevier Ltd. Selection under responsibility of the National Toxicology Program © 2011 Published by Elsevier Ltd. Selection and/orpeer-review peer-review under responsibility of the National Toxicology Program Center for the Evaluation ofand/or Alternative Toxicological Methods (NICEATM). Interagency Center for the Evaluation Alternative of Toxicological (NICEATM) Program Interagency Center for theofEvaluation AlternativeMethods Toxicological Methods (NICEATM). Keywords: veterinary vaccines; post-licensing safety test; regulatory requirements; alternative methods

1. Introduction Post-licensing batch release safety tests for veterinary vaccines are quality control tests that are conducted on samples from individual batches of a licensed (registered) vaccine, as part of a comprehensive quality assurance monitoring system [2, 6, 7, 8, 28, 29, 30, 39, 43]. These tests serve as broad-spectrum bioassay to assess the biological properties of each batch of a licensed (registered) vaccine, to detect any signs of abnormal toxicity that #

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The National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods and the Interagency Coordinating Committee on the Validation of Alternative Methods *Corresponding Corresponding author author e-mail e-mailaddress: address:[email protected] [email protected]

1877-282X 1877-282X©©2011 2011Published PublishedbybyElsevier ElsevierLtd. Ltd.Selection Selectionand/or and/orpeer-review peer-reviewunder underresponsibility responsibilityof ofthe theNational National Toxicology Toxicology Program Program Interagency Interagency Center Center for forthe theEvaluation EvaluationofofAlternative AlternativeToxicological ToxicologicalMethods Methods(NICEATM). (NICEATM). doi:10.1016/j.provac.2011.10.025 doi:10.1016/j.provac.2011.10.00110.1016/j.provac.2011.10.025

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may have been overlooked by manufacturing controls and in-process tests, which focus on specific quality parameters. Veterinary vaccines are complex mixtures of biological components, and are subject to some inherent batch-tobatch variability that might affect the safety of the product. Accordingly, in addition to conducting pre-licensing safety testing, manufacturers are also generally required to conduct animal-based safety tests on each batch of finished product prior to release [6, 10, 43, 44], unless it can be clearly demonstrated that this end-product testing is not necessary [10, 25]. These tests are conducted on the finished vaccine (bulk containers or final bottles), and can therefore potentially assess the cumulative effects of any minor deviations in the formulation of the product. The batch safety tests are ordinarily conducted by the manufacturer, and are not usually repeated by the regulatory agencies, except possibly to conform with import requirements [37, 40]. Table 1 provides some examples of the potential applications of in vitro or in vivo batch release safety tests for veterinary vaccines. Table 1. Examples of vaccine safety parameters that could be assessed by in vitro or in vivo batch release safety tests. Residual viable vaccine organisms in inactivated viral or bacterial vaccines Contamination of the finished product by pathogenic extraneous agents Elevated levels of bacterial endotoxin (lipopolysaccharide) in vaccines derived from Gram negative bacteria Elevated levels of biologically active tetanus neurotoxin in tetanus toxoid vaccines Increased neurovirulence in attenuated live virus vaccines Residual neurovirulence in inactivated virus vaccines Elevated levels of residual formaldehyde in inactivated bacterial vaccines.

In comparison with pharmaceutical products, finished product testing is relatively more important for veterinary vaccines because these products are complex biological products which are comprised of a mixture of relatively uncharacterized antigens originating from pathogenic organisms and their potentially toxic by-products [2, 28, 29, 30, 35, 36, 39]. In comparison with a pharmaceutical product, the protective components and potentially toxic factors in a veterinary vaccine are much less well characterized, and the composition of the finished vaccine is less uniform than a pharmaceutical product. Although the manufacturing methods for veterinary vaccines are precisely controlled, due to the nature of the products, it is possible that there could be undetected variations in the composition of the finished vaccine that might adversely affect product safety. These batch-to-batch variations could involve toxic elements or contaminants that are difficult to detect by the manufacturers’ in vitro quality control testing. Veterinary vaccines also tend to be relatively less purified than vaccines intended for human use, which leads to a higher risk for contamination by toxic elements than comparable human vaccines. Consequently, in addition to meeting licensing requirements through comprehensive pre-licensing safety, efficacy, and quality testing, and conformance with good manufacturing practice (GMP) principles or equivalent standards, as a supplemental precautionary measure, veterinary vaccines are usually also required to undergo quality control tests to verify the potency and safety of each batch of licensed vaccine. The current regulatory standards for veterinary vaccines in Europe, the United States, and other countries include general provisions for development, validation, and approval of alternative methods to reduce, refine, or replace the use of animals for batch release safety testing [10, 11, 12, 24, 25, 42, 43]. In Europe, there are specific provisions for elimination of animal-based batch release safety tests for veterinary vaccines, if other compensatory regulatory controls and quality control tests are implemented to provide assurances about batch-to-batch consistency and safety of the product [10, 11, 12, 25]. This strategy for improving quality control and implementing the 3Rs is known as the consistency approach [11, 16]. This paper will provide an overview of currently utilized batch release safety tests for veterinary vaccines, and the associated regulatory requirements. It will also review some potentially acceptable alternative approaches for reducing, refining, and replacing the use of animals in these tests.

