ECBS 2016 BS2300 WHO Guidelines Antivenom Clean1

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3 WHO/BS/2016.2300
4 ENGLISH ONLY
6 WHO Guidelines for the Production Control and

7 Regulation of Snake Antivenom Immunoglobulins
8 NOTE:
9 This document has been prepared for the purpose of inviting comments and suggestions on the proposals
10 contained therein, which will then be considered by the Expert Committee on Biological Standardization
11 (ECBS). Publication of this early draft is to provide information about the proposed WHO Guidelines for
12 the Production Control and Regulation of Snake Antivenom Immunoglobulins, to a broad audience and to
13 improve transparency of the consultation process.
14 The text in its present form does not necessarily represent an agreed formulation of the Expert Committee.
15 Written comments proposing modifications to this text MUST be received by 30 September 2016 in
16 the Comment Form available separately and should be addressed to the World Health Organization,
17 1211 Geneva 27, Switzerland, attention: Department of Essential Medicines and Health Products (EMP).
18 Comments may also be submitted electronically to the Responsible Officer: Dr C Micha Nübling at email:
19 [email protected].
20 The outcome of the deliberations of the Expert Committee on Biological Standardization will be published
21 in the WHO Technical Report Series. The final agreed formulation of the document will be edited to be in
22 conformity with the "WHO style guide" (WHO/IMD/PUB/04.1).
23
24 © World Health Organization 2016
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28 addressed to WHO Press, at the above address (fax: +41 22 791 4806; e-mail: [email protected]).
29 The designations employed and the presentation of the material in this publication do not imply the expression of any opinion
30 whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its
31 authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for
32 which there may not yet be full agreement.
33 The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by
34 the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the
35 names of proprietary products are distinguished by initial capital letters.
36 All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication.
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39 arising from its use.
40 Second edition, draft (August 28, 2016)

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2 CONTENTS
3 1 Introduction ......................................................................................................................... 7
4 2 List of abbreviations and definitions used............................................................................ 9
5 3 General considerations ..................................................................................................... 14
6 3.1 Historical background ................................................................................................ 14
7 3.2 The use of serum versus plasma as source material ................................................. 14
8 3.3 Antivenom purification methods and product safety ................................................... 14
9 3.4 Pharmacokinetics and pharmacodynamics of antivenoms ......................................... 15
10 3.5 Need for national and regional reference venom preparations ................................... 15
11 4 Epidemiological Background ............................................................................................. 16
12 4.1 Global burden of snakebites ...................................................................................... 16
13 4.2 Main recommendations.............................................................................................. 16
14 5 Worldwide distribution of venomous snakes...................................................................... 18
15 5.1 Taxonomy of venomous snakes ................................................................................ 18
16 5.2 Medically important venomous snakes ...................................................................... 21
17 5.3 Minor venomous snake species ................................................................................. 23
18 5.4 Sea snake venoms .................................................................................................... 24
20 6 Antivenoms design: selection of snake venoms ................................................................ 25
21 6.1 Selection and preparation of representative venom mixtures ..................................... 25
22 6.2 Manufacture of monospecific or polyspecific antivenoms ........................................... 25
23 6.2.1 Monospecific antivenoms .................................................................................... 25
24 6.2.2 Polyspecific antivenoms...................................................................................... 26
26 7 Preparation and storage of snake venom .......................................................................... 28
27 7.1 Production of snake venoms for immunization ........................................................... 28
28 7.1.1 Quarantine of snakes .......................................................................................... 28
29 7.1.2 Maintenance of captive snakes for venom production ......................................... 29
30 7.1.3 General maintenance of a serpentarium ............................................................. 30
31 7.1.4 Snake venom production .................................................................................... 31
32 7.2 Staff responsible for handling snakes ........................................................................ 33
33 7.2.1 Safety and health considerations ........................................................................ 33
34 7.2.2 Personal Protective Equipment (PPE) for snake or venom handling ................... 33
35 7.2.3 Procedures to be followed if a bite occurs ........................................................... 34
37 8 Quality control of venoms.................................................................................................. 36
38 8.1 Records and traceability ............................................................................................ 36
39 8.2 National reference materials ...................................................................................... 36
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1 8.3 Characterization of venom batches ............................................................................ 36
2 8.4 Main recommendations .............................................................................................. 37
3 9 Overview of the production process of antivenoms............................................................ 38
4 10 Selection and veterinary health care of animals used for production of antivenoms ....... 40
5 10.1 Selection and Quarantine period ................................................................................ 40
6 10.2 Veterinary care, monitoring and vaccinations ............................................................. 40
7 10.3 Animal health and welfare after inclusion in the herd .................................................. 40
9 11 Immunization regimens and use of adjuvant .................................................................. 43
10 11.1 Animals used in antivenom production ....................................................................... 43
11 11.2 Venoms used for immunization .................................................................................. 43
12 11.3 Preparation of venom doses....................................................................................... 43
13 11.4 Detoxification of venom .............................................................................................. 44
14 11.5 Immunological adjuvants ............................................................................................ 45
15 11.6 Preparation of immunogen in adjuvants ..................................................................... 45
16 11.7 Immunization of animals ............................................................................................. 45
17 11.8 Traceability of the immunization process .................................................................... 48
19 12 Collection and control of animal plasma for fractionation ............................................... 50
20 12.1 Health control of the animal prior to and during bleeding sessions ............................. 50
21 12.2 Premises for blood or plasma collection ..................................................................... 50
22 12.3 Blood or plasma collection session............................................................................. 50
23 12.4 Labelling and identification ......................................................................................... 51
24 12.4.1 Collection and storage of whole blood ................................................................. 51
25 12.4.2 Plasma collection by automatic apheresis and storage ....................................... 52
26 12.5 Pooling ....................................................................................................................... 52
27 12.6 Control of plasma prior to fractionation ....................................................................... 53
29 13 Purification of immunoglobulins and immunoglobulin fragments in the production of ..... 55
30 13.1 Good manufacturing practices.................................................................................... 55
31 13.2 Purification of the active substance ............................................................................ 55
32 13.2.1 Purification of intact IgG antivenoms ................................................................... 55
33 13.2.2 Purification of F(ab')2 antivenoms ....................................................................... 56
34 13.2.3 Purification of Fab antivenoms ............................................................................ 57
35 13.2.4 Optional additional steps used by some manufacturers....................................... 62
36 13.2.5 Formulation ......................................................................................................... 62
37 13.2.6 Analysis of bulk product before dispensing.......................................................... 62
38 13.2.7 Dispensing and labelling of final product ............................................................. 63
39 13.2.8 Use of preservatives ........................................................................................... 64
40 13.2.9 Freeze-drying ...................................................................................................... 64
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1 13.2.10 Inspection of final container ............................................................................. 64
2 13.2.11 Archive samples of antivenoms ....................................................................... 65
3 13.3 Pharmacokinetic and pharmacodynamic properties of IgG, F(ab')2 and Fab ............. 65
5 14 Control of infectious risks .............................................................................................. 68
6 14.1 Background ............................................................................................................... 68
7 14.2 Risk of viral contamination of the starting plasma ...................................................... 68
8 14.3 Viral validation of manufacturing processes ............................................................... 68
9 14.3.1 Down-scale experiments..................................................................................... 71
10 14.3.2 Selection of viruses for the validation of antivenom production processes .......... 71
11 14.4 Viral validation studies of antivenom immunoglobulins............................................... 72
12 14.4.1 Caprylic acid treatment ....................................................................................... 73
13 14.4.2 Acid pH treatment ............................................................................................... 74
14 14.4.3 Filtration steps .................................................................................................... 75
15 14.4.4 Validation of dedicated viral reduction treatments ............................................... 75
16 14.4.5 Other viral inactivation treatments currently not used in antivenom manufacture 75
17 14.4.6 Possible contribution of phenol and cresols ........................................................ 75
18 14.5 Production-scale implementation of process steps contributing to viral safety ........... 76
19 14.6 Transmissible spongiform encephalopathy ................................................................ 77
21 15 Quality control of antivenoms ........................................................................................ 79
22 15.1 Routine assays .......................................................................................................... 79
23 15.1.1 Appearance ........................................................................................................ 79
24 15.1.2 Solubility (freeze-dried preparations) .................................................................. 79
25 15.1.3 Extractable volume ............................................................................................. 79
26 15.1.4 Venom-neutralizing efficacy tests ....................................................................... 79
27 15.1.5 Osmolality ........................................................................................................... 79
28 15.1.6 Identity test ......................................................................................................... 80
29 15.1.7 Protein concentration .......................................................................................... 80
30 15.1.8 Purity and integrity of the immunoglobulin........................................................... 80
31 15.1.9 Molecular-size distribution .................................................................................. 80
32 15.1.10 Test for pyrogen substances ........................................................................... 80
33 15.1.11 Abnormal toxicity test ...................................................................................... 81
34 15.1.12 Sterility test ..................................................................................................... 81
35 15.1.13 Concentration of sodium chloride and other excipients.................................... 81
36 15.1.14 Determination of pH ........................................................................................ 81
37 15.1.15 Concentration of preservatives ........................................................................ 81
38 15.1.16 Chemical agents used in plasma fractionation................................................. 81
39 15.1.17 Residual moisture (freeze-dried preparations) ................................................. 82
40 15.2 Antivenom reference preparations ............................................................................. 82
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2 16 Stability, storage and distribution of antivenoms ............................................................ 84
3 16.1 Stability ...................................................................................................................... 84
4 16.2 Storage ...................................................................................................................... 84
5 16.3 Distribution ................................................................................................................. 85
7 17 Preclinical assessment of antivenom efficacy ................................................................ 86
8 17.1 Ethical considerations for the use of animals in preclinical testing of antivenoms ....... 86
9 17.2 Preliminary steps which may limit the need for animal experimentation...................... 86
10 17.3 Essential preclinical assays to measure antivenom neutralisation of venom-induced
11 lethality ................................................................................................................................. 87
12 17.3.1 LD50 range-finding test: ....................................................................................... 88
13 17.3.2 The Median Lethal Dose (LD50) assay: ................................................................ 88
14 17.3.3 Antivenom Efficacy Assessment: ........................................................................ 88
15 17.3.4 General recommendations .................................................................................. 89
16 17.3.5 Development of alternative assays to replace murine lethality testing ................. 89
17 17.4 Supplementary preclinical assays to measure antivenom neutralisation of specific
18 venom-induced pathologies .................................................................................................. 90
19 17.4.1 Neutralization of venom haemorrhagic activity .................................................... 90
20 17.4.2 Neutralization of venom necrotizing activity ......................................................... 91
21 17.4.3 Neutralization of venom procoagulant effect........................................................ 91
22 17.4.4 Neutralization of in vivo venom defibrinogenating activity .................................... 92
23 17.4.5 Neutralization of venom myotoxic activity ............................................................ 92
24 17.4.6 Neutralization of venom neurotoxic activity.......................................................... 93
25 17.5 Limitations of preclinical assays ................................................................................. 93
27 18 Clinical assessment of antivenoms ................................................................................ 95
28 18.1 Introduction ................................................................................................................ 95
29 18.1.1 Identification of biting species in clinical studies of antivenoms ........................... 95
30 18.1.2 Phase I studies.................................................................................................... 96
31 18.1.3 Phase II and III studies ........................................................................................ 96
32 18.1.4 Phase IV studies ................................................................................................. 96
33 18.2 Clinical studies of antivenom ...................................................................................... 97
34 18.2.1 Dose-finding studies ............................................................................................ 97
35 18.2.2 Randomized controlled trials ............................................................................... 97
36 18.2.3 Effectiveness end-points for antivenom trials ...................................................... 98
37 18.2.4 Safety end-points for antivenom trials ................................................................. 98
38 18.2.5 Challenges in clinical testing of antivenoms ........................................................ 98
39 18.3 Post-marketing surveillance ....................................................................................... 99
40 18.3.1 Possible approaches ........................................................................................... 99
41 18.3.2 Responses to results of post-marketing studies .................................................. 99
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1 18.4 Main recommendations.............................................................................................100
2 19 Role of national regulatory authorities ..........................................................................101
3 19.1 Regulatory evaluation of antivenoms ........................................................................101
4 19.2 Establishment licencing and site inspections ............................................................101
5 19.3 Impact of good manufacturing practices....................................................................102
6 19.4 Inspections and audit systems in the production of antivenoms ................................103
7 19.5 Antivenom licensing ..................................................................................................104
8 19.6 National Reference Venoms .....................................................................................104
9 19.7 Main recommendations.............................................................................................104
10 20 Authors and acknowledgements...................................................................................106
11 20.1 First Edition...............................................................................................................106
12 20.1.1 WHO Secretariat................................................................................................108
13 20.2 Second Edition..........................................................................................................108
14 21 References ...................................................................................................................110
15 22 Annex 1 ........................................................................................................................118
16 Worldwide distribution of medically important venomous snakes .........................................118
17 23 Annex 2 ........................................................................................................................143
18 Summary protocol for production and testing of snake antivenom immunoglobulins ...........143
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1
2 1 Introduction
3 Snake antivenom immunoglobulins (antivenoms) are the only therapeutic products for the
4 treatment of snakebite envenoming. The unavailability of effective snake antivenom
5 immunoglobulins to treat envenoming by medically important venomous snakes encountered in
6 various regions of the world has become a critical health issue at global level. The crisis has
7 reached its greatest intensity in sub-Saharan Africa, but other regions, such as south and south-
8 east Asia, are also suffering from a lack of effective and affordable products.
9 The complexity of the production of efficient antivenoms, in particular the importance of
10 preparing appropriate snake venom mixtures for the production of hyperimmune plasma (the
11 source of antivenom immunoglobulins), the decreasing number of producers and the fragility of
12 the production systems in developing countries further jeopardize the availability of effective
13 antivenoms in Africa, Asia, the Middle East and South America. Most of the remaining current
14 producers are located in countries where the application of quality and safety standards needs
15 to be improved.
16 In October 2005, the WHO Expert Committee on Biological Standardization (ECBS) recognized
17 the extent of the problem and asked the WHO Secretariat to support and strengthen world
18 capacity to ensure long-term and sufficient supply of safe and efficient antivenoms. In March
19 2007, snake antivenom immunoglobulins were included in the WHO Model List of Essential
20 Medicines [1], acknowledging their role in a primary health care system.
21 WHO recognises that urgent measures are needed to support the design of immunizing snake
22 venom mixtures that can be used to make the right polyspecific antivenoms for various
23 geographical areas of the world. Sustainable availability of effective and safe antivenom
24 immunoglobulins must be ensured and production systems for these effective treatments must
25 be strengthened at global level. Meaningful preclinical assessment of the neutralizing capacity
26 of snake antivenom immunoglobulins needs to be done before these products are used in
27 humans and medicines regulatory authorities should enforce the licensing of these products in
28 all countries, before they are used in the population.
29 The first edition of the “WHO Guidelines for the production, control and regulation of snake
30 antivenoms immunoglobulins” were developed in response to the above-mentioned needs and
31 approved by the ECBS in October 2008. These Guidelines covered all the steps involved in the
32 production, control and regulation of venoms and antivenoms. The guidelines are supported by
33 a WHO Antivenoms Database Website that features information on all the venomous snakes in
34 Annex 1, including distributions and photographs, as well as information about available
35 antivenoms: http://apps.who.int/bloodproducts/snakeantivenoms/database/. It is hoped that
36 these guidelines, by comprehensively describing the current existing experience in the
37 manufacture, preclinical and clinical assessment of these products will serve as a guide to
38 national control authorities and manufacturers in the support of worldwide production of these
39 essential medicines. The production of snake antivenoms following good manufacturing
40 practices should be the aim of all countries involved in the manufacture of these life-saving
41 biological products.
42 In addition to the need to produce appropriate antivenoms, there is a need to ensure that
43 antivenoms are appropriately used and that outcomes for envenomed patients are improved.
44 This entails improving availability and access to antivenoms, appropriate distribution policies,
45 antivenom affordability, and training of health workers to allow safe and effective use of
46 antivenoms and effective management of snakebite envenoming. These important issues are
47 beyond the scope of this document and will not be further addressed specifically here, but
48 should be considered as vital components in the care pathway for envenoming.
49 This second edition was prepared in 2016 in order to ensure that the information contained in
50 these chapters remains relevant to the production of snake antivenom immunoglobulins and
51 their subsequent control and regulation. The revisions bring the taxonomic names used for
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1 snake species into line with current nomenclature, provide information on new methods of
2 production, new preclinical evaluation technologies, and address important ethical issues
3 relating to animal use. Specific information for national control authorities has been expanded.
4
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1 2 List of abbreviations and definitions used
2 The definitions given below apply to the terms used in these Guidelines. They may have
3 different meanings in other contexts.
4 Antivenom (also called antivenin, anti-snake bite serum, anti-snake venom or ASV): A purified
5 fraction of immunoglobulins or immunoglobulin fragments fractionated from the plasma of
6 animals that have been immunized against one or more snake venoms.
7 Apheresis: Procedure whereby blood is removed from the donor, separated by physical means
8 into components and one or more of them returned to the donor.
9 ASV: anti-snake venom.
10 Batch: A defined quantity of starting material or product manufactured in a single process or
11 series of processes so that it is expected to be homogeneous.
12 Batch records: All documents associated with the manufacture of a batch of bulk product or
13 finished product. They provide a history of each batch of product and of all circumstances
14 pertinent to the quality of the final product.
15 Blood collection: A procedure whereby a single donation of blood is collected in an
16 anticoagulant and/or stabilizing solution, under conditions designed to minimize microbiological
17 contamination of the resulting donation.
18 BVDV: Bovine viral diarrhoea virus.
19 Bulk product: Any product that has completed all processing stages up to, but not including,
20 aseptic filling and final packaging.
21 CK: Creatine kinase
22 Clean area: An area with defined environmental control of particulate and microbial
23 contamination, constructed and used in such a way as to reduce the introduction, generation,
24 and retention of contaminants within the area.
25 Combined antivenoms: Antivenoms directed against several venoms, prepared by mixing
26 different monospecific plasma prior to the plasma fractionation process, or purified monospecific
27 antivenom fractions prior to the aseptic filling stage.
28 Contamination: The undesired introduction of impurities of a microbiological or chemical nature,
29 or of foreign matter, into or on to a starting material or intermediate during production, sampling,
30 packaging, or repackaging, storage or transport.
31 Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES): An
32 international agreement between governments that aims to ensure that international trade in
33 specimens of wild animals and plants does not threaten their survival.
34 Cross-contamination: Contamination of a starting material, intermediate product or finished
35 product with another starting material or product during production.
36 Cross-neutralization: The ability of an antivenom raised against a venom, or a group of venoms,
37 to react and neutralize the toxic effects of the venom of a related species not included in the
38 immunizing mixture.
39 Common Technical Document (CTD) format: A specific format for product dossier preparation
40 recommended by WHO and the ICH.
41 CPD: Citrate phosphate dextrose solution, an anticoagulant agent.
42 Desiccation: A storage process where venoms are dehydrated under vacuum in the presence of
43 calcium salts or phosphoric acid.
44 dsDNA: Double strand deoxyribonucleic acid
45 dsRNA: Double strand ribonucleic acid
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1 EMCV: Encephalomyocarditis virus.
2 EIA: Enzyme immunoassay.
3 ELISA: Enyyme-linked immunosorbent assay.
4 Envenoming: Process by which venom is injected into a human by the bite of a venomous
5 snake, leading to pathological manifestations (also called envenomation).
6 Fab: An antigen-binding fragment (Fab) of an immunoglobulin comprised of heavy chain and a
7 light chain that each have a single constant domain and a single variable domain. Fab
8 fragments result from the proteolytic digestion of immunoglobulins by papain.
9 F(ab')2: An immunoglobulin fragment comprising a pair of Fab fragments connected by a protein
10 hinge, and produced by proteolytic digestion of whole immunoglobulins with pepsin.
11 FCA: Freund`s complete adjuvant
12 FIA: Freund`s incomplete adjuvant
13 Fractionation: Large-scale process by which animal plasma is separated to isolate the
14 immunoglobulin fraction, that is further processed for therapeutic use or may be subjected to
15 digestion with pepsin or papain to generate immunoglobulin fragments. The term fractionation is
16 generally used to describe a sequence of processes, generally including plasma protein
17 precipitation and/or chromatography, ultrafiltration and filtration steps.
18 Freund`s complete adjuvant (FCA): An adjuvant that may be used in the immunization process
19 of animals to enhance the immune response to venoms. It is composed of mineral oil, an
20 emulsifier and inactivated Mycobacterium tuberculosis.
21 Freund`s incomplete adjuvant (FIA): An adjuvant that may be used in the immunization process
22 of animals to enhance the immune response to venoms. It is composed of mineral oil and an
23 emulsifier.
24 Good Clinical Practice (GCP): An international standard for rigorous, ethical and high quality
25 conduct in clinical research, particularly in relation to all aspects of the design, conduct,
26 analysis, record keeping, auditing and reporting of clinical trials involving human subjects. GCP
27 standards are established by the ICH under Topic E 6 (R1).
28 Good manufacturing practice (GMP): That part of quality assurance which ensures that
29 products are consistently produced and controlled to the quality standards appropriate to their
30 intended use and as required by the marketing authorization or product specification. It is
31 concerned with both production and quality control.
32 Hgb: Haemoglobin.
33 HPLC: High-performance liquid chromatography.
34 ICH: International Council on Harmonization of Technical Requirements for Registration of
35 Pharmaceuticals.
36 IgG: Immunoglobulin G.
37 IgM: Immunoglobulin M.
38 Immunization process: A process by which an animal is injected with venom(s) to produce a
39 long-lasting and high-titre antibody response against the lethal and other deleterious
40 components in the immunogen.
41 Immunoglobulin: Immune system protein produced by B cells in plasma that can recognise
42 specific antigens. These can be generated by immunizing an animal (most often a horse)
43 against a snake venom or a snake venom mixture. Immunoglobulin G (IgG) is the most
44 abundant immunoglobulin fraction.
45 In-process control: Checks performed during production to monitor and, if necessary, to adjust
46 the process to ensure that the antivenom conforms to specifications. The control of the
47 environment or equipment may also be regarded as part of in-process control.
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1 Manufacture: All operations of purchase of materials and products, production, quality control,
2 release, storage and distribution of snake antivenom immunoglobulins, and the related controls.
3 MCD-F-effective dose (MCD-F100) or MCD-P-effective dose (MCD-P100): The minimum volume
4 of antivenom or venom/antivenom ratio, which completely prevents clotting induced by either
5 one MCD-F or MCD-P dose of venom.
6 MDD-effective dose (MDD100): The minimum volume of antivenom or venom/antivenom ratio, at
7 which the blood samples of all injected mice show clot formation after administration of one or
8 more MDD doses of venom.
9 Median effective dose (or effective dose 50%) (ED50): The quantity of antivenom that protects
10 50% of test animals injected with a number of LD50 of venom.
11 Median lethal dose or lethal dose 50% (LD50): The quantity of snake venoms, injected
12 intravenously or intraperitoneally, that leads to the death of 50% of the animals in a group after
13 an established period of time (usually 24–48 hrs).
14 MHD-median effective dose (MHD50): The minimum volume of antivenom (in µl) that reduces
15 the diameter of haemorrhagic lesions by 50% compared to those induced in animals who
16 receive a control solution of venom/saline.
17 Minimum Haemorrhagic Dose (MHD): The minimum amount of venom (in µg) that when
18 injected intradermally in mice, causes a 10-mm haemorrhagic lesion within a predefined time
19 interval (e.g.: 2-3 hours).
20 Minimum Necrotizing Dose (MND): The minimum amount of venom (in µg) that when injected
21 intradermally in groups of lightly anaesthetized mice, results in a necrotic lesion 5-mm in
22 diameter within 72 hours.
23 Minimum Coagulant Dose (MCD): The minimum amount of venom (in mg/l or µg/ml) that clots
24 either a solution of bovine fibrinogen (2.0 g/l) in 60 seconds at 37 ˚C (MCD-F) and/or a standard
25 citrated solution of human plasma (2.8 g/l fibrinogen) under the same conditions (MCD-P).
26 Minimum Defibrinogenating Dose (MDD): The minimum amount of venom that produces
27 incoagulable blood in all mice tested within one hour of intravenous injection.
28 Minimum Myotoxic Dose (MMD): The minimum amount of venom that produces a fourfold
29 increase in serum or plasma creatine kinase (CK) activity above that of control animals.
30 MMD-median effective dose (MMD50): The minimum amount of antivenom (in µl or the
31 venom/antivenom ratio) that reduces the serum or plasma CK activity by 50% compared to
32 those induced in animals who receive a control solution of venom/saline.
33 MND: Minimum necrotizing dose
34 MND-median effective dose (MND50): The minimum amount of antivenom (in µl or the
35 venom/antivenom ratio) that reduces the diameter of necrotic lesions by 50% compared to
36 those induced in animals who receive a control solution of venom/saline.
37 Monospecific antivenom: Defines antivenoms that are limited in use to a single species of
38 venomous snake or to a few closely related species whose venoms show clinically effective
39 cross-neutralization with the antivenom. The term “monovalent” is often used and has the same
40 meaning.
41 Mr: Relative molecular mass.
42 Nanofilter: Filters, most typically with effective pore sizes of 50 nm or below, designed to
43 remove viruses from protein solutions.
44 National Regulatory Authority (NRA): WHO terminology to refer to national medicines regulatory
45 authorities. Such authorities promulgate medicine regulations and enforce them.
46 PCV: Packed cell volume.
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1 Plasma: The liquid portion remaining after separation of the cellular elements from blood
2 collected in a receptacle containing an anticoagulant, or separated by continuous filtration or
3 centrifugation of anticoagulated blood in an apheresis procedure.
4 Plasmapheresis: Procedure in which whole blood is removed from the donor, the plasma is
5 separated from the cellular elements by sedimentation, filtration, or centrifugation, and at least
6 the red blood cells are returned to the donor.
7 Polyspecific antivenom: Defines antivenoms that are obtained by fractionating the plasma from
8 animals immunized by a mixture of venoms from several species of venomous snakes. The
9 term “polyvalent” is often used and has the same meaning.
10 Prion: A particle of protein that is thought to be able to self-replicate and to be the agent of
11 infection in a variety of diseases of the nervous system, such as mad cow disease and other
12 transmissible spongiform encephalopathies (TSE). It is generally believed not to contain nucleic
13 acid.
14 Production: All operations involved in the preparation of snake antivenom immunoglobulins,
15 from preparation of venoms, immunization of animals, collection of blood or plasma, processing,
16 packaging and labeling, to its completion as a finished product.
17 PRV: Pseudorabies virus.
18 Quality Manual (QM): An authorized, written controlled document that defines and describes the
19 quality system, the scope and operations of the quality system throughout all levels of
20 production, management responsibilities, key quality systems processes and safeguards
21 Quarantine: A period of enforced isolation and observation typically to contain the spread of an
22 infectious disease among animals. The same terminology applies to the period of isolation used
23 to perform quality control of plasma prior to fractionation, or of antivenom immunoglobulins prior
24 to release and distribution.
25 RCT: Randomized controlled trial of a pharmaceutical substance or medical device.
26 SDS–PAGE: Sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
27 Serpentarium: A place where snakes are kept, e.g. for exhibition and/or for collection of
28 venoms.
29 Serum: A liquid portion remaining after clotting of the blood. Serum has a composition similar to
30 plasma (including the immunoglobulins) apart from fibrinogen and other coagulation factors
31 which constitute the fibrin clot.
32 Site Master File (SMF): An authorized, written controlled document containing specific factual
33 details of the GMP production and quality control manufacturing activities that are undertaken at
34 every site of operations linked to products that a company produces.
35 ssDNA: Single strand deoxyribonucleic acid
36 ssRNA: Single strand ribonucleic acid
37 Standard Operating Procedure (SOP): An authorized written procedure giving instructions for
38 performing operations not necessarily specific to a given product or material (e.g. equipment
39 operation, maintenance and cleaning; validation; cleaning of premises and environmental
40 control; sampling and inspection). Certain SOPs may be used to supplement product-specific
41 master and batch production documentation.
42 Toxin: A toxic substance, especially a protein, which is produced by living cells or organisms
43 and is capable of causing disease when introduced into the body tissues. It is often also
44 capable of inducing neutralizing antibodies or antitoxins.
45 TPP: Total plasma protein.
46 Traceability: Ability to trace each individual snake, venom, immunized animal, or unit of blood or
47 plasma used in the production of an antivenom immunoglobulin with each batch of the final
48 product. The term is used to describe forward and reverse tracing.
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1 TSE: Transmissible spongiform encephalopathy.
2 Validation: Action of proving, in accordance with the principles of GMP, that any procedure,
3 process, equipment, material, activity, or system actually leads to the expected results.
4 Venom: The toxic secretion of a specialized venom gland which, in the case of snakes, is
5 delivered through the fangs and provokes deleterious effects. Venoms usually comprise many
6 different protein components of variable structure and toxicity.
7 Venom extraction (venom collection, “milking”): The process of collecting venom from live
8 snakes.
9 Viral inactivation: A process of enhancing viral safety in which viruses are intentionally “killed”.
10 Viral reduction: A process of enhancing viral safety in which viruses are inactivated and/or
11 removed.
12 Viral removal: A process of enhancing viral safety by partitioning viruses from the components
13 of interest.
14 VSV: Vesicular stomatitis virus.
15 WNV: West Nile virus.
16
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1 3 General considerations
2 Snake antivenom immunoglobulins (antivenoms, antivenins, anti-snake bite serum, anti-snake
3 venom, ASV) are the only specific treatment for envenoming by snakebites. They are produced
4 by the fractionation of plasma usually obtained from large domestic animals hyper-immunized
5 against relevant venoms. Important but infrequently used antivenoms may be prepared in
6 smaller animals. When injected into an envenomed human patient, antivenom will neutralize
7 any of the venoms used in its production, and in some instances will also neutralize venoms
8 from closely related species.
9 3.1 Historical background

10 Shortly after the identification of diphtheria and tetanus toxins, von Behring and Kitasato
11 reported the antitoxic properties of the serum of animals immunized against diphtheria or
12 tetanus toxins and suggested the use of antisera for the treatment of these diseases [2]. In
13 1894, von Behring diphtheria antitoxin was first successfully administered by Roux to save
14 children suffering from severe diphtheria. Thus, serum therapy was born and the antitoxin was
15 manufactured by Burroughs Wellcome in the United Kingdom. The same year, Phisalix and
16 Bertrand [3] and Calmette [4] simultaneously, but independently, presented during the same
17 session of the same meeting their observations on the antitoxic properties of the serum of
18 rabbits and guinea-pigs immunized against cobra and viper venoms, respectively. Immediately
19 after his discovery of “antivenin serum-therapy”, Albert Calmette was actively involved in proving
20 its effectiveness in the treatment of human envenoming. The first horse-derived antivenom sera
21 that he prepared were already in clinical use in 1895 by Haffkine in India and by Lépinay in Viet
22 Nam. The latter reported the first successful use of antivenin serum therapy in patients in 1896
23 [5].
24 3.2 The use of serum versus plasma as source material

25 Historically, the pioneers Calmette, Vital Brazil and others, used serum separated from the
26 blood of hyperimmunized horses for the preparation of antivenom (“antivenin serum-therapy”).
27 Later, antibodies (immunoglobulins) were demonstrated to be the active molecules responsible
28 for the therapeutic action of “antivenom serum”. Subsequently, immunoglobulins, or
29 immunoglobulin fragments (F(ab')2, Fab), purified from serum were used instead of crude serum
30 [6, 7].
31 Nowadays, plasmapheresis, whereby erythrocytes are re-injected into the donor animal within
32 24 hours of blood collection, is commonly employed to reduce anaemia in the hyperimmunized
33 animal that donates the plasma. Accordingly, it is almost exclusively, plasma rather than serum
34 that is used as the starting material for the extraction of the immunoglobulin or its fragments [8-
35 10]. Thus “snake antivenom immunoglobulin” is the preferred term, rather than “anti-snakebite
36 serum” or “antiserum” which are no longer accurate.
37 3.3 Antivenom purification methods and product safety

38 Purification methods were introduced to reduce the frequency of antivenom reactions by
39 removing the Fc fragment from IgG, thus preventing complement activation and perhaps
40 reducing the intensity of immune-complex formation responsible for late antivenom reactions
41 (serum sickness). For 60–70 years, immunoglobulin F(ab')2 fragments have been widely used.
42 However, antivenom protein aggregation, and not Fc-mediated complement activation, was
43 increasingly identified as a major cause of antivenom reactions. Thus, a critical issue in
44 antivenom safety probably lies in the physicochemical characteristics of antivenoms and not
45 exclusively in the type of neutralizing molecules constituting the active substance. It is also
46 important to ensure that the current methodologies to produce antivenoms provide a sufficient
47 margin of safety with regard to the potential risk of transmission of zoonosis.
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1 3.4 Pharmacokinetics and pharmacodynamics of antivenoms
2 Rapid elimination of some therapeutic antivenoms (e.g. when Fab fragments are used) has led
3 to recurrence of envenoming in patients. However, the choice of preparing specific IgG or
4 fragments appears to depend on the size and toxicokinetics of the principal toxin(s) of the
5 venoms. Large relative molecular mass (Mr) bivalent antibodies (IgG and F(ab')2 fragments)
6 may be effective for the complete and prolonged neutralization of intravascular toxins (e.g.
7 procoagulant enzymes) which have a long half-life in envenomed patients, whereas low Mr and
8 monovalent IgG fragments such as Fab may be more appropriate against low-molecular-mass
9 neurotoxins which are rapidly distributed to their tissue targets and are rapidly eliminated from
10 the patient’s body [11].
11 3.5 Need for national and regional reference venom preparations

12 Antivenom production is technically demanding. The need to design appropriate monospecific
13 or polyspecific antivenoms (depending on the composition of the snake fauna) is supported by
14 the difference in venom composition among venomous animals, associated with the fact that:
15  many countries can be inhabited by several medically important species
16  there may be wide variation in venom composition (and hence antigenicity) through the
17 geographical range of a single species; and
18  in some circumstances there is no distinctive clinical syndrome to direct the use of
19 monospecific antivenoms.
20 However, similarities in the venom toxins of closely related venomous species may result in
21 cross-neutralization (para-specific neutralisation), thus reducing the number of venoms required
22 for the preparation of polyspecific antivenoms. Cross-neutralization should be tested in animal
23 models and ideally by clinical studies in envenomed patients. Preclinical testing of antivenoms
24 against medically important venoms present in each geographical region or country is a
25 prerequisite for product licences and batch approval, and should always precede clinical use in
26 envenomed patients. This requires efforts by manufacturers and/or regulators to establish
27 regional or national reference venom preparations that can be used to test the neutralization
28 capacity of antivenoms. The national control laboratory of the country where the antivenom will
29 be used, or the manufacturer seeking a licence for the antivenom, should perform such
30 preclinical testing using reference venom preparations relevant to the country or the
31 geographical area.
32
33
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1 4 Epidemiological Background
2 The incidence of snakebites in different parts of the world and the recognition of the particular
3 species of greatest medical importance is fundamental to the appropriate design of
4 monospecific and polyspecific antivenoms in countries and regions. Up-to-date epidemiological
5 and herpetological information is therefore highly relevant to antivenom manufacturers and
6 regulators, especially for the selection of the most appropriate venoms, or venom mixtures, to
7 be used in the production and quality control of antivenoms.
8 4.1 Global burden of snakebites

9 Envenoming and deaths resulting from snakebites are a particularly important public health
10 problem in rural tropical areas of Africa, Asia, Latin America and Papua New Guinea [12].
11 Agricultural workers and children are the most affected groups. Epidemiological assessment of
12 the true incidence of global mortality and morbidity from snakebite envenoming has been
13 hindered by several well recognized problems [13, 14]. Snakebite envenoming and associated
14 mortality are under-reported because many victims (20–70% in some studies) do not seek
15 treatment in government dispensaries or hospitals and hence are not recorded. This is
16 compounded by the fact that medical posts in regions of high incidence are unable to keep
17 accurate records of the patients who do present for treatment, and because death certification
18 of snakebite is often imprecise [15, 16]. Correctly designed population surveys, in which
19 questionnaires are distributed to randomly selected households in demographically well-defined
20 areas, are the only reliable method for estimating the true burden of snakebites in rural areas.
21 The results of the few such surveys that have been performed have shown surprisingly high
22 rates of bites, deaths and permanent sequelae of envenoming [16-20]. However, because of the
23 heterogeneity of snakebite incidence within countries, the results of surveys of local areas
24 cannot be extrapolated to give total national values. Most of the available data suffer from these
25 deficiencies and, in general, should be regarded as underestimates and approximations.
26 However, the true burden of national snakebite morbidity and mortality has recently be revealed
27 by results of three well designed community-based studies. In India, a direct estimate of 46,000
28 snakebite deaths in 2005 was derived from the Million Death Study [21], in Bagladesh there
29 were an estimated 589,919 snakebites resulting in 6,041 deaths in 2009 [22], and in Sri Lanka
30 in 2012-13, 80,000 bites, 30,000 envenomings and 400 deaths in one year [23]. Published
31 estimates of global burden suggest a range from a minimum of 421,000 envenoming and
32 20,000 deaths up to as high as 2.5 million cases and over 100,000 deaths each year [14, 24]. In
33 view of the recent data from South Asia, these figures would seem to be underestimates. In
34 addition, the number of people left with permanent sequelae as a result of these envenoming is
35 likely to be higher than the number of fatalities [12]. As already identified, most of the estimated
36 burden of snakebite is from sub-Saharan Africa, south and south-east Asia and central and
37 south America.
38 The current literature on snakebite epidemiology highlights the inadequacy of the available data
39 on this neglected tropical disease. There is clearly a need to improve reporting and record-
40 keeping of venomous bites in health facilities, to support high-quality epidemiological studies of
41 snakebite in different regions, and to improve the training of medical personnel. Wherever
42 possible, recording the species that caused the bite as well as death or injury would greatly
43 assist in documenting which species are of clinical significance in individual countries. Making
44 venomous bites notifiable and fully implementing the use of the International Statistical
45 Classification of Diseases and Related Health Problems 10th Revision [25] in official death
46 certification (e.g. T 63.0 snake venom) would further help to determine the burden of snakebite
47 more accurately.
48 4.2 Main recommendations

49 In most parts of the world, snakebites are under-reported and in some parts are
50 completely unreported. This deficiency in surveillance and the paucity of properly
51 designed epidemiological studies explain why the impact of this important public health
52 problem has remained for so long unrecognized and neglected.
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1 National health authorities should be encouraged to improve the scope and precision of
2 their epidemiological surveillance of this disease by:
3  improving the training of all medical personnel so that they are more aware of the
4 local causes, manifestations and treatment of venomous bites;
5  making venomous bites notifiable diseases;
6  setting up standardized and consistent epidemiological surveys;
7  improving the reporting and record keeping of venomous bites by hospitals, clinics,
8 dispensaries and primary health care posts, and relating the bites to the species of
9 venomous snake that caused the bite wherever possible; and
10  fully implementing the use of the International Statistical Classification of Diseases
11 and Related Health Problems 10th Revision (2007) (22) in official death certification
12 (e.g. T 63.0 snake venom ).1
13
14
1
http://www.who.int/classifications/apps/icd/icd10online/
WHO/BS/2016.2300
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1 5 Worldwide distribution of venomous snakes
2 5.1 Taxonomy of venomous snakes
3 Recognizing the species causing the greatest public health burden, designing and
4 manufacturing antivenoms and optimizing patient treatment are all critically dependent on a
5 correct understanding of the taxonomy of venomous snakes. Like other sciences, the field of
6 taxonomy is constantly developing. New species are still being discovered, and many species
7 formerly recognized as being widespread have been found to comprise multiple separate
8 species as scientists obtain better information, often with new technologies. As the
9 understanding of the relationships among species is still developing, the classification of
10 species into genera is also subject to change. The names of venomous species used in these
11 guidelines conform to the taxonomic nomenclature that was current at the time of publication.
12 Some groups of venomous snakes remain under-studied and poorly known. In these cases, the
13 classification best supported by what evidence exists is presented with the limitation that new
14 studies may result in changes to the nomenclature.
15 Clinicians, toxinologists, venom producers and antivenom manufacturers should endeavour to
16 remain abreast of these nomenclatural changes. These changes often reflect improved
17 knowledge of the heterogeneity of snake populations, and may have implications for venom
18 producers, researchers and antivenom manufacturers. Although taxonomic changes do not
19 necessarily indicate the presence of “new” venoms, they strongly suggest that toxinological and
20 epidemiological research into these “new” taxa may be required to establish their medical
21 relevance, if any.
22 Since some of the names of medically important species have changed in recent years, the
23 following points are intended to enable readers to relate the current nomenclature to information
24 in the former literature.
25  The large group of Asian arboreal pit vipers, which in recent years had been split from a
26 single genus (Trimeresurus), into a number of new genera (e.g. Cryptelytrops, Parias,
27 Peltopelor, Himalayophis, Popeia, Viridovipera, Ovophis and Protobothrops, with a few
28 species retained in Trimeresurus) based on prevailing views of the inter-relationships
29 between these groups, have now largely been returned to Trimeresurus. There are divergent
30 views on this approach to the taxonomy of these snakes, and interested parties should
31 consult the literature. Some changes which occurred in the early 1980s have gained
32 acceptance and been retained (i.e. Protobothrops). Medically important species formerly
33 classified in Cryptelytrops include Trimeresurus albolabris, T. erythrurus and T. insularis.
34 Viridovipera stejnegeri has been returned to Trimeresurus.
35  It is likely that new species of cobra (Naja spp.) will be identified within existing taxa in both
36 Africa and Asia; three new species (N. ashei, N. mandalayensis and N. nubiae) have been
37 described and several subspecies elevated to specific status since 2000 (e.g. Naja
38 annulifera and N. anchietae, from being subspecies of N. haje), in addition to the
39 synonymization of the genera Boulengerina and Paranaja within the Naja genus. Such
40 changes may hold significance for antivenom manufacturers and should stimulate further
41 research to test whether existing antivenoms cover all target snake populations.
42  Several medically important vipers have been reclassified: Daboia siamensis has been
43 recognized as a separate species from Daboia russelii; Macrovipera mauritanica and M.
44 deserti have been transferred to Daboia; the Central American rattlesnakes, formerly
45 classified with Crotalus durissus, are now Crotalus simus; and Bothrops neuwiedi has been
46 found to consist of a number of different species, three of which (B. neuwiedi, B. diporus and
47 B. mattogrossensis) may be of public health importance.
48 It is recognized that there have been many accepted revisions of taxonomy over the past few
49 decades. These Guidelines are aimed at a very wide range of readers, and to assist in matching
50 some old and familiar names with the current nomenclature, Tables 1 and 2 summarize major
51 changes between 1999-2016. References are listed at the end of Annex 1.
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1 Table 1
2 Genus-level name changes (1999–2016)
Currently accepted name Previous name/s
Bothrocophias hyoprora Bothrops hyoprora
Bothrocophias microphthalmus Bothrops microphthalmus
Trimeresurus albolabris Cryptelytrops albolabris
Trimeresurus erythrurus Cryptelytrops erythrurus
Cryptelytrops insularis,
Trimeresurus insularis
Trimeresurus albolabris insularis
Trimeresurus macrops Cryptelytrops macrops
Trimeresurus purpureomaculatus Cryptelytrops purpureomaculatus
Cryptelytrops septentrionalis,
Trimeresurus septentrionalis
Trimeresurus albolabris septentrionalis
Macrovipera deserti, Vipera mauritanica deserti,
Daboia deserti
Vipera lebetina deserti
Macrovipera mauritanica,
Daboia mauritanica
Vipera lebetina mauritanica
Daboia palaestinae Vipera palaestinae
Daboia russelii Vipera russelii
Himalayophis tibetanus Trimeresurus tibetanus
Montivipera raddei Vipera raddei
Montivipera xanthina Vipera xanthina
Naja annulata Boulengerina annulata
Naja christyi Boulengerina christyi
Trimeresurus flavomaculatus Parias flavomaculatus
Trimeresurus sumatranus Parias sumatranus
Zhaoermia mangshanensis, Ermia mangshanensis,
Protobothrops mangshanensis
Trimeresurus mangshanensis
Trimeresurus stejnegeri Viridovipera stejnegeri
3
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1 Table 2
2 Changes resulting from new species descriptions, or redefinitions (1999–2016)
Currently accepted name Previous name(s)
Acanthophis crytamydros Previously part of Acanthophis rugosus
Acanthophis antarcticus laevis, confused with A.
Acanthophis laevis
antarcticus or A. praelongus
Acanthophis antarcticus rugosus, confused with A.
Acanthophis rugosus (New Guinea)
antarcticus or A. praelongus
Agkistrodon howardgloydi Agkistrodon bilineatus howardgloydi
Agkistrodon russeolus Agkistrodon bilineatus russeolus
Agkistrodon taylori Agkistrodon bilineatus taylori
Bitis gabonica Bitis gabonica gabonica
Bitis harenna New species
Bitis rhinoceros Bitis gabonica rhinoceros
Bothrops diporus Bothrops neuwiedi diporus
Bothrops mattogrossensis Bothrops neuwiedi mattogrossensis, B.n. bolivianus
Bothrops pubescens Bothrops neuwiedi pubescens
Bungarus persicus New species
Cerrophidion sasai Previously part of Cerrophdion godmani
Cerrophidion wilsoni Previously part of Cerrophidion godmani
Crotalus oreganus Previously considered part of Crotalus viridis
Crotalus ornatus Previously considered part of Crotalus molossus
Crotalus durissus durissus (Central American
Crotalus simus
populations of C. durissus complex)
Crotalus totonacus Crotalus durissus totonacus
Crotalus tzabcan Crotalus simus tzabcan, Crotalus durissus tzabcan
Daboia russelii Daboia russelii russelii, Daboia r. pulchella
Daboia russelii siamensis, D.r. limitis, D.r. sublimitis,
Daboia siamensis
D.r. formosensis
Echis borkini Previously part of Echis pyramidum
Echis omanensis Previously known as NE population of Echis coloratus
Gloydius intermedius Previously named Gloydius saxatilis
Hypnale zara New species
Lachesis acrochorda Previously part of Lachesis stenophrys
Naja arabica Previously part of Naja haje
Naja anchietae Naja annulifera anchietae, Naja haje anchietae
Naja ashei Previously part of Naja nigricollis
Naja nigricincta Naja nigricollis nigricincta, Naja nigricollis woodi
Naja nubiae Previously part of Naja pallida
Naja senegalensis Previously part of Naja haje
Pailsus rossignolii, previously part of Pseudechis
Pseudechis rossignolii
australis
Pseudonaja aspidorhyncha Previously part of Pseudonaja nuchalis
Pseudonaja mengdeni Previously part of Pseudonaja nuchalis
Thelotornis mossambicanus Thelotornis capensis mossambicanus
Thelotornis usambaricus Thelotornis capensis mossambicanus
Trimeresurus cardamomensis Previously part of Trimeresurus macrops
Trimeresurus rubeus Previously part of Trimeresurus macrops
Tropidolaemus philippensis Previously part of Tropidolaemus wagleri
Tropidolaemus subannulatus Previously part of Tropidolaemus wagleri
Vipera renardi Previously part of V. ursinii
Walterinnesia morgani Previously part of Walterinnesia aegyptia
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1 5.2 Medically important venomous snakes
2 Based on current herpetological and medical literature, it is possible to partially prioritize the
3 species of snakes that are of greatest medical importance in different regions. Detailed statistics
4 on the species of snakes responsible for morbidity and mortality throughout the world are
5 lacking, except for a few epidemiological studies which include rigorous identification of the
6 biting snake in a few scattered localities. Thus, establishing a list of medically important species
7 for different countries, territories and other areas relies, at least in part, on extrapolation from
8 the few known studies, as well as on the biology of the snake species concerned: e.g. where
9 species of a group of snakes are known to be of public health importance, based on
10 epidemiological studies, it seems reasonable to deduce that closely related species with similar
11 natural history occurring in hitherto unstudied regions are also likely to be medically important.
12 Examples include Asian cobras in several under-studied regions of Asia, lowland Bungarus
13 species in Asia, and spitting cobras in Africa.
14 Tables 3–6 list the species of venomous snakes of greatest medical importance in each of four
15 broad geographical regions. Species listed in these tables are either:
16  those which are common or widespread in areas with large human populations and which
17 cause numerous snakebites, resulting in high levels of morbidity, disability or mortality
18 among victims; or
19  poorly known species that are strongly suspected of falling into this category; or
20  species which cause major and life-threatening envenoming responsive to antivenom, but
21 are not common causes of bites.
22 The venoms of these species should be considered a starting point for establishing the most
23 important targets for antivenom production. The need for additional epidemiological and
24 toxinological research to better define which venoms to include and exclude for antivenom
25 production in various regions, territories and countries around the world is emphasized.
26 Detailed data regarding countries, territories and other areas on species believed to contribute
27 most to the global burden of injury, and/or which pose the most significant risk of morbidity or
28 mortality are provided in Annex 1.
29
30
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1 Table 3
2 Medically important venomous snakes: Africa and the Middle East
North Africa/Middle East
Atractaspididae: Atractaspis andersonii; Elapidae: Naja arabica, Naja haje, Naja oxiana;
Viperidae: Bitis arietans; Cerastes cerastes, Cerastes gasperettii; Daboia mauritanica1,
Daboia palaestinae1; Echis borkini, Echis carinatus, Echis coloratus, Echis omanensis2,
Echis pyramidum; Macrovipera lebetina, Montivipera xanthina1; Pseudocerastes persicus
Central sub-Saharan Africa
Elapidae: Dendroaspis jamesoni, Dendroaspis polylepis; Naja anchietae1, Naja haje, Naja
melanoleuca, Naja nigricollis; Viperidae: Bitis arietans, Bitis gabonica1, Bitis nasicornis;
Echis leucogaster, Echis ocellatus, Echis pyramidum
Eastern sub-Saharan Africa
Elapidae: Dendroaspis angusticeps, Dendroaspis jamesoni, Dendroaspis polylepis; Naja
anchietae1, Naja annulifera, Naja ashei1, Naja haje, Naja melanoleuca, Naja
mossambica, Naja nigricollis; Viperidae: Bitis arietans, Bitis gabonica1, Bitis nasicornis;
Echis pyramidum
Southern sub-Saharan Africa
Elapidae: Dendroaspis angusticeps, Dendroaspis polylepis; Naja anchietae1, Naja
annulifera, Naja mossambica, Naja nigricincta1, Naja nivea; Viperidae: Bitis arietans
Western sub-Saharan Africa
Elapidae: Dendroaspis jamesoni, Dendroaspis polylepis, Dendroaspis viridis; Naja haje,
Naja katiensis, Naja melanoleuca, Naja nigricollis, Naja senegalensis; Viperidae: Bitis
arietans, Bitis gabonica1, Bitis nasicornis, Bitis rhinoceros1; Cerastes cerastes; Echis
jogeri, Echis leucogaster, Echis ocellatus
3 Table 4
4 Medically important venomous snakes: Asia and Australasia
Central Asia
Elapidae: Naja oxiana; Viperidae: Echis carinatus; Gloydius halys; Macrovipera lebetina
East Asia
Elapidae: Bungarus multicinctus; Naja atra; Viperidae: Trimeresurus albolabris3; Daboia
russelii1; Deinagkistrodon acutus; Gloydius blomhoffii, Gloydius brevicaudus;
Protobothrops flavoviridis, Protobothrops mucrosquamatus; Trimeresurus stejnegeri1
South Asia
Elapidae: Bungarus caeruleus, Bungarus ceylonicus, Bungarus niger, Bungarus
sindanus, Bungarus walli; Naja kaouthia, Naja naja, Naja oxiana; Viperidae: Trimeresurus
erythrurus1; Daboia russelii1; Echis carinatus; Hypnale hypnale; Macrovipera lebetina
South-East Asia (excluding Indonesian West Papua)
Elapidae: Bungarus candidus, Bungarus magnimaculatus, Bungarus multicinctus,
Bungarus slowinskii; Naja atra, Naja kaouthia, Naja mandalayensis, Naja philippinensis,
Naja samarensis, Naja siamensis, Naja sputatrix, Naja sumatrana; Viperidae:
Calloselasma rhodostoma; Trimeresurus albolabris1, Trimeresurus erythrurus1,
Trimeresurus insularis1; Daboia siamensis1; Deinagkistrodon acutus
Australo-Papua (includes Indonesian West Papua)
Elapidae: Acanthophis laevis1; Notechis scutatus; Oxyuranus scutellatus; Pseudechis
australis4, Pseudonaja affinis, Pseudonaja mengdeni, Pseudonaja nuchalis, Pseudonaja
textilis
5
2
Recent nomenclatural change. Refer to Tables 1 and 2 for details of previous names.
3
4
Pseudechis australis is common and widespread and causes numerous snakebites; bites may be
severe, although this species has not caused a death in Australia since 1968.
WHO/BS/2016.2300
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1
2 Table 5
3 Medically important venomous snakes: Europe
Central Europe
Viperidae: Vipera ammodytes
Eastern Europe
Viperidae: Vipera berus
Western Europe
Viperidae: Vipera aspis, Vipera berus
4 Table 6
5 Medically important venomous snakes: the Americas
North America
Viperidae: Agkistrodon bilineatus, Agkistrodon contortrix, Agkistrodon piscivorus,
Agkistrodon taylori5; Bothrops asper, Crotalus adamanteus, Crotalus atrox, Crotalus
horridus, Crotalus oreganus2, Crotalus simus2, Crotalus scutulatus, Crotalus totonacus2,
Crotalus viridis
Caribbean
Viperidae: Bothrops cf. atrox (Trinidad), Bothrops caribbaeus (St Lucia), Bothrops
lanceolatus (Martinique); Crotalus durissus (Aruba)
Central America
Viperidae: Bothrops asper; Crotalus simus2
South America
Viperidae: Bothrops alternatus, Bothrops asper, Bothrops atrox, Bothrops brazili,
Bothrops bilineatus, Bothrops diporus2, Bothrops jararaca, Bothrops jararacussu,
Bothrops leucurus, Bothrops matogrossensis2, Bothrops moojeni, Bothrops pictus,
Bothrops venezuelensis; Crotalus durissus; Lachesis muta
6 5.3 Minor venomous snake species

7 In many countries, territories and other areas there are species of snakes that rarely bite
8 humans but are capable of causing severe or fatal envenoming. Their medical importance may
9 not justify inclusion of their venoms in the immunizing mixture for production of polyspecific
10 antivenoms but the need to make antivenoms against these species needs to be carefully
11 analysed.
12 In some cases, such as with some Central American pit vipers (genera Agkistrodon, Porthidium,
13 Bothriechis, Atropoides among others), there is clinically effective cross-neutralization of
14 venoms by standard national polyspecific antivenoms [26].
15 In other cases, there is no effective cross-neutralization and manufacturers may therefore
16 consider that the production of a monospecific antivenom is justified for use in potentially fatal
17 cases of envenoming, provided that such cases can be identified. Such antivenoms are
18 currently available for envenoming by the boomslang (Dispholidus typus), desert black snake
19 (Walterinnesia aegyptia), Arabian burrowing asp (Atractaspis andersonii) [27], king cobra
20 (Ophiophagus hannah), Malayan krait (Bungarus candidus) [27] “yamakagashi” (Rhabdophis
21 tigrinus) and red-necked keelback (R. subminiatus), Martinique’s “Fer-de-lance” (Bothrops
22 lanceolatus), St Lucia’s B. caribbaeus, and some species of American coral snake (Micrurus).
23 No antivenoms are currently available for envenoming by species such as African bush vipers
24 (e.g. Atheris, Proatheris), berg adder (Bitis atropos) and several other small southern African
25 Bitis spp. (e.g. B. peringueyi), Sri Lankan and south-west Indian humpnosed vipers (Hypnale
5
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1 spp.) [28, 29], many Asian pit vipers (“Trimeresurus” sensu lato), some species of kraits (e.g. B.
2 niger) and all but one species of burrowing asp (genus Atractaspis).
3 An alternative to antivenom production against species that cause few, but potentially severe
4 accidents, is to manufacture polyspecific antivenoms for broadly distributed groups that have
5 similar venom compositions (e.g. African Dendroaspis and Atractaspis; Asian “green pit vipers”;
6 American Micrurus). This may result in antivenoms that offer broad protection against venoms
7 from minor species within genera, or species whose bites are less frequent than those of others
8 in the same taxonomic groups (i.e. genus, sub-family or family).
9 5.4 Sea snake venoms

10 Although venomous marine sea snakes have not been included in the tables of medically
11 important venomous snakes, it should be recognized that there are a number of species of
12 marine snakes with potent venoms that can cause illness or death. Available evidence,
13 particularly clinical experience, indicates that the major sea snake antivenom that is currently
14 commercially available, which uses venom of a single sea snake, Hydrophis schistosus
15 (previously known as Enhydrina schistosa), in the immunizing venoms mixture, is effective
16 against envenoming by other sea snake species for which there are clinical data. Further
17 research would be needed to better define the full extent of cross-neutralization offered by this
18 antivenom against other sea snake species.

