Containment Standards for Laboratories, Animal Facilities and Post Mortem Rooms Handling Prion Disease Agents

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Table of Contents

Glossary

Chapter 1 - Introduction

Chapter 2 - Physical Requirements

• 2.1 Facility Location and Access
• 2.2 Surfaces
• 2.3 Containment Perimeter
• 2.4 Air Handling System
• 2.5 Facility Services

Chapter 3 - Confinement versus Containment of Large Animals

• 3.1 Confinement Requirements
• 3.2 Containment versus Confinement

Chapter 4 - Operational Practices

• 4.1 General Requirements
• 4.2 Laboratory
• 4.3 Small Animal Containment Facility
• 4.4 Large Animal Containment Facility
• 4.5 Large Animal Post Mortem Rooms

Chapter 5 - Decontamination

• 5.1 Recommended Methods
• 5.2 Chemicals

Chapter 6 - Facility Certification

• 6.1 Certification
• 6.2 Re-certification
• 6.3 Verification and Performance Testing

Acknowledgements

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Glossary:

Anteroom: a room that separates the laboratory, animal facility, or post mortem room from the hallway in order to separate street clothing from dedicated facility clothing.

BCS: Biohazard Containment and Safety

BSC: biological safety cabinet

BSE: bovine spongiform encephalopathy

CJD: Creutzfeldt-Jakob disease

CL: containment level

Containment of large animals: The physical requirements and operational practices described herein which enable the collection and appropriate disposal of excreta from TSE infected large animals and minimize contact with these animals.

Confinement of large animals: During certain periods, natural excretions and casual contact with TSE infected animals would not pose a risk for pathogen transmission. Thus, while the animals must remain adequately confined, they do not have to be maintained in a manner or facility that limits contact and enables the collection and disposal of excreta.

Containment perimeter: the boundary between the laboratory, animal facility or post mortem room and outside of containment. An anteroom, if present, is considered to be within the containment perimeter.

CWD: chronic wasting disease

HEPA Filter: high efficiency particulate air filter

ID: identification

Large animals: This term pertains to the type of housing not the size of the animal. Large animals are those animals housed in such a way that the room provides primary containment.

NaOH: sodium hydroxide

NaOCl: sodium hypochlorite

PM: post mortem

Post Mortem Facility: The area where animals are necropsied. The room provides primary containment.

PPE: personal protective equipment

Prion: small proteinaceous particle thought to be responsible for transmissible spongiform encephalopathy (TSE) diseases.

Small animals: This term pertains to the type of caging not the size of the animal. Small animals are those animals housed in microisolators or other primary containment devices.

TSE: transmissible spongiform encephalopathy

vCJD: variant Creutzfeldt-Jakob disease

VHP: Vapor Hydrogen Peroxide

Waste: Any solid or liquid material generated from the facility that is to be disposed of.

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Chapter 1 Introduction

There are a number of diseases caused by prions, also known as transmissible spongiform encephalopathies (TSE), which affect animals and humans. Bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD), feline spongiform encephalopathy, transmissible mink encephalopathy and scrapie are prion diseases which affect animals. Creutzfeldt-Jakob disease (CJD), variant Creutzfeldt-Jakob disease (vCJD), Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia and Kuru are examples of prion diseases which affect humans.

To date neither the Containment Standards for Veterinary Facilities⁽¹⁾ nor the Laboratory Biosafety Guidelines⁽²⁾ have addressed the unique requirements for prion diseases. The Containment Standards for Veterinary Facilities⁽¹⁾ provide guidance on the design and operational requirements for facilities (ie. laboratories, small and large animal facilities as well as PM areas) handling animal pathogens. Likewise the Laboratory Biosafety Guidelines⁽²⁾ provide guidance on the design and operational requirements for laboratories handling human pathogens. These documents must be referred to for facilities handling pathogens other than TSEs as additional requirements may be necessary. Health Canada's Infection Control Guideline: Classic Creutzfeldt-Jakob Disease in Canada⁽³⁾ should be consulted for health care facilities examining patients and conducting surgeries and autopsies.

The Containment Standards for Laboratories, Animal Facilities and Post Mortem Rooms Handling Prion Disease Agents are intended to describe the minimum requirements for government, university, industry and other facilities in Canada known TSE infected materials and animals are handled.

There is no evidence to suggest that prion diseases are transmitted by inhalation⁽⁴⁾. This does not preclude the need to mitigate possible exposure when aerosol or splash generating procedures are being conducted. The short and long term consequences of gross contamination of mucosa in the nasal, olfactory and oral cavities as well as possible secondary ingestion are not known. Training of personnel who work in such facilities is paramount. Personnel must be aware of the risks associated when working with TSE agents. The most likely route of transmission for prion disease agents to humans is through ingestion or inoculation. Procedures to avoid accidental cuts and punctures as well as proper hygiene are the best approaches to protect workers. Personal protective equipment (PPE) such as cut resistant gloves should be used and care must be taken to avoid punctures.

TSE diagnostic laboratories must at a minimum meet the physical requirements for containment level 2 as per the Containment Standards for Veterinary Facilities⁽¹⁾ plus the following physical requirements. The entrance to the laboratory should provide for the separation of PPE from staff clothing, preferably in a separate anteroom. New construction should consider the requirements for surface finishes described in section 2.2. The intent is for surfaces to be non-porous and cleanable and able to withstand the chemicals used for decontamination. Penetrations in areas where spills may be likely should be sealed (eg. caulked) to allow for containment and thorough surface decontamination. Bag-in / bag-out HEPA biological safety cabinets are recommended. If they are not available then a procedure must be in place for the contained removal of HEPA filters. The autoclave should ideally be located in the laboratory. If the autoclave is located elsewhere in the facility then protocols should be in place that will allow for proper identification of the waste (including treatment parameters) and the secure transport within the building. Operational procedures listed in chapter 4 of the Containment Standards for Laboratories, Animal Facilities and Post Mortem Rooms Handling Prion Disease Agents must be followed. The laboratory and equipment that will be used for work with prion agents should, where possible, be dedicated. If it is not possible to use dedicated equipment then an effective decontamination protocol must be in place.

