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

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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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To assess the potency of diphtheria vaccine by serology as an alternative procedure to the guinea pig challenge method (Section II.2), antibody responses to diphtheria toxoid induced in guinea pigs after 5 to 6 weeks are compared relative to the antibody response induced by the reference vaccine. For this comparison, a reference preparation of diphtheria toxoid calibrated in International Units (IU) and a suitable antibody detection method is required. The Vero cell toxin neutralisation test (TNT, Section II.1.3.2) and ELISA (Section II.1.3.3) are described in this chapter as suitable antibody detection methods [1-3].

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Because mice are not sensitive to diphtheria toxin, challenge with toxin is not possible. The mouse potency assay therefore involves the detection of functional antibodies against diphtheria toxoid induced in mice by observing neutralizing effect of sera on diphtheria toxin in a Vero cell culture model [2-4]. The potency of the test vaccine is calculated by the parallel line analysis comparing the scores obtained for test and reference vaccines at each dilution. A minimum of 3 serial dilutions of test and reference vaccine are injected in groups of mice. Experience confirmed that 8-12 animals per group are likely to be sufficient to enable a valid calculation of potency. Mice are bled after 4-5 weeks.

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The ability of diphtheria toxin to cause an erythematic reaction when injected intradermally into the skin of animals can also be used to determine the neutralising capacity of an antibody preparation. The in vivo toxin neutralisation test (TNT) can thus be performed on the depilated skin of guinea pigs using general principles described for potency testing of Diphtheria Antitoxin products [1]. Rabbit is also used for toxin neutralization test. Different dilutions of serum are mixed with fixed amounts of diphtheria toxin and injected intradermally into the depilated skin. The antitoxin protective concentration is estimated by ability to protect inflammatory erythema reaction of the fixed dose of toxin. The TNT test is able to confirm functional capacity of serum antibody and can thus be used to confirm relevance of in vitro serological methods, as part of the validation process [2].

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The purpose of the specific toxicity test for diphtheria toxin is to confirm freedom from residual toxin and reversion to toxicity in final bulk vaccines and/or bulk purified toxoid. The in vivo assay remains the method of choice for routine testing or validation of production processes. The toxicity reversal test for diphtheria toxin is also suitable for the assessment of concentrated toxoid intermediate and is based on the measurement of specific toxicity following incubation of the test toxoid for a prolonged period of time at high temperature to ensure that no reversion of toxoid to toxin has occurred. The WHO specifies the use of the specific toxicity test for the control of purified toxoid bulk and final bulk vaccine, whereas the toxicity reversal assay is only used for the control of purified toxoid bulk [1]. The in vivo tests for specific toxicity and toxicity reversal are usually performed in guinea pigs by subcutaneous injection. However, the induction of specific erythema following intradermal injections of at least 20 Lf of purified toxoid can also be used in rabbits and guinea pigs.

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A Vero cell culture system may be used as an alternative to in vivo tests for specific toxicity and reversion to toxicity as long as sensitivity of the assay is shown to be comparable to the guinea pig test [1].
WHO recommends the use of Vero cell culture assay provided that the test is validated against the guinea pig test. During such validation studies recommendations regarding pass/fail requirements can be made.

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The purpose of the potency test is to assess in a suitable animal model the capacity of the product being tested to induce a protective response analogous to that shown to be efficacious in humans.

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The challenge potency test for tetanus vaccine (adsorbed) is determined by comparing the dose of the vaccine to that of a reference preparation required to protect guinea pigs or mice from either a lethal or paralytic toxic effect following subcutaneous (s.c.) challenge with tetanus toxin

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To assess the potency of tetanus vaccine by serology as an alternative to the guinea pig challenge method (Section III.1.2), antibody responses to tetanus toxoid induced in guinea pigs after 5 to 6 weeks are compared relative to the antibody response induced by a reference vaccine.

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According to the latest revision of WHO recommendations, potency test for routine lot release can be performed by immunising mice as well as guinea pigs with appropriate dilutions of the calibrated reference preparation and the test vaccine.

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