Part 3
Containment and Control Measures
for Experimental Laboratory Work with TSE Agents, Materials and Infected Animals
Introduction
Scope
3.1 This section gives advice on the prevention and control measures for work involving the deliberate use of, or exposure to, TSE agents. It covers:
- all experimental work with preparations, body fluids or tissues known or likely to contain the agents of both human and animal TSEs;
- work with experimentally infected animals;
- work with preparations of purified prion proteins;
- any hosts or vectors in which TSE-associated material has been cloned by techniques of genetic modification and in which expression may be achieved.
3.2 The guidance and information given in this document is provided to help employers arrive at safe working practices, but it is emphasised that this does not negate the responsibility of the employer to carry out a full risk assessment of all individual work situations.
3.3 The COSHH Regulations employ a hierarchy of controls to prevent or, if this is not reasonably practicable, to control exposure to biological agents. The preferred method of control is to substitute a safer biological agent, or to use engineering controls for primary containment. Personal protective equipment, especially respiratory protective equipment (RPE), should always be seen as a last resort.
Basic precautions to avoid exposure
3.4 The use of basic hygiene precautions are generally applicable wherever there is a risk of exposure to potentially infected material. These are summarised in Table 2. In the context of TSEs, they are particularly important when the work may involve exposure to high or medium risk tissues (see Annex A, table A.1) from TSE infected individuals or animals or extracts prepared from them. Other measures may be required in certain occupational settings and there are, in any case, more stringent requirements under law for laboratory work with biological agents.
3.5 There is no evidence that the TSEs are transmitted by aerosols from contaminated material. Accidental ingestion should be readily avoidable by the use of basic hygiene measures. Where the agent is in high concentration and/or likely to be actively dispersed during, for example, some laboratory operations such as homogenisation of tissue, there might be a need to prevent inhalation and/or splashing of mucous membranes by the use of a microbiological safety cabinet or other primary
enclosure.
Table 2
General basic protective measures
(see also Annex B for advice on cleaning, decontamination and waste disposal) |
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- Adhere to safe working practices, e.g. do not eat, drink, smoke or take medication in the laboratory; remove protective clothing and wash hands before leaving the laboratory.
- Protect skin wounds such as cuts, abrasions, eczematous lesions, e.g. by the use waterproof dressings.
- Wear appropriate protective clothing routinely, e.g. consider use of disposable gowns and aprons.
- Wear disposable gloves for all work with TSE material.
- If there is a possibility of splashing, protect eyes and mucous membranes - use eye protection or full face visor where appropriate.
- Avoid active uncontrolled dispersal of material - take care when mixing, centrifuging or homogenising material to avoid splashing. In the laboratory, use enclosed systems (e.g. sealed centrifuge buckets or, where appropriate, a microbiological safety cabinet).
- Avoid or minimise the use of sharps wherever possible (needles, knives, scissors and laboratory glassware) use plastic single-use disposable items (e.g. containers, pipettes, inoculating loops and other such instruments).
- Consider use of suitable hand protection, such as armoured glove(s) where use of sharp instruments is essential, e.g. in post mortem examinations or collection of human or animal brain/spinal cord.
- Record all accidents involving parenteral exposure to TSE material or contaminated wastes.
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Experimental Laboratory Work
Containment of laboratory work with the TSE agents and associated materials
3.6 Those involved in laboratory work with biological agents should be familiar with the detailed advice on containment and control measures given in the 1995 ACDP publication “Categorisation of biological agents according to hazard and categories of containment”. It is not the intention to re-iterate that advice here but to highlight those aspects which are particularly relevant for work with TSEs, and to give guidance on the meaning of derogation in this context.
3.7 The Containment Level at which a biological agent is to be handled usually corresponds with its categorisation, but this may not necessarily be the case for TSE agents because of their unique features. The Hazard Group of the agent forms the basis of a risk assessment to determine the appropriate containment and control measures.
3.8 The main precautions that must be emphasised for laboratory work with human or animal TSEs are:
- a high standard of information, instruction and training for staff, together with effective supervision;
- the laboratory should be dedicated to TSE work and separated from other activities taking place in the same building;
- due to the unusual resistance properties of TSE agent, special decontamination procedures are required (see Annex B);
- because of the need for special decontamination procedures, single-use disposable items or dedicated equipment should be used wherever practicable. In the case of large items this could be interpreted as specified parts of the item, e.g. dedicated ultracentrifuge rotors or electron microscope grids;
- access is restricted to authorised individuals.
3.9 Based on the current Hazard Categorisation of the TSE agents, the recommended overall Containment Levels are given in Table 3. For some work derogation from full Containment Level 3 may be allowed, but this will depend on the nature of the work and the results of the local risk assessment.
