Guia Sheep Animal Laboratory30

Guidelines for Biomedical Facilities using
Sheep as Research Animals

(December 2000)
Introduction
Q fever is a zoonotic disease caused by the rickettsial organism Coxiella burnetii. Cattle, sheep and goats are the most
common reservoirs of C. burnetii and large numbers of organisms (up to 109 organisms per gram of tissue) may be
present in placenta, birth tissues and amniotic fluids of infected animals(1, 2). The organisms are also highly resistant to
heat, dessication, many common disinfectants and can persist for months in contaminated soils (2).
Human infection usually occurs through inhalation of contaminated dusts and aerosols generated by infected animals,
their waste products, placental tissues and fluids, and contaminated straw or bedding (1, 2, 3). Only a single inhaled
organism may be sufficient to cause infection in a susceptible host (1, 2). About one-half of all people infected with C.
burnetii show signs of clinical illness. Atypical pneumonia will develop in approximately 50% of clinical cases and liver
involvement is common(3). Mortality is less than 1% and is generally related to endocarditis (3). Most patients will
recover to good health within several weeks without any treatment. Persons at risk (i.e. those with valvular heart
disease, persons who are immunosuppressed, pregnant women) should be advised of the risk of serious illness that may
result from Q fever. Chronic Q fever, characterized by infection that persists for more than 6 months is uncommon but
is a much more serious disease.
Exposure to naturally infected, often asymptomatic sheep and their birth products is a documented occupational hazard
in biomedical facilities using sheep as research animals(2, 4-7). Institutional outbreaks of Q fever have occurred not only
in those researchers working directly with sheep, but also in persons such as janitors, secretaries and others who
worked in the same facility and who had no direct contact with the animals.
The protective efficacy of Q fever vaccines for human use has been demonstrated and used successfully in Australia(8).
However, this vaccine is not commercially available in Canada. Vaccination of persons at high risk of exposure who are
without demonstrated sensitivity to Q fever antigen is currently only available in Canada through the Special Access
Program (SAP) administered by the Therapeutic Products Programme of Health Canada. To initiate a request for
vaccine a physician may write, telephone, fax or email the SAP:
An overview of many aspects of Q fever including information on the organism, disease, signs and symptoms in
humans, diagnosis, and treatment can be found at the Centers for Disease Control and Prevention's Q fever web page as
follows: http://www.cdc.gov/ncidod/dvrd/qfever Included on the suggested reading page is a list of references and
reports on cases associated with institutional use of sheep in research.
Scope
The first line of defence is to reduce the risk of bringing infected animals into the research facility. Unfortunately, it has
been established that seronegative sheep can still shed rickettsiae(2, 4, 8). Until new testing methods such as polymerase
chain reaction can be well validated, it would be premature to recommend the use of animals documented to be
serologically Q-fever free as a safe alternative to containment precautions. The risk of Q fever in a flock or herd can
however be dramatically reduced through the use of a diligent surveillance and certification program. While no flock
can be guaranteed to be and stay "Q-fever free" an ongoing surveillance program can markedly reduce the likelihood of
infection. The use of open flocks with unknown Q fever status should be avoided.
These Guidelines for Biomedical Facilities using Sheep as Research Animals are intended to specifically address the
task of containing pregnant and periparturient research animals where the occupational hazard is well documented.
The use of males or nonpregnant female animals does reduce the risk and the specific containment precautions outlined
below are not applicable. However, it is still advisable to purchase all animals from flocks with a well-documented Q
fever surveillance program to reduce the risk even further.
Farms, barns and other open animal husbandry or farm facilities present different public health problems for which
these Guidelines are not specifically applicable. Where possible, the separation of research facilities using sheep,
especially pregnant ewes, from buildings housing unassociated research and teaching activities, laboratories, hospitals
and patient areas is preferable. Such physical separation is not always possible. Some flexibility is provided for in the
Guidelines with respect to the facility design requirements where physically separate facilities are used.
These Guidelines are not designed for large animal facilities specifically manipulating C. burnetii infected animals. Such
studies must be performed in a level 3 containment facility that is designed and operated in accordance with the
Containment Standards for Veterinary Facilities. A copy of this document is available on the Canadian Food Inspection
Agency's web site as follows: www.cfia-acia.agr.ca/english/lab/bioe.shtml
These Guidelines were developed in consultation with experts in this field, including those in the medical community
currently working with sheep.
Operational Practices
The following operational practices are required:
• A documented procedural manual for the sheep facility outlining the safety and containment practices (e.g.
entry/exit protocols for persons, animals, equipment, samples, waste) should be written and followed. General
protocols should be supplemented with protocols specific for each project in process. Emergency procedures for
entry/exit, air handling failure, fire, animal escape and other emergencies should also be written.
• Staff, including animal handlers and maintenance personnel, should receive training on the potential hazards
associated with the work involved, the necessary precautions to prevent exposure to Q fever, the practices to
prevent the release of infectious agents from the facility, the operational protocols for the project in process,
and emergency procedures. Staff should show evidence that they understood the training provided. Training
should be documented and signed by both the employee and supervisor.
• Staff working with and around sheep and sheep products (including bedding, excrement, birth products, and
animals or animal tissues) should be enrolled in an occupational health and safety program.
• Only persons meeting specific entry requirements (including medical surveillance requirements as dictated by
the facility occupational health and safety program) should enter the sheep facility unless the facility has been
appropriately decontaminated. Access should be restricted to authorized personnel only. Where necessary,
maintenance and service staff may enter the unit under other conditions (e.g. without decontamination) when
accompanied by trained facility staff and provided with appropriate personal protective equipment.
• All accidents, overt or potential exposures to infectious materials, breaches of containment, seroconversions,
suspected cases of Q fever, and other hazardous occurrences should be reported immediately to the facility
supervisor. Written records of such incidents should be maintained.
• Researchers using sheep should follow good microbiological practices and perform a risk assessment designed
to minimize contact with infectious agents, minimize the creation of infectious aerosols, and reduce the
opportunity for exposure of staff and the environment.
• Staff working in the containment area should have general knowledge of the physical operational and design
features of the facility (e.g. negative air pressure gradients, directional airflow patterns, alarm signals for air
handling failure).
• Traffic flow patterns from clean to dirty areas should be established and adhered to (i.e. move from least to
most contaminated areas). Where this is not possible, operational procedures should be in place (e.g.
disinfection/decontamination barriers) to prevent the transfer of contamination to clean areas of the facility.
• Staff entering the sheep facility should wear dedicated protective clothing (i.e. scrubs) to that area. Additional
protective clothing may also include solid-front or wrap-around gowns, coveralls, gloves, boots, and disposable
shoe covers. Outer gowns should have a liquid proof protective surface. None of this clothing should be worn
outside the designated area and should be decontaminated prior to laundering and/or disposal.
• For non-vaccinated staff and those with no demonstrated immunity to Q fever, an N-95 respirator should be
used when attending parturient ewes or during surgical procedures that may generate infectious aerosols.
• At the end of high-risk procedures (e.g. when attending parturient ewes or during surgical procedures), staff
leaving the area should shower in a designated area before going anywhere else, particularly if primary clothing
becomes soiled with potentially infected material. Entry into low-risk areas of the facility with no direct sheep
contact (i.e. to read chart records) would not necessitate a shower on exit providing protocols are in place to
prevent contamination of such areas.
• Potentially contaminated items (including paperwork) to be removed from sheep holding and surgery areas
should be decontaminated on exit from the facility. Alternatively, such items can be double-bagged or placed in
impervious containers for processing in a central decontamination area. The exterior surface of such
bags/containers and transport containers containing items for further study (e.g. tissue samples) should be
disinfected on exit from the facility. Items from low-risk areas of the facility with no direct sheep contact (ie.
chart records) can be removed without further decontamination provided they have been handled in a manner
that prevents their contamination.
• At the end of the experiment all supplies remaining in the animal room (e.g. feed, bedding) should be removed
and decontaminated.
• Animal carcasses and tissues should be incinerated or processed through new technology proven to be effective
(e.g. tissue autoclave).
• Potentially contaminated items to be removed from the facility and surfaces in surgical or laboratory areas can
be disinfected with a fresh 1:100 dilution of household bleach, 5% solution of H2O2, or a 1:100 dilution of LysolR
(3).
• Areas that have held parturient ewes should be cleaned and decontaminated at the end of an experiment (i.e.
when practical and not necessarily at the end of an experiment involving only one of several sheep in the
facility) using a fresh 1:100 dilution of household bleach, 5% solution of H2O2, or a 1:100 dilution of LysolR (3).
Decontamination can also be achieved by spraying with a liquid formaldehyde disinfectant or fumigating with
paraformaldehyde.
• Smoke testing (i.e. with a smoke pencil) should be done periodically by staff to verify correct airflow and results
documented.
• Water seals in floor drains and other drainage traps should be maintained (i.e. through regular usage and/or by
filling traps in areas that are not being used).
• Sheep should never be transported through hospital patient-care areas. Transfer of sheep through corridors
and other areas not specifically designated as part of the sheep facility should be done using containment
transport carts.
• An effective rodent and insect control program should be maintained.
Physical Facilities
In addition to the requirements provided below, the facility requirements and environmental conditions suitable to
sheep as recommended by the Canadian Council for Animal Care (CCAC) should be followed.
• The sheep facility should be located away from areas that are open to unrestricted personnel traffic within the
building.
• Animal entry to the facility should be provided away from public entrances.
• Entry to the sheep facility should be labelled with appropriate signage (i.e. biohazard identification, name and
phone number of contact person, specific entry requirements).
• Office areas should be located outside of the sheep facility. Paperwork areas for researchers and animal
handlers are permitted within the facility but should be located away from animal holding and surgery areas.
• A double-door entry/egress to sheep holding and surgery rooms should be provided with an area designed to
don protective clothing dedicated to the sheep facility. A protocol should be in place to prevent the opening of
both entry doors at the same time, or, preferably be equipped with interlocking doors. The exterior entry door
should control access by means of a key lock, card key, or proximity reader.
• The area should be designed to facilitate cleaning and disinfection. Interior surface coatings (ie. floors, walls,
ceilings) should be impervious to liquids and chemicals, and penetrations in the containment barrier should be
sealed, to facilitate cleaning and decontamination of the area.
• Any windows, although not recommended, should be resistant to breakage and sealed shut.
• A handwashing sink with hands-free capability should be provided near the exit door.
• The sheep facility should be maintained at negative air pressure with respect to adjoining corridors and
facilities. Visual monitoring devices that confirm directional inward airflow should be located at the entry to the
sheep facility.
• The exhaust air should not be recirculated to any other areas of the building unless it has passed through HEPA
filtration.
• Sealed ductwork should be used where sheep facilities are not physically separated from other building
activities (i.e. potentially contaminated ductwork passes through occupied areas).
• Exhaust air from the sheep facility should be HEPA filtered where physically separate sheep facilities are not
used. Filtration of the exhaust air should be located as near as practicable to the source in order to minimize the
length of potentially contaminated ductwork, or, alternatively, sealed ductwork should be considered
• A ventilation control system and equipment should be installed where physically separate sheep facilities are
not used. (e.g. redundant exhaust fan, supply isolation damper to prevent sustained positive pressurization and
backdraft of contaminated air). An alarm system to notify personnel of ventilation systems failure should be
installed.
• The performance of critical containment components (e.g. testing of HEPA filters, integrity of containment
perimeter, verification of HVAC control systems and alarms) and operational parameters should be verified
prior to operation. Re-verification should also be performed as required by operational experience. Detailed
records of the verification process and test results should be maintained.
References
1. Richmond, J.Y and McKinney, R.W. (eds.). 1999. Biosafety in Microbiological and Biomedical Laboratories. 4th
edition. U.S. Government Printing Office, Washington, D.C.
2. Bernard, K.W., Parham, G.L., Winkler, W.G., and Helmick, C.G. 1982. Q fever control measures: recommendations
for research facilities using sheep. Infection Control. #:461-465.
3. Chin, J. (ed.). 2000. Control of Communicable Diseases Manual. 17th edition. American Public Health Association.
Washington, D.C.
4. Centers for Disease Control. 1979. Q fever at a university research center- California. MMWR. 28.
5. Simor, A.E., Brunton, J.L., Salit, I.E., Vellend, H., Ford-Jones, L. and Spence, L.P. 1984. Q fever: hazard from sheep
used in research. Can. Med. Assoc. J. 130: 1013-1016.
6. Spinelli, J.S., Ascher, M.S., Brooks, D.L., Dritz, S.K., Lewis, H.A., Morrish, R.H., Rose, L., and Ruppanner, R. 1981. Q
fever crisis in San Fransisco: Controlling a sheep zoonosis in a lab animal facility. Lab. Anim. 10:24-27.
7. Ruppanner, R., Brooks, D., Morrish, D., Spinelli, J., Franti, C.E., and Behymer, D.E. 1982. Q fever hazards from sheep
and goats used in research. Archives. 37:103-110.
8. Grant, C.G., Ascher, M.S., Bernard, K.W., Ruppanner, R., and Vellend, H. 1985. Q fever and experimental sheep.
Infect. Control. 6:122-123.
CHAPTER 1
INTRODUCTION
1.1 PREFACE
In February 1977, the Medical Research Council of Canada (MRC) published Guidelines for the
Handling of Recombinant DNA Molecules and Animal Viruses and Cells. The MRC Guidelines
went further than similar documents in the United States of America or the United Kingdom, by
addressing the laboratory safety of animal viruses and cells in culture as well as the potential safety
problems raised by the application of the new genetic techniques.
MRC undertook this task because of its support of research in universities and their affiliated
teaching hospitals. These Guidelines rapidly caused many research institutions to establish
biohazard or biosafety committees. The Natural Sciences and Engineering Research Council
(NSERC) and the National Research Council of Canada (NRC) adopted and implemented the
Guidelines, as did a number of provincial and private research funding agencies. Moreover, the
Minister of National Health and Welfare Canada stated that the Guidelines would apply to all
research carried out or supported by the federal government. Also, while there was no formal
legislative or regulatory enforcement, many industries adopted the Guidelines.
MRC published two revisions in 1979 and 1980, under the advice of its Biohazards Committee.
These changes markedly reduced the containment requirements for research using recombinant
DNA technology, in line with the rapidly changing perceptions of risk posed by these techniques.
Limited changes were made to the containment requirements for the use of animal viruses and cells
in 1990.
The 1990 Laboratory Biosafety Guidelines were developed by MRC and the Laboratory Centre for
Disease Control (LCDC) who formed a joint Working Group consisting of the following persons: Dr.
Lorne A. Babiuk (Chairman), Dr. Fraser E. Ashton, Dr. W.A. Black, Dr. Clarence Fuerst, Mrs. M.E.
Kennedy, Dr. M.S. Mahdy, Mr. C. Blakeway Millar, Mr. S.T. Morawski, Dr. Marc Quevillon, Dr.
Francis Rolleston and Dr. K.R. Rozee.
The Working Group's objective was to provide a technical document for those who design, build,
operate or work in laboratories in which human pathogens are grown for research or development
purposes. The focus of this document is therefore on the use of bacteria, viruses, parasites, fungi
and other infectious agents which are pathogenic to humans. These agents may be encountered in
laboratories, in universities, hospitals and their affiliated institutions, as well as in governmental,
industrial, research, diagnostic and teaching laboratories.
If a microbial pathogen is isolated or suspected to be present in a specimen, it then must be

