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

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Efficacy testing of antivenoms is one of a suite of assessments required for the quality control of antivenoms (see section 17 where further quality control tests are described) performed for each new batch. Efficacy testing of antivenoms is also part of the preclinical programme to be performed for new antivenoms, where the respective data are used for licensing or registration of antivenoms by regulatory agencies. The details of efficacy testing in the preclinical phase of antivenoms and for quality control purposes are described below. The testing of antivenoms on animals raises important ethical considerations (section 4) and it is essential that manufacturers and others apply the highest standards of ethical conduct, including appropriate 3R steps, and use of analgesia or anaesthesia for the minimization of pain and discomfort.

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To prevent unnecessary animal use, careful perusal of existing literature for data on venom lethality may help to refine the experimental design and thereby reduce the number of experimental animals required. Manufacturers may also investigate the immunological venom-binding capability of an antivenom by performing immunological assays (for example, ELISA, to identify, and exclude from experimentation, antivenoms that do not possess the requisite titre of venom-binding immunoglobulins. It is crucial to note, however, that: (a) a high venom-binding titre in an ELISA result for an antivenom cannot be used to infer venom-neutralizing efficacy; and (b) the failure of an antivenom to bind venom in an ELISA result suggests very strongly that the antivenom should be considered ineffective at neutralizing the effects of that venom – and withdrawn from ED50 testing. This step can further limit nonproductive animal experimentation. There is no single ELISA metric that enables stop/go decisions to be made for all the possible snake venom and antivenom combinations. These will therefore be in-house decisions.

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An additional immunological cross-reactivity technology that can inform the preclinical assessment process before animal experiments are undertaken is the use of a proteomics-centred platform, termed antivenomics, which has been developed to assess the immunological reactivity of antivenoms against homologous and heterologous venoms (136–139). Antivenomics complements the in vitro and in vivo venom activity neutralization assays and substitute for the traditional, essentially qualitative, immunological methods, such as ELISA and Western blotting.

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For venoms whose LD50 is unknown, it is recommended that a range dosefinding study, using one mouse per venom dose, is performed to set a narrow range of dose parameters for the full LD50 test – reducing the total number of animals required.

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For venoms whose LD50 is unknown, and where a non-animal alternative potency test is not available/appropriate, it is recommended that a range dosefinding study, using one mouse per venom dose, is performed to set a narrow range of dose parameters for the full LD50 test – reducing the total number of animals required.

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Venom doses are prepared in saline and intravenously injected (maximum 0.2 mL) into the tail vein of groups of 5–6 mice (of a defined weight range). A group size of five mice is the smallest number recommended for obtaining a statistically significant result. In some laboratories the LD50 is estimated by the intraperitoneal route using an injection volume of a maximum of 0.5 mL. Deaths are recorded at 24 hours (for assays involving intravenous injections) or at 48 hours (when intraperitoneal injections are used), and LD50 is estimated by Probit analysis (141), Spearman-Karber (11) or alternative procedures (such as non-parametric methods). One venom LD50 dose is defined as the amount of venom causing death in 50% of injected mice.

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The venom LD50 results provide the information necessary to test the venomneutralizing efficacy of an antivenom – using the median effective dose (ED50) assay. It is important that the venoms used in the ED50 assays are from the same batch (lot) as that used to determine the venom LD50 result. It is equally important that all the LD50 and ED50 assays utilize mice of identical strain and weight.

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The venom lethal potency (determined by a suitable in vitro or in vivo assay) results provide the information necessary to test the venomneutralizing efficacy of an antivenom – using the median effective dose (ED50) assay. It is important that the venoms used in the ED50 assays are from the same batch (lot) as that used to determine the venom lethal potency result. It is equally important that all the lethal potency and ED50 assays are approprately standardised.

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The minimum haemorrhagic dose of a venom (MHD) quantifies this venom-induced pathology, and is defined as the amount of venom (in μg dry weight) which, when injected intradermally, induces in mice a 10 mm haemorrhagic lesion after a predefined
time interval, usually 2–3 hours, after injection (145, 146).

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The minimum necrotizing dose (MND) of a venom is defined as the smallest amount of venom (in μg dry weight) which, when injected intradermally into groups of five lightly anaesthetized mice (18–20 g body weight), results in a necrotic lesion of 5 mm diameter 3 days later.

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This test is a direct measure of the in vivo defibrinogenating effect of certain venoms. To measure the minimum venom defibrinogenating dose (MDD), a wide range of venom doses is selected and each dose, in a volume of 0.2 mL, is injected intravenously into five mice (18–20 g body weight). One hour after injection, the mice are placed under terminal general anaesthesia and bled by cardiac puncture.

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Venom myotoxic activity is determined by injecting mice with various doses of venom in a constant volume of 50 μL (using saline solution as diluent) into the right gastrocnemius muscle. Groups of five animals of 18–20 g body weight are used per dose. Control animals are injected with the same volume of saline solution. Tail-snip blood samples are collected after a specified time interval (3 hours in mice), and the CK activity of serum or plasma is determined using commercially available diagnostic kits (155, 156).

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