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2. Regulations and technical standards To meet licensing (registration) requirements, veterinary vaccines must be shown in comprehensive pre-licensing testing to be pure, potent, safe and effective when used in the target species according to the approved label recommendations [6, 7, 9, 10, 21, 23, 24, 26, 44, 45]. Manufacturers are also required to demonstrate that they can consistently produce batches that meet the established quality control criteria [6, 7, 9, 10, 21, 44, 45]. The regulatory requirements for batch release safety testing are outlined in legislation, regulations, and technical standards that are implemented by government regulatory agencies, such as the United States Department of Agriculture Center for Veterinary Biologics (USDA CVB) which regulates veterinary vaccines in the United States [43], and the European Medicines Agency (EMA) which serves as the centralized product registration agency for member states of the European Union [7]. The regulatory agencies (competent authorities) of other countries tend to have regulatory frameworks based on either the European or American standards. Several standard-setting bodies also play an important role in establishing common technical standards for veterinary vaccines. Examples include the World Organisation for Animal Health-Office Internationale des Epizooties (OIE) [40], the Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme (PIC/S), the Veterinary International Cooperation for Harmonization (VICH) [41], and the International Association for Biologicals (IABS) [34]. The European requirements for batch release safety testing are outlined in Eudralex Volume 7 Scientific guidelines for medicinal products for veterinary use [7], the European Pharmacopoeia (Ph. Eur.) General Monograph on Vaccines for Veterinary Use [10], and individual Ph. Eur. Monographs for vaccines for specific diseases, such as anthrax [12], salmonellosis [13], tetanus [14], and vibriosis of salmonids [15]. The USDA CVB requirements for batch release safety testing are based on the Virus Serum Toxin Act, the United States Code of Federal Regulations Title 9 Animal Products (9 CFR), and a series of associated guidance documents (Veterinary Services Memorandums, Center for Veterinary Biologics Public Notices, and Supplemental Assay Methods) [42, 43]. 3. Good manufacturing practices The manufacturing standards and associated inspection procedures for veterinary vaccine manufacturing facilities are outlined in various regulatory documents and technical documents, including the 9 CFR [44], the Rules Governing Medicinal Products for the European Union [7] and harmonized GMP protocols established by the Pharmaceutical Inspection Convention (PIC) and the Pharmaceutical Inspection Co-operation Scheme (PIC Scheme), which function jointly as the PIC/S [41]. The PIC/S is an independent standard-setting body whose mission is "to lead the international development, implementation and maintenance of harmonised Good Manufacturing Practice (GMP) standards and quality systems of inspectorates in the field of medicinal products" [41]. 4. Animal-based batch safety testing protocols – 9 CFR and Ph. Eur. Manufacturers, in accordance with standardized testing protocols that are documented in an approved production outline or special outline, or product master file, ordinarily conduct batch release safety tests. Manufacturers located in the United States or countries whose system is based on 9 CFR standards use production outlines and special outlines. A product master file is prepared by manufacturers located in the European Union or countries whose regulatory system is based on European Union Good Manufacturing Practices (GMP) standards and the Ph. Eur. The formats for the production outline and product master file are different, however they serve the same purpose – to document the materials and methods that are used to manufacture and test the regulated product. For products manufactured to conform to requirements of the 9 CFR, the currently employed in vivo batch release safety tests involve administration of vaccine to small numbers of animals of the species for which the product is intended (target species), or laboratory animals [44, 45]. Batch release safety tests conducted in the target species (i.e., dogs, cats, cattle, horses, pigs) generally use two animals, while safety tests conducted in laboratory animals generally use eight mice or two guinea pigs. If a product may be used in various ages, classes, or species of

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animals, the tests are ordinarily conducted in the youngest age animal that would receive the vaccine since they would probably be the most susceptible to adverse effects. To meet the 9 CFR requirements, the test dose for products tested in the target species is typically either a single (1x) dose, a 2x overdose (for inactivated vaccines), or a 10x overdose (for live attenuated vaccines) [44, 45]. The 2x and 10x overdose tests are intended to increase the likelihood of detecting unsatisfactory serials by (1) amplifying the effects of any toxic factor, and (2) increasing the volume of the innoculum for enhanced detection of low level contamination with residual viable vaccine organisms or infectious contaminants. A 1x dose is typically used when the batch safety test is conducted as the preliminary phase of an animal potency test. The test vaccine is ordinarily administered by the recommended route(s); however it may be administered by other routes or to additional species for specific purposes. For example, inactivated rabies vaccines are tested by intracerebral inoculation of mice and rabbits as well as intramuscular administration in the most susceptible target species [44]. In most protocols, only one test dose is administered, however some protocols include a booster dose. For overdose tests, the vaccine is administered at multiple injection sites. Since the protective immune responses in fish are not as well characterized as those in domestic mammals and poultry, the batch release protocols for fish vaccines often require testing groups of vaccinates and control fish in a vaccination-challenge test to demonstrate protection from challenge as a measure of potency through a parameter known as relative percent survival (RPS) [15, 31, 37]. To minimize the numbers of animals required for batch release testing, the vaccinated fish that are used in potency testing can also be used to generate safety information, based on their response to vaccination during the pre-challenge phase of the batch release potency test [15, 31, 37]. The sample sizes for batch safety tests are relatively small to minimize the use of experimental animals. To conform with the 9 CFR and the Ph. Eur., the batch release safety tests for products intended for use in mammals are usually conducted in two animals, while products intended for use in birds or fish are tested in 10 or more birds or fish [8, 10, 11, 12, 13, 14, 15, 21, 37, 44, 45]. In some safety test protocols, the batches are tested in the target species at the recommended dose during the post-vaccination phase of an animal batch release potency test. It has been noted that, when safety tests are conducted by vaccinating target species animals according to the label recommendations, there is usually no major animal suffering associated with these tests [3]. In Europe, batch release safety testing for veterinary vaccines usually involves administration of a 2x overdose of an inactivated vaccine or a 10x overdose of a live vaccine by the recommended route [12, 13, 14, 15]. To meet the Ph. Eur. requirements, the tests must be conducted in target species animals, using at least two mammals or 10 birds or fish. In Europe, the batch release safety tests are not ordinarily conducted in laboratory animals. The observation period for batch release safety tests, to conform to 9 CFR and Ph. Eur. Requirements, is usually 7, 14, or 21 days. During the observation period, animals are examined daily for evidence of local or systemic adverse reactions [10, 12, 13, 14, 15, 44]. Some regulatory agencies, including the USDA CVB and the Canadian Food Inspection Agency Canadian Centre for Veterinary Biologics (CFIA CCVB), allow batch release safety testing in species other than the target species (e.g., sheep, laboratory mice, rabbits, guinea pigs, chickens) [6, 44, 45]. The tests conducted in laboratory animals are primarily intended to detect significant changes in acute toxicity, rather than being a precise assessment of local injection site reactions. Consequently, the test dose for laboratory animals is standardized at a predetermined volume, as prescribed in regulations and guidance documents, and is based on established generic protocols, rather than requiring manufacturers to calibrate an appropriate test dose for each product. The test dose is usually less than the dose for the target species, and in some protocols, a different route of administration is used. For example, to conform to the 9 CFR 113.33 requirements, bacterins may be tested in mice by administration of 0.5 mL of the test bacterin via the subcutaneous or intraperitoneal route [44]. Title 9 of the CFR includes standard requirement protocols for batch release safety tests to be conducted in target animals or laboratory animals [44]. Target animals and laboratory animals may be used to test some products. Manufacturers may implement the standard tests as described in the 9 CFR, or they may implement alternative methods that must be documented and approved in the production outline. To reduce the use of animals for regulatory testing, both the 9 CFR and the Ph. Eur. have provisions for conducting safety tests on bulk samples from bulk containers that are used to formulate multiple batches of vaccine [10, 44]. Where multiple batches are prepared from the same bulk, if the safety tests are satisfactory on the first batch, the tests may be omitted for subsequent batches produced from the same bulk [10, 44, 45]. Similarly, if