Effluent treatment systems are not required for PM rooms where the animals necropsied are not known to be infected with TSE agents. However, operational procedures should be in place to collect and treat liquid wastes in the event that a positive animal is detected. For example, plastic or absorbent pads may be used to contain liquids during necropsy of animals that exhibit signs of neurological disease. Tissues should be incinerated and surfaces decontaminated.

The Biohazard Containment and Safety (BCS) Unit reviews permit applications for the importation of animal and zoonotic pathogens, assesses containment facilities for import purposes, and issues import permits. The Health of Animals Act⁽⁵⁾ and its Regulations⁽⁶⁾, provide the legislative authority to control the use of imported animal pathogens as well as pathogens associated with reportable animal diseases. BSE, CWD and Scrapie are reportable disease under the Health of Animals Act⁽⁵⁾. The BCS Unit must be contacted before diagnostic testing, or research is conducted with these agents. The BCS Unit works closely with the CFIA's Animal Products Directorate to determine safe handling practices for animal pathogens and prevent release to the environment. The Office of Laboratory Security at the Public Health Agency of Canada reviews permit applications for the importation of human and zoonotic pathogens, assesses containment facilities for import purposes, and issues import permits. The Human Pathogens Importation Regulations⁽⁷⁾ (SOR/94-558) govern the import of human pathogens into and transfer within Canada.

References:

1. Containment Standards for Veterinary Facilities. Ottawa: Agriculture and Agri-Food Canada, Minister of Supply and Services Canada, No. 1921/E, 1996.
2. Laboratory Biosafety Guidelines. Ottawa: Public Health Agency of Canada, Minister of Health, 2004.
3. Infection Control Guidelines, Classic Creutzfeldt-Jakob Disease in Canada. Ottawa: Public Health Agency of Canada, Volume: 28S5, November 2002.
4. Prusiner, S.B. Prion Biology and Diseases. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 2004; 189-190.
5. Health of Animals Act. (1990, c.21)
6. Health of Animals Regulations. SOR/91-525, s. 2.
7. Human Pathogens Importation Regulations (SOR/94-558)

Chapter 2 Physical Requirements

The physical requirements described below are for the containment of both human and animal prion disease agents. Both the Containment Standards for Veterinary Facilities⁽¹⁾ and the Laboratory Biosafety Guidelines⁽²⁾ have been referred to in development of these requirements.

In addition to these biocontainment requirements, work with animals must comply with all applicable laws and guidelines within the province where the research will be conducted, including Canadian Council on Animal Care guidelines, provincial welfare acts and provincial research animal acts.

For cattle infected with BSE or sheep infected with scrapie there are periods of time after experimental inoculation where infected animals do not shed the agent and can therefore be held in a confined area. Confinement requirements are addressed in Chapter 3. The mode of transmission for CWD between animals is not fully understood. Therefore, to protect the environment the following containment measures apply for large animal trials with CWD for the duration of the experiment. TSE experiments in non-host species must also follow the containment requirements described in this chapter.

Key:
- Mandatory
- Recommended

2.1 Location and Access

2.1	Location & Access	Laboratory	Small Animal Facility	Large Animal Facility	Post Mortem Room
1	Separated from public areas by a door.
2	Access limited to authorized personnel.
3	Doors to provide restricted access by a controlled access system (eg. key card, locking hardware) or equivalent. Does not apply to areas within the facility.
4	Room doors to have appropriate signage (ie. biohazard sign, containment level, contact information, entry requirements).
5	Entry to be provided via an anteroom
6	Entry to allow for separation of personal clothing from dedicated facility clothing (ie. "clean" change area separated from "dirty" change area).
7	Office areas to be located outside of containment perimeter. Paperwork stations for data collection can be within containment provided they are located away from laboratory work areas.
8	Door openings to allow passage of required equipment (ie. may be greater than standard width and height in accordance with equipment size).

2.2 Surfaces Finishes

2.2	Surfaces Finishes (ie. Floors; walls - at and below work surface; benches)	Laboratory	Small Animal Facility	Large Animal Facility	Post Mortem Room
1	Doors and frames to be non-absorptive and have solid finishes (ie. unfinished wood is not acceptable); hollow doors must be sealed.
2	Interior surfaces (eg. floors, walls, benches, casework, etc.) must be non-absorptive and able to withstand decontamination methods (eg. sodium hypochlorite or sodium hydroxide).
3	Interior coatings to be cleanable.
4	Interior surfaces to be continuous (flooring with welded seams is acceptable).
5	Interior surfaces to provide impact resistance in accordance with function.
6	Surfaces to be continuous and compatible with adjacent and overlapping materials to maintain adhesion and a continuous perimeter.
7	Continuity of seal to be maintained between the floor and wall (a cove floor finish up the wall is recommended).
8	Floors to be slip-resistant.
9	Bench tops to have no open seams.
10	Bench tops to contain spill of material (eg. with marine edges and drip stops).

2.3 Containment Perimeter

2.3	Containment Perimeter	Laboratory	Small Animal Facility	Large Animal Facility	Post Mortem Room
1	All mechanical, electrical and service piping penetrations to be sealed at and below the work surface.
2	Windows positioned on containment perimeter to be sealed in place; window glazing material to provide required level of security.
3	Autoclave capable of achieving at least 134°C or 121°C if a two-step (chemical then heat) process is used.
4	Double door autoclave located on the containment perimeter or autoclave within containment perimeter.
5	If it is not possible to locate the autoclave at or within the containment perimeter, then strict waste control procedures must be implemented for the transport of waste and equipment in leakproof and impact resistant containers to a suitable autoclave within the facility.
6	Body of autoclave should be located, for ease of maintenance, outside of containment.
7	Autoclave located at the perimeter should be equipped with an interlocking mechanism to prevent both doors from opening at the same time.
8	Autoclave to be equipped with a cycle log recorder (ie. to record time, temperature, and pressure).
9	Area for chemical decontamination of materials and equipment.
10	Other accepted technologies for decontamination, (eg. incineration, chemical dunk tank, alkaline hydrolysis) to be provided at or within the containment perimeter.
11	If not possible to install other accepted technologies on the perimeter or inside, then strict waste control procedures must be implemented for the transport of waste to such systems.

