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

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Secondary reference preparations (regional, national, working or product-specific standards) should be calibrated with a multiple dilution protection assay (of at least three dilutions), using either guinea-pigs or mice. Standards calibrated in IUs in guinea-pigs are considered to be suitable for use in guinea-pig potency assays, and standards calibrated using mouse assays are considered to be suitable for use only in mouse-potency tests.

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For routine testing, the potency of every new bulk of tetanus vaccine is determined by the immunization of guinea-pigs or mice with appropriate dilutions of the calibrated reference preparation and test vaccine. Care should be taken to ensure that dilutions are inert (phosphate may interfere with the adsorption of toxoid) and not pyrogenic. Between four and six weeks after immunization, animals may be directly challenged with tetanus toxin by the subcutaneous route, or they may be bled for titration of immune serum.
Titration of immune serum samples may be performed using an in vivo TNT or by using a suitably validated in vitro method, such as an enzyme-linked immunosorbent assay (ELISA), the toxin-binding inhibition (ToBI) test or the particle agglutination test (30, 54, 61, 62). If in vitro serological assays are used, they should show that the product induces an appropriate antibody response in animals when compared with the reference preparation.

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As biological assays (e.g. humoral antibody response in sera from a suitable species) with the titration of Ab by in vitro methods (in vitro TNT, ELISA, MIT depending on the component tested) and/or physicochemical tests have been developed and are considered to be more precise and reproducible than the challenge test, a biological and/or physicochemical assay should be used if approved by the NRA. In some countries the titration of Abs are performed using multiplex immunological methods for combined DTaP vaccines. If several final lots are issued from one final bulk product, the potency assay should be carried out on the final bulk product and omitted on the final lots. After the demonstration of consistency of production by the biological and/or physicochemical assay on an appropriate number of final bulk products, a single dilution assay approved by the NRA should be carried out. Biological and/or physicochemical assays are preferred. However, if an in vivo challenge test for Diphtheria, Pertussis & Tetanus is carried out, it must be scientifically justified and should be approved by the NRA. A single dilution assay may be implemented for Diphtheria, Pertussis & Tetanus components after demonstration of consistency of production on an appropriate number of final bulk products and should be approved by the NRA. In the context of a 3Rs strategy, development of a package of appropriately validated in vitro assays for the characterization of the drug product is strongly encouraged in order to replace animal models. For all components, in vitro antigenicity assays are being developed and may be considered as potency assays once they are appropriately validated.

Original text

Once consistency in production has been demonstrated for the vaccine, the potency assay (using the challenge or serology model) may, with the approval of the NRA, be performed using a reduced number of animals or doses, or both.
If one-dilution assays are not advantageous, a reduction in animal usage may nevertheless be achieved by using two-dilution assays or another suitable design modification.

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A potency test should be carried out on each final lot as outlined in Part A, section A.3.5.2.6, if such a test has not been performed on the final bulk.

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Each final lot should be tested for innocuity. This test is also referred to as the abnormal toxicity test or the general safety test. One human dose, but not more than 1 ml, of the final lot is injected by the intraperitoneal route into each of five mice (weighing 17–22 g) and each of two guinea-pigs (weighing 250–350 g). The test should be approved by the NRA. The final product is considered to be innocuous if the animals survive for at least seven days without showing significant signs of toxicity. If the NRA approves, the innocuity test on the final lot may be omittedfrom routine lot release once the consistency of production has been
demonstrated.

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Several studies conducted during the last decade have provided useful information on the value and potential use of in vitro immunochemical assays for measurement of the toxoid antigen content and degree of adsorption in the final bulk or final fill of tetanus vaccines (47, 48). The results obtained using these methods will not necessarily correlate with measurements of vaccine potency that have been determined in vivo, particularly for complex combinations of vaccines, but the value of these in vitro methods for monitoring trends (47) and stability (27) has been well documented. When a new bulk lot of vaccine is made, it is essential to confirm its safety (i.e. the absence of toxin and reversion to toxicity) and potency. Potency is measured using an in vivo challenge test, or a validated alternative,

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Reference to mouse and guinea pig challenge assays is made repeatedly throughout this section in regard to standards and potentcy testing. It talks about the differences in results obtained between species and regional preferences of a particular species, e.g EU relies largely on mouse-protection assays. Also goes on to state that the relevance of animals for prediciting an effective protective response in humans has been questioned and that over last decade there have been significant efforts to reduce animal use and refine end-points (e.g.paralysis) or by using validated serology assays.

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The use of animals bred in a carefully controlled colony, especially those that are SPF, is strongly recommended. Nevertheless, as suitable alternative cell substrates become available, PCCs are less likely to be used in the future. WHO has promoted the replacement of animals for experimental purposes, both for ethical reasons (18) and in the interests of progressive improvement in product safety and quality.

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no specific test is mentioned, only the logistical approach how to exclude and detect adventitious agents in starting materials, during production and in the final product. There is no specific method mentioned. As there are still old fashioned methods used for the detection of adventitious agents, like mice and guinea pigs, this problem should be mentioned explicitely including description of in vitro methods (not tests using antibodies or other animal material) which are proven to detect adventitious agents better, more sensitive, cheaper etc.

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Recognition/acceptance of lot release certificates from the NRA/NCL of the country where the vaccine is manufactured, or from another competent NRA/NCL, should also be considered as an alternative (see section 7.1).

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