handled at the appropriate containment level. Every pathogen isolated is to be handled according
to the risk category.
After revision on the basis of their review, the report was submitted to MRC and Health Canada
(HC) for their approval as a draft to be published for public comment. The public responses were
analyzed by an editorial sub-committee comprised of Dr. L. Babiuk, Dr. C.M. Johnson-Lussenburg,
Mrs. M.E. Kennedy, Dr. J. Penner, Dr. K. Rozee and Dr. P. Stockdale. The needed changes were
made and the report was submitted to MRC and HC for final approval.
1.2 SCOPE OF THE DOCUMENT
The second edition of these guidelines has been updated to reflect currently recognized
containment requirements and operational practices and is consistent with such practices
worldwide. Additionally, current legislation relevant to microbiological laboratories is included in
this edition. While this edition is published by Health Canada, Medical Research Council of Canada
and NSERC will continue to use these Guidelines as a requirement in their granting process.
The draft of the second edition was distributed widely for comment and review. An editorial
committee consisting of Ms. M.E.Kennedy (Chair), Dr. F. Ashton, Mr. Adrian Delaat, Dr. C.M.
Johnson-Lussenburg, Ms. Anne Monteath and Dr. Jim Talbot was convened to review the
comments received from the public discussion draft and prepare the final document.
We extend thanks to Liliane Leroux and Danielle Plouffe for their clerical assistance in the
preparation of this document and to Eleanor Paulson for preparing the bibliography.
CHAPTER 2
CONTAINMENT OF BIOHAZARDS
2.1 INTRODUCTION
Bacteria, viruses, fungi or other infectious agents are studied because they may cause disease, they
can help us understand the natural world, and for many other reasons including the possibility of
industrial applications. Since many of the agents can be pathogenic to humans, animals or other
forms of life, their use poses risks which vary with each agent and the way it is used.
Safety standards are designed to reduce to an acceptable level the risks inherent in the use of
dangerous materials. Stringent standards are set for hazardous agents and less stringent ones for
agents which cause only minor problems. Safety standards are therefore compromises designed to
allow needed work to proceed without exposing those involved or others to more than minimal risk.
2.2 SAFETY PRACTICES