2.4 Air Handling System

Prion disease agents are not known to be transmitted by inhalation, nevertheless inward directional airflow is highly recommended to prevent spreading of air contaminants to other occupied parts of the building. Other reasons for maintaining inward directional airflow include odor and allergen control from animals and chemicals. In general, when an aerosol generating procedure is performed outside a primary containment device (such as a BSC) it is prudent practice to ensure that the aerosol generated does not spread to other occupied areas (eg. hallways and offices).

Where inward directional airflow is deemed critical, by local risk assessment or applicable code, a magnehelic gauge may also be installed to ensure appropriate pressure is maintained⁽³⁾. Users should verify the functioning by visual inspection with a smoke pencil or other visual aid.

When choosing a BSC one must consider the type of work to be done (ie. will volatile toxic chemicals or radionuclides be handled). When installing a BSC one must also consider the ability to remove the HEPA filter safely from the BSC, the ability to decontaminate the BSC for servicing and the location in the laboratory. For more information about BSCs please refer to the Laboratory Biosafety Guidelines⁽²⁾.

Formaldehyde gas is not an effective method for inactivating prions. Where facilities choose gas decontamination for other reasons, backdraft prevention devices on supply and exhaust air ducts should be considered to isolate the facility.

2.4	Air Handling System:	Laboratory	Small Animal Facility	Large Animal Facility	Post Mortem Room
1	Provide appropriate ventilation for use of caustic chemicals.
2	Laboratory to be equipped with a BSC.
3	Risk assessment to be done for the type of BSC required (ie. hard ducted vs. recirculating cabinet).
4	BSCs to have bag in/bag out HEPA filters.
5	Where bag in/bag out HEPA filtered BSC's are not available, then procedure must be in place for the safe removal of HEPA filters*.
6	Supply and exhaust air diffusers must not interfere with the operation of BSCs and fume hoods.
7	Exhaust air system to be independent of other areas.

*Bag-in/bag-out filters are recommended because formaldehyde fumigation is ineffective against prions. A recent study demonstrates that vapour phase hydrogen peroxide (VHP) provides a significant reduction in infectivity (6). Decontamination of filters with VHP followed by incineration is considered to be an acceptable option for safe removal and disposal of filters.

2.5 Facility Services

2.5	Facility Services (ie. water, compressed gases, electricity):	Laboratory	Small Animal Facility	Large Animal Facility	Post Mortem Room
1	Drains and associated piping (including autoclave chamber condensate) to be separated from lower levels of containment (ie. connected to a liquid effluent treatment system as appropriate).
2	If effluent treatment is present, drains connected to effluent treatment should be sloped towards treatment system to ensure gravity flow; consideration should be given to the installation of valves to isolate sections for decontamination; piping to be heat and chemical resistant consistent with application; joints should be made by thermo/chemical fusible means or welding to ensure integrity of entire system in accordance with pressure decay testing specified in the Containment Standards for Veterinary Facilities(1)).
3	Floor drains should be installed only when essential.
4	Water supply control valve to be located outside Facility.
5	Domestic water branch piping to be provided with backflow prevention, in accordance with CAN/CSA-B64.10- 01/B64.10.1-01(4) and isolation valve, to be located in close proximity to the containment perimeter.
6	Compressed gas cylinders (with the exception of fire extinguishers) to be located outside the containment perimeter.
7	If required, portable vacuum pumps are to be provided with device to minimize internal contamination of vacuum pump (eg. equivalent of HEPA filtration of vacuum line, use of disinfectant traps).
8	Handwashing sinks to be provided near the point of exit or in anteroom.
9	Handwashing sinks to be provided with "hands-free" capability.
10	Emergency eyewash stations to be provided in containment in accordance with activities and applicable regulations (ie. ANSI Z358.1-2004(5) ).
11	Emergency shower equipment to be provided in containment in accordance with activities and applicable regulations (ie. ANSI Z358.1-2004(5) ).
12	Water seals to be maintained in drainage traps through regular sink usage, seal primer, or by filling traps in areas that are not used regularly.
13	Plumbing vent lines to be heat or chemical resistant consistent with application.
14	Supply conduit and wiring to be sealed at and below the work surface to ensure surface decontamination is possible.
15	Power system circuit breakers to be located outside containment perimeter.
16	Life-safety systems, lighting, BSCs and other essential equipment to be supported by emergency power. If BSCs are not supported by emergency power then procedures should be in place for exiting.
17	Communication system to be provided between inside and outside of containment perimeter.
18	System (eg. fax, computer) to electronically transfer information and data to outside containment.

References:

1. Containment Standards for Veterinary Facilities. Ottawa: Agriculture and Agri-Food Canada, Minister of Supply and Services Canada, No. 1921/E, 1996.
2. Laboratory Biosafety Guidelines. Ottawa: Public Health Agency of Canada, Minister of Health, 2004.
3. Standard for Laboratory Ventilation. ANSI/AIHA Z9.5-2003, Arlington, Virginia: American National Standards Institute, Inc., 1992
4. Manual for the selection and installation of backflow prevention devices/manual for the maintenance and field testing of backflow prevention devices. CAN/CSA-B64.10-01/B64.10.1-01. Toronto, ON: Canadian Standards Association, 2001.
5. American national standard for emergency eyewash and shower equipment. ANSI Z358.1-2004. Arlington, Virginia: American National Standards Institute, Inc., 2004.
6. Fichet G., Comoy E., Duval C., Antloga K., Dehen C., Charbonnier A., McDonnell G., Brown P., Lasmézas C., Deslys JP., Novel Methods for disinfection of prion-contaminated medical devices. Lancet 2004; 364:521-26.

Chapter 3 Confinement versus Containment of Large Animals

Although scrapie has been described as a disease of sheep and goats since the 1700s, the overwhelming focus on research into TSEs since the discovery of BSE in Britain has resulted in significant recent scientific advances in the understanding of the epidemiology of both BSE and scrapie While genetic susceptibility has been documented as playing a large role in the distribution of scrapie within sheep populations, horizontal transmission of scrapie has been confirmed. Recent research documents that pregnancy triggers the migration of abnormal prion protein to the reproductive tract. Fluids and tissues associated with parturition, such as placenta from infected females, contain large quantities of the scrapie agent. Healthy animals become infected by eating or licking contaminated materials in the lambing or kidding environment^{(1, 2)}.