20  Clinicians, toxinologists, poison centres, regulators, venom producers and antivenom
21 manufacturers should be well-informed about current nomenclature and new changes
22 to taxonomy, so as to ensure the currency of information, correct identification of
23 species in their countries, and correct selection and sourcing of venoms used in the
24 manufacture of antivenoms.
25  Identification of the medically important venomous snakes (those that cause the
26 greatest burden of injury, disability and/or mortality) is a critical prerequisite to
27 meeting the need for efficacious antivenom. Improving the quality of the available
28 data and correcting and amplifying the level of geographical detail and precision of
29 attribution should be important priorities.
30  Support for establishment of local capacity for venom production as a means of
31 ensuring that venom immunogens from geographically representative populations of
32 medically important snake species are used in antivenom production would improve
33 antivenom specificity.
34
35
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1 6 Antivenoms design: selection of snake venoms
2 Venomous snakes exhibit significant species and genus specific variation in venom protein
3 composition [30]. The efficacy of antivenom is therefore largely restricted to the venom/s used in
4 its manufacture.
5 It is therefore imperative that antivenom manufacturers carefully consider the venoms used in
6 antivenom manufacture by first defining the geographical area where the antivenom will be
7 deployed, and sequentially:
8  Identifying the most medically-important snakes in that region;
9  Examining the venom protein composition of the snakes, including information from relevant
10 literature;
11  Conducting antivenom preclinical efficacy tests on venoms of all the most medically-
12 important snakes in that region.
13 6.1 Selection and preparation of representative venom mixtures

14 Annex 1 presents an up-to-date list of the most medically-important venomous snake species
15 by country, region and continent. The venoms from Category 1 snakes must be included for
16 antivenom production and venoms from Category 2 snakes only excluded after careful
17 assessment of risk/benefit considerations.
18 It is important to appreciate that there are variations in venom composition and antigenicity (i)
19 within the geographical range of a single species and (ii) within snakes of different ages [31,
20 32]. Therefore, venom should be collected from specimens of different geographic origins and
21 ages, and mixed before being used for immunisation (see section 7 on venom preparation). The
22 greater the intra-specific variation, the more snake specimens of distinct origin and age are
23 required to create an adequate venom immunisation mixture.
24 Cross-neutralization of venoms with similar protein-composition profiles as the venoms used for
25 immunization may extend the efficacy range of some antivenoms, but requires, minimally,
26 preclinical efficacy testing to identify the potential cross-neutralization capacity of an antivenom.
27 In vitro immunological cross-reactivity testing is NOT an adequate measure of antivenom
28 efficacy.
29 6.2 Manufacture of monospecific or polyspecific antivenoms

30 Antivenom manufacturers face an early, critical decision as to whether the antivenom should
31 possess monospecific or polyspecific efficacy.
32 6.2.1 Monospecific antivenoms
33 Monospecific antivenoms are manufactured with venoms from a single venomous snake
34 species, and their efficacy is largely restricted to that snake species. These conditions apply in
35 areas where:
36  there is only one medically-important species (e.g. Vipera berus in the United Kingdom and
37 Scandinavia) or where one species is responsible for the majority of cases (e.g. Oxyuranus
38 scutellatus in southern Papua New Guinea);
39  a simple blood test, suitable for use even in under-resourced health care centres, can define
40 the biting species (e.g. detection of incoagulable blood by the 20-minute whole blood clotting
41 test in the northern third of Africa where only Echis spp. cause coagulopathy);
42  a simple algorithmic approach allows the species to be inferred from the pattern of clinical
43 and biological features;
44  there is a reliable and affordable rapid immunodiagnostic test readily available allowing the
45 toxins to be identified unambiguously (currently only available in Australia).
46 Monospecific antivenoms can be effective in treating envenoming by a few closely related
47 species whose venoms show clinically effective cross-neutralization – but this requires
48 preclinical and clinical confirmation.
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1 6.2.2 Polyspecific antivenoms
2 Most tropical countries are inhabited by several medically-important snake species, and it is
3 commercially unrealistic to develop multiple monospecific antivenoms. In these cases, the
4 manufacture of polyspecific antivenoms is highly recommended. Polyspecific antivenoms are
5 designed to contain IgG effective against venoms from multiple species or genera of venomous
6 snakes in a defined region. Manufacturing protocols of polyspecific antivenom include:
7  Mixing venoms from multiple snake species/genera (sometimes in amounts quantitatively
8 associated with medical importance, immunogenicity etc) and immunizing donor animals
9 with this mixture. Immunizing an animal with venoms from several taxonomically-related
10 snakes (e.g. different vipers) can have the advantage over monospecific antivenom of
11 increasing the titre of neutralizing IgG to any one snake venom [33].
12  Immunizing groups of donor animals with distinct venom mixtures and then mixing the
13 hyperimmune plasma from each group of animals.
14  Immunizing groups of donor animals with distinct venom mixtures and then mixing the
15 monospecific antivenom IgGs to formulate the final polyspecific antivenom.
16 When using the latter two options it is important to monitor the efficacy for each monospecific
17 antivenom to ensure that the efficacy of the mixed final product is consistent, reproducible and
18 in line with the product specification for each individual venom. This ‘combined monospecific
19 antivenoms’ approach anticipates that the amount of neutralizing IgG targeting each individual
20 venom will be proportionally diluted – necessitating administration of more vials to reverse
21 venom pathology, which in turn increases the risks of adverse reactions.
22 In some regions, it is possible to differentiate envenoming by detecting distinct clinical
23 syndromes: neurotoxicity, haematological disturbances (haemorrhage or coagulopathy) and/or
24 local tissue damage. Such situations justify the preparation of syndrome-specific polyspecific
25 antivenoms by immunizing donor animals with mixtures of either neurotoxic venoms or venoms
26 inflicting haemorrhage and/or coagulopathy and local tissue damage.
27 In most tropical regions where snakebite is a significant medical burden, polyspecific
28 antivenoms offer significant clinical advantages and their production should be encouraged.
29 They can also offer greater commercial-manufacturing incentives (economies of scale) than
30 monospecific antivenoms because of their significantly greater geographic and snakes-species
31 cover – increasing the likelihood of their delivery to victims residing in regions where antivenom
32 manufacture is not government subsidised.

34  National Health authorities should, prior to importing antivenoms, carefully consider
35 their regional threat from venomous snakes to inform their antivenom requirements.
36  The design of the venom mixture used in immunization, and the decision to prepare
37 monospecific or polyspecific antivenoms, must be informed by the epidemiological
38 and clinical information on snakebites in the defined country, region or continent.
39  In most tropical countries polyspecific antivenoms are likely to have significant
40 clinical and logistic advantages over monospecific antivenoms, particularly in the
41 absence of rapid, affordable snake venom diagnosis.
42  Polyspecific antivenom may be prepared from IgG of donor animals immunised with a
43 mixture of venoms, or by mixing monospecific antivenoms.
44  Manufacturers seeking marketing authorization for antivenoms in a given country
45 should provide experimental evidence from preclinical testing that the product
46 exhibits a neutralization capacity against different local venoms (see section 17).
47  National Health authorities should organise for independent preclinical efficacy
48 testing prior to importation of any antivenom - to avoid national distribution of
49 dangerously ineffective therapies.
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1
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1 7 Preparation and storage of snake venom
2 Venom preparations are used both to hyper-immunize animals, as part of antivenom production,
3 and to provide reference venom samples for routine and/or preclinical potency assessment of
4 antivenoms. Under GMP for pharmaceutical products, snake venoms are starting materials, and
5 therefore ensuring their quality is critical, and their preparation should follow the principles and
6 recommendations stated below. The essential principles of quality systems should be followed
7 in venom production including traceability, reproducibility, taxonomic accuracy, and hygiene
8 control. Manufacturers of snake venoms used in antivenom production should strive to comply
9 with WHO’s Guidelines on GMP for Biological Products and Guidelines for Good Manufacturing
10 Practices for Pharmaceutical Products.
11 Venoms used for antivenom manufacture should be representative of the snake population
12 living in the area where the antivenom is to be used. To take account of the variability in venom
13 composition within a species [34-38], it is imperative that the venom of an adequate number of
14 individual snakes (generally not less than 20 specimens, including males and females) collected
15 from various regions covering the entire geographical distribution of the particular venomous
16 snake species should be collected together. Consideration should also be given to including
17 venom from juvenile or sub-adult snakes in these venom pools as there is strong evidence of
18 age-related venom variation within individual specimens and populations [39]. A similar
19 approach should be used in the preparation of Standard Reference Venoms (national or
20 regional) for use in the validation of antivenom products by reference laboratories and
21 regulatory agencies (see Section 8) or in preclinical testing of antivenoms by manufacturers
22 (see section 17).
23 Venom producers should ensure that they fully document, and can provide evidence of:
24  geographical origin and the size or age (juvenile or adult) of each individual snake used for
25 venom production;
26  taxonomic details of each snake species used;
27  correct implementation of compliance with local wildlife legislation, and the Convention on
28 International Trade in Endangered Species (CITES) documents in the case of endangered
29 species;
30  application of appropriate withholding rules (e.g.: not collecting venom from animals under
31 quarantine, or which are gravid, injured, sick or in poor condition);
32  individual identification of snake specimens contributing to each venom batch;
33  traceability of each venom batch;
34  appropriate handling and stabilization of venoms (e.g.: rapid freezing of the venom after
35 collection and lyophilisation for long-term stable storage6);
36  quality control confirmation of batch-to-batch consistency of venoms of each species/country
37 of origin (e.g.: SDS-PAGE or HPLC profiling of venoms, measurement of residual moisture
38 in lyophilized venom); and
39  confirmation of batch-to-batch similarity of venom of the same origin.
40 7.1 Production of snake venoms for immunization

41 The maintenance of a serpentarium and the handling of snakes used for antivenom production
42 should comply with quality systems principles.
43 7.1.1 Quarantine of snakes
44 All new accessions should be quarantined for at least 2 months in a special “quarantine room”
45 which should be located as far as possible from the “production rooms” where snakes qualified
46 for venom production are kept.
6
Desiccation or vacuum-drying may be acceptable if proven to ensure stability of the preparation.
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1 On arrival, snakes should be examined by a specialized veterinary surgeon (or experienced
2 person) for ectoparasites, wounds and fractures. Endoparasites (nematodes, cestodes,
3 trematodes and pentastomids) should be eliminated using broad-spectrum antiparasitic drugs
4 and any injury must be adequately treated by a veterinarian [40-42]. Some viruses can be
5 transmitted between different species, and between different Families of snakes. Therefore,
6 different Families should be kept in different rooms.
7 Sick snakes should be treated and their quarantine extended for 1-2 months after complete
8 clinical recovery. Sick animals found in “production rooms” may be treated in situ (although
9 quarantine is preferable) but they cannot be used for venom production. If an antibiotic
10 treatment is given, the snake should not be used to obtain venom for 4 weeks following the end
11 of the treatment. When housed in good conditions, adult snakes collected from the wild can live
12 in captivity for 10 years or more. When handling snakes, the risk of infection with human
13 mosquito-borne viruses such as Japanese encephalitis should be prevented, since arbovirus
14 infections have been reported in some snakes [43].
15 7.1.2 Maintenance of captive snakes for venom production
16 Individual snakes should preferably be housed in separate cages large enough to allow them to
17 move about, according to local and international standards. There are several acceptable
18 options for the design of the cages. Transparent or black (for burrowing snakes) plastic boxes
19 are recommended. Cage materials should be impermeable, free from fissures, and inert to
20 disinfectants, cleaning chemicals and common solvents. The selection of cleaning and
21 disinfecting agents should be carefully considered to ensure they do not have adverse effects
22 on the snakes. Cages should be adequately ventilated but perforations or mesh small enough to
23 prevent escape. Ventilation holes should be clearly marked as hazard areas since there is a risk
24 of accidental envenoming (e.g.: spitting cobras have been known to spray venom through such
25 openings, and large vipers have fangs which can extend through a small hole if the snake
26 strikes). In the case of gravid viviparous snakes, the ventilation holes or mesh should be
27 sufficiently fine to prevent escape of their tiny, live-born offspring. The cage interior should be
28 visible from the outside to allow safe maintenance and handling. Access to cages through
29 doors, lids or sliding panels should facilitate management without compromising safety or
30 allowing snakes to escape. Be wary of cages with internal ledges or lips above doors, as some
31 snakes can conceal themselves above them out of sight of the keepers. A disposable floor
32 covering (e.g. newspaper) is recommended. Cryptic and nocturnal species should be provided
33 with a small shelter where they can hide.
34 The use of “hide boxes” is increasingly common as these provide both a more reassuring
35 environment for the snake, and increased safety for keepers. Hide boxes should be designed to
36 be slightly larger than the curled snake, with an entrance/exit hole, large enough to allow a
37 recently fed snake easy access, plus some simple closure device to lock the snake in the hide
38 box. This will allow removal of the snake from the cage without hazard to the keeper, making
39 routine cage maintenance simpler and safer. Hide boxes can be plastic, wooden, or even made
40 from cardboard (which is inexpensive and can be discarded and replaced regularly). Permanent
41 hide boxes should be readily cleanable or autoclavable. The roof, or side of the hide box should
42 be removable, to allow easy, safe extraction of the snake, when required.
43 Cages should be thoroughly cleaned and disinfected when soiled (daily if necessary). Faeces
44 and uneaten or regurgitated food items should be removed as soon as possible. To avoid
45 misidentification of the snake, a microchip should be implanted in the hypodermal layer of the
46 snake’s posterior region and a label bearing its individual data should be attached to the cage
47 and transferred with the snake when it is moved to another cage. Water should be provided ad
48 libitum and for species from humid climates, more frequent watering or misting may be required,
49 particularly when sloughing. Water should be changed regularly and as soon as it becomes
50 contaminated. Water treatment by ultraviolet (UV) sterilization or acidification may be
51 considered.
52 Tens of cages may be accommodated in the same “production room”, provided that there is
53 enough space for maintenance and venom extraction. This room should be kept as clean as
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1 possible at all times, and thoroughly cleaned at least weekly. Measures should be taken to
2 minimise or eliminate contamination or spread of diseases. The use of antiseptic hand washes,
3 disposable over-clothing, antiseptic foot wash trays at entry and exit points, and other measures
4 should be routine. The temperature and humidity of the snake room should be controlled
5 according to the climatic requirements of the particular snake species. Ventilation should be
6 ensured using fans, air conditioning, or air renewing systems.
7 Access to snake rooms should be restricted to personnel responsible for their maintenance.
8 They should be kept locked, with any windows permanently closed or protected by bars and
9 mosquito proofing. Access should be via a safety porch not allowing simultaneous door opening
10 and with a transparent panel allowing a view of the entire snake room for pre-entry safety
11 inspections. The spaces below the doors should be less than 3 mm and all openings to the
12 exterior (e.g. water pipes, drainage conduits, ventilation entrances and exits) should be
13 protected by grilles having holes smaller than 3 mm. Natural light is often used; however, when
14 not available, artificial light should be turned on for 12 hours during the day and turned off
15 during the night for tropical species, but species from temperate zones may have different
16 requirements. Snakes of the same species, collected at the same time in the same area should
17 be placed in the same racks. The same “production room” can contain snakes of different
18 species, provided that they have similar living requirements (i.e. temperature and humidity).
19 When kept under favourable housing and climatic conditions, and if left undisturbed, snakes will
20 reproduce in captivity [44]. Animals should be mated only with specimens from the same
21 species, subspecies and local origin [45, 46]. Sexing can be difficult, but is helped by the use of
22 intra-cloacal probes. The male and the female should be individually identified and separated
23 soon after copulation. The female should be kept under careful surveillance. Eggs from
24 oviparous snakes and neonates from viviparous snakes should be removed from the females
25 cage as soon as possible. Differences in the venom composition of adult and juvenile snakes
26 have been reported in some species [34, 39, 47-49], and where this is known to occur or is
27 suspected, the venom of a certain proportion of juvenile snakes might be mixed with that of
28 adults during the production of venom batches.
29 The ideal frequency of feeding captive snakes depends on the species and age of the snake,
30 varying from twice per week to once per month. Snakes are usually fed after venom extraction,
31 ideally with dead mice or other appropriate prey according to the snake species. Animals such
32 as rats and mice that are raised to feed snakes should be produced under appropriate
33 quarantine standards in facilities designed for this task. Humane euthanasia should be
34 employed in the killing of food animals, and ideally these food animals should be frozen for at
35 least 7 days before being thawed for use. Some snakes will only accept living prey, but attempts
36 should be made to wean them onto dead prey, and all local ethical standards should be
37 followed in the production and use of food animals. Snake-eating species, such as kraits, coral
38 snakes and king cobras, can be enticed to take dead mice if the prey is first flavoured with
39 snake tissue fluids, although any such material should be frozen first for at least 7 days to kill
40 parasites, before it is thawed for use. Living, dead or regurgitated prey should not be left in the
41 cage for more than a few hours. Force-feeding may be necessary for neonates and snakes that
42 persistently refuse to feed. Feeding time affords an opportunity to carefully check the snake for
43 abnormal behaviour, wounds, and possible infections and to give dietary supplements when
44 necessary. Individual feeding records are crucial. They should include details of what, when and
45 how prey was offered, when it was consumed and whether it was regurgitated. The health of
46 captive snakes can be estimated and recorded by observing regular feeding and by measuring
47 their weight and length. These data are best stored on a computer system, using a “bar code”
48 for each snake, or, alternatively, using a reliable manual recording system, and constitute useful
49 records related to the venom batches produced. Venom extraction rooms should be equipped
50 with emergency eyewash stations and safety showers as is the case in laboratories where there
51 is a risk of chemical contact hazards.
52 7.1.3 General maintenance of a serpentarium
53 Serpentariums should be designed to comply with appropriate GMP principles. Quarantine
54 facilities should be isolated in all respects from the main animal housing area, and should have
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1 separate air-handling systems, or be in a separate building. Maintenance areas such as
2 storerooms, rooms for cleaning and sanitizing cages and racks, animal houses for production of
3 food animals (e.g.: rodents or invertebrates), and rooms used for administration or for venom
4 processing, venom quality control and secure storage of venoms, should also be separated by
5 appropriate barrier systems from the main snake housing and venom extraction rooms. The
6 main housing rooms for snakes used in venom production should be designed with security,
7 hygiene and disease control needs in mind. Separate rooms for accommodation of snake egg
8 incubators and both neonates and juvenile snakes should be included in the design of the
9 serpentarium.
10 The cage cleaning rooms should be large enough to hold all the cages that are being cleaned
11 and sanitized. Dirty cages and other items should be kept separate from clean cages and
12 equipment being stored ready for use. Furthermore it is desirable to have two sets of washing
13 and sanitizing rooms, a larger one for equipment from the venom production room and a smaller
14 one for equipment from the quarantine area. These rooms should be secure in case a snake is
15 inadvertently left in its cage when the container is placed in the cleaning room. The cleaning
16 procedures for production rooms and for cages in which snakes are kept, and the cleaning
17 schedule, should be established and documented.
18 Food animals, usually rodents, should be purpose-bred in clean conventional animal houses,
19 and kept, handled and sacrificed in accordance with ethical principles. The rooms, exclusively
20 used for rodent production, should be large enough to provide sufficient numbers of rats or mice
21 to feed the snakes. Alternatively, rodents can be purchased from qualified commercial sources.
22 Breeding of rats and mice cannot take place in the same room, because of the stress induced
23 by the rats in the mice. The diets required by young snakes may differ from those of adults (for
24 instance, frogs and tadpoles are preferred to rodents by some species), and facilities for
25 producing these food animals may also be required.
26 When possible, it is useful to have a small laboratory for performing quality control on the
27 venoms. All serpentariums need to be designed with separate laboratories where venom can be
28 processed after extraction and quality control performed (see section 8). An area for repairing
29 broken equipment and for other miscellaneous purposes is also required. The administrative
30 area should be sufficiently large and adequately equipped with computer facilities so that the
31 traceability requirements needed for venom production can be met. The whole venom
32 production facility should be made secure against unauthorized intrusion.
33 7.1.4 Snake venom production
34 The collection of venom is an inherently dangerous task therefore specific safety protocols for
35 operators must be applied and rigidly enforced (see section 7.2). All operations should be fully
36 described in written procedures and SOPs, which should be checked and revised periodically
37 according to a written master document. Pools of venom require unique batch numbers, and
38 should be traceable to the individual specimens from whom venom was collected for that batch.
39 7.1.4.1 Venom collection in serpentariums
40 Venom can be extracted from snakes according to a regular schedule, depending on the
41 species. The interval between extractions varies among producers and ranges from every 2 or 3
42 weeks to every 3 months. Specimens that are undergoing quarantine, or are gravid, undergoing
43 treatment for sickness or injury, or in the process of sloughing their skins should not be used for
44 venom production.
45 Handling equipment must be appropriate for the particular species of snake to minimize risk of
46 stress, discomfort and injury to both the snake and the operator. Staff must be familiar with the
47 equipment and properly training in its use. Common methods of restraint include gently
48 removing the snake from its cage with a hook and either placing it on a foam rubber pad before
49 being pinned behind the head, or encouraging the snake to crawl into a transparent plastic tube
50 in which it can be restrained. Developing innovative methods that enable safe restraint of
51 venomous snakes that minimize the risk of injury to both operators and specimens is strongly
52 recommended. For very dangerous species, the use of short-acting general anaesthesia or
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1 moderate cooling (15°C) during venom extraction can be considered (e.g. inhaled isoflurane or
2 sevoflurane or even carbon dioxide) as it reduces the risk of accidents both to the snake and to
3 the snake-handler. Excessive cooling of the snake in a refrigerator is potentially harmful and is
4 not recommended. For the collection of venom, the snake’s head is grasped in one hand just
5 behind the angle of the jaw, while the snake’s body is held with the other hand, or by an
6 assistant snake handler. Individual techniques for holding the head of the snake vary and each
7 operator should use the method that works best for them. An assistant should gently occlude
8 the snake’s cloaca to prevent messy contamination of the locality by spraying of faeces.
9 Different techniques are used to collect venom. Many rely on encouraging the snake to open its
10 mouth and either bite through a plastic/parafilm covered membrane, which provides a barrier to
11 contaminants such as saliva and blood (from minor oral trauma), or to release venom into a
12 container over which the fangs have been hooked by the operator. In the case of large vipers,
13 the dental sheath may be retracted when necessary with sterile forceps. Although it is common
14 practice to squeeze the sides of the snakes head to try to force venom from the glands, this
15 may cause traumatic bruising to the animal and should be avoided. The use of brief electrical
16 impulses of moderate intensity to stimulate venom secretion is not recommended. Any venom
17 sample contaminated with blood should be centrifuged. After venom extraction, the fangs are
18 carefully withdrawn from the collection vessel, while preventing damage to the mouth and
19 dentition and avoiding the snake impaling itself with its own fangs. Then, the oral cavity should
20 be sprayed with an antiseptic solution to avoid stomatitis. After each venom extraction, all
21 materials used in the process should be sterilized.
22 Peptides and proteins in venom are amphiphatic and will absorb to most common surfaces
23 including glass and plastic [50] resulting in the potential loss of toxins from the venom used to
24 produce hyperimmune plasma. The use of polypropylene vessels and the addition of 1% bovine
25 serum albumin (BSA) can help reduce such losses, but different peptides may have variable
26 affinity for being retained on vessel surfaces regardless of the approach taken to minimize loss.
27 Special procedures that avoid direct handling should be employed in the case of burrowing asps
28 (genus Atractaspis) because they cannot be held safely in the way described above [51]. For
29 some species with small fangs and small venom yields the use of sterile pipette tips or capillary
30 tubes which are slipped over each fang one at a time, and pressure applied to the base of the
31 fang to stimulate venom release into the tube is recommended. In the case of colubrid snakes,
32 special techniques are required, such as application of foam rubber pads (from which venom is
33 recovered in the laboratory) or pipette tips/capillary tubes to the posteriorly-placed fangs and the
34 use of secretagogue drugs. Similarly, some elapid snakes have only small fangs and the pipette
35 tip/capillary tube technique is required to collect venom. At the time of venom extraction, there is
36 an opportunity to remove broken or diseased fangs and to examine the snake for ectoparasites
37 (e.g. ticks and mites), wounds, dermatitis, areas of adherent dead skin and retained spectacles
38 over the snake’s eyes. The snake can be treated with drugs and/or vitamins at the same time
39 and, if necessary, can be force-fed. When force-fed with rodents, the rodents incisors must be
40 cut out so as not to cause any injury in the snakes’ oesophagus. The process of venom
41 extraction is often combined with cage cleaning and disinfection and the feeding of the snake.
42 Avoiding trauma to the snake's mouth and dentition is critical to prevent infection and “mouth
43 rot” and the venom extraction process should be performed following clean practices.
44 Several snakes from the same group (same species and subspecies collected at the same time
45 in the same area) can be milked into the same venom collection vessel. The vessel should be
46 kept in an ice bath between individual extractions, and the venom aliquoted into labelled storage
47 tubes/vials and snap-frozen at -20°C or colder within 1 hour. For venoms with high proteolytic
48 activity, the collected venom pool should be transferred into a vial maintained at ultra-low
49 temperature (-70 to -80°C) or at least -20°C, every 10-30 minutes, before continuing
50 extractions from that group of specimens. Another method is to transfer the collected venom
51 into a vial maintained in an ice bath. Refrigerated centrifugation of freshly collected venom is
52 recommended, for instance at 1000 g for 5 minutes (4ºC), to remove cellular debris.
53 It is important to identify the vial into which the venom has been collected or transferred for
54 storage, with an appropriate reference number. Primary indelible identification must be on the
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1 vial. This allows the identification of all the snakes used during venom extraction, the name of
2 the operator and any other relevant information. To obtain large venom batches for the
3 preparation of antivenom, especially from species with low yields, one approach is to use the
4 same vial over several months for extractions performed with the the same specimens,
5 providing the cold chain is never broken. Pools of venom require unique batch numbers, and
6 the individual venom extractions contributing to the pool must be traceable. When a pool is
7 sufficient in volume, the venom should be either freeze- or vacuum-dried and kept in the dark at
8 a low temperature (either –20°C or 4°C) in a well-sealed flask, precisely identified with a
9 number, up to the time of delivery. Some producers use an alternative system, keeping dried
10 venom at 20–25°C in a desiccator. Regardless of the method used, the procedures for drying
11 venom should be well-established, documented, validated and incorporate appropriate quality
12 control steps (e.g.: periodic determination of residual moisture against established standards).
13 Venom stored for considerable periods of time should be tested to ensure that no degradation
14 or loss of activity has occurred (see section 8).
15 The equipment used for storage of frozen venom (freezers) and for venom drying, should be
16 cleaned using established procedures, and the cleaning documented, in order to minimize
17 cross-contamination. Likewise, equipment requiring calibration, such as freezers, balances and
18 freeze-driers, should be calibrated as per a defined schedule.
19 7.1.4.2 Venom collection from wild snakes
20 The practice of collecting venoms from wild-caught snakes that are subsequently released in
21 either the same or a different location should be discontinued, and is not recommended due to
22 the lack of traceability and difficulties posed for ensuring effective quality control of venoms.
23 There is also evidence that indicates high levels of mortality among relocated snake species
24 particularly if they are released distant to the capture site [52-55]. In jurisdictions where it is
25 current practice for collectors to go to designated localities in the wild, catch snakes and collect
26 venom before releasing them elsewhere, strong efforts must be made to replace this approach
27 with regulated production using captive snakes maintained in well-designed serpentariums.
28 7.2 Staff responsible for handling snakes

29 7.2.1 Safety and health considerations
30 Handling and extracting venom from snakes is a dangerous operation. One envenoming
31 occurred every two years in each of the 15 extraction facilities reviewed by Powell et al. [56]. At
32 a commercial venom production plant in Uberlândia, Brazil between 1981 and 1999, 25
33 technicians performed 370,768 venom extractions from Bothrops moojeni. Twelve bites were
34 recorded, 10 with envenoming, and one case of venom being squirted into the eye of a worker
35 [57].
36 Venom extractions should be performed according to well-designed and documented SOPs by
37 well-trained snake handlers. All personnel involved in snake handling and venom collection
38 should be fully informed about the potential dangers of being bitten and envenomed. They
39 should be thoroughly trained, and the training procedures must be documented and specific
40 protocols practiced as a team. A minimum of two people should be present during snake
41 handling for venom collection. For safety reasons, it is recommended that venom extraction
42 sessions should be interrupted at least every 2 hours for a rest period, before re-starting the
43 process.
44 Personnel involved in snake handling and venom extraction should observe established
45 hygiene standards (see below) to minimize the impact on snakes and the potential transfer of
46 pathogens between snakes.
47 7.2.2 Personal Protective Equipment (PPE) for snake or venom handling
48 Protective clothing should include appropriate eye protection (safety glasses or face shields),
49 face masks, nitrile gloves and a laboratory coat or gown. Eye protection is especially important
50 when handling spitting elapids capable of squirting their venom. The wearing of puncture
51 resistant gloves designed to prevent an effective bite is unpopular among many keepers who
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1 fear that it impairs manual dexterity and sense of touch, but the use of nitrile gloves is advisable
2 to prevent cross-contamination. Puncture resistant gloves should be mandatory as protective
3 equipment for assistants helping to restrain or handle snakes during procedures such as venom
4 extraction.
5 When lyophilized or desiccated venom is being handled, the safety of operators is paramount,
6 since dried venom can easily be aerosolized and affect people through skin breaks, eyes or
7 mucous membranes, or by sensitizing them to the venom [58]. Appropriate gowning is
8 necessary when handling dried or liquid venom, to prevent contact of the venom with skin or
9 mucous membranes. It is highly recommended that a biological safety cabinet (e.g.: Class II,
10 B2), be used while handling lyophilized or desiccated venom.
11 7.2.3 Procedures to be followed if a bite occurs
12 There are several important measures to be put in place for dealing with a bite [59], as
13 described below.
14 7.2.3.1 Procedures and alarms
15 Clearly defined, prominently displayed, well understood and regularly rehearsed procedures
16 should be in place in case of a bite. An alarm should be sounded to summon help, the snake
17 returned safely to its cage or box and the victim should withdraw to an area designated for first
18 aid.
19 7.2.3.2 First-aid protocols
20 Clearly understandable first-aid protocols should be established for each species. These should
21 be available in printed form adjacent to each cage. Immediate application of pressure-
22 immobilization may be appropriate for treating the bites of rapidly neurotoxic elapids. However,
23 the technique is not easy and, if they are to use the method properly, staff will need extensive
24 training and regular practice, and must be provided with the necessary materials (a number of
25 crepe bandages, 10 cm wide × 4.5 m long, and splints). Analgesia should only be provided for
26 pain during the pre-hospital period upon the advice of an attending physician. Provision of
27 appropriate analgesia for first aid should be considered. If venom enters the eyes, immediate
28 irrigation with generous volumes of clean water is an urgent necessity.
29 7.2.3.3 Hospital admission
30 As a precaution, all victims of bites, scratches by snakes’ fangs or teeth, and those in whom
31 venom has entered the eye. Anyone suspected of a snake bite or venom exposure injury (e.g.:
32 aerosolized dried venom) should be transferred as quickly as possible to the designated local
33 hospital, by prearranged transport, for medical assessment. It may be helpful to remove from
34 the cage, and take to the hospital with the victim, the label identifying the snake responsible for
35 the bite, so that accurate identification of the snake species and of the antivenom to be
36 administered is ensured. Staff members should wear, or carry a card detailing their personal
37 medical information (including drug allergies) at all times which should be taken with them to
38 hospital in the event of an injury. The contact details of a recognised clinical toxinology expert
39 should be included on this card.
40 It is highly recommended that all serpentarium`s stock in-date supplies of antivenom
41 appropriate to the species of snakes being held are accessible, so that an adequate supply of
42 the correct antivenom can accompany the victim to hospital. Hospital staff should be warned in
43 advance by telephone of the arrival of the casualty and informed about the species responsible
44 and any background medical problems and relevant medical history, such as past reactions to
45 antivenom or other equine sera (e.g. anti-tetanus serum), and known allergies.
46 7.2.3.4 Snake venom hypersensitivity
47 Snake venom hypersensitivity is an occupational hazard of snake handlers that occurs due to
48 sensitization to venom proteins. Two out of 12 snakebites in a commercial venom production
49 plant in Brazil resulted in venom-anaphylaxis [57](47). Hypersensitivity is usually acquired by
50 mucosal contact with aerosolized dried venom. Important early evidence of evolving
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1 sensitization is sneezing, coughing, wheezing, itching of the eyes or weeping when working
2 around snakes and snake enclosures, or even upon entering the snake room. No one with
3 established venom allergy should be permitted to continue working with snakes. Venom-
4 induced anaphylaxis should be treated with self-injectable adrenaline (epinephrine) 0.5 ml of
5 0.1% solution by intramuscular injection (adult dose) which should be stocked in adequate
6 doses in each room holding snakes, or where snakes are used for procedures such as venom
7 extraction.
8 7.2.3.5 Medico-legal and health insurance aspects
9 The occupational exposure to venomous snakebites in commercial venom production units is
10 the responsibility of the employers and requires their formal attention.

12  Well-managed serpentariums are a key element in the production of venom
13 preparations meeting the quality requirements for the production of effective
14 antivenoms.
15  The quality of snake venoms used for animal immunization, as material for preclinical
16 assessment of neutralization efficacy, or for the development of national or regional
17 reference preparations is of critical importance.
18  The procedures used in snake maintenance, handling and venom extraction, as well
19 as in all aspects of venom collection should be properly documented and scheduled.
20  Venoms used for antivenom preparations should be representative of the entire snake
21 population living in the area for which the polyspecific and/or monospecific
22 antivenoms are intended to be used. Because of regional and individual variations in
23 venom composition within snake species, the venoms used for immunization should
24 be collected from a large number of individuals (generally at least 20, including males
25 and females of different ages) collected from various regions covering the entire
26 geographical distribution of the particular venomous snake species.
27  Venom producers should adhere to the following recommendations rigorously and
28 should be able to demonstrate their application:
29 — Taxonomic identity and geographical origin of each individual animal used for
30 venom production should be known and recorded.
31 — Housing, feeding, and handling of snakes should meet the highest veterinary and
32 ethical standards, and follow documented protocols.
33 — Adequate training should be provided to personnel involved in venom production
34 in all procedures, and implementation of health and safety measures.
35 — Formal guidelines and procedures for emergency response in the event of any
36 suspected snake bite or venom exposure should be established and well
37 documented.
38 — Venom should not be collected from animals under quarantine, or which are
39 gravid, injured, sick or in poor condition.
40 — Full traceability of each venom batch should be ensured.
41 — Venoms should be frozen as soon as possible after collection, and at least within 1
42 hour.
43 — Freeze-drying or desiccation of the venoms should be done under conditions that
44 ensure stability for long-term storage.
45 — Batch-to-batch consistency of venoms of the same origin should be confirmed.
46
47
48
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1 8 Quality control of venoms
2 8.1 Records and traceability
3 It is critical to accurately identify the species (and subspecies, if any) of each individual snake
4 used for venom production and the taxonomic status should be validated by a competent
5 herpetologist. Increasingly, DNA taxonomy is replacing conventional morphological methods,
6 but this technique is impracticable in most venom production units which will continue to rely on
7 well-established physical features such as colour pattern and scale count to distinguish the
8 principal medically important species.
9 Internationally recognized scientific names should be used and the bio-geographical origin of
10 each snake should be specified, since differences in venom composition may occur between
11 different populations of the same species or subspecies [34-39, 60]. Venom producers can
12 consult academic zoologists who have appropriate skill and experience.
13 Data pertaining to each numbered venom batch should include the information considered to be
14 key for traceability, quality and specificities of the venom (e.g. identification of all the snakes
15 used, the species, subspecies and biogeographical origin, feeding, health care, date of each
16 milking, number of specimens used to prepare the batch, and quantity of venom produced).
17 This information should be made available upon request to any auditor or control authority.
18 For long-term storage, venoms may be regularly re-lyophilized and, when not possible, re-dried
19 by desiccation to ensure minimum water content, as this is critical to their long-term stability.
20 Liquid venoms should be stored frozen at -80 ˚C, while lyophilized or dried venoms may be
21 stored at -20 ˚C.
22 8.2 National reference materials

23 The quality of snake venoms used as a reference standard by quality control laboratories and
24 national regulatory authorities is crucial. WHO recommends that national reference venom
25 collections be established and that these cover each medically important snake species used in
26 antivenom production. Such reference venoms should be prepared as described elsewhere in
27 this document (see sections 7 and 19).
28 Due to the large variations in venom composition even within a single species it is
29 recommended that national reference venom collections should be established, which cover the
30 entire interspecies variability. Regional reference materials could be used when countries within
31 the region share similar distribution of venomous snakes.
32 Establishing a collection of reference venoms ensures that the antivenoms produced will be
33 tested against the same relevant venoms in the specific countries or regions. The
34 characterization and maintenance of reference venom collections should be performed with
35 oversight from national regulatory authorities and other competent agencies with technical
36 expertise to ensure that reference venoms are produced to international reference material
37 standards.
38 Venom batches may be prepared following the procedure outlined in section 7. Whatever their
39 origin, the snakes used for these reference standards should be accurately authenticated
40 (species, subspecies) by a qualified person and the place of capture recorded. Genetic
41 samples (e.g. tissue, blood) should be routinely collected from all specimens for DNA analysis if
42 questions arise regarding the validity of the identification of specimens. Photographs of
43 individual specimens may also have value.
44
45 8.3 Characterization of venom batches

46 It is the responsibility of the venom producer to provide clear information pertaining to the
47 species, the subspecies and the geographical origin of the snakes used for the production of the
48 venoms supplied for antivenom production, quality control and preclinical studies. This
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1 information should be included in the technical dossier supporting the marketing authorization of
2 any antivenom. In addition to the certificate which details the scientific name of the snake
3 species (and subspecies if any), the geographical origin and the number of animals used for
4 preparing the batch, and the date of collection of the venom, additional biochemical and
5 biological information may be provided for each venom batch as evidence of consistency.
6 This information may include analysis of:
7  Biochemical characteristics of the venom:
8 — protein concentration per gram;
9 — scans or pictures of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–
10 PAGE) (in reducing and non-reducing conditions);
11 — size-exclusion or reverse-phase chromatographic profiles (e.g. reverse-phase high-
12 performance liquid chromatography (HPLC);
13  Enzymatic and toxicological activities of the venoms:
14 — e.g. median lethal dose (LD50) and, depending on the particular venom, in vitro
15 procoagulant activity, proteinase activity or phospholipase A2 activity.
16  For lyophilized, vacuum-dried or dessicated venoms, analysis of residual moisture.
17 If the venom producer is not able to perform these determinations they can be subcontracted.
18 Alternatively, (depending on the agreement) the antivenom manufacturer can perform relevant
19 assays to confirm compliance of venoms with specifications as part of the quality control of the
20 raw material.