Table 3
Containment Levels recommended for experimental work with the agents of TSEs |
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| Laboratory work with: | Overall Laboratory
Containment Level | Animal Containment Level |
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| Human TSE agents(a) | 3(b) | 3(b) for small animal work |
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| BSE and other related animal TSE agents (FSE, SE in captive exotic ungulates TME and CWD(d)) | | 1 for large animal work(c) |
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| Scrapie(d) | 2 | 2 for small animal work
1 for large animal work(c) |
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| Notes |
| (a) | This includes primary sources and any sub-passages of human derived agents in other species. The inclusion of work with any animal TSE agents passaged in primates or in genetically modified mice with the human PrP gene should also be considered. The containment measures apply to experimental work with tissues or preparations unless they are known not to contain infectivity. |
| (b) | Subject to local risk assessment, derogation from full Containment Level 3 may be applied, see paragraph 3.11. |
| (c) | The risk of exposure to TSE agents from large intact animals (e.g. sheep or cattle) is considered to be remote and therefore Containment Level 1 is generally considered appropriate for experimental work with large animals. Smaller animals (e.g. mice) generally present a higher risk of biting or scratching and should usually be handled at the Containment Level equivalent to the Hazard Group of the agent. |
| (d) | The European Directive 97/65/EC on the adaptation of the European classification of biological agents includes the following footnote: |
| “There is no evidence in humans of infections caused by the agents responsible for other animal TSEs. Nevertheless, the containment measures for agents categorised in risk group 3(**) are recommended as a precaution for laboratory work, except for laboratory work relating to an identified agent of scrapie where Containment Level 2 is sufficient.” |
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Human TSEs, BSE and other animal TSEs (except scrapie)
3.10 For work with the TSE agents classified in Hazard Group 3 (agents of CJD, GSS, kuru, FFI, BSE and related TSEs) it may not always be appropriate to work in conditions that relate strictly to the hazard grouping. Transmission of TSEs is thought most likely to occur by the percutaneous route, and to a lesser extent by ingestion, so derogation from full Containment Level 3 may normally be permitted, subject to local risk assessment. The TSEagents classified in Hazard Group 3 are specified in the exemption certificate accompanying the 1998 edition of the Approved List.
Derogation from Containment Level 3
3.11 The following derogations from full Containment Level 3 may be considered as part of any risk assessment process for work with TSE agents classified in Hazard Group 3.
Negative air pressure
Subject to local risk assessment, it may not be necessary to maintain the laboratory at an air pressure negative to atmosphere, as would normally be required for work at Containment Level 3. However, if the laboratory is mechanically ventilated, it must be maintained at an air pressure negative to atmosphere while work is in progress. In practice, when in use, the air flow through a microbiological safety cabinet will usually mean that the laboratory is maintained at negative pressure.
Sealability
The TSE agents are largely unaffected by normal fumigants, therefore, for the purposes of TSE containment, there is little practical benefit to be gained by making the laboratory sealable for fumigation. However, the local assessment should consider what decontamination methods would be used, particularly in the event of a major spillage.
HEPA filtration
Where a laboratory is mechanically ventilated, it may not be necessary for all extract air to be HEPA filtered, subject to local risk assessment. (Any air exhausted through a microbiological safety cabinet would in any case be HEPA filtered).
Scrapie
3.12 Work with the agent of scrapie can be conducted at lower levels of containment. Containment Level 2 is considered sufficient for experimental work involving tissues from scrapie infected animals. Special attention should be given to the use of dedicated equipment where practicable and the application of special decontamination requirements (see Annex B).
Work with disrupted tissues and concentrated TSE agents
3.13 There may be experimental situations where the amount of TSE agent is likely to be significantly higher than levels normally encountered in naturally occurring disease. The risk of exposure may also be increased when tissue known to carry TSE infectivity is disrupted or concentrated (e.g. by homogenisation and centrifugation procedures). Such operations should be appropriately assessed and contained. Modified containment with additional precautions may be required on the basis of a local risk assessment. For example, the derogations from Containment Level 3 suggested above may not be appropriate for such work with CJD infected material.
Research work with animals
Containment of animals experimentally infected with TSE
3.14 Detailed guidance on handling infected animals can be found in the publication “Working safely with research animals: management of infection hazards” (ACDP 1997). It supplements the guidance in the “Categorisation of biological agents according to hazard and categories of containment” (ACDP 1995).
3.15 In general, live animals infected experimentally with TSEs do not pose a significant risk of exposure to TSE agents. However, because of the possibility of maternal transmission, parturient animals infected with TSE agents should be safely isolated and the non-viable products of parturition and other contaminated material destroyed by incineration. A local risk assessment should be made and local rules drawn up to ensure worker safety. The principles of safe working practice, including handling and restraint of animals by fully trained staff, good husbandry, basic hygiene precautions and the use of appropriate personal protection apply.
3.16 Derogation from full Containment Level 3 may be considered for experimental work with live animals inoculated with TSE agents in Hazard Group 3. The following broad principles for assigning containment may be considered, subject to a local risk assessment:
For small animals (e.g. rodents, rabbits, cats, dogs) Animal Containment Level 3 with derogations is generally acceptable. The derogated measures are the same as those for laboratory work (paragraph 3.11) except that the requirement for negative air pressure may be balanced by the need for positive pressure for animal husbandry purposes. In such cases the use of simple engineering controls (e.g. flexible barriers) or RPE may be necessary and should be carefully considered in the local risk assessment. In cases of doubt, further information can be obtained from HSE.
Animal Containment Level 2 is considered sufficient for small animals inoculated with the scrapie agent, with the proviso above about the need to balance the requirements for inward air flow against the need for positive pressure.
For large domestic animals (e.g. sheep and cattle) Animal Containment Level l is normally sufficient. However, to prevent cross-contamination and/or cross-infection, local guidelines may insist on a higher level of isolation and containment. Additional measures may be required in particular circumstances, for example, if there was a risk of exposure from bites, scratches, abrasions or contact with fluids or tissues, additional containment precautions would be required.
Animal Containment Level l is considered adequate for large animals inoculated with the agent of scrapie.
3.17 The preferred method of disposal of carcasses and other waste materials from all animals experimentally infected with TSE is incineration. Bedding and faeces from large animal accommodation should be incinerated if the results of the assessment indicate that TSE infectivity may be shed (e.g. for a period following oral challenge). After the initial shedding phase it can be disposed of in the normal way (e.g. by landfill burial or discharge to the sewer system) subject to the requirements of MAFF and the Environment Agency.
3.18 Additional containment precautions need to be considered for procedures and experiments where the following special circumstances apply:
- concentrations of infectivity above those found naturally could be expected;
- routes of inoculation are used in which leakage of infectious material externally could occur, or during the oral dosing phase when the feed material remains exposed;
- there are other experimental circumstances that might enable external release of infectivity;
- experiments with genetically modified animals.
Post mortem examination of animals with TSE
3.19 Before post mortem examinations are performed on animals with natural or experimental TSE, a risk assessment must be carried out to establish whether it is appropriate to do a post mortem (i.e. can exposure be prevented) and to assess the appropriate level of containment and controls necessary for the procedure. The principles described above for derogation from Containment Level 3, and the use of additional precautions where necessary, apply here also. The following procedures for small animal post mortem should serve as a guide to drawing up local codes of practice. If it is necessary to undertake a large animal post mortem, it is recommended that specialist expert advice is sought from the Health and Safety Executive.
3.20 Basic precautions for small animal post mortems:
- ensure that the Containment Level of the post mortem area is appropriate for the agent involved. Where it is not possible to use a dedicated room, an area of the post mortem room should be set aside;
- consideration should be given to the subsequent disinfection of working surfaces, for example, work may be conducted in a stainless steel or enamel tray which can then be autoclaved. Other working surfaces should be protected by disposable coverings;
- the procedure should be planned so that all equipment required is readily to hand and work should be organised so that there are no interruptions (e.g. to answer the telephone);
- single-use disposable items should be used wherever practicable; alternatively a set(s) of dedicated instruments may be used;
- protective clothing including gloves, gowns, masks and visors or safety spectacles should be worn;
- a ‘clean’ assistant should be available to take care of record-keeping, and handing over instruments etc;
- procedures for disinfection and decontamination as described in Annex B should be followed.
Animal neuropathology
3.21 Neuropathological examination of unprocessed experimentally infected animal TSE material (except scrapie and related agents) should be conducted at Containment Level 3. Precautions must be taken to prevent dispersal of infected material. Extra care will be needed to avoid penetrating injuries, and eye protection should be used to avoid splashing on to the conjunctiva. Autoclaving and disinfection procedures should be as recommended in Annex B. Further guidance on the decontamination of formalin fixed tissue for neuropathology is also given in Annex B.
Use of bovine eyes in research
3.22 Infectivity has been detected in the retina taken from animals clinically affected with BSE. There is a potential risk that researchers working with bovine eyes from apparently healthy cattle may be handling material containing BSE infectivity.
3.23 As a precautionary measure, eyes from healthy animals over 6 months of age are subject to the Specified Bovine Material Order 1997. Before collection, prior approval should be sought in writing from the Regional Manager of the Meat Hygiene Service.
3.24 If research work with bovine eyes is essential, it is recommended that standard basic precautions to prevent infection are strictly adhered to (see Table 2). If a choice can be made, it would be preferable to use eyes from beef breed calves less than 6 months old. If the eyes are from animals known or suspected of being infected with BSE, the earlier recommendations for work with BSE must be adopted (see Table 3).
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