In contrast, no evidence has been found to indicate that horizontal transmission of BSE occurs. Likewise, ongoing long-term research studies in the United Kingdom have indicated that transmission of BSE to offspring does not occur^{(3, 4)}. Ingestion of BSE infected tissue in feed (meat and bone meal) remains the single documented route of transmission for BSE.

While CWD of deer and elk is known to be transmitted horizontally, both animal to animal, as well as by contact with a heavily contaminated environment, it is still not established by which route the abnormal prion is excreted from the body. Until this essential information regarding the epidemiology of CWD is available, all bodily excretion and secretions of infected deer and elk must be treated as potentially infectious ^{(5, 6)}.

Due to the prolonged incubation periods of TSEs in host species (several years), it is logical to examine the prolonged time of captivity to determine if the research animal poses the same level of risk for the TSE pathogens during the entire period. In light of the recent advances in the understanding of the epidemiology of both BSE and scrapie it was determined that these pathogens inoculated into host species only presented significant risk for pathogen transmission during specific periods of time as defined in the chart below. During these specific periods of time the infected animals must be maintained in appropriate containment which enables the collection and appropriate disposal of excreta as well as minimizing contact with these animals. During the remaining periods of time, natural excretions and casual contact with these animals would not pose a risk for pathogen transmission. Thus, while the animals must remain adequately confined, they do not have to be maintained in a manner or facility that limits contact and enables the collection and disposal of excreta.

3.1 Confinement Requirements

The requirements listed below are the minimal conditions for confining natural hosts infected with BSE or Scrapie. Experimental design or other factors (eg. value of animals) may dictate tighter controls for housing.

1. Observation and counting of animals in confinement must occur daily. The number of animals must be recorded daily.
2. Single point access control must be adequate for the local environment to prohibit unauthorized personnel or animals from the entering area and to confine experimental animals. Access must be verified to function as intended.
3. A double identification (ID) system for animals must be in place and verification of ID must occur daily. If an ID tag or other device is missing then it must be replaced immediately.
4. Materials (manure and bedding) from pens can be dealt with by normal composting and disposal.
5. Pastures are subject to normal pasture management.

3.2 Containment versus Confinement

The following table outlines the time frame in which the natural hosts of the corresponding diseases must be housed in containment as described in Chapter 2 or may be confined with the appropriate measures listed in Chapter 3.

Experimental time frame	BSE	Scrapie	CWD
post inoculation	TSE containment for at least 4 weeks	TSE containment for at least 4 weeks	TSE containment
incubation	confine	confine	TSE containment
clinical*	TSE containment	TSE containment	TSE containment
parturition	confine	TSE containment	TSE containment

* at first sign of clinical disease animals are to be contained and waste treated. Pregnant female small ruminants should be moved into containment at 140 days gestation. Placentas and all bedding contaminated with birthing fluids must be collected and decontaminated.

References:

1. S. Ryder, G. Dexer, S. Bellworthy, S. Tongue Demonstration of lateral transmission of scrapie between sheep kept under natural conditions using lymphoid tissue biopsy. Research in Veterinary Science 76 (2204) 211-217
2. Baylis M, Houston F, Kao RR, McLean AR, Hunter N, Gravenor MB. BSE -a wolf in sheep's clothing? Trends Microbiol. 2002 Dec;10(12):563-70. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&
   db=pubmed&dopt=Abstract&list_uids=12564992
3. Wrathall AE, Brown KF, Sayers AR, Wells GA, Simmons MM, Farrelly SS, Bellerby P, Squirrell J, Spencer YI, Wells M, Stack MJ, Bastiman B, Pullar D, Scatcherd J, Heasman L, Parker J, Hannam DA, Helliwell DW, Chree A, Fraser H. Studies of embryo transfer from cattle clinically affected by bovine spongiform encephalopathy (BSE). Vet Rec. 2002 Mar 23;150(12):365-78. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&
   db=pubmed&dopt=Abstract&list_uids=11936410
4. Wilesmith JW, Ryan JB. Absence of BSE in the offspring of pedigree suckler cows affected by BSE in Great Britain. Vet Rec. 1997 Sep 6;141(10):250-1.
5. Michael W. Miller, Elizabeth S. Williams, N. Thompson Hobbs, and Lisa L. Wolfe Environmental Sources of Prion Transmission in Mule Deer. Emerging Infectious Diseases. 2004 Jun; Vol 10, No 6
6. Sigurdson CJ, Miller MW. Other animal prion diseases. Br Med Bull. 2003;66:199-212.

Chapter 4 Operational Practices

Facility personnel, animal care staff and pathologists handling infected, or potentially infected tissues could be exposed to infectious prions through accidental inoculation, cuts or punctures with contaminated instruments, contact with open wounds, contact with mucous membranes and/or accidental ingestion. The most likely route of transmission for prion disease agents to humans is through ingestion or inoculation. Procedures to avoid accidental cuts and punctures as well as proper hygiene are the best approaches to protect workers. PPE such as cut resistant gloves should be used and care must be taken to avoid punctures.

Biosafety practices should be written and implemented for facilities handling infected animals and their tissues, blood and body fluids as well as infected human tissue to prevent transmission and worker exposure to TSE contaminated material.

Other countries with guidance documents for handling TSEs are the United Kingdom⁽³⁾, Germany^{(4, 5)} and Australia^{(6, 7)}. Health Canada(8) and the World Health Organization ⁽⁹⁾ have also produced infection control guidelines for health care facilities handling TSE infected patients and tissues.

4.1 General Requirements

General practices are required when working in any containment laboratory or animal facility. These can be referred to in the Containment Standards for Veterinary Facilities⁽¹⁾ and the Laboratory Biosafety Guidelines⁽²⁾.

4.2 Laboratory

In addition to the general practices, the following describes the minimum operational practices required for TSE laboratory containment.