22  Quality control of snake venoms is essential to provide assurance that the venoms
23 are representative of venomous snakes inhabiting the region for which the
24 antivenoms are manufactured.
25  National Reference Venom Collections should be established covering each of the
26 medically important snake species for which antivenoms are produced.
27  Traceability of each venom batch is important for rapid detection of any errors which
28 might occur during the preparation process.
29  For each venom batch, a certificate stating the scientific names of the snake species
30 (and subspecies if any), their geographical origin and the number of animals used in
31 collecting the batch, the date of collection of the venom, and any other relevant
32 information, must be provided by the venom supplier to the antivenom manufacturer
33 and also to the regulatory authority if required.
34  Consistency (within established limits of composition and quality) of venom batches
35 produced over time for the same venomous species of the same origin should be
36 guaranteed. Specific tests should be performed in each venom sample, and data
37 recorded for traceability, including: the protein concentration per g (or mg), an
38 assessment of biochemical or biological activity, scans or pictures from SDS–PAGE
39 (in reducing and non-reducing conditions), and/or size-exclusion or reverse-phase
40 HPLC chromatographic profiles of the venom sample. This information has proved
41 useful to confirm the origin and the integrity of the venom.
42
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1 9 Overview of the production process of antivenoms
2 Antivenoms are obtained following a complex production process (Figure 1), which involves
3 several steps critical to efficacy, quality and safety [61]. These steps are summarized below:
4  Collection of representative venom pools from correctly identified individual venomous
5 snakes which have been confirmed to be in good health. They should be representative of
6 the snake populations (e.g.: males/females, adults/juveniles) and region(s) where the
7 resulting antivenom immunoglobulins are intended to be used.
8  Preparation of the venom(s) mixtures used for the programme of immunization of animals.
9  Immunization regimens of animals (most often horses). Animals should be selected and
10 controlled carefully, and subjected to continuous health surveillance.
11  Collection of blood or plasma from the immunized animals, once the immune response to
12 the immunizing venom mixture has yielded satisfactory antibody levels.
13  Preparation of the pool of plasma for fractionation.
14  Fractionation of the plasma to extract the antivenom immunoglobulins.
15  Formulation of the bulk antivenom immunoglobulins and aseptic filling.
16  Quality control tests, including potency assessment by in vivo assay.
17  Labelling, packaging, boxing and release.
18  Distribution within the region(s) where snakes used to prepare the venoms to immunize the
19 animals are prevalent.
20
21
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1
2
3
4
5
6 Veterinary Surveillance
7
8 Serpentarium
9
10 Collection of venoms (milking)
Selection of animals (eg
11 horses)
12 Preparation of venom mixtures

Quarantine, vaccination & Veterinary
13 control
Quality control of venom

14 mixtures On-going veterinary
surveillance
15
Preparation of immunizing doses of
16 venoms
Inclusion in the herd
17
18
Immunization programme of each animal
19
20 Control of animal immune responses
21
22 Collection of blood or plasma
23
Storage and pooling of plasma for
24 fractionation
25
Quality control of plasma for fractionation
26
27
Fractionation of plasma to isolate the antivenom
28 immunoglobulins
29 Formulation and filling

30
31 Quality control of antivenom immunoglobulins
32
33 Labelling, packaging, boxing and release
34
35
36 Figure 1: General manufacturing process of antivenoms
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1 10 Selection and veterinary health care of animals used for production of
2 antivenoms
3 10.1 Selection and Quarantine period
4 Animals selected for antivenom production should comply to specific selection criteria relating to
5 animals breed, size, age, health status and history, preferably animals should be purchased
6 from known accredited suppliers. The use of animals in the production of hyperimmune plasma
7 should follow strict ethical standards in accordance with national and international conventions
8 for the use and welfare of animals. Animals must be transported according to local transport
9 standards. Before an animal is introduced into the herd used for a production programme, it
10 should be subjected to a period of quarantine (which, in most countries, is from 6 to 12 weeks),
11 depending upon the source of the animal, during which an appropriate veterinarian assessment
12 is performed to ensure its suitability for the programme. The quarantine facility should be
13 separate from the main animal housing facility or farm and a biosecurity plan for all animal
14 promises is recommended. Each animal should have an individual monitoring record system
15 created on entry into the quarantine facility which will remain with the animal throughout its life
16 at the facility or farm. All activities and information including husbandry, health, antivenom
17 immunization, bleeding and emergency care must be recorded on this file which should be
18 accessible for external review.
19 When an animal is imported from a country or region with different ecological characteristics, a
20 period of acclimatization to the local environment of about 3 months is needed. Each individual
21 animal should be unambiguously identified using, for example, a microchip, branding or ear-
22 clipping.
23 In the case of horses and other equines, animals between 3 and 10 years are usually included
24 in an immunization programme, but in some cases older animals may also be suitable as long
25 as they exhibit a satisfactory immune response to the immunization programme. In the case of
26 sheep, animals retired from wool production have proved capable of useful antibody production
27 for a number of years (beyond the age of 10 years). No particular breed is preferred, but in
28 general large horses or sheep are preferred because they yield larger individual volumes of
29 blood.
30 10.2 Veterinary care, monitoring and vaccinations

31 The veterinary examination will include a complete physical examination and blood tests
32 including serological testing for the most prevalent infectious diseases for that type of animal in
33 that particular geographical location (e.g. Equine Infectious Anaemia).
34 Depending upon the local epidemiological situation, animals should be vaccinated against
35 tetanus and, possibly other endemic diseases, such as rabies, equine influenza, anthrax,
36 brucellosis, glanders, African horse sickness and equine encephalitides. Animals should go
37 through a programme to eliminate gut helminths and other locally prevalent parasites. All
38 vaccinations and health information will be recorded on the animal’s individual record.
39 Staff who are in regular contact with the animals should be vaccinated against tetanus and
40 rabies.
41 10.3 Animal health and welfare after inclusion in the herd

42 After the quarantine period, if the animal is in good health according to a veterinaryexamination
43 and blood parameters and body condition score, and the results of relevant serological tests are
44 negative, the animal may be incorporated into the herd of animals used for immunization.
45 An individual record should be kept for each animal being used in an immunization programme
46 for antivenom production. In addition to surveillance by a veterinary professional, the staff in
47 charge of the animals should be well-trained, and the operations related to animal care,
48 emergency care and use should be clearly specified in the standard operating procedure.
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1 During the time an animal is used for immunization aimed at antivenom production, careful
2 veterinarymonitoring should be maintained, including continued vaccination regimes, and the
3 performance of regular clinical examinations, together with clinical laboratory tests such as
4 packed cell volume, haemogram, clotting tests and other tests associated with the possible
5 clinical effects of venoms [62] and of successive large volume blood collection [63].
6 Possible anaemia, resulting from excessive volume or frequency of bleeding (when erythrocytes
7 are not re-infused into the animals after the whole blood bleeding session) or from the
8 deleterious action of venoms should also be tested for.
9 The immune response against venom components should, when feasible, be followed
10 throughout the immunization schedule, in order to detect when animals reach an acceptable
11 antivenom titre. However, the monitoring of the immune response can be done on a pool of sera
12 from various animals. This response may be followed by in vivo potency assays of
13 neutralization of lethality or by in vitro tests, such as enzyme immunoassays (EIAs) (provided
14 that a correlation has been demonstrated between these tests and the in vivo potency tests).
15 Whenever an animal develops any manifestation of sickness, it must be temporarily withdrawn
16 from immunization programmes to allow it to receive appropriate veterinary examination and
17 treatment. If the disease is controlled, the animal may return to the immunization programme
18 after a suitable length of time, usually 4 weeks. If an animal is receiving any type of antibiotic or
19 drug, it should be withdrawn from the immunization programme for a period that would depend
20 on the elimination kinetics of the particular drug(s) concerned. In the case of vaccination, this
21 withdrawal period should not be shorter than 1 month.7 Any blood/plasma /serum obtained from
22 the animal in the incubation period of any contracted disease should be excluded from use for
23 the production of antivenoms. Animals should have appropriate physical exercise and routine
24 husbandry (hoof care, teeth rasping etc). Their feed should originate from a controlled source
25 and should be free of ruminant-derived material. Ideally, the diet should include both hay and
26 grass, or alternative plant material, and concentrated food preparations containing vitamins
27 including folic acid, iron and other mineral supplements. The routine quality control of the food
28 and water is recommended, in order to assure a consistent composition and adequate level of
29 nutrients.
30 As a consequence of immunization with venoms (see section 11) a common problem in
31 antivenom-producing animals is the development of local ulcers or abscesses (sterile and
32 infected) at sites of venom injection. This is a particular problem when necrotic venoms and
33 complete Freund’s adjuvant are used. All injections should be given under aseptic conditions
34 and given subcutaneously. There should be a limit to the total volume and dose of venom
35 injected at a single site. Infected or ulcerated areas should be treated appropriately with
36 abscesses lanced and drained and the skin site not be used again. In the event of the death of
37 an animal being used for antivenom production, a careful analysis of the causes of death should
38 be performed, including, when necessary, the performance of a necropsy and histopathology.
39 All deaths should be recorded including the necropsy report and made available for external
40 review.
41 Some animals show declining titres of specific venom antibodies over time, despite rest or
42 increasing doses of immunizing venoms. Such animals should be retired from the immunization
43 programme. In agreement with GMP principles and to avoid impact on the composition and
44 consistency of the antivenom produced, it is, in principle, not considered good practice to move
45 animals from a given venom immunization programme to another one, unless the animal has
46 been used in the preparation of a monospecific antivenom that is included into a polyspecific
47 preparation, or if it was used for the production of other animal-derived antisera (e.g. anti-rabies,
48 anti-tetanus, or anti-botulism).
49 When an animal is withdrawn from the herd, it could be either kept on the horse farm or if sold,
50 continued good care should be ensured.
7
In some areas, legislation stipulates that animals used for production of plasma cannot be treated with
penicillin or streptomycin.
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2  A thorough biosecurity plan should be developed and implemented for each farm and
3 facility.
4  All staff working with the animals should be trained and qualified to care for the
5 animals. Staff training records and history should be available for review.
6  An emergency care protocol is essential especially during procedures e.g.
7 sensitization to venoms, blood collection, post plasmapheresis. Adverse events must
8 be reported and tracked appropriately.
9  Animals intended for antivenom production programmes should be identified to
10 ensure full traceability and health monitoring.
11  Animals should go through a quarantine period of 6–12 weeks during which they are
12 submitted to veterinary scrutiny and are vaccinated against specific diseases and
13 treated for internal and external parasites.
14  Following the quarantine period, they are introduced into the immunization
15 programme. Animals should be appropriately housed, fed, and managed according to
16 best practice in veterinary, animal welfare and ethical standards.
17  During immunization, the clinical status of each animal must be followed by a
18 veterinarian through clinical and laboratory assessments which are recorded on the
19 animal records. If an animal develops clinical signs of disease, it should be
20 temporarily separated from the immunization programme to receive appropriate care
21 and treatment. Particular care must be paid to the local lesions that develop at the site
22 of venom injections and to development of anaemia.
23  The immune response to venoms of each animal should, when possible, be
24 monitored during the immunization schedule (alternatively, the antivenom titres can
25 be monitored indirectly by testing the plasma pool).
26  An animal receiving an antibiotic or drug should be withdrawn from the immunization
27 programme for a period depending on the elimination kinetics of each drug. In the
28 case of vaccination, this withdrawal period should not be shorter than 1 month.
29
30
31
32
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1 11 Immunization regimens and use of adjuvant
2 One of the most crucial steps in antivenom production involves the immunization of animal with
3 venom(s) to produce a long-lasting and high titre antibody response against the lethal and other
4 deleterious components in the immunogenic toxins. To achieve this goal, the following
5 considerations are important:
6  Venom(s) used should be prepared as described in section 7, and should be in an optimal
7 condition for inducing specific and neutralizing antibodies.
8  Immunogen and the immunization regimens used should not seriously affect the health of
9 the animal.
10  Preparation of immunogens and the immunization protocol should be technically simple and
11 economical and use a minimal amount of venom. The procedures followed must be included
12 in a protocol and their performance must be documented.
13 The antivenom manufacturer is responsible for defining the appropriate immunization
14 programme (choice of doses, selection of adjuvants, sites of immunization, and bleeding
15 schedule) able to generate the best immune response and plasma production, while also
16 ensuring optimal animal care. Good manufacturing practices (GMP) principles should be
17 applied in the preparation of the immunizing doses as well as in the immunization process.
18 11.1 Animals used in antivenom production

19 Numerous animal species have been used on various scales in antivenom production (horse,
20 sheep, donkey, goat and rabbit) or for experimental purposes (camel, llama, dog and hen) [64,
21 65]. However, the production of large volumes of antivenom from large animals such as equines
22 is an advantage compared to the smaller species. The selection of the animal species should
23 be based on several considerations, such as locally prevalent diseases, availability in the
24 region, adaptation to the local environment, and cost of maintenance. The information in these
25 Guidelines refers mostly to horse-derived immunoglobulins.
26 The horse is the animal of choice for commercial antivenom production. Horses are docile,
27 thrive in most climates and yield a large volume of plasma. Antivenoms made from horse
28 plasma have proven over time to have a satisfactory safety and efficacy profile [66]. Sheep
29 have also been used as an alternative source for antivenom production because they are
30 cheaper, easier to raise, can better tolerate oil-based adjuvant than horses, and their antibodies
31 may be useful in patients who are hypersensitive to equine proteins. However, increasing
32 concern about prion diseases may limit the use of the sheep as an animal for commercial
33 antivenom production. Larger animals are preferable to smaller ones because of their greater
34 blood volume, but breed and age are less important. Any animals used should be under
35 veterinary supervision (see section 10). When sheep or goats are to be used, manufacturers
36 should comply with regulations to minimize the risk of transmissible spongiform
37 encephalopathies to humans, such as the WHO Guidelines on tissue infectivity distribution in
38 transmissible spongiform encephalopathies [67].
39 11.2 Venoms used for immunization

40 Venoms used as immunogens in antivenom production are chosen based on criteria discussed
41 in section 6. Priority should be given to venoms from snakes responsible for frequent
42 envenomings. The quality, quantity, and biological variation of venoms are important
43 considerations (see sections 7 and 8).
44 11.3 Preparation of venom doses

45 Venom doses used for the immunization of animals should be prepared carefully in a clean
46 environment, with an established, scheduled and documented cleaning regime. All venom
47 manipulations should be performed using aseptic techniques under a hood; for highly toxic
48 venoms, a cytotoxic cabinet may be used. Batch process records should be completed for each
49 dose preparation session. The venom batches used and the animals to be immunized should
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1 be recorded and the containers in which the venom is dissolved should be appropriately
2 identified. Ideally, the calculations and operations related to the dose of venom to be used, as
3 well as dilutions, require verification by a second person to ensure accuracy and to prevent
4 errors that may lead to animals receiving overdoses.
5 Venoms, when freeze-dried, are highly hygroscopic and allergenic, thus care should be taken
6 when manipulating them. When taken out of the refrigerator or freezer, the venom should be
7 allowed to warm up to room temperature before the bottle is opened, otherwise condensation
8 may occur causing inaccuracy in weighing and, more seriously, proteolytic degradation of the
9 venom proteins by venom enzymes. Venom should be dissolved in distilled water or buffer, but
10 care should be taken not to shake the solution too vigorously since excessive foaming may
11 cause protein denaturation.
12 The solvents used to dissolve venoms should be sterile and within established expiry periods. A
13 stock solution of each venom should be prepared separately, rather than being mixed with other
14 venoms. This is to allow flexibility of dosage and to avoid proteolytic degradation by one venom
15 component of other venom proteins. Venom solutions can be sterile-filtered where this is known
16 not to affect the potency of the preparation, aliquoted, labelled and stored appropriately (e.g.
17 refrigerated, frozen at –15 to –20 °C, or deep frozen at –70 °C) for a short time (less than 1
18 month). However, it is recommended that venoms used for immunization be freshly prepared at
19 the time of use.
20 All the equipment used for venom storage (freezers and refrigerators) and preparation (e.g.
21 balances) should be calibrated and validated for their intended purpose. Balances should be
22 calibrated at least annually and calibration should be checked daily. Where possible, laboratory
23 items used in venom preparation, i.e. pipettes, syringes and other such items should be pre-
24 sterilized, single-use, disposable items. The siliconization of venom solution containers may be
25 considered to avoid the adherence of venom components to the surfaces of containers.
26 Transport of venom solutions/suspension to the facilities where animals are going to be injected
27 should be done in a safe manner while the venom solutions/suspensions are kept cold at about
28 5-10 °C.
29 Care should be taken to avoid accidents that may result in envenoming of the persons
30 preparing the venom solutions. Protective equipment (e.g. eyewear, gloves and gowns) should
31 be worn by personnel preparing venom solutions. Procedures for cleaning up broken glass or
32 plastic containers that have held venom should be established and the personnel should be
33 trained to follow them.
34 11.4 Detoxification of venom

35 Some snake venoms can cause local and/or systemic toxicity when injected into naive horses at
36 the beginning of an immunization course. Various physical or chemical means have been
37 adopted to decrease venom toxicity, for example, treatment with aldehydes (formaldehyde or
38 glutaraldehyde), hypochlorite, ultraviolet or gamma radiation, and heat, among others. However,
39 in most cases, not only the toxic sites, but also the antigenic sites of the toxins are destroyed
40 after these treatments [68]. For example, when glutaraldehyde is used, the protein
41 polymerization is often extensive and is difficult to control and reproduce. Thus, although the
42 detoxified toxin (toxoid or venoid) induces vigorous antibody response, the antibodies usually
43 fail to neutralize the native toxin. In fact, no detoxification is usually necessary if inoculation is
44 made with a small dose of venom well-emulsified in an adjuvant such as Freund’s complete or
45 incomplete adjuvants.
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1 11.5 Immunological adjuvants
2 Various types of immunological adjuvants have been tested, for example, Freund’s complete
3 and incomplete adjuvants, aluminium salts (hydroxide and phosphate), bentonite and liposomes
4 [69]. The choice of adjuvant is determined by its effectiveness, side-effects, ease of preparation,
5 especially on a large scale, and cost. It may vary depending upon the type of venoms and
6 following manufacturers’ experience. Freund's incomplete adjuvant (FIA) contains mineral oil
7 and an emulsifier. Freund’s complete adjuvant (FCA), which contains mineral oil, an emulsifier
8 and inactivated Mycobacterium tuberculosis, has been shown in experimental animals to be one
9 of the most potent adjuvants known. However, horses are quite sensitive to FCA which tends to
10 cause granuloma formation. For this reason, some producers prefer to use other adjuvants. It is
11 recommended that when using FCA and FIA, they be utilised only at the beginning of the
12 immunization schedule, and not during the rest of the immunization, nor during booster
13 injections of venom; this significantly reduces the formation of granulomas in the horses.
14 It has been noted that the granuloma caused by FCA is due to injection of a large volume (5–10
15 ml) of the emulsified immunogen at 1 or 2 sites. The large granuloma formed usually ruptures,
16 resulting in a large infected wound. If the emulsified immunogen is injected subcutaneously in
17 small volumes (50–200 µl/site) at multiple sites of injection, granuloma formation may be
18 avoided. Manufacturer’s are also encouraged to adopt an innovative approach with regard to
19 adjuvants used for antivenom production, and should strive to replace FCA/FIA with new
20 compounds of low toxicity and high adjuvant effect. The advances in the vaccine field
21 concerning new adjuvants should be transferred to the antivenom field, such as for example the
22 use of microbial-derived products of low toxicity or of Toll-like Receptor 4 (TLR4) ligand-based
23 adjuvants [70].
24 11.6 Preparation of immunogen in adjuvants

25 To minimize infection at the immunization sites, all manipulations should be carried out under
26 aseptic conditions. Venom solutions are prepared in distilled water or phosphate-buffered saline
27 solution (PBS) and filtered through a 0.22-µm membrane. The venom solution is then mixed
28 and/or emulsified with adjuvant, according to the instructions of the supplier. An example for the
29 preparation of venom immunogen in FCA/FIA and aluminium salts is described in Box 1. To
30 facilitate the injections, the immunogen suspension is filled in tuberculin syringes as shown in
31 Fig. 1.
32 11.7 Immunization of animals

33 The areas to be immunized should be thoroughly scrubbed with a disinfectant, shaved and
34 rubbed with 70% ethanol before venom immunogen injection.
35 In general, the sites of immunization (Figure 2) should be in areas close to major lymph nodes,
36 preferably on the animal’s neck and back, while the route of injection should be subcutaneous
37 so as to recruit a large number of antigen presenting cells and consequently resulting in high
38 antibody response. Some procedures call for a small volume of injection at each site (50–200
39 l) so that the total surface area of the immunogen droplets is maximized, enhancing the
40 interaction with the antigen presenting cells and the immune response [71, 72]. An example of
41 immunization of a horse using venom emulsified in FCA is described in Box 2.
42 Other immunization protocols, using larger amounts of venoms devoid of local tissue-damaging
43 activity (such as those of some elapids) and/or adjuvants other than FCA may be used with
44 satisfactory results, as long as the schedule does not compromise the health of the animals. In
45 situations where the main toxins of a given venom have a low molecular mass and would not
46 induce a sufficient immune response if injected together with the other venom components,
47 isolating such toxins using mild chromatographic procedures or ultrafiltration can be beneficial.
48 Such isolated fractions can then be used for immunization.
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Box 1
Example of preparation of venom immunogen in FCA, FIA and aluminium salts
Since FCA can cause severe irritation, precautions should be taken to avoid contact
with the eyes, and protective eyewear and gloves are recommended. The vial
containing FCA is shaken to disperse the insoluble Mycobacterium tuberculosis. The
venom solution is mixed in a stainless steel container with an equal volume of FCA at 4
ºC. The emulsification is achieved by vigorous blending in a high-speed blender at a
speed of approximately 3000 rpm for 15 minutes. The container is put in ice water to
dissipate the heat generated. The resultant emulsion should be quite thick and remains
stable when dropped on the surface of cold water. The highly viscous emulsion is then
transferred into a sterile 50-ml glass syringe with the plunger removed. The plunger is
then put into the syringe to expel any air pocket inside. By means of a three-way
stopcock, the emulsion from the 50-ml syringe is then transferred into tuberculin
syringes to give a volume of 0.1-0.2 ml/syringe. After the tuberculin syringe is fitted
with a 38 mm no. 21 gauge disposable needle, the needle cover with its end cut off is
attached so that only 2-3 mm of the needle tip is exposed and penetrated the horse
skin (Fig 1). With each filled tuberculin syringe, immunization at a site could be
performed by injection and expulsion of the immunogen almost simultaneously in one
single step. This immunization procedure makes multiple subcutaneous injections with
small immunogen volume easier, faster and with minimal restrain on the horse.
Immunogen in FIA is prepared by a process similar to that described above except that
FIA is used in place of FCA. Both the FCA and FIA emulsified immunogens may, if
necessary, be stored at 4 ºC, preferably for a maximum of 2 weeks but re-
emulsification is needed before their injection.
When the immunogen is prepared in Al(OH)3 (aluminium hydroxide) or Al(PO)4
(aluminium phosphate), a sterile venom solution and a suspension of aluminium salts
are mixed in a ratio of 1:3 (v/v) and homogenized. When using other adjuvants, the
preparation of the solution or emulsion should follow the manufacturer’s instructions for
that type of adjuvant.
1
2
3 Figure 1 Tuberculin syringes are filled with immunogen suspension and used for the
4 subcutaneous injection of the horse.
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1
2 Figure 2 Recommended areas of immunization in horses.
3
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Box 2
Example of immunization of horses using FCA, FIA and aluminium salts
The primary immunization could be made with venom(s) mixed with (FCA) as described in
Box 1. The initial dose of each venom could be as low as 1–4 mg/horse with a total
combined volume of injection of about 2 ml. The immunogen is filled in several 1-ml
tuberculin syringes with 21G needles as described in Box 1 and Fig 1 above. Subcutaneous
injections of 100–200 µl of immunogen are made at each site, up to as many as 8–12 sites,
although some producers may use only 3–4 injection sites. The neck of the horse, supplied
with extensive lymphatic vessels and large lymph nodes, is a preferred area for
immunization. If inoculation is made on the lateral sides of the neck, the animal tends to rub
itself causing skin blisters. Thus, injections should be made to the upper (dorsal) part of the
neck, close to the mane. About 4–6 injections can be made at each side of the neck. If
injection in the rump is possible, 1–2 injections can be made in the area between the outer
hip bone and the top of the thigh bone. The scratching of injected sites by animals can be
partially alleviated by massaging the injection site after venom injection to disperse the dose
material.
Immunization using Freund’s complete adjuvant is usually made only once; repeated use of
this adjuvant may in most cases cause serious reactions which can affect the horse’s health.
After 2 weeks, the horses should receive a booster injection with the same venom(s) well
emulsified in Freund’s incomplete adjuvant. Similar volume and areas of injection to those
described above can be made. Subsequent booster immunizations at 2-week intervals can
be made with higher doses (5–10 mg) of venom(s) in saline or mixed with aluminium salts or
any other adjuvant selected. In this case, subcutaneous injections of 1 ml of immunogen at
each site in a total of 4 sites are recommended.
Blood (10–20 ml) should be drawn before each immunization. Serum or plasma is prepared
and EIA (enzyme immunoassay) titres and/or lethality potency are determined. When the
EIA titres reach a plateau, usually about 8–10 weeks after the primary immunization, an in
vivo potency assay may be performed to confirm that the horse could be bled. After bleeding
for antivenom production, the horses are allowed 3–8 weeks rest, depending on their
physical condition. After the rest period, a new round of immunization can be made as
described above, but without the use of Freund’s complete adjuvant.
1
2 11.8 Traceability of the immunization process

3 The traceability of the immunization process is critical for the quality control of the antivenoms
4 produced and the steps to ensure it should be performed very accurately. Each immunized
5 animal should be identified by its code number (see section 10). The details of each
6 immunization step should be recorded precisely. The details to be recorded include:
7  date of immunization;
8  batch(es) of venom(s) used with its (their) reference number(s) (see section 8);
9  venom dose(s);
10  adjuvant and/or salt used;
11  names of the veterinary and supporting staff in charge of the immunization;
12  eventual reaction and/or sickness.
13 The antivenom titre of the immunized animals should be followed throughout the immunization
14 procedure either in vitro, using EIA, during the immunization phase, or in vivo, by neutralization
15 potency assays of lethality when the immunization plateau is reached or before each blood
16 collection.
17 Each plasma batch should be assigned a unique reference number (e.g. a bar code), which
18 should allow complete traceability to the donor animal. Information (such as the date of
19 collection, the unique identification number of the immunized donor animal, and the reference
20 number of the venom(s) used for immunization) should be recorded to allow traceability to all
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1 venoms. Computer-based databases are very useful for properly recording these data, which
2 are crucial for the traceability of the antivenoms produced. Standard procedures should be used
3 to protect the integrity of data stored on a computer, including regular, frequent backup,
4 protection from unauthorized access, and storing backup copies securely off-site.

6  Venom solutions should be prepared in such a way as to minimize proteolytic
7 digestion and denaturation of the venom proteins. Venom solution should be
8 prepared under aseptic conditions to avoid infection at the injection sites.
9  The type of adjuvant used is selected on the basis of its effectiveness, side-effects,
10 ease of preparation and cost.
11  Primary immunization should be made by subcutaneous injections of small volumes
12 at multiple sites close to the animal’s lymphatic system to favour the recruitment of
13 antigen presenting cells and involving anatomically different groups of lymph nodes
14 for antibody production.
15  Subsequent booster injections can be made using venom immunogen doses, at
16 volumes and intervals depending on the type of adjuvant used, until the antivenom
17 titre reaches a plateau or a pre-established minimum accepted titre.
18  After collection of blood for antivenom production, animals should have a resting
19 period of 3–8 weeks. After this, a new round of immunization can be performed as
20 above without the use of Freund’s complete adjuvant.
21  All steps in the immunization of the immunized donor animal, as well as the collection
22 of blood or plasma, should be traceable.
23
24
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1 12 Collection and control of animal plasma for fractionation
2 Historically, serum separated from the blood of hyperimmunized horses was the basis of
3 “antivenin serum-therapy”, but today plasma is used, almost exclusively, as the starting material
4 and undergoes a fractionation process for the separation of purified antivenoms. Thus
5 “antivenom immunoglobulins” is the preferred term, rather than “anti-snakebite serum” or
6 “antiserum”, which are imprecise and confusing terms that refer to a crude therapeutic
7 preparation.
8 Plasma as a starting material is preferred to serum largely because erythrocytes can be
9 returned to the animal, thus preventing anaemia and hypovolaemia in the donor animal and
10 allowing more frequent bleeding. Some laboratories have found that using plasma enables
11 higher recovery of antibodies per donation and it is less contaminated with haemoglobin than
12 serum. Separation of plasma from anticoagulated blood is much more rapid than separation of
13 serum from clotted blood. Plasma for fractionation can be obtained either from the collection of
14 whole blood or by the apheresis procedure.
15 12.1 Health control of the animal prior to and during bleeding sessions
16 When an immunized animal has developed an antivenom antibody titre that meets the
17 necessary specifications, it can be bled, provided the animal is in a satisfactory clinical condition
18 and blood parameters and biochemistry are within normal range for animal type and breed.
19 Before bleeding is performed, the animals should be evaluated by a veterinarian or other
20 qualified person and declared healthy. Individual blood chemistry parameters (packed cell
21 volume, PCV; haemoglobin, Hgb; total plasma protein, TPP) must be within specified
22 parameters. Animals showing evidence of clinical deterioration, such as weight loss, altered
23 horse body condition score,drop in haemoglobin or serum protein concentration below a critical
24 predefined value for animal type and breed, or evidence of infections, should not be bled. It is
25 recommended that animals to be bled have no contact with potentially infectious animals.
26 Human beings can be a potential source of fomite infection to horses therefore a biosecurity
27 plan is essential.
28 12.2 Premises for blood or plasma collection

29 The bleeding of animals should be performed in designated rooms or areas dedicated to this
30 activity and equipped with appropriate restraining devices. Some producers may design the
31 bleeding rooms so that they can be closed, if needed, during the bleeding sessions, but this is
32 not general practice. The rooms or areas should be thoroughly washed and cleaned before and
33 after each bleeding session and their design should facilitate such cleaning procedures, which
34 should be clearly established. The room or area should be inspected before the confinement of
35 the animal. Animals need to be made as safe and comfortable as possible, in a quiet
36 environment, during bleeding to minimize the chance of injury to the animal or its handlers.
37 Individual animals should be confined in circumstances that reduce the potential for stress as
38 much as possible. It is recommended that these rooms allow the simultaneous bleeding of
39 various animals to reduce the time required for this operation as well as the stress.
40 To avoid bacteria and fungi contamination, animals should be cleaned and injection sites and
41 jugular catheter area clipped in a separated room before bleeding. Humidity control of the
42 surrounding bleeding area should be ensured.
43 12.3 Blood or plasma collection session

44 Animals are bled by venipuncture from the external jugular vein. The area surrounding the
45 venipuncture site should be clipped before bleeding and thoroughly cleaned and disinfected,
46 using a disinfectant that has not reached the end of its recommended shelf-life, and, depending
47 on the type of disinfectant, it should be allowed to dry. The disinfected area should not be
48 touched or palpated before the needle has been inserted.
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1 Before venipuncture all containers and tubing should be inspected for defects (for example,
2 abnormal moisture or discoloration as these may suggest a defect). There should be means to
3 determine the volume of blood or plasma collected (such as a weighing machine).
4 When using disposable plastic bags to collect blood (which may take about one hour) it is
5 recommended that blood to be gently and continuously mixed to ensure a homogenous
6 distribution of the anticoagulant and avoid formation of clots.
7 Horses should be weighed before bleeding if possible and their weight recorded on their record.
8 The clinical condition of the animals being bled should be closely monitored at the time of
9 bleeding and during the days that follow, and bleeding should be suspended in the event of any
10 adverse effect on the animal. If an animal shows signs of distress during the operation, the
11 collection procedure should be terminated. In addition, animals should be kept under
12 observation for at least 1 hour after the bleeding to allow any evidence of physical alterations to
13 be detected. Horses can be fed during blood collection depending on the horse crush set-up.
14 For animal’s welfare, a resting period of 45 to 60 days is ideal for the animal to recover its
15 weight and health condition before the next immunization cycle.
16 12.4 Labelling and identification

17 The identity of the animal should be recorded immediately before venipuncture. Labels on all
18 bottles or bags of blood or plasma should be marked with the animal’s unique identification
19 number. The label should be waterproof and heat-resistant, and contain the following
20 information: specificity of antivenom, plasma unit number and date of collection.
21 A document to register all steps of the production of the plasma lot should be maintained to
22 guarantee traceability of the process.
23 12.4.1 Collection and storage of whole blood
24 12.4.1.1 Collection
25 The volume of blood to be obtained depends on the species and size of the immunized animal.
26 It is recommended that around 13–15 ml of blood per kilogram body weight are collected in one
27 bleeding session, or 1.5 to 2% of the weight of the animal. For sheep, 0.5 l is a typical yield,
28 whereas in the case of horses, the volume of blood may range between 3 and 6 l, depending on
29 the size of the animal.
30 Blood is collected, ideally, in disposable plastic bags containing sterile citrate anticoagulant or
31 other preparations containing citrate phosphate dextrose solution (CPD), to prolong the
32 durability of red blood cells. Usually, the volume ratio of anticoagulant to blood is 1:9 to 1:15,
33 depending on the anticoagulant. Use of double plastic bags containing anticoagulant is
34 recommended to avoid bacterial contamination and for ease of use. When plastic bags are not
35 available, disposable polypropylene plastic bottles, or sterilized glass bottles containing
36 anticoagulant may be considered.
37 While the bleeding is taking place, a constant flow of blood should be ensured. Blood should be
38 gently and continuously mixed with the anticoagulant solution to ensure a homogeneous
39 distribution of the anticoagulant, to avoid the risks of activation of the coagulation cascade and,
40 therefore, avoid the formation of clots. The duration of a bleeding session per animal is usually
41 between 30 and 45 minutes depending upon the weight of the animal and the total volume
42 collected. Care should be taken to avoid contamination of the blood by exposing the needle to
43 contaminated surfaces. It is recommended to seal or occlude the device before removing the
44 needle from the animal.
45 12.4.1.2 Storage
46 The bags or bottles in which the whole blood has been collected should be appropriately
47 cleaned and sanitized on their external surfaces. They should be put into a refrigerated room
48 (2–8 °C) for the plasma and blood cells separation procedure. They should be stored for not
49 more than 24 hours before the reinfusion of the red cells, unless CPD is used. In this case,
50 blood cells may be stored for up 72 hours
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1 Alternatively, aseptically-collected blood can be stored for a maximum of 7 hours at 20–25 °C to
2 allow for sedimentation. Under such circumstances, great care should be taken to avoid
3 bacterial contamination.
4 12.4.1.3 Separation of plasma from whole blood
5 Hyperimmune plasma should be separated from blood cells under aseptic conditions and
6 should be transferred into sterile containers (plastic bags, bottles, or stainless steel containers).
7 A designated room, designed to allow proper cleaning and sanitization, should be used for
8 separation. When bottles are used, separation of plasma from blood cells should be performed
9 in a laminar flow cabinet located in a room separated from the plasma fractionation area.
10 12.4.1.4 Reinfusion of the erythrocytes
11 Reinfusion of the erythrocytes after whole blood collection is recommended.
12 Blood cells, most specifically erythrocytes (red blood cells), should be separated from plasma
13 by validated centrifugation or sedimentation procedures. Erythrocyte reinfusion should take
14 place within 24 hours after blood collection (or 72 hours if CPD anticoagulant is used), and after
15 being suspended in sterile saline solution at room temperature for one hour, or 32–37 °C prior
16 to infusion. This procedure in which whole blood is collected and erythrocytes are reinfused to
17 the animal is commonly referred to as “manual apheresis”.
18 12.4.2 Plasma collection by automatic apheresis and storage
19 12.4.2.1 Plasma collection
20 In some laboratories, plasmapheresis machines are used to perform automatic plasma
21 collection. This has proved a useful investment in some facilities; it ensures that the animal
22 does not become hypovolaemic and it reduces the risks of handling errors, in particular during
23 re-infusion of the erythrocytes to the donor. Plasma from automatic apheresis tends to be less
24 contaminated by blood cells (red blood cells, leukocytes and platelets) and in the experience of
25 some laboratories is easier to fractionate, as the filtration steps, in particular, are more readily
26 performed, resulting in higher yields.
27 In such procedures, whole blood is collected from the animal, mixed with anticoagulant, and
28 passed through an automated cell separator. The plasma is separated from the cellular
29 components of the blood, which are returned to the animal in a series of collection/separation
30 and return cycles. The plasma is separated from the red blood cells by centrifugation or
31 filtration, or a combination of the two. The operational parameters of the plasmapheresis
32 equipment are provided by the manufacturers of the equipment. In general, the anticoagulant is
33 delivered at a rate yielding a specified ratio of anticoagulant to blood. The anticoagulant
34 solutions used include AB16 (35.6 g sodium citrate, 12.6 g citric acid monohydrate, 51.0 g
35 glucose monohydrate per 1 litre using water for injection) and anticoagulant citrate dextrose
36 formula A (ACDA) (22.0 g sodium citrate, 8.0 g citric acid, 24.5 g dextrose monohydrate, per 1
37 litre using water for injection). The number of collection/separation and return cycles for each
38 donor animal depends on the total volume of plasma that is to be harvested. For horses, the
39 average volume of plasma collected may be about 6 litres per session. The number of cycles
40 ranges from 10 to 20 depending upon the haematocrit of the horses. The collection process
41 lasts for 1–4 hours. The apheresis equipment and apheresis procedures should be validated,
42 maintained and serviced. Machine plasmapheresis can take several hours and animals can be
43 fed during the operation.
44 12.4.2.2 Plasma storage
45 Apheresis plasma: bags or bottles should be stored in a refrigerated room (2–8 °C) in the dark
46 until the fractionation process starts. This storage room should be designed to allow proper
47 cleaning and sanitization.
48 12.5 Pooling
49 Plasma from individual animals should be pooled into sterile and sanitized containers before
50 fractionation. For traceability purposes each plasma pool should be identified with a unique
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1 number. The number of plasma units collected from individual animals and used in the pool
2 should be recorded. Before the large pool of plasma is prepared, it is recommended to prepare
3 a small volume pool and to test it for microbial contamination. If there is no contamination, the
4 large pool can be prepared. If microbial contamination is detected, plasma of individual animals
5 should be checked, and the contaminated ones should be discarded, to ensure that the pool is
6 prepared with plasma free of microbial contamination.
7 Such pooling should be performed in an environment suitable to prevent microbial
8 contamination, like classified areas (class D [73]) and pools should be adequately identified.
9 The room should be designed to allow for appropriate cleaning and sanitization of all surfaces.
10 Individual or pooled plasma should be stored at 2–8 °C in a room dedicated for this purpose. To
11 ensure the prevention of microbial contamination of plasma, preservatives (phenol or cresols)8
12 can be added at a dose of less than 3 g/l at this stage and kept during storage of plasma. Care
13 should be taken to dilute the phenol or cresols with water or saline solution before they are
14 added to plasma, to avoid denaturation of plasma proteins. The transportation of containers or
15 bottles containing pooled plasma within the production facility or between facilities should be
16 performed in such a way that contamination is avoided and the cold chain is maintained.
17 To avoid the risk of contamination, it is recommended that individual or pooled plasma is not
18 stored for too long before fractionation, i.e. the plasma should be fractionated as soon as
19 possible after pooling. In the event that plasma is stored for prolonged periods of time (for
20 instance 6 months), the storage time and conditions should be validated to ensure that there is
21 no detrimental impact on the quality of the plasma material, on the fractionation process, or on
22 the quality, efficacy and stability of the antivenoms.
23 It is also the experience of some manufacturers that plasma can be stored frozen at −20 °C,
24 particularly if no preservative is added.
25 12.6 Control of plasma prior to fractionation

26 Before fractionation, pools of plasma should be checked for macroscopically evident
27 precipitates, gross haemolysis and bacterial contamination (bioburden assay). The neutralizing
28 potency of the starting plasma should be ensured so that the resulting antivenoms will be within
29 potency specifications. Additional checks may include, when relevant, a test for pyrogenic
30 substances and total protein content.
31 Plasma pools should be discarded if the bioburden exceeds a defined limit stated in the
32 marketing dossier or if the neutralizing potency is below a minimum limit established by the
33 producer. Cloudy plasma, below this defined bioburden limit, may still be used for fractionation
34 provided the fractionation process and product quality has been proven not to be impaired.
35 Grossly haemolysed plasma should not be used for fractionation.

37  When animals have developed an adequate immune response against venoms, and if
38 they are in good health, they can be bled for antivenom production. Bleeding should
39 be performed in enclosed rooms which should be kept scrupulously clean.
40 Traceability of the donations should be ensured.
41  Plasma is preferred to serum as a source material. Animals should be bled from the
42 external jugular vein. Plasma can be obtained either from whole blood or by
43 automated plasmapheresis and using approved anticoagulants. Blood or plasma
44 should ideally be collected into closed plastic bags. When this is not possible, glass
45 or plastic bottles can be used, if they can be readily cleaned and sterilized.
46  Plasmapheresis is recommended using either automatic or manual procedures. When
47 manual apheresis is used, blood cells should be sedimented, separated from the
8
In these guidelines, cresol isomers are referred to as cresols.
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1 plasma, resuspended in saline solution and returned to the animals within 24 to 72
2 hours. Plasma separation should be performed in a designated room with a controlled
3 environment.
4  Plasma containers should be thoroughly cleaned on their external surfaces,
5 adequately identified and stored in refrigerated rooms for further fractionation.
6  Plasma should be checked prior to fractionation to establish compliance with relevant
7 acceptance criteria for fractionation, in particular the neutralizing potency and lack of
8 bacterial contamination.
9  Special attention should be paid to ensuring traceability between individual animal
10 donors and the plasma pool.
11  A certificate from a veterinarian or other qualified person should be issued stating
12 that the donor animals were checked periodically to ensure that they were in good
13 health at the time of plasma collection and during the follow-up observation period.
14
15
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1 13 Purification of immunoglobulins and immunoglobulin fragments in the
2 production of
3 13.1 Good manufacturing practices
4 The purification of immunoglobulins and immunoglobulin fragments for the production of
5 antivenoms should aim at obtaining products of consistent quality, safety and efficacy. The
6 fractionation processes used should adhere to the GMP principles developed for medicinal
7 products. All operations should therefore be carried out in accordance with an appropriate
8 system of quality assurance and GMP. This covers all stages leading to the finished
9 antivenoms, including the production of water, the production of plasma (animal selection and
10 health control, production of venoms and immunization protocols, containers used for blood and
11 plasma collection, anticoagulant solutions and quality control methods) and the purification,
12 storage, transport, processing, quality control and delivery of the finished product. Of particular
13 relevance is the control of microbiological risks, contamination with particulates and pyrogens,
14 and the existence of a documentation system that ensures the traceability of all production
15 steps. To establish satisfactory traceability of the antivenom produced, all the steps of the
16 purification procedure used for the preparation of the antivenom batch should be recorded
17 carefully in pre-established and approved batch record documents, and sampling should be
18 done at established critical steps for in-process quality control tests.
19 WHO Guidelines on good manufacturing practices for medicinal products are available [73] and
20 the main principles of GMP for the manufacture of blood plasma products of human origin have
21 also been published [74, 75]. These Guidelines can serve as a general guide for manufacturing
22 practices in the production of antivenoms. A useful reference in the field of antivenoms is also
23 the Note for guidance on production and quality control of animal immunoglobulins and
24 immunosera for human use (CPMP/BWP/3354/99) [76].
25 13.2 Purification of the active substance

26 Antivenoms are prepared from the starting plasma pool using diverse methods to obtain one of
27 the following active substances:
28  intact IgG molecules;
29  F(ab')2 fragments; or
30  Fab fragments.
31 In general, fractionation procedures should not impair the neutralizing activity of antibodies; they
32 should yield a product of acceptable physicochemical characteristics and purity with a low
33 content of protein aggregates, which is non-pyrogenic and which should provide good recovery
34 of antibody activity. If possible, the process should be simple (with few steps) and economical.
35 The characteristics of a batch of plasma to be fractionated should be clearly established. The
36 methods used to purify the active substance and the in-process controls should be described in
37 detail in standard operating procedures (SOP). In the following sections, examples of basic
38 protocols used for the production of IgG, F(ab')2 and Fab antivenoms are described. Some
39 additional methodologies introduced to further purify the active substance of antivenoms are
40 also discussed. Variations in these manufacturing procedures have often been developed by
41 individual fractionators and should be considered as acceptable when shown to yield
42 consistently safe and effective preparations of antivenoms.
43 13.2.1 Purification of intact IgG antivenoms
44 13.2.1.1 Ammonium sulfate precipitation
45 In the past, most laboratories that produced whole IgG antivenoms have used fractionation
46 protocols based on salting-out procedures employing ammonium sulfate or sodium sulfate [77].
47 Two precipitation steps are included using two different salt concentrations in addition to the
48 elimination of “euglobulins” by precipitation in a diluted acidic solution.
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1 Such fractionation protocols generally lead to a recovery of antibodies of between 40 and 50%
2 and to the formation of protein aggregates. The final product of this procedure used to contain a
3 relatively high proportion of contaminating proteins, such as albumin [78]. This compromised the
4 safety of the product, since a high incidence of early adverse reactions has been described in
5 response to such intact IgG antivenoms [79].
6 13.2.1.2 Caprylic acid precipitation
7 The use of caprylic acid (octanoic acid) as an agent for precipitating proteins from animal
8 plasma has been described in the literature [80]. Several procedures for the purification of
9 whole IgG antivenoms with a good physicochemical profile and purity using caprylic acid
10 precipitation of non-immunoglobulin proteins have been developed [78, 81, 82] and are now
11 used for the production of licensed antivenoms.
12 Figure 3 illustrates a particular process in which caprylic acid is added slowly to undiluted
13 plasma, with constant stirring, to reach a concentration of 5% (v/v) and pH 5.5. The mixture is
14 stirred at 22–25 °C for a minimum of 1 hour. The precipitated proteins are removed by filtration
15 or centrifugation and discarded. The filtrate or the supernatant containing the immunoglobulins
16 is then submitted to tangential flow filtration to remove residual caprylic acid and low-molecular-
17 mass proteins, depending on the molecular cut-off of the ultrafiltration membranes, and to
18 concentrate the proteins. The immunoglobulin solution is then formulated by adding sodium
19 chloride solution (NaCl), an antimicrobial agent and any other excipient(s) needed, such as
20 stabilizers. The pH is then adjusted to a neutral value and finally subjected to sterile filtration
21 through a filter of pore size 0.22 µm, and dispensed into final containers (vials or ampoules).
22 Variations of this procedure have been introduced by various manufacturers, and include
23 dilution of plasma, changes in caprylic acid concentration, pH, and temperature among others.
24 Caprylic acid fractionation allows the production of antivenoms of relatively high purity and with
25 a low protein aggregate content, because the immunoglobulins are not precipitated during the
26 process. The yield may reach up to 60–75% of the activity in the starting plasma, depending
27 upon the details of the procedure and/or the equipment used. The efficacy and safety profiles of
28 caprylic acid-fractionated antivenom immunoglobulins have been demonstrated in clinical trials
29 [79, 83, 84].
30 13.2.2 Purification of F(ab')2 antivenoms
31 Many manufacturers follow the classical protocol for F(ab')2 antivenom production developed by
32 Pope [6, 7], with a number of recent modifications [9, 10, 85].
33 The method of pepsin digestion (see Figure 4) involves the digestion of horse plasma proteins
34 by pepsin, leading to the degradation of many non-IgG proteins, and to the cleavage of IgG into
35 bivalent F(ab')2 fragments by removal and digestion of the Fc fragment into small peptides. A
36 heating step and the purification of F(ab')2 fragments by salting-out using ammonium sulfate are
37 also key elements of this methodology. Some procedures involve performing the pepsin
38 digestion step on a pre-purified IgG fraction that is obtained by treatment of plasma with
39 ammonium sulfate to obtain an IgG-enriched precipitate, whereas albumin is not precipitated.
40 Pepsin digestion is accomplished at a pH of 3.0–3.5. A typical protocol is based on incubation at
41 pH 3.3 for 1 hour, at 30–37 °C in a jacketed tank, with a pepsin concentration of 1.0 g/l. Other
42 procedures can be used which give similar results. Each manufacturer should adjust the pepsin
43 concentration to achieve the required enzymatic activity.
44 13.2.2.1 Downstream processing using ammonium sulfate
45 After pepsin digestion, the pH is adjusted to 4.5–5.0, by adding NaOH or a weak alkaline buffer;
46 then ammonium sulfate is added with stirring to a final concentration usually close to 12% (w:v).
47 The precipitate is eliminated by filtration or centrifugation, and the filtrate, or supernatant, is
48 heat-treated (usually at 56 °C for 1 hour; this is known as “thermocoagulation”). After
49 thermocoagulation, the preparation is cooled down to less than 30 °C, e.g. by passing cold
50 water through a jacketed vessel. The resulting fraction is filtered or centrifuged to remove the
51 precipitate. The pH is then adjusted to 7.0–7.2 with NaOH, and a solution of ammonium sulfate
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1 is added with stirring to a final concentration high enough to precipitate the F(ab') 2 fragments
2 (usually 23% (w:v) or higher). After an additional filtration step, or following centrifugation, the
3 F(ab')2 precipitate is dissolved, and then desalted (to remove the ammonium sulfate) and
4 concentrated preferentially by tangential flow diafiltration. Care should be taken to avoid
5 aggregate formation by ensuring gentle mixing and rapid dissolving of the precipitate.
6 Alternatively, the 23% (w:v) step is bypassed by some manufacturers and, directly after the
7 heating step, the filtrate obtained is subjected to ultrafiltration. Additional precipitation could also
8 be applied on the starting material at a low ionic strength and acid pH to remove "euglobulins"
9 (10).
10 The F(ab')2 solution is then formulated by adding NaCl, an antimicrobial agent, and any other
11 excipient needed for formulation, such as protein stabilizers, and the pH is adjusted, generally
12 to a neutral value. Finally, the preparation is sterilized by filtration through 0.22-µm filters, and
13 dispensed into final containers (vials or ampoules). Such a process, or similar ones developed
14 by other manufacturers, using pepsin digestion, ammonium sulfate precipitation and tangential
15 diafiltration, is the most often used for the manufacture of F(ab') 2 fragments. The yield of this
16 fractionation protocol usually ranges between 30% and 40%.
17 13.2.2.2 Downstream processing using caprylic acid
18 Purification of F(ab')2 has also been shown, on an experimental scale, to be achievable by
19 caprylic acid precipitation of non-F(ab')2 proteins after pepsin digestion, with an improved yield
20 (60%) [86]. However, the yield obtained on a large scale has not been reported. Figure 5
21 shows a fractionation scheme of F(ab')2 using caprylic acid. F(ab')2 is not precipitated, therefore
22 reducing the formation of aggregates. Some manufacturers have introduced additional
23 processing steps such as ion-exchange chromatography or ultrafiltration to eliminate low-
24 molecular-mass contaminants.
25 13.2.3 Purification of Fab antivenoms
26 Production of monovalent Fab fragments is performed by some manufacturers [87], currently
27 using hyper-immunized sheep plasma. Papain is used to carry out the enzymatic digestion, and
28 the process of preparation of the fragment may use ammonium sulfate, sodium sulfate or
29 caprylic acid. Figure 6 shows a process in which immunoglobulins are precipitated from plasma
30 by adding ammonium sulfate or sodium sulfate to a concentration of 23%. After filtration the
31 filtrate is discarded and the immunoglobulin precipitate is dissolved in a sodium chloride solution
32 at pH 7.4. Papain is added and digestion performed at 37 °C for 18–20 hours in a jacketed tank.
33 Reaction is stopped by adding iodoacetamide. The product is then applied to a diafiltration
34 system to remove iodoacetamide, salts and low-molecular-mass peptides and equilibrated with
35 a buffered isotonic NaCl solution. The preparation is then chromatographed on an anion-
36 exchanger (usually in quaternary aminoethyl (QAE)-based or diethylaminoethyl (DEAE)-based
37 media). Fc fragments and other impurities are bound on the column, whereas Fab fragments
38 pass through. After an additional diafiltration/dialysis step, the product is formulated by adding
39 NaCl, antimicrobial agents (when used) and any other excipients needed, and the pH is
40 adjusted. Finally, the preparation is sterile-filtered and dispensed into the final containers.
41
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1
2 Figure 3
3 Example of a fractionation process in which intact IgG is prepared by caprylic acid
4 precipitation of non-immunoglobulin proteins
5
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1
2 Figure 4
3 Example of a fractionation process in which F(ab')2 fragments are prepared by pepsin
4 digestion and ammonium sulfate precipitation
5
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1
2 Figure 5
3 Example of a fractionation process in which F(ab')2 fragments are prepared by pepsin
4 digestion and caprylic acid precipitation
5
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1
2 Figure 6
3 Example of a fractionation process in which Fab fragments are prepared by papain
4 digestion and ammonium sulfate precipitation
5
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1 13.2.4 Optional additional steps used by some manufacturers
2 When performed following GMP and using validated fractionation protocols, the basic
3 methodologies described above for the manufacture of IgG, F(ab')2 and Fab antivenoms allow
4 the production of antivenoms of adequate purity, safety and efficacy. Nevertheless, some
5 manufacturers include additional steps to enhance product purity. The methodologies include
6 those described below.
7 13.2.4.1 Ion-exchange chromatography
8 Ion-exchange chromatography can be successfully used for antivenom purification based on
9 charge differential with the contaminants. Anion-exchange columns of DEAE or QAE gels or
10 membranes, such as quaternary ammonium cellulose microporous membranes, can be used at
11 neutral pH to adsorb protein contaminants [10, 85, 88]. Alternatively, cation-exchange columns,
12 e.g. carboxymethyl or sulfopropyl gels, have been used for purification of IgG or F(ab') 2
13 fragments [86]. The column is equilibrated at acid pH, e.g. pH 4.5, to bind the antivenom IgG or
14 its fragments, whereas protein contaminants are eluted in the break-through.
15 Chromatographic procedures should be applied following GMP. Columns should be adequately
16 regenerated, sanitized, and stored to prolong their useful lifetime. The reproducibility of columns
17 over cycles should be validated. Measures to avoid batch to batch contamination should be in
18 place. Specific standard operating procedures should be developed and followed.
19 13.2.4.2 Affinity chromatography
20 Affinity chromatography using either immobilized venom or other ligands can be designed to
21 bind immunoglobulins or their fragments [89]. However, columns usually deteriorate rather
22 rapidly, and meticulous care should be taken to wash, sanitize and store them under
23 appropriate conditions. Procedures should be followed to ensure that any substances leaching
24 from the columns do not affect the quality and safety of the product or else are completely
25 removed during downstream processing; this is especially critical in affinity chromatography
26 using immobilized venom. Affinity processes may affect recovery and high-affinity antibodies
27 may be lost and/or denatured owing to the harsh elution conditions needed to elute them from
28 the chromatographic material.
29 13.2.4.3 Process improvement
30 Some manufacturers have introduced process improvements to enhance the quality or the yield
31 of antivenoms. These include the use of a depth filtration system combined with filter-aids to
32 facilitate filtration steps and improve antivenom recovery. In addition, other manufacturing steps
33 may be introduced to ensure inactivation or removal of infectious agents (see section 14).
34 13.2.5 Formulation
35 During formulation of antivenoms after diafiltration steps one should consider the addition of
36 salts to adjust the osmolality, addition of preservatives, other excipients, if needed for protein
37 stability, and the adjustment of pH.
38 In general, antivenoms are formulated at neutral pH (pH 7.0 ± 0.5) although some
39 manufacturers are exploring the feasibility of formulation at more acidic pHs to improve stability
40 and/or to reduce aggregate formation.
41 Formulation at pH higher than 7.5 may not be recommended, since the stability of
42 immunoglobulins and their fragments at alkaline pH may be poor, and the formation of
43 aggregates may be favoured.
44 13.2.6 Analysis of bulk product before dispensing
45 The biological, physical and chemical characteristics of the final bulk product should meet pre-
46 established specifications before dispensing. Analysis may include tests required to
47 demonstrate:
48  the purity and potency of the product;
49  the sterility;
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1  the compliance with the specifications for the aggregate content;
2  the pyrogen limit and/or the bacterial endotoxin content; and
3  the formulation, i.e. the concentration of excipients and the pH.
4 When the product is a stored liquid, some of these tests (such as the potency assay) may not
5 need to be duplicated on the final container if the processing after the bulk preparation has been
6 validated and shown not to alter this activity.
7 The sterilization equipment and the integrity of the membrane should be guaranteed before
8 sterilization; moreover, the aseptic filling should be validated.
9 13.2.7 Dispensing and labelling of final product
10 Once compliance of the final bulk product with the quality control specifications is established,
11 the final product is bottled. For this, final glass containers (vials or ampoules) should be used.
12 General principles prevailing for the dispensing of parenteral medicinal products should be
13 applied. The dispensing should be performed in class A [73] clean room conditions, usually
14 under a laminar flow hood. The equipment used for dispensing should be calibrated beforehand
15 to ensure that the correct volume is delivered.
16 In the case of ampoules, the dispensing system should ensure an aseptic closure and the
17 sealing of the ampoule should prevent risk of protein denaturation due to heat. For vials,
18 insertion of rubber stoppers should be done inside this clean dispensing area. The quality of the
19 rubber stoppers should be such as to guarantee inertness and to prevent leaching. Thereafter,
20 aluminium seals should be placed on each vial in a clean area outside the class A area.
21 Ampoules or vials containing the final product should then be properly identified and stored in a
22 quarantine area maintained under proper storage conditions. Samples of the antivenoms should
23 be sent to the quality control laboratory for analysis.
24 When an antivenom complies with all the quality control tests established for the final product, it
25 should be properly labelled and identified.
26  The vial or ampoule should be labelled with, at least, the following information:
27 — name of the product and of the producer;
28 — animal species used to produce the antivenom;
29 — batch number;
30 — pharmaceutical presentation (liquid or freeze-dried);
31 — volume content;
32 — administration route;
33 — specificity (venoms neutralized by the antivenom, including both the common and the
34 scientific name of the snake(s)9);
35 — neutralizing potency;
36 — storage conditions; and
37 — expiry date.
38 Additional information may be requested by the national regulatory authorities.
39  The package, which is usually a cardboard box, in which the vials or ampoules are packed,
40 should include the same information as is given on the primary container.
41  The package insert should include all the information relating to the product, as established
42 by national regulatory agencies, including:
43 — the neutralizing potency;
44 — the recommended dosage;
45 — reconstitution procedure, if lyophilized;
46 — the mode of administration (e.g. the dilution of antivenom in a carrier fluid such as
47 saline);
9
Special care should be taken considering the frequent changes in snake species taxonomy.
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1 — the rate of administration;
2 — details on the symptoms and treatment of early and delayed adverse reactions;
3 — snake species against which the antivenom is effective;
4 — recommended storage conditions, and
5 — an indication that the product is for single use.
6 13.2.8 Use of preservatives
7 The addition of preservatives to prevent bacterial and fungal contamination should be kept to a
8 minimum during plasma storage and during fractionation. Their inclusion during the
9 manufacturing process should be clearly justified, and should never substitute for any aspect of
10 GMP. Preservatives can be considered in the final product, especially if it is manufactured in
11 liquid form, and most specifically preservatives are required for multiple-dose presentations.
12 Antimicrobial agents currently used in antivenom formulation include phenol and cresols. In
13 general, phenol concentration is adjusted to 2.5 g/l, and concentration of cresols should be less
14 than 3.5 g/l. The concentration of preservatives should be validated by each production
15 laboratory on the basis of assays to test their efficacy and keeping in mind that they may
16 degrade with time and cease to be effective. It is necessary to ascertain that any agent used
17 has no potential detrimental interaction with the active substance and excipients of antivenoms.
18 Any change in the formulation involving preservatives, or the elimination of preservatives from
19 the final product, requires a very careful risk–benefit assessment on various microbial safety
20 aspects, as well as a detailed validation procedure. Mercury-containing preservatives are not
21 recommended in antivenom manufacture. The volume of antivenom required for the treatment
22 of envenoming (in excess of 50 ml) might lead to an exposure to mercury far higher than the
23 amounts currently used for other biological preparations and the toxic levels at which they are
24 toxic, especially in young children, are not known [90, 91].
25 13.2.9 Freeze-drying
26 Antivenoms are available either as liquid or as freeze-dried preparations. Freeze-dried
27 antivenoms, which may usually be stored at a temperature not exceeding 25°C, are generally
28 distributed to markets where the cold chain cannot be guaranteed, such as in many tropical
29 regions of the world. The absence of guarantee of a cold chain during distribution highlights the
30 need for manufacturers to demonstrate the stability of the antivenoms under the high
31 temperatures found in tropical climates.
32 Freeze-drying is a critical operation. Careful attention should be given to the rate of freezing as
33 well as to the protocol used for the primary and secondary drying cycles [92]. The details of the
34 freeze-drying protocols are product-specific and should be adjusted according to the particular
35 formulation of each antivenom. Inadequate freeze-drying protocols may affect the
36 physicochemical quality of the product, inducing protein precipitation and denaturation, as well
37 as aggregate formation, and altering stability and reconstitution. Specific stabilizers, such as
38 sugars or polyols, aimed at protecting proteins from denaturation and aggregation, may be
39 added to the final formulation of the antivenom [93]. Bulking agents, frequently used for some
40 biological products, are generally not required in the case of antivenoms owing to their relatively
41 high protein concentration; however they are sometimes used for high-titre monospecific
42 antivenoms.
43 13.2.10 Inspection of final container
44 All the vials or ampoules of each batch of liquid antivenoms should be inspected, either visually,
45 or using a mechanical device. Any vial or ampoule presenting turbidity, abnormal coloration,
46 presence of particulate matter, or defects of the vial, stopper, or capsule should be discarded. In
47 the case of freeze-dried products, a representative sample of the whole batch should be
48 dissolved in the solvent and inspected as described. Turbidity can be assessed quantitatively by
49 using a turbidimeter.
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1 13.2.11 Archive samples of antivenoms
2 In compliance with GMP, manufacturing laboratories should archive a number of vials of each
3 antivenom batch, under the recommended storage conditions, in an amount that would enable
4 the repetition of all quality control tests, when required.
5 13.3 Pharmacokinetic and pharmacodynamic properties of IgG, F(ab')2

6 and Fab
7 Owing to their different molecular mass, the pharmacokinetics of heterologous IgG molecules
8 (approximately 150 kDa) and F(ab')2 (approximately 100 kDa) and Fab (approximately 50 kDa)
9 fragments differ significantly. In envenomed patients, Fab fragments have the largest volume of
10 distribution and readily reach extravascular compartments. Fab fragments are, however, rapidly
11 eliminated, mainly by renal excretion, thus having a short elimination half-life (from 4–24 hours)
12 [94, 95]. In contrast, F(ab')2 fragments and intact IgG molecules are not eliminated by the renal
13 route and therefore have a more prolonged elimination half-life (between 2 and 4 days) [11, 96,
14 97]. Such different pharmacokinetic profiles have important pharmacodynamic implications, and
15 the selection of the ideal type of active substance in an antivenom should rely on a careful
16 analysis of the venom toxicokinetics and antivenom pharmacokinetics.
17 Another difference between low-molecular-mass fragments, such as Fab and those with a
18 higher molecular mass, such as F(ab')2 and IgG, is the number of paratopes of each molecule:
19 Fab has one antigen binding site while IgG and F(ab')2 each have two binding sites. Thus they
20 will be able to form large and stable complexes or precipitates with antigens carrying several
21 epitopes, while the former will form small, reversible non-precipitable complexes.
22 Ideally, the volume of distribution of an antivenom should be as similar as possible to the
23 volume of distribution of the main toxins in a particular venom; however, this is rarely the case.
24 In venoms composed of low-molecular-mass toxins, such as some elapid snake venoms, low-
25 molecular-mass neurotoxins are rapidly absorbed into the bloodstream and are rapidly
26 distributed to the extravascular spaces where toxin targets are located. Furthermore, low-
27 molecular-mass toxins are eliminated from the body in a few hours. In these cases, an
28 antivenom of high distribution volume that readily reaches extravascular spaces, such as Fab,
29 might be convenient, although its action is then eliminated within a few hours. It should be
30 noted, however, that a number of elapid venoms contain some high-molecular-mass toxins of
31 great clinical significance, such as procoagulants and pre-synaptic phospholipase A2
32 neurotoxins.
33 In contrast, in the case of viperid snake venoms and other venoms made up of toxins of larger
34 molecular mass, including a number of elapid venoms, many of which act intravascularly to
35 provoke bleeding and coagulopathy, the situation is different. The time required for toxins to
36 distribute to extravascular spaces is longer than in the case of low-molecular-mass neurotoxins,
37 and the targets of some of these toxins are present in the vascular compartment. In addition,
38 the toxins of viperid venoms have a long half-life in vivo and can remain in the body for several
39 days [98, 99]. In this case, an antivenom made by Fab fragments neutralizes the toxins that
40 reach the circulation but, after a certain time has elapsed, the Fab fragments are eliminated and
41 non-neutralized toxins reach the circulation, thus giving rise to the well-known phenomenon of
42 recurrent envenoming, i.e. the reappearance of signs and symptoms of envenoming at later
43 time intervals after an initial control of envenoming. This situation demands repeated
44 administration of antivenom to maintain therapeutic levels of Fab in the circulation [100].
45 Therefore, in such envenomings, antivenoms made of IgG or F(ab') 2 may be more appropriate
46 because of their longer elimination half-lives. Moreover, it has been proposed that formation of
47 venom–antivenom complexes in the circulation results in the redistribution of venom
48 components from the extravascular space to the blood compartment, where they are bound and
49 neutralized by circulating antivenom, provided that the dose of antivenom is sufficient [101,
50 102]. Consequently, the maintenance of a high concentration of specific antivenom antibodies in
51 the circulation for many hours is required for complete neutralization of toxins reaching the
52 bloodstream during both early and late phases of envenoming (redistribution of toxins) present
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1 in the extravascular space. In conclusion, IgG and F(ab')2 antivenoms have a pharmacokinetic
2 profile that makes them more effective in many types of snakebite envenoming.
3
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2  Antivenoms should be manufactured using fractionation procedures that are well
3 established, validated, and shown to yield products with proven safety and efficacy.
4 Fractionation processes used for the manufacture of antivenoms should adhere to
5 the principles of GMP for parenteral medicinal products.
6  Antivenoms can be composed of intact IgG molecule, F(ab')2 fragments or Fab
7 fragments. Intact IgG antivenoms are mainly produced by caprylic acid precipitation
8 of non-IgG plasma proteins, leaving a highly purified IgG preparation in the
9 supernatant or filtrate.
10  F(ab')2 fragment antivenoms are produced by pepsin digestion of plasma proteins, at
11 acidic pH, usually followed by F(ab')2 purification by salting out with ammonium
12 sulfate solutions or by caprylic acid precipitation. Fab monovalent fragments are
13 obtained by papain digestion of IgG at neutral pH.
14  Further to ultrafiltration to remove low-molecular-mass contaminants, preparations
15 are formulated, sterilized by filtration and dispensed in the final containers.
16 Formulations of antivenoms may include preservative agents. Additional steps, such
17 as chromatography, can be added to the fractionation protocols to enhance purity.
18  Antivenoms can be presented as liquid or freeze-dried preparations. Freeze-drying of
19 antivenoms should be performed in conditions that ensure no denaturation of
20 proteins and no formation of protein aggregates.
21  IgG, F(ab')2 and Fab antivenoms exhibit different pharmacokinetic profiles: Fab
22 fragments have a larger distribution volume and a much shorter elimination half-life.
23 Thus, for viperid envenomings, IgG or F(ab')2 antivenoms have a more suitable
24 pharmacokinetic profile, whereas Fab fragments may be useful for the neutralization
25 of venoms rich in low-molecular-mass neurotoxins which are rapidly distributed to
26 the tissues. However, in general terms, IgG and F(ab')2 antivenoms have shown a
27 better pharmacokinetic profile than Fab antivenoms.
28
29
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1 14 Control of infectious risks
2 14.1 Background
3 The viral safety of any biological product results from a combination of measures to ensure a
4 minimal risk of viral contamination in the starting raw material (plasma), together with steps to
5 inactivate or remove potential contaminating viruses during processing.
6 There are currently several recognized complementary approaches used for virus risk reduction
7 for biological products. These are:
8  minimizing the potential initial virus content by implementing a quality system for the
9 production of the raw material;
10  contribution of the manufacturing processes to inactivating and/or removing residual viruses
11 during manufacture of the biological product; such a contribution can be inherent to the
12 existing production technology or may result from the introduction of dedicated viral
13 reduction steps;
14  adhesion to GMP at all steps of the manufacturing process;
15  appropriate and timely response to any infectious events recognized during the clinical use
16 of the product.
17 Production steps to inactivate and/or remove viruses have long been shown to play a powerful
18 role in ensuring safety of biologicals [74]. Similarly, keeping to a minimum the potential viral load
19 at the stage of the plasma pool, through appropriate epidemiological surveillance and health
20 control of the donor animals, is also an important safety measure (see section 10).
21 Based on experience with human plasma products, a production process for antivenoms that
22 includes two robust steps for viral reduction (comprising preferably at least one viral inactivation
23 step) should provide a satisfactory level of viral safety. However, it should be kept in mind that
24 non-enveloped viruses are more difficult to inactivate or remove than lipid-enveloped viruses.
25 14.2 Risk of viral contamination of the starting plasma

26 The main structural characteristics of viruses reported to possibly infect horses, sheep and
27 goats are presented in Tables 7 and 8. They include viruses with a DNA or RNA genome, with
28 and without a lipid envelope, and varying widely in size (22 to 300 nm).
29 A few of these viruses have been identified as possibly present, at least at some stages of the
30 infection cycle, in the blood, or are considered as being pathogenic to humans. Special attention
31 should be paid to these viruses.
32 14.3 Viral validation of manufacturing processes

33 Understanding how much a manufacturing process may contribute to the viral safety of
34 antivenoms is fundamental to both manufacturers and regulators. Such an understanding can
35 only be achieved by viral validation studies. These studies are complex and require well-
36 established virology laboratory infrastructure and cell culture methodologies. They are usually
37 carried out by specialized laboratories, outside the manufacturing facilities. The principles
38 guiding such studies have been described in WHO Guidelines [74] and are summarized below.
39
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1 Table 7
2 Viruses identified in horses [76, 103]
Virus Family Size Genome Presence Classified as
(nm) in blood pathogenic
a
reported to humans
[76]
Lipid-enveloped viruses
b
Borna virus Bornaviridae 70–130 ss-RNA Yes
Equine arteritis virus Arteriviridae 50–60 ss-RNA
Equine encephalitis virus, Togaviridae 40–70 ss-RNA Yes
Eastern and Western
Equine coronavirus Coronaviridae 75–160 ss-RNA
Equine foamy virus Retroviridae 80–100 ss-RNA Yes
Equine herpes virus 1–5 Herpesviridae 125–150 ds-DNA Yes
Equine infectious Lentiviridae 80–100 ss-RNA Yes
anaemia virus
Equine influenza virus Orthomyxoviridae 80–120 ss-RNA Yes
Equine morbillivirus Paramyxoviridae 150 ss-RNA Yes
(Hendra virus)
Japanese encephalitis Flaviviridae 40–70 ss-RNA Yes
virus
Equine hepacivirus Flaviviridae 40-70 ss-RNA Yes [104]
Equine pegivirus Flaviviridae 40-70 ss-RNA Yes [105]
Nipah virus Paramyxoviridae 150–300 ss-RNA Yes
Rabies virus Rhabdoviridae 75–180 ss-RNA Yes
Salem virus Paramyxoviridae 150–300 ss-RNA
St Louis encephalitis Flaviviridae 40–70 ss-RNA Yes
virus
Theiler’s disease- Flaviviridae 40-70 ss-RNA No [104]
associated virus
Tick-borne encephalitis Flaviviridae 40-70 ss-RNA Yes
virus [106, 107]
Venezuelan equine Togaviridae 40–70 ss-RNA Yes Yes
encephalitis virus
Vesicular stomatitis virus Rhabdoviridae 50–80 ss-RNA Yes Yes
West Nile virus Flaviviridae 40–70 ss-RNA Yes Yes
Non-lipid enveloped viruses
Equine encephalosis Reoviridae 80 ds-RNA
viruses
Equine rhinitis A and B Picornaviridae 22–30 ss-RNA
viruses
Equine rotavirus Reoviridae 60–80 ds-RNA
a
3 Absence of a report does not mean that the virus may not be found in the blood at certain
4 stages of the cycle of infection.
b
5 Recent studies have suggested that Borna virus is non-pathogenic to humans [108].
6
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1 Table 8
2 Viruses identified in sheep and goats [76]
Virus Family Size (nm) Genome Reported Classified as
presence pathogenic to
a
in blood humans [76]
Lipid-enveloped viruses
Adenovirus Adenoviridae 80–110 ds-DNA
Akabane virus Bunyaviridae 80–120 ss-RNA
Bluetongue virus Reoviridae 80 ds-RNA Yes
Border disease virus Flaviviridae 40–70 ss-RNA
b
Borna virus Bornaviridae 70–130 ss-RNA Yes
Bovine herpes virus types Herpesviridae 120–200 ds-DNA
1, 2, 4
Bovine viral diarrhoea virus Togaviridae 40–60 ss-RNA
Loiping ill virus Flaviviridae 40–50 ss-RNA Yes
Nairobi sheep disease Bunyaviridae 80–120 ss-RNA
Ovine/bovine Papillomaviridae 40–55 ds-DNA
papillomavirus
Ovine herpes virus 2 Herpesviridae 120–200 ds-DNA
Parainfluenza virus type 3 Paramyxoviridae 150–300 ss-RNA Yes
Peste des petits ruminants Paramyxoviridae 150–300 ss-RNA
(Morbillivirus)
Poxviruses (Parapox, Poxviridae 140–260 ds-DNA Yes
Capripox,
Cowpox)
Respiratory syncytial virus Paramyxoviridae 150–300 ss-RNA
Retroviruses (Caprine 80–100 ss-RNA
arthritis encephalitis virus, Retroviridae
Maedi-Visna virus,
Jaagsiekte virus, Bovine
leukaemia virus
Rift Valley fever complex Bunyaviridae 80–120 ss-RNA Yes
Ross river virus Togaviridae 70 ss-RNA
Rotavirus Reoviridae 80 ds-RNA
Tick-borne encephalitis Flaviviridae 40–50 ss-RNA Yes
virus
Vesicular stomatitis virus Rhabdoviridae 50–380 ss-RNA Yes Yes
Wesselbron virus Flaviviridae 40–50 ss-RNA Yes
Non-lipid enveloped viruses
Epizootic haemorrhagic Reoviridae 80 ds-RNA
disease virus
Foot and mouth disease Picornaviridae 27–30 ss-RNA Yes
virus
Reovirus 1-3 Reoviridae 60–80 ds-RNA
a
3 Absence of report does not mean that the virus may not be found in the blood at certain stages
4 of the cycle of infection.
b
5 Recent studies have suggested that Borna virus is non-pathogenic to humans [108].
6
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1 14.3.1 Down-scale experiments
2 The contribution of manufacturing processes towards inactivation and/or removal of potential
3 viral contamination should be demonstrated. For this purpose, viral validation studies should be
4 performed using at least three viruses exhibiting different structural characteristics. The
5 antivenom manufacturer should first identify the steps that, based on the existing literature, are
6 likely to remove or inactivate viruses and, then, provide evidence and quantitative assessment
7 of the extent of virus reduction achieved for the specific process evaluated.
8 Validation should be done by down-scale experiments. The accuracy of the down-scale process
9 should be assessed by comparing the characteristics of the starting intermediate and the
10 fraction resulting from that step, for both the laboratory and the production scales. Selected
11 physical factors (e.g. temperature, stirring or filtration conditions) and chemical factors (e.g. pH
12 or concentration of precipitating agents such as caprylic acid) should be as close as possible to
13 those used at manufacturing scale.
14 Once the step is accurately modelled, the antivenom fraction derived from the fractionation
15 process just prior to the step being evaluated (e.g. the starting plasma to be subjected to a low
16 pH treatment, or to caprylic acid precipitation, or a F(ab')2 fragment fraction to be subjected to
17 an ammonium sulfate-heat treatment) should be spiked with one of the model viruses selected.
18 Viral infectivity, most often determined using cell culture assays (less frequently animal models),
19 should be quantified before (e.g. prior to pH adjustment and addition of pepsin) and immediately
20 after (e.g. following low pH adjustment and incubation at that pH for a known period of time in
21 the presence of pepsin) the steps evaluated to determine the viral clearance achieved. The
22 results are conventionally expressed as logarithm (log) of the reduction in infectivity that is
23 observed. Total infectivity or viral load is calculated as the infectious titre (infectious units per
24 ml) multiplied by the volume. For a viral inactivation step, it is highly recommended that the
25 kinetics of the virus kill be evaluated. Such inactivation kinetics of the infectivity provides an
26 important indication of the virucidal potential of the step and enables comparison of the data
27 obtained to those from published studies.
28 Typically, a viral reduction of 4 logs or more is considered to represent an effective and reliable
29 viral safety step.
30 Establishing the relative insensitivity of a manufacturing step to changes or deviatons in process
31 conditions is also important to evaluate its robustness, in addition to adding to the level of
32 understanding of its contribution to the overall viral safety of the preparation. This can be
33 achieved by validating the same step using a range of conditions deviating from those used in
34 production (such as an upper pH limit applied to a pepsin digestion or to a caprylic acid
35 precipitation step).
36 Virus validation studies are subject to a number of limitations [74], which should be considered
37 when interpreting the results.
38 14.3.2 Selection of viruses for the validation of antivenom production processes
39 Viruses selected for viral validation studies should resemble as closely as possible those which
40 may be present in the starting animal plasma material (Tables 7 and 8). When possible, viruses
41 known to potentially contaminate animal plasma (called “relevant viruses”) should be used.
42 Table 9 gives examples of a few viruses that have been used for the validation of animal-
43 derived immunoglobulins. Vesicular stomatitis virus (VSV) and West Nile virus (WNV) are
44 relevant lipid-enveloped horse plasma-borne viruses. Bovine viral diarrhoea virus (BVDV), a
45 lipid-enveloped flavivirus, can be used as a model for West Nile virus (WNV), tick-born
46 encephalitis virus, and for the Eastern, Western, and Venezuelan equine encephalitis
47 togaviruses. Pseudorabies virus (PRV) is a lipid-enveloped virus that can serve as a model for
48 pathogenic equine herpesvirus. Encephalomyocarditis virus (EMCV), a picornavirus, can serve
49 as a model for non-lipid-enveloped viruses. Porcine parvovirus can also be selected as a model
50 for small resistant non-lipid-enveloped viruses or as a relevant virus when pepsin of porcine
51 origin is used in the manufacture of F(ab')2 fragments.
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1 This list is not exhaustive and other model viruses can be used for validation studies of animal-
2 derived antivenoms, in particular taking into account the characteristics of the viruses that may
3 be present in the animal species used to generate antivenoms.
4 Table 9
5 Examples of laboratory model viruses that can be used for validation studies of horse-
6 derived antivenoms
Virus Family Lipid- Size Genom Resistanc Model for
enveloped (nm) e e
Animal Parvoviridae No 18–26 ss-DNA High Relevant virus
Parvovirus (e.g. (when pepsin of
porcine) porcine origin is
used)
Bovine virus Togaviridae Yes 40–60 ss-RNA Low Eastern, Western
Diarrhoea virus and
Venezuelan equine
encephalitis virus,
Tick-borne
encephalitis virus
Parainfluenza Paramyxoviridae Yes 100–200 ss-RNA Low Hendra virus;
virus Nipah virus; Salem
virus
Poliovirus; Picornaviridae No 25–30 ss-RNA Medium- Equine rotavirus
Encephalomyoca high
rditis virus;
Hepatitis A virus
Pseudorabies Herpes Yes 100–200 ds-DNA Medium Equine herpes
virus virus
Reovirus type 3 Reoviridae No 60–80 ds-RNA Medium Equine
encephalosis virus
Sindbis virus Togaviridae Yes 60–70 ss-RNA Low Eastern, Western
and
Venezuelan equine
encephalitis virus
Vesicular Rhabdoviridae Yes 50–200 ss-RNA Low Relevant virus
stomatitis virus
West Nile virus Flaviridae Yes 40–70 ss-RNA Low Relevant virus and
model for
Eastern equine
encephalitis
Virus
7
8 14.4 Viral validation studies of antivenom immunoglobulins

9 There is no documented case of transmission of zoonotic infections, including viral diseases, by
10 antivenom immunoglobulins, or any other animal-derived immunoglobulins. Absence of reports
11 of viral transmission may result from a lack of long-term surveillance of the patients receiving
12 antivenoms. Alternatively, this may reveal that current processes for the manufacturing of
13 antivenoms include processing steps that contribute to the viral safety.
14 Among the various processing steps used in the production of antivenoms, caprylic acid and
15 low pH treatments are known to contribute to safety against lipid-enveloped viruses. This
16 information is based on well-established experience in the fractionation of human plasma with a
17 production step comprising caprylic acid [109-111] or low pH treatment [74, 112-114].
18 Although information is still limited, there is now growing evidence that similar steps used in the
19 production of animal derived immunoglobulins may also inactivate or remove viruses. In
20 addition, some manufacturers have implemented dedicated viral reduction procedures.
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1 14.4.1 Caprylic acid treatment
2 The conditions used for caprylic acid treatment of antivenoms [78, 103] and of human
3 immunoglobulins [109-111] are similar, in particular the pH range, duration of treatment,
4 temperature, and the caprylic acid/protein ratio, as summarized in Table 10.
5 Table 10
6 Comparison of conditions for caprylic acid treatment used for human immunoglobulin
7 preparations and antivenoms [103]
Product Protein Caprilic acid pH Temperature Duration
concentration (g/l) (g/kg solution) (°C) (hr)
Human IgG 35 7.45 5.5 22 1
Human IgM- 43 15 4.8 20 1
enriched
Human IgM 25 20 5.0 20 1
Antivenoms 60–90 50 5.5–5.8 18–22 1
8
9 14.4.1.1 Validation studies with human immunoglobulins
10 Unsaturated fatty acids, most specifically caprylic acid, have long been known to have the
11 capacity to inactivate lipid-enveloped viruses in human plasma protein fractions [115, 116]. The
12 non-ionized form of caprylic acid is thought to disrupt the lipid bilayer and membrane-associated
13 proteins of enveloped viruses. Utilizing the dissociation reaction and varying the concentration
14 of the ionized form of caprylate, a specific amount of the non-ionized form of caprylate can be
15 maintained over a wide pH range.
16 The robustness of a caprylic acid treatment applied to human immunoglobulin G (IgG), human
17 immunoglobulin M (IgM) and IgM-enriched preparations has been investigated using various
18 enveloped viruses (human immunodeficiency virus (HIV), BVDV, Sindbis virus and
19 Pseudorabies) [110]. Under the conditions applied during manufacture, caprylic acid leads to
20 robust inactivation of lipid-enveloped viruses; pH is a particularly critical parameter and should
21 be less than 6.
22 Another investigation studied the viral reduction achieved during treatment by caprilate of a
23 human IgG product [109]. At pH 5.1, at 23 °C, and in the presence of 9 mM caprylate, ≥ 4.7 and
24 ≥ 4.2 log of HIV and PRV, respectively, were inactivated during the 1-hour treatment, but only
25 1.5 log for BVDV was inactivated. At 12 mM caprylate, ≥ 4.4 log of BVDV were inactivated
26 within this time period. At pH 5.1, 24 °C, and 19 mM caprylate, and pH 5.1, 24 °C, and 12 mM
27 caprylate, complete inactivation of BVDV and of HIV and PRV was achieved in less than 3 min.
28 Treatment of cryoprecipitate-poor plasma with 5% caprylic acid/pH 5.5 at 31+/-0.5°C (a
29 condition closed to that used to prepare antivenoms) was shown to inactivate ≥ 5 log of HIV,
30 BVDV, and PRV in less than 5 min [117].
31 14.4.1.2 Validation studies with antivenom immunoglobulins
32 Virus inactivation studies have been carried out on a F(ab')2 fraction obtained from pepsin
33 digested plasma subjected to ammonium sulfate precipitation. The F(ab')2 fraction was
34 subjected to precipitation by drop-wise addition of caprylic acid to 0.5% (final concentration) and
35 the mixture was maintained under vigorous stirring for 1 hour at 18 °C. Rapid and complete
36 reduction of BVDV, PRV and VSV (> 6.6 log10, > 6.6 log10, and > 7.0 log10, respectively) was
37 observed. No significant reduction (0.7 log10) of the non-enveloped EMCV [116] was observed.
38 In another process used to prepare equine immunoglobulins, in which serum is thawed at 4 °C,
39 subjected to heating at 56° C for 90 min, brought to 20 ± 5 °C, adjusted to pH 5.5 and subjected
40 to 5% caprylic acid treatment for 1 hour, fast reduction of infectivity of > 4.32 and > 4.65 log10
41 was found for PRV and BVDV, respectively. The caprylic acid step was confirmed to have
42 limited impact on the infectivity of EMCV and Minute virus of mice (MVM) non-lipid enveloped
43 viruses [118]. Data suggest that significant reduction in the infectivity of lipid-enveloped viruses
44 can be obtained during caprylic acid treatment of antivenoms. The reduction of viral infectivity
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1 may result from both viral inactivation and partitioning during the precipitation step. No
2 significant inactivation of non-enveloped viruses is expected.
3 14.4.1.3 Recommended actions
4 Further studies of the viral reduction achieved during caprylic acid treatment of antivenoms are
5 recommended; in particular, robustness studies to define the impact on process variations
6 should also be performed.
7 14.4.2 Acid pH treatment
8 The conditions used for low pH treatment of equine derived antivenom immunoglobulins and of
9 human immunoglobulins are summarized in Table 11.
10 Table 11
11 Typical conditions for acid pH treatment of human IgG preparations and equine
12 antivenoms [103]
Product Protein pH Temperature (°C) Duration (hrs)
concentration (g/l)
Human IgG 40–60 4.0 30–37 20–30
Antivenoms 60–90 3.1–3.3 30–37 0.6–24
13 14.4.2.1 Validation studies with human immunoglobulins

14 Many studies have demonstrated that the low pH 4 treatment used in the manufacture of human
15 intravenous IgG has the capacity to inactivate lipid-enveloped viruses [112-114]. The rate and
16 extent of inactivation may differ depending upon the virus. Inactivation is temperature-
17 dependent, and is influenced by the formulation of the IgG solution. Pepsin is sometimes added
18 in traces (to reduce anticomplementary activity and content of aggregates) but, at this low
19 concentration, contributes little to virus kill [76]. Most non-lipid enveloped viruses are resistant to
20 acid pH treatment.
21 14.4.2.2 Virus inactivation studies performed with antivenom immunoglobulins
22 As described in section 13, peptic cleavage of horse plasma IgG at pH 3.0–3.3 for 60 min is a
23 common procedure for the preparation of F(ab')2. More than 4 logs of inactivation of WNV and
24 of Sindbis has been found when horse plasma was subjected to peptic digestion at pH 3.2 for
25 60 min [119]. WNV was very sensitive whether pepsin was added or not, whereas the rate and
26 extent of inactivation of Sindbis was higher in the presence of pepsin. This suggests that pH 3.2
27 alone inactivates WNV, while other phenomena involving the action of pepsin contribute to
28 Sindbis inactivation at low pH.
29 Confirmation of the significant inactivation of lipid-enveloped viruses during peptic cleavage of
30 plasma at pH 3.2 was obtained by another group [116]. In this process, plasma is diluted with
31 two volumes of saline, pH is adjusted to 3.3, and pepsin is added to a final concentration of 1g/l.
32 The mixture is incubated at pH 3.3 for 1 hour. Inactivation of PRV > 5.1 log10 occurred in less
33 than 6 minutes and > 7.0 log10 in 60 min. There was > 3.1 log10 and > 4.5 log10 inactivation of
34 VSV after 6 and 20 min, respectively. The reduction of infectivity of BVDV was less: 1.7 log 10
35 after 60 min. Inactivation of EMC, a non-enveloped virus, was relatively slow but reached
36 between 2.5 and 5.7 log10 after 60 min of pepsin incubation. This showed that reduction of
37 infectivity of at least some non-lipid enveloped viruses may take place during peptic digestion of
38 diluted horse plasma. This does not mean, however, that other non-lipid enveloped viruses
39 would be inactivated to the same extent under such conditions.
40 14.4.2.3 Recommended actions
41 Manufacturers of F(ab')2 antivenoms are encouraged to validate the pepsin digestion process
42 since virus inactivation is likely to be influenced by pH, time, temperature, pepsin content, and
43 protein content. Robustness studies to define the impact on process variations are also
44 recommended.
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1 14.4.3 Filtration steps
2 Other steps used in antivenom production may contribute to viral safety through non-specific
3 viral removal. The virus removal capacity of two depth-filtration steps performed in the presence
4 of filter aids and used in the production of equine derived immunoglobulins prepared by
5 ammonium sulfate precipitation of pepsin-digested IgG has been evaluated [120]. Clearance
6 factors of 5.7 and 4.0 log10 have been found for two lipid-enveloped viruses (infectious bovine
7 rhinotraceheitis virus and canine distemper virus, respectively) and of 5.3 and 4.2 log 10 for two
8 non-lipid enveloped viruses (canine adenovirus virus and poliovirus type I, respectively).
9 However, it should be kept in mind that viral reductions obtained by non-dedicated removal
10 steps are usually regarded as less robust than those resulting from dedicated viral inactivation
11 or removal steps [74].
12 14.4.4 Validation of dedicated viral reduction treatments
13 14.4.4.1 Pasteurization
14 Pasteurization is defined as the treatment of a liquid protein fraction for 10 hours, usually at 60
15 °C. It is a well-established viral inactivation treatment of human plasma products, such as
16 immunoglobulin G [74]. It is being used in the production process of a few equine-derived
17 immunoglobulins [10].
18 Validation studies showed that heating a purified equine immunoglobulin at 58 °C ± 0.1 °C
19 without stabilizers inactivates ≥ 4.8 log10 of PRV and ≥ 4.3 log10 of BVDV in less than 30 min,
20 and > 4.7 log of EMCV in less than 1 hour. In contrast, infectivity of MVM, a non-enveloped
21 virus, was still detected after 9 h and 30 min of treatment; only 1.59 log10 were inactivated [118].
22 14.4.4.2 Nanofiltration
23 Nanofiltration is a technique of filtration specifically designed to remove viruses, based on size,
24 while permitting flow-through of the desired protein [121]. Effective viral removal requires, in
25 principle, that the pore size of the filter be smaller than the effective diameter of the virus
26 particles.
27 14.4.5 Other viral inactivation treatments currently not used in antivenom
28 manufacture
29 Other methods of viral inactivation have been developed to ensure the viral safety of biological
30 products. These include, in particular, a treatment with a combination of an organic solvent (tri-
31 n-butyl phosphate or TnBP) at concentrations between 0.3 and 1%, and detergents such as
32 Triton X-100 or Tween 80, also at concentrations generally between 0.3% and 1%. Such
33 solvent–detergent (S/D) procedures have proven to be very efficient and robust in the
34 inactivation of lipid-enveloped viruses in human plasma products [74]. However, use of this
35 method for antivenoms has not been reported.
36 Implementation of dedicated viral inactivation treatments, such as S/D or other methods, should
37 be encouraged for processes which, based on risk assessment, would offer an insufficient
38 margin of viral safety. Process changes associated with the introduction of new viral reduction
39 steps, and the subsequent removal of any toxic compounds needed for viral inactivation, should
40 be demonstrated not to affect the quality and stability of antivenoms, and most particularly the
41 neutralization efficacy of venoms. Preclinical assessment of the possible impact of newly
42 introduced viral inactivation treatments should be mandatory.
43 14.4.6 Possible contribution of phenol and cresols
44 The anti-bacterial agents, phenol or cresols, and more rarely formaldehyde, are added by most
45 manufacturers to the starting plasma donations as well as to the final liquid antivenom
46 preparations, at a maximum final concentration of 0.25–0.35%. Compounds like phenol are
47 known to be very lipid-soluble and lipophilic.
48 Addition of 0.25% (final concentration) phenol to concentrated antivenom bulk at pH 6.5-6.7,
49 prior to its dilution and formulation, was found to inactivate eight enveloped and non-enveloped
50 viruses very efficiently within 30 minutes [122].
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1 Performing additional validations of the virucidal effect of antimicrobial agents as added to the
2 starting hyperimmune plasma and to the final antivenom preparations is encouraged. More
3 information is needed on the potential impact of these antimicrobial agents on the viral safety of
4 antivenoms.
5 14.5 Production-scale implementation of process steps contributing to

6 viral safety
7 As there is increasing, although preliminary, evidence that at least some of the existing steps in
8 the production of antivenoms contribute to viral reduction, it is already recommended that
9 specific care should be taken to ensure their appropriate industrial implementation so as not to
10 compromise any possible benefits they provide on viral safety.
11 Measures should therefore be taken to ensure that such steps are correctly carried out in a
12 manufacturing environment and that cross-contamination and downstream-contamination are
13 avoided. Such important aspects of product safety have been highlighted recently in WHO
14 Guidelines [74] and should also be taken into consideration for large-scale manufacture of
15 antivenoms. Specific attention should be given to:
16  Process design and layout, in particular the production floor area needed to carry out such
17 treatment safely, minimizing the chance of cross contamination between pre viral treatment
18 steps product and post treatment product; the justification for creating a safety zone to avoid
19 risk of down-stream contamination, and the procedures used for cleaning and sanitization of
20 the equipment to avoid batch-to-batch cross-contamination.
21  Equipment specifications, having in mind the potential contribution to viral reduction. For
22 instance, vessels used for low pH incubation or caprylic acid treatment should be fully
23 enclosed and temperature-controlled. There should be no “dead points” where the
24 temperature defined in the specification or the homogeneity of mixing cannot be ensured. A
25 poor equipment design could compromise the viral safety potentially afforded by a given
26 production step.
27  Qualification and validation: should verify that the equipment conforms to predefined
28 technical specifications and relevant GMP.
29  Process implementation: production steps contributing to viral safety such as low pH
30 treatment and caprylic acid treatments could be implemented in two stages performed in two
31 distinct enclosed tanks. Care should be taken to ensure complete process segregation
32 before and after the completion of these treatments to avoid risks of downstream
33 contamination.
34  Process control: is a critical part of the manufacturing process since completion of viral
35 inactivation and removal cannot be guaranteed by testing the final product. Samples should
36 be taken to confirm that the process conditions of claimed inactivation steps meet the
37 specified limits (e.g. for pH, concentration of stabilizers and concentration of virus
38 inactivating agent, such as caprylate). When this is technically feasible and intermediates
39 are stable, samples can be kept frozen for possible additional analysis prior to the release of
40 the batch. It is the responsibility of the quality assurance department to ensure that the
41 execution of steps contributing to virus inactivation and removal in a production setting
42 conforms to the conditions that contribute to such virus reduction.
43  Standard operating procedures: steps contributing to viral reduction should be described in
44 approved standard operating procedures. These should contain critical process limits for the
45 viral inactivation and removal methods.
46  Role of the quality assurance department: because of the critical nature of the viral
47 inactivation and removal steps, quality assurance personnel should review and approve the
48 recorded conditions for viral inactivation and removal while the batch is being processed; i.e.
49 not just as part of the final overall review of the batch file.
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1 14.6 Transmissible spongiform encephalopathy
2 Transmissible Spongiform Encephalopathy (TSE) has not been identified in any equine species.
3 There has been no case of transmission of TSE linked to antivenoms or other equine-derived
4 blood products.
5 Of particular concern, however, is the fact that TSEs include scrapie in sheep, a ruminant
6 species that is used, although much less frequently than the horse, in the manufacture of
7 antivenoms. Scrapie is a disease similar to bovine spongiform encephalopathy (BSE or “mad-
8 cow” disease), but is not known to infect humans. However, the blood of sheep with
9 experimental BSE or natural scrapie can be infectious and, because scrapie and BSE prion
10 agents behave similarly in sheep and goats, the use of the blood of small ruminants should
11 either be avoided in preparing biologicals or be selected very carefully from sources known to
12 be free of TSEs. The recent findings of disease-associated proteins in muscle tissue of sheep
13 with scrapie and the recognition of BSE itself in a goat, reinforce the need for manufacturers of
14 biologicals, including antivenoms, to maintain the precautionary safety measures recommended
15 in the WHO guidelines on TSE tissue infectivity [67].
16 According to these recommendations, the use of tissues or body fluids of ruminant origin should
17 be avoided in the preparation of biological and pharmaceutical products. When sheep materials
18 must be used, they should therefore be obtained from sources assessed to have negligible risk
19 from the infectious agent of scrapie. Documented surveillance records should be available. The
20 feed of animals used for production of hyperimmune plasma should be free of ruminant-derived
21 material.
22 The infectious agent is thought to be a misfolded, abnormal, prion protein (PrPTSE). It is not yet
23 known whether manufacturing processes used to produce antivenoms from sheep plasma
24 include steps that can contribute to the removal of PrPTSE. Experimental prion clearance studies,
25 based on spiking experiments, can be performed to assess the capacity of the process to
26 remove prions. However, there is still uncertainty about the validity of such experimental studies
27 since the biochemical features of PrPTSE in blood and plasma are not known.

29  The viral safety of antivenoms results from a combination of measures:
30 — to ensure satisfactory health status of the animals;
31 — to reduce the risk of contamination in the starting raw material;
32 — to ensure the contribution of the manufacturing process towards inactivation
33 and/or removal of viruses; and
34 — to ensure compliance with GMP all along the chain of production.
35  Manufacturing processes should include at least one, and, preferably, two steps
36 contributing to robust viral reduction. A virus inactivation step that can be easily
37 monitored is usually preferred to other means of viral reduction, such as non-specific
38 removal.
39  Manufacturers are encouraged to evaluate and validate the capacity of their current
40 manufacturing processes (in particular low pH pepsin digestion, caprylic acid
41 treatment, ammonium sulfate or heat precipitation, and possibly other steps) to
42 inactivate or remove viruses. These studies should be done following existing
43 international guidelines and using relevant and/or model viruses that are
44 representative of the viruses that could affect the animals used for the production of
45 the antivenom immunoglobulins.
46  The removal of antimicrobial agents from the final formulation of antivenoms should
47 be carefully weighed against the likely benefits these agents may have on the viral
48 safety.
49  Should the viral reduction processes used be found to be insufficient to ensure a
50 margin of safety, the introduction of dedicated viral reduction methods should be
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1 considered. The impact of such process changes on product efficacy and safety
2 should be carefully analysed in vitro as well as in preclinical studies before
3 performing clinical evaluations in humans.
4  Great attention should be paid to the production-scale implementation of all steps
5 contributing to viral safety to ensure a consistent and reproducible batch-to-batch
6 viral reduction and an absence of risks of cross-contamination and downstream re-
7 contamination that would jeopardize the viral safety of the product.
8  When materials originating from sheep must be used for the production of plasma,
9 they should be obtained from sources assessed to have negligible risk from the
10 infectious agent of scrapie.
11
12
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1 15 Quality control of antivenoms
2 Quality control of the final product is a key element ensuring the quality assurance of
3 antivenoms. Quality control tests should be performed by the manufacturer or under its
4 responsibility before the product is released. In addition, relevant analyses should be performed
5 on any intermediate steps of the manufacturing protocol as part of the in-process quality control
6 system.
7 The results obtained should meet the specifications approved for each antivenom product or its
8 intermediates, and constitute part of the batch record. For a liquid preparation, some quality
9 control tests, such as the venom-neutralising efficacy test or the detection of residual reagents
10 used during fractionation, can be performed on the final bulk and may not need to be repeated
11 on the final bottled product if the processing after the bulk preparation has been validated and
12 shown not to have any impact. Quality control assessments of the final antivenom product
13 includes the tests described below.
14 15.1 Routine assays

15 15.1.1 Appearance
16 The appearance of the product (e.g. colour and clarity of the liquid, appearance of the powder)
17 should comply with the description in the marketing dossier.
18 15.1.2 Solubility (freeze-dried preparations)
19 The time from the addition of solvent to the complete dissolution of freeze-dried antivenom,
20 under gentle mixing, should be determined. Antivenoms should be completely dissolved within
21 10 minutes at room temperature. The solution should not be cloudy. Shaking of the container
22 should be avoided to prevent the formation of foam.
23 15.1.3 Extractable volume
24 The volume of product extractable from the container should be in compliance with that
25 indicated on the label.
26 15.1.4 Venom-neutralizing efficacy tests
27 These tests determine the capability of an antivenom to neutralize the lethal effect of the snake
28 venom(s) against which the antivenom is designed. It is first necessary to determine the lethal
29 potency of the venom, using the median lethal dose (LD50) assay. The exact volume of
30 antivenom required to neutralise venom lethality can then be determined using the antivenom
31 Effective Dose (ED50) assay.
32 The outputs of these tests provide globally-applicable standard metrics of (i) venom lethality and
33 (ii) antivenom efficacy, which enable internal monitoring and external/independent auditing of
34 antivenom efficacy – thereby preventing the distribution of ineffective antivenom.
35 Consistent use of outbred strains of mice, of a defined weight range (e.g. 18–20 g), are
36 recommended for all the assays. Some producers use other test animals, such as guinea-pigs.
37 While weights will clearly vary between animal species, the following principles, specified for
38 mice, will still apply to these alternative test animals. It should be borne in mind that there are
39 variations in the susceptibility of various strains of mice to the lethal effect of venoms.
40 The protocol details and ethical issues are described in detail in section 17.
41 15.1.5 Osmolality
42 Osmolality is used to measure the tonicity of the antivenom solution, and should be at least 240
43 mosmol/kg. Determination of osmolality is also an indirect means to determine the quantity of
44 salts or excipients added for formulating the batch.
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1 15.1.6 Identity test
2 When several types of antivenoms are produced by a single production facility, a system to
3 identify each batch of antivenom should be established for monitoring and auditing purposes.
4 Identity tests may include biological assays as well as physicochemical and immunological
5 tests. Double immunodiffusion assays, confronting the antivenom with the venoms against
6 which the antivenom is designed to act, are often used. In the case of laboratories that use
7 various animal species to raise antivenoms, i.e. horses and sheep, an immunological identity
8 test should be used to identify the mammalian species in which the antivenoms are produced.
9 15.1.7 Protein concentration
10 The total protein concentration of antivenoms is performed using (i) the Kjeldahl method to
11 determine nitrogen content, (ii) several colorimetric procedures and (ii) spectrophotometric (280
12 nm) assays can be used. The presence of preservatives should be taken into account since
13 they may interfere with some protein determination methods [123].
14 The total concentration of proteins in antivenoms should preferably not exceed 10 g/dl, unless a
15 higher protein content is justified and authorized by the competent authority.
16 15.1.8 Purity and integrity of the immunoglobulin
17 The purity and integrity of the active substance, i.e. intact immunoglobulin or immunoglobulin
18 fragments, should be assessed to identify contaminants and immunoglobulin degradation. The
19 active ingredient should constitute the great majority of the preparation, ideally greater than
20 90%.
21 Electrophoretic methods in polyacrylamide gels (SDS–PAGE run under reducing or non-
22 reducing conditions) are suitable for this purpose, since these techniques allow the detection
23 and monitoring of IgG, F(ab')2, Fab, non-immunoglobulin plasma protein contaminants (in
24 particular albumin), and degradation products. The electrophoretic pattern should be compared
25 to that of a reference preparation. A semi-quantification can be performed by calibration of the
26 procedure. Of particular relevance is the assessment of the albumin content which ideally
27 should not exceed 1% of total protein content. The following approach can serve as a guide in
28 assessing the purity of antivenoms:
29  SDS–PAGE under non-reducing conditions. This analysis provides qualitative (or, at best,
30 semiquantitative) information on the amounts of intact immunoglobulins, digestion products
31 and, importantly, on the presence of high-molecular-mass oligomers (soluble aggregates)
32 and low-molecular-mass contaminants (which are expected in the case of enzymatically-
33 digested antivenoms).
34  SDS–PAGE under reducing conditions. Analysis under these conditions can provide
35 information on the amount of immunoglobulins and their fragments by direct visualization of
36 intact and/or digested immunoglobulin heavy chains.
37 15.1.9 Molecular-size distribution
38 The presence of aggregates and other components in antivenoms can be assessed by size-
39 exclusion liquid chromatography (gel filtration) in FPLC or HPLC systems.
40 Densitometric analyses of chromatographic profiles allow the quantification of protein
41 aggregates and of the relative abundances of: intact immunoglobulins, divalent immunoglobulin
42 fragments (F(ab')2), monovalent immunoglobulin fragments (Fab) and dimers, as well as low-
43 molecular-mass enzymatic digestion products.
44 In intact immunoglobulin-based antivenoms this method allows quantitation of albumin as its
45 molecular mass (~66 kDa) can be resolved from the ~160 kDa peak of intact immunoglobulins.
46 15.1.10 Test for pyrogen substances
47 Antivenoms should comply with the rabbit pyrogen test where required by the local regulations.
48 This test is based on intravenous injection of antivenoms in the ear vein of rabbits (usually 1.0
49 ml per kg body weight, although other doses might be used depending on the Pharmacopeia),
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1 followed by the measurement of rectal temperature at various time intervals after injection. The
2 detailed procedures are described in various pharmacopoeias. Bacterial lipopolysaccharides
3 can also be detected by the Limulus amoebocyte lysate (LAL) test. The test should be validated
4 for each type of antivenom, since there have been reports of false-positive and false-negative
5 reactions when testing antivenoms and other plasma-derived products. The sensitivity of this
6 LAL test should be correlated with the rabbit pyrogen test, and the endotoxin limits established
7 [124]. When regulation allows, a validated LAL test is used in place of the rabbit pyrogen test.
8 15.1.11 Abnormal toxicity test
9 The abnormal toxicity test (7 day observation of the effects of intraperitoneal injection of 0.2 ml
10 and 0.5 ml antivenom into mice and guinea pigs, respectively, is still required by some
11 pharmacopoeias and is performed at the stage of product development.
12 However, because of the very limited quality control value of this assay, it is increasingly being
13 abandoned by most regulatory authorities. Correct implementation of GMP should provide
14 evidence that the product would comply with the test for abnormal toxicity.
15 15.1.12 Sterility test
16 Antivenoms should be free of bacteria and fungi, i.e. they should be sterile. The sterility test is
17 performed following methodologies specified in various pharmacopoeias such as the European
18 pharmacopoeia.
19 Since antivenoms may contain preservatives in their formulation, it is necessary to “neutralize”
20 the preservatives before the samples are added to culture media. This is usually performed by
21 filtering a volume of antivenom through a 0.45-μm pore membrane, and then filtering through
22 the same membrane a solution that neutralizes the bacteriostatic and fungistatic effects of the
23 preservatives used in antivenom. The membrane is then aseptically removed and cut into two
24 halves. One half is added to trypticase soy broth and the other is added to thioglycolate
25 medium. Control culture flasks are included for each medium. Flasks are incubated at 20–25 ºC
26 (trypticase soy broth) or at 30–35 ºC (thioglycolate) for 14 days. Culture flasks are examined
27 daily for bacterial or fungal growth. The number of vials tested per batch should be in
28 compliance with local regulations.
29 15.1.13 Concentration of sodium chloride and other excipients
30 The concentration of the various excipients or stabilizers added for formulation should be
31 determined using appropriate chemical methods.
32 15.1.14 Determination of pH
33 The pH of antivenom should be determined using a potentiometer.
34 15.1.15 Concentration of preservatives
35 .Phenol concentration should not exceed 2.5 g/l and cresols 3.5 g/l.
36 Phenol concentration can be determined spectrophotometrically on the basis of the reactivity of
37 phenol with 4-aminoantipyrine, under alkaline conditions (pH 9.0–9.2) in the presence of
38 potassium ferrocyanide as oxidant. Other methods are also available. Cresols can be
39 determined by HPLC methods.
40 15.1.16 Chemical agents used in plasma fractionation
41 The chemical reagents used in the precipitation and purification of antivenoms, such as
42 ammonium sulfate, caprylic acid and others, should be removed from the final product during
43 diafiltration or dialysis. Limits should be established and their residual amount quantified in the
44 final product. Likewise, the elimination of pepsin or papain from the final preparations should be
45 guaranteed, especially for preparations that are maintained in liquid form, to avoid proteolytic
46 activity that may damage the antivenoms.
47 The determination of the residual amount of agents used in plasma fractionation could be
48 excluded from routine release testing if the process of manufacturing has been validated to
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1 eliminate these reagents. The detection of residual reagents can also be performed on the final
2 bulk rather than in the final product.
3 15.1.17 Residual moisture (freeze-dried preparations)
4 Residual moisture content can be determined by several methodologies, such as:
5  a gravimetric method assessing the loss of weight on heating;
6  the Karl-Fischer titration, based on the principle that iodine, together with pyridine, sulfur
7 dioxide and methanol from the reagent react quantitatively with water; and
8  thermogravimetric methods.
9 The methodology most commonly recommended is the Karl-Fischer titration. Every
10 manufacturing and quality control laboratory must establish the accepted maximum residual
11 moisture for their antivenom ensuring the stability of the product over its claimed shelf-life. A
12 residual moisture content of less than 3% is usually recommended for most freeze-dried
13 therapeutic biological products.
14 15.2 Antivenom reference preparations

15 The use of in-house reference preparations of antivenoms, instead of international standards, is
16 recommended, since venom-neutralising efficacy, specificity, and purity can only be compared
17 with antivenoms of similar specificity and neutralizing profile. An in-house reference preparation
18 should be obtained from a suitable batch of the product that has been fully evaluated by the
19 quality control laboratory. For assays not related to venom-neutralising efficacy or specificity,
20 such as the quantification of proteins, preservatives and excipients, international standards can
21 be used.

23  Quality control of antivenom preparations, both for intermediate and final products,
24 as part of the batch release, must be performed by the manufacturers and results
25 disclosed in the documentation.
26  Results from the following quality control test need to be provided by the
27 manufacturer as part of the batch release documentation:
28 — venom-neutralising efficacy test against the most relevant venoms to be
29 neutralized,
30 — identity test,
31 — protein concentration,
32 — purity of the active substance,
33 — content of protein aggregates and non-IgG contaminants,
34 — pyrogen test,
35 — sterility test,
36 — concentration of excipients,
37 — osmolality,
38 — pH,
39 — concentration of preservatives,
40 — determination of traces of agents used in plasma fractionation,
41 — visual inspection, and, for freeze-dried preparations, residual moisture and
42 solubility.
43  Antivenom reference preparations reflecting specific characteristics of antivenoms
44 produced should be prepared by each manufacturer to be used as standards in their
45 laboratory settings, in particular to measure neutralization capacity of their specific
46 antivenom products against targeted venoms. When possible, a national reference
47 antivenom should be established.
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1  It is the ethical responsibility of the manufacturer to use only the minimum number of
2 experimental animals to measure the efficacy of an antivenom.
3
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1 16 Stability, storage and distribution of antivenoms
2 16.1 Stability
3 Stability studies should be performed to determine the stability of antivenoms. These studies
4 should be done when a new product, a process change, or a new formulation is developed.
5 They are essential to define the shelf-life of the product and are intended to prove that the
6 antivenom remains stable and efficacious until the expiry date. During the developmental
7 stages, stability studies should be included, taken into consideration they are a complex set of
8 procedures involving considerable cost, time consumption and expertise in order to build in
9 quality, efficacy and safety of a product.
10 It has long been considered, somewhat empirically, that liquid preparations have a shelf-life of
11 up to 3 years at +2 to +8 °C, and freeze-dried preparations up to 5 years, when kept in the dark
12 at room temperature. Nevertheless, the actual stability of each antivenom formulation should be
13 appropriately determined by each manufacturer. It is highly recommended that manufacturers
14 perform stability studies to evaluate the possibility that their preparations could be stored for a
15 long period under non-refrigeration (for instance at 30 °C).
16 Real-time stability tests should be performed under the expected storage conditions of the
17 antivenom. In addition, these tests could be performed under worst-case storage conditions.
18 Quality control parameters are determined at regular pre-established time intervals, normally for
19 longer duration of the test period in order to allow significant product degradation under
20 recommended storage conditions. Essential parameters include venom neutralization potency,
21 turbidity and content of aggregates, among others, since these are especially prone to alter
22 upon storage.
23 Accelerated stability studies may be performed to provide early useful information on the
24 product stability profile, but are not a substitute for real-time data. The antivenom is exposed to
25 harsher conditions than usual, such as a higher temperature, and the stability is assessed over
26 a shorter timespan. This is done to assess the conditions that accelerate degradation of the
27 product and this information is then projected to predict shelf life.
28 Retained sample stability testing is a usual practice for every product for which stability data are
29 required. In this study, stability samples, for retained storage for at least one batch a year are
30 selected and tested at predetermined intervals i.e. if a product has shelf life of 5 years, it is
31 conventional to test samples at 3, 6, 9, 12, 18, 24, 36, 48, and 60 months.
32 Cyclic temperature stress testing is not a routine testing method but it may be useful since it is
33 designed so as to mimic likely conditions in field place storage. The minimum and maximum
34 temperatures are recommended to be selected on a product-by-product basis and considering
35 factors like recommended storage temperatures specific chemical and physical degradation
36 properties.
37 16.2 Storage
38 Antivenoms should be stored at a temperature within the range that assures stability, as found
39 by stability tests. This is particularly critical for liquid formulations, which usually require storage
40 at between 2 and 8 °C. Therefore, deviations from this temperature range, due to interruptions
41 in the cold chain during transportation or storage, are likely to result in product deterioration.
42 The design of adequate cold chain programmes, as part of the public health systems in every
43 country, is critical, and national protocols should be developed. The distribution policies for
44 national vaccination programmes can be adopted for the transportation and storage of
45 antivenoms. The stability of liquid preparations at temperatures higher than 2–8 °C should be
46 evaluated and, if needed, new formulations allowing such storage conditions should be
47 developed.
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1 16.3 Distribution
2 Adequate distribution of antivenoms is a matter of great concern in many regions of the world.
3 Since most of the antivenoms available are liquid preparations, the maintenance of an adequate
4 cold chain must be guaranteed, despite the difficulties to be encountered in rural areas of some
5 developing countries. National and regional health authorities should develop distribution
6 strategies to ensure that antivenoms are allocated to the areas where they are needed or use
7 the distribution channels in place for other national primary health care programmes. Both the
8 specificity of the antivenom and the number of vials or ampoules to be distributed should be
9 taken into consideration. This is particularly relevant in countries that use monospecific
10 antivenoms, since distribution of these products should be guided by the known distribution of
11 the species and epidemiological data. To ensure an appropriate supply for clinical use,
12 inventories should be in excess of the estimated number of cases, to allow for unpredictable
13 surges in local demand, accepting that some antivenoms will not have been used by the time of
14 their expiry date.

16  The quality control of each antivenom batch prepared by a manufacturer should
17 include the potency test for neutralization of lethality (ED50).
18  In general, liquid preparations require a cold chain, whereas freeze-dried preparations
19 do not. However, storage conditions are product- or formulation-specific and may
20 vary. Manufacturers should therefore determine the stability of each antivenom
21 pharmaceutical preparation by conducting real-time stability studies.
22  Manufacturers should study the stability of antivenoms at the ambient temperatures
23 in the areas where the product will be used.
24  The distribution of antivenoms by health authorities should rely on a proper
25 assessment of the epidemiology of snakebite envenomings, and on the proper
26 knowledge of the geographical distribution of the most relevant venomous species.
27 This is particularly important for monospecific antivenoms.
28  National regulatory authorities should ask manufacturers to provide information
29 obtained from the preclinical assessment of all antivenom used in their territories
30 against the venoms found in the region or country where the product is intended to
31 be used.
32
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1 17 Preclinical assessment of antivenom efficacy
2 Preclinical efficacy testing of antivenoms is one of a suite of assessments required for the
3 quality control of antivenoms (see chapter 15 where also further QC tests are described). The
4 details of preclinical efficacy testing of antivenoms and ethical considerations are described
5 below.
6 It is a fundamental regulatory and ethical requirement that all new therapeutic agents for human
7 use are tested for their safety and efficacy - initially by in vitro LD50 laboratory, and then in vivo
8 ED50 preclinical tests and, if the results of these prove satisfactory, by clinical trials in human
9 patients. The preclinical efficacy tests must therefore be performed on new antivenoms, and
10 newly-manufactured batches of existing antivenoms.
11 17.1 Ethical considerations for the use of animals in preclinical testing of

12 antivenoms
13 The preclinical testing of new or existing antivenoms necessitates the use of experimental
14 animals, typically rodents, particularly for essential median lethal venom dose (LD50) and
15 median effective antivenom dose (ED50) determination. Despite reservations over the
16 physiological relevance of these animal models to human envenoming and the harm that these
17 in vivo assays cause in the animals (sections 17.4 and 17.5), their use for determining venom
18 lethality (LD50) and antivenom neutralizing capacity (ED50) are currently the only validated
19 means of assessing venom toxicity and antivenom neutralizing potency by both manufacturers
20 and regulatory authorities worldwide. Non-sentient or in vitro alternative assays to the standard
21 venom LD50 and antivenom ED50 in vivo tests have been promoted in the past [125].
22 Unfortunately, such systems have not been developed to the point where they can replace the
23 above preclinical assays. In the absence of effective alternatives, the continued use of
24 experimental animals is justified by the considerable benefits to human health of these
25 preclinical assays.
26 It is imperative that laboratories that make use of animals in research or in the preclinical
27 evaluation of antivenoms adhere to the highest ethical standards. The “International Guiding
28 Principles for Biomedical Research Involving Animals (2012)” developed by the International
29 Council for Laboratory Animal Science (ICLAS) and the Council for International Organization of
30 Medical Sciences (CIOMS) provide an international benchmark for the use of animals in
31 research. Compliance with national guidelines, laws and regulations is also essential. All animal
32 experimentation should be subject to regulatory oversight at an institutional and national level.
33 In many jurisdictions the 3R principles of Replacement, Reduction and Refinement have been
34 adopted as cornerstones of ethical use of animals in experimentation, and WHO strongly
35 recommends that every effort be made to reduce pain, distress and discomfort to experimental
36 animals.
37 17.2 Preliminary steps which may limit the need for animal
38 experimentation
39 To prevent unnecessary use of animals careful use of existing literature for data on venom
40 lethality may help to refine the experimental design and thereby reduce the number of
41 experimental animals required.
42 Manufacturers may also examine the immunological venom-binding capability of an antivenom
43 by performing immunological assays (e.g.: ELISA) to identify, and exclude from
44 experimentation, antivenoms that do not possess the requisite titre of venom-binding
45 immunoglobulins. It is very important to note however, that (i) although a high venom-binding
46 titre in an ELISA result for an antivenom cannot be used to infer venom-neutralising efficacy, (ii)
47 a failure of an antivenom to bind venom in an ELISA result suggests very strongly that the
48 antivenom be considered ineffective at neutralising the effects of that venom – and withdrawn
49 from ED50 testing. This step can further limit non-productive animal experimentation. There is no
50 single ELISA metric enabling Stop/Go decisions to be made for all the possible snake venom
51 and antivenom combinations. These will therefore be in-house decisions.
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1 An additional immunological cross-reactivity technology that can inform the preclinical
2 assessment process before animal experiments are undertaken is the use of a proteomics-
3 centred platform, termed antivenomics, has been developed to assess the immunological
4 reactivity of antivenoms against homologous and heterologous venoms [126-129]. Antivenomics
5 complements the in vitro and in vivo venom activity neutralization assays and substitute the
6 traditional, essentially qualitative, immunological methods, such as ELISA and Western blotting.
7 Antivenomics uses an affinity chromatography approach to investigate the immuno-capturing
8 ability of immobilized IgG, F(ab')2, or Fab antibody molecules followed by the proteomic
9 identification of the venom components recovered in both the retained and the non-bound
10 fractions. The fraction of non-immuno-captured protein “i” (%NRi) is estimated as the relative
11 ratio of the chromatographic areas of the same protein recovered in the non-retained (NRi) and
12 retained (Ri) affinity chromatography fractions using the equation:
13 %NRi = 100 - [(Ri/(Ri + NRi)) x 100]
14 The antivenomic analysis provides both qualitative and quantitative information on the types of
15 venom proteins presenting antivenom-recognized epitopes and those exhibiting impaired
16 immunoreactivity. Although the level of immune recognition gathered from antivenomics should
17 not be absolutely relied upon to predict the in vivo neutralization capacity of an antivenom (since
18 both experiments involve radically different protocols), an immuno-capture capability of ≥25%
19 generally correlates with a good outcome in homologous in vivo neutralization tests. If immuno-
20 capture via this method is <25% the further testing of an antivenom using in vivo methods
21 should be reconsidered.
22 As the degree of immuno-recognition of a given toxin by the immunoglobulins present in
23 antivenom represents a measure of the capability of that particular antivenom to neutralize the
24 toxic activity of that toxin, the antivenomics analysis may assist in assessing the range of clinical
25 applications of current commercial or experimental antivenoms, and in the development of
26 improved antivenoms on an immunologically-sound basis. Growing evidence shows the
27 potential of the combination of antivenomics and neutralization assays for analyzing at the
28 molecular level the preclinical efficacy of antivenoms against homologous and heterologous
29 venoms. This is particularly so where antivenoms from more than one manufacturer, or from
30 more than one batch of antivenom produced by a manufacturer require preclinical evaluation
31 against the same venoms [126, 130].
32 Manufacturers should take steps to incorporate these approaches to their preliminary screening
33 of antivenoms before in vivo animal testing is conducted.
34 17.3 Essential preclinical assays to measure antivenom neutralisation of

35 venom-induced lethality
36 These tests determine the capability of an antivenom to neutralize the lethal effect of the snake
37 venom(s). It is first necessary to determine the lethal potency of the venom, using the median
38 Lethal Dose (LD50) assay. The exact volume of antivenom required to neutralise venom lethality
39 can then be determined using the antivenom Effective Dose (ED50) assay.
40 Preclinical testing of antivenom is required:
41  for routine quality control of efficacy of each newly-manufactured batch of an existing
42 antivenom;
43  to test the ability of a new antivenom to neutralize the lethal effects of venoms from snakes
44 from the country or region where it is going to be introduced; and,
45  to test the ability of an existing antivenom to neutralise the lethal effects of venoms for which
46 no prior ED50 data exists (e.g.: prior to introducing an antivenom to treat envenoming in a
47 new geographical region/country).
48 The outputs of these tests provide globally-applicable standard metrics of (i) venom lethality and
49 (ii) antivenom efficacy, which enable internal/manufacturer monitoring and external/independent
50 auditing of antivenom efficacy – thereby providing manufacturers and National Regulatory
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1 Authorities worldwide with a standardised mechanism preventing the distribution of dangerously
2 ineffective antivenom.
3 Consistent use of venom standards and outbred strains of mice, of a defined weight range (e.g.
4 18–20 g), are recommended for all the assays. Some producers use other test animals, such as
5 guinea-pigs. While weights will clearly vary between animal species, the following principles,
6 specified for mice, will still apply to these alternative test animals. It should be borne in mind that
7 there are variations in the susceptibility of various strains of mice to the lethal effect of venoms.
8 17.3.1 LD50 range-finding test:
9 For venoms whose LD50 is unknown, it is recommended that a range dose-finding study, using
10 one mouse per venom dose, is performed to set a narrow range of dose parameters for the full
11 LD50 test - reducing the total number of animals required. Range finding tests are not required
12 for venoms from validated suppliers where the lethal potency across successive batches of
13 venom is established. If venom is sourced from a new supplier, re-establishment of LD50 should
14 be performed.
15 Various venom doses are prepared using saline as diluent, and aliquots of a precise volume
16 (maximum 0.2 ml) of each dose are injected, using one mouse per dose, by the intravenous
17 route, in the tail vein (or, alternatively, by the intraperitoneal route (using injection volumes of
18 maximum 0.5 ml)). Deaths are recorded at 24 hours (intravenous test) or at 48 hours
19 (intraperitoneal test). On the basis of this preliminary dose-finding experiment, a range of venom
20 doses causing 0% to 100% lethality is established and thus narrows the range of venom doses
21 required for the full venom LD50 assay.
22 17.3.2 The Median Lethal Dose (LD50) assay:
23 Venom doses are prepared in saline and intravenously injected (maximum 0.2 ml) into the tail
24 vein of groups of 5–6 mice (of a defined weight range). A group size of 5 mice is the smallest
25 number recommended for obtaining a statistically significant result. In some laboratories the
26 LD50 is estimated by the intraperitoneal route using an injection volume of a maximum of 0.5 ml.
27 Deaths are recorded at 24 hours (for assays involving intravenous injections) or at 48 hours
28 (when intraperitoneal injections are used), and LD50 is estimated by Probit analysis [131],
29 Spearman-Karber [8] or alternative procedures (such as non-parametric methods). One venom
30 LD50 dose is defined as the minimal amount of venom causing death in 50% of injected mice.
31 17.3.3 Antivenom Efficacy Assessment:
32 The venom LD50 results provide the information necessary to test the venom-neutralising
33 efficacy of an antivenom - using the median Effective Dose (ED50) assay. It is important that the
34 venoms used in the ED50 assays are from the same batch (lot) as that used to determine the
35 venom LD50 result. It is equally important that all the LD50 and ED50 assays utilise mice of
36 identical strain and weight.
37 17.3.3.1 ED50 range-finding test:
38 For an antivenom whose ED50 against a specific venom is unknown, it is recommended that a
39 range dose-finding study, using one mouse per venom dose, is performed to set a narrow range
40 of dose parameters for the full ED50 test - reducing the total number of animals required. Range
41 finding tests are not required for antivenom whose venom-neutralising efficacy is established.
42 The selected multiple of the venom LD50 (3 – 6 LD50) is mixed with different doses of antivenom,
43 incubated at 37oC for 30 minutes and each mixture intravenously injected into a single mouse.
44 This preliminary test establishes a range of antivenom volumes that result in 100% survival and
45 100% death of the injected mice and thus narrows the range of doses required for the formal
46 antivenom ED50 test.
47 17.3.3.2 The median effective dose (ED50) assay:
48 A fixed amount of venom (“challenge dose”, usually corresponding to 3 – 6 LD50) is mixed with
49 various volumes of the antivenom and adjusted to a constant final volume with saline [66, 132,
50 133]. The mixtures are incubated for 30 minutes at 37°C, and then aliquots of a precise volume
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1 (maximum 0.2 ml intravenously; maximum 0.5 ml intraperitoneally) of each mixture are injected
2 into groups of 5-610 mice (of the same strain and weight range used for the LD50 assay). A
3 control group injected with a mixture of the venom “challenge dose” with saline solution alone
4 (no antivenom) should be included to confirm that the venom “challenge dose” induces 100%
5 lethality. Centrifugation of the antivenom–venom mixtures prior to use is not recommended
6 because residual venom toxicity may remain in the immuno-precipitate.
7 After injection, deaths are recorded at 24 hours (intravenous test) or at 48 hours (intraperitoneal
8 test) and the results analysed using Probit analysis, Spearman-Karber or alternative procedures
9 (such as non-parametric methods). One antivenom ED50 dose is defined as the minimal amount
10 of antivenom resulting in the survival of 50% of mice injected with a mixture of antivenom and a
11 lethal quantity of venom.
12 The ED50 result can be expressed in various ways:
13  mg of venom neutralized by ml of antivenom;
14  µl antivenom required to neutralize the “challenge dose” of venom used; and
15  µl of antivenom required to neutralize one mg of venom; and
16 The practice of defining the ED50 by the number of murine LD50s of venom neutralized per ml of
17 antivenom is inaccurate and has little clinical usefulness. Since LD50 values for the same venom
18 may vary from one manufacturer to another, this representation of ED 50 should be avoided in
19 favour of one of the approaches shown above.
20 17.3.4 General recommendations
21 Before any antivenom is used therapeutically in humans, it’s efficacy against the relevant snake
22 venoms should be confirmed in the essential preclinical LD50 and ED50 assays. Where minimum
23 standards for venom-neutralizing efficacy exist in geographically relevant Pharmacopeia, or
24 have been established by NRAs, these requirements must be met.
25 In regions where no minimum acceptable levels of therapeutic efficacy that are clinically
26 relevant to human envenoming have been established which take into account the need to
27 deliver a therapeutic dose of antivenom in a realistic volume for administration, NRAs in
28 consultation with other organizations should establish such standards as a matter of priority for
29 the various antivenoms being produced or distributed in these jurisdictions.
30 The essential tests of preclinical efficacy, the venom LD50 and antivenom ED50, should be
31 standardized by NRAs and National Quality Control laboratories, and common protocols
32 adopted to avoid variation in methodology between production facilities. Therefore,
33 manufacturers should disclose details of their ED50 protocol to the corresponding National
34 Regulatory Authority as part of the licensing/registration application to demonstrate compliance.
35 Quality control laboratories need to establish national reference venom collections (venoms
36 representing the taxonomic and geographical range of snake species in a country), and these
37 must be independently evaluated periodically to ensure that they have not deteriorated (see
38 section 8 on quality control of venoms).
39 17.3.5 Development of alternative assays to replace murine lethality testing
40 In vivo murine assays cause considerable suffering and a 3R approach involving innovation and
41 validation should be applied in the development of standardized LD50 and ED50 test protocols.
42 Designing protocols that use the minimal number of animals necessary and introducing
43 procedures to minimize pain and suffering is essential. The development of alternative methods
44 to animal testing in the preclinical evaluation of antivenoms, should be encouraged and when
45 live animals are absolutely necessary, anaesthesia or analgesia should be considered and
46 evaluated to ensure that the humane benefits of anaesthesia or analgesia to the experimental
47 animals do not invalidate the objectives of the assay by altering relevant physiological
48 processes [66]. In particular, the use of analgesia is recommended when working with venoms
10
10 mice may be needed for some venoms.
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1 that induce tissue damage [134]. The establishment of humane end-points to reduce suffering
2 and limiting the duration of the assays to reduce the period of animal suffering is also
3 encouraged, but also requires appropriate standardization and validation within a quality
4 assurance framework.
5 17.4 Supplementary preclinical assays to measure antivenom

6 neutralisation of specific venom-induced pathologies
7 Snake venoms generate a wide range of systemic pathologies, including a variety of
8 haemostasis-disruptive (haemorrhage, pro- and anti-coagulopathic effects), neurotoxic,
9 myotoxic, nephrotoxic and cardiac effects. Supplementary tests are therefore recommended for
10 new antivenoms and for new applications of existing antivenoms to determine whether they are
11 effective in eliminating the most clinically-relevant pathophysiological effects induced by the
12 specific venom(s) of interest.
13 For example, a new antivenom developed against Echis ocellatus envenoming should, in
14 addition to preclinical LD50 and ED50 testing, be tested for its ability to eliminate venom-induced
15 coagulopathy and haemorrhage – the most medically important effects of envenoming by E.
16 ocellatus.
17 In this context it may be useful to consider that post-mortem observations of mice from LD50 and
18 associated ED50 experiments can provide a wealth of pathophysiological information as to
19 antivenom neutralisation of venom-induced pathology. Post-mortem observations from LD50 and
20 associated ED50 experiments may prove useful in reducing the needs for/frequency of some of
21 the supplementary assays recommended here, however their use requires the same degree of
22 scientific and procedural validation as other supplementary preclinical assays.
23 These supplementary preclinical tests are listed below.
24 17.4.1 Neutralization of venom haemorrhagic activity
25 Many venoms, especially those of vipers, exert powerful local and systemic haemorrhagic
26 activity effected primarily by snake venom zinc-dependent metalloproteinases (SVMPs). These
27 enzymes damage the basement membrane that surrounds the endothelial cells of capillary
28 blood vessels resulting in bleeding into the tissues. Bleeding into the brain and other major
29 organs is considered to be the major lethal effect of envenoming by many viperid species [135].
30 The minimum haemorrhagic dose of a venom (MHD) quantifies this venom-induced pathology,
31 and is defined as the amount of venom (in g dry weight) which, when injected intradermally,
32 induces in mice causes a 10-mm haemorrhagic lesion at a predefined interval, usually 2-3
33 hours, after injection [136, 137].
34 The venom MHD test is carried out by preparing aliquots of 50 l of physiological saline solution
35 containing a range of venom doses. Mice (18–20 g body weight; 5 mice per group) are held
36 securely and the hair surrounding the injection site is shaved. Venom solutions (50 l) are
37 injected intradermally in the shaved skin. After a particular time interval (usually 2-3 hours), mice
38 are killed using an approved humane procedure, the area of the injected skin is removed, and
39 the size of the haemorrhagic lesion in the inner side of the skin is measured using calipers in
40 two directions with background illumination. Care should be taken not to stretch the skin. The
41 mean diameter of the haemorrhagic lesion is calculated for each venom dose and the MHD
42 estimated by plotting mean lesion diameter against venom dose and reading off the dose
43 corresponding to a 10-mm diameter [136, 137].
44 The assay measuring the efficacy of antivenom to neutralize venom-induced haemorrhage is
45 termed the MHD-median effective dose (MHD50), and is defined as the volume of antivenom, in
46 microlitres, which reduces the diameter of haemorrhagic lesions by 50% when compared with
47 the diameter of the lesion in animals injected with the control venom/saline mixture [137]. A
48 “challenge dose” of venom is selected - between one and five venom MHDs have been used as
49 the challenge dose by different laboratories. The test is carried out as above, using 5 mice per
50 group. Mixtures of a fixed amount of venom and various dilutions of antivenom are prepared so
51 that the challenge dose of venom is contained in 50 l. Controls must include venom solutions
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1 incubated with physiological saline solution alone. Mixtures are incubated at 37 °C for 30 min,
2 and aliquots of 50 l are injected intradermally in lightly anaesthetized mice. The diameter of
3 haemorrhagic lesions is quantified as described above, and the neutralizing ability of antivenom
4 is expressed as the MHD50.
5 17.4.2 Neutralization of venom necrotizing activity
6 Venom-induced local dermonecrosis is a major problem in human victims of snakebite and it
7 has long been considered important to have an assay system to evaluate the effect of an
8 antivenom on this pathology. However, it should be noted that the value of antivenoms in
9 overcoming the cytolytic effects of venoms has not yet been established; indeed, there is
10 considerable doubt whether antivenom is useful in obviating such effects in human victims of
11 snakebite. This is because venom-induced dermonecrosis occurs quickly after a bite and there
12 is usually a considerable delay between the envenoming of a victim and his or her arrival in
13 hospital for treatment. Consequently, antivenom therapy can have little or no effect in reversing
14 the damage [138, 139]. Animal experiments in which the antivenom was administered to the
15 animal at different times after the venom support this opinion [139-141]).
16 The minimum necrotizing dose (MND) of a venom is defined as the least amount of venom (in
17 μg dry weight) which, when injected intradermally into groups of five lightly anaesthetized mice
18 (18–20 g body weight), results in a necrotic lesion of 5 mm diameter 3 days later. The method
19 used is the same as that for the MHD, except that the skin is examined 3 days after the
20 intradermal injection of the venom [136].
21 The assay measuring the ability of an antivenom to neutralize venom-induced dermonecrosis is
22 termed the MND-median effective dose (MND50), and is defined as the volume of antivenom, in
23 microliters or the venom/antivenom ratio, which reduces the diameter of necrotic lesions by 50%
24 when compared with the diameter of the lesion in mice injected with the control venom/saline
25 mixture. A challenge dose of venom is selected, usually between one and two MNDs. The test
26 is carried out as above, using 5 mice per group. Mixtures of a fixed concentration of venom and
27 various dilutions of antivenom are prepared so that the venom challenge dose is contained in 50
28 l. Controls include venom solutions incubated with physiological saline solution alone. Mixtures
29 are incubated at 37 °C for 30 min, and aliquots of 50 l are injected intradermally in lightly
30 anaesthetized mice [142, 143]. The diameter of dermonecrotic lesions is quantified 3 days after
31 injection, as described above, and the neutralizing ability of antivenom, expressed as the
32 MND50..
33 17.4.3 Neutralization of venom procoagulant effect
34 Many venoms, especially from some vipers, cause consumption of coagulation factors which
35 results in incoagulable blood. This, combined with the haemorrhagic nature of some of these
36 venoms, can result in a very poor prognosis for severely envenomed patients. Simple in vitro
37 methods exist to measure this venom-induced pathophysiological effect and the ability of an
38 antivenom to eliminate it.
39 The minimum coagulant dose (MCD) of a venom is defined as the least amount of venom (in
40 mg dry weight per litre of test solution or μg/ml) that clots either a solution of bovine fibrinogen
41 (2.0 g/l) in 60 sec at 37 oC (MCD-F) and/or a standard citrated solution of human plasma
42 (fibrinogen content 2.8 g/l) under the same conditions (MCD-P).
43 For measurement of the MCD-F, 50 μl of physiological saline with final venom concentrations
44 ranging from 240 to 0.5 mg/l is added to 0.2 ml of bovine fibrinogen solution (2.0 g/l) at 37 oC in
45 new glass clotting tubes. The solutions are mixed thoroughly and the clotting time recorded. The
46 MCD-P is estimated by adding the same venom concentrations to 0.2 ml of the standard
47 citrated human plasma solution under identical conditions and recording the clotting time. In
48 each case, the MCD is calculated by plotting clotting time against venom concentration and
49 reading off the level at the 60-second clotting time [136].
50 To estimate the ability of an antivenom to neutralize venom procoagulant activity, a challenge
51 dose of venom is selected, which corresponds to one MCD-P or one MCD-F. Mixtures of a fixed
52 concentration of venom and various dilutions of antivenom are prepared so that the challenge
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1 dose of venom is contained in 50 l. Controls include venom solutions incubated with
2 physiological saline solution alone. Mixtures are incubated at 37 °C for 30 min, and aliquots of
3 50 l are added to 0.2 ml of plasma or fibrinogen solution, as described. The formation or
4 absence of clots is observed during a maximum of 30 min. The minimum volume of antivenom,
5 or venom/antivenom ratio, which completely prevents clotting is estimated and is known as
6 either the MCD-F-effective dose (MCD-F100) or MCD-P-effective dose (MCD-P100).
7 17.4.4 Neutralization of in vivo venom defibrinogenating activity
8 This test is a direct measure of the in vivo defibrinogenating effect of certain venoms. To
9 measure the minimum venom defibrinogenating dose (MDD), a wide range of venom doses is
10 selected and each dose, in a volume of 0.2 ml, is injected intravenously into 5 mice (18–20 g
11 body weight). One hour after injection, the mice are placed under terminal general anaesthesia
12 and bled by cardiac puncture. The blood from each animal is placed in a new glass clotting
13 tube, left at room temperature for 1 hour and the presence/absence of a clot recorded by gently
14 tilting the tube. The MDD is defined as the minimum dose of venom that produces incoagulable
15 blood in all mice tested within 1 hour of intravenous injection.
16 Antivenom neutralization of the venom component(s) responsible for in vivo defibrinogenation is
17 estimated by incubating a challenge dose of venom, corresponding to one or more MDD, with
18 different dilutions of the antivenom. Controls should include venom solutions incubated with
19 saline solution instead of antivenom. Mixtures are incubated at 37 °C for 30 min before injection
20 of 0.2 ml by the intravenous route in groups of 5 mice (18–20 g body weight). After 1 hour, mice
21 are bled as described above, the blood is placed in new glass clotting tubes and left undisturbed
22 for 1 hour at room temperature, after which the presence or absence of a clot is recorded.
23 Neutralizing ability of antivenoms is expressed as MDD-effective dose (MDD100), corresponding
24 to the minimum volume of antivenom, or venom/antivenom ratio in which the blood samples of
25 all injected mice showed clot formation [143, 144].
26 17.4.5 Neutralization of venom myotoxic activity
27 The presence of myotoxic components in a venom results in the degeneration of skeletal
28 muscle by myonecrosis of muscle fibres. Damage is characterized by the disruption of the
29 muscle cell plasma membranes, myofilament hypercontraction, local infiltration of inflammatory
30 cells and oedema. Myotoxicity is characterized by the appearance of myoglobin in urine and by
31 increments in the serum levels of muscle-derived enzymes, such as creatine kinase (CK).
32 Myotoxic phospholipase A2 (PLA2) enzymes are found in a wide range of snake venoms. Some
33 of these PLA2s may be primarily myotoxic, or neurotoxic, or both. Cytotoxic proteins of the
34 three-finger toxin family present in some elapid venoms also cause myonecrosis. In addition,
35 myotoxicity may occur as a consequence of ischaemia induced in muscle fibres by the effect of
36 haemorrhagic venom components in the microvasculature [145].
37 Venom myotoxic activity is determined by injecting mice with various doses of venom in a
38 constant volume of 50 l (using saline solution as diluent) into the right gastrocnemius muscle.
39 Groups of 5 animals of 18–20 g body weight are used per dose. Control animals are injected
40 with the same volume of saline solution. Tail-snip blood samples are collected at a specific time
41 interval (3 hours in mice), and the CK activity of serum or plasma is determined using
42 commercially-available diagnostic kits [146, 147]. Myotoxic activity is expressed as the minimum
43 myotoxic dose (MMD), defined as the amount of venom that induces an increment in serum or
44 plasma CK activity corresponding to four times the activity in serum or plasma of animals
45 injected with saline solution alone. Myotoxicity can also be assessed by histological evaluation
46 of muscle damage after venom injection, although this is a more expensive and more time
47 consuming method than the CK determination.
48 To estimate the ability of an antivenom to neutralize venom myotoxicity, a challenge dose of
49 venom is selected, which corresponds to 3 MMDs. The test is carried out as above, using 5
50 mice per group. Mixtures of a fixed concentration of venom and various dilutions of antivenom
51 are prepared so that the challenge dose of venom is contained in 50 l. Controls include venom
52 solutions incubated with physiological saline solution alone. Mixtures are incubated at 37 °C for
53 30 min, and aliquots of 50 l are injected into the gastrocnemius muscle, as described above.
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1 Blood samples are collected 3 hours after injection (in the case of mice) and serum or plasma
2 CK activity is quantified. The neutralizing ability of antivenom, expressed as MMD-median
3 effective dose (MMD50) is estimated as the volume of antivenom in microlitres, or the
4 venom/antivenom ratio, which reduces the serum or plasma CK activity by 50% when compared
5 to the activity of animals injected with venom incubated with saline solution only [133].
6 17.4.6 Neutralization of venom neurotoxic activity
7 Several laboratory methods for assessing venom-induced neurotoxicity have been developed
8 (e.g. chick biventer cervicis nerve-muscle preparation [148, 149]; mouse hemidiaphragm
9 phrenic nerve preparation [125, 150-153], but they are difficult to perform, require costly
10 equipment and expert technological help and are unlikely to be practicable for most antivenom
11 producers. Mouse lethality tests are usually reliable in predicting the neutralization of neurotoxic
12 effects of venoms.
13 17.5 Limitations of preclinical assays

14 It is acknowledged that the in vivo and in vitro essential and supplementary preclinical tests
15 have physiological limitations since venom and venom/antivenom injection protocols do not
16 represent the natural situation, and the physiological responses of rodents to envenoming and
17 treatment may differ from those of humans. Even comparing the levels of immune recognition
18 gathered from antivenomic or ELISA data with the in vivo neutralization capacity of an
19 antivenom is not straightforward. Such limitations make the rodent model of human
20 envenoming and treatment less than ideal. Care should therefore be taken to avoid simplistic
21 extrapolations from this assay to the clinical situation. Nevertheless, the LD50 and ED50 tests
22 represent the methods most widely used for assessment of antivenom potency, and a number
23 of clinical trials have demonstrated that the ED50 test is useful [125, 154], but not infallible [155,
24 156], at predicting the efficacy of antivenoms in the clinical setting. In some cases, it is
25 recommended to test the ability of antivenoms to neutralize pathophysiologically-relevant effects
26 different from the lethal effect. Examples are the neutralization of hemorrhagic and in vitro
27 coagulant effects in the case of Echis sp venoms, and of dermonecrotising effect in the case of
28 cytotoxic Naja sp venoms. An additional value of these tests is the assurance that antivenoms
29 are manufactured with an accepted, quantifiable and uniform neutralizing potency.

31  The estimation of the ability of an antivenom to neutralize the lethal activity of
32 venom(s) (LD50 and ED50) is a critical preclinical assessment and should be performed
33 by manufacturers for all antivenoms, and enforced by the National Regulatory
34 Authorities as part of the antivenom-licensing procedure.
35  All practitioners of these preclinical tests must prioritise the implementation of 3R
36 into these tests to reduce the substantial number of mice used, and their collective
37 pain, harm and distress.
38  In vitro methods such as ELISA, antivenomics or other emerging technologies that
39 enable antivenoms to be screened for immune-recognition of venom components
40 prior to in vivo evaluation should be adopted by manufacturers.
41  The development of improved in vivo assay protocols to reduce pain and suffering of
42 animals, and of in vitro alternatives to the in vivo assays to reduce the number of
43 animals used in preclinical testing is encouraged. The results of any modified in vivo,
44 or new in vitro protocols, should be rigorously compared with results from existing
45 protocols and validated to ensure statistical reliability of the newly developed
46 methods.
47  All new antivenoms, as well as existing antivenoms to be used in new geographical
48 areas, should furthermore be assessed for their ability to eliminate specific
49 pathologies caused by the venoms of the snakes for which the antivenom has been
50 designed.
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1  The selection of which preclinical supplementary test(s) to perform will depend on the
2 predominant pathophysiological effects induced by the specific snake venom and be
3 appropriately adapted for each antivenom. These supplementary tests are not
4 required for quality control assessment of subsequent batches of antivenom.
5
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1 18 Clinical assessment of antivenoms
2 18.1 Introduction
3 Antivenoms are unusual among pharmaceutical agents in that they have been used in human
4 patients since 1896 with little attention being paid to clinical trials of their effectiveness and
5 safety. However, since the 1970s it has been clearly demonstrated that it is possible to carry out
6 dose-finding and randomized controlled trials in human victims of snakebite envenoming. These
7 studies have yielded invaluable information, as in the case of clinical trials of other therapeutic
8 agents for which clinical trials are generally regarded as the essential basis for regulatory
9 approval.
10 The conduct of clinical studies is guided by the principles set down in the international
11 regulations governing good clinical practice (see http://www.therqa.com/committees-working-
12 parties/good-clinical-practice/regulations-and-guidelines/), comprising EU, UK and USA
13 regulations, summarised in ICH Topic E 6 (R1) Guideline for Good Clinical Practice (GCP), an
14 international ethical and scientific quality standard for designing, conducting, recording
15 and reporting trials that involve the participation of human subjects (see
16 http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC5000
17 02874.pdf). These principles emphasize the responsibilities of the researcher and of the
18 organization sponsoring the research to protect participants in the research and to ensure that
19 the conduct of the trial is likely to lead to reliable results.
20 Clinical trials should be registered with an appropriate registration body, prior to their
21 commencement:
22  https://clinicaltrials.gov/ct2/manage-recs/how-register;
23  http://www.isrctn.com/;
24  https://www.clinicaltrialsregister.eu/;
25  http://www.umin.ac.jp/ctr/;
26  http://www.anzctr.org.au/)
27 The conventional pathway for clinical evaluation of new therapeutic products is:
28  Phase I: healthy volunteer studies – detection of unanticipated adverse events
29  Phase II: limited effectiveness and safety studies, often dose-finding
30  Phase III: full-scale clinical evaluation, often randomized controlled trials
31  Phase IV: postmarketing surveillance
32 The appropriateness of this pathway for antivenoms depends upon a number of factors,
33 including whether an antivenom is new or has been previously used in human patients, the
34 ethical basis for the study, the trial’s practicability as well as ethical and national regulatory
35 considerations. So far, most antivenoms have been registered without prior clinical studies. This
36 situation should not persist: it is desirable, firstly, to collect the existing clinical data regarding
37 antivenoms already marketed, and secondly to promote phase II or III clinical trials before
38 registering new antivenoms. In the absence of clinical data for antivenoms already in use,
39 appropriate clinical trials should be quickly implemented.
40 18.1.1 Identification of biting species in clinical studies of antivenoms
41 It is absolutely essential that all clinical studies of antivenom effectiveness or safety, including
42 clinical trials incorporate robust methodologies for ensuring the identification of the biting
43 species. This can be achieved through:
44  Expert identification of the dead snake responsible for the bote, or of a photographic image
45 of that snake; or,
46  the identification of specific venom components unique to particular species (from bite site
47 swabs, wound exudate, serum or urine samples) through the use of enzyme immunoassays
48 (ELISA) or other immunological methods.
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1 Failure to properly identify the species of snakes that are responsible for cases of envenoming
2 included in clinical trials and other studies of snake antivenoms, significantly diminishes the
3 value of the research, and renders the results unreliable. National regulatory authorities should
4 be cautious about accepting the results of clinical evaluations of antivenom where robust,
5 reliable identification of the biting species is not available.
6 18.1.2 Phase I studies
7 Conventional clinical studies using healthy volunteers are not appropriate in the case of
8 antivenoms11 because of the risk of anaphylactic and other reactions (e.g. pyrogenic or serum
9 sickness and, rarely, hypersensitivity reactions to equine/ovine plasma proteins) and the risk of
10 sensitisation to equine/ovine plasma proteins in the volunteers. Phase I studies are primarily
11 designed to detect unanticipated adverse reactions. This can be done only in human subjects
12 as it is not possible in an animal model. Such studies are an essential protection against severe
13 and even fatal effects of a new medication, before it is tested in the much larger numbers
14 demanded for Phase II and III studies. Recent disasters or near-disasters during Phase I
15 studies of new therapeutic monoclonal antibodies have emphasised both the need for such
16 studies but also their potential dangers. A similar situation exists in the early testing of cytotoxic
17 drugs and antibodies used in oncology. A preliminary open-label dose-finding study can
18 establish both the effectiveness and safety of an initial dose of a new antivenom in small groups
19 of adult, non-pregnant patients with systemic envenoming, but excluding those with features of
20 severe envenoming. The aim is to assess clinical safety and effectiveness, as a prelude to full-
21 scale Phase II/III randomised controlled trials. This modified Phase I approach can be combined
22 with a preliminary dose-finding study. The ‘‘3 x 3’’ dose escalation design [157] can be used to
23 determine the minimum dose capable of achieving a defined end-point in two-thirds or more of a
24 small group of patients. An additional stopping rule is added to ensure that patients are not
25 exposed to doses likely to cause reactions in more than one third of them (for details see
26 Abubakar et al., 2010)[84]. Currently, there is no alternative for ethical Phase I studies.
27 18.1.3 Phase II and III studies
28 Phase II studies are usually conducted to optimize doses, establish/confirm the relative safety of
29 a product and give an indication of effectiveness. Phase III studies are normally used to
30 establish effectiveness of a product, often in comparison with an existing product, or
31 occasionally a placebo. Since antivenoms are so well established in the treatment of snake bite
32 envenoming, the use of placebo controls is ethically acceptable only where there is genuine
33 uncertainty about whether the benefit (degree of clinical improvement) from the antivenom
34 outweighs the risk (potential incidence and severity of adverse events). Depending on the
35 speed of evolution of envenoming, immediate treatment might be compared to delayed
36 antivenom treatment. A new antivenom with demonstrable pre-clinical potency (see above) can
37 be compared with an established product, or two markedly different initial doses or regimens of
38 the same antivenom can be compared. In randomised controlled trials (RCTs), non-inferiority,
39 rather than superiority of a new antivenom or regimen, compared to an existing treatment,
40 requires smaller numbers of trial participants to achieve acceptable power [84]. Basic
41 requirements for any clinical antivenom RCT are that the participants should be reasonably
42 homogeneous as far as the species of snake responsible and their pre-treatment characteristics
43 (e.g. interval between bite and treatment) are concerned, and that an objective clinical end-point
44 should be selected to judge effectiveness. of adverse events).
45 18.1.4 Phase IV studies
46 Phase IV studies are clinical surveillance studies that occur after market authorization of the
47 product. In view of the difficulty in performing standard clinical trials of antivenom in some
48 situations, this may be the only way to study safety and effectiveness of an antivenom in a large
49 number of patients. In practice, such studies have rarely if ever been attempted for antivenoms,
50 but they are strongly recommended for the future.
11
Immunoglobulins derived from animal plasma.
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1 18.2 Clinical studies of antivenom
2 Although preclinical testing may be valuable in ensuring that antivenoms neutralize the venoms
3 of interest, the complex effects of venoms in humans and the need to consider venom
4 pharmacokinetics mean that, ultimately, the effectiveness and safety of antivenoms for the
5 treatment of human envenoming can only be determined by well-designed clinical studies.
6 Clinical studies of antivenoms primarily address three main issues:
7  assessment of the optimal initial dose of antivenom;
8  assessment of effectiveness of the antivenom; and
9  assessment of the safety of an antivenom, particularly the incidence and severity of early
10 and late reactions.
11 Antivenom safety and tolerance depend on manufacturing factors (immunoglobulins
12 composition, purification of immunoglobulin fragments, protein concentration, and presence of
13 preservatives) [158]. Consequently, incidence and severity of adverse reactions for similar
14 doses of a given batch of antivenom are unlikely to vary in different geographical locations.
15 Conversely, the effectiveness depends on both manufacturing factors (choice of venoms,
16 immunological title) and also circumstantial factors (quality and quantity of inoculated venom,
17 patient's physical condition, delay of treatment, etc.). However, following initial preclinical
18 testing, both effectiveness and dose-finding studies may need to be repeated for a new
19 geographical location, depending upon the similarity of the snake species in the new place with
20 those where the antivenom was initially tested. If the species are similar, preclinical testing
21 indicates good neutralization, and if evidence of clinical effectiveness exists in other places,
22 post marketing surveillance studies may be adequate.
23 18.2.1 Dose-finding studies
24 Dose-finding studies seek to establish the optimum initial dose of an antivenom required to
25 control envenoming in patients with different severities of envenoming. The therapeutic dose of
26 an antivenom administered by intravenous route depends on:
27  the quantity of venom injected (assessed by clinical and laboratory outcomes);
28  the neutralizing potency of the antivenom (given by preclinical tests); and
29  the dosage of the antivenom administered to the patient.
30 The dose is calculated to neutralize a certain amount of venom and does not vary between
31 adults and children. Preclinical testing may be used to estimate starting doses and these
32 dosage regimens may be evaluated in a number of ways using standard effectiveness and
33 safety end-points. Dose regimens can be assessed approximately by using prospective
34 observational studies [95].
35 In these, the proportion of patients with good clinical outcomes (for example, restoration of
36 blood coagulability or failure to develop local wound necrosis) can be observed with different,
37 escalating or de-escalating doses of antivenom.
38 As part of the design of the study, it is important to determine the minimum number of patients
39 required to establish meaningful results by using sample size calculations [159]. Results may
40 sometimes be compared to those of previous studies (historical controls) to determine how the
41 effectiveness or safety of a newly introduced antivenom compares with previously used
42 antivenoms [160]. However, such comparisons are liable to many kinds of confounding
43 variables and are inherently unreliable. Subsequently, the minimum dose that appears to be
44 effective can be evaluated in larger phase II trials or compared to another antivenom or a
45 different dose in phase III randomized controlled trials.
46 18.2.2 Randomized controlled trials
47 Definitive phase III randomized controlled trials may require large numbers of patients because
48 of considerable individual variation in the clinical manifestation of envenoming (or the great
49 variability in the quantity and quality of venom injected in different patients). The new antivenom
50 is compared with the existing standard antivenom treatment or, if none exists, two different
51 doses of the test antivenom may be compared. Placebo controls are rarely justified unless there
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1 is genuine uncertainty about the risk and benefits of antivenom treatment. In this situation, as a
2 safeguard against unnecessary morbidity in either treatment group, a restricted sequential plan
3 might be incorporated [161] which allows evaluation of results as the trial progresses, as in the
4 early trials of therapeutic tetanus antitoxin [162].
5 To avoid bias, patients should be randomly allocated to the groups and the study should be
6 blinded, at a minimum to those research personnel who are assessing the clinical response and
7 ideally to both investigators and participants. There should be a calculation of the number of
8 patients required in each trial arm to give the study sufficient statistical power. These power
9 calculations are based on the expected difference in outcome between the treatment groups (if
10 designed to demonstrate superiority of one treatment over another) or predefined limits of the
11 acceptable performance compared to an existing product (if designed to demonstrate that the
12 new antivenom is not worse than existing products (non-inferiority)).
13 18.2.3 Effectiveness end-points for antivenom trials
14 The assessment criteria (end-points) used for antivenom studies should be predefined a-priori
15 and objective. They may be clinical or assessed by laboratory investigations. Common end-
16 points include mortality, development of local tissue effects of envenoming such as necrosis,
17 time taken to restore blood coagulability (assessed by the 20-minute whole blood clotting test)
18 [163], other laboratory parameters such as the prothrombin time, halting of bleeding or objective
19 clinical improvement in neurotoxicity. Surrogate markers such as platelet count are less suitable
20 as they may be affected by complement activation resulting from antivenom treatment itself.
21 Patients should be observed carefully for long enough to reveal evidence of recurrent
22 envenoming (seen particularly with short half-life Fab antivenoms) [164].
23 However, due to the high variability of the mode of action of venoms, that of the individual
24 patient's responses and diagnostic capacity of health centres, particularly in developing
25 countries, it is necessary to promote clinical researches to identify appropriate clinical and
26 laboratory criteria.
27 18.2.4 Safety end-points for antivenom trials
28 Because antivenoms consist of foreign proteins/fragments that are liable to aggregation,
29 adverse effects are an inevitable risk in therapy. Appropriate manufacturing steps can reduce
30 the rate of adverse reactions. Rates of reaction are correlated with the purity of the antivenom
31 product and the amount of protein infused. Continuous clinical observation at the bedside is
32 necessary for several hours after treatment to detect acute reactions; late adverse reactions
33 may occur several weeks later. Accurate reaction rates can only be assessed prospectively.
34 Reaction rates may differ considerably between different antivenoms, but only in most cases
35 only a small proportion are life-threatening. Although there is no consensus on classifying or
36 grading early adverse reactions (EAR), studies should aim to detect both early adverse events
37 (anaphylaxis and pyrogenicity) occurring at the time of, or within 24 hours of, antivenom
38 administration (such as urticaria itching, fever, hypotension or bronchospasm) and late
39 reactions such as serum sickness occurring between 5 and 24 days of antivenom administration
40 (e.g. fever, urticaria, arthralgia, lymphadenopathy, proteinuria, or neuropathy).
41 18.2.5 Challenges in clinical testing of antivenoms
42 Several particular features of snakebite make clinical testing of antivenoms challenging. These
43 features include the large variation in the consequences of envenoming between individuals
44 making it necessary to study large number of patients, difficulties in identification of the species
45 responsible for envenoming and the inaccessibility and logistical challenges of areas where
46 snakebite is sufficiently common to provide sufficient numbers of patients to study. Clinical
47 studies may also be expensive, particularly if they need to be multicentre with the attendant
48 additional complexity and logistics of between-centre variations. However, despite these
49 difficulties, a number of randomized controlled trials have been undertaken and published since
50 1974 [79, 83, 94, 163, 165-169].
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1 18.3 Post-marketing surveillance
2 Phase IV studies may be of much greater importance for antivenoms than is the case for other
3 products. A period of active post-licensing surveillance should follow:
4  the introduction of a new antivenom (often a regulatory requirement);
5  the introduction of an established antivenom into a new geographical area.
6 Post marketing studies of antivenoms examine efficacy as well as the frequency of immediate
7 or delayed side-effects. The combination of preclinical testing and post marketing surveillance
8 studies is a minimum acceptable clinical evaluation when an existing antivenom is used in a
9 new region.
10 18.3.1 Possible approaches
11 Passive surveillance is currently practised by some antivenom manufacturers. However,
12 approaches that rely upon voluntary return of questionnaires about safety and efficacy are
13 unlikely to provide the high quality data that are necessary. There are two potential approaches
14 to obtaining such data.
15 18.3.1.1 National or regional system for post-marketing surveillance
16 Countries using antivenoms should establish a national or regional system for the post
17 marketing surveillance of antivenoms. Clinicians and health workers (such as those working in
18 poison centres) should be encouraged to report actively to national control authorities and
19 manufacturers any unexpected lack of clinical efficacy and adverse reactions. These should
20 include both early adverse events, occurring at the time of, or within 24 hours of, antivenom
21 administration, and, late reactions between 5 and 24 days. The mechanism for reporting (such
22 as the use of standardized forms), the receiving body (e.g. the national control authority), the
23 deadline for reporting, and the type of adverse events reportable need to be clearly defined by
24 the authority and will depend on its structure and resources. The manufacturer of the antivenom
25 and the authorities should assess these reports and, in consultation with one another and with
26 specialists in the field, attempt to evaluate their significance. This assessment may require the
27 testing of products already released and the inspection of production, control facilities and local
28 distribution channels. If an imported product is associated with adverse reactions, the
29 manufacturer and the national control authorities both in the country of distribution and from the
30 country of origin should be notified.
31 18.3.1.2 Observational studies
32 In certain situations, for example, the first use of an established antivenom in a new
33 geographical area, or when routine surveillance has identified safety or effectiveness concerns,
34 there is a rationale for setting up observational studies to ensure adequate effectiveness and
35 safety. In the case of first use of an established antivenom in a new geographical area, such
36 studies should follow preclinical testing that ensures neutralization of locally important venoms.
37 Observational studies should carefully document the clinical responses to antivenom, the
38 clinical outcomes and the frequency of reactions in a substantial cohort of patients [170].
39 18.3.1.3 Sentinel sites
40 In some settings, where post marketing surveillance of the whole of a country may be
41 problematic, the use of sentinel sites may allow focusing of limited resources to maximize
42 surveillance effectiveness.
43 18.3.2 Responses to results of post-marketing studies
44 High quality post marketing studies will allow clinicians, public health officials and manufacturers
45 to identify antivenoms with poor effectiveness, between batch variations in potency/safety,
46 instances of incorrect use and dosage of antivenoms and serious safety issues arising from the
47 use of antivenoms. In some situations, these issues may be addressed by improving training of
48 staff in the management of snakebite, but these studies may also allow identification of the use
49 of an inappropriate antivenom [171].
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2  Preclinical and clinical testing of antivenoms has been largely neglected in the past.
3 Despite challenges, clinical trials of antivenoms in human patients have proved
4 feasible and useful. As far as possible, trials should adhere to the principles of WHO
5 and International Conference on Harmonisation (ICH) good clinical practice and
6 should measure robust, objective end-points.
7  National regulatory bodies should expect producers either to provide data confirming
8 the clinical efficacy and safety of their antivenoms against envenoming by local
9 species of venomous snakes or, to support in-country clinical testing of these
10 products.
11  Incorporating robust methodologies for reliable identification of the biting snake
12 species is absolutely essential to the design of all clinical trials and other clinical
13 studies of antivenoms.
14  Prospective observational studies are of some use in monitoring the effectiveness
15 and safety of an antivenom when first used in a new geographical region.
16  Post marketing surveillance studies should play a major role in the evaluation of
17 efficacy and safety of antivenoms.
18
19
20
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1 19 Role of national regulatory authorities
2 National Regulatory Authorities (NRAs) or Medicines Regulatory Authorities (MRAs) play a
3 crucial role in ensuring that pharmaceuticals, vaccines, biological and other medicinal products
4 that are available for use in a country have been carefully and thoroughly evaluated against
5 internationally recognised standards of safety and quality. These agencies of government are
6 vital to the process of strengthening health systems by providing regulatory controls based on
7 legislative frameworks and technical expertise. NRAs therefore have a pivotal role in ensuring
8 the quality, safety, and efficacy of antivenoms.
9 WHO Guidelines for national regulatory authorities on quality assurance of biological products
10 [172, 173] state that national regulatory authorities should ensure that available biological
11 products, whether imported or manufactured locally, are of good quality, safe and efficacious,
12 and should thus ensure that manufacturers adhere to approved standards regarding quality
13 assurance and good manufacturing practice. The responsibilities should also include the
14 enforcement and implementation of effective national regulations, and the setting of appropriate
15 standards and control measures. The evaluation and control of the quality, safety and
16 consistency of production of animal-derived blood products involve the evaluation of the starting
17 material, production processes and test methods to characterize batches of the product.
18 This requires the regulatory authorities to have appropriate expertise. WHO provides Member
19 States with support in the establishment of NRAs and with the development of regulatory
20 functions, technical abilities and adoption of standards and best practice guidelines, such as this
21 document. An example of a summary protocol for production and testing of snake antivenom
22 immunoglobulins to assist national regulatory authorities in reviewing the quality of antivenom
23 batches is shown in Annex 2.
24 19.1 Regulatory evaluation of antivenoms