The attitudes and actions of those who work in the laboratory determine their own safety, and that
of their colleagues and of the community. Laboratory equipment and design can contribute to
safety only if they are used properly by people who are genuinely concerned and knowledgeable
about safety issues.
The Canada Labour Code requires that each employer provide safe working conditions and that
employees be informed about all hazards they will face in the course of their duties. The employee
is also given the right to withdraw from the workplace if faced with an unsafe condition. Other
federal legislation such as Workplace Hazardous Materials Information System (WHMIS) requires
that all hazardous substances, including microorganisms, be labelled in a specified manner and
that there be a Material Safety Data Sheet (MSDS) available to accompany each hazardous
substance. Employers are also required to provide all training necessary to work with the hazardous
substance and to keep a written record of their employee education program. For more
information, contact Labour Canada, tel: (613) 997-3520.
The following requirements are basic for any laboratory using infectious or toxic agents.
1. All laboratory personnel and others whose work requires them to enter the laboratory must
understand the biological and other hazards with which they will come in contact through
their normal work in the laboratory, and be trained in appropriate safety precautions and
procedures. A laboratory safety manual must beprepared and adopted. And it is the
responsibility of the laboratory director/principal investigator to ensure it identifies known
and potential biohazards and specifies practices and procedures to eliminate or minimize
such risks. The manual must also contain an emergency response plan. Personnel must be
required to know, understand, and follow standard practices and procedures. Training in
laboratory safety must be provided and competence in safe technique demonstrated before
work is allowed with hazardous agents or toxins.
2. Laboratories should have a biological safety officer (BSO) and/or a biological safety
committee whose responsibility include ensuring that all work is carried out in accordance
with safety practices established at the Institution.
The duties of the BSO should include providing technical advice on safety procedures and
equipment, developing emergency plans, conducting safety inspections, providing biosafety
training, conducting or supervising testing of containment systems, and providing guidance
and information related to compliance with pertinent regulations.
3. The laboratory must be kept neat, orderly and clean, and storage of materials not pertinent
to the work should be minimized.
4. Protective laboratory clothing (uniforms, coats, gowns) must be available and worn properly
fastened by all personnel, including visitors, trainees and others entering or working in the
laboratory. Protective laboratory clothing must not be worn in non-laboratory areas.
Suitable footwear with closed toes and heels and preferably with non-slip soles must be worn
in all laboratory areas.
5. Gloves must be worn for all procedures that might involve direct skin contact with toxins,
blood, infectious materials, or infected animals. Rings or hand jewelry which would interfere
with glove functioning should be removed before gloving. Gloves should be removed
carefully and decontaminated with other laboratory wastes before disposal. Reusable gloves
(e.g. insulated, chemical resistant, etc.) may be used only where necessary and must be
appropriately decontaminated.
6. Safety face and eyewear (e.g. glasses, goggles, face shields, or other protective devices) must
be worn when necessary to protect the face and eyes from splashes, impacting objects,
harmful substances, UV light, or other rays.
7. Eating, drinking, smoking, storing food, personal belongings or utensils, applying cosmetics,
and inserting or removing contact lenses are not permitted in any laboratory work area.
Contact lenses should be worn only when other forms of corrective eyewear are not suitable.
The wearing of jewelry should be discouraged in the laboratory.
8. Oral pipetting of any substance is prohibited in any laboratory.
9. Long hair must be tied back or restrained.
10. Hands must be washed after gloves are removed, before leaving the laboratory, and at any
time after handling materials known or suspected to be contaminated.
11. Work surfaces must be cleaned and decontaminated with a suitable disinfectant at the end of
the day and after any spill of potentially dangerous material. Loose or cracked work surfaces
must be replaced or repaired.
12. All technical procedures must be performed in a manner that minimizes the creation of
aerosols.
13. All contaminated or infectious liquid or solid materials must be decontaminated before
disposal or reuse. Contaminated materials that are to be autoclaved or incinerated at a site
away from the laboratory must first have the outside of the container disinfected chemically
or be double-bagged.
14. Access to the laboratories must be strictly limited (containment Levels 3 and 4). Decisions
on entry into containment Level 1 and 2 laboratories should be at the discretion of the
laboratory director/principal investigator (e.g. only persons who have been advised of the
potential hazards and meet any specific entry requirements such as immunization should be
allowed to enter the laboratory area). Children under the age of 16 years old should not be
permitted in the laboratory or support areas. Pregnant women or immunocompromised
people who work in or enter the laboratory should be advised of the associated risks.
15. Hazard warning signs, indicating the risk level of the agents being used, must be posted
outside each laboratory. Where infectious agent(s) used in the laboratory require special
provisions for entry, the relevant information must be included in the sign. The agent must
be identified, and the name of the laboratory supervisor and other responsible person(s) as
well as any special conditions for staff entry must be listed.
16. The use of needles, syringes and other sharp objects should be strictly limited. Needles and
syringes should be used only for parenteral injection and aspiration of fluids from laboratory
animals and diaphragm bottles. Extreme caution should be used when handling needles and
syringes to avoid autoinoculation and the generation of aerosols during use and disposal.
Procedures should be performed in a biological safety cabinet. Needles should not be bent or
sheared. They should not be replaced in the sheath or guard. They should be promptly
placed in a puncture-proof container and decontaminated, preferably by incineration or
autoclaving, before disposal.
17. All spills, accidents, and overt or potential exposures must be reported in writing to the
laboratory supervisor or acting alternate as soon as circumstances permit; this person
should file this report with management and the appropriate biosafety officer or committee.
Appropriate medical evaluation, surveillance, and treatment should be sought and provided
as required. Actions taken to prevent future occurrences should be documented.
18. Baseline serum for laboratory and other at-risk personnel (eg. laboratory support and
maintenance staff) should be collected and stored. Additional serum specimens may be
collected periodically, depending on the agents handled or the function of the facility.
19. Laboratory workers should be protected by appropriate immunization where possible.
Levels of antibody considered to be protective should be documented. Particular attention
must be given to individuals who are or may become immunocompromised, as vaccine
administration may be different than for immunologically competent adults.
2.3 USE OF LABORATORY ANIMALS
Naturally occurring or experimentally induced infections in laboratory animals may be transmitted

to other laboratory animals, invertebrates and laboratory workers. Animals infected or challenged
experimentally with organisms in any of the risk groups may be small (e.g. mice) or large (e.g.
livestock), have unique housing requirements (e.g. fish) or have uncharacterized susceptibilities.
The requirements for maintenance of the animals may differ therefore in scale and degree but the
basic principles for microbiological safety will be similar to those outlined in Section 2.2 and must
be followed.
In addition, the following requirements and conditions must be satisfied:
- All aspects involved in the proposed use of animals in research must meet the standards
and regulations for the care and maintenance of experimental animals as described by the
Canadian Council on Animal Care, relevant provincial legislation and local animal care
authorities.
-The appropriate species must be selected for animal experiments to reduce potential
biohazards.
-The investigator and/or person(s) responsible for the animal experiment must ensure that
all those having contact with the animals and waste materials are familiar with and aware of
any special precautions and procedures that may be required. Where possible, personnel
should be protected by immunization with appropriate vaccines.
-It is essential that all accidents, including animal bites and scratches or cuts from cages or
other equipment, be reported and recorded.
-Small laboratory rodents or other small animals that escape from their cages should be
killed when captured, their carcasses incinerated and the area should be fully
decontaminated. In the event that animals escape the containment perimeter, the relevant
authorities must be notified promptly and appropriate action initiated.
-Unexpected illness or deaths among animals must be reported to both the researcher and
head of animal services without delay; instructions for dealing with such animals should be
available. However, animals should not be touched until instructions are given by the person
in charge.
CHAPTER 3
REGULATIONS
3.1 IMPORTATION
The importation of human pathogens is regulated by the Importation of Human Pathogens

Regulations (1994). Permits are required for the importation of all infectious substances into
Canada regardless of whether they infect humans, animals or plants. The importation of infectious
agents which are predominantly pathogenic to animals is regulated by means of the Health of
Animals Act and Regulations administered by Agriculture and Agri-Food Canada. It may also be
necessary to obtain permission to transfer listed pathogens within Canada from one scientist or
laboratory to another.
Requests for single-entry and long-standing permits to import infectious substances affecting
humans should be directed to Director, Office of Biosafety, Laboratory Centre for Disease Control,
Ottawa, Ontario K1A 0L2, tel: (613) 957-1779. Information regarding veterinary pathogens and
permits may be obtained from Agriculture and Agri-Food Canada, 59 Camelot Drive, Nepean,
Ontario K1A 0Y9, tel.: (613) 952-8000.
3.2 EXPORTATION
Permits are required for the export from Canada of certain microorganisms and associated
equipment. Canada presently controls certain toxicological and biological agents, as well as their
related equipment, components, materials and technology under item 2007 of the Export Control
List. For assistance or advice, contact Foreign Affairs and International Trade Canada, Export
Control Division, Lester B. Pearson Building, 125 Sussex Drive, Ottawa, Ontario K1A 0G2, tel: (613)
996-2387.
3.3 TRANSPORTATION
The careful handling, transfer and shipment of diagnostic specimens and infectious agents is
absolutely essential if Canada is to maintain an effective health care system. Transportation
methods must minimize risks to employees of the carrier, the public and the staff of the receiving
laboratory. Hazards are compounded by improper packaging; a broken specimen container may
lead to contamination of both laboratory and non-laboratory personnel, and an improperly labelled
package may be opened inadvertently by secretarial, clerical or other untrained staff.
In Canada, effective July 1, 1985, Transport Canada has become responsible for regulations
concerning the transportation of dangerous goods. Any person handling, offering for transport or
transporting dangerous goods must comply with the Transportation of Dangerous Goods Act and
Regulations, RegistrationSOR 85-77 as amended in 1994. Inquiries regarding these Regulations
should be directed to the Director General of the Transport of Dangerous Goods Directorate of
Transport Canada, Canada Building, 344 Slater Street, 14th Floor, Ottawa, Ontario K1A 0N5, tel.:
(613) 998-0517.
The efficient and safe transfer of infectious substances requires good coordination between the
sender, carrier, and receiver to ensure safe and prompt transport and arrival in proper condition. It
is important that the sender make advance arrangements with the carrier and the receiver to
ensure that specimens will be accepted and promptly processed. In addition, the sender must
prepare the appropriate dispatch documents according to the Transportation of Dangerous Goods
Act and Regulations. The sender should also forward all transportation data to the receiver. No
infectious substances should be dispatched before advance arrangements have been made between
the sender, the carrier and the receiver, or before the receiver has confirmed with national
authorities that the substance can be imported legally and that no delay will be incurred in the
delivery of the consignment to its destination.
In addition, information may be routinely obtained from the Canadian Transport

Emergency Centre (CANUTEC) during working hours at (613) 992-4624 and IN AN
EMERGENCY at (613) 996-6666 (24 hours per day).
3.4 THE HEALTH OF ANIMALS ACT 1990
The Health of Animals Act 1990 and its Regulations gives Agriculture and Agri-Food Canada
(AAFC) the legislative authority to control the distribution and use of any pathogen which may
cause infectious or contagious disease in animals. This includes materials of animal origin which
contain potential pathogens. In practical terms, this means that AAFC approval must be obtained
for the importation of every animal pathogen. In the case of pathogens which affect both humans
and animals, importation permits are required from both Health Canada and AAFC. If an agent was
brought into Canada under an import permit which restricts its distribution, further approval must
be obtained before transferring it to another scientist or laboratory. Recombinant organisms and
their release into the environment may also be restricted. AAFC will also establish for animal
pathogens, the conditions under which they will be maintained and work will be carried out. It is
necessary to consider not only the risk to human health, but also the level of containment needed to
prevent escape of an animal pathogen into the environment where it may constitute a risk to any
indigenous animal species.
Animal pathogens, including pathogens which affect both humans and animals, under the control
of AAFC, are listed in a database maintained by the Animal & Plant Health Directorate, AAFC. This
is a dynamic listing which is continuously amended to include emerging pathogens that may
require restriction. Animal disease agents considered as not indigenous to Canada forma portion of
this database and are severely restricted. Table 1 provides a partial list of these organisms. For each
animal pathogen, AAFC must be consulted for its importation, use and distribution. Information on
the status of veterinary pathogens may be obtained from AAFC, Animal Health Division, 59
Camelot Drive, Nepean, ON K1A 0Y9, (613) 952-8000.
3.4.1 NON-INDIGENOUS AGENTS