1. Personnel must have demonstrated proficiency in handling infectious tissues and materials.
2. All protocols specific to the operation of the laboratory must be developed, read and understood by personnel. Employees must certify in writing that they have understood the material in the protocols. Protocols should include entry and exit procedures for people, equipment, samples and waste. General protocols must be supplemented with protocols specific to each project in progress.
3. Protocols are to be in place for the effective decontamination of all laboratory waste, re-usable equipment, and laboratory space. If contaminated materials or liquids (such as reagents for washing, staining, etc.) are to be released to sanitary sewer then they must be decontaminated before release.
4. Laboratory personnel must be trained in, and follow, the safe use of laboratory equipment, biological safety cabinets, procedures to minimize the production of aerosols, decontamination and emergency response.
5. An exposure protocol is to be developed, posted and communicated to staff in the event of accidental inoculation or splashing into mouth or eyes. Training in the application of this protocol must be documented.
6. Open wounds, cuts, scratches and grazes must be covered with waterproof dressings.
7. Eating, chewing gum, drinking, smoking, storing food, and applying cosmetics are prohibited in the containment area.
8. Personal items such as, purses and outdoor clothing must not be brought into the containment laboratory.
9. Long hair should be tied back so that it cannot come into contact with hands, specimens, containers or equipment.
10. Laboratory doors must be kept closed to control access. Entry to the laboratory should be documented (eg. logbook).
11. Where possible, the laboratory should be a dedicated facility.
12. Equipment that will be used for work with TSE agents should, where possible, be dedicated. If dedicated equipment is not available, then an effective decontamination protocol must be in place. Consideration should be given to disposable equipment.
13. Personal clothing should be kept separate from dedicated facility clothing.
14. Selection of appropriate PPE will depend on the amount and nature of infectious material as well as the procedures being used. A job hazard analysis (JHA) should be conducted in the facility to examine each procedure, identify risks and develop safe work practices that include appropriate PPE.
15. Personnel entering the laboratory must remove jewellery and should don solid-front gowns with tight-fitting cuffs, gloves, shoecovers or dedicated footwear. In general, solid front gowns are preferable to lab coats for preventing contamination of clothing. Consideration should be given to disposable labwear. Double gloves must be worn when handling infectious materials. Disposable sleeve covers are recommended for handling and manipulating infectious tissue.
16. Full face protection, such as dedicated glasses or eye protection with a mask or a face shield, should be worn for any procedures in which splashing and/or flying particles may be a hazard. If respirators are used then a respiratory protection program must be in place as per the applicable Provincial and Federal (eg. Canada Labor Code regulations).
17. In case of a spill or splash, face shields and dedicated eye protection should be decontaminated.
18. Cuts and punctures are to be avoided by minimizing the use of sharp knives, scalpels and blades; wearing cut resistant gloves; substituting blunt cannulas for needles and plasticware for glassware where possible. Cut resistant gloves do not protect against punctures.
19. Procedures which generate, or may generate, aerosols or splashes (eg. homogenization, sonication, titration, centrifugation and micro centrifugation, preparation of dilutions of inoculum, vortexing, pipetting (recommend using plugged tips), cell sorting, tissue trimming, necropsy procedures) must be conducted in a BSC; if this is not possible, other primary containment devices in combination with personal protective clothing and equipment must be used. Care must be taken not to overcrowd or block the grills of BSCs. This will ensure that the appropriate airflow patterns are not compromised.
20. Enclosed homogenization systems may be placed in a BSC where possible.
21. Centrifugation of infectious materials must carried out in sealed safety cups or rotors that are loaded and unloaded in a BSC.
22. To minimize contamination of work surface in the BSC, work should be conducted within a tray or container, or on absorbent coverings provided they do not interfere with the functioning of the BSC. Consideration should be given to absorbent coverings that can be incinerated after use.
23. When handling known positive material consideration should be given to the use of disposable laboratory supplies (eg. forceps, embedding molds).
24. Outer gloves must be removed prior to exiting the BSC.
25. Work surfaces must be decontaminated after work with contaminated material is completed. Where absorbent coverings are used they should discarded as waste once work is completed. Where absorbent coverings are used they must be decontaminated before disposal.
26. Microtome work should be done in a dedicated low traffic area. The area on the floor around the microtome should be taped off and in this area disposable shoecovers worn and removed as exiting, or other effective measure to prevent tracking of wax shavings. Troughs may be installed on the edge of the work bench to contain any excess shavings.
27. Routine laboratory cleaning must be done by personnel using the containment facility or by specific personnel dedicated and trained for this task.
28. The laboratory work area must be kept free from materials that are not required and cannot be easily decontaminated (eg. journals, books, correspondence).
29. Paperwork and report writing should be kept separate from areas where infectious materials are handled. Data transfer should occur by electronic devices.
30. All contaminated material is to be transported in closed, break resistant carriers when not in a BSC.
31. Infectious agents should be stored inside the laboratory zone in leakproof containers. Agents stored outside the laboratory must be kept in leakproof containers in a locked freezer within a secure zone.
32. Hands should be washed after removing inner gloves, and before leaving the laboratory.
33. Where disposable gowns are not worn, contaminated clothing must be decontaminated prior to laundering.
34. Efficacy monitoring of autoclaves using an appropriate temperature indicator or internal load probe must be used in every load. Records of the results must be kept on file including cycle log records (ie. time, temperature and pressure).
35. Heat sensitive materials that cannot be autoclaved must be decontaminated at the containment perimeter (eg. decontaminated using liquid chemicals, or other effective technology).
36. An inventory of positive control material is to be maintained and documented. Access to this material must also be controlled.

4.3 Small Animal Containment Facility

Work with small animals poses a variety of special hazards including exposure to animal bites and scratches.

Allergic conditions can result from contact with animal fur or hair, bedding, and animal wastes⁽¹⁾. At least one-fifth of people who work with laboratory rodents, guinea pigs and rabbits develop allergies. Protection from allergens must be provided through engineering controls, ventilation, use of isolators and cages with filter tops and appropriate use of respiratory protection.

Animal handlers must have knowledge of the species' general characteristics such as behaviour, instincts and physical attributes. Consideration should also be given to their natural ecto- and endoparasites and the zoonotic diseases to which they are susceptible including their route of excretion and dissemination.

Small animal containment pertains to the type of caging not the size of the animal. Small animals are those animals housed in microisolators or other primary containment devices.

