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

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Several laboratory methods for assessing venom-induced neurotoxicity have been developed (for example, chick biventer cervicis nerve-muscle preparation (157, 158) and the mouse hemidiaphragm phrenic nerve preparation (2,159–162). However, they are difficult to perform, require costly equipment and technological expertise and are unlikely to be practicable for most antivenom producers. Mouse lethality tests are usually reliable in predicting the neutralization of neurotoxic effects of venoms.

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Suggest the following: Several laboratory methods for assessing venom-induced neurotoxicity have been developed (for example, chick biventer cervicis nerve-muscle preparation (157, 158) and the mouse hemidiaphragm phrenic nerve preparation (2,159–162). Manufacturers are encourage to use these methods (or to develop and validate other alternatives). Mouse lethality tests should be avoided where more humane alternatives are available and appropriate.

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It is acknowledged that the in vivo and in vitro essential and supplementary preclinical tests have physiological limitations. Venom and venom/antivenom injection protocols do not represent the natural situation, and the physiological responses of rodents to envenoming and treatment may differ from those of humans. Even comparing the levels of immune recognition gathered from antivenomic or ELISA data with the in vivo neutralization capacity of an antivenom, is not straightforward. Such limitations make the rodent model of human envenoming and treatment less than ideal.

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The estimation of the ability of an antivenom to neutralize the
lethal activity of venom(s) (LD50 and ED50) is a critical preclinical assessment and should be performed by manufacturers for all antivenoms, and enforced by the NRAs as part of the antivenom licensing procedure.
-All practitioners of these preclinical tests must prioritize the implementation of 3R to reduce the substantial number of mice used, and their collective pain, harm and distress.
-In vitro methods such as ELISA, antivenomics or other emerging technologies that enable antivenoms to be screened for immune recognition of venom components prior to in vivo evaluation should be adopted by manufacturers.
-The development of improved in vivo assay protocols to reduce pain and suffering of animals, such as routine use of opioids or other analgesics, and of in vitro alternatives to the in vivo assays to reduce the number of animals used in preclinical testing, is encouraged. The results of any modified in vivo, or new in vitro protocols, should be rigorously compared with results from existing protocols and validated to ensure statistical reliability of the newly developed methods.

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The estimation of the ability of an antivenom to neutralize the lethal activity of venom(s) is a critical preclinical assessment and should be performed by manufacturers for all antivenoms, and enforced by the NRAs as part of the antivenom licensing procedure. -All practitioners of these preclinical tests must prioritize the implementation of 3R to reduce the substantial number of mice used, and their collective pain, harm and distress. -In vitro methods such as ELISA, antivenomics or other emerging technologies that enable antivenoms to be screened for immune recognition of venom components prior to in vivo evaluation should be adopted by manufacturers. -The development of improved in vivo assay protocols to reduce pain and suffering of animals, such as routine use of opioids or other analgesics, and of in vitro alternatives to the in vivo assays to reduce the number of animals used in preclinical testing, is encouraged. The results of any modified in vivo, or new in vitroprotocols, should be rigorously compared with results from existing protocols and validated to ensure statistical reliability of the newly developed methods.

Original text

Current methods of antivenom production rely on the use of animals to manufacture these life-saving products. For all animals, whether they are venomous snakes from which venom is obtained for use as an immunogen; the horses, sheep or other large animals that are injected with the venom, and serve as living antibody factories, producing hyperimmune plasma from which antivenom is derived; or the small laboratory animals sacrificed in order to test the preclinical efficacy and safety of antivenoms, there is an absolute necessity for all manufacturers to use animals humanely and ethically. It is imperative that venom producers, antivenom manufacturers and quality control laboratories that make use of animals in venom or antivenom research, production, or in the preclinical evaluation of antivenoms adhere to the highest ethical standards. The International guiding principles for biomedical research involving animals (2012) developed by the International Council for Laboratory Animal Science and the Council for International Organization of Medical Sciences provide an international benchmark for the use of animals in research. Compliance with national guidelines, laws and regulations is also essential. All animal experimentation should be subject to regulatory oversight at an institutional and national level. In many jurisdictions, the 3R principles of
Replacement, Reduction and Refinement have been adopted as cornerstones of ethical use of animals, and WHO strongly recommends that every effort be made to reduce pain, distress and discomfort to experimental animals – for example, by the routine use of analgesia in mice used in these assays.