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animal-based tests are used for batch release potency tests, the post-vaccination phase of the batch potency test can be used to generate batch safety data [10, 44, 45]. 5. World Organisation for Animal Health – Office Internationale des Epizooties (OIE) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals Part 2 Part 2 of the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals [40] describes (1) specific test methods for diagnostic tests for infectious agents, and (2) procedures that have been adopted for preparing and testing veterinary vaccines and diagnostic tests for use in prevention or diagnosis against infectious agents that are of concern for international trade in livestock and poultry. In the OIE Manual, the prescribed safety tests for inactivated vaccines involve inoculating two animals with a 2x overdose via each recommended route. A 10x overdose is recommended for live vaccines. For some vaccines, the tests may be conducted in laboratory species (i.e., mice or guinea pigs) however the OIE Manual does not specify precise testing protocols. Generally, the OIE batch release safety test protocols recommend using seronegative animals and the test animals are observed for 14 days for signs of local or systemic reactions, because one of the main objectives of the OIE vaccine tests is to identify residual infectious material or extraneous agents in attenuated or inactivated vaccines. Some OIE product batch release safety testing protocols recommend testing in laboratory animals, including administration by the intracerebral route in mice or chicks, to test for presence of neuropathogenic viruses such as Newcastle Disease virus, rabies virus, and Japanese encephalitis virus. However, the OIE Manual also allows for substitution of alternative methods where appropriate. For example, safety tests to detect residual live rabies virus in batches of inactivated rabies virus vaccines may be carried out either by intracerebral injection into mice, or inoculation of cell culture. To conform to the OIE Manual [40], batch release safety tests for live attenuated rabies vaccines must be carried out on each lot of vaccine in the intended host species, because there are species differences in susceptibility to rabies virus infection and clinical disease. According to the prescribed protocol, at least three, preferably five or six animals of the intended host species should be given a dose equivalent to 10 times the recommended field dose, by the recommended route of administration. The animals should be observed for 90 days for adverse reactions attributable to the vaccine. 6. International harmonization Regulatory agencies, international standard setting bodies, and industry associations are actively involved in development of harmonized technical standards for veterinary vaccines, including alternatives to animal testing [17, 21, 26, 27, 34, 36, 37, 38, 39, 40, 44, 45, 46]. Current activities include applied research on humane endpoints [3,4,5], standardization of laboratory test methods [46], production and exchange of reference reagents [45, 46], development of harmonized standards for batch release safety testing [46], information exchange [34, 35, 37], and publication of technical reference documents [4, 38, 39, 40, 41]. Table 2 lists some of the groups involved in these initiatives. 7. Predictive capacity of current animal-based batch release safety methods Although animal-based batch release safety tests would be expected to generate some level of supplemental information about the safety of a specific batch of vaccines, the incremental added value of a specific test is difficult to quantify in practical terms. The overall utility of an animal-based batch release safety test, its predictive capabilities, and its relative importance for quality monitoring depend on several factors, including the following: x Inherent safety of the product, and likelihood of producing batches with abnormally high toxicity x Consistency of manufacturing processes x Potential batch-to-batch variability of the specific product x Reliability of manufacturing controls, in-process tests, or finished product tests x Potential impacts of inadvertent release of unsatisfactory batches

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Table 2. Examples of organizations involved in the development and implementation of harmonized technical standards and regulatory approval procedures for veterinary vaccines. World Organisation for Animal Health - Office Internationale des Epizooties (OIE) Veterinary International Cooperation for Harmonisation of Technical Requirements for Veterinary Medicinal Products (VICH) European Directorate for the Quality of Medicines & HealthCare (EDQM) Paul Ehrlich Institute (PEI) Committee of the Americas for Veterinary Medicines (CAMEVET). International Association for Biologicals (IABS) European Centre for the Validation of Alternative Methods (ECVAM) European Partnership for Alternative Approaches to Animal Testing (EPAA) National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) Institute for International Cooperation in Animal Biologics (IICAB) International Federation for Animal Health (IFAH) Pharmaceutical Inspection Convention (PIC) and the Pharmaceutical Inspection Co-operation Scheme (PIC Scheme), collectively known as PIC/S.