The following partial list is provided as an example of animal pathogens under AAFC control and is
not complete.
TABLE 1
AGENTS NOT INDIGENOUS TO CANADA
BACTERIA
Mycoplasma agalactiae
Mycoplasma mycoides
Rickettsia ruminantium
PARASITES
Besnoitia besnoiti
Theileria annulata
Theileria bovis
Theileria hirci
Theileria lawrencei
Theileria parva
Trypanosoma equiperdum
Trypanosoma evansi
Trypanosoma vivax
VIRUSES
Bornaviridae
Borna disease virus
Bunyaviridae
Nairobi sheep disease virus
Rift Valley fever virus
Caliciviridae
Swine vesicular disease
Vesicular exanthema virus
Herpesviridae
Pseudorabies virus
Iridoviridae
African swine fever virus
Orthomyxoviridae
Fowl plague virus
Paramyxoviridae
Rinderpest, Newcastle disease virus (mesogenic, velogenic strains),
Peste des petits ruminants
Picornaviridae
Genus Aphthovirus: Foot-and-mouth disease virus
Genus Enterovirus: Teschen disease virus
Poxviridae
Chordopoxvirinae (poxviruses of vertebrates),
Small pox (Alastrim)
Genus Capripoxvirus
Sheeppox
Goatpox
Lumpy skin disease
Genus Suipoxvirus
Swinepox
Camelpox virus
Reoviridae
Genus Orbivirus
Bluetongue virus
African horsesickness virus
Rhabdoviridae
Genus Vesiculovirus
Ephemeral fever virus
Vesicular stomatitis virus (Animal inoculation)
Togaviridae
Hog Cholera virus
Louping III virus (Animal inoculation)
Wesselsbron disease virus
Venezuelan Equine Encephalitis (VEE)
CHAPTER 4
CLASSIFICATION OF BIOLOGICAL AGENTS ACCORDING TO RISK
4.1 GENERAL
Judgements of the inherent risks of a pathogen are made on the basis of such factors as the severity
of the disease it causes, the routes of infection, its virulence and infectivity. This judgement should
take into account the existence of effective therapies (e.g. antibiotic resistance), immunization, the
presence (or absence) of vectors, quantity of agent and whether the agent is indigenous to Canada
as well as possible effects on other species, including plants and animals. Emerging pathogens and
novel agents, because of their unknown characteristics, may require specialized practices and
procedures for handling.
With these factors as the prime consideration, biological agents are classified according to risk
groups which are analogous to the levels of containment described in section 4.6. These
classifications presume ordinary circumstances in the research laboratory, or growth in small
volumes for diagnostic and experimental purposes.
The classifications of biological agents primarily reflect the judgements made on

their inherent risks. Section 4.5 lists the general criteria. The agents listed in Section
4.6 were chosen because they seem to be frequently used in Canada. Agents not listed
should be classified on the basis of similarity to those listed; risk groups for others
will be identified on request to the Office of Biosafety, Laboratory Centre for Disease
Control.
4.2 RECOMBINANT DNA AND GENETIC MANIPULATION
Genetic methods such as natural selection, cross breeding, conjugation and transformation have
been used for many years to change biological species and organisms. These methods have recently
been supplemented by newer and much more efficient ones, of which the best known are the
techniques of recombinant DNA. This technology allows scientists to transfer genes between
unrelated organisms and species, and has spawned the recent surge in biotechnology.
The initial fear of possible risks arising from organisms altered by this technology led Canada, the
United States and Great Britain, among other countries, to develop stringent biosafety guidelines.
Experience rapidly showed that the initial fears were not justified. By 1980, many of the
containment requirements of 1975-1977 had been removed.
Guidance in how to assess potential risks in recombinant DNA research can only be very general;
each case needs individual assessment. It is not realistic to try to define in advance all of the
possible genetically engineered organisms which might be created or used in the laboratory. The
vast majority of this research involves only the remotest possibility of creating a hazard because the
source of the DNA being transferred, the vector and the host are all innocuous. However, some
genetic manipulation does raise significant possibility of risk. In general, if none of the components
of the genetic manipulation presents any known hazard, and none can be reasonably foreseen in
their combination, then no biohazard restrictions are needed. If one of the components of the
reaction is hazardous, then, in general, discussion of the containment level required should start at
the level appropriate to the known hazard. Its containment level might be increased or decreased
according to such considerations as: the particular gene being transferred; the expression of the
gene in the recombinant organism; the biological containment offered by the host vector system;
the envisaged interactions between the gene being transferred and the host vector system; and
other such factors. In any research with genes coding for hazardous products, host vector systems
of limited ability to survive outside the laboratory (i.e. offering biological containment) should be
used; their use will reduce the level of containment required.
4.3 USE OF MAMMALIAN CELLS IN CULTURE

The biological hazards of mammalian cells arise from the possibility that they might contain or
transmit infectious agents. It is prudent to consider all cell lines to be potentially infectious. Cells
known or suspected to contain such agents, or primary cultures from animals and humans known
or reasonably suspected to be infected, should be in the risk group for the suspected agent. Primate
cell lines derived from lymphoid or tumor tissue, all cell lines exposed to or transformed by a
primate oncogenic virus, all samples of human tissues and fluids, all primate tissue, all cell lines
new to the laboratory (until proven to be free of adventitious agents), all virus-containing primate
cell lines, and all mycoplasma-containing cell lines should be handled at containment Level 2.
4.4 RISK LEVELS ASSOCIATED WITH THE USE OF LABORATORY ANIMALS
The use of experimental animals and insects poses special problems. Animals can harbour
infectious organisms which are acquired naturally. These infections can give rise to a chronic
carrier state, or the agent might persist in a latent non-infective form which can be reactivated
periodically or as a result of certain stimuli. If the possibility that such an agent may be excreted by
an animal during the course of an experiment cannot be excluded, all those animals should be kept
at a containment level appropriate to the risk.
Animals may also be deliberately inoculated with microorganisms in each of the four risk groups or
with viable materials (i.e. transformed cells) suspected of containing these organisms. Under these
circumstances, the animal should be kept at the containment level appropriate to the risk of the
organism, recognizing that, in some cases, in vivo work may increase that risk.
In all situations, it is the responsibility of the scientist and the host institution in consultation with
the Government and the Animal Care authorities, to determine the risk levels inherent in the
proposed activity.
4.5 CRITERIA FOR CLASSIFICATION OF BIOLOGICAL AGENTS BY RISK GROUP
A biological agent that is unlikely to cause disease in healthy workers or animals.
A pathogen that can cause human or animal disease but, under normal circumstances, is unlikely to
be a serious hazard to laboratory workers, the community, livestock, or the environment.
Laboratory exposures rarely cause infection leading to serious disease; effective treatment and
preventive measures are available and the risk of spread is limited.
Risk Group 3 (high individual risk, low community risk)
A pathogen that usually causes serious human or animal disease, or which can result in serious
economic consequences but does not ordinarily spread by casual contact from one individual to
another, or that can be treated by antimicrobial or antiparasitic agents.
Risk Group 4 (high individual risk, high community risk)
A pathogen that usually produces very serious human or animal disease, often untreatable, and
may be readily transmitted from one individual to another, or from animal to human or vice-versa
directly or indirectly, or by casual contact.
4.6 CATEGORIES OF PATHOGENS
As a general precaution, the risk group for agents should be raised when manipulation may result
in the production of infectious droplets and aerosols. Agents of similar pathogenic characteristics
not included in these lists should be considered in the same risk category. It must also be
understood that this is not a complete list - many agents are referred to in the literature by a
variety of names and before assuming that an unlisted organism is classified in Risk Group 1, its
characteristics, and pathogenicity must be verified in consultation with the Office of Biosafety,
Health Canada.
4.6.1 RISK GROUP 1 AGENTS: REQUIRING CONTAINMENT LEVEL 1

R community risk)
This group includes those microorganisms, bacteria, fungi, viruses and parasites, which are
unlikely to cause disease in healthy workers or animals.

A pathogen that can cause human or animal disease but under normal circumstances, is unlikely to
be a serious hazard to healthy laboratory workers, the community, livestock, or the environment.
Laboratory exposures rarely cause infection leading to serious disease; effective treatment and
preventive measures are available and the risk of spread is limited.
Risk Group 3 (high individual risk, low community risk)
A pathogen that usually causes serious human or animal disease, or which can result in serious
economic consequences but does not ordinarily spread by casual contact from one individual to
another, or that can be treated by antimicrobial or antiparasitic agents.
Risk Group 4 (high individual risk, high community risk)
A pathogen that usually produces very serious human animal disease, often untreatable, and may
be readily transmitted from one individual to another, or from animal to human or vice-versa
directly or indirectly, or casual contact.
CHAPTER 5
PHYSICAL CONTAINMENT LEVELS
Four levels of containment (1-4) appropriate to the four risk groups for infectious agents are
defined below. These levels of containment are to be regarded as adequate for most laboratory uses
of the particular agents. It remains the responsibility of the principal investigator or laboratory
director and the institution to require a higher level of containment for specific manipulations, if
these appreciably increase the hazard of infection.
5.1 CONTAINMENT LEVEL 1

This level applies to the basic laboratory for the handling of Risk Group 1 agents. Containment
Level 1 (CL1) requires no special design features beyond those suitable for a well designed and
functional laboratory. Biological safety cabinets are not required. Work may be done on an open
bench top and containment is achieved through the use of practices normally employed in a basic
microbiology laboratory.
5.1.1 PHYSICAL REQUIREMENTS

-A room separated from public areas by a door is required. There are no particular
restrictions on locating the facility near public or heavily travelled corridors; however, doors
should remain closed.
-Coatings on walls, ceilings, furniture, and floors should be cleanable. Windows that can be
opened should not be near working areas or containment equipment and should be
equipped with fly screens.
-There are no special air handling requirements beyond those concerned with proper
functioning of the biological safety cabinets, if used, and those required by building codes.
-Handwashing facilities must be provided, preferably near the point of exit to public areas.
-Separate hanging areas should be provided for street clothing and laboratory coats.
-Eye wash stations may be required by local statute.
5.1.2 OPERATIONAL REQUIREMENTS
Basic safety practices as described in Chapter 2 must be followed.
In addition, where chemical disinfection procedures are practised, effective concentrations and
contact times must be employed. Chemical disinfectants used to decontaminate materials to be
removed from the laboratory must be replaced regularly.

Containment Level 2 (CL2) is suitable for work with agents in Risk Group 2. In addition to the
requirements of containment Level 1, the following are required:
-The laboratory should be located away from public areas, general offices, and patient care
areas.
-A biohazard sign with appropriate information must be posted on the entrance to the
laboratory.
-Laboratory furnishings and work surfaces should be impervious and readily cleanable.
-Coat hooks must be provided for laboratory coats near the exit.
-An autoclave must be available in or near the laboratory.
-Laboratory doors should be self-closing.

-Class I or II biological safety cabinets (see Appendix B) are required for all manipulations of
agents which may create an aerosol. The biological safety cabinet must have been tested and
certified within the previous 12 months according to accepted standards (see Appendix B).
-Air from these cabinets may be recirculated to the room only after passage through a high
efficiency particulate air (HEPA) filter.
-Centrifugation must be carried out using closed containers or aerosol proof safety heads or
cups. These should be opened only in the biological safety cabinet described above.
-Animals or insects which have been experimentally infected must remain in the laboratory
or appropriate animal containment facility.
-An emergency plan for handling spills of infectious materials must be developed and be
ready for use whenever needed. Laboratory workers must be educated and drilled in the
emergency plans.
-Vacuum lines used for work involving the agent must be protected from contamination by
HEPA filters or equivalent.
-A laboratory coat to be worn only in the laboratory area is required. Coats that fasten on the
front are permissible. These coats shall not be worn outside the containment laboratory.
-Special care should be taken to avoid contamination of the skin with infectious materials;
gloves should be worn when handling infected animals or when skin may be exposed to
infectious materials.
-Contaminated glassware must not leave the facility; decontamination must be carried out
using procedures demonstrated to be effective. If there is no autoclave or incinerator in the
laboratory, contaminated materials must be disinfected chemically or double bagged and
transported to the autoclave or incinerator in durable, leakproof containers which are closed
and wiped on the outside with disinfectant before leaving the laboratory.
-Service personnel and cleaning staff who enter the facility must be informed of the hazards
that might be encountered. Cleaning staff should clean only the floors. The laboratory
personnel have the responsibility for rendering the facility safe for routine cleaning. Periodic
intensive cleaning must be done at regular intervals. Cleaning and maintenance staff should
receive appropriate immunization and medical surveillance if appropriate.
Containment Level 3 (CL3) is suitable for work with agents in Risk Group 3. The operational
requirements for the Level 3 laboratory are substantially greater than those for Levels 1 and 2 and
the laboratory staff must receive specific training in the safe handling and manipulation of the
agents used in this laboratory. Because the laboratory is designed to minimize environmental
release of hazardous materials and provide enhanced worker protection the containment level 3
laboratory must undergo annual performance, testing and verification (see 7.8).
A Level 3 containment laboratory requires specialized design and construction. Those responsible
for biosafety in an institution should maintain close control and seek expert advice and remain in
close communication for all phases of design, construction, performance, verification and testing,
operation and maintenance, and annual testing.

The following are required in addition to the requirements for containment level 1 and containment
level 2.
-The laboratory must be located away from general work areas and have controlled access
from other areas. This is accomplished by entry through a lockable changing room with self-
closing doors. A body shower should be provided within the containment perimeter.
-The laboratory must be held at a negative pressure relative to the surrounding areas at all
times such that a directional airflow is created by air ingressing through all entry and exit
areas. The laboratory should be provided with a dedicated supply and exhaust system which
is sealed. The air discharged from the laboratory cannot be recirculated back into either the
air supply system of the laboratory itself or into the building or adjacent buildings. Provided
there is a dedicated sealed exhaust system, air may be exhausted from the laboratory to the
exterior of the building without HEPA filtration. At the discharge point the exhausted air
must be dispersed away from air intake or populated areas. However, when the air is not
exhausted by means of a dedicated exhaust system, it must be passed through a HEPA
filtered exhaust before discharging into the main building exhaust air ventilation system.
This exhaust housing must be designed to allow in situ decontamination and must pass
annual testing and certification by aerosol challenge and scan techniques. A control system
must be provided to ensure that the Level 3 laboratory does not become positively
pressurized relative to the surrounding area. When the supply air is not provided by a
dedicated system, air-tight back draft dampers or HEPA filters must be installed in the
supply system. The supply must be interlocked with the exhaust system.
-Biological safety cabinets must be installed in a manner which does not interfere with the
air balance of the cabinet or room. Thimble unit connections are recommended (see
Appendix B).
-The laboratory must have a dedicated handwashing sink with foot, knee or automatic
controls, located near the exit.
-The laboratory must have a pass-through or stand alone autoclave located in the work zone.
Where physical constraints preclude the installation of an autoclave, in an existing level 3,
alternative technologies may be used for sterilization of contaminated materials.
-Laboratory furnishings should be kept to a minimum. Work surfaces should be impervious,
readily cleanable, and resistant to chemical disinfectants.
-All penetrations for services in the floors, walls, and ceiling of the laboratory must be
sealed. The air supply/exhaust system should be provided with manual dampers at the room
perimeter that may be closed as required to permit gas decontamination.
-Water supplied to the laboratory must be provided with reduced pressure back flow
preventers.
-HEPA filters or equivalent should be provided on all ventlines.
-Dunk tanks may be provided at the containment perimeter.
-Sink and floor drains from this suite should be piped separately to the main building drain
and be appropriately labelled. Floor drains are not generally recommended. Infectious
materials must never be placed in sinks or floor drains.
-Autoclave condensate drains should have closed connections and go directly to sanitary
sewer.
-In animal care facilities for small animals, the disposal of wastes will not differ from other
contaminated laboratory materials. Large animals producing quantities of infectious wastes
require special facilities which must be designed accordingly.
-Portable vacuum pumps must be fitted with in-line HEPA filters or equivalent equipment.
No vacuum lines may exit the containment perimeter.
-Laboratory windows must be sealed and unbreakable.
-Backup power should be provided to critical items such as biological safety cabinets, fume
hoods , freezers etc.
The following are required in addition to those stated for CL1 and CL2:
-Laboratory staff must be fully trained in the handling of pathogenic and other hazardous
material and in the use of safety equipment, disposal techniques, handling of contaminated
waste, and emergency response.
-Staff are required to change into dedicated solid front laboratory clothing on entry to the
facility. This laboratory clothing must be removed on completion of work and autoclaved
prior to laundering.
-Personal protective clothing, which may include head covers and dedicated shoes or
impervious foot covers must be used while in the containment facility and removed on
leaving.
-Appropriate respiratory protection should be considered depending on the infectious agents
in use.
-Showers may be required depending on infectious agents used and manipulations involved.
-Personal effects may not be taken into or stored in the laboratory.
-Gloves must be worn when handling infective or potentially infective materials, including
animals or waste.
-All activities involving infectious materials are conducted in biological safety cabinets or
other appropriate combinations of personal protective and physical containment devices.
-Centrifugation must be carried out in closed containers using aerosol proof safety heads or
cups which are loaded and unloaded in the biological safety cabinet.
-Effective disinfectants must be available at all times in the laboratory.
-All Risk Group 3 agents must be stored within the containment level 3 facility.
-An effective pest control program must be in effect.
-Written protocols must be provided and posted within the laboratory outlining operational
protocols, waste disposal, disinfection procedures, and emergency response.
-The facility must have a medical surveillance program appropriate to the agents used which
includes serum storage for all personnel working in the containment laboratory.
-The laboratory must have a reporting system for accidents and exposures to infective agents
or other incidents or unusual occurrences in the operation of the laboratory.
-Authorized maintenance and service personnel must abide by the same operational
protocols as laboratory staff and be accompanied by laboratory personnel when entering and
working within the laboratory.
-The containment level 3 facility and its systems must be tested for containment capability
upon completion of construction and at least annually thereafter (see 7.8).
The physical and operational requirements of containment level 4 are highly specialized and any
Institution undertaking level 4 work must seek experienced assistance in developing the lab design
and operational protocols.
Containment Level 4 is the highest level of containment and represents an isolated unit
functionally independent of other areas.
This level of containment requires an air lock for entry and exit, Class III biological safety cabinets
or positive pressure ventilated suits, a laboratory support area, and a separate ventilation system in
addition to the physical and operational requirements of containment levels 1 to 3.

-The laboratory must be physically separated from other laboratories or consist of an
isolated zone which is monolithic in construction with all penetrations to floors, walls, and
ceilings sealed with non-shrinking sealant.
-The laboratory must be designed to accommodate a minimum of two persons at all times,
all laboratory equipment, long term storage of cultures and maintenance of infected animals.
-Entry must be through an airlock system with doors that are electrically interlocked.
Manual alarm overrides must be provided.
-Change rooms must be contiguous with the containment perimeter of the structure and
have a personnel shower and /or chemical shower (depending on the mode of operation).
-All drain traps must be kept filled with an effective disinfectant and connected to a liquid
waste effluent system.
-All air access to any sewer and ventilation lines must be fitted with HEPA filters or
equivalent.
-All gas services must be fitted with HEPA filters, equivalent equipment, or back-flow
preventers to prevent egress of contaminated material.
-The water supply systems for the laboratory must be provided with back-flow preventers.
-All windows must be sealed and provided with break-resistant glass.
-The laboratory must be provided with a double-door autoclave preferably serviceable from
outside the facility. The autoclave should have read-out charts inside and outside the
containment laboratory and have operating controls inside the laboratory.
-Dunk tank(s) which are chemically resistant and of a suitable size for the passage of
anticipated laboratory materials may be required for Class III cabinet lines laboratories.
-A dedicated handwashing sink with foot, knee or automatic controls must be located at the
exit of each laboratory room in the containment level 4 laboratory (not applicable for suit
laboratory).
-The facility must be equipped with a two-way intercom system.
-A closed circuit television system should be considered.
-All liquid effluents from the facility must enter a waste effluent treatment system for
sterilization which is mechanically and biologically monitored.
-The facility must be ventilated by an independent, dedicated, sealed supply and exhaust air
system which is not recirculated. Exhaust air must be passed through at least 2 HEPA filters
mounted in series in sealed housings designed to allow in situ decontamination and testing
by aerosol challenge techniques. Supply and exhaust ventilation systems must be designed to
maintain directional (inward) airflows and pressure differentials, and interlocked to prevent
pressurization in the event of exhaust fan failure. The supply air system must be equipped
with a HEPA filter. The facility must be fully equipped with manometers and/or other
monitoring devices and audible and visual alarms capable of being monitored by both
laboratory and maintenance staff.
-The laboratory must be provided with Class I, II or III tested and certified biological safety
cabinets which must be installed in a manner that does not interfere with the air balance of
the cabinet or room.
-If a positive-pressure-ventilated-suit type of operation is used, a life-support system with
full alarming, backup breathing air, emergency power and a chemical shower facility are
required. Positive-pressure suits must be used whenever agents are worked with outside a
Class III cabinet.
-Alarms, ventilation and other critical systems must be on separate electrical circuits with
emergency backup.
-A laboratory support area should be provided adjacent to the Level 4 containment facility
for all non-hazardous laboratory manipulations.

-Only fully authorized personnel may enter the Level 4 containment laboratory. A logbook
(signed by all personnel) or electronic entry record must be maintained.
-At all times when work is undertaken in the laboratory, there must be a competent person
available outside containment who will be able to assist in case of emergency.
-Protective clothing, gloves, and impermeable footwear are required. No street clothing will
be worn under the protective clothing. On exit from the area, personnel will shower and re-
dress in street clothing.
-Small laboratory animals or insects under experimentation must be held in this area in
ventilated cabinets having all output air HEPA filtered.
-Large animals require specialized care and handling not dealt with by these guidelines.
-All level 4 agents must be stored within the containment zone.
-All materials shall be removed from the contaminated zone through an autoclave or placed
in a double, unbreakable, sealed container, the outside of which will be disinfected. Where
equipment and apparatus are not compatible with heat sterilization, materials may be
removed via dunk tanks or air locks with suitable decontamination procedures.
-All manipulations with agents in containment Level 4 must be performed in Class III
biological safety cabinets (see Appendix A) or in Class I or II biological safety cabinets used
in conjunction with one-piece, positive-pressure-ventilated suits.
-Contingency plans for emergencies which may include responses to biological, toxic or
hazardous spills, fire, or life-threatening situations must be prepared, and reviewed by all
personnel.
-A written reporting system for laboratory accidents and exposures must be in effect. A
serum storage program for all laboratory and support personnel and a full medical
surveillance and treatment program must be implemented.
-The Level 4 facility and its systems must be tested for containment capability upon
completion of construction and annually thereafter (see 7.8).
5.5 ANIMAL BIOHAZARD CONTAINMENT FACILITIES
Laboratory facilities must provide containment for laboratory animals exposed to or harbouring
infectious agents which is appropriate to the risk level of the infectious agents involved. In addition
to the physical requirements identified in Sections 5.1 to 5.4, special equipment (e.g. filter cages,
partial or isolation caging systems) appropriate to the animal species as well as to the level of risk
must be used.
Operational procedures for the care and maintenance of the infected animals must satisfy the
Guidelines of the Canadian Council on Animal Care (ref. 10) as well as the Laboratory Biosafety
Guidelines in order to ensure not only protection for laboratory personnel and the environment but
to ensure that every care is taken to avoid causing the animals unnecessary pain or suffering and to
provide the animals with the highest quality care.
CHAPTER 6
LARGE SCALE PRODUCTION OF MICROORGANISMS
6.1 INTRODUCTION
Since Canada is rapidly expanding its industrial base in the area of biotechnology, it is imperative
that industrial fermentation and large scale manipulation of microorganisms be addressed so that
all manipulations be conducted under conditions of containment that will ensure minimal risk to
workers and to the environment. Large scale work is not necessarily more hazardous than
laboratory scale work.
In fact, it is often less of a concern since most of the procedures in large scale fermentation are
automated, and are often conducted in closed systems, thereby reducing the probability of exposure
of the operator.
This has been adequately demonstrated with many highly infectious agents used for vaccine
production. However, all organizations working with large scale fermentation must ensure that the
appropriate equipment for such procedures and contingency plans are in place to minimize risks
should there be a malfunction in the process.
This chapter is to be used as a guideline for work involving large scale processes. Previous chapters
contain information that may be relevant. Large scale cultivation of microorganisms may involve
hazards in addition to those that are discussed in this chapter. These must be identified and
considered.
6.2 SCOPE
The working group's objective was to provide a technical document for those who design, build,
operate or work in research and industrial facilities where large scale manipulation of
microorganisms is performed. The focus of this document is therefore on the use of
microorganisms under conditions other than those described in previous chapters. The committee
considers large scale to include fermenters and equipment that cannot be easily moved and
sterilized in an autoclave, therefore, requiring in situ sterilization. This would normally apply to
volumes greater than 10 litres.
A number of countries have addressed large scale work with recombinant organisms. The objective
of this working group was to present guidelines to parallel those previously adopted by other
countries, but which include large scale fermentation of all microorganisms.
The Committee is of the opinion that because a microorganism has been manipulated genetically, it
does not necessarily pose an increased risk. For example, it is possible that a genetically altered
microorganism (deletion mutants) may have a reduced potential to cause infections in humans or
animals as a result of the deletion of a specific virulence gene. Similarly, insertion of foreign genes
into an organism at a specific site may either reduce or increase its virulence. Each genetic
manipulation must be evaluated on a case by case basis. If possible, an organism should be
modified in such a way that its pathogenicity, dissemination and survival in the environment are
reduced. Once the level of risk is established, each organism is required to be handled according to
the criteria for that risk group.
Although this chapter does not address issues relating to the intentional environmental release of
microorganisms, containment practices recommended should be sufficient to reduce the release of
microorganisms which may be harmful to the environment.
6.3 SPECIAL CONSIDERATIONS
6.3.1 Biological Safety Officer:
When a production facility is engaged in activities with organisms in Risk Groups 2-4, it must
appoint a Biosafety Officer (BSO), experienced and knowledgeable in handling pathogenic
microorganisms and eukaryotic cells, with expertise in large scale containment.
6.3.2 Transgenic Plants and Animals
There is considerable potential for commercial production of bioproducts in transgenic plants or

animals. The potential release of transgenics into the environment and transmission of novel genes
to other plants and animals need to be considered when designing both the production system and
facilities to contain the transgenics. In each case, the risk level needs to be determined in
consultation with the appropriate Government agency.
This section will focus on transgenic plants and animals that are used for production of bioproducts
and the containment required for these activities.
In the case of transgenic animals, the first consideration is that they be handled according to the
Guidelines set forth by the Canadian Council for Animal Care. An important consideration is the
ability of the animal to transmit genes by interbreeding with the same species or any related
species. Under these conditions, it is important that the transgenics are well-contained to prevent
the spread of genetic modifications. It is recommended that, if at all possible, transgenics be
created using methodology which restricts the potential for transmission of the genes from one host
to another.
Transgenic plants may transmit novel characteristics to other plants, thereby modifying the gene
pool of existing species. Since this transmission is mediated by pollen, transgenic plants should be
made sterile or contained in a growth chamber or greenhouse designed to prevent pollen release
either by air or insects. If plants are allowed to mature, care must be taken to contain the seeds in
the green house or growth chamber.
If live microorganisms are used as vehicles for transfection, the containment level for the plants or
animals inoculated with these viable recombinant microorganisms must be at least as high as that
required for work with that specific microorganism. Transgenics (eg. produced by micro-injection,
by use of replication defective vectors, or other sequences that are not horizontally transmitted) can
be normally handled at Containment Level 1.
The following recommendations should be considered prior to the initiation of transgenic studies:
a. Complete copies of the genome or replication competent genome should not be used.
b. The constructs should not contain genes capable of causing neoplastic transformation of
animals.
c. The probability of recombination with extraneous microorganisms should be minimal or
non-existent.
6.3.3 Cell Lines
The biological hazards associated with the use of mammalian or other cells in culture fall into 3
categories:
a. Primary cultures of mammalian or other cells may harbour infectious agents or integrated
DNA originally present in the animal or person from which the cultures were derived.
Whenever possible, the donor should be tested for any suspect pathogens prior to the
preparation of the culture, and the culture should be considered to be contaminated until
proven to be free of suspect agents. Such primary cultures should be handled in a manner
appropriate to the risk class of the suspected contaminant, and precautions should be taken
against parenteral or other means of exposure of laboratory personnel.
b. Cell lines known to contain infectious agents or integrated DNA should be handled
according to the risk class of the agent.
c. Cell lines that are deemed to be free of infectious agents would, rarely, pose a biological
hazard. If there is unintentional parenteral inoculation, normal immune response should
provide protection, prevent progressive growth and cause rejection of accidentally
transplanted cells.
6.4 CONTAINMENT LEVELS AND PRACTICES
6.4.1 Containment Level Large Scale 1 (LS1)
Microorganisms that are demonstrated to be non-pathogenic, containing no adventitious agents

