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

Original text

When a new working seed lot is established, a suitable test for delayed hypersensitivity in guinea-pigs is carried out; the vaccine is shown to be not significantly different in activity from the in-house reference.

New text

Original text

The test for absence of virulent mycobacteria, described in Part A, section 4.2.3, should be made in at least 10 healthy guinea-pigs injected with a quantity o fvaccine not less than 50 single human doses and should be observed for at least six weeks. If none of the animals shows signs of progressive TB and at least 90% survive the observation period (i.e. should one of the 10 animals die), the seed lot should be considered to be free from virulent mycobacteria. If more than 10% of the guinea-pigs die during the observation period (i.e. should two out of 10 animals die) and freedom from progressive TB disease is verified, the test should be repeated on at least 10 more guinea-pigs. On the second occasion, the seed lot passes the test if not more than 10% of the animals die during the observation period (i.e. should one of the 10 animals die) and the autopsy does not reveal any sign of TB.

New text

A test for the absence of virulent mycobacteria should be performed. Where available and appropriately validated, an in vitro test should be used (for example a validated nucleic acid amplification test or culture method). If in vitro assays are not available or appropriate, a suitable compendial in vivo test may be used.

Original text

The test for excessive dermal reactivity, described in Part A, section 6.4.2, should be made in six healthy guinea-pigs, each weighing not less than 250 g and having received no treatment likely to interfere with the test. Each guineapig should be injected intradermally, according to a randomized plan, with 0.1 ml of the reconstituted vaccine and of vaccine dilutions 1:10 and 1:100. The same dilutions of the appropriate international Reference Reagent or in-house reference should be injected into the same guinea-pigs at randomly selected sites. The guinea-pigs should be observed for at least four weeks. The vaccine complies with the test if the reactions it produces at the sites of injection are not markedly different from those produced by the appropriate international Reference Reagent or in-house reference.

New text

A test for excessive dermal reactivity should be performed. Where available and appropriately validated, an in vitro test should be used. If in vitro assays are scientifically justified as unavailable and inappropriate, a suitable compendial in vivo test may be used.

Original text

At least six healthy guinea-pigs, all of the same sex, each weighing 250–400 g should be used. They should not have received any treatment or diet, such as antibiotics, that is likely to interfere with the test. A sample of the final bulk intended for this test should be stored at 4 °C for not more than 72 hours after harvest. A dose of BCG organisms corresponding to at least 50 single human doses of vaccine intended for intradermal injection should be injected into each guinea-pig by the subcutaneous or intramuscular route.1 The guinea-pigs should be observed for at least six weeks. If, during that time, they remain healthy, gain weight, show no signs of progressive TB and not more than one dies, the final bulk should be considered to be free from virulent mycobacteria.

New text

A test for the absence of virulent mycobacteria should be performed. Where available and appropriately validated, an in vitro test should be used (for example a validated nucleic acid amplification test or cell culture method). If in vitro assays are scientifically justified as unavailable and inappropriate, a suitable compendial in vivo test may be used.

Original text

Provided the test for virulent mycobacteria has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. If the test for the absence of virulent mycobacteria, applied to the final bulk, is unsatisfactory (and freedom from progressive TB disease is verified), it should be repeated with a sample of a final lot (see Part A, section 4.2.3).

New text

A test for the absence of virulent mycobacteria should be performed. Where available and appropriately validated, an in vitro test should be used (for example a validated nucleic acid amplification test or cell culture method). If in vitro assays are scientifically justified as unavailable and inappropriate, a suitable in vivo test may be used.

Original text

Provided the test has been carried out with satisfactory results on the working seed lot and on at least three consecutive final lots produced from it, the test may be omitted on the final lot.

New text

Original text

If the working virus seed lot is derived from mouse brain or primary cell cultures, it should be tested for adventitious agents as in section A.3.2.3.3. If working virus seed lots are produced in cells derived from a validated cell bank where a master virus seed lot was tested for adventitious agents, these tests do not have to be repeated.
A.3.2.3.3 - Tests for adventitious agentsThe master virus seed lot should be tested for adventitious agents. For these tests the virus should be neutralized by a specific anti-Japanese-encephalitis serum. The specificity and sensitivity of assays should be defined and approved by the national regulatory authority.

New text

A strategy for testing adventitious viruses in vaccines must be developed based on a risk assessment. Relevant culture methods and/or specific molecular biology or broad molecular methods should be part of the overall testing package with the agreement of the NRA. In vivo tests may only be used if the risk assessment indicates that this test provides an additional risk mitigation taking into account the overall testing package.

Original text

A sample removed from each virus harvest should be tested for virus content using suitable methods approved by the national regulatory authority. Both mice and cell culture with defined sensitivity are suitable for testing infectivity.

New text

A sample removed from each virus harvest should be tested for virus content using suitable methods approved by the national regulatory authority. Cell culture methods with defined sensitivity are suitable for testing infectivity.

Original text

Each bulk suspension should be tested in an appropriate test system for effective inactivation of the virus before the addition of preservatives and other substances. The sensitivity of the assay should be determined according to the JE virus used for production and the most sensitive assay should be used.
In some countries the test involves direct inoculation intracerebrally into mice followed by three blind passages.

New text

Each bulk suspension should be tested in an appropriate test system for effective inactivation of the virus before the addition of preservatives and other substances. The sensitivity of the assay should be determined according to the JE virus used for production and the most sensitive assay should be used.

Original text

Each final lot should be tested for pyrogenic substances. The test procedures
should be approved by the national regulatory authority.

New text

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.