In vivo batch release safety tests have been widely used for many years, and it would be expected that they would provide important safety information. Nonetheless, because batch release test results are generally conducted in manufacturer’s quality control laboratories, the individual test reports and summary data tends to be retained in manufacturers’ confidential product files, and used only for reporting batch release test results to regulatory agencies. Consequently, there is relatively little published summary data available to characterize the actual predictive value of the animal-based batch release safety tests that are currently being used for veterinary vaccines. In the absence of supporting data, several authors have questioned the predictive capacity of these test protocols, and recommended that alternative methods be employed [1, 16, 17, 18, 19, 20, 21, 22, 31, 32, 34, 37, 42, 43]. The authors note the following main limitations of the currently utilized in vivo batch release safety tests as follows: x Small sample sizes (as few as two animals per test), which may limit the statistical validity x Potential variability of individual animal responses to vaccination x Rudimentary pass/fail criteria (observation of clinical signs, visible/palpable injection site reactions, survival) x Lack of validation data for laboratory animal tests to establish the biological relevance, statistical validity, and predictive value of the laboratory animal test. This leads to uncertainty about the correlation of the laboratory animal test results with potential effects in the target species. The above factors may limit the capacity of the animal-based tests to detect unsatisfactory batches, except for those batches with major deviations in the product composition due to formulation errors or introduction of contaminants. Due to animal welfare concerns, which are compounded by questions about the biological relevance of the tests, and uncertainty about their predictive value, the appropriateness of animal-based batch release safety tests is increasingly being questioned [1, 16, 17, 18, 19, 20, 21, 22, 25, 28, 31, 32, 33, 34, 37, 42, 43]. To address this issue, several groups have proposed that the current test methods be refined or replaced by alternative methods such as the consistency approach [19], for reasons such as those listed in Table 3. 8. Alternative approaches As part of a broadly based “3R” initiative to reduce, refine, and replace the use of animals for research and regulatory testing, proceedings from several international forums have recommended that industry and regulatory agencies should implement alternative methods to reduce, refine, and replace the use of animals in batch release testing of veterinary vaccines [1, 3, 16, 17, 18, 19, 20, 21, 22, 28, 31, 32, 33, 34, 37, 42, 43]. Considerable progress

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has been made toward improvement of manufacturing standards and development of new test methods to characterize veterinary vaccines. These advances have helped to reduce and refine animal-based safety tests for batch release. However, animal-based batch release safety tests are still employed for many products – frequently as a component of animal-based batch release potency tests. Table 3. Reasons cited for moving to reducing, refining or (preferably) replacing animal-based batch release safety tests for veterinary vaccines Improved consistency of veterinary vaccine manufacturing procedures has diminished the batch-to-batch variability of licensed vaccines Lack of validation data for current batch release safety tests to characterize their predictive value for detecting unsatisfactory batches Availability of improved in vitro methods to assess product composition and verify freedom from microbial contamination Low overall rate of adverse reactions associated with use of licensed veterinary biologics Animal welfare concerns for test animals

The European and American regulatory systems both include provisions for approval of 3R alternatives to reduce, refine, or replace the use of animals for post-licensing batch release safety testing of veterinary vaccines [10, 25, 44, 45]. Manufacturers may implement batch release test protocols based on standard requirements, as listed in the Ph. Eur. or 9 CFR; or they may propose alternative test methods in the licensing (registration) documentation. The Ph. Eur. Monograph 62 (Vaccines for Veterinary Use) states that the routine application of the batch release safety test may be waived for established vaccines provided the consistency of production has been demonstrated for at least 10 consecutive batches [10]. The EMA has informed manufacturers, through a guidance document [25], that the EMA will consider requests for exemptions to eliminate the requirement for in vivo batch release testing, provided the manufacturers can furnish supporting data to demonstrate consistency and safety for multiple consecutive batches. Manufacturers in the United States, Canada, and other countries can also propose alternative methods and request exemptions from prescribed standard test requirements under existing regulations and policies [6, 44, 45]. For example, the United States 9 CFR Chapter 113 (Standard Requirements) includes provisions for authorizing exemptions from standard requirements (see 9 CFR 113.4 Exemptions to Tests) [44]. Manufacturers that are regulated by the USDA CVB are required to document their manufacturing and testing procedures in an approved production outline or special outlines that can either cite standard batch safety testing protocols as outlined in 9 CFR, or describe alternative methods [44, 45]. For regulatory approval, the proposed alternative methods would need to be biologically relevant and appropriately validated. 9. Regulatory approval procedures for European Union – consistency approach The EudraLex guidelines entitled “Rules Governing Medicinal Products in the European Union Volume 7 Scientific Guidelines” outline the requirements for medicinal products for veterinary use [7]. A series of corresponding European Pharmacopoeia (Ph. Eur.) notices and monographs [8, 9, 10, 11, 12, 13, 14, 15] describe the technical specifications for licensing veterinary vaccines in European Union countries through the centralized procedure that is administered by the EMA, including standards for pre-licensing safety testing and post-licensing safety testing. The Ph. Eur. notices and monographs also outline the procedures to follow to request an exemption from batch release safety testing [10, 11). The requirements are further clarified in a Position Paper [25]. The Ph. Eur. Chapter 5.2.6 (Evaluation of Safety of Veterinary Vaccines and Immunosera) [9], describes recommended protocols for conducting comprehensive pre-licensing laboratory safety tests under the following headings: x Safety of administration of one dose x Examination of reproductive performance x Safety of one administration of an overdose (2x for inactivated vaccines and 10x overdose for live attenuated vaccines)

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x Safety of repeated administration of one dose; residues; adverse effects on immunological functions, and adverse effects from interactions Chapter 5.2.6 of the Ph. Eur. also lists additional special requirements for testing live vaccines under the following headings: x Spread of the vaccine strain; x Dissemination in vaccinated animal; x Increase in virulence; biological properties of vaccine strain; and x Recombination or genomic re-assortment of the vaccine strain [9]. Specific post-licensing batch release safety testing requirements are noted in Chapter 5.2.9 (Evaluation of Safety of Each Batch of Veterinary Vaccines and Immunosera) [8]. Additional information is provided in individual monographs for each major class of veterinary vaccine, such as vaccines for anthrax [12], salmonellosis [13], tetanus [14], and vibriosis in salmonids [15]. These monographs typically specify that post-licensing safety tests must be conducted in target animals, by administering a 2x or a 10x overdose to either two or 10 test animals, depending on the species. Tests in mammals require two animals per test. Tests in birds and fish require at least ten birds or fish. Tetanus vaccines are an exception, as they are tested in five guinea pigs, rather than using the target species [14]. For licensing (registration) in Europe, to meet the requirements of the Ph. Eur. monographs, all products must ordinarily be tested in the target species, unless a waiver has been granted, as noted below. The Ph. Eur. includes guidance in a General Notice, which allows for use of alternative methods of analyses [11]. In addition, the Ph. Eur. General Monograph on Vaccines for Veterinary Use [10] includes the following description of the circumstances where the consistency approach may be implemented: “It is recognized that in accordance with the General Notices (section 1.1. General statements), for an established vaccine the routine application of the safety test will be waived by the competent authority in the interests of animal welfare when a sufficient number of consecutive production batches have been produced and found to comply with the test, thus demonstrating consistency of the manufacturing process. Significant changes to the manufacturing process may require resumption of routine testing to re-establish consistency. The number of consecutive batches to be tested depends on a number of factors such as the type of vaccine, the frequency of production of batches and experience with the vaccine during development safety testing, and during application of the batch safety test. Without prejudice to the decision of the competent authority in light of information available for a given vaccine, testing of 10 consecutive batches is likely to be sufficient for most products. For products with an inherent safety risk, it may be necessary to continue to conduct the safety test on each batch” [10]. A position paper of the EMEA Committee for Veterinary Medicinal Products (CVMP) outlines the specific data requirements for removing the target animal batch safety test for immunological veterinary products in the European Union [25]. The CVMP guidance document [25] cites the provisions in the Ph. Eur. General Monograph on Vaccines for Veterinary Use [10]. It explains that the option of waiving this batch release safety testing requirement relies on the assurances that are provided by comprehensive pre-licensing safety testing to meet the current European Union requirement for pre-licensing safety tests (single dose, overdose, repeat dose), as well as documented consistency of the manufacturing process, and a retrospective analysis of batch release test results and pharmacovigilance data to demonstrate the overall safety of the product and the reliability of other controls. An independent third party expert report must be filed in support of the application [25]. The expert opinion must address topics such as the inherent variability of the product, the intrinsic safety margin, post-licensing pharmacovigilance data, and pertinent validation data to provide the necessary assurance that the product would always be manufactured to an acceptable level of quality and safety [25]. 10. Discussion The current animal-based batch release safety tests probably serve an important precautionary function for some veterinary vaccines, however they have some inherent deficiencies that limit their utility. Without systematic validation data, it is difficult to assess the predictive value of these tests. Consequently, manufacturers, regulators, and animal welfare advocacy groups have shown considerable interest in developing alternatives to the use of animals for these tests, such as the consistency approach. Continued progress toward development and adoption of alternative methods depends on several factors, as noted below.

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A key prerequisite will be implementation of appropriate comprehensive manufacturing controls to ensure consistency of manufacturing processes and uniformity of the end product. Biologically relevant test methods and reference standards will need to be developed and validated, preferably through collaborative efforts involving manufacturers and regulatory agencies. It will also be important for regulatory agencies to develop clear, harmonized parameters for approval of alternative methods. This will help address concerns among manufacturers and the international regulatory community about the risk of implementing changes that might lead to inadvertent release of unsafe batches of veterinary vaccines. The European veterinary vaccine regulations and technical standards clearly define the documentation requirements and approval procedures for the consistency approach. In other regions, the consistency approach could also be applied on a case-by-case basis, provided sufficient supporting data is available; however, the acceptance criteria for implementation of alternative methods for replacement of animal safety tests are not clearly defined. It is likely that improved in vitro safety tests will become available in the future, especially if (1) manufacturers are encouraged to work collaboratively to develop alternatives such as improved manufacturing controls, improved vaccinovigilance, and in vitro tests as part of a consistency approach, and (2) the manufacturers have some assurance that these alternative approaches would be acceptable to meet international regulatory requirements. Another important element will be to establish rigorous pre-licensing safety testing protocols and post-licensing quality controls, such as GMP or equivalent standards. It will also be important to establish rigorous batch safety test pass/fail criteria with clearly defined maximum tolerances for key product components. For example, production outlines for bacterial vaccines should clearly define upper limits for key product components such as total antigen content, endotoxin levels, or potency. These limits should be within the ranges that were shown to be safe in prelicensing safety testing. When considering modifications to existing regulatory controls and manufacturing practices, manufacturers and regulatory agencies are proceeding cautiously, because they are accountable for implementing the appropriate science-based regulations and corresponding science-based quality controls to help ensure that the regulated products are as safe as possible. Although manufacturers and regulators recognize the need to conform to 3R principles and encourage development of alternative methods, they must also fulfil their fundamental regulatory obligations. Vaccine manufacturers are understandably reluctant to invest in development of alternative methods unless there is some reassurance that the changes in test procedures would not diminish the overall quality of veterinary vaccines and that they would be sufficient to meet international regulatory requirements. There is currently very little publicly available data on the predictive value of the currently employed safety tests, despite the fact that some of these tests have been used to test numerous batches of vaccine over an extended period of time. These tests may, therefore, not have appropriate baseline references for validation of alternative methods. Conversely, there is also a risk that the predictive value of some of the currently utilized animal-based tests has been underestimated. An increased availability of summary information on the currently used safety assessment procedures would greatly assist the decision process. Manufacturers and regulatory agencies should, therefore, be encouraged to conduct a retrospective review of their collective experiences with batch release safety tests. This would provide baseline data and a context for discussions regarding the utility of specific currently used tests, as well as establishing a benchmark against which future modifications could be assessed. The principal regulatory agencies in the countries where international manufacturing facilities are located, and the regulatory agencies of importing countries, can influence the development and adoption of alternative methods. This can be accomplished through proactive information sharing and collaboration to promote development, validation, and implementation of alternatives to reduce, refine, and replace the use of animals for batch release safety tests for veterinary vaccines. In some circumstances, such as for those types of products that have demonstrated a high level of batch-to-batch consistency and freedom from adverse events, it may be feasible to eliminate the traditional animal-based safety tests, and replace them with alternative methods of characterizing the uniformity and safety of each batch and freedom from deleterious factors. In the future, it may also be possible to collaboratively develop in vitro bioassays that utilize cell cultures, blood samples or biopsy specimens to measure specific factors or assess non-specific cytotoxicity, rather than relying on animal testing.