The following, in addition to sections 4.1 and 4.2 describes the minimum operational practices required in small animal facilities:

1. Personnel entering the facility must remove jewelry and don dedicated lab clothing such as scrubs, solid-front gowns with tight-fitting cuffs (disposable if possible), bonnets, shoecovers or dedicated footwear.
2. Double gloves must be worn when handling infected animals or materials. Outer gloves to be removed after handling animals.
3. Full face protection, such as face shields or eye protection and masks are required for handling animals where infectious aerosols, splashing or flying particles may be a hazard.
4. Each animal room must be labelled with unique hazards and entry requirements.
5. Animal room doors must be kept closed to control access.
6. Cages housing infected animals must be appropriately labelled.
7. Containment caging systems should be used to contain all materials and aerosols (eg. solid wall and bottomed cages, covered with filter bonnets).
8. Careful handling procedures must be employed so as to minimize the creation of aerosols and dissemination of dust from cages, refuse and animals.
9. Proper methods of restraint must be used to minimize scratches, bites and accidental self-inoculations.
10. Inoculation and necropsy of infected animals must be conducted in a BSC; animals must be securely transported to the BSC.
11. Animal bedding must be removed in a manner that minimizes the generation of aerosols and dust; cages must either be decontaminated prior to removing bedding (usually by autoclaving), or bedding must be removed at a ventilated cage changing station or BSC.
12. Animal cages must be decontaminated prior to washing.
13. Animal carcasses, tissues and contaminated bedding must be incinerated or processed through other effective technologies. Material to be transported from the animal room for disposal must be in leakproof, break resistant containers that are appropriately labelled.

4.4 Large Animal Containment Facility

Work with large animals poses a variety of special hazards including kicking, crushing, fight or flight responses (depending on species) and/or accidental self inoculations. Large animals should undergo a transitional period to their new environment and surroundings before a trial begins.

Animal handlers must have knowledge of the species' general characteristics such as behaviour, instincts and physical attributes. Consideration should also be given to their natural ecto- and endoparasites and the zoonotic diseases to which they are susceptible including their route of excretion and dissemination.

Large animal containment pertains to the type of housing not the size of the animal. Large animals are those animals housed in either a room, barn or cage, where the room provides primary containment.

The following, in addition to 4.1- 4.2 describes the minimum operational practices required in large animal facilities:

1. Personnel entering facility must remove street clothing and jewellery, and change into dedicated facility protective clothing, equipment and footwear (eg. face protection such as face shields or safety goggles with masks, rubber boots, outer gloves, disposable coveralls), this layer must be removed on exit from contaminated area. Where dedicated facility clothing is used (eg. scrubs or farm clothing) then a complete clothing change may not be required if a secondary layer of dedicated clothing is worn in the animal facility.
2. Entering more than one animal cubicle from a clean corridor is generally not acceptable unless appropriate decontamination or change in protective clothing, equipment and footwear has occurred; entering more than one animal cubicle from a dirty corridor may be acceptable depending on the project (eg. moving between contaminated areas of equal status).
3. Face shields and safety goggles should be decontaminated after use.
4. Hands must be washed after removing gloves and before leaving the facility.
5. Where dedicated eye glasses are not worn, eye glasses should be decontaminated at the containment perimeter.
6. Animal cubicle doors must be kept closed to control access.
7. Each animal cubicle must be labelled with unique hazards and entry requirements (eg. additional PPE or other requirements).
8. Proper methods of restraint must be used to minimize kicks, crushing injuries and accidental self-inoculations.
9. The exterior surfaces of containers of biological samples to be removed from contaminated animal rooms must be decontaminated. Heat sensitive samples can be chemically decontaminated (eg. immersion in disinfectant on the perimeter).
10. At the end of the experiment all supplies remaining in the animal cubicles must be removed and decontaminated. Feed remaining in the animal cubicles must be decontaminated and destroyed appropriately.
11. Animal carcasses and tissues must be incinerated or processed through other effective technology; carcasses must be transported from the animal cubicle for disposal via the dirty corridor (alternatively, leakproof containers may be used for transport).
12. Animal cubicles and the dirty corridor must be cleaned and decontaminated at the end of an experiment using an appropriate procedure.
13. Animal faeces and bedding must be decontaminated using appropriate methods.
14. Sheep used in scrapie experiments must be held in containment while lambing. Bedding, placental fluids and any other waste must be collected and decontaminated. Lambing area must be decontaminated upon removal of the ewe and lamb.

4.5 Large Animal Post Mortem Rooms

As with the other types of facilities, protocol development, training and documentation are required for PM areas. Hazards in the PM room are not limited to splashes and aerosols of infectious materials. Accidents can be caused by cutting instruments, sharp ends of cracked bones, slippery floors, electrical equipment, chemical fixatives and disinfectants.

The following operational practices, in addition to the requirements outlined in the Containment Standards for Veterinary Facilities⁽¹⁾, must be followed for PM rooms.

1. The minimal PPE to be worn while working with infectious materials is full protection clothing (eg. scrubs, disposable impermeable suits, waterproof aprons, head covers, dedicated footwear or disposable impermeable footwear) and double gloves (when using sharps also use cut proof gloves). Exposure to mucous membranes or accidental ingestion of contaminated tissues must be prevented by use of full face shields or safety goggles and surgical masks. A respirator is not a requirement, however when respirators are used a respiratory protection program must be in place as per the applicable Provincial and Federal (eg. Canada Labor Code;) regulations.
2. Where a PM room is not equipped with an effluent treatment system, work with infectious tissue must be done in an isolated area where fluids and tissues can be contained and treated appropriately before release.
3. The use of equipment that will minimize the generation of aerosols is preferred (eg. manual saws).
4. Gross contamination on equipment and surfaces must be removed and treated appropriately prior to decontamination of surfaces. Instruments should be kept moist until decontamination has occurred.
5. Procedures must be in place where decontamination of personnel is required. Showering must not be used as a method of decontamination where liquid effluent treatment is not available.