25 The regulatory evaluation and control of the quality, safety and consistency of production of
26 antivenoms is summarized in Figure 7 and involves the evaluation and approval of:
27  the preparation of the starting plasma material from immunized animals, including the
28 preparation of snake venom batches representative of the poisonous animals of the
29 geographical region the antivenom is made for, and the animal husbandry, control and
30 traceability of the immunized animals and of the immunization process;
31  the fractionation process used to produce the antivenoms;
32  the test methods used to control batches of the product including realistic and validated
33 potency tests based on neutralisation of likely maximal envenomation;
34  shelf-life and stability testing of intermediates and final product
35  the preclinical data supporting the expected efficacy of the products for treatment of local
36 envenomings;
37  the clinical efficacy of locally manufactured or imported antivenoms against the species of
38 snakes found in the country, through active marketing surveillance.
39 19.2 Establishment licencing and site inspections

40 Many NRAs implement control systems based on licensing manufacturing establishments,
41 inspecting them regularly, and enforcing the implementation of the legal requirements and
42 applicable standards. This applies to the preparation of snake venoms, production of animal
43 hyperimmune plasma for fractionation, and the manufacturing process of the antivenoms.
44 Establishments involved in any or all stages of the manufacture of antivenoms need to have an
45 establishment licence and to be inspected by the competent NRA before operations commence.
46 To obtain the licence, the establishments have to fulfil a defined set of requirements to
47 guarantee that their operation ensures the safety, quality and efficacy of the antivenoms.
48
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Venom producer
G
M specifications
P A
u
I Plasma producer d
n i
s t
p specifications s
e
c
t Fractionator of antivenoms
i
o
n specifications
Antivenom
Post-marketing
surveillance
Antivenom marketing file evaluation and authorization
National regulatory authority

1
2 Figure 7
3 Schematic diagram for the regulatory evaluation and control of the quality, safety and standards
4 of production of antivenoms.
5 19.3 Impact of good manufacturing practices

6 Implementing the principles of GMP in the production of therapeutic products is acknowledged
7 as essential for assuring the quality and safety of biological medicinal products. For antivenoms,
8 GMP becomes even more important and more complex due to the biological nature of the
9 production process and the complexity and local specificities of snake envenoming.
10 Therefore, taking into account the principles of GMP and the existence of an appropriate quality
11 assurance system to address and implement these requirements at all stages of manufacture,
12 should be a pivotal element in ensuring the quality and safety of antivenoms. The following
13 benefits are expected:
14  ensures the application of quality assurance principles at all steps involved in the
15 preparation of snake venoms, the production of animal plasma and the fractionation process
16 of antivenoms;
17  reduces errors and technical problems at all stages of manufacture of plasma for
18 fractionation and antivenoms;
19  contributes to the release of products which comply with quality and safety requirements;
20  ensures adequate documentation and full traceability of plasma for fractionation and
21 antivenom production stages;
22  enables continuous improvement in production of plasma for fractionation and antivenoms;
23  provides suitable tools for the national regulatory authorities to assess the compliance status
24 of a manufacturer of antivenoms, either local or abroad;
25  supports regional cooperation networks that may result in the formation of competence
26 centres by centralizing activities to enable compliance to be achieved at the required level.
27 An establishment licensing system for antivenom manufacturers by the competent national
28 regulatory authorities should therefore exist. The main requirements to be met to obtain an
29 establishment licence may include especially:
30  quality assurance system and GMP applied to all steps of venom and antivenom production;
31  personnel directly involved in collection, testing, processing, storage and distribution of
32 antivenoms are appropriately qualified and provided with timely and relevant training;
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1  adequate premises and equipment are available;
2  an adequate control system to ensure traceability of antivenoms manufacture is to be
3 enforced through accurate identification procedures, through record maintenance, and
4 through an appropriate labelling system;
5  a postmarketing information system exists.
6 19.4 Inspections and audit systems in the production of antivenoms

7 The ongoing operations of antivenom manufacturers need to be subject to regulatory authority
8 control and supervision in accordance with legislation. Regulatory authorities can also make use
9 of WHO technical support services, monographs and guidelines to assist them in developing the
10 capacity to undertake ongoing inspection activities, audits and reviews of manufacturing, quality
11 control and other production process systems.
12 All manufacturers must have in place a quality assurance system for manufacture of animal-
13 derived plasma products that comprehensively covers all stages leading to the finished product,
14 from production of plasma (including venom sourcing and preparation, production animal
15 sourcing, selection, immunization, and animal health control) to the collection and fractionation
16 of the plasma into the finished products and their control. Manufacturers of antivenoms must
17 maintain complete site master files (SMFs) containing specific, factual details of the GMP
18 production and quality control manufacturing activities that are undertake at every site of
19 operations linked to the products they produce. Manufacturers should also maintain Quality
20 Manuals that define and describe the quality system, the scope and operations of the quality
21 system throughout all levels of production, management responsibilities, key quality systems
22 processes and safeguards. For individual products a Product Dossier (PD) in common technical
23 document (CTD) format as recommended by the WHO and International Conference on
24 Harmonization (ICH) may also be required. National regulatory authorities should make full use
25 of these three forms of production documentation in preparing for and conducting site
26 inspections and audits.
27 For local producers, the National Regulatory Authority should enforce the implementation of
28 GMP with the aim of ensuring the compliance of the manufacturer with the existing provisions. It
29 is the responsibility of the inspector from the national regulatory authority to ensure that
30 manufacturers adhere to the approved standards of GMP and quality assurance. The inspection
31 and enforcement of control measures for venom producers, immune plasma producers,
32 fractionation facilities and final product producers and distributors should be carried out by
33 officials representing the competent national regulatory authority. They should be familiar with
34 biological product technologies and trained in GMP inspections.
35 Inspections may follow common inspection procedures, including an opening meeting, a tour of
36 the facility, inspection of main areas and activities (such as serpentariums; animal husbandry
37 practices; animal identification and suitability for blood or plasma collection; process of
38 collection and storage of blood or plasma; plasma fractionation process; testing and availability
39 of test results for venoms, antivenoms, and raw materials; storage, transportation and shipment;
40 quality assurance (including internal audits and change control), documentation (standard
41 operating procedure, records, donor record files, log books), personnel and organization,
42 qualification and process validations, error and corrective action system, recalls and complaints,
43 product quality controls), and a final meeting summarizing the inspection outcome. A thorough
44 inspection includes the observation of staff during performance of operations and comparison
45 with established standard operating procedures. The inspection should not only be considered
46 as checking compliance with good manufacturing procedures, but also as an indirect product
47 quality assessment by checking product-specific validation and quality control data.
48 A written report should summarize the main findings of the inspection including its scope, a
49 description of the company, the deficiencies listed, specified and classified (e.g. as critical –
50 major – minor), and a conclusion. The written report is sent to the manufacturer. The
51 manufacturers are requested to notify the national regulatory authority about the specific steps
52 which are being taken or are planned to correct the failures and to prevent their recurrence. If
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1 necessary, follow-up inspections should be performed e.g. to check the successful
2 implementation of specific corrective actions.
3 The National Regulatory Authority should have the mandate to withdraw an establishment
4 licence in a case where inspection results show critical noncompliance with the requirements or
5 product specifications. In the procedure for granting marketing authorization for an antivenom,
6 information on the collection and control of the venoms and of the starting animal blood or
7 plasma needs to be documented as part of the dossier.
8 In summary, the enforcement and implementation of licensing and inspection regulatory
9 systems for antivenoms constitute fundamental tools to ensure the quality of antivenoms
10 produced or distributed to treat envenomings in a country.
11 19.5 Antivenom licensing

12 All antivenoms that are in use in a country must be approved and licensed by the appropriate
13 NRA or another competent authority with legal jurisdiction. The process for applying for,
14 considering and making a decision on the merits of an application should follow established
15 processes and be subject to transparency and review. The process of product dossier
16 assessment and review should be defined by legislation and appropriate regulations. Marketing
17 authorization (licensing) of antivenoms should be subject to a thorough review of the product
18 dossier, site master file and quality management system, and where the product is to be
19 imported, NRAs should communicate directly with the licensing authority in the country of
20 manufacturer to ensure that claims in the documents are factual and the product meets country
21 of origin licensing requirements.
22 19.6 National Reference Venoms

23 As discussed in Section 8.2 countries or regions should establish collections of reference
24 venoms (“standard venoms”) against which antivenom products and the venoms used by
25 manufacturers can be assessed. The establishment of reference venoms for release control of
26 final product should be reviewed and monitored by the regulatory authority or by other
27 competent authorities with technical expertise in the production of international reference
28 material standards. Antivenom manufacturers should not be involved in the production of
29 reference standards in order to ensure transparency. The task should be assigned to a central
30 quality control laboratory, or to a third-party organization with specific expertise. The potency of
31 each batch of final product should be confirmed by specific neutralization of a standard venom
32 of the (each) species of snake for which the antivenom is indicated.
33 A system of control for the reference venoms and for the design of the venom pools (e.g.: the
34 geographical selection of animals) should be in place as part of the procedures for the
35 management of reference venom collections.

37  National regulatory authorities (NRAs) should regulate and supervise local antivenom
38 manufacturers
39  National regulatory authorities are responsible for market authorization of antivenoms
40 distributed in the country.
41  Inspection and audit processes are fundamental to the effective regulation and
42 control of antivenoms and NRAs should seek appropriate assistance to develop both
43 the legislative and technical expertise necessary to undertake these functions.
44  Only antivenoms that pass stringent applications processes should be granted
45 market authorization.
46  National reference collections of “standard venoms” should be established according
47 to accepted international reference material standards, and used to independently
48 assess antivenoms, or to validate venoms used by manufacturers.
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1
2
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1 20 Authors and acknowledgements
2 20.1 First Edition
3 The first edition of these Guidelines was developed under the coordination of Dr A. Padilla,
4 Scientist, Quality Assurance and Safety: Blood Products and Related Biologicals, World Health
5 Organization, Geneva.
6 The development of this evidence-based document has been achieved thanks to the
7 collaboration of a large number of experts supporting specific areas of the Guidelines. The
8 experts who contributed to the drafting of the document are listed below:
9 The first draft was prepared by the late Dr Cassian Bon, Museum National d'Histoire Naturelle,
10 France; Dr Thierry Burnouf, Consultant, World Health Organization; Dr Jose-Maria Gutierrez,
11 Instituto Clodomiro Picado, Costa Rica; Dr A. Padilla, Quality Assurance and Safety of Blood
12 Products and Related Biologicals, World Health Organization, Switzerland; Professor Kavi
13 Ratanabanangkoon, Chulabhorn Research Institute, Thailand; and Professor DA Warrell,
14 University of Oxford, England.
15 The draft was submitted for preliminary discussion to the Expert Committee on Biological
16 Standardization (58th Meeting). This was followed by a detailed discussion within the World
17 Health Organization Blood Regulators Nerwork (BRN), the members of which were: Dr J.
18 Epstein, Food and Drug Administration (FDA), USA; Dr A. Farrugia, Therapeutic Goods
19 Administration (TGA), Australia; Dr P. Ganz, Health Canada, Canada; Dr E. Griffiths, Health
20 Canada, Canada; Dr M. Heiden, Paul-Ehrlich Institute (PEI), Germany; Dr I. Sainte Marie,
21 Agence française de Sécurité sanitaire des Produits de Santé (Afssaps), France; Dr C. Schärer,
22 Swissmedic, Switzerland; Dr R. Seitz, Paul-Ehrlich Institute (PEI), Germany (Chairman); Dr P.
23 Zorzi, Agence française de Sécurité sanitaire des Produits de Santé (Afssaps), France.
24 Because of the importance of these Guidelines in improving production and control of
25 antivenoms worldwide, two World Health Organization Bi-Regional Workshops were organized
26 in Asia and Africa, these being the Regions where snakebite envenomings constitute a major
27 public health problem. The Workshops aimed to discuss the draft Guidelines with clinical
28 toxinologists, manufacturers, national poison centres and regulators directly involved in
29 manufacture and regulation of antivenoms and in the treatment of snakebite envenomings in
30 those Regions. The Workshops were conducted by the World Health Organization with the
31 collaboration of the following facilitators:
32 Dr T. Burnouf, Human Protein Process Sciences, France; Dr J.P. Chippaux, Institut de
33 Recherche pour le Développement (IRD), Bolivia; Dr J.M. Gutierrez, Instituto Clodomiro Picado,
34 University of Costa Rica, Costa Rica; Dr G. Müller, University of Stellenbosch, South Africa
35 (retired); Dr A. Padilla, World Health Organization, Switzerland; Professor H.J. de Silva, Faculty
36 of Medicine, University of Kelaniya, Sri Lanka; Professor P. Gopalakrishnakone, Yong Loo Lin
37 School of Medicine, National University of Singapore, Singapore; Dr R. Harrison, Liverpool
38 School of Tropical Medicine, England; Dr D. Lalloo, Liverpool School of Tropical Medicine,
39 England; Prof. K. Ratanabanangkoon, Chulabhorn Research Institute, Thailand; Professor D.
40 Theakston, Liverpool School of Tropical Medicine, England (retired); Mr D. Williams, Australian
41 Venom Research Unit/Nossal Institute for Global Health, School of Medicine, University of
42 Melbourne, Australia.
43 Acknowledgements are due to the following participants of the World Health Organization Bi-
44 Regional South-East Asian and Western Pacific Workshop on Production, Control and
45 Regulation of Antivenoms in Jakarta, Indonesia, for their professional contributions and useful
46 discussions:
47 Mrs A. Abas, Centre for Product Registration, National Pharmaceutical Control Bureau, Ministry
48 of Health, Malaysia; Mrs S.S. Akter, Institute of Public Health, Bangladesh; Professor R. Alam,
49 Dhaka Medical College, Bangladesh; Dr S. Amudhavalli, King Institute of Preventive Medicine,
50 India; Mr A. Azhari, Bio Farma, Indonesia; Mr I. Baru, Drug Registration, Department of Health,
51 Papua New Guinea; Dr B. Bissumbhar, World Health Organization, Switzerland; Dr Chhuo
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1 Meng, Poisons Centre, Calmette Hospital, Cambodia; Mr M. C. Dancel, Research Institute for
2 Tropical Medicine, Philippines; Mr A. Fernandes, Bharat Serum and Vaccines Ltd, India; Dr S.
3 Gnanaiah, King Institute of Preventive Medicine, India; Dr A. Gnanathasan, Department of
4 Clinical Medicine, Faculty of Medicine, Sri Lanka; Ms L. St. Halimah, Bio Farma, Indonesia; Dr
5 Heng Bun Kiet, Department of Drugs and Food, Ministry of Health, Cambodia; Dr T.N. Huu,
6 Pasteur Institute, Viet Nam; Mrs W. Jariyapan, Department of Medical Sciences, Ministry of
7 Public Health, Thailand; Dr Jing Zhang, Lanzhou Institute of Biological Products, People's
8 Republic of China; Dr M.V. Khadilkar, Haffkine Biopharmaceutical Corporation Ltd, India;
9 Professor S. Khomvilai, Queen Saovabha Memorial Institute, Thai Red Cross, Thailand; Dr M.
10 Kuppusamy, VINS Bioproducts Ltd, India; Mrs D. Kusmiaty, National Agency of Drug and Food
11 Control, Indonesia; Dr Li Jingyu, Shanghai Serum Biological Technology Co Ltd, People's
12 Republic of China; Dr F. Malbas, Research Institute of Tropical Medicine, Muntinlupa,
13 Philippines; Brig. Gen. S. M.A. Matin, Directorate of Drug Administration, Bangladesh; Ms
14 M.T.S. Modina, Bureau of Food and Drugs, Philippines; Ms A. Mohamed Ariff, National Poison
15 Center, Penang, Malaysia; Mr A. Nair, VINS Bioproducts Ltd, India; Mr N. T. Nguyen, Poison
16 Control Centre, Viet Nam; Ms L. Nitisaporn, The Government Pharmaceutical Organization,
17 Thailand; Dr L.R. Panganiban, National Poison Management and Control Center, University of
18 the Philippines, Philippines; Dr V.V. Pillay, Amrita Institute of Medical Sciences & Research,
19 India; Mr K. Ragas, CSL Biotherapies, Victoria, Australia; Dr Y. Sano, World Health
20 Organization Regional Office for the Western Pacific, the Philippines; Dr B. Santoso, World
21 Health Organization Regional Office for the Western Pacific, the Philippines; Dr M. Shahjahan,
22 World Health Organization, Country Office of Indonesia; Dr Shumin Zhang, National Institute for
23 the Control of Pharmaceutical and Biological Products, People's Republic of China; Dr L.S.
24 Slamet, National Agency of Drug and Food Control, Indonesia; Mr J. Smith, CSL Biotherapies,
25 Victoria Australia; Dr D. Sundari, Poison Information Center, Indonesia; Dr M. Takahashi,
26 National Institute of Infectious Diseases, Japan; Mr B. Thapa, Ministry of Health and Population,
27 Nepal; Dr C.L. Thapa, Ministry of Health and Population, Dumkauli Primary Health Care Center,
28 Nepal; Dr M. Toriba, Japan Snake Institute, Japan; Mrs D. Vu Bach, Drug Registration
29 Department, Ministry of Health, Viet Nam; Professor J. White, Women's and Children's Hospital,
30 Australia; Dr Zhou Jing, Center for Acute Poisoning Control, Beijing, People’s Republic of
31 China.
32 Acknowledgements are also due to the participants at the World Health Organization Bi-
33 Regional African and Eastern Mediterranean Workshop on Production, Control and Regulation
34 of Antivenoms in Addis Ababa, Ethiopia for their contributions and willingness to share expertise
35 and information:
36 Dr F. Aljenoobi, Food and Drug Authority, Saudi Arabia; Dr B. Allali Kouadio, Pasteur Institute,
37 Côte d'Ivoire; Dr S. Ansar Ahmad, Ministry of Health, Pakistan; Dr M. Atef Abd-Elsalam,
38 National Antivenom & Vaccine Production Center, Saudi Arabia; Dr S. Bah, Institut National de
39 Recherche en Santé publique, Mali; Dr S. Bokata Masika, University of Kinshasa, Democratic
40 Republic of Congo; Dr C.M. Baldé, Ministry of Health, Pasteur Institute, Guinea; Dr B.
41 Bissumbhar, World Health Organization, Switzerland; Dr M. Chisale, World Health Organization,
42 Regional Office for Africa, Congo; Professor A. Diouf, Poison Centre, Health Ministry and
43 Prevention, Senegal; Dr N. Durfa, Federal Ministry of Health, Nigeria; Dr B. Ed Nignpense,
44 Poisons Information & Control Centre, Ridge Hospital, Ghana; Dr M. El-Sayed Aly Ibrahim,
45 National Regulatory Authority, Egypt; Dr A.G. Etoundi Mballa, Hôpital Central de Yaoundé,
46 Cameroon; Dr N.D. Fall Diop, Ministry of Health and Prevention, Senegal; Dr V. Gomwe,
47 Medicines Control Authority, Zimbabwe; Mr M. Haladou, Ministry of Health, Niger; Dr G. Habib,
48 Organization for Biological Products and Vaccines, Egypt; Dr C. Ilonze, National Agency for
49 Food and Drug Administration and Control, Nigeria; Mrs N. Khalid Nomani, National Institute of
50 Health, Pakistan; Dr F. Kolon Diallo, Direction Nationale de la Pharmacie et du Laboratoire,
51 Ministry of Health, Guinea; Dr H. Langar, World Health Organization, Regional Office for the
52 Eastern Mediterranean, Cairo; Mr T. Makoe, South African Vaccine Producers Ltd, South Africa;
53 Ms M. Masanja, Food and Drugs Authority, United Republic of Tanzania; Professor A.
54 Massougbodji, Unit of Parasitology, Benin; Dr A. Mhenni, Pasteur Institute, Tunisia; Dr A.
55 Mohanna Mohamed, Egyptian Organization for Biological Products and Vaccines, Egypt; Dr F.
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1 Moin Siyoi, Pharmacy and Poisons Board, Ministry of Health, Kenya; Professor C. Nhachi,
2 University of Zimbabwe Medical School, Zimbabwe; Dr G. Noreddine, Pasteur Institute,
3 Morocco; Mr A. Okello Okonye, National Drug Authority, Uganda; Mr K.C. Yemoa, Direction des
4 Pharmacies et du Médicament, Ministère de la Santé, Benin.
5 The second draft of these Guidelines was prepared, taking into consideration the information
6 and discussions held at the abovementioned Workshops. The following experts contributed to
7 the preparation of this draft: Dr T. Burnouf, Human Protein Process Sciences, France; Dr J. M.
8 Gutiérrez, Instituto Clodomiro Picado, University of Costa Rica, Costa Rica; Professor Geoff
9 Isbister, Menzies School of Health Research, Australia; Dr A. Padilla, World Health
10 Organization, Switzerland; Professor H. J. de Silva, Faculty of Medicine, University of Kelaniya,
11 Sri Lanka; Professor P. Gopalakrishnakone, Yong Loo Lin School of Medicine, National
12 University of Singapore, Singapore; Dr Rafael Otero, Programa de Ofidismo/Escorpionismo y
13 Grupo Toxaven, Facultad de Medicina, Universidad de Antioquia Medellín, Colombia; Dr K.
14 Ragas, CSL Biotherapeutics, Australia; Dr R. Harrison, Liverpool School of Tropical Medicine,
15 England; Dr D. Lalloo, Liverpool School of Tropical Medicine, England; Professor K.
16 Ratanabanangkoon, Chulabhorn Research Institute, Thailand; Professor D. Theakston,
17 Liverpool School of Tropical Medicine, England (retired); Professor Julian White, Women's and
18 Children Hospital, Australia.
19 Chapter 5 and Annex 1 of these Guidelines, on distribution of the venomous snakes of the
20 highest medical importance worldwide, provides extremely valuable and detailed information
21 that will assist manufacturers, regulators, public health officials, governments and
22 nongovernmental organizations, as well as international procurement agencies, to make
23 informed decisions with regard to the antivenoms to be considered within a particular region,
24 country or territory. This Appendix was prepared by Mr D. Williams, Australian Venom Research
25 Unit/Nossal Institute for Global Health, School of Medicine, University of Melbourne; Dr M.
26 O’Shea, Australian Venom Research Unit, School of Medicine, University of Melbourne and Dr
27 W. Wüster, School of Biological Sciences, University of Wales. The final Draft of the Annex was
28 reviewed by Dr D. Broadley, The National Museum of Zimbabwe, Zimbabwe; Dr J.P. Chippaux,
29 Institut de Recherche pour le Développement (IRD), La Paz, Bolivia; Dr B. Currie, Menzies
30 School for Health Research, Darwin, Australia; Dr J.M. Gutiérrez, Instituto Clodomiro Picado,
31 University of Costa Rica, San José, Costa Rica; Dr S. Seifert, USA; Professor D.A. Warrell,
32 University of Oxford, England; Professor J. White, Women's and Children Hospital, Adelaide,
33 Australia.
34 20.1.1 WHO Secretariat
35 Dr B. Bissumbhar, Quality Assurance and Safety: Blood Products and Related Biologicals,
36 World Health Organization, Geneva, Switzerland; Dr M. Chisale, World Health Organization,
37 Regional Office for Africa; Dr H. Langar, World Health Organization, Regional Office for the
38 Eastern Mediterranean; Dr F. Nafo-Trafore, World Health Organization Country Office of
39 Ethiopia; Dr A. Padilla, Quality Assurance and Safety: Blood Products and Related Biologicals,
40 World Health Organization, Geneva, Switzerland; Dr Y. Sano, World Health Organization,
41 Regional Office for the Western Pacific; Dr B. Santoso, World Health Organization, Regional
42 Office for the Western Pacific; Dr M. Shahjahan, World Health Organization, Country Office of
43 Indonesia; Dr K. Weerasuriya, World Health Organization, Regional Office for South East Asia.
44 20.2 Second Edition