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Timely regulatory review and approval of alternative approaches should be identified as a high priority activity for regulatory agencies, and consideration should be given to reducing or waiving the applicable regulatory fees, if the fees are a significant impediment to implementation of the necessary regulatory approvals. Despite progress in developing alternative methods, in vivo batch release safety tests will likely continue to be required for some classes of products, especially those that are potentially hazardous, and for which the potential toxicity-eliciting factors are not known. In situations where animal safety testing is unavoidable, regulatory agencies should continue to encourage development and adoption of the most biologically relevant and humane batch release safety test protocols. In these circumstances, the batch release safety tests should be conducted in target animals or laboratory animals, using fully validated test protocols with appropriate humane endpoints. In the longer term, once useful alternatives are identified and validated, the pertinent legislation, regulations, and technical guidance should be amended to facilitate adoption of appropriate alternative methods, and to minimize and eventually eliminate use of redundant or obsolete animal-based batch release safety tests. The eventual objective should be global standardization of the technical requirements for development and validation of batch release safety testing of veterinary vaccines and timely adoption of appropriate manufacturing controls and alternative in vitro test methods as a component of a consistency approach. Continued future progress toward reduction, refinement, and replacement of animals for safety testing of veterinary vaccines will require an ongoing commitment and collaboration from industry, regulators, and animal welfare advocacy groups to address the technical, regulatory, and financial challenges. References [1]

Advisory Group on Alternatives to Animal Testing in Immunobiologicals. The target animal safety test – is it still relevant? Biologicals 2002;30:277-87. [2] Allende RM, Mendes da Silva AJ, Comparsi Darsie G. South American standards for foot-and-mouth disease vaccine quality [Internet]. In: Dodet B and Vicari M, editors. Foot-and-Mouth Disease: Control Strategies. Paris, Elsevier, 2003: 331-36. [Accessed 9 March 2011]. Available at: http://bvs1.panaftosa.org.br/local/File/textoc/AllendeSouthAmericanLyon2002.pdf [3] Bruckner L. Application of humane endpoints in veterinary vaccine testing in target species [Internet]. 2nd International Conference on the Use of Humane Endpoints in Animal Experiments for Biomedical Research, 20-21 August 2005, Berlin, Germany. [Accessed 9 March 2011]. Available at: http://www.humane-endpoints.org [4] Canadian Council on Animal Care. CCAC guidelines on: choosing an appropriate endpoint in experiments using animals for research, teaching and testing [Internet]. Ottawa, Ontario, Canada, CCAC, 1998. [Accessed 9 March 2011]. Available at: http://www.ccac.ca/en/CCAC_Programs/Guidelines_Policies/GDLINES/ENDPTS/APPOPEN.HTM [5] Canadian Council on Animal Care. CCAC Three Rs Microsite. Testing and Three Rs. Biologics and Vaccines [Internet]. Ottawa, Ontario, Canada, CCAC, 2008-2009. [Accessed 9 March 2011]. Available at: http://www.ccac.ca/en/alternatives/tests/biologicsvaccines_produits-vaccins.html [6] Canadian Food Inspection Agency, Canadian Centre for Veterinary Biologics. Veterinary Biologics Guideline 3.29: Safety Requirements for Veterinary Biologics [Internet]. 15 December 2010. [Accessed 9 March 2011]. Available at: http://www.inspection.gc.ca/english/anima/vetbio/info/vb329e.shtml [7] Committee for Medicinal Products for Human Use (CHMP), European Commission. The Rules Governing Medicinal Products in the European Union. Volume 7: Scientific guidelines for medicinal products for veterinary use. Volume 7B–Immunologicals, quality. Guidelines for production and control of immunological veterinary medicinal products [Internet]. [Accessed 9 March 2011]. Available at: http://ec.europa.eu/health/documents/eudralex/vol-7/index_en.htm [8] Council of Europe. Evaluation of Safety of Each Batch of Veterinary Vaccines and Immunosera [ch. 5.2.9]. In: European Pharmacopoeia, 7th ed. Strasbourg, France, European Department for the Quality of Medicines & HealthCare (EDQM), Council of Europe, 2010. [9] Council of Europe. Evaluation of Safety of Veterinary Vaccines and Immunosera [ch. 5.2.6]. In: European Pharmacopoeia, 7th ed. Strasbourg, France, European Department for the Quality of Medicines & HealthCare (EDQM), Council of Europe, 2010. [10] Council of Europe. General Monographs. Vaccines for Veterinary Use. Monograph 62. In: European Pharmacopoeia, Supplement 7.2 to the 7th ed. Strasbourg, France, European Department for the Quality of Medicines & HealthCare (EDQM), Council of Europe, 2010. [11] Council of Europe. General Notices. In: European Pharmacopoeia, 7th ed. Strasbourg, France, European Department for the Quality of Medicines & HealthCare (EDQM), Council of Europe, 2010. [12] Council of Europe. Vaccines for veterinary use. Anthrax spore vaccine (live) for veterinary use. Monograph 441. In: European Pharmacopoeia, 7th ed. Strasbourg, France, European Department for the Quality of Medicines & HealthCare (EDQM), Council of Europe, 2010.