References:

1. Containment Standards for Veterinary Facilities. Ottawa: Agriculture and Agri-Food Canada, Minister of Supply and Services Canada, No. 1921/E, 1996.
2. Laboratory Biosafety Guidelines. Ottawa: Public Health Agency of Canada, Minister of Health, 2004.
3. Guidance from the Advisory Committee on Dangerous Pathogens and the Spongiform Encephalopathy Advisory Committee. Transmissble Spongiform Encephalopathy Agents: Safe Working and the Prevention of Infection. Part 3. December 15, 2003,
4. Recommendations of the Federal Animal Viral Diseases Research Institute for the taking of samples and conduct of diagnostic procedures under the BSE and scrapie epidemiological monitoring program and the examination of the actual suspects. Decision of the Biological Agents Board, ABAS 603. February 2001; Federal Labour Gazette, 2001-02, p.97
5. Special measures for the protection of employees against infection by BSE or scrapie pathogens. Decision of the Biological Agents Board, ABAS 602. Federal Labour Gazette, 2002-12, p.92.
6. AUSVETPLAN: Disease Stratagey; Bovine Spongiform Encelalopathy - PDF (152 kb). Version 3, 2003.
7. Creutzfeldt-Jakob Disease and Other Human Transmissible Spongiform Encephalopathies: Guidelines on patient management and infection control. Commonwealth of Australia. 1995. http://www.nhmrc.gov.au/publications/pdf/ic5.pdf
8. Infection Control Guidelines, Classic Creutzfeldt-Jakob Disease in Canada. Ottawa: Public Health Agency of Canada, Volume: 28S5, November 2002.
9. WHO Infection Control Guidelines for Transmissible Spongiform Encephalopathies. Report of a WHO consultation. Genva, Switzerland, 23-26 March 1999.

Chapter 5 Decontamination

TSE agents are particularly resistant to standard physical and chemical methods of decontamination⁽¹⁾. Complete inactivation may not always be possible, however all attempts should be made to ensure that adequate decontamination is met. This chapter describes decontamination methods that have been accepted internationally (eg. WHO Infection Control Guidelines for Transmissible Spongiform Encephalopathies - PDF (194 kb): http://whqlibdoc.who.int/hq/2000/WHO_CDS_CSR_APH_2000.3.pdf). These recommendations may change as new information becomes available. Treatment technologies described in this document are limited to those intended to decontaminate waste from laboratories, animal facilities and post mortem areas working with prion disease agents. From this perspective, new technologies will require further evaluation including evidence of prion inactivation published in peer reviewed scientific journals.

Marketed liquid chemical disinfectants are evaluated by Health Canada for safety, efficacy and quality. There are currently no validated methods for testing "disinfectants" against prions. Manufacturers seeking label claims against prions should contact Health Canada's Therapeutic Products Directorate, For decontamination methods appropriate for use in human health care facilities please refer to Health Canada's Infection Control Guidelines: Classic Creutzfeldt-Jakob Disease in Canada⁽²⁾.

5.1 Recommended Decontamination Methods

Infected tissue and animal carcasses:

• Incineration at 850°C ^(1,3)
• Alkaline hydrolysis⁽⁴⁾, consult municipal, provincial and federal environmental regulations

Contaminated Small Animal waste and bedding :

• Incineration at 850°C is highly recommended ⁽³⁾
• Autoclave at 134°C for 1 hour⁽⁵⁾

Contaminated Large Animal waste and bedding :

• For BSE in cattle, waste and bedding must be decontaminated for at least 4 weeks after inoculation, and again when clinical signs are observed. Incineration at 850°C ⁽³⁾ is recommended. Liquid effluent treatment at 134°C for 1 hour is acceptable.
• For scrapie in sheep, waste and bedding must be decontaminated for at least 4 weeks after inoculation, and again when clinical signs are observed. Incineration at 850°C ⁽³⁾  is recommended. Liquid effluent treatment at 134°C for 1 hour is acceptable. Pregnant ewes must be contained so that placental materials may be incinerated and birthing fluids decontaminated.
• For CWD in deer, all waste and bedding must be incinerated at 850°C ⁽³⁾.
• For non-host species inoculated with a prion disease agent, it may be unclear whether there is shedding in animal waste (eg. CWD in cattle, or BSE in deer). All animal waste and bedding must therefore be incinerated. at 850°C ⁽³⁾.

Solid Waste (disposable PPE, garbage etc.)

• Incineration at 850°C is highly recommended^{(3, 5, 6)}
• Autoclave at 134°C for 1 hour⁽⁵⁾
• Chemical treatment with sodium hydroxide (NaOH) or sodium hypochlorite (NaOCl) then transfer to water before autoclaving at 121°C

Instruments intended for disposal:

• Incineration at 850°C ^{(2, 3, 5)} (may be inappropriate for some materials, eg. plastics)
• Wipe clean, soak in 2% available chlorine for 1 hour at 20°C⁽⁵⁾, then disposal
• Wipe clean, soak in 2M NaOH for 1 hour at 20°C⁽⁵⁾, then disposal
• Wipe clean, autoclave at 134-138°C for 1 hour⁽⁵⁾, then disposal

Paper towels used to wipe instruments clean must be treated as solid waste.

Instruments intended for re-use :

Instruments and materials subject to re-use should be kept moist between the time of exposure to infectious materials and subsequent cleaning and decontamination⁽⁵⁾.

• Wipe clean thoroughly, then soak in 2% available chlorine for 1 hour at 20°C⁽⁵⁾
• Wipe clean thoroughly, then soak in 2M NaOH for 1 hour at 20°C⁽⁵⁾ rinse with water and then autoclave at 134-138°C for 18 min⁽⁵⁾
• Wipe clean thoroughly, immerse in 1N NaOH or 2% available chlorine for 1 hour, rinse with water, transfer to water, autoclave at 121°C ⁽⁷⁾ for 1 hour ⁽⁵⁾.

Paper towels used to wipe instruments clean must be treated as solid waste.

Liquids:

• Autoclave at 134°C for one hour.
• Incineration at 850°C ⁽³⁾
• Mix with bleach for a final concentration of 2% available chlorine for one hour

Surfaces:

• Clean thoroughly, then flood surfaces with 2% available chlorine for 1 hour at 20°C⁽¹⁾, then rinse with water
• Clean thoroughly, then flood surfaces with 2M NaOH for 1 hour at 20°C^(1,5), then rinse with water

5.2 Chemicals

Be familiar with (and observe) safety guidelines for working with chemicals. Read labels and material safety data sheets carefully. Wear appropriate PPE to prevent chemical exposure. NaOH reacts with CO₂ to form carbonates that neutralize NaOH and diminish its decontamination effectiveness.

The exposure of NaOCl solutions to light and air increases the loss of available chlorine. Therefore it is appropriate to:

• keep these solutions in closed light-tight containers
• prepare fresh solutions prior to each use
• label containers appropriately

The following examples show how to prepare solutions to be used for chemical decontamination:

To prepare a 2% (20,000ppm) solution of free available chlorine:

The concentration of NaOCl in commercially available bleach products varies. Therefore check the guarantee on the product label before calculating the dilution ratio.