and having a long history of safe industrial use are not considered in this containment level.
As the volume of cultivation and the quantity of biomass increases from laboratory to development
or production scales, increased care must be taken to achieve control over the dispersal of Risk
Group 1 agents, which is consistent with current large scale microbiological practice.
For genetically engineered organisms certain criteria are necessary to ensure continued safety. The
host organism should be a non-pathogen with no adventitious agents, a history of safe use, and
have limited capacity to survive in the environment. Vectors with known inserts should be well
characterised and free from sequences that result in adverse effects to humans, animals, plants or
the environment. The genomic insert should be limited in size to the smallest sequence required
and should not increase the stability of the gene product in the environment.
Resistance markers should be transferred with caution to organisms, to prevent acquisition of

resistance that might compromise therapeutic use of antimicrobials. The resulting recombinant
organism should be non-pathogenic or alternatively possess limited survival characteristics and be
without adverse environmental consequences.
Containment Level LS1 is suitable for large scale work with Risk Group 1 agents. In addition to the
requirements of Containment Level 1, the following are required:
Physical Requirements
-The work area and equipment should be well planned and appropriate for the intended use.
-Process equipment should be designed to minimize aerosol generation.
-Process equipment must contain organisms within a closed system.
-Process equipment and unit operations must be designed to minimize environmental
effects.
Operational Requirements
-All personnel should be adequately trained and knowledgable in workplace safety and
protocols.
-A plan for the clean-up and, if required, disinfection of large scale spills must be developed
and ready for use when required.
Large scale cultivation or processing of Risk Group 2 agents requires greater containment than
laboratory scale operations.
Containment Level LS2 is suitable for large scale work with Risk Group 2 agents. In addition to
both the requirements for Containment Level LS1 and for Containment Level 2, the following are
required:
-Process equipment must contain the organisms within a closed system, and must be
provided with HEPA or equivalent filters, which have been tested for integrity to prevent
releases of aerosols of the organism being processed. Alternatively equivalent procedures
(i.e. incineration or off gassing through chemical disinfectants) may be employed to prevent
release of microorganisms.
-Process equipment must be capable of being decontaminated with a validated inactivation
procedure.
-Unit operations and transfers between operations must be designed to prevent the release
of aerosols.
-Seals and mechanical devices associated with the process equipment shall be designed to
prevent leakage or shall be enclosed in HEPA or equivalent filtered housings.
-Decontamination of process equipment and process effluent must be performed by a
validated inactivation procedure.
-Decontamination must precede breach of containment.
-Sampling is to be performed in a controlled manner which prevents the release of infectious
materials.
-Process equipment should be tested regularly for integrity of containment capability (i.e.
after each use or operation) and records maintained of such testing.
-Unauthorized personnel are not permitted to enter process areas.
-Staff must be fully acquainted with emergency procedures to deal with spills or accidental
release of viable organisms. Procedures should be posted and training documented.
-Equipment for emergency and decontamination response must be readily available in the
process area and be maintained for immediate and effective use.
Large scale production and processing of Risk Group 3 agents may result in serious hazard to
people, animals and the environment. Containment Level LS3 is characterized by design features
which involve primary and secondary levels of physical containment.
Containment Level LS3 is suitable for large scale work with Risk Group 3 agents. In addition to
both the requirements for Containment Level LS2 and for Containment Level 3, the following are
required:
-Air leaving the containment area is to be HEPA filtered.
-Provision must be made to contain the full volume of a complete release of all process fluids
in the process area.
-All potentially contaminated liquids must be transferred in closed piping.
-Effluent must be taken to a decontamination system which is regularly validated to ensure
efficacy of the process.
-Showers and change rooms must be provided.
-Entry to process area should be restricted to essential personnel while production is in
progress.
-Upon entry, all personnel should exchange their outer clothing for dedicated laboratory
clothing and shoes. Laboratory clothing shall be decontaminated before laundering or
disposal.
- A personal shower should be used on egress.
-It is recommended that provision should be made for electronic transfer of all data from the
containment area (e.g. computer networking).
Specific requirements have not been outlined for large scale research or production of viable
organisms classified in Risk Group 4. These requirements will be established on an individual basis,
through consultation with the Office of Biosafety, Laboratory Centre for Disease Control, Health
Canada.
6.5 TRANSPORTATION OF LARGE SCALE PRODUCTS
6.5.1 Transportation
Requirements for the shipment of infectious agents are outlined in Section 3.3. Specifically, all
microorganisms classified in Risk Groups 2, 3 or 4 must be packaged, labelled and transported in
accordance with the Transportation of Dangerous Goods Act and Regulations.
Further information may be obtained from the Transport Dangerous Goods Directorate of
Transport Canada, 344 Slater Street, 14th Floor, Ottawa, Ontario K1A 0N3, tel.: (613) 958-0517.
6.5.2 Transportation On-site Between Containment Areas
Transportation of microorganisms of Risk Group 2 or higher within a facility must be accomplished

within a closed system. Alternatively, process piping or a secure unbreakable, sealed container
must be used. Containers or transfer lines must be capable of being decontaminated.
The facility's emergency response plan must include provision to respond to a spill, leak or accident
during transportation between containment areas. All emergency response materials and
equipment must be ready for use whenever needed.
CHAPTER 7
LABORATORY DESIGN
This chapter is designed to inform building operators, physical plant departments, laboratory
management designers and cost estimators of the construction features required to achieve the four
levels of containment outlined in Chapter 5.
A matrix system is used to present detailed information on the variety of design elements necessary
for required biosafety standards, and to allow rapid comparison of features required to design or
upgrade existing facilities.
7.1 LABORATORY LOCATION
The location of laboratories relative to other facilities in a building is one factor in containment.
Proper location can help control entry by uninformed people. Lower and higher containment areas
can be integrated to concentrate the necessary support services close to sterilizing and waste
disposal facilities including exhaust air.
MATRIX NO 1.- LABORATORY LOCATION

• - MANDATORY
o – RECOMMENDED
Containment Levels
1 2 3 4 Laboratory Location
• • • • Separated from public areas by door.
Laboratory doors labelled with biohazard
• • •
signs.
o • • Access limited to authorized personnel.
In a separate sealed room(s) with restricted
• •
access away from public thoroughfares.
In a separate building or sealed room with
o • independent air supply and exhaust -
restricted access.
Containment labs located away from outside
o o •
building envelope walls.
Containment labs located adjacent to or
o • nearby mechanical rooms to minimize lengths
of containment ducts.
Office areas can be located within lab if next to
• •
access or egress door.
Office areas must be outside laboratory
• •
containment zone.
Facility must be kept locked when not in use
• • (consistent with local fire and safety
regulations).
7.2 PHYSICAL CONSTRUCTION OF CONTAINMENT BARRIERS
In addition to providing physical separation, the construction of the laboratory perimeter can also
contribute to containment by providing surfaces that are easy to clean and disinfect. Chemical
resistance, impermeability, durability and compatibility with other construction materials must be
considered in choosing finishes and sealants of the containment perimeter. Careful consideration of
long term uses at the time of design and construction can allow for cost-effective upgrading, at
minimal cost.
For containment Level 3 and 4 laboratories, the requirement for negative air pressure must be
considered in the construction of walls and ceilings.
Users, designers and builders of these laboratories should be aware of the high costs involved in the
construction and long-term maintenance of these facilities.
MATRIX NO 2.- LABORATORY CONTAINMENT PERIMETER

• - MANDATORY
o – RECOMMENDED
Containment Levels
1 2 3 4 Laboratory Containment Perimeter
A. WALLS
o • Reinforced structural masonry.
o • Reinforced non-load-bearing masonry.
Steel frame reinforced non-load-bearing
o •
masonry.
o • Reinforced concrete.
B. CEILINGS
Steel frame gypsum partition or impervious
• •
ceiling acoustic tile.
Reinforced steel frame and gypsum ceiling,
• •
filler primer and paint finish.
C. COATINGS AND SEALANTS
Seamless, gas- and chemical-resistant wall
o • •
and ceiling coatings.
Chemical- and gas-resistant (disinfectant),
o • •
non-hardening sealants.
Containment seals for mechanical/electrical
o • •
service openings.
D. DOORS
o • • Doors lockable.
o • • Doors self-closing.
Doors to provide restricted access via keycard
o •
system or equivalent.
Ventilated airlock required for the separation
of higher and lower containment areas with
•
interlocking pneumatic or compressible sealed
doors.
o • • Doors and frames of solid finish construction.
Door openings should be of sizes to allow
• • • •
passage of all anticipated equipment.
Doors to have fire ratings as required and be
• • • •
located as per fire safety standards.
Entrance doors to be interlocked with manual
o •
overrides.
• • • • All exits marked and illuminated.
Egress to fire exits set out so that travel
• • • • through any high-hazard areas is minimized
or to conform to applicable codes.
E. WINDOWS
Windows, if openable, protected by fly
• •
screens.
Windows of safety glass, of proven
• •
performance, solid stops sealed in place.
F. FLOORS
• • • • Slip-resistant flooring.
Seamless, gas- and chemical-resistant (e.g.
o o • •
epoxy) coating with integral cove base.
Seamless, rolled or resilient tile flooring (e.g.
• •
vinyl).
7.3 AIR-HANDLING SYSTEMS
Because of their insidious, pervasive nature, aerosols are a major factor in the dissemination of
hazardous agents. They pose a risk to both the laboratory worker and the outside environment.
Contamination by aerosols can be minimized by use of proper laboratory techniques, biological
containment cabinets, primary containment devices, setting and balancing the room air supply and
exhaust systems and the use of HEPA filters.
It is of particular importance that purchased containment equipment (e.g. biological cabinets) be

tested to meet specified standards after installation and that the connections between such
equipment and room air handling systems should meet the standards required.
The following matrices indicate the HEPA filtration requirements for each containment level for
supply and exhaust air from the laboratory and from biocontainment cabinet interior
environments.
MATRIX NO 3.- AIR HANDLING

