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

Original text

Any scientist carrying out bioassays using animals should be aware of the 3Rs, as described by Russell and Burch (1959). Thus, in vivo bioassays should only be used if scientifically valid in vitro or other techniques are not available. Refinement should be introduced as far as possible in in vivo bioassays. For example, several of the assays described here employ ‘humane endpoints’. Techniques are being developed which allow use of the same animals for serologically based testing of more than one component in some combined vaccines. Replacement of in vivo bioassays is encouraged, but it must be based on sound scientific principles and requires stringent validation. Reduction, a key element of the 3Rs has been described as the mode of progress ‘most obviously, immediately, and universally advantageous in terms of efficiency’ without loss of scientific information. Good experimental design is a key element in reduction. Thus, in vivo assays should be designed to use the minimum number of animals consistent with giving an estimate with the required precision.

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The analyses and examples given here are for the more straight forward designs, and typically for the completely randomized design. If the total group of animals is reasonably homogeneous, then allocation of treatments to animals is made randomly, subject to the restriction that each treatment is as far as possible allocated to the same number of animals. Where the animals can be divided into recognizable sub-groups, then these sub-groups should be considered separately, and treatments allocated randomly within the sub-group. For example, for the challenge assay for tetanus vaccine potency, if both male and female animals are used, then there must be equal numbers of males and females in each treatment group. The experimental records should include details of the sex with the response, so that data for male and female animals can be separately analyzed, or so that the difference in response between them can be included in the analysis of variance. Animals might similarly be put into sub-groups on the basis of weight or age, if these factors have not been strictly limited when the total group of animals is selected. These identified sub-groups must also be equally distributed among the treatment groups.

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The need to test the final lots of the Ebola vaccine for unexpected toxicity (also
known as abnormal toxicity) should be discussed and agreed with the NRA.
Some countries no longer require this test (88, 89).

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Each final lot should be tested for pyrogenic substances through intravenous injection into rabbits. A Limulus amoebocyte lysate (LAL) test may be used in lieu of the rabbit pyrogen test if it has been validated and the presence of non-endotoxin pyrogens has been ruled out. A suitably validated monocyte-activation test may also be considered as an alternative to the rabbit pyrogen test. The endotoxin content or pyrogenic activity should be consistent with levels found to be acceptable in vaccine lots used in clinical trials and should be approved by the NRA.

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The need for pyrogenicity testing should be assessed during the manufacturing development process and be re-evaluated following any significant changes in the production process or relevant reported production inconsistencies that may influence pyrogenicity. A risk-based approach should be implemented which is suitable to the manufacturing process and the product depending on the potential presence of endotoxins and non-endotoxin pyrogens. The endotoxin content of the final product should be determined using a suitable in vitro assay, such as the recombinant factor C (rFC) or limulus/tachypleus amoebocyte lysate (LAL/TAL) tests. The rFC method is strongly recommended due to concerns over the impact on the sustainability of limulus stocks. The endotoxin content should be consistent with levels found to be acceptable in final product lots used in clinical trials and within the limits agreed upon with the NRA. A monocyte activation test (MAT) may be used for pyrogen testing after a product-specific validation. The use of the rabbit pyrogen test should be avoided due to its inherent variability, high retesting rates, and interspecies differences in pyrogenic responses as compared to humans.

Original text

At the end of the observation period a fraction of control cells comprising not less than 25% of the total should be tested for the presence of haemadsorbing viruses, using guinea-pig red blood cells. If the red blood cells have been stored prior to use in the haemadsorption assay, the duration of storage should not have exceeded 7 days and the temperature of storage should have been in the range of 2–8 °C.

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At the end of the observation period a fraction of culture comprising not less than 25% of the total should be tested for the presence of haemadsorbing viruses, using red blood cells from guinea-pig or other suitable red blood cells. It is not necessary to use red blood cells from multiple species. If the red blood cells have been stored prior to use in the haemadsorption assay, the duration of storage should not have exceeded 7 days and the temperature of storage should have been in the range of 2–8 °C.

Original text

At the end of the observation period a fraction of control cells comprising not less than 25% of the total should be tested for the presence of haemadsorbing viruses, using guinea-pig red blood cells. If the red blood cells have been stored prior to use in the haemadsorption assay, the duration of storage should not have exceeded 7 days and the temperature of storage should have been in the range of 2–8 °C.

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At the end of the observation period a fraction of culture comprising not less than 25% of the total should be tested for the presence of haemadsorbing viruses, using red blood cells from guinea-pig or other suitable red blood cells. It is not necessary to use red blood cells from multiple species. If the red blood cells have been stored prior to use in the haemadsorption assay, the duration of storage should not have exceeded 7 days and the temperature of storage should have been in the range of 2–8 °C.

Original text

Antigenic specificity may be confirmed by an immunodiffusion or haemagglutination-inhibition technique using appropriate specific immune sera. The tests for haemagglutinin content (A.3.4.2) and presence of neuraminidase (A.3.4.3) also serve as identity tests.
Reference viruses for identity tests may be obtained from reference laboratories (Appendix 2).
Alternatively antigenic identity may be confirmed by:
— injection of vaccine into mice, chickens or other suitable animals and demonstration of the production of antibodies to the haemagglutinin of the influenza virus used to produce the vaccine. In addition, demonstration of production of antibody to neuraminidase may also be performed; or
— suitable genetic tests.
With split and subunit vaccines, the identity test may be performed before virus disruption.

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Antigenic specificity may be confirmed by an immunodiffusion or haemagglutination-inhibition technique using appropriate specific immune sera. The tests for haemagglutinin content (A.3.4.2) and presence of neuraminidase (A.3.4.3) also serve as identity tests. Reference viruses for identity tests may be obtained from reference laboratories (Appendix 2).

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Each filling lot should be tested for unexpected toxicity (sometimes called abnormal toxicity) using a general safety (innocuity) test approved by the national regulatory authority.
This test may be omitted for routine lot release once consistency of production has been well established to the satisfaction of the national regulatory authority and when good manufacturing practices are in place. Each lot, if tested, should pass a test for abnormal toxicity.

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A test for endotoxin should be included, e.g. the Limulus amoebocyte lysate test.
The permissible level of endotoxin is determined by the national regulatory authority. It is likely that the permissible level of endotoxin for mammaliancell-derived vaccine will be lower than that for egg-derived vaccine.

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The need for pyrogenicity testing should be assessed during the manufacturing development process and be re-evaluated following any significant changes in the production process or relevant reported production inconsistencies that may influence pyrogenicity. A risk-based approach should be implemented which is suitable to the manufacturing process and the product depending on the potential presence of endotoxins and non-endotoxin pyrogens. The endotoxin content of the final product should be determined using a suitable in vitro assay, such as the recombinant factor C (rFC) or limulus/tachypleus amoebocyte lysate (LAL/TAL) tests. The rFC method is strongly recommended due to concerns over the impact on the sustainability of limulus stocks. The endotoxin content should be consistent with levels found to be acceptable in final product lots used in clinical trials and within the limits agreed upon with the NRA. A monocyte activation test (MAT) may be used for pyrogen testing after a product-specific validation. The use of the rabbit pyrogen test should be avoided due to its inherent variability, high retesting rates, and interspecies differences in pyrogenic responses as compared to humans.

Original text

For influenza vaccine (human, live attenuated) for intranasal administration prepared in vaccine-quality embryonated eggs, a sample of 0.25 ml of allantoic fluid taken from each control egg should be tested for haemagglutinating agents by the addition of chick erythrocytes,.......
For influenza vaccine (human, live attenuated) for intranasal administration prepared in cell culture, testing for the presence of haemadsorbing viruses at the end of the observation period or at the time the virus is harvested from the production substrate, whichever is later, should include at least 25% of control cells. The control cells should be tested using guinea-pig red blood

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