Example 1:	Example 2:
Household bleach typically contains 5.25% NaOCl 2/5 X 5.25% = 2.1% NaOCl Add 2 parts household bleach to 3 parts water.	Bleach sold for swimming pools contains 10-12% NaOCl 1/5 X 10% = 2% NaOCl Add 1 part commercial bleach to 4 parts water.

To prepare a 2M NaOH solution:

The molarity(M) and normality(N) of NaOH are equal. In other words, a 2N NaOH solution is the same as a 2M NaOH solution. (M = mol/L, 1 mol NaOH = 40g)

Caution: Use care while mixing NaOH and water as an exothermic reaction, releasing heat, occurs.

References:

1. Guidance from the Advisory Committee on Dangerous Pathogens and the Spongiform Encephalopathy Advisory Committee. Transmissble Spongiform Encephalopathy Agents: Safe Working and the Prevention of Infection - Part 3. December 15, 2003
2. Infection Control Guidelines, Classic Creutzfeldt-Jakob Disease in Canada. Ottawa: Public Health Agency of Canada, Volume: 28S5, November 2002.
3. Animal Health Risk Assessment Unit (AHRA). (2005). Disposal of Specified Risk Materials Through Controlled Incineration - Draft, N5, Canadian Food Inspection Agency, Ottawa.
4. European Comission, Scientific Steering Committee. Final Opinion and Report on: A Treatment of Animal Waste by Means of High Temperature(150°C, 3 hours) and High Pressure Alkaline Hydrolysis. April 10-11, 2003.
5. World Health Organization Communicable Disease Surveillance and Control. WHO Infection Control Guidelines for Transmissible Spongiform Encephalopathies; Report of a WHO Consultation Geneva, Switzerland, 23-26 March 1999. http://www.who.int/emc
6. Guidance from the Advisory Committee on Dangerous Pathogens and the Spongiform Encephalopathy Advisory Committee. Decontamination and Waste Disposal. Appendix C. December 15, 2003.
7. Taylor DM. Inactivation of prions by physical and chemical means. J Hosp Infect. 1999 Dec;43 Suppl:S69-76. Review.

Chapter 6 Facility Certification

6.1 Certification

Certification will be conducted by the Canadian Food Inspection Agency (BSE, CWD, scrapie) or the Publich Health Agency of Canada (CJD) depending on the pathogens that will be manipulated. Where animal and human prion disease agents are manipulated a lead will be identified. The critical containment components to be verified during initial certification are provided in section 6.3. A floor plan of the facility with specifications of surfaces should be submitted for review. Operational protocols must also be established and submitted for review before work with TSE agents can be carried out. Training of personnel is a critical aspect of this process and may involve initial work with non-infected tissue. Users must understand the containment systems and their operation in addition to scientific procedures. Detailed records of the certification process and test reports must be maintained. A site inspection may be conducted prior to certification.

6.2 Re-certification

Re-certification should be carried out every two years. Detailed records of the certification process and test reports must be maintained. Before program changes are implemented operational procedures should be submitted for review.

6.3 Verification and Performance Testing

Surface finishes

Benches, casework, walls and floors to be visually inspected to determine whether they are cleanable and can withstand decontamination methods. Surfaces should be continuous and without seams. Penetrations at and below the work surface should be sealed to allow for thorough cleaning and decontamination.

Communication devices

Communication and electronic paper transfer systems (eg. intercom, telephone, fax) to be verified to ensure that systems will operate as specified.

Access control/security devices

Security systems (eg. controlled access) to be verified to ensure that systems will operate as specified.

Autoclaves & Decontamination Systems

All treatment systems (eg. autoclaves, liquid effluent treatment systems, etc.) to be verified for operation as specified and to be tested using representative loads.

Biological indicators are inactivated at temperatures below those required for prion disease agents (ie. 134-138°C). Use of biological indicators may therefore provide a false assurance of prion inactivation. Therefore, specially designed chemical indicators for prions or an internal load temperature probe should be used to confirm that treatment parameters have been achieved. For technologies based on chemicals, follow chapter 5. For novel products provide adequate literature for verification of inactivation of prions. All other decontamination systems (eg. dunk tanks) to be verified for operation as specified. A description of the procedure to be followed must be provided.

Biological safety cabinets:

Testing and certification of BSCs shall be performed in accordance with CSA Z316.3-95⁽¹⁾ or the applicable NSF Standard⁽²⁾.

Interlocks (ie. Class II Type B2 BSC internal cabinet supply fan and exhaust fan) to be tested in accordance with the applicable NSF standard⁽³⁾.

Backflow preventors:

Water supply backflow prevention to be tested in accordance with CAN/CSA-B64.10-94⁽⁴⁾.

Effluent Treatment (where required):

Drains and associated piping leading to liquid effluent treatment systems (including associated vent lines) to be tested in accordance with Section 3.6 of the Canadian Plumbing Code⁽⁵⁾, Testing of Drainage and Venting Systems (1990); pressure for air test on drainage system shall be at a factor of safety beyond standard code requirements of 35 kPa (eg. 2 X code).

Effluent treatment to be appropriate for prions.

Standard Operating Procedures:

Standard operating procedures for facility must be submitted for review.

References:

1. Biological containment cabinets: installation and field testing. CSA Z316.3-95. Toronto, ON: Canadian Standards Association, 1995.
2. Class II (laminar flow) biohazard cabinetry. Standard 49. Ann Arbor, Michigan: NSF International, 2002.
3. Class II (laminar flow) biohazard cabinetry. Standard 49. Ann Arbor, Michigan: NSF International, 2002.
4. Manual for the selection and installation of backflow prevention devices/manual for the maintenance and field testing of backflow prevention devices. CAN/CSA-B64.10-01/B64.10.1-01. Toronto, ON: Canadian Standards Association, 2001.
5. National plumbing code of Canada. Ottawa, ON: Canadian Commission on Building and Fire Codes, National Research Council, 1995.

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Acknowledgements

This document was developed by the Biohazard Containment & Safety Unit at the Canadian Food Inspection Agency.

A committee consisting of a Scientific Advisory Group and Biosafety Specialists reviewed the document. The group was comprised of the following individuals: