Review of animal use requirements in WHO biologics guidelines – database of suggested guideline revisions

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Sharing of test results can contribute to reducing the number of animals used for testing

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When animals are used for testing, the NCL should be aware of the potential variability of the source, housing and handling of animals. It is desirable to apply the “3R” principles (reduction, replacement, refinement) to minimize the use of animals, for ethical reasons. Validated in vitro alternatives should be favoured wherever possible. However, the type of testing should be driven by the scientific need for valid relevant data. Moreover, in the spirit of minimizing animal testing worldwide, agreements should be sought with the NCL of the exporting country or with other NCLs, in a mutual recognition or collaborative agreement, in order to utilize the results of animal testing already performed by another NCL.

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Appendices illustrate model approaches and examples. The model approaches may need to be revised to reflect 3Rs

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Chapter 7 mentions experimental design 'sound statistical design principles'. This could be streathened and extended to include a general statement about model selection and the 3Rs.

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This is a general guideline describing strategies for the assessment and monitoring of vaccine stability, both during the development process and post-licensure. The selection of assays for stability studies would be dependent on the vaccine product, but should include safety- and potency-indicating parameters. Whether those assays are in vivo or in vitro is not defined or required within the document (as that would be dependent on the vaccine).

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Parameters other than potency-indicating ones should also be considered since they indicate changes in vaccine quality with unknown effects on efficacy and safety. Such parameters may include, in addition to in vivo and in vitro potency, antigen content, appearance, pH, general safety, specific toxicity, antimicrobial agent content, completeness of adsorption, sterility, adjuvant (adsorbent) content and changes in physicochemical properties.

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Bioassays performed on living animals are still the method of choice for evaluating the safety and potency of many bacterial vaccines. Before any new procedure is introduced it should be validated against the standard method to ensure at least non-inferiority of performance. Most bio-assays use either guinea-pigs or mice often in quite large numbers. The main shortcoming of such tests is the result of individual variation between animals which can result in low precision and poor reproducibility. More than any other system used for testing, animals have to be handled and maintained appropriately to generate accurate, reliable, and reproducible results. It is essential to be aware of all the factors that may affect the biological functions of the test animals, and thus interfere with the outcome of a potency, safety or toxicity test.

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Tests performed to assure the safety and potency of diphtheria and tetanus vaccines should ideally be performed in guinea-pigs. However, these animals have become increasingly expensive and in some countries their supply and maintenance is problematic.
For pertussis vaccine potency and safety tests, mice have to be used. In a study on the possible influence of mouse strain on the assayed potency of tetanus vaccines, Hardegree et al. (4) tested a batch of tetanus vaccine in parallel with the International Standard for Tetanus Toxoid in five different mouse strains. A significant difference in relative potency between the two vaccines was found for the individual strains. This is a recurrent problem and the difficulty of relating potency test data obtained in mice to results obtained by the traditional method in guinea-pigs has not been completely resolved.

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Whatever the disadvantages and problems associated with the use of animals as a “measuring device” in the quality control of vaccines, no test system is known which can replace animal models completely. The use of a reference can partly overcome the effect of external factors on test results. However, attention should be paid to the care and handling of laboratory animals to minimize effects of environment and nutrition and to maximize efficacy in their use, particularly in the quality control of bacterial vaccines. Animals should be bred and maintained in such a way that the maximum possible standardization and reproducibility are obtained.

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The challenge potency test for diphtheria vaccine (adsorbed) is determined by comparing the dose of the vaccine to that of a reference preparation required to protect guinea pigs from either an erythrogenic toxic effect (toxin administered intradermally, i.d.) or a lethal toxic effect (toxin administered subcutaneously, s.c.) [1,2]. For this comparison, a reference preparation of diphtheria toxoid (adsorbed) calibrated in International Units (IU) and a suitable preparation of purified diphtheria toxin is required. The current International Standard for diphtheria toxoid with an assigned activity of
213 IU/ampoule (based on results obtained in guinea pig lethal and i.d. challenge assays, NIBSC code 07/216) was established in 2009 for determining the potency of vaccines containing diphtheria toxoid [3]

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