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Venomous snakes kept in serpentariums for use in venom production should be maintained according to nationally and internationally accepted ethical standards. All relevant local regulations should be strictly adhered to, and where required the use of venomous snakes in venom production should be conducted in accordance with ethics approvals obtained from responsible authorities in the jurisdiction.
The types of animals used as food, their production, humane euthanasia, or in some cases, presentation to snakes as live prey, require appropriate ethical considerations, and specific licences and ethics approvals may be required to keep, breed and use some animals as sources of food for venomous snakes. Venom producers must ensure that their operations comply with all necessary regulations and requirements in this regard.

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The use of large animals (for example, horses, ponies, mules and sheep) in the production of hyperimmune plasma requires constant veterinary supervision and strict adherence to approved national and international ethical standards for these animals.
Equines are the most commonly used for production of hyperimmune plasma in antivenom production and have specific physiological and psychological requirements for good health and the minimization of pain and distress. Manufacturers must recognize these needs and structure their use of animals to ensure that their social, physical and environmental needs are appropriately met.
Animals used in plasma production may suffer considerable distress, pain or discomfort as a result of the immunization process and all manufacturers have an obligation to strictly comply with animal welfare and ethical use requirements and actively work to minimize these deleterious effects. Similarly, the bleeding of animals to collect hyperimmune plasma can be traumatic for donor animals if appropriate techniques are not used to minimize negative effects, including fear, pain, distress and physical harm. Manufacturers are encouraged very strongly to proactively improve the welfare of large animals used in plasma production, and to develop protocols that reduce suffering and improve the health of animals.

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The preclinical testing of new or existing antivenoms necessitates the use of experimental animals, typically rodents, particularly for essential median lethal venom dose (LD50) and median effective antivenom dose (ED50) determination. Despite reservations about the physiological relevance of these animal models to human envenoming and the harm that these in vivo assays cause to the animals (sections 19.2 and 19.3), they are used by both manufacturers and regulatory authorities worldwide for determining venom lethality (LD50) and antivenom neutralizing capacity (ED50) as these are currently the only validated means of assessing venom toxicity and antivenom neutralizing potency. Non-sentient or in vitro assays as alternatives to the standard venom LD50 and antivenom
ED50 in vivo tests have been promoted (2). Unfortunately, such systems have not been developed to the point where they can fully replace the above-mentioned preclinical assays.

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The use of animals for potency testing of antivenoms raises important ethical considerations and it is essential that 3Rs principles are applied, including use of appropriate analgesia, anaesthesia, humane endpoints, high standards of animal housing husbandry and care and optimization of experimental design to use the minimum number of experimental animals to measure the potency of an antivenom. In this context, developments of appropriate in vitro immunochemical methods validated for replacing animal experiments are strongly encouraged. If an in vitro assay has been developed it should be implemented as the potency test if approved by the NRA.

Original text

In vivo murine assays cause considerable suffering and a 3R approach involving innovation and validation should be applied in the development of standardized LD50 and ED50 test protocols. Designing protocols that use the minimal number of animals necessary and introducing procedures to minimize pain and suffering is essential. The development of alternative methods to replace animal testing in the preclinical evaluation of antivenoms should be encouraged.
The establishment of humane end-points to reduce suffering and limiting the duration of the assays to reduce the period of animal suffering is encouraged; this requires appropriate standardization and validation within a quality assurance framework.

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The substantial suffering caused to small animals by the preclinical assays is outweighed by the considerable benefits to human health. Nevertheless, WHO strongly encourages that opportunities to implement alternatives to the essential and supplementary tests, according to the 3R, to reduce pain, harm and distress be tested. Thus, designing protocols that use the minimum number of animals necessary and introducing procedures to minimize pain and suffering is essential.
Analgesia should be considered, and evaluated to ensure that the humane benefits of analgesia to the experimental animals do not invalidate the objectives of the assay by altering relevant physiological processes (3). In particular, the use of analgesia is recommended when working with venoms that induce tissue damage (4). The establishment of humane end-points, instead of using survival/death as the assay metric, is encouraged to reduce suffering and limit the duration of the assays. The use of humane end-points also offers the opportunity to introduce ‘dose-staging’ into the experimental design (in which multiple doses are prepared for the assays, one dose given and the next dose(s) selected based on the results of giving the previous dose) to reduce the number of mice required for these assays. All such efforts towards 3R require appropriate standardization and validation within a quality assurance framework.

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