45 The second edition of these Guidelines was revised and redrafted in 2016 under the
46 coordination of Dr C. M. Nuebling, Scientist, Blood Products and Related Biologicals, World
47 Health Organization, Geneva. Dr D. J. Williams, Australian Venom Research Unit, University of
48 Melbourne, Australia, led the editing of the new draft. Reviewers included: Emeritus Professor
49 D. A. Warrell, Oxford University, United Kingdom; Professor A. Habib, Bayero University,
50 Nigeria; Dr W. Wüster, University of Bangor, United Kingdom; K. Ragas, CSL Limited, Australia;
51 Dr R. Harrision, Liverpool School of Tropical Medicine, United Kingdom; Mrs D. Barr, University
52 of Melbourne, Australia; Dr K. Grego, Instituto Butantan, Brazil; Professor J-M. Gutierrez,
53 Instituto Clodomiro Picado, Costa Rica; Dr T. Burnouf, Human Protein Process Sciences,
WHO/BS/2016.2300
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1 France; Professor K. Ratanabanangkoon, Mahidol University, Thailand; Dr Fan Hui Wen,
2 Instituto Butantan, Brazil; Dr A. Britton, Ultimate Efficacy Consulting Pty Ltd, Australia; Dr G.
3 Leon, Instituto Clodomiro Picado, Costa Rica; Dr J. Marcelino, Instituto Butantan, Brazil; Dr R.
4 Ferreira, Instituto Butantan, Brazil; Dr C. Graner, Instituto Butantan, Brazil; Dr F. Petitto de
5 Assis, Instituto Butantan, Brazil; Professor J. J, Calvete, Instituto de Biomedicinia de Valencia,
6 Spain; Dr G. Alcoba, Geneva University Hospitals, Switzerland; Dr F. Chappuis, Geneva
7 University Hospitals, Switzerland; Dr J-P. Chippaux, Institut de Recherche pour le
8 Développement (IRD), Benin; Dr C Rolls, Australia; and Dr J. Southern, South Africa.
9
WHO/BS/2016.2300
Page 110
1 21 References
2 1. World Health Organization. WHO Model List of Essential Medicines. 2007 [cited 2016
3 17/08/16]; 15th:[Available from:
4 http://www.who.int/medicines/publications/08_ENGLISH_indexFINAL_EML15.pdf.
5 2. Von Behring, E. and S. Kitasato, Über das Zustande-kommen der Diphtherie-Immunität
6 und der Tetanus-Immunität bei Thieren. . Deutsche Medizinische Wochenschrif, 1890.
7 16: p. 1113-1145.
8 3. Phisalix, C. and G. Bertrand, Sur la propriété antitoxique du sang des animaux vaccinés
9 contre le venin de vipère. Comptes Rendus de la Société de Biologie, 1894. 46: p. 111-
10 113.
11 4. Calmette, A., Contribution à l'étude du venin des serpents. Immunisation des animaux et
12 traitement de l'envenimation. . Annales de l'Institut Pasteur, VIII, 1894: p. 275-291.
13 5. Calmette, A., Sur le venin des serpents et sur l'emploi du sérum antivenimeux dans la
14 thérapeutique des morsures venimeuses chez l'homme et chez les animaux. Annales de
15 l'Institut Pasteur XII., 1897: p. 214-237.
16 6. Pope, C.G., The action of proteolytic enzymes on the antitoxins and proteins in immune
17 sera. I. true digestion of the proteins. . British Journal of Experimental Pathology., 1939.
18 20: p. 132-149.
19 7. Pope, C.G., The action of proteolytic enzymes on the antitoxins and proteins in
20 immmune sera. II. Heat denaturation after partial enzyme action. . British Journal of
21 Experimental Pathology., 1939. 20: p. 201-212.
22 8. World Health Organization, Progress in the characterization of venoms and
23 standardization of antivenoms. . 1981 Geneva.
24 9. Raw, I.e.a., Antivenins in Brazil: Preparation. In: (ed), , in Handbook of Natural Toxins,
25 Vol 5, Reptile venoms and toxins., A.T. Tu, Editor. 1991, Marcel Dekker: New York. p.
26 557-581.
27 10. Grandgeorge, M.e.a., Preparation of improved F(ab’)2 antivenoms. An example: new
28 polyvalent European viper antivenom (equine). in Envenomings and their Treatments. ,
29 C. Bon and M. Goyffon, Editors. 1996, Fondation Marcel Mérieux: Lyon. p. 161-172.
30 11. Gutierrez, J.M., G. Leon, and B. Lomonte, Pharmacokinetic-pharmacodynamic
31 relationships of immunoglobulin therapy for envenomation. Clin Pharmacokinet, 2003.
32 42(8): p. 721-41.
33 12. World Health Organization. Rabies and envenomings, a neglected public health issue.
34 Report of a Consultative Meeting. . 2007; Available from:
35 http://www.who.int/bloodproducts/animal_sera/Rabies.pdf.
36 13. Swaroop, S. and B. Grab, Snakebite mortality in the world. Bull World Health Organ,
37 1954. 10(1): p. 35-76.
38 14. Chippaux, J.P., Snake-bites: appraisal of the global situation. Bull World Health Organ,
39 1998. 76(5): p. 515-24.
40 15. Snow, R.W., et al., The prevalence and morbidity of snake bite and treatment-seeking
41 behaviour among a rural Kenyan population. Ann Trop Med Parasitol, 1994. 88(6): p.
42 665-71.
43 16. Fox, S., et al., Underestimation of snakebite mortality by hospital statistics in the
44 Monaragala District of Sri Lanka. Trans R Soc Trop Med Hyg, 2006. 100(7): p. 693-5.
45 17. Hati, A.K., et al., Epidemiology of snake bite in the district of Burdwan, West Bengal. J
46 Indian Med Assoc, 1992. 90(6): p. 145-7.
47 18. Pugh, R.N. and R.D. Theakston, Incidence and mortality on snake bite in savanna
48 Nigeria. Lancet, 1980. 2(8205): p. 1181-3.
49 19. Sharma, S.K., et al., Impact of snake bites and determinants of fatal outcomes in
50 southeastern Nepal. Am J Trop Med Hyg, 2004. 71(2): p. 234-8.
51 20. Trape, J.F., et al., High mortality from snakebite in south-eastern Senegal. Trans R Soc
52 Trop Med Hyg, 2001. 95(4): p. 420-3.
53 21. Mohapatra, B., et al., Snakebite mortality in India: a nationally representative mortality
54 survey. PLoS Negl Trop Dis, 2011. 5(4): p. e1018.
WHO/BS/2016.2300
Page 111
1 22. Rahman, R., et al., Annual incidence of snake bite in rural bangladesh. PLoS Negl Trop
2 Dis, 2010. 4(10): p. e860.
3 23. Ediriweera, D.S., et al., Mapping the Risk of Snakebite in Sri Lanka - A National Survey
4 with Geospatial Analysis. PLoS Negl Trop Dis, 2016. 10(7): p. e0004813.
5 24. Kasturiratne, A., et al., The global burden of snakebite: a literature analysis and
6 modelling based on regional estimates of envenoming and deaths. PLoS Med, 2008.
7 5(11): p. e218.
8 25. World Health Organization. International statistical classification of diseases and related
9 health problems. 2007; 10th:[Available from:
10 http://www.who.int/classifications/icd/en/index.html.
11 26. Theakston, R.D. and D.A. Warrell, Antivenoms: a list of hyperimmune sera currently
12 available for the treatment of envenoming by bites and stings. Toxicon, 1991. 29(12): p.
13 1419-70.
14 27. Ismail, M., et al., Preparation of a novel antivenom against Atractaspis and Walterinnesia
15 venoms. Toxicon, 2007. 49(1): p. 8-18.
16 28. Joseph, J.K., et al., First authenticated cases of life-threatening envenoming by the
17 hump-nosed pit viper (Hypnale hypnale) in India. Trans R Soc Trop Med Hyg, 2007.
18 101(1): p. 85-90.
19 29. Ariaratnam, C.A., et al., Frequent and potentially fatal envenoming by hump-nosed pit
20 vipers (Hypnale hypnale and H. nepa) in Sri Lanka: lack of effective antivenom. Trans R
21 Soc Trop Med Hyg, 2008. 102(11): p. 1120-6.
22 30. Chippaux, J.P., V. Williams, and J. White, Snake venom variability: methods of study,
23 results and interpretation. Toxicon, 1991. 29(11): p. 1279-303.
24 31. Warrell, D.A., Geographical and intraspecies variation in the clinical manifestations of
25 envenoming by snakes., in Venomous snakes. Ecology, evolution and snakebite., R.S.
26 Thorpe, Wüster. W., and M. A., Editors. 1997, Clarendon Press: Oxford. p. 189-203.
27 32. Fry, B.G., et al. , Effectiveness of snake antivenom: species and regional variation and
28 its clinical impact. . Journal of Toxicology: Toxin Reviews, 2003. 22: p. 23-34.
29 33. Raweerith, R. and K. Ratanabanangkoon, Immunochemical and biochemical
30 comparisons of equine monovalent and polyvalent snake antivenoms. Toxicon, 2005.
31 45(3): p. 369-75.
32 34. Saravia, P., et al., Geographic and ontogenic variability in the venom of the neotropical
33 rattlesnake Crotalus durissus: pathophysiological and therapeutic implications. Rev Biol
34 Trop, 2002. 50(1): p. 337-46.
35 35. Faure, G. and C. Bon, Several isoforms of crotoxin are present in individual venoms
36 from the South American rattlesnake Crotalus durissus terrificus. Toxicon, 1987. 25(2):
37 p. 229-34.
38 36. Creer, S., et al., Genetic and ecological correlates of intraspecific variation in pitviper
39 venom composition detected using matrix-assisted laser desorption time-of-flight mass
40 spectrometry (MALDI-TOF-MS) and isoelectric focusing. J Mol Evol, 2003. 56(3): p. 317-
41 29.
42 37. Tan, K.Y., et al., Geographical venom variations of the Southeast Asian monocled cobra
43 (Naja kaouthia): venom-induced neuromuscular depression and antivenom
44 neutralization. Comp Biochem Physiol C Toxicol Pharmacol, 2016. 185-186: p. 77-86.
45 38. Tan, K.Y., et al., Venomics, lethality and neutralization of Naja kaouthia (monocled
46 cobra) venoms from three different geographical regions of Southeast Asia. J
47 Proteomics, 2015. 120: p. 105-25.
48 39. Calvete, J.J., et al., Snake population venomics and antivenomics of Bothrops atrox:
49 Paedomorphism along its transamazonian dispersal and implications of geographic
50 venom variability on snakebite management. J Proteomics, 2011. 74(4): p. 510-27.
51 40. Reichenbach-Klinke, H. and E. Elkan, Diseases of reptiles. . 1965, Neptune New
52 Jersey.: TFH Publications.
53 41. Frye, F.L., Reptile care. An atlas of diseases and treatments, 2 vols. 1991, Neptune,
54 New Jersey: TFH Publications.
55 42. Cooper, J.E. and O.F. Jackson, Diseases of the reptilia, 2 Vols. . 1981, London:
56 Academic Press.
WHO/BS/2016.2300
Page 112
1 43. Shortridge, K.F., et al., Arbovirus infections in reptiles: immunological evidence for a high
2 incidence of Japanese encephalitis virus in the cobra Naja naja. Trans R Soc Trop Med
3 Hyg, 1974. 68(6): p. 454-60.
4 44. Gans, C., K.A. Gans, and L. P., Various aspects of venomous snake breeding on a large
5 scale. . Acta Zoologica et Pathologica Antverpiensia., 1984, . 78: p. 177-198.
6 45. Mitchell, M.A., Snake care and husbandry. , . Veterinary Clinics of North America: Exotic
7 Animal Practice, 2004. 7: p. 421-446.
8 46. Chanhome, L., et al., Venomous snake husbandry in Thailand. Wilderness Environ Med,
9 2001. 12(1): p. 17-23.
10 47. Gutierrez, J.M., F. Chaves, and R. Bolanos, [Comparative study of venoms of newborn
11 and adult specimens of Bothrops asper]. Rev Biol Trop, 1980. 28(2): p. 341-51.
12 48. Furtado, M.F., et al., Comparative study of nine Bothrops snake venoms from adult
13 female snakes and their offspring. Toxicon, 1991. 29(2): p. 219-26.
14 49. Alape-Giron, A., et al., Snake venomics of the lancehead pitviper Bothrops asper:
15 geographic, individual, and ontogenetic variations. J Proteome Res, 2008. 7(8): p. 3556-
16 71.
17 50. Goebel-Stengel, M., et al., The importance of using the optimal plasticware and
18 glassware in studies involving peptides. Anal Biochem, 2011. 414(1): p. 38-46.
19 51. Kurnik, D., Y. Haviv, and E. Kochva, A snake bite by the Burrowing Asp, Atractaspis
20 engaddensis. Toxicon, 1999. 37(1): p. 223-7.
21 52. Harvey, D.S., et al., Moving massasaugas: Insight into rattlesnake relocation using
22 Sistrurus c. catenatus. Herpetological Conservation and Biology, 2014. 9(1): p. 67-75.
23 53. Roe, J.H., et al., No place like home: an experimental comparison of reintroduction
24 strategies using snakes. Journal of Applied Ecology, 2010. 47: p. 1253-1261.
25 54. Butler, H., B. Malone, and N. Clemann, The effects of translocation on the spatial
26 ecology of tiger snakes (Notechis scutatus) in a suburban landscape. Wildlife Research,
27 2005. 32: p. 165-171.
28 55. Reinert, H.K. and R.R. Rupert, Impacts of translocation on behaviour and survival of
29 timber rattlesnakes, Crotalus horridus. Journal of Herpetology, 1999. 33(1): p. 45-61.
30 56. Powell, R.L., E.E. Sanchez, and J.C. Pérez, Farming of venom: Survey of snake venom
31 extraction facilities worldwide. . Applied Herpetology., 2006. 3: p. 1-10.
32 57. Nishioka, S.A., et al., Occupational injuries with captive lance-headed vipers (Bothrops
33 moojeni): experience from a snake farm in Brazil. Trop Med Int Health, 2000. 5(7): p.
34 507-10.
35 58. de Medeiros, C.R., et al., Predictors of Bothrops jararaca venom allergy in snake
36 handlers and snake venom handlers. Toxicon, 2008. 51(4): p. 672-80.
37 59. Warrell, D.A., Envenoming, poisoning, hypersensitivity and injuries caused by animals,
38 in Oxford Textbook of Medicine, D.A. Warrell, T.M. Cox, and J.D. Firth, Editors. 2005,
39 Oxford University Press: Oxford.
40 60. Wüster, W. and C.J. McCarthy, Venomous snake systematics: Implications for snakebite
41 treatment and toxinology. , in In: Envenomings and their Treatments., C. Bon and M.
42 Goyffon, Editors. 1996, Fondation Mérieux.: Lyon. p. 13-23.
43 61. León, G., et al., Industrial production and quality control of snake antivenoms. , in
44 Venom Genomics and Proteomics. , P. Gopalakrishnakone, Calvete, J.J, Editor. 2016,
45 Springer Netherlands. p. 1-22.
46 62. Angulo, Y., R. Estrada, and J.M. Gutierrez, Clinical and laboratory alterations in horses
47 during immunization with snake venoms for the production of polyvalent (Crotalinae)
48 antivenom. Toxicon, 1997. 35(1): p. 81-90.
49 63. Malikides, N., et al., Cardiovascular, haematological and biochemical responses after
50 large volume blood collection in horses. Vet J, 2001. 162(1): p. 44-55.
51 64. Landon, J., J.A. Woolley, and C. McLean, Antibody production in the hen. , in
52 Therapeutic antibodies. , J. Landon and T. Chard, Editors. 1995:, Springer-Verlag:
53 London. p. 47-68.
54 65. Landon, J. and D. Smith, Merits of sheep antisera for antivenom manufacture. . Journal
55 of Toxicology: Toxin Reviews, 2003, . 22: p. 15-22.
WHO/BS/2016.2300
Page 113
1 66. Theakston, R.D., D.A. Warrell, and E. Griffiths, Report of a WHO workshop on the
2 standardization and control of antivenoms. Toxicon, 2003. 41(5): p. 541-57.
3 67. World Health Organization. Guidelines on tissue infectivity distribution in transmissible
4 spongiform encephalopathies. . 2006 [cited 2016 17/08/16]; Available from:
5 http://www.who.int/bloodproducts/cs/TSEPUBLISHEDREPORT.pdf.
6 68. Moroz-Perlmutter, C., et al. , Detoxification of snake venoms and venom fractions by
7 formaldehyde. Proceedings of the Society for Experimental Biology and Medicine., 1963.
8 112: p. 595-598.
9 69. Freitas, T.V. and e. al., Immunization of horses with Crotalus durissus terrificus (South
10 American rattlesnake) venom. A comparison of four different procedures. . Revista
11 brasileira de pesquisas medicas e biologicas/Sociedade Brasileira de Biofisica, 1991.
12 24: p. 281-290.
13 70. Reed, S.G., et al., The science of vaccine adjuvants: advances in TLR4 ligand
14 adjuvants. Curr Opin Immunol, 2016. 41: p. 85-90.
15 71. Pratanaphon, R., et al., Production of highly potent horse antivenom against the Thai
16 cobra (Naja kaouthia). Vaccine, 1997. 15(14): p. 1523-8.
17 72. Chotwiwatthanakun, C., et al., Production of potent polyvalent antivenom against three
18 elapid venoms using a low dose, low volume, multi-site immunization protocol. Toxicon,
19 2001. 39(10): p. 1487-94.
20 73. World Health Organization, Quality assurance of pharmaceuticals : a compendium of
21 guidelines and related materials. Volume 2, Good manufacturing practices and
22 inspection. 2nd updated ed. 2007, Geneva: World Health Organization. iv, 409 p.
23 74. World Health Organization, Guidelines on viral inactivation and removal procedures
24 intended to assure the viral safety of human blood plasma products. . 2004: Geneva, .
25 75. World Health Organization, Recommendations for the production, control and regulation
26 of human plasma for fractionation. . 2007: Geneva. p. Annex 4: 189-246.
27 76. European Agency for the Evaluation of Medicinal Products (EMEA), CPMP. Note for
28 guidance on production and quality control of animal immunoglobulins and immunsera
29 for human use. . 2002: London. p. 1-14.
30 77. Bolanos, R. and L. Cerdas, [Production and control of antivenin sera in Costa Rica]. Bol
31 Oficina Sanit Panam, 1980. 88(3): p. 189-96.
32 78. Rojas, G., J.M. Jimenez, and J.M. Gutierrez, Caprylic acid fractionation of hyperimmune
33 horse plasma: description of a simple procedure for antivenom production. Toxicon,
34 1994. 32(3): p. 351-63.
35 79. Otero, R., et al., A randomized blinded clinical trial of two antivenoms, prepared by
36 caprylic acid or ammonium sulphate fractionation of IgG, in Bothrops and Porthidium
37 snake bites in Colombia: correlation between safety and biochemical characteristics of
38 antivenoms. Toxicon, 1999. 37(6): p. 895-908.
39 80. Steinbuch, M. and R. Audran, The isolation of IgG from mammalian sera with the aid of
40 caprylic acid. Arch Biochem Biophys, 1969. 134(2): p. 279-84.
41 81. Gutierrez, J.M., et al., Pan-African polyspecific antivenom produced by caprylic acid
42 purification of horse IgG: an alternative to the antivenom crisis in Africa. Trans R Soc
43 Trop Med Hyg, 2005. 99(6): p. 468-75.
44 82. dos Santos, M.C., et al., Purification of F(ab')2 anti-snake venom by caprylic acid: a fast
45 method for obtaining IgG fragments with high neutralization activity, purity and yield.
46 Toxicon, 1989. 27(3): p. 297-303.
47 83. Otero-Patino, R., et al., A randomized, blinded, comparative trial of one pepsin-digested
48 and two whole IgG antivenoms for Bothrops snake bites in Uraba, Colombia. The
49 Regional Group on Antivenom Therapy Research (REGATHER). Am J Trop Med Hyg,
50 1998. 58(2): p. 183-9.
51 84. Abubakar, I.S., et al., Randomised controlled double-blind non-inferiority trial of two
52 antivenoms for saw-scaled or carpet viper (Echis ocellatus) envenoming in Nigeria.
53 PLoS Negl Trop Dis, 2010. 4(7): p. e767.
54 85. Jones, R.G. and J. Landon, A protocol for 'enhanced pepsin digestion': a step by step
55 method for obtaining pure antibody fragments in high yield from serum. J Immunol
56 Methods, 2003. 275(1-2): p. 239-50.
WHO/BS/2016.2300
Page 114
1 86. Raweerith, R. and K. Ratanabanangkoon, Fractionation of equine antivenom using
2 caprylic acid precipitation in combination with cationic ion-exchange chromatography. J
3 Immunol Methods, 2003. 282(1-2): p. 63-72.
4 87. Al-Abdulla, I., et al., Formulation of a liquid ovine Fab-based antivenom for the treatment
5 of envenomation by the Nigerian carpet viper (Echis ocellatus). Toxicon, 2003. 42(4): p.
6 399-404.
7 88. Saetang, T., et al., Quantitative comparison on the refinement of horse antivenom by salt
8 fractionation and ion-exchange chromatography. J Chromatogr B Biomed Sci Appl,
9 1997. 700(1-2): p. 233-9.
10 89. Sullivan, J.B., Jr. and F.E. Russell, Isolation and purification of antibodies to rattlesnake
11 venom by affinity chromatography. Proc West Pharmacol Soc, 1982. 25: p. 185-92.
12 90. Magos, L., Review on the toxicity of ethylmercury, including its presence as a
13 preservative in biological and pharmaceutical products. J Appl Toxicol, 2001. 21(1): p. 1-
14 5.
15 91. World Health Organization, Guidelines on regulatory expectations related to the
16 elimination, reduction or replacement of thiomersal in vaccines. . 2004: Geneva. p.
17 Annex 4.
18 92. Pikal, M.J., Freeze drying. , in Encyclopedia of pharmaceutical technology. 2002, Marcel
19 Dekker: New York. p. 1299-1326.
20 93. Herrera, M., et al., Freeze-dried snake antivenoms formulated with sorbitol, sucrose or
21 mannitol: comparison of their stability in an accelerated test. Toxicon, 2014. 90: p. 56-63.
22 94. Meyer, W.P., et al., First clinical experiences with a new ovine Fab Echis ocellatus snake
23 bite antivenom in Nigeria: randomized comparative trial with Institute Pasteur Serum
24 (Ipser) Africa antivenom. Am J Trop Med Hyg, 1997. 56(3): p. 291-300.
25 95. Ariaratnam, C.A., et al., A new monospecific ovine Fab fragment antivenom for
26 treatment of envenoming by the Sri Lankan Russell's viper (Daboia Russelii Russelii): a
27 preliminary dose-finding and pharmacokinetic study. Am J Trop Med Hyg, 1999. 61(2):
28 p. 259-65.
29 96. Scherrmann, J.M., Antibody treatment of toxin poisoning--recent advances. J Toxicol
30 Clin Toxicol, 1994. 32(4): p. 363-75.
31 97. Ho, M., et al., Pharmacokinetics of three commercial antivenoms in patients envenomed
32 by the Malayan pit viper, Calloselasma rhodostoma, in Thailand. Am J Trop Med Hyg,
33 1990. 42(3): p. 260-6.
34 98. Choumet, V., et al., New approaches in antivenom therapy. Adv Exp Med Biol, 1996.
35 391: p. 515-20.
36 99. Audebert, F., et al., Viper bites in France: clinical and biological evaluation; kinetics of
37 envenomations. Hum Exp Toxicol, 1994. 13(10): p. 683-8.
38 100. Boyer, L.V., S.A. Seifert, and J.S. Cain, Recurrence phenomena after immunoglobulin
39 therapy for snake envenomations: Part 2. Guidelines for clinical management with
40 crotaline Fab antivenom. Ann Emerg Med, 2001. 37(2): p. 196-201.
41 101. Riviere, G., et al., Absorption and elimination of viper venom after antivenom
42 administration. J Pharmacol Exp Ther, 1998. 285(2): p. 490-5.
43 102. Riviere, G., et al., Effect of antivenom on venom pharmacokinetics in experimentally
44 envenomed rabbits: toward an optimization of antivenom therapy. J Pharmacol Exp
45 Ther, 1997. 281(1): p. 1-8.
46 103. Burnouf, T., et al., Assessment of the viral safety of antivenoms fractionated from equine
47 plasma. Biologicals, 2004. 32(3): p. 115-28.
48 104. Lu, G., et al., First Description of Hepacivirus and Pegivirus Infection in Domestic Horses
49 in China: A Study in Guangdong Province, Heilongjiang Province and Hong Kong
50 District. PLoS One, 2016. 11(5): p. e0155662.
51 105. Kapoor, A., et al., Identification of a pegivirus (GB virus-like virus) that infects horses. J
52 Virol, 2013. 87(12): p. 7185-90.
53 106. Rushton, J.O., et al., Tick-borne encephalitis virus in horses, Austria, 2011. Emerg Infect
54 Dis, 2013. 19(4): p. 635-7.
WHO/BS/2016.2300
Page 115
1 107. Klaus, C., et al., Tick-borne encephalitis virus (TBEV) infection in horses: clinical and
2 laboratory findings and epidemiological investigations. Vet Microbiol, 2013. 163(3-4): p.
3 368-72.
4 108. Durrwald, R., et al., Meta-analysis of putative human bornavirus sequences fails to
5 provide evidence implicating Borna disease virus in mental illness. Rev Med Virol, 2007.
6 17(3): p. 181-203.
7 109. Korneyeva, M., et al., Enveloped virus inactivation by caprylate: a robust alternative to
8 solvent-detergent treatment in plasma derived intermediates. Biologicals, 2002. 30(2): p.
9 153-62.
10 110. Dichtelmuller, H., D. Rudnick, and M. Kloft, Inactivation of lipid enveloped viruses by
11 octanoic Acid treatment of immunoglobulin solution. Biologicals, 2002. 30(2): p. 135-42.
12 111. Parkkinen, J., et al., A modified caprylic acid method for manufacturing immunoglobulin
13 G from human plasma with high yield and efficient virus clearance. Vox Sang, 2006.
14 90(2): p. 97-104.
15 112. Reid, K.G., et al., Potential contribution of mild pepsin treatment at pH4 to the viral safety
16 of human immunoglobulin products. Vox Sang, 1988. 55(2): p. 75-80.
17 113. Omar, A., et al., Virus inactivation by pepsin treatment at pH 4 of IgG solutions: factors
18 affecting the rate of virus inactivation. Transfusion, 1996. 36(10): p. 866-72.
19 114. Bos, O.J., et al., Virus validation of pH 4-treated human immunoglobulin products
20 produced by the Cohn fractionation process. Biologicals, 1998. 26(4): p. 267-76.
21 115. Lundblad, J.L. and R.L. Seng, Inactivation of lipid-enveloped viruses in proteins by
22 caprylate. Vox Sang, 1991. 60(2): p. 75-81.
23 116. Burnouf, T., et al., Assessment of viral inactivation during pH 3.3 pepsin digestion and
24 caprylic acid treatment of antivenoms. Biologicals, 2007. 35(4): p. 329-34.
25 117. El-Ekiaby, M., et al., Minipool caprylic acid fractionation of plasma using disposable
26 equipment: a practical method to enhance immunoglobulin supply in developing
27 countries. PLoS Negl Trop Dis, 2015. 9(2): p. e0003501.
28 118. Mpandi, M., et al., Partitioning and inactivation of viruses by the caprylic acid
29 precipitation followed by a terminal pasteurization in the manufacturing process of horse
30 immunoglobulins. Biologicals, 2007. 35(4): p. 335-41.
31 119. Lazar, A., et al., Inactivation of West-Nile virus during peptic cleavage of horse plasma
32 IgG. Biologicals, 2002. 30(2): p. 163-5.
33 120. Cameron-Smith, R., et al., The removal of viruses during the purification of equine
34 antisera using filtration aids Hyflo Super-Cel and Fulmon Super A. Biologicals, 2000.
35 28(3): p. 169-74.
36 121. Burnouf, T., et al. , Place of nanofiltration for assuring viral safety of biologicals. . Current
37 Nanoscience, 2005. 1: p. 189-201.
38 122. Caricati, C.P., et al., Safety of snake antivenom immunoglobulins: efficacy of viral
39 inactivation in a complete downstream process. Biotechnol Prog, 2013. 29(4): p. 972-9.
40 123. Tuck, S. and R. Ng, Protein assays., in Analytical techniques for biopharmaceutical
41 development., R. Rodriguez-Diaz and T. Wehr, Editors. 2005, Marcel Dekker.: New
42 York. p. 14-24.
43 124. Solano, G., A. Gomez, and G. Leon, Assessing endotoxins in equine-derived snake
44 antivenoms: Comparison of the USP pyrogen test and the Limulus Amoebocyte Lysate
45 assay (LAL). Toxicon, 2015. 105: p. 13-8.
46 125. Sells, P.G., Animal experimentation in snake venom research and in vitro alternatives.
47 Toxicon, 2003. 42(2): p. 115-33.
48 126. Sanchez, L.V., et al., Evaluation of the preclinical efficacy of four antivenoms, distributed
49 in sub-Saharan Africa, to neutralize the venom of the carpet viper, Echis ocellatus, from
50 Mali, Cameroon, and Nigeria. Toxicon, 2015. 106: p. 97-107.
51 127. Pla, D., J.M. Gutierrez, and J.J. Calvete, Second generation snake antivenomics:
52 comparing immunoaffinity and immunodepletion protocols. Toxicon, 2012. 60(4): p. 688-
53 99.
54 128. Gutierrez, J.M., et al., Immunological profile of antivenoms: preclinical analysis of the
55 efficacy of a polyspecific antivenom through antivenomics and neutralization assays. J
56 Proteomics, 2014. 105: p. 340-50.
WHO/BS/2016.2300
Page 116
1 129. Calvete, J.J., et al., Omics meets biology: application to the design and preclinical
2 assessment of antivenoms. Toxins (Basel), 2014. 6(12): p. 3388-405.
3 130. Calvete, J.J., et al., Preclinical evaluation of three polyspecific antivenoms against the
4 venom of Echis ocellatus: Neutralization of toxic activities and antivenomics. Toxicon,
5 2016. 119: p. 280-8.
6 131. Finney, D.J., Probit analysis. 3rd ed. 1971, Cambridge: Cambridge University Press.
7 132. World Health Organization, The biological potency assay for antivenom preparations. .
8 1990, Expert Committee on Biological Standardization.: Geneva.
9 133. Rojas, E., et al., Neutralization of four Peruvian Bothrops sp. snake venoms by
10 polyvalent antivenoms produced in Peru and Costa Rica: preclinical assessment. Acta
11 Trop, 2005. 93(1): p. 85-95.
12 134. Gutierrez, J.M. and C. Herrera, The analgesics morphine and tramadol do not alter the
13 acute toxicity induced by Bothrops asper snake venom in mice. Toxicon, 2014. 81: p. 54-
14 7.
15 135. Reid, H.A. and R.D. Theakston, The management of snake bite. Bull World Health
16 Organ, 1983. 61(6): p. 885-95.
17 136. Theakston, R.D. and H.A. Reid, Development of simple standard assay procedures for
18 the characterization of snake venom. Bull World Health Organ, 1983. 61(6): p. 949-56.
19 137. Gutierrez, J.M., et al., Neutralization of proteolytic and hemorrhagic activities of Costa
20 Rican snake venoms by a polyvalent antivenom. Toxicon, 1985. 23(6): p. 887-93.
21 138. Reid, H.A., Cobra-Bites. Br Med J, 1964. 2(5408): p. 540-5.
22 139. Gutierrez, J.M., et al., Neutralization of local tissue damage induced by Bothrops asper
23 (terciopelo) snake venom. Toxicon, 1998. 36(11): p. 1529-38.
24 140. Iddon, D., R.D. Theakston, and C.L. Ownby, A study of the pathogenesis of local skin
25 necrosis induced by Naja nigricollis (spitting cobra) venom using simple histological
26 staining techniques. Toxicon, 1987. 25(6): p. 665-72.
27 141. Rivel, M. and e. al., Pathogenesis of dermonecrosis induced by venom of the spitting
28 cobra, Naja nigricollis: An experimental study in mice. . Toxicon, , , 2016. 119: p. 171-
29 179.
30 142. Laing, G.D., et al., Comparison of the potency of three Brazilian Bothrops antivenoms
31 using in vivo rodent and in vitro assays. BIASG (Butantan Institute Antivenom Study
32 Group). Toxicon, 1992. 30(10): p. 1219-25.
33 143. Theakston, R.D.G., Characterization of venoms and standardization of antivenoms., in
34 Natural toxins, animal, plant and microbial. , J.B. Harris, Editor. 1986, Clarendon Press,
35 Oxford Science Publications: Oxford. p. 287-303.
36 144. Gene, J.A., et al., Comparative study on coagulant, defibrinating, fibrinolytic and
37 fibrinogenolytic activities of Costa Rican crotaline snake venoms and their neutralization
38 by a polyvalent antivenom. Toxicon, 1989. 27(8): p. 841-8.
39 145. Gutierrez, J.M., et al., Skeletal muscle necrosis and regeneration after injection of BaH1,
40 a hemorrhagic metalloproteinase isolated from the venom of the snake Bothrops asper
41 (Terciopelo). Exp Mol Pathol, 1995. 62(1): p. 28-41.
42 146. Gutierrez, J.M., et al., Experimental myonecrosis induced by the venoms of South
43 American Micrurus (coral snakes). Toxicon, 1992. 30(10): p. 1299-302.
44 147. Alam, J.M., et al. , Myotoxic characteristics of snake venoms; myonecrosis induced in
45 albino rats by crude venoms and the purified myotoxic component, phospholipase A2. .
46 Journal of College of Physicians and Surgeons Pakistan, 2002. 12: p. 398-404.
47 148. Ginsborg, B.L. and J. Warriner, The isolated chick biventer cervicis nerve-muscle
48 preparation. Br J Pharmacol Chemother, 1960. 15: p. 410-1.
49 149. Harvey, A.L., et al., Screening of snake venoms for neurotoxic and myotoxic effects
50 using simple in vitro preparations from rodents and chicks. Toxicon, 1994. 32(3): p. 257-
51 65.
52 150. Bulbring, E., Observations on the isolated phrenic nerve diaphragm preparation of the
53 rat. Br J Pharmacol Chemother, 1946. 1: p. 38-61.
54 151. Jones, R.G., L. Lee, and J. Landon, The effects of specific antibody fragments on the
55 'irreversible' neurotoxicity induced by Brown snake (Pseudonaja) venom. Br J
56 Pharmacol, 1999. 126(3): p. 581-4.
WHO/BS/2016.2300
Page 117
1 152. Crachi, M.T., L.W. Hammer, and W.C. Hodgson, A pharmacological examination of
2 venom from the Papuan taipan (Oxyuranus scutellatus canni). Toxicon, 1999. 37(12): p.
3 1721-34.
4 153. Kitchen, I., Neuromuscular blocking drugs and the rat phrenic nerve hemidiaphragm
5 preparation., in Textbook of In Vitro Pharmacology. 2009., Blackwell Scientific: Oxford.
6 p. 79-83.
7 154. Theakston, R.D., et al., Treatment of snake bites by Bothrops species and Lachesis
8 muta in Ecuador: laboratory screening of candidate antivenoms. Trans R Soc Trop Med
9 Hyg, 1995. 89(5): p. 550-4.
10 155. Keegan, H.E., et al. , Southeast Asian snake bite antivenin studies. 1964, Japan: 406th
11 Medical Laboratory, Control Department of Entomology, US Army Medical Command.
12 156. Warrell, D.A., et al., Comparison of Pasteur and Behringwerke antivenoms in
13 envenoming by the carpet viper (Echis carinatus). Br Med J, 1980. 280(6214): p. 607-9.
14 157. Rosenberger, W.F. and L.M. Haines, Competing designs for phase I clinical trials: a
15 review. Stat Med, 2002. 21(18): p. 2757-70.
16 158. Chippaux, J.-P., R.P. Stock, and A. Massougbodji, Antivenom Safety and Tolerance for
17 the Strategy of Snake Envenomation Management., in Snake Venom Toxinology, P.
18 Gopalakrishnakone, et al., Editors. 2015, Springer Netherlands.: Dordrecht p. 1-16.
19 159. Machin, D., et al. , Sample size tables for clinical studies. 3rd ed. 2009, Chichester:
20 Wiley Blackwell.
21 160. Visser, L.E., et al., Failure of a new antivenom to treat Echis ocellatus snake bite in rural
22 Ghana: the importance of quality surveillance. Trans R Soc Trop Med Hyg, 2008. 102(5):
23 p. 445-50.
24 161. Armitage, P., Sequential medical trials. 2nd ed. 1975, New York: John Wiley.
25 162. Vakil, B.J., et al., Therapeutic trial of intracisternal human tetanus immunoglobulin in
26 clinical tetanus. Trans R Soc Trop Med Hyg, 1979. 73(5): p. 579-83.
27 163. Smalligan, R., et al., Crotaline snake bite in the Ecuadorian Amazon: randomised double
28 blind comparative trial of three South American polyspecific antivenoms. BMJ, 2004.
29 329(7475): p. 1129.
30 164. Ariaratnam, C.A., et al., An open, randomized comparative trial of two antivenoms for the
31 treatment of envenoming by Sri Lankan Russell's viper (Daboia russelii russelii). Trans R
32 Soc Trop Med Hyg, 2001. 95(1): p. 74-80.
33 165. Warrell, D.A., et al., Randomized comparative trial of three monospecific antivenoms for
34 bites by the Malayan pit viper (Calloselasma rhodostoma) in southern Thailand: clinical
35 and laboratory correlations. Am J Trop Med Hyg, 1986. 35(6): p. 1235-47.
36 166. Warrell, D.A., et al., Bites by the saw-scaled or carpet viper (Echis carinatus): trial of two
37 specific antivenoms. Br Med J, 1974. 4(5942): p. 437-40.
38 167. Jorge, M.T., et al., A randomized 'blinded' comparison of two doses of antivenom in the
39 treatment of Bothrops envenoming in Sao Paulo, Brazil. Trans R Soc Trop Med Hyg,
40 1995. 89(1): p. 111-4.
41 168. Cardoso, J.L., et al., Randomized comparative trial of three antivenoms in the treatment
42 of envenoming by lance-headed vipers (Bothrops jararaca) in Sao Paulo, Brazil. Q J
43 Med, 1993. 86(5): p. 315-25.
44 169. Otero, R., et al., Efficacy and safety of two whole IgG polyvalent antivenoms, refined by
45 caprylic acid fractionation with or without beta-propiolactone, in the treatment of
46 Bothrops asper bites in Colombia. Trans R Soc Trop Med Hyg, 2006. 100(12): p. 1173-
47 82.
48 170. Bush, S.P., et al., Crotalidae polyvalent immune Fab (ovine) antivenom is efficacious for
49 envenomations by Southern Pacific rattlesnakes (Crotalus helleri). Ann Emerg Med,
50 2002. 40(6): p. 619-24.
51 171. Warrell, D.A., Unscrupulous marketing of snake bite antivenoms in Africa and Papua
52 New Guinea: choosing the right product--'what's in a name?'. Trans R Soc Trop Med
53 Hyg, 2008. 102(5): p. 397-9.
54 172. World Health Organization, Guidelines for national authorities on quality assurance for
55 biological products., in WHO Expert Committee on Biological Standardization. . 1992:
56 Geneva, . p. Annex 2
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1 173. World Health Organization, Regulation and licensing of biological products in countries
2 with newly developing regulatory authorities., in WHO Expert Committee on Biological
3 Standardization 1995: Geneva. p. Annex 1.
4
5 22 Annex 1
6 Worldwide distribution of medically important venomous snakes
7 Venomous snakes are widely distributed especially in tropical countries, from sea level to
8 altitudes of up to 4 900 metres (Gloydius himalayanus). The European adder (Vipera berus)
9 enters the Arctic Circle, and the Argentine Yararanata (Bothrops ammodytoides) occurs to 47 ˚S
10 and is the most southerly occurring venomous snake. No other venomous species occur in cold
11 regions such as the Arctic, Antarctic and north of about latitude 51 ºN in North America
12 (Newfoundland, Nova Scotia).
13 This schedule lists venomous snake species considered to represent the greatest threat
14 to public health in various countries, territories and other areas or regions around the
15 world. Only species which fall into one of the two categories listed below are shown, and
16 category listings are in alphabetical order according to taxonomic family, genus and species.
17 The intention in categorizing these medically important snakes into two groups is to provide
18 users of the Guidelines with a prioritized listing. Snakes in both Category 1 and Category 2 are
19 species for which antivenom production is important; however species listed in Category 1
20 within a country, territory or area should be considered as being of highest priority for antivenom
21 production on the basis that available knowledge implicates them as being responsible for the
22 greater burden in that particular setting.
23 Definitions of the categories used in this listing are:
24 CATEGORY 1: Highest medical importance
25 Definition: Highly venomous snakes which are common or widespread and cause
26 numerous snakebites, resulting in high levels of morbidity, disability or mortality.
27 CATEGORY 2: Secondary medical importance
28 Definition: Highly venomous snakes capable of causing morbidity, disability or death, but
29 for which:
30  exact epidemiological or clinical data may be lacking; and/or
31  are less frequently implicated (due to their activity cycles, behaviour, habitat
32 preferences or occurrence in areas remote to large human populations).
33 There are numerous other venomous species that rank as lesser threats in countries territories
34 and other areas listed here, and interested readers should refer to herpetological references in
35 these guidelines. It should be noted that over time, as more information becomes available, new
36 species will doubtlessly be added to these lists, and some species, currently defined within
37 Category 1 or Category 2 will be re-ranked.
38 It should also be noted that the organization of countries, territories and other areas in this
39 Annex does not follow the WHO regional organization, but is instead arranged bio-
40 geographically in alphabetical order of country, territory or geographical area. This approach
41 was necessary to reflect the geographical distribution of major groups of venomous snakes
42 throughout the world. For example, the venomous snakes of the eastern Indonesian Province of
43 Papua have bio-geographical origins in Australo-Papua, and are evolutionarily distinct from the
44 venomous snakes of Asian origin that occur west of Wallace’s Line which runs south of the
45 Philippines, between Borneo and Sulawesi, and between Bali and Lombok separates the
46 zoogeographical regions of Asia and Australia. For this reason, we have listed the medically
47 important snakes of Indonesian Papua in the Australo-Papuan region, rather than the South-
48 East Asian region.
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1 Users of this Annex should also recognize that the relative risk of injury from a particular
2 species may vary from one country, territory or area to another. For this reason, some species
3 that have been listed under Category 1 in one country, territory or area may have been listed
4 under Category 2 in another country territory or area, as a reflection of the different risk posed
5 by that species in different locations. Assignment to Category 1 or Category 2 was based in
6 some cases on the relative importance of a species as a cause of snakebite. In Europe for
7 example, the overall incidence of snakebite is trivial compared to that in West Africa or India,
8 but where a European species (such as Vipera berus) is a major (or sole) cause of envenoming
9 where it occurs, this warrants ranking it as a medically important species in that setting.
10 AFRICA AND THE MIDDLE EAST
11 Island populations
12 Off the coast of Africa, there are no medically important snakes in Mauritius, Réunion,
13 Rodrigues, the Comoros, the Canary Islands, the Cape Verde Islands or the Seychelles. The
14 islands that do have venomous snakes include the Lamu group, Zanzibar, Pemba and Mafia
15 Islands, the Bazaruto Archipelago and Inhaca Island, São Tomé, Principe, Bioko (Fernando Po)
16 and Dahlak Islands. The venomous snakes on these islands tend to be similar to those on the
17 adjacent mainland. A colubrid, Madagascarophis meridionalis, and perhaps other species of the
18 same genus, are the only terrestrial snakes of possible, if minimal, medical importance found in
19 Madagascar.
20 North Africa/Middle East
21 Algeria:
Cat 1: Elapidae: Naja haje; Viperidae: Cerastes cerastes; Daboia mauritanica12
Cat 2: Viperidae: Daboia deserti1; Echis leucogaster; Macrovipera lebetina; Vipera latastei
22 Cyprus:
Cat 1: None
Cat 2: Viperidae: Macrovipera lebetina
23 Egypt:
Cat 1: Elapidae: Naja haje; Viperidae: Cerastes cerastes; Echis coloratus (east), Echis
pyramidum;
Cat 2: Atractaspididae: Atractaspis engaddensis (Sinai); Elapidae: Naja nubiae1;
Walterinnesia aegyptia (Sinai); Viperidae: Pseudocerastes fieldi
24 Iraq:
Cat 1: Viperidae: Echis carinatus; Macrovipera lebetina
Cat 2: Elapidae: Walterinnesia morgani1; Viperidae: Cerastes gasperettii; Pseudocerastes
fieldi, Pseudocerastes persicus
25 Iran (Islamic Republic of):
Cat 1: Elapidae: Naja oxiana; Viperidae: Echis carinatus; Macrovipera lebetina;
Pseudocerastes persicus
Cat 2: Elapidae: Bungarus persicus (south-east); Walterinnesia morgani1 (west); Viperidae:
Eristicophis macmahonii (east); Gloydius halys caucasicus; Montivipera raddei;
Vipera spp.
26 Israel
Cat 1: Viperidae: Daboia palaestinae1; Echis coloratus
Cat 2: Atractaspididae: Atractaspis engaddensis; Elapidae: Walterinnesia aegyptia;
Viperidae: Cerastes cerastes, Cerastes gasperettii; Pseudocerastes fieldi
12
Recent nomenclatural changes. Refer to Tables 1 and 2 of the main text for details of previous names.
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1 Jordan:
Viperidae: Cerastes gasperettii; Macrovipera lebetina; Pseudocerastes fieldi
2 Kuwait and Qatar:
Cat 1: Viperidae: Cerastes gasperettii
Cat 2: Elapidae: Walterinnesia morgani1 (Kuwait only)
3 Lebanon:
Cat 1: Viperidae: Daboia palaestinae1; Macrovipera lebetina
Cat 2: None
4 The Libyan Arab Jamahiriya:
Cat 1: Elapidae: Naja haje; Viperidae: Cerastes cerastes; Echis pyramidum
Cat 2: Viperidae: Daboia deserti1
5 Morocco:
Cat 1: Elapidae: Naja haje; Viperidae: Bitis arietans; Cerastes cerastes; Daboia
mauritanica1
Cat 2: Viperidae: Echis leucogaster; Vipera latastei
6 Oman:
Cat 1: Atractaspididae: Atractaspis andersonii (south-west); Viperidae: Bitis arietans
(south-west); Echis coloratus (south-west), Echis carinatus, Echis omanensis1
(north)
Cat 2: Elapidae: Naja arabica1 (south-west); Viperidae: Cerastes gasperettii; Echis
khosatzkii (south-west); Pseudocerastes persicus
7 Saudi Arabia:
Cat 1: Atractaspididae: Atractaspis andersonii (south-west); Viperidae: Cerastes
gasperettii; Echis coloratus, Echis borkini1 (south-west)
Cat 2: Atractaspididae: Atractaspis engaddensis (north-west); Elapidae: Naja arabica
(south-west); Walterinnesia aegyptia (west), Walterinnesia morgani1 (central &
south); Viperidae: Bitis arietans (south-west); Cerastes cerastes (south-west);
Pseudocerastes fieldi
8 The Syrian Arab Republic:
Cat 1: Viperidae: Daboia palaestinae1; Macrovipera lebetina
Cat 2: Viperidae: Pseudocerastes fieldi
9 Tunisia:
Cat 1: Viperidae: Daboia mauritanica1
Cat 2: Elapidae: Naja haje; Viperidae: Cerastes cerastes; Daboia deserti1; Echis
leucogaster; Macrovipera lebetina; Vipera latastei
10 Turkey:
Cat 1: Viperidae: Macrovipera lebetina; Montivipera xanthina1
Cat 2: Elapidae: Walterinnesia morgani1 (south); Viperidae: Montivipera raddei1; Vipera
ammodytes; Vipera eriwanensis; Vipera spp.
11 The United Arab Emirates:
Cat 1: Viperidae: Echis carinatus (east); Echis omanensis1
Cat 2: Viperidae: Cerastes gasperettii; Pseudocerastes persicus
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1 West Bank and Gaza Strip:
Viperidae: Cerastes cerastes, Pseudocerastes fieldi
2 Western Sahara:
Cat 1: Viperidae: Cerastes cerastes
Cat 2: Elapidae: Naja haje; Viperidae: Bitis arietans
3 Yemen:
Cat 1: Atractaspididae: Atractaspis andersonii; Elapidae: Naja arabica1; Viperidae: Bitis
arietans; Echis borkini1, Echis coloratus
Cat 2: Viperidae: Cerastes cerastes, Cerastes gasperettii; Echis khosatzkii
4 Central sub Saharan Africa
5 Angola:
Cat 1: Elapidae: Dendroaspis jamesoni, Dendroaspis polylepis; Naja anchietae1, Naja
melanoleuca, Naja nigricollis; Viperidae: Bitis arietans, Bitis gabonica1
Cat 2: Atractaspididae: Atractaspis bibronii, Atractaspis irregularis; Colubridae: Dispholidus
typus; Thelotornis capensis, Thelotornis kirtlandii (north); Elapidae: Naja christyi1
(Cabinda), Naja mossambica (south), Naja nigricincta1 (south-west); Pseudohaje
goldii; Viperidae: Atheris squamigera; Bitis nasicornis (Cabinda)
6 Burundi:
Cat 1: Elapidae: Naja nigricollis; Naja melanoleuca; Viperidae: Bitis arietans
typus; Thelotornis mossambicanus1; Elapidae: Dendroaspis jamesoni; Viperidae:
Bitis gabonica1, Bitis nasicornis
7 The Central African Republic:
Cat 1: Elapidae: Dendroaspis jamesoni, Dendroaspis polylepis; Naja haje, Naja
nigricollis; Viperidae: Bitis arietans, Bitis gabonica1; Echis ocellatus, Echis
pyramidum
Cat 2: Atractaspididae: Atractaspis irregularis; Colubridae: Dispholidus typus; Thelotornis
kirtlandii; Elapidae: Naja annulata1, Naja melanoleuca13; Pseudohaje goldii;
Viperidae: Atheris broadleyi, Atheris squamigera; Bitis nasicornis1
8 Chad:
Cat 1: Elapidae: Naja haje, Naja nigricollis; Viperidae: Bitis arietans (south); Echis
ocellatus (south)
Cat 2: Colubridae: Dispholidus typus; Elapidae: Naja katiensis, Naja nubiae1; Viperidae:
Cerastes cerastes
9 The Congo:
Cat 1: Elapidae: Dendroaspis jamesoni; Naja melanoleuca; Viperidae: Bitis gabonica1,
Bitis nasicornis
kirtlandii; Elapidae: Naja annulata1, Naja christyi1, Naja nigricollis; Pseudohaje goldii;
Viperidae: Atheris squamigera; Bitis arietans
13
The medical importance of this species may be higher in the primary forest zone of the south-western Central
African Republic, and in some secondary forest mosaic zones elsewhere in the Central African Republic.
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1 The Democratic Republic of the Congo:
Cat 1: Elapidae: Dendroaspis jamesoni; Naja melanoleuca, Naja nigricollis; Viperidae:
Bitis arietans, Bitis gabonica1, Bitis nasicornis
typus; Thelotornis capensis, Thelotornis kirtlandii; Elapidae: Dendroaspis polylepis;
Naja anchietae1 (Katanga pedicle), Naja annulata1, Naja christyi1, Naja haje (north);
Pseudohaje goldii; Viperidae: Atheris squamigera
2 Equatorial Guinea:
Cat 1: Elapidae: Dendroaspis jamesoni; Naja melanoleuca; Viperidae: Bitis gabonica1,
Bitis nasicornis
Cat 2: Atractaspididae: Atractaspis irregularis; Colubridae: Thelotornis kirtlandii; Elapidae:
Naja annulata1; Pseudohaje goldii; Viperidae: Atheris squamigera
3 Gabon:
Cat 1: Elapidae: Dendroaspis jamesoni; Naja melanoleuca, Naja nigricollis; Viperidae:
Bitis gabonica1, Bitis nasicornis
Cat 2: Viperidae: Atractaspis irregularis; Colubridae: Thelotornis kirtlandii; Elapidae: Naja
annulata1; Pseudohaje goldii; Viperidae: Atheris squamigera; Bitis arietans
4 Rwanda:
Cat 1: Elapidae: Dendroaspis jamesoni; Naja nigricollis; Viperidae: Bitis arietans
typus; Thelotornis kirtlandii; Elapidae: Dendroaspis polylepis; Naja annulata1, Naja
melanoleuca; Pseudohaje goldii; Viperidae: Bitis gabonica1, Bitis nasicornis
5 East sub Saharan Africa
6 Djibouti:
Cat 1: Viperidae: Echis pyramidum
Cat 2: Atractaspididae: Atractaspis fallax; Colubridae: Dispholidus typus; Elapidae: Naja
pallida; Viperidae: Bitis arietans
7 Eritrea:
Cat 1: Elapidae: Dendroaspis polylepis; Naja haje; Viperidae: Bitis arietans; Echis
pyramidum
Cat 2: Atractaspididae: Atractaspis irregularis; Colubridae: Dispholidus typus; Elapidae:
Naja nubiae1; Viperidae: Echis megalocephalus
8 Ethiopia:
Cat 1: Elapidae: Dendroaspis polylepis; Naja ashei1 (south-east), Naja haje, Naja
nigricollis; Viperidae: Bitis arietans; Echis pyramidum
Cat 2: Atractaspididae: Atractaspis fallax, Atractaspis irregularis (Mt Bizen); Colubridae:
Dispholidus typus; Elapidae: Naja melanoleuca, Naja pallida; Viperidae: Bitis
parviocula, Bitis harenna.
9 Kenya:
Cat 1: Elapidae: Dendroaspis angusticeps, Dendroaspis polylepis; Naja ashei1 (north
& east), Naja haje, Naja nigricollis; Viperidae: Bitis arietans; Echis pyramidum
Cat 2: Atractaspididae: Atractaspis bibronii, Atractaspis fallax, Atractaspis irregularis;
Colubridae: Dispholidus typus; Thelotornis mossambicanus1, Thelotornis
usambaricus1 (east coast); Elapidae: Dendroaspis jamesoni; Naja melanoleuca
(west & coastal forest), Naja pallida (north & east); Pseudohaje goldii; Viperidae:
Atheris squamigera; Bitis nasicornis, Bitis gabonica1 (west)
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1 Malawi:
Cat 1: Elapidae: Dendroaspis angusticeps, Dendroaspis polylepis; Naja annulifera1,
Naja mossambica, Naja nigricollis; Viperidae: Bitis arietans
Cat 2: Atractaspididae: Atractaspis bibronii; Colubridae: Dispholidus typus; Thelotornis
capensis, Thelotornis mossambicanus1; Elapidae: Naja melanoleuca; Viperidae:
Proatheris superciliaris
2 Mozambique:
Cat 1: Elapidae: Dendroaspis angusticeps, Dendroaspis polylepis; Naja annulifera1,
Naja mossambica; Viperidae: Bitis arietans, Bitis gabonica1
capensis, Thelotornis mossambicanus1; Elapidae: Hemachatus haemachatus, Naja
melanoleuca; Viperidae: Proatheris superciliaris
3 Somalia:
Cat 1: Elapidae: Dendroaspis polylepis; Naja ashei1 (south); Naja haje; Viperidae: Bitis
arietans; Echis pyramidum
Cat 2: Atractaspididae: Atractaspis fallax; Colubridae: Dispholidus typus; Thelotornis
mossambicanus1; Elapidae: Naja pallida, Naja melanoleuca; Viperidae: Echis
hughesi (north)
4 South Sudan
Cat 1: Elapidae: Naja haje, Naja nigricollis; Viperidae: Bitis arietans; Echis pyramidum
Cat 2: Atractaspididae: Atractaspis fallax, Atractaspis irregularis; Colubridae: Dispholidus
typus; Elapidae: Dendroaspis jamesoni, Dendroaspis polylepis; Naja melanoleuca,
Naja nubiae1, Naja pallida; Viperidae: Bitis gabonica1, Bitis nasicornis
5 The Sudan:
Cat 1: Elapidae: Naja haje Viperidae: Bitis arietans; Echis pyramidum
Cat 2: Colubridae: Dispholidus typus; Elapidae: Dendroaspis polylepis (east); Naja
nubiae1, Viperidae: Cerastes cerastes, Echis coloratus (east)
6 The United Republic of Tanzania:
Cat 1: Elapidae: Dendroaspis angusticeps, Dendroaspis polylepis; Naja mossambica
(including Pemba Island), Naja nigricollis; Viperidae: Bitis arietans
Cat 2: Atractaspididae: Atractaspis bibronii, Atractaspis fallax (north), Atractaspis
irregularis (north-east); Colubridae: Dispholidus typus; Thelotornis capensis,
Thelotornis kirtlandii (Mahali and Udzungwa Mountains), Thelotornis
mossambicanus1, Thelotornis usambaricus1 (East Usambara Mts); Elapidae: Naja
ashei1 (poss. north-east), Naja annulata1, Naja haje (north), Naja melanoleuca (west
and coast, including Mafia Island), Naja pallida; Viperidae: Atheris squamigera; Bitis
gabonica1 (west and south-east), Bitis nasicornis (north); Proatheris superciliaris
7 Uganda:
Cat 1: Elapidae: Naja ashei1 (north-east), Naja haje (north), Naja nigricollis;
Dendroaspis jamesoni, Dendroaspis polylepis; Viperidae: Bitis arietans, Bitis
gabonica1
kirtlandii; Elapidae: Naja melanoleuca; Pseudohaje goldii; Viperidae: Atheris
squamigera; Bitis nasicornis
8 Zambia:
Cat 1: Elapidae: Dendroaspis polylepis; Naja anchietae1, Naja annulifera1, Naja
mossambica, Naja nigricollis; Viperidae: Bitis arietans, Bitis gabonica1
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capensis, Thelotornis kirtlandii, Thelotornis mossambicanus1; Elapidae: Naja
annulata1, Naja melanoleuca
1
2
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1 South sub Saharan Africa
2 Botswana:
Cat 1: Elapidae: Dendroaspis polylepis; Naja anchietae1 (west), Naja annulifera1 (east),
Naja mossambica, Naja nivea (south-west); Viperidae: Bitis arietans
capensis
3 Lesotho:
Cat 1: Elapidae: Naja nivea; Viperidae: Bitis arietans
Cat 2: Elapidae: Hemachatus haemachatus
4 Namibia:
Cat 1: Elapidae: Dendroaspis polylepis; Naja anchietae1, Naja nivea (central &
southern), Naja mossambica (north-east), Naja nigricincta1; Viperidae: Bitis
arietans
capensis; Elapidae: Naja nigricollis (Caprivi)
5 South Africa:
Cat 1: Elapidae: Dendroaspis angusticeps (Natal), Dendroaspis polylepis; Naja
annulifera1 (north-east), Naja nivea, Naja mossambica (north-east); Viperidae:
Bitis arietans
capensis; Elapidae: Hemachatus haemachatus; Naja melanoleuca (KwaZulu-Natal),
Naja nigricincta1 (north-west) Viperidae: Bitis gabonica1 (KwaZulu-Natal);
6 Swaziland:
Cat 1: Elapidae: Dendroaspis polylepis; Naja annulifera1, Naja mossambica; Viperidae:
Bitis arietans
capensis; Elapidae: Hemachatus haemachatus
7 Zimbabwe:
Cat 1: Elapidae: Dendroaspis polylepis; Naja anchietae1 (west), Naja annulifera1, Naja
mossambica; Viperidae: Bitis arietans
capensis, Thelotornis mossambicanus1; Elapidae: Dendroaspis angusticeps (east);
Hemachatus haemachatus (Nyanga Mts); Naja melanoleuca (east); Viperidae: Bitis
gabonica1 (east)
8 West sub Saharan Africa
9 Benin:
Cat 1: Elapidae: Naja nigricollis; Viperidae: Bitis arietans; Echis ocellatus
Cat 2: Atractaspididae: Atractaspis irregularis; Colubridae: Dispholidus typus; Elapidae:
Dendroaspis jamesoni; Naja katiensis, Naja melanoleuca, Naja senegalensis1;
Pseudohaje nigra; Viperidae: Bitis rhinoceros, Echis leucogaster (far north)
10 Burkina Faso:
Cat 1: Elapidae: Naja nigricollis, Naja katiensis; Viperidae: Bitis arietans; Echis
ocellatus
Cat 2: Colubridae: Dispholidus typus; Elapidae: Dendroaspis polylepis; Naja melanoleuca,
Naja senegalensis1; Viperidae: Echis leucogaster
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1 Cameroon:
Cat 1: Elapidae: Dendroaspis jamesoni; Naja haje, Naja nigricollis, Naja
melanoleuca14; Viperidae: Bitis arietans, Bitis gabonica1, Bitis nasicornis;
Echis ocellatus
kirtlandii; Elapidae: Dendroaspis polylepis; Naja annulata1, Naja katiensis;
Pseudohaje goldii; Viperidae: Atheris broadleyi (East Province), Atheris squamigera
2 Côte d’Ivoire:
Cat 1: Elapidae: Dendroaspis viridis; Naja nigricollis, Naja melanoleuca, Naja
senegalensis1; Viperidae: Bitis arietans, Bitis nasicornis, Bitis rhinoceros1;
Echis ocellatus
kirtlandii; Elapidae: Dendroaspis polylepis; Naja katiensis; Pseudohaje goldii,
Pseudohaje nigra; Viperidae: Atheris chlorechis
3 The Gambia:
Cat 1: Elapidae: Dendroaspis viridis; Naja nigricollis; Viperidae: Bitis arietans; Echis
jogeri
Cat 2: Colubridae: Dispholidus typus; Elapidae: Naja katiensis, Naja melanoleuca, Naja
senegalensis1
4 Ghana:
Cat 1: Elapidae: Dendroaspis viridis, Naja nigricollis, Naja senegalensis1; Viperidae:
Bitis arietans; Echis ocellatus
Cat 2: Atractaspididae: Atractaspis irregularis; Colubridae: Dispholidus typus, Thelotornis
kirtlandii; Elapidae: Naja katiensis, Naja melanoleuca15; Pseudohaje goldii,
Pseudohaje nigra; Viperidae: Atheris chlorechis; Bitis nasicornis, Bitis rhinoceros1
5 Guinea:
Cat 1: Elapidae: Dendroaspis polylepis, Dendroaspis viridis; Naja katiensis, Naja
nigricollis, Naja melanoleuca, Naja senegalensis1; Viperidae: Bitis arietans;
Echis jogeri
kirtlandii; Elapidae: Pseudohaje nigra; Viperidae: Atheris chlorechis; Bitis nasicornis,
Bitis rhinoceros1
6 Guinea-Bissau:
Cat 1: Elapidae: Dendroaspis viridis; Naja nigricollis, Naja melanoleuca, Naja
senegalensis1; Viperidae: Bitis arietans; Echis jogeri
Cat 2: Colubridae: Dispholidus typus; Thelotornis kirtlandii; Viperidae: Bitis rhinoceros1
7 Liberia:
Cat 1: Elapidae: Dendroaspis viridis; Naja melanoleuca, Naja nigricollis
Cat 2: Atractaspididae: Atractaspis irregularis; Colubridae: Thelotornis kirtlandii; Elapidae:
Pseudohaje nigra; Viperidae: Atheris chlorechis; Bitis nasicornis, Bitis rhinoceros1
14
This large, highly venomous snake is common in forested areas of south-west Cameroon and a high burden of
injury may be expected, although clinical data with direct attribution are not yet available.
15
The medical importance of this species may be higher in the forested zone of southern Ghana.
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1
2 Mali:
Cat 1: Elapidae: Naja katiensis, Naja nigricollis, Naja senegalensis1; Viperidae: Bitis
arietans; Echis jogeri (west); Echis leucogaster; Echis ocellatus
Cat 2: Colubridae: Dispholidus typus; Elapidae: Naja melanoleuca; Viperidae: Cerastes
cerastes
3 Mauritania:
Cat 1: Elapidae: Naja senegalensis1 (south-east); Viperidae: Cerastes cerastes; Echis
leucogaster
Cat 2: Viperidae: Bitis arietans;
4 The Niger:
Cat 1: Elapidae: Naja nigricollis; Viperidae: Bitis arietans; Echis leucogaster, Echis
ocellatus
Cat 2: Colubridae: Dispholidus typus; Elapidae: Naja haje (south-central), Naja katiensis,
Naja nubiae1 Naja senegelensis1 (south-west); Viperidae: Cerastes cerastes
5 Nigeria:
Cat 1: Elapidae: Dendroaspis jamesoni; Naja haje (north-east), Naja nigricollis;
Viperidae: Bitis arietans, Bitis gabonica1; Echis ocellatus
kirtlandii; Elapidae: Naja katiensis, Naja melanoleuca16; Naja senegalensis (north-
west); Pseudohaje goldii, Pseudohaje nigra; Viperidae: Atheris squamigera; Bitis
nasicornis; Echis leucogaster (north)
6 Sao Tome and Principe:
Cat 1: Elapidae: Dendroaspis jamesoni; Naja melanoleuca
Cat 2: None
7 Senegal:
Cat 1: Elapidae: Naja katiensis; Naja nigricollis; Viperidae: Bitis arietans; Echis
leucogaster; Echis jogeri
Cat 2: Colubridae: Dispholidus typus; Elapidae: Dendroaspis polylepis; Dendroaspis
viridis; Naja melanoleuca, Naja senegalensis
8 Sierra Leone:
Cat 1: Elapidae: Dendroaspis viridis; Naja nigricollis; Viperidae: Bitis arietans
kirtlandii; Elapidae: Naja melanoleuca17; Pseudohaje nigra; Viperidae: Atheris
chlorechis; Bitis nasicornis, Bitis rhinoceros1
9 Togo:
Cat 1: Elapidae: Naja nigricollis, Naja senegalensis1; Viperidae: Bitis arietans (south);
Echis ocellatus
kirtlandii; Elapidae: Dendroaspis jamesoni, Dendroaspis viridis; Naja katiensis, Naja
melanoleuca; Pseudohaje goldii, Pseudohaje nigra; Viperidae: Atheris chlorechis;
Bitis nasicornis, Bitis rhinoceros1
16
The medical importance of this species may be higher in the southern rainforest belt of Nigeria, from Ibadan in
the west to Oban and Eket in the east, and in the forested southern quarter of Sierra Leone.
17
The medical importance of this species may be higher in the forested southern quarter of Sierra Leone.
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1 ASIA AND AUSTRALASIA
2 Central Asia
3 Armenia:
Cat 1: Viperidae: Macrovipera lebetina;
Cat 2: Viperidae: Montivipera raddei1; Vipera eriwanensis, Vipera spp.
4 Azerbaijan:
Cat 1: Viperidae: Macrovipera lebetina
Cat 2: Viperidae: Gloydius halys; Vipera eriwanensis; Vipera spp.
5 Georgia:
Cat 1: Viperidae: Macrovipera lebetina; Vipera ammodytes
Cat 2: Viperidae: Vipera kaznakovi, Vipera renardi, Vipera spp.
6 Kazakhstan, Kyrgyzstan, Tajikistan, Uzbekistan and Turkmenistan:
Cat 1: Elapidae: Naja oxiana (except Kazakhstan & Kyrgyzstan); Viperidae: Echis
carinatus (except Kyrgyzstan); Macrovipera lebetina (except Kazakhstan &
Kyrgyzstan); Gloydius halys (throughout)
Cat 2: Viperidae: Vipera renardi (except Turkmenistan)
7 Mongolia:
Cat 1: Viperidae: Gloydius halys
Cat 2: Viperidae: Vipera berus, Vipera renardi
8 East Asia
9 China:
10 China Mainland
Cat 1: Elapidae: Bungarus multicinctus; Naja atra; Viperidae: Trimeresurus
albolabris1; Daboia siamensis1; Deinagkistrodon acutus; Gloydius
brevicaudus; Protobothrops mucrosquamatus
Cat 2: Colubridae: Rhabdophis tigrinus; Elapidae: Bungarus bungaroides (south-east
Tibet), Bungarus fasciatus; Naja kaouthia; Ophiophagus hannah; Viperidae:
Trimeresurus septentrionalis (south Tibet); Gloydius halys, Gloydius intermedius1,
Gloydius ussuriensis; Himalayophis tibetanus (south Tibet); Protobothrops jerdonii,
Protobothrops kaulbacki, Protobothrops mangshanensis1; Vipera berus (Jilin,
western Xinjiang); Vipera renardi (western Xinjiang); Trimeresurus stejnegeri1
11 Hong Kong, Special Administrative Region
Cat 1: Elapidae: Bungarus multicinctus; Naja atra; Viperidae: Trimeresurus albolabris1
Cat 2: None
12 Taiwan Province
Cat 1: Elapidae: Bungarus multicinctus; Naja atra; Viperidae: Protobothrops
mucrosquamatus; Trimeresurus stejnegeri1
Cat 2: Viperidae: Deinagkistrodon acutus; Daboia siamensis1
13 The Democratic People’s Republic of Korea:
Cat 1: Viperidae: Gloydius brevicaudus
Cat 2: Viperidae: Gloydius intermedius1, Gloydius ussuriensis; Vipera berus
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1
2 Japan (including Ryukyu Islands):
Cat 1: Viperidae: Gloydius blomhoffii (main islands); Protobothrops flavoviridis
(Ryukyu Islands)
Cat 2: Colubridae: Rhabdophis tigrinus; Viperidae: Gloydius tsushimaensis (Tsushima);
Protobothrops elegans;
3 The Republic of Korea:
Cat 1: Viperidae: Gloydius brevicaudus
Cat 2: Colubridae: Rhabdophis tigrinus; Viperidae: Gloydius intermedius1, Gloydius
ussuriensis
4 South Asia
5 Afghanistan:
Cat 1: Elapidae: Naja oxiana; Viperidae: Echis carinatus; Macrovipera lebetina
Cat 2: Elapidae: Bungarus caeruleus (east), Bungarus sindanus (east), Naja naja (poss.
south-east); Viperidae: Eristicophis macmahonii (south-west); Gloydius halys (north)
6 Bangladesh:
Cat 1: Elapidae: Bungarus caeruleus, Bungarus niger, Bungarus walli; Naja kaouthia;
Viperidae: Trimeresurus erythrurus1
Cat 2: Elapidae: Bungarus bungaroides,Bungarus fasciatus, Bungarus lividus; Naja naja;
Ophiophagus hannah; Viperidae: Trimeresurus albolabris1 (far north-west); Daboia
russelii1 (west)
7 Bhutan:
Cat 1: Elapidae: Bungarus niger; Naja naja
Cat 2: Elapidae: Bungarus caeruleus, Bungarus fasciatus; Bungarus lividus; Naja kaouthia;
Ophiophagus hannah; Viperidae: Trimeresurus erythrurus1; Daboia russelii1;
Protobothrops jerdonii
8 India:
Cat 1: Elapidae: Bungarus caeruleus; Naja kaouthia (east), Naja naja (throughout);
Viperidae: Daboia russelii1; Echis carinatus; Hypnale hypnale (south-west)
Cat 2: Bungarus bungaroides, Bungarus fasciatus, Bungarus lividus, Bungarus niger,
Bungarus sindanus, Bungarus walli; Naja oxiana (west), Naja sagittifera (Andaman
Islands); Ophiophagus hannah (south, north-east, Andaman Islands); Viperidae:
Trimeresurus albolabris1, Trimeresurus erythrurus1, Trimeresurus septentrionalis1;
Gloydius himalayanus; Protobothrops jerdonii, Protobothrops kaulbacki,
Protobothrops mucrosquamatus; Trimeresurus gramineus (south India),
Trimeresurus malabaricus (south-west),
9 Nepal:
Cat 1: Elapidae: Bungarus caeruleus, Bungarus niger; Naja naja, Naja kaouthia;
Viperidae: Daboia russelii1
Cat 2: Elapidae: Bungarus bungaroides, Bungarus fasciatus; Bungarus lividus, Bungarus
walli; Ophiophagus hannah; Viperidae: Trimeresurus septentrionalis1; Gloydius
himalayanus; Himalayophis tibetanus1; Protobothrops jerdonii;
10 Pakistan:
Cat 1: Elapidae: Bungarus caeruleus, Bungarus sindanus; Naja naja, Naja oxiana;
Viperidae: Daboia russelii1; Echis carinatus
Cat 2: Viperidae: Eristicophis macmahonii (west); Gloydius himalayanus (north);
Macrovipera lebetina (west)
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1 Sri Lanka:
Cat 1: Elapidae: Bungarus caeruleus; Naja naja; Viperidae: Daboia russelii1; Hypnale
hypnale
Cat 2: Elapidae: Bungarus ceylonicus; Viperidae: Echis carinatus; Hypnale nepa, Hypnale
zara; Trimeresurus trigonocephalus
2 South-east Asia
3 Brunei Darussalam:
Cat 1: Elapidae: Naja sumatrana
Cat 2: Elapidae: Bungarus fasciatus, Bungarus flaviceps; Calliophis bivirgatus, Calliophis
intestinalis; Ophiophagus hannah; Viperidae: Trimeresurus sumatranus1;
Tropidolaemus subannulatus
4 Cambodia:
Cat 1: Elapidae: Bungarus candidus; Naja kaouthia, Naja siamensis; Viperidae:
Calloselasma rhodostoma; Trimeresurus albolabris1; Daboia siamensis1
Cat 2: Elapidae: Bungarus fasciatus, Bungarus flaviceps; Ophiophagus hannah; Viperidae:
Trimeresurus cardamomensis
5 Indonesia (Sumatra, Java, Borneo, Sulawesi & Lesser Sunda Islands)
Cat 1: Elapidae: Bungarus candidus (Sumatra & Java); Naja sputatrix (Java & Lesser
Sunda Islands), Naja sumatrana (Sumatra & Borneo); Viperidae: Calloselasma
rhodostoma (Java); Trimeresurus albolabris1; Daboia siamensis1
Cat 2: Elapidae: Bungarus fasciatus, Bungarus flaviceps (Sumatra & Borneo); Calliophis
bivirgatus, Calliophis intestinalis; Ophiophagus hannah (Sumatra, Borneo & Java);
Viperidae: Trimeresurus insularis1, Trimeresurus purpureomaculatus1 (Sumatra);
Trimeresurus sumatranus; Tropidolaemus subannulatus
6 The Lao People’s Democratic Republic:
Cat 1: Elapidae: Bungarus candidus, Bungarus multicinctus; Naja atra (north), Naja
siamensis1 (south & east); Viperidae: Calloselasma rhodostoma; Trimeresurus
albolabris1
Cat 2: Elapidae: Bungarus fasciatus; Naja kaouthia (south & east); Ophiophagus hannah
Viperidae: Trimeresurus macrops; Protobothrops jerdonii; Protobothrops
mucrosquamatus
7 Malaysia:
Cat 1: Elapidae: Bungarus candidus (Peninsular Malaysia); Naja kaouthia (northern
Peninsular Malaysia), Naja sumatrana (Peninsular Malaysia, Sabah & Sarawak);
Viperidae: Calloselasma rhodostoma
Cat 2: Elapidae: Bungarus fasciatus, Bungarus flaviceps; Calliophis bivirgatus; Calliophis
intestinalis; Ophiophagus hannah; Viperidae: Trimeresurus purpureomaculatus1;
Trimeresurus hageni1; Tropidolaemus subannulatus
8 Myanmar:
Cat 1: Elapidae: Bungarus magnimaculatus, Bungarus multicinctus; Naja kaouthia,
Naja mandalayensis; Viperidae: Trimeresurus albolabris1, Trimeresurus
erythrurus1; Daboia siamensis1
Cat 2: Elapidae: Bungarus bungaroides (Chin State), Bungarus candidus (Thaninthayi
Div.); Bungarus flaviceps (east Shan State), Bungarus niger; Ophiophagus hannah;
Viperidae: Calloselasma rhodostoma (Thaninthayi Div.); Trimeresurus
purpureomaculatus; Protobothrops jerdonii, Protobothrops kaulbacki, Protobothrops
mucrosquamatus (Kachin)
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1 The Philippines:
Cat 1: Elapidae: Naja philippinensis (Luzon), Naja samarensis (Mindanao), Naja
sumatrana (Palawan)
Cat 2: Elapidae: Calliophis intestinalis; Ophiophagus hannah; Viperidae: Trimeresurus
flavomaculatus1; Tropidolaemus philippensis1; Tropidolaemus subannulatus1
2 Singapore:
Cat 1: Elapidae: Bungarus candidus; Naja sumatrana
Cat 2: Elapidae: Bungarus fasciatus; Calliophis bivirgatus, Calliophis intestinalis;
Ophiopahgus hannah; Viperidae: Trimeresurus purpureomaculatus1
3 Thailand:
Cat 1: Elapidae: Bungarus candidus; Naja kaouthia, Naja siamensis1; Viperidae:
Calloselasma rhodostoma; Trimeresurus albolabris1, Daboia siamensis1
Cat 2: Elapidae: Bungarus fasciatus, Bungarus flaviceps; Calliophis bivirgatus, Calliophis
intestinalis; Naja sumatrana; Ophiophagus hannah; Viperidae: Trimeresurus
macrops1; Trimeresurus hageni
4 Timor-Leste:
Cat 1: Viperidae: Trimeresurus insularis1
Cat 2: None
5 Viet Nam:
Cat 1: Elapidae: Bungarus candidus, Bungarus multicinctus, Bungarus slowinskii
(north); Naja atra (north), Naja kaouthia (south); Viperidae: Calloselasma
rhodostoma; Trimeresurus albolabris1 (throughout);
Cat 2: Elapidae: Bungarus fasciatus, Bungarus flaviceps (south); Naja siamensis (south);
Ophiophagus hannah; Viperidae: Trimeresurus rubeus; Protobothrops jerdonii,
Protobothrops mucrosquamatus (north); Trimeresurus stejnegeri1; Deinagkistrodon
acutus
6 Australo-Papua (including Pacific Islands):
7 There are no medically important land snakes in American Samoa; Cook Islands; Fiji; French
8 Polynesia; Guam; Kiribati; Marshall Islands; Nauru; New Caledonia; New Zealand; Northern
9 Mariana Islands; Pitcairn Island; Samoa; Tokelau; Tonga; Tuvalu; or Wallis and Futuna Islands.
10 Fiji possesses a single terrestrial venomous snake species (Ogmodon vitianus) while the
11 Solomon Islands possess three terrestrial venomous species (Salomonelaps par; Loveridgelaps
12 elapoides and Parapistocalamus hedigeri) with no and few snakebites, respectively.
13 Australia:
Cat 1: Elapidae: Notechis scutatus; Pseudechis australis18; Pseudonaja affinis,
Pseudonaja mengdeni1, Pseudonaja nuchalis, Pseudonaja textilis;
Cat 2: Elapidae: Acanthophis antarcticus, Acanthophis cryptamydros, Acanthophis spp.;
Austrelaps spp.; Hoplocephalus spp.; Oxyuranus microlepidotus, Oxyuranus
scutellatus, Oxyuranus temporalis; Pseudechis spp.; Pseudonaja aspidorhyncha1,
Pseudonaja spp.; Tropidechis carinatus
14 Indonesia (West Papua and Maluku):
Cat 1: Elapidae: Acanthophis laevis1
Cat 2: Elapidae: Acanthophis rugosus1; Micropechis ikaheka; Oxyuranus scutellatus;
Pseudechis papuanus, Pseudechis rossignolii1; Pseudonaja textilis
18
Pseudechis australis is common and widespread and causes numerous snakebites; bites may be
severe, although this species has not caused a fatality in Australia since 1968.
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1 Papua New Guinea:
Cat 1: Elapidae: Acanthophis laevis1; Oxyuranus scutellatus
Cat 2: Elapidae: Acanthophis rugosus1; Micropechis ikaheka; Pseudonaja textilis;
Pseudechis papuanus, Pseudechis rossignolii1
2 EUROPE
3 There are no venomous snakes in Iceland, Ireland, Isle of Man, Outer Hebrides, Orkney or
4 Shetland Islands. Crete and most of the islands of the western Mediterranean are also without
5 venomous snakes.
6 Central Europe
7 Albania; Bulgaria; Romania; Serbia; Montenegro; Slovenia; The former Yugoslav
8 Republic of Macedonia:
Cat 1: Viperidae: Vipera ammodytes
Cat 2: Viperidae: Vipera berus
9 Bosnia and Herzegovina:
10 Croatia:
11 The Czech Republic:
Cat 1: None
12 Greece:
Cat 2: Viperidae: Macrovipera schweizeri; Montivipera xanthina1; Vipera berus
13 Hungary:
Cat 1: None
14 Poland:
Cat 1: None
15 Slovakia:
Cat 1: None
16 Eastern Europe
17 Belarus; Estonia; Latvia; Lithuania; The Republic of Moldova:
Cat 1: None
Cat 2: Viperidae: Vipera berus, Vipera nikolskii (Moldova
18 The Russian Federation:
Cat 2: Viperidae: Gloydius halys, Gloydius intermedius1, Gloydius ussuriensis; (far-east
Russia); Macrovipera lebetina (Dagestan); Vipera nikolskii; Vipera renardi, Vipera
spp.
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1 Ukraine:
Cat 1: None
Cat 2: Viperidae: Vipera berus, Vipera nikolskii, Vipera renardi
2 Western Europe
3 Austria:
Cat 1: None
Cat 2: Viperidae: Vipera ammodytes, Vipera berus
4 Belgium:
Cat 1: None
5 Denmark:
Cat 1: None
6 Finland:
Cat 1: None
7 France:
Cat 1: Viperidae: Vipera aspis
8 Germany:
Cat 1: None
9 Italy:
Cat 1: Viperidae: Vipera aspis
Cat 2: Viperidae: Vipera ammodytes, Vipera berus
10 The Netherlands:
Cat 1: None
11 Norway:
Cat 1: None
12 Portugal:
Cat 1: None
Cat 2: Viperidae: Vipera latastei, Vipera seoanei
13 Spain:
Cat 1: None
Cat 2: Viperidae: Vipera aspis, Vipera latastei, Vipera seoanei
14 Sweden:
Cat 2: None
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1
2 Switzerland:
Cat 1: None
Cat 2: Viperidae: Vipera aspis, Vipera berus
3 The United Kingdom of Great Britain and Northern Ireland:
Cat 1: Viperidae: Vipera berus (not Northern Ireland)
Cat 2: None
4 THE AMERICAS
5 North America
6 Canada:
Cat 1: None
Cat 2: Viperidae: Crotalus oreganus1, Crotalus viridis, Sistrurus catenatus
7 Mexico:
Cat 1: Viperidae: Agkistrodon bilineatus, Agkistrodon taylori1; Crotalus atrox,
Crotalus scutulatus, Crotalus simus1, Crotalus molossus; Bothrops asper
Cat 2: Elapidae: Micruroides euryxanthus, Micrurus nigrocinctus, Micrurus tener, Micrurus
spp.; Viperidae: Agkistrodon contortrix, Agkistrodon russeolus; Atropoides
mexicanus, Atropoides occiduus, Atropoides spp.; Bothriechis schlegelii, Bothriechis
spp.; Cerrophidion godmani, Cerrophidion spp.; Crotalus basiliscus, Crotalus
totonacus1, , Crotalus oreganus1, Crotalus ruber, Crotalus tzabcan1, Crotalus viridis,
Crotalus spp.; Ophryacus spp.; Porthidium nasutum, Porthidium spp.; Sistrurus
catenatus
8 The United States of America:
Cat 1: Viperidae: Agkistrodon contortrix, Agkistrodon piscivorus; Crotalus
adamanteus, Crotalus atrox, Crotalus horridus, Crotalus oreganus1, Crotalus
scutulatus, Crotalus viridis
Cat 2: Elapidae: Micrurus fulvius, Micrurus tener; Viperidae: Crotalus molossus, Crotalus
ornatus, Crotalus ruber, Crotalus spp., Sistrurus catenatus, Sistrurus miliarius
9 Central America
10 The most medically important species are Crotalus simus1 and Bothrops asper.
11 Belize:
Cat 1: Viperidae: Bothrops asper
Cat 2: Elapidae: Micrurus spp.; Viperidae: Agkistrodon russeolus; Atropoides mexicanus;
Bothriechis schlegelii; Crotalus tzabcan1; Porthidium nasutum
12 Costa-Rica:
Cat 1: Viperidae: Bothrops asper; Crotalus simus1
Cat 2: Elapidae: Micrurus nigrocinctus, Micrurus spp.; Viperidae: Agkistrodon
howardgloydi; Atropoides mexicanus, Atropoides picadoi.; Bothriechis schlegelii,
Bothriechis lateralis, Bothriechis spp.; Cerrophidion sasai; Lachesis melanocephala,
Lachesis stenophrys; Porthidium nasutum, Porthidium ophrymegas, Porthidium spp.
13 El Salvador:
Cat 1: Viperidae: Crotalus simus1
Cat 2: Elapidae: Micrurus nigrocinctus Micrurus spp.; Viperidae: Agkistrodon bilineatus;
Atropoides occiduus; Bothriechis spp.; Cerrophidion wilsoni; Porthidium
ophryomegas
WHO/BS/2016.2300
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1 Guatemala:
Cat 1: Viperidae: Bothrops asper; Crotalus simus
Cat 2: Elapidae: Micrurus nigrocinctus, Micrurus spp.; Viperidae: Agkistrodon bilineatus,
Agkistrodon russeolus; Atropoides mexicanus, Atropoides occiduus; Bothriechis
schlegelii, Bothriechis spp.; Cerrophidion godmani; Crotalus tzabcan1, Porthidium
nasutum, Porthidium ophryomegas
2 Honduras:
howardgloydi; Atropoides mexicanus, Atropoides spp.; Bothriechis marchi,
Bothriechis schlegelii, Bothriechis spp.; Cerrophidion wilsoni; Crotalus simus1;
Porthidium nasutum, Porthidium ophryomegas
3 Nicaragua:
Cat 1: Viperidae: Bothrops asper; Crotalus simus1
howardgloydi; Atropoides mexicanus; Bothriechis schlegelii; Cerrophidion godmani;
Lachesis stenophrys; Porthidium nasutum, Porthidium ophryomegas
4 Panama:
Cat 2: Elapidae: Micrurus mipartitus, Micrurus nigrocinctus, Micrurus spp.; Viperidae:
Atropoides mexicanus, Atropoides spp.; Bothriechis lateralis, Bothriechis schlegelii,
Bothriechis spp.; Cerrophidion sasai; Lachesis acrochorda, Lachesis stenophrys;
Porthidium nasutum, Porthidium lansbergii, Porthidium spp.
5 Caribbean
6 No medically important snakes occur naturally in Anguilla; Antigua and Barbuda; the Bahamas;
7 Barbados; Bermuda; The British Virgin Islands; Cayman Islands; Cuba; Dominica; the
8 Dominican Republic; Grenada; Guadeloupe; Haiti; Jamaica; Montserrat; the Netherlands
9 Antilles; Saint Kitts and Nevis; Saint Vincent and the Grenadines; and Turks and Caicos
10 Islands.
11 Aruba; Martinique; Saint Lucia; Trinidad and Tobago, and offshore islands:
Cat 1: Viperidae: Bothrops cf. atrox (Trinidad), Bothrops caribbaeus (St Lucia),
Bothrops lanceolatus (Martinique); Crotalus durissus (Aruba)
Cat 2: Elapidae: Micrurus circinalis (Trinidad), Micrurus lemniscatus (Trinidad); Viperidae:
Lachesis muta (Trinidad)
12 South America
13 No venomous snakes are naturally occurring in the Falkland Islands; and no dangerously
14 venomous snakes are naturally occurring in Chile.
15 Argentina:
Cat 1: Viperidae: Bothrops alternatus, Bothrops diporus1; Crotalus durissus
Cat 2: Elapidae: Micrurus corallinus, Micrurus lemniscatus, Micrurus spp.; Viperidae:
Bothrops ammodytoides, Bothrops jararaca, Bothrops jararacussu, Bothrops
mattogrossensis, Bothrops neuwiedi, Bothrops pubescens
16 Plurinational State of Bolivia:
Cat 1: Viperidae: Bothrops atrox, Bothrops mattogrossensis1; Crotalus durissus
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Cat 2: Elapidae: Micrurus lemniscatus, Micrurus spixii, Micrurus surinamensis, Micrurus
spp.; Viperidae: Bothrocophias hyoprora, Bothrocophias microphthalmus1; Bothrops
bilineatus, Bothrops brazili, Bothrops jararacussu, Bothrops jonathani, Bothrops
moojeni, Bothrops sanctaecrucis, Bothrops spp., Bothrops taeniatus; Lachesis muta
1 Brazil:
Cat 1: Viperidae: Bothrops atrox, Bothrops jararaca, Bothrops jararacussu, Bothrops
leucurus, Bothrops moojeni; Crotalus durissus
Cat 2: Elapidae: Micrurus corallinus, Micrurus lemniscatus, Micrurus spixii, Micrurus
surinamensis, Micrurus spp.; Viperidae: Bothrocophias hyoprora1; Bothrocophias
microphthalmus1; Bothrops alternatus, Bothrops bilineatus, Bothrops brazili,
Bothrops diporus, Bothrops mattogrossensis, Bothrops neuwiedi, B. pubescens,
Bothrops taeniatus, Bothrops spp.; Lachesis muta
2 Colombia:
Cat 1: Viperidae: Bothrops asper, Bothrops atrox, Bothrops bilineatus; Crotalus
durissus
Cat 2: Elapidae: Micrurus lemniscatus, Micrurus mipartitus, Micrurus nigrocinctus, Micrurus
spixii, Micrurus surinamensis, Micrurus spp.; Viperidae: Bothriechis schlegelii;
Bothrocophias hyoprora1, Bothrocophias microphthalmus1, Bothrocophias spp.;
Bothrops brazili, Bothrops taeniatus, Bothrops spp.; Lachesis acrochorda1, Lachesis
muta; Porthidium nasutum, Porthidium lansbergii
3 Ecuador:
Cat 1: Viperidae: Bothrops asper, Bothrops atrox, Bothrops bilineatus; Lachesis
muta
Cat 2: Elapidae: Micrurus lemniscatus, Micrurus mipartitus, Micrurus spixii, Micrurus
surinamensis, Micrurus spp.; Viperidae: Bothriechis schlegelii; Bothrocophias
hyoprora1, Bothrocophias microphthalmus1, Bothrocophias spp.; Bothrops brazili,
Bothrops taeniatus, Bothrops spp.; Lachesis acrochorda1; Porthidium nasutum,
Porthidium spp.
4 French Guiana (France):
Cat 1: Viperidae: Bothrops atrox, Bothrops bilineatus; Crotalus durissus
Cat 2: Elapidae: Micrurus lemniscatus, Micrurus surinamensis, Micrurus spp.; Viperidae:
Bothrops brazili, Bothrops taeniatus; Lachesis muta
5 Guyana:
6 Paraguay:
Cat 1: Viperidae: Bothrops alternatus; Crotalus durissus
Cat 2: Elapidae: Micrurus corallinus, Micrurus lemniscatus, Micrurus spixii, Micrurus spp.;
Viperidae: Bothrops diporus, Bothrops jararaca, Bothrops jararacussu, Bothrops
mattogrossensis, Bothrops moojeni, Bothrops neuwiedi, Bothrops spp.
7 Peru:
Cat 1: Viperidae: Bothrops atrox, Bothrops bilineatus, Bothrops pictus; Crotalus
durissus; Lachesis muta
Cat 2: Elapidae: Micrurus lemniscatus, Micrurus mipartitus, Micrurus spixii, Micrurus
surinamensis, Micrurus spp.; Viperidae: Bothriechis schlegelii; Bothrocophias
hyoprora, Bothrocophias microphthalmus; Bothrops asper; Bothrops brazili,
Bothrops mattogrossensis, Bothrops taeniatus, Bothrops spp.
WHO/BS/2016.2300
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1 Suriname:
2 Uruguay:
Cat 1: Viperidae: Bothrops alternatus; Bothrops pubescens1
Cat 2: Elapidae: Micrurus corallinus, Micrurus spp.; Viperidae: Crotalus durissus
3 Bolivarian Republic of Venezuela:
Cat 1: Viperidae: Bothrops atrox, Bothrops cf. atrox, Bothrops venezuelensis;
Crotalus durissus (including Isla de Margarita)
Cat 2: Elapidae: Micrurus circinalis, Micrurus lemniscatus, Micrurus mipartitus, Micrurus
spixii, Micrurus surinamensis, Micrurus spp.; Viperidae: Bothriechis schlegelii;
Bothrops asper, Bothrops brazili, Bothrops bilineatus; Lachesis muta; Porthidium
lansbergii
4
5
WHO/BS/2016.2300
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1 Herpetological references19
2 Abtin E, Nilson G, Mobaraki A, Hosseini AA, Dehgannejhad M. A New Species of Krait,
3 Bungarus (Reptilia, Elapidae, Bungarinae) and the First Record of that Genus in Iran. Russ. J.
4 Herpetol. 2014. 21(4):243-250
5 Ananjeva NB et al. The reptiles of Northern Eurasia. Sofia, Pensoft, 2006.
6 Anderson, CG, Greenbaum E. Phylogeography of Northern Populations of the Black-Tailed
7 Rattlesnake (Crotalus molossus Baird And Girard, 1853), With the Revalidation of C. ornatus
8 Hallowell, 1854. Herpetological Monographs. 2012. 26(1):19-57.
9 Ashton KG, de Querioz A. Molecular systematics of the western rattlesnake, Crotalus viridis
10 (Viperidae), with comments on the utility of the D-loop in phylogenetic studies of snakes.
11 Molecular Phylogenetics and Evolution, 2001, 21:176–189.
12 Babocsay G. A new species of saw-scaled viper of the Echis coloratus complex (Ophidia:
13 Viperidae) from Oman, Eastern Arabia. Systematics and Biodiversity, 2004, 1:503–514.
14 Basoglu M, Baran I. The reptiles of Turkey. Part II. The Snakes. Izmir, University of Matbaasi,
15 1980.
16 Buys PJ, Buys PJC. Snakes of Namibia. Windhoek, Gamsberg Macmillan. 1983.
17 Bons J, Geniez P. Amphibiens et Reptiles du Maroc. Barcelona, Asociación Herpetológica
18 Española. 1996.
19 Boycott RC. A herpetological survey of Swaziland [MSc thesis]. Durban, University of Natal,
20 1992.
21 Branch B. Field guide to the snakes and other reptiles of Southern Africa. Cape Town, Sruik
22 1988.
23 Broadley DG et al. Snakes of Zambia: An atlas and field guide. Frankfurt am Main, Edition
24 Chimaira, 2003.
25 Broadley DG. FitzSimons snakes of Southern Africa. Cape Town, Delta Books, 1983.
26 Broadley DG. The herpetofaunas of the islands off the coast of south Moçambique. Arnoldia
27 Zimbabwe, 1990, 9:469–493.
28 Broadley DG. Reptiles and amphibians from the Bazaruto Archipelago, Mozambique. Arnoldia
29 Zimbabwe, 1992, 9:539–548.
30 Broadley DG. Review of the Dispholidini, with the description of a new genus and species from
31 Tanzania (Serpentes, Colubridae). Bulletin of the Natural History Museum London (Zoology),
32 2002, 68:57–74.
33 Broadley DG. A review of the genus Thelotornis A. Smith in eastern Africa, with the description
34 of a new species from the Usambara Mountains (Serpentes: Colubridae: Dispholidini). African
35 Journal of Herpetology, 2001, 50:53–70.
36 Broadley DG. The herpetofaunas of the islands off the coast of south Moçambique. Arnoldia
37 Zimbabwe, 1990, 9:469–493.
38 Campbell JA, Lamar WW. The venomous snakes of the western hemisphere. Vols. I and II.
39 Ithaca, NY, Comstock-Cornell, 2004.
40 Creer S et al. Genetic and ecological correlates of intraspecific variation in pitviper venom
41 composition detected using matrix-assisted laser desorption time-of-flight mass spectrometry
42 (MALDI-TOF-MS) and isoelectric focusing. Journal of Molecular Evolution. 2003, 56:317–329.
19
Major regional guides have author names italicized.
WHO/BS/2016.2300
Page 139
1 da Silva VX. The Bothrops neuwiedi Complex. In: Campbell JA, Lamar WW (eds) The
2 venomous reptiles of the Western Hemisphere. Vol. I. Ithaca, NY, Comstock Publishing
3 2004:410–422.
4 David P, Ineich I. Les serpents venimeux du monde: systématique et répartition. Dumerilia,
5 1999, 3:3–499.
6 David P, Vogel G, Dubois A. On the need to follow rigorously the Rules of the Code for the
7 subsequent designation of a nucleospecies (type species) for a nominal genus which lacked
8 one: the case of the nominal genus Trimeresurus Lacépède, 1804 (Reptilia : Squamata :
9 Viperidae). Zootaxa. 2011, 2992:1-51.
10 De Smedt J. The vipers of Europe. Hablech, JDS Verlag, 2001.
11 Dobiey M, Vogel G. Venomous snakes of Africa. Frankfurt am Main, Terralog Edition Chimaira,
12 2006.
13 El Din SB. A guide to the reptiles and amphibians of Egypt. Cairo, American University of Cairo
14 Press, 2006.
15 Gasperetti J. Snakes of Arabia. Fauna of Saudi Arabia, 1988, 9:169–450.
16 Geniez P et al. The amphibians and reptiles of the Western Sahara. Frankfurt am Main, Edition
17 Chimaira, 2004.
18 Gloyd HK, Conant R. Snakes of the Agkistrodon complex. Oxford, OH, SSAR, 1990.
19 Gower DJ, Wade EOZ, Spaels S, Böhme W, Buechley ER, Sykes D, Colston TJ. A new
20 large species of Bitis Gray, 1842 (Serpentes: Viperidae) from the Bale Mountains of Ethiopia.
21 Zootaxa. 2016. 4093(1):41–63.
22 Guo P et al. New evidence on the phylogenetic position of the poorly known Asian pitviper
23 Protobothrops kaulbacki (Serpentes: Viperidae: Crotalinae) with a redescription of the species
24 and a revision of the genus Protobothrops. The Herpetological Journal 2007, 17:237–246.
25 Gumprecht A et al. Asian Pitvipers. Berlin, Geitje Books, 2004.
26 Gutberlet RL Jr, Campbell JA. Generic recognition of a neglected lineage of South American
27 pitvipers (Squamata: Viperidae: Crotalinae), with the description of a new species from the
28 Colombian Chocó. American Museum Novitates, 2001, 3316:1–15.
29 Jadin, RC., Townsend, JH., Castoe, TA. & Campbell, JA. Cryptic diversity in disjunct
30 populations of Middle American Montane Pitvipers: a systematic reassessment of Cerrophidion
31 godmani. Zoologica Scripta 2012. doi:10.1111/j.1463-6409.2012.00547.x
32 Khan MS. A guide to the snakes of Pakistan. Edition Chimaira, 2002.
33 Kreiner G. The snakes of Europe. Edition Chimaira, 2007.
34 Latifi M. The snakes of Iran. Oxford, OH, SSAR, 1985.
35 Le Berre M. Faune du Sahara 1: Poissons-amphibiens-reptiles. Editions Raymond Chabaud,
36 1989.
37 Lenk P et al. Phylogeny and taxonomic subdivision of Bitis (Reptilia: Viperidae) based on
38 molecular evidence. In: Joger U (ed) Phylogeny and systematics of the Viperidae. Kaupia,
39 Darmstädter Beiträge zur Naturgeschichte, 1999, 8:31–38.
40 Lenk P et al. Evolutionary relationships among the true vipers (Reptilia: Viperidae) inferred from
41 mitochondrial DNA sequences. Molecular Phylogenetics and Evolution, 2001, 19:94–104.
42 Leviton AE et al. Handbook to Middle East amphibians and reptiles. Oxford, OH, SSAR, 1992.
43 Leviton AE et al. The dangerously venomous snakes of Myanmar. Illustrated checklist with
44 keys. Proceedings of the California Academy of Sciences, 2003, 54:407–462.
WHO/BS/2016.2300
Page 140
1 Maddock ST, Ellis RJ, Doughty P, Smith LA, Wuster W. A new species of death adder
2 (Acanthophis: Serpentes: Elapidae) from north-western Australia. Zootaxa. 2015. 4007(3):301–
3 326
4 Maduwage K, Silva A, Manamendra-Arachchi K, Pethiyagoda R. A taxonomic revision of the
5 South Asian hump-nosed pit vipers (Squamata: Viperidae: Hypnale). Zootaxa. 2009. 2232:1-28.
6 Malhotra A, Thorpe RS. A phylogeny of four mitochondrial gene regions suggests a revised
7 taxonomy for Asian pitvipers (Trimeresurus and Ovophis). Molecular Phylogenetics and
8 Evolution. 2004, 32:83–100.
9 McDiarmid RW et al. Snake species of the world: A taxonomic and geographical reference.
10 Vol.1. 1999.
11 Murphy JC. Amphibians and Reptiles of Trinidad and Tobago. Malabar, FL, Krieger, 1997.
12 Nilson G, Rastegar-Pouyani N. Walterinnesia aegyptia Lataste, 1887 (Ophidia: Elapidae) and
13 the status of Naja morgani Mocquard 1905. Russian Journal of Herpetology, 2007, 14:7–14.
14 Orlov NL, Barabanov AV. Analysis of nomenclature, classification, and distribution of the
15 Agkistrodon halys – intermedius complexes: a critical review. Russian Journal of Herpetology
16 1999, 6:167–192.
17 O’Shea M. A guide to the snakes of Papua New Guinea. Port Moresby, Independent
18 Publishing, 1996.
19 Parkinson CL et al. Phylogeography of the pitviper clade Agkistrodon: historical ecology,
20 species status, and conservation of cantils. Molecular Ecology, 2000, 9:411–420.
21 Pitman CRS. A guide to the snakes of Uganda, 2nd ed. Codicote, Wheldon & Wesley, 1974.
22 Porras LW, Wilson LD, Schuett GW, Reiserer R.S. A taxonomic reevaluation and
23 conservation assessment of the common cantil, Agkistrodon bilineatus (Squamata: Viperidae):
24 a race against time. Amphibian & Reptile Conservation 2013. 7(1):48–73.
25 Pyron RA, et al. The phylogeny of advanced snakes (Colubroidea), with discovery of a new
26 subfamily and comparison of support methods for likelihood trees. Molecular Phylogenetics and.
27 Evolution. 2010. doi:10.1016/j.ympev.2010.11.006
28 Pyron RA, Dushantha Kandambi HK, Hendry CR, Pushpamal V, Burbrink FT, Somaweera
29 R. Genus-level phylogeny of snakes reveals the origins of species richness in Sri Lanka.
30 Molecular Phylogenetics and Evolution 2013. 66(3):969-978
31 Pyron RA, Burbrink FT, Wiens JJ. A phylogeny and revised classification of Squamata,
32 including 4161 species of lizards and snakes. BMC Evol Biol 2013.13:93-.
33 Quijada-Mascareñas JA et al. Phylogeographic patterns of trans-Amazonian vicariants and
34 Amazonian biogeography: the Neotropical rattlesnake (Crotalus durissus complex) as an
35 example. Journal of Biogeography, 2007, 34:1296–1312.
36 Roman B. Serpents de Haute-Volta. Ouagadougou, CNRST, 1980.
37 Schleich HH et al. Amphibians and reptiles of Nepal. Ruggall, Gantner Verlag, 2002.
38 Schleich HH et al. Amphibians and reptiles of North Africa. Koenigstein, Koeltz. 1996.
39 Spawls S, Branch B. The dangerous snakes of Africa. London, Blandford, 1995.
40 Spawls S, et al. A field guide to the reptiles of East Africa: Kenya, Tanzania, Uganda, Rwanda
41 and Burundi. London, Academic Press, Natural World, 2002.
42 Steward JW. The snakes of Europe. Newton Abbott David & Charles, 1971.
43 Street D. Reptiles of northern and central Europe. London, Batsford 1979.
44 Sweeney RCH. Snakes of Nyasaland. Amsterdam, Asher & Co, 1971.
WHO/BS/2016.2300
Page 141
1 Thorpe RS, Pook CE, Malhotra A. Phylogeography of the Russell's viper (Daboia russelii)
2 complex in relation to variation in the colour pattern and symptoms of envenoming. The
3 Herpetological Journal, 2007, 17:209–218.
4 Visser J, Chapman DS. Snakes and snakebite. Cape Town, Struik, 1978.
5 Vogel G. Venomous snakes of Asia. Frankfurt am Main, Terralog Edition Chimaira, 2006.
6 Vogel G et al. Revision of the Tropidolaemus wagleri-complex (Serpentes: Viperidae:
7 Crotalinae). I. Definition of included taxa and redescription of Tropidolaemus wagleri (Boie,
8 1827). Zootaxa, 2007, 1644:1–40.
9 Wallach V, Williams KL, Boundy J. Snakes of the World: A Catalogue of Living and Extinct
10 Species. CRC Press, London, United Kingdom. 2014. 1,237 p.
11 Whitaker R, Captain A. Snakes of India: The field guide. Chennai, Draco Books, 2004.
12 Williams DJ et al. Origin of the eastern brownsnake, Pseudonaja textilis (Duméril, Bibron and
13 Duméril) (Serpentes: Elapidae: Hydrophiinae) in New Guinea: evidence of multiple dispersals
14 from Australia, and comments on the status of Pseudonaja textilis pughi Hoser 2003. Zootaxa,
15 2008, 1703:47–61.
16 Williams DJ, Wüster W. Snakes of Papua New Guinea. In: Williams DJ et al (eds) Venomous
17 bites and stings in Papua New Guinea. Melbourne, AVRU University of Melbourne, 2005:33–64.
18 Wüster W et al. Snakes across the strait: trans-Torresian phylogenetic relationships in three
19 genera of Australo-Papuan snakes (Serpentes: Elapidae: Acanthophis, Oxyuranus and
20 Pseudechis). Molecular Phylogenetics and Evolution, 2005, 34:1–14.
21 Wuster W, Broadley DG. A new species of spitting cobra from north-eastern Africa (Serpentes:
22 Elapidae: Naja). Journal of Zoology, London, 2003, 259:345–359.
23 Wüster W, Broadley DG. Get an eyeful of this: a new species of giant spitting cobra from
24 eastern and north-eastern Africa (Squamata: Serpentes: Elapidae: Naja). Zootaxa, 2007,
25 1532:51–68.
26 Wüster W et al. Origin and evolution of the South American pitviper fauna: evidence from
27 mitochondrial DNA sequence analysis. In: Schuett GW, Höggren M, Douglas ME, Greene HW
28 (eds) Biology of the vipers. Eagle Mountain, Utah, Eagle Mountain Publishing, 2002:111–128.
29 Wüster W et al. Origin and phylogenetic position of the Lesser Antillean species of Bothrops
30 (Serpentes: Viperidae): biogeographical and medical implications. Bulletin of the Natural History
31 Museum London (Zoology), 2002, 68:101–106.
32 Wüster W et al. Redescription of Naja siamensis Laurenti, 1768 (Serpentes: Elapidae), a
33 widely overlooked spitting cobra from Southeast Asia: geographic variation, medical importance
34 and designation of a neotype. Journal of Zoology, 1997, 243:771–788.
35 Wüster W et al. Systematics of the Bothrops atrox species complex: insights from multivariate
36 analysis and mitochondrial DNA sequence information. In: Thorpe RS, Wüster W, Malhotra A.
37 (eds) Venomous snakes: ecology, evolution and snakebite. Oxford, Clarendon Press, 1997:99–
38 113 (Symposia of the Zoological Society of London, No. 70).
39 Wüster W et al. The phylogeny of cobras inferred from mitochondrial DNA sequences:
40 evolution of venom spitting and the phylogeography of the African spitting cobras (Serpentes:
41 Elapidae: Naja nigricollis complex). Molecular Phylogenetics and Evolution, 2007, 45:437–453.
42 Wüster W et al. Synopsis of recent developments in venomous snake systematics. Toxicon,
43 1997, 35: 319–340.
44 Wüster W et al. Synopsis of recent developments in venomous snake systematics, No. 2.
45 Toxicon, 1998, 36:299–307.
46 Wüster W et al. Synopsis of recent developments in venomous snake systematics, No. 3.
47 Toxicon, 1999, 37:1123–1129.
WHO/BS/2016.2300
Page 142
1 Wüster W, McCarthy CJ. Venomous snake systematics: Implications for snake bite treatment
2 and toxinology. In: Bon C, Goyffon M (eds) Envenomings and their treatments. Lyon, Fondation
3 Mérieux, 1996:13–23.
4 Zhao E, Adler K. Herpetology of China. Oxford, OH, SSAR 1993.
5
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1 © World Health Organization
2
3 23 Annex 2
4 Summary protocol for production and testing of snake antivenom
5 immunoglobulins
6 Identification of the lot
Name and address of manufacturer ………………………………………………………………
Lot number of antivenom ………………………………………………………………
Date of filling ………………………………………………………………
Liquid or freeze-dried ………………………………………………………………
Expiry date ………………………………………………………………
Number of vials or ampoules ………………………………………………………………
Temperature of storage ………………………………………………………………
7 Control of the venom batch(es) used for animal immunization