246 246

Glen Gifford et et al. al.//Procedia ProcediaininVaccinology Vaccinology5 5(2011) (2011236 ) 236 – 247 Glen Gifford – 247

[13] Council of Europe. Vaccines for veterinary use. Salmonella enteriditis vaccine (inactivated) for chickens. Monograph 1947. In: European Pharmacopoeia, 7th ed. Strasbourg, France, European Department for the Quality of Medicines & HealthCare (EDQM), Council of Europe, 2010. [14] Council of Europe. Vaccines for veterinary use. Tetanus vaccine for veterinary use. Monograph 697. In: European Pharmacopoeia, 7th ed. Strasbourg, France, European Department for the Quality of Medicines & HealthCare (EDQM), Council of Europe, 2010. [15] Council of Europe. Vaccines for veterinary use. Vibriosis vaccine (inactivated) for salmonids. Monograph 1581. In: European Pharmacopoeia, 7th ed. Strasbourg, France, European Department for the Quality of Medicines & HealthCare (EDQM), Council of Europe, 2010. [16] Cussler K. Overview of the 3Rs Concept in Vaccine Quality Control—Approaches and Goals [Internet]. Presented at: IICAB Meeting—Technology and Approaches to Reduce, Refine and Replace Animal Testing, 5-7 April 2004, Ames, Iowa. [Accessed 9 March 2011]. Available at: http://www.cfsph.iastate.edu/IICAB/meetings/april2004/Cussler.pdf [17] Cussler K, Kulpa J, Calver J. The international symposium on regulatory testing and animal welfare: recommendations on best scientific practices for biologicals: safety and potency evaluations) [Internet]. ILAR J 2002;3(suppl):S126-28. [Accessed 9 March 2011]. Available at: http://dels-old.nas.edu/ilar_n/ilarjournal/43_supp/v43supCussler.pdf [18] De Boo J, Knight A. Use of laboratory animals: revisiting reduction and refinement strategies [Internet]. Vet Times [U.K.] 2010; 40(8):6, 8-9. [Accessed 9 March 2011]. Available at: http://www.aknight.info/publications/anim_expts_overall/summaries/JdeB%20et%20al%203Rs%20Vet%20Times%202010%2040(8) %206,8-9.pdf [19] De Mattia F, Chapsal JM, Descamps J, Halder M, Jarrett N, Kross I, Mortiaux F, Ponsar C, Redhead K, McKelvie J, Hendricksen C. Meeting Report: The consistency approach for quality control of vaccines – A strategy to improve quality control and implement the 3Rs. Biologicals 2011;39:59-65. [20] Dhruvakumar S (PETA). The Reduction of Animal Use in the Critical Path to Vaccines [Internet]. Presented at: Vaccines and Related Biological Products Advisory Committee (VRBPAC) Meeting, 17 February 2005. [Accessed 9 March 2011]. Available at: http://webcache.googleusercontent.com/search?q=cache:lS82xZOSeF0J:www.fda.gov/ohrms/dockets/ac/05/slides/2005087oph2.ppt+veterinary+vaccine+batch+safety+test+consistency+alternative&cd=15&hl=en&ct=clnk&gl=ca [21] European Centre for the Validation of Alternative Methods (ECVAM) and Paul Ehrlich Institute. Animal welfare aspects in the quality control of immunobiologicals: a critical evaluation of animal tests in pharmacopoeial monographs [Internet]. Krys Bottril, editor. Fund for the Replacement of Animals in Medical Experiments (FRAME), 1997. [Accessed 9 March 2011]. Available at: http://www.frame.org.uk/page.php?pg_id=184 [22] European Centre for the Validation of Alternative Methods (ECVAM). Statement on the Relevance of the Target Animal Safety Test for Batch Release Safety Testing of Vaccines for Veterinary Use [Internet]. Ispra, Italy, ECVAM, 2002. [Accessed 9 March 2011]. Available at: http://ecvam.jrc.it/publication/TargetAnimalSafetyT_statement.PDF [23] European Agency for the Evaluation of Medicinal Products (EMEA), Committee for Veterinary Medicinal Products (CVMP). Note for Guidance: Requirements for Combined Veterinary Vaccines. CVMP/IWP/52/97-Final. London, EMEA, 2000. Available at: http://www.ema.europa.eu/pdfs/vet/iwp/005297en.pdf [24] European Agency for the Evaluation of Medicinal Products (EMEA), Committee for Veterinary Medicinal Products (CVMP). Position Paper on Compliance of Veterinary Vaccines with Veterinary Vaccine Monographs of the European Pharmacopoeia. EMEA/CVMP/140/97-Final. London, EMEA, 1999. Available at: http://www.ema.europa.eu/pdfs/vet/press/pp/014097en.pdf [25] European Agency for the Evaluation of Medicinal Products (EMEA), Committee for Veterinary Medicinal Products (CVMP). Position Paper on Data Requirements for Removing the Target Animal Batch Safety Test for Immunological Veterinary Medicinal Products in the EU. EMEA/CVMP/865/03-Final. London, EMEA, 2004. Available at: http://www.ema.europa.eu/pdfs/vet/iwp/086503en.pdf [26] European Medicines Agency (EMA). Veterinary medicines [Internet]. [Accessed 14 March 2011]. Available at: http://www.ema.europa.eu/ema/index.jsp?curl=pages/regulation/landing/veterinary_medicines_regulatory.jsp&murl=menus/regulatio ns/regulations.jsp&mid=WC0b01ac058001ff8a [27] European Medicines Agency (EMA), Committee for Medicinal Products for Veterinary Use (CVMP). Reflection paper on control of the active substance in the finished product for immunological veterinary medicinal products (IVMPs). London, EMA, 2010. Available at: http://www.ema.europa.eu/pdfs/vet/iwp/58297009en.pdf [28] European Vaccine Manufacturers. Animal Welfare and Vaccine Development [Internet]. EVM, 2008. [Accessed 9 March 2011]. Available at: http://www.evm-vaccines.org/pdfs/vaccines_and_animal_welfare_fin.pdf [29] Food and Agriculture Organization. FAO Manual for the production of anthrax and blackleg vaccines. Part I: Production of anthrax spore vaccine – General laboratory procedures (FAO Animal Production and Health Paper 87) [Internet]. FAO, 1991. [Accessed 9 March 2011]. Available at: http://www.fao.org/DOCREP/004/T0278E/T0278E01.htm [30] Food and Agriculture Organization. FAO Manual for the production of anthrax and blackleg vaccines. Part II: Production of blackleg vaccine – General laboratory procedures (FAO Animal Production and Health Paper 87) [Internet]. FAO, 1991. [Accessed 9 March 2011]. Available at: http://www.fao.org/DOCREP/004/T0278E/T0278E02.htm [31] Fyrand K, Gravningen K. Vaccine Testing: How can we reduce fish numbers and/or avoid the use of fish? [Internet] Presented at: NORECOPA Conference, 22-24 September 2009, Gandermoen, Norway. [Accessed 9 March 2011]. Available at: http://www.norecopa.no/norecopa/vedlegg/Fyrand.pdf

