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

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The purpose of the tetanus toxin neutralisation test (TNT) is to determine the functional potency of tetanus antitoxin by comparing the dose required to protect mice against the paralytic effects of a fixed dose of tetanus toxin, with the quantity of reference tetanus antitoxin required to give the same protection.

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The purpose of the specific toxicity test for tetanus is to confirm freedom from residual toxin and reversion to toxicity in final bulk vaccines and/or bulk purified toxoid. The toxicity reversal test for tetanus is also suitable for the assessment of concentrated toxoid intermediate product, 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 tests for specific toxicity and toxicity reversal are usually performed in guinea pigs by subcutaneous injection. Although mice are not as sensitive to tetanus toxin as guinea pigs, WHO allows the use of mice for the toxicity reversal test, subject to approval by the National Regulatory Authority.

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The MWGT is considered as a general, non-specific test measuring overall toxicity of pertussis whole cell vaccine, since a number of B. pertussis toxins may induce weight loss in mice. Correlation of the results of the MWGT with adverse reactions in children has been reported [3-6]. It is a test used to assess the toxicity of whole cell pertussis containing vaccines, and it is based on the ability of certain toxins or components from B. pertussis to cause weight loss in young mice

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Mice inoculated with pertussis toxin become highly sensitive to a histamine challenge. The effects include reduction in body temperature and in the severe cases death. The reduction in body temperature occurs within 30 minutes after histamine challenge, but in the non-lethal situations it returns to normal levels after 30 minutes. Therefore, reduction in body temperature 30 minutes following histamine challenge is directly proportional to the dose of active PT present in the vaccine. This method is highly sensitive, it can detect levels of PT activity that do not induce lethal effects following histamine challenge. Body temperature in mice can be assessed by measuring rectal or dermal temperature using either an electric thermometer with a probe specific for mice or an infrared thermometer, respectively. Results are obtained as continuous variables so as to allow calculating mean and variance for each group. Rectal temperature method has been used in Japan since 1981 [2, 3]. Assessment of body temperature by both methods (rectal and dermal) correlates with PT toxicity in animals. However, for practical reasons dermal measurements are preferred in some countries.

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An assay to assess the active pertussis toxin (PT) content of pertussis containing vaccines on the basis of the histamine sensitising effect of active PT on mice. Pertussis toxin increases the sensitivity of mice to histamine. The exact mode of action is not yet fully understood. Even when small amounts of active PT are present in a vaccine, mice will become vulnerable to challenge with histamine, resulting in anaphylactic shock and inevitable death. The amount of histamine sensitisation factor (HSF) activity in a vaccine can be quantified in a parallel-line assay in comparison with a reference vaccine. In this assay the reference and test vaccine doses, which induce a histamine sensitisation in 50% of the animals, as measured by death after challenge with histamine, are compared and a relative HSF activity is calculated for the vaccine. Different mouse strains may show different sensitivity to the test, laboratories are recommended to set up their own experimental conditions.

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Heat-labile toxin (HLT) is a heat labile protein toxin of B. pertussis which can be inactivated in 10 minutes at 56°C. HLT is dermonecrotizing, lethal, and causes spleen atrophy in experimental animals. Since its discovery by Bordet and Gengou (1909), the toxin has been considered to play an important role in pathogenicity, most probably in the initial stage of whooping cough. The toxin is produced by all phase I B. pertussis strains. Pertussis vaccine should not contain biologically active HLT. Absence of HLT is not considered to be a product release criterion but validation of the manufacturing process should demonstrate the absence of HLT in the pertussis bulk after inactivation. Suckling mice are most responsive to the lethal or dermonecrotizing activity of HLT after subcutaneous injection into the nuchal area. Both lethality and dermonecrocity, can be used as parameters. As a negative control saline may be used or heat inactivated sample (56°C, 10 minutes). Usually one to three dilutions per sample are tested.

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Pertussis vaccine contains pertussis toxin, formerly Leukocytosis Promoting Factor (LPF) that enhances the number of circulating leukocytes. Although most methods applied for determining the pertussis toxin-induced leukocytosis do not discriminate between leukocytes and lymphocytes some people persist in calling it lymphocytosis promoting factor. The LP activity of a pertussis vaccine is estimated by counting the number of circulating leukocytes 7 days after injection of mice with the test vaccine. Counting the number of circulating leukocytes can be done either by haemocytometer or electronic cell counter. In some laboratories, this is done in combination with MWGT.

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The endotoxin assay is a test to detect or quantify bacterial endotoxins using a reagent prepared from lysate extract of blood of horse shoe crab (Limulus polyphemus or Tachypleus tridentatus). The test is based on highly sensitive clotting of horse shoe crab blood in the presence of endotoxin.

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This is prepared in-house by injecting a rabbit intramuscularly into the rear leg with one single human dose (0.5 ml) of pertussis whole cell vaccine (NIBSC 88/522). Boost after 4 weeks with the same dose by sub-cutaneous route. Bleed after a further 2 weeks and store sera infinitely as 200 μl aliquots at -20°C. When thawed, store at +4°C for up to 1 year with the expiry date written on the container. There is no requirement for a set titre.

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In the ideal situation, an assay would give the ‘true’ potency of the test vaccine without error. However, the biological systems which form the basis of bioassays are inherently variable, reflecting the responsiveness of biological functions to a wide variety of environmental and other factors. This responsiveness gives bioassays their high specificity and sensitivity for measuring the biological activity of vaccines. This responsiveness also means that good experimental design is essential to ensure that all factors apart from the applied treatment are carefully controlled in order to reduce variability and the possibility of bias. ....//.... Many differences between animals can be controlled by selecting healthy animals with restricted sex, age, weight range and other characteristics. Genetically homogeneous inbred strains might reduce animal variation, but in practice healthy out-bred strains are widely used without apparent problems.....//..... Some differences will inevitably remain. The role of randomization of the animals is to ensure that any remaining differences and any uncontrolled factors are spread with equal probability among all treatments. This avoids bias and the effects of any unknown or unspecified factors to link to particular treatments. Thus only the specified treatments differentially influence the responses. Complete randomization of all animals or experimental units may not be practical or feasible. Nevertheless, highly structured and systematic designs should be avoided. Similarly, it may not be feasible to use a completely randomized design for some in vivo assays. As far as possible, animals should be randomly assigned to cages, treatments should be blinded and assigned to cages in randomized order, and replicate cages should be used to provide some indication of the magnitude of any cage effects. It may be possible to design experiments in which cages are ‘blocks’ with the number of animals in a cage being the same as the total number of treatments, and with each animal in the cage receiving a different treatment. A simple example of a ‘block’ design is given. Professional advice should be taken to ensure the best analysis if more complex designs are used."

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