Producer of venom and location ………………………………………………………………
Information on the snake contributing ………………………………………………………………
to the venom batch
Scientific names of the snake species ………………………………………………………………
Number of snakes ………………………………………………………………
Geographical origins of the snakes ………………………………………………………………
Dates of collection of the venoms ………………………………………………………………
Expiry date of the venoms preparation ………………………………………………………………
Biochemical and biological ………………………………………………………………
characterization of the venoms
- Test performed ………………………………………………………………
- Results ………………………………………………………………
8 Control of plasma donor animals

Location of the animal herd ………………………………………………………………
Animal species used for immunization ………………………………………………………………
Vaccinations performed on animals ………………………………………………………………
Dates of immunization ………………………………………………………………
Control of antivenom antibody titre of ………………………………………………………………
animal
Veterinary certificate of health of ………………………………………………………………
animal donor
9
10
WHO/BS/2016.2300
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1 Collection and storage of plasma
Method of collection ………………………………………………………………
Date of collection ………………………………………………………………
Date of storage ………………………………………………………………
Type of containers ………………………………………………………………
Temperature of storage ………………………………………………………………
Type and content of preservatives ………………………………………………………………
added (if any)
2 Transport of plasma to fractionation facility

Date of transport ………………………………………………………………
Temperature of transport ………………………………………………………………
Date of arrival ………………………………………………………………
3 Plasma pooling and fractionation
Temperature of plasma storage at ………………………………………………………………
fractionation facility
Volume of plasmas of different ………………………………………………………………
specificity pooled for the production of
polyspecific antivenoms (if applicable)
Date of plasma pooling ………………………………………………………………
Volume of the manufacturing plasma ………………………………………………………………
pool
Number of animal donors contributing ………………………………………………………………
to the manufacturing plasma pool
Quality control of manufacturing ………………………………………………………………
plasma pool
- Test performed ………………………………………………………………
- Results ………………………………………………………………
Type of active substance (intact IgG, ………………………………………………………………
fragments)
4 Preparation and control of final bulk

Volume of bulk antivenoms of different ………………………………………………………………
specificity pooled for the production of
polyspecific antivenoms (if applicable)
Concentration of preservatives (if used) ………………………………………………………………
- Type ………………………………………………………………
- Method ………………………………………………………………
- Result ………………………………………………………………
Quality control of manufacturing ………………………………………………………………
plasma pool
- Test performed ………………………………………………………………
- Results ………………………………………………………………
WHO/BS/2016.2300
Page 145
1 Filling and containers
Date of filling ………………………………………………………………
Quantity of containers ………………………………………………………………
Volume of antivenoms per container ………………………………………………………………
Date of freeze-drying (if any) ………………………………………………………………
2 Control tests on final product

Appearance ………………………………………………………………
Solubility (freeze-dried product) ………………………………………………………………
Extractable volume ………………………………………………………………
Venom-neutralizing potency assay
- Method ………………………………………………………………
- Venom used ………………………………………………………………
- Results ………………………………………………………………
Osmolality ………………………………………………………………
Identity test
- Method ………………………………………………………………
- Result ………………………………………………………………
Protein concentration
- Method ………………………………………………………………
- Result ………………………………………………………………
Purity
- Method ………………………………………………………………
- Result ………………………………………………………………
Molecular size distribution
- Method ………………………………………………………………
- Result ………………………………………………………………
Test for pyrogenic substances
- Method ………………………………………………………………
- Result ………………………………………………………………
Sterility test
- No. of containers examined ………………………………………………………………
- Method ………………………………………………………………
- Date at start of test ………………………………………………………………
- Date at end of test ………………………………………………………………
Concentration of sodium chloride
and other excipients
- Method ………………………………………………………………
- Result ………………………………………………………………
WHO/BS/2016.2300
Page 146
Determination of pH
- Result ………………………………………………………………
Concentration of preservatives (if
used)
- Type ………………………………………………………………
- Method ………………………………………………………………
- Result ………………………………………………………………
Chemical agents used in plasma
fractionation
- Type ………………………………………………………………
- Method ………………………………………………………………
- Result ………………………………………………………………
Inspection of final containers
- Results ………………………………………………………………
Residual moisture in freeze-dried
antivenoms
- Method ………………………………………………………………
- Result ………………………………………………………………
1 Internal certification
2 Certification by person taking overall responsibility for production of the antivenom
3 I certify that Batch No. ………………………. of ………………………… snake antivenom
4 immunoglobulin satisfies the WHO Guidelines for the Production, Control and Regulation of
5 Snake Antivenom Immunoglobulins.
6
Signature ………………………………………………………………
Name (typed) ………………………………………………………………
Date ………………………………………………………………
7

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1

6 WHO Guidelines for the Production Control and

24 © World Health Organization 2016

40 Second edition, draft (August 28, 2016)

9 3.1 Historical background

24 3.2 The use of serum versus plasma as source material

37 3.3 Antivenom purification methods and product safety

11 3.5 Need for national and regional reference venom preparations

8 4.1 Global burden of snakebites

48 4.2 Main recommendations

6 5.3 Minor venomous snake species

9 5.4 Sea snake venoms

19 5.5 Main recommendations

13 6.1 Selection and preparation of representative venom mixtures

29 6.2 Manufacture of monospecific or polyspecific antivenoms

33 6.3 Main recommendations

40 7.1 Production of snake venoms for immunization

28 7.2 Staff responsible for handling snakes

11 7.3 Main recommendations

22 8.2 National reference materials

45 8.3 Characterization of venom batches

21 8.4 Main recommendations

12 Preparation of venom mixtures

Quality control of venom

29 Formulation and filling

30 10.2 Veterinary care, monitoring and vaccinations

41 10.3 Animal health and welfare after inclusion in the herd

18 11.1 Animals used in antivenom production

39 11.2 Venoms used for immunization

44 11.3 Preparation of venom doses

34 11.4 Detoxification of venom

24 11.6 Preparation of immunogen in adjuvants

32 11.7 Immunization of animals

2 11.8 Traceability of the immunization process

5 11.9 Main recommendations

28 12.2 Premises for blood or plasma collection

43 12.3 Blood or plasma collection session

16 12.4 Labelling and identification

25 12.6 Control of plasma prior to fractionation

36 12.7 Main recommendations

25 13.2 Purification of the active substance

5 13.3 Pharmacokinetic and pharmacodynamic properties of IgG, F(ab')2

25 14.2 Risk of viral contamination of the starting plasma

32 14.3 Viral validation of manufacturing processes

8 14.4 Viral validation studies of antivenom immunoglobulins

13 14.4.2.1 Validation studies with human immunoglobulins

5 14.5 Production-scale implementation of process steps contributing to

28 14.7 Main recommendations

14 15.1 Routine assays

14 15.2 Antivenom reference preparations

22 15.3 Main recommendations

15 16.4 Main recommendations

11 17.1 Ethical considerations for the use of animals in preclinical testing of

34 17.3 Essential preclinical assays to measure antivenom neutralisation of

5 17.4 Supplementary preclinical assays to measure antivenom

13 17.5 Limitations of preclinical assays

30 17.6 Main recommendations

24 19.1 Regulatory evaluation of antivenoms

39 19.2 Establishment licencing and site inspections