GlenGifford Giffordetetal. al./ /Procedia Procediain in Vaccinology Vaccinology 55 (2011) Glen (2011) 236 236––247 247

247 247

[32] Hendriksen C. Refinement, reduction, and replacement of animal use for regulatory testing: current best scientific practices for the evaluation of safety and potency of biologicals [Internet]. ILAR J 2002;43(suppl):S43-S48. [Accessed 9 March 2011]. Available at: http://dels-old.nas.edu/ilar_n/ilarjournal/43_supp/v43supHendriksen.pdf [33] Hendriksen C, Arciniega J, Bruckner L, Chevalier M, Coppens E, Descamps J, Duchene M, Dusek D, Halder M, Kreeftenberg H, Maes A, Redhead K, Ravetkar S, Spieser J, Swam H. Short communication. The consistency approach for the quality control of vaccines. Biologicals 2008;36:73-77. [34] International Association for Biologicals (IABS) Symposium: Practical Alternatives to Reduce Animal Testing in Quality Control of Veterinary Biologicals in the Americas [Internet]. 18–19 February 2010, Buenos Aires, Argentina. [Accessed 9 March 2011]. Available at: http://www.iabs.org/index.php/conferences/iabs-conferences/past-iabs-conferences [35] International Federation for Animal Health. About IFAH [Internet]. [Accessed 10 March 2011]. Available at: http://www.ifahsec.org [36] Krug M. Prevention of adverse effects in pigs after vaccination [Internet]. 3R Info Bulletin 18. September 2001. [Accessed 9 March 2011]. Available at: http://www.forschung3r.ch/de/publications/bu18.html [37] Midtlyng P, Hendriksen C, Balks E, Bruckner L, Elsken L, Evensen O, Fyrand K, Guy A, Halder M, Hawkins P, Kisen G, Berit Romstad A, Salonius K, Smith P, Sneddon L. Meeting report: Three Rs approaches in the production and quality control of fish vaccines. Biologicals 2011;39:117-128. [38] OIE (World Organisation for Animal Health). Animal vaccination – Part 1: development, production, and use of vaccines [Internet]. Pastoret P-P, Lombard M, Schudel AA, editors. Sci Tech Rev 2007;26(1). [Accessed 9 March 2011]. Available at: http://www.oie.int/publications-and-documentation/scientific-and-technical-review-free-access/list-of-issues [39] OIE (World Organisation for Animal Health). Animal vaccination – Part 2: scientific, economic, regulatory and socio-ethical aspects [Internet]. Pastoret P-P, Lombard M, Schudel AA, editors. Sci Tech Rev 2007;26(2). [Accessed 9 March 2011]. Available at: http://www.oie.int/publications-and-documentation/scientific-and-technical-review-free-access/list-of-issues [40] OIE (World Organisation for Animal Health). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2010 [Internet]. [Accessed 9 March 2011]. Available at: http://www.oie.int/en/international-standard-setting/terrestrial-manual/access-online [41] Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme (PIC/S). Homepage [Internet]. [Accessed 9 March 2011]. Available at: http://www.picscheme.org/ [42] Pöbetanecker A, Cubetaler K. Evaluation of the relevance of the target animal safety test for the quality control of veterinary immunological medicinal products [Internet]. Abstract. ALTEX 1998;15(5):71-75. [Accessed 9 March 2011]. Available at: http://www.ncbi.nlm.nih.gov/pubmed/11178548 [43] Roberts B, Lucken RN. Reducing the use of the target animal batch safety test for veterinary vaccines [Internet]. Abstract. Dev Biol Stand 1996;86:97-102. [Accessed 9 March 2011]. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8785997 [44] United States Code of Federal Regulations. 9 CFR 113. Title 9–Animals and Animal Products; Chapter 1–Animal and Plant Health Inspection Service, Department of Agriculture; Subchapter E–Viruses, Serums, Toxins, and Analogous Products: Organisms and Vectors; Part 113–Standard Requirements [Internet]. [Accessed 9 March 2011]. Available at: http://ecfr.gpoaccess.gov/cgi/t/text/textidx?c=ecfr&tpl=/ecfrbrowse/Title09/9cfr113_main_02.tpl [45] United States Department of Agriculture, Animal Plant Health Inspection Service, Animal Health, Veterinary Biologics. Biologics Regulations and Guidance [Internet]. [Accessed 9 March 2011]. Available at: http://www.aphis.usda.gov/animal_health/vet_biologics/vb_regs_and_guidance.shtml [46] Veterinary International Cooperation for Harmonization (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Veterinary Medicinal Products). What is VICH [Internet]? [Accessed 10 March 2011]. Available at: http://www.vichsec.org/en/what-is.htm