• - MANDATORY
o – RECOMMENDED
Containment Levels
1 2 3 4 Air Handling
A. ROOM AIR SUPPLY
Air supply independent from adjoining
o •
laboratory zones.
Air supply HEPA-filtered or provided with
o •
bubble tight dampers.
Equipped with pressure maintaining gauges at
o •
entry (e.g. magnehelics).
o • • Directional inward, non-recirculated airflow.
Interlocked with exhaust ventilation to
• •
prevent positive pressurization.
Equipped with audible alarms to detect
o • depressurization (i.e. failure of the exhaust
system).
Air supply ductwork sealed airtight and
o • independent from other laboratory zones and
accessible from outside the containment zone.
Equipped with bubble tight damper to permit
• •
sealing for decontamination procedures.
B. ROOM EXHAUST VENTILATION
Room equipped with magnehelic gauges or
o •
pressure monitoring devices at entry.
Sealed airtight independent-exhaust ductwork
and fan system accessible from outside the
o •
containment zone which meets performance
and verification testing requirement.
All exhaust ventilation HEPA-filtered and
o • connected to audible alarm to detect failure of
exhaust system.
Interlocked with air supply to prevent positive
o • •
pressurization of the laboratory.
Equipped with bubble tight damper to permit
o • •
sealing for decontamination.
Exhaust from laboratory at a minimum of ten
o • •
room volumes per hour.
Air vertically discharged to the outside, clear
o o o o of buildings or supply air intakes, at 12
metres/second.
Recirculated HEPA-filtered room air
o •
permitted.
Minimization of dead spaces where
o o • •
contaminated air can accumulate.
Ventilation sufficient to remove vapours of
• • • • flammable liquids and dangerous chemicals
before they reach hazardous concentrations.
Exposed ductwork to stand clear of walls to
• • allow access for maintenance, leak testing and
access to equipment filters and lighting.
C. BIOLOGICAL SAFETY CABINETS
o • Class I
o • • Class II
• Class III
Class I and II cabinets permitted with use of
•
positive-pressure suits.
Cabinet air can be recirculated in laboratory if
• •
HEPA-filtered.
D. FUME HOODS
o o o o Recommended when necessary.
o • HEPA and charcoal filters (if required).
o o o o Air flow alarms.
7.3.1 REQUIREMENTS FOR BIOLOGICAL SAFETY CABINETS IN ALL LEVELS OF

LABORATORIES
1. Biological safety cabinets should not be installed as an integral part of a room(s) supply and
exhaust system in such manner that fluctuations of the room supply and exhaust air cause
the biological safety cabinets to operate outside their design parameters for containment.
2. It is recommended that biological safety cabinets or fume hoods not be used as the sole
source of room exhaust.
3. Room supply air system equipped with dampers to prevent backflow if biological safety
cabinets are connected to exhaust ductwork.
4. If biological safety cabinets are connected to exhaust ductwork, connections are by thimble
units where appropriate and room exhaust ducts are equipped with manual dampers to
permit sealing for decontamination (ref. no. 42). Manufacturers recommendations for
installation should be carefully followed.
5. Cabinets to be located away from doors, from windows that can be opened, from room
supply air and from heavily-travelled laboratory areas.
6. A minimum of 30cm clearance is recommended, where possible, behind each side of the
cabinet to permit cleaning and testing and a minimum unobstructed clearance of 40cm at
the exhaust filter discharge to permit testing.
7. Biological safety cabinets must be installed and tested in accordance with CSA Z316.3-95 or
NSF Standard 49.
7.3.2 REQUIREMENTS FOR HEPA FILTERS IN LEVELS 2-4 BIOCONTAINMENT

LABORATORIES
1. All HEPA filters to have minimum particulate removal of 99.97% for particles of 0.3 µm.
2. HEPA filters installed as close as possible to the source of hazard to minimize length of
contaminated ductwork.
3. HEPA filters to be installed in housings with leakproof junctions between filter frame and
ducting.
4. HEPA filters to be installed in housings equipped with airtight dampers which will allow
decontamination and particle testing (e.g.: Dioctylphthalate [DOP]) in situ (i.e. access to
both upstream and downstream face of filter) by the scanning method.
5. HEPA filters to be monitored by magnehelic gauges or other appropriate devices.
7.4 DECONTAMINATION, STERILIZATION AND WASTE-DISPOSAL SYSTEMS
Biocontainment barriers should be maintained to the level required in all supply, drainage and
waste-disposal systems, decontamination systems, sampling ports, and condensation collection
and disposal systems as part of sterilization equipment.
In Canada, there are numerous pieces of legislation which control the disposal of infectious or
contaminated biomedical waste. The waste generator is held directly accountable for ensuring that
all stages of transportation and disposal are carried out in a safe and legal manner. Following
decontamination, solid wastes may be disposed of through burial in a landfill site. This process is
usually regulated and controlled by the province. Liquid wastes which are released to the local
sanitary sewer are controlled and regulated by municipal by-laws. When the effluent is released
into the environment from a drain, septic tank, the local sanitary sewer or a sewage treatment
plant, it comes under the jurisdiction of the province. The federal government has legislation which
covers all areas not controlled by existing municipal or provincial legislation. In order to find out
which rules apply to your operation, check with your municipal, provincial and federal authorities.
Most legislation does not refer specifically to communicable disease organisms by name. However,
there is usually some general requirement that wastes shall not be, or become, a hazard to persons,
animals or property. It is left to the waste generator to evaluate each risk and to ensure that it is not
significant.
MATRIX NO 4.- DECONTAMINATION, STERILIZATION AND WASTE DISPOSAL

SYSTEMS
• - MANDATORY
o - RECOMMENDED
Containment Levels
1 2 3 4 Decontamination, Sterilization and
Waste
Disposal Systems
A. DECONTAMINATION
Laboratory floors, walls and ceilings to be
o o • • treated with disinfectant-resistant, cleanable
coatings.
All laboratory furnishings and surface
materials disinfectant-resistant and cleanable.
o o • • Bench tops with coved splash backs, seamless,
or with unions sealed with non shrinking
sealant.
o o • Surfaces of plastic laminate
o • Surfaces of stainless steal
Laboratory perimeter sealed to allow gaseous
• •
decontamination of whole room.
B. STERILIZATION
Interlocking double-door pass-through
o •
autoclave in laboratory.
• • Autoclave in laboratory.
o • • • Autoclave in building.
Exposed steam pipes covered with insulating
• • • •
material.
o Incinerator in building.
C. WASTE DISPOSAL
LIQUIDS
Drainage traps filled with disinfectant
o • •
specified by laboratory operator.
All liquid effluents must be sterilized in
mechanically and biologically monitored tanks
•
located adjacent to the containment area prior
to disposal.
SOLIDS
Provide space for support stands for
• • • •
biomedical waste bags.
Provide refrigerated space for lockable, closed
o o o • storage for biomedical waste which will be
disposed of off site.
Provide disinfectant dunk tanks for all non-
autoclavable materials and equipment exiting
the containment area as a part of the cabinet
o • line. In the case of a suited lab, this function
may be provided by a dunk tank or via the
chemical shower at the containment
perimeter.
7.5 PERSONAL HYGIENE AND SAFETY FACILITIES
Good laboratory practice and protective clothing minimize the contamination of laboratory
personnel with infectious agents. The laboratory must, however, incorporate facilities for changing
into protective clothing, and for its eventual disposal, and also provide emergency personal
decontamination and washing facilities required in the event of a spill or accident.
MATRIX NO 5.- PERSONAL HYGIENE AND SAFETY FACILITIES

• - MANDATORY
o – RECOMMENDED
Containment Levels
1 2 3 4 Personal Hygiene and Safety Facilities
• • • • Handwashing facilities in laboratory.
Dedicated handwashing facilities with foot,
o • • knee or automatic controls in laboratory (not
applicable for positive-pressure-suit mode).
Chemical deluge shower at the laboratory
•
perimeter (for positive-pressure-suit mode).
Eye/face wash facilities equipped with in-use
o o o • audio/visual alarm (not applicable for positive
pressure suit mode).
o • Body shower in containment area.
Clothing change area adjacent to containment
• •
area (0.5 m2 per person).
Provide storage space for laboratory clothing
• • • • in lab or adjacent change area (minimum 300
linear mm. for each peg).
Provide space adjacent to exit door for laundry
hamper (minimum 0.900m2) for used
o o • •
laboratory clothing to be autoclaved prior to
laundering.
7.6 BUILDING SERVICES
Plumbing and drainage, electrical, fire alarm and communications systems, other than those
concerned with air handling, are covered in this section.
MATRIX No 6
BUILDING SERVICES
• - MANDATORY
o - RECOMMENDED
Containment Levels
1 2 3 4 Building Services
A. PLUMBING AND DRAINAGE
• All drains connected to sterilization system.
o All drains connected directly to sanitary sewer.
Autoclave chamber condensate directed to
o •
sewer through a closed system.
All piping penetrations sealed with non-
• •
shrinking sealant at laboratory perimeter.
All supply water services fitted with backflow
• •
preventers.
Piping to be exposed and stand clear of walls
• • within high containment area to allow access
for maintenance.
Main water supply control to be located
o •
outside laboratory perimeter.
All exposed hot and cold water pipes are to be
• • • • covered with insulating material and protected
from movement.
All vent lines equipped with HEPA filters or
o •
equivalent.
B. COMPRESSED GASES
Air supply lines HEPA filtered or equivalent as
o • •
backflow protection.
• • All gas lines to have backflow preventers.
All vacuum lines HEPA-filtered or
o • •
equivalent*.
No vacuum lines should lead from facility
• •
(needs to be met by pumps inside laboratory).
All supply line penetrations sealed with non-
• •
shrinking sealant at laboratory perimeter.
Compressed gas cylinder storage outside
o •
laboratory.
*Vacuum lines must not leave Containment Level 3 or 4.
C. ELECTRICAL
All supply conduit and wiring to be sealed at
o • the containment barrier with non-shrinking
sealant.
Fluorescent light ballasts and starters to be
o o
located outside containment area.
Circuit breakers located outside
o •
biocontainment area.
Building security systems integrated with
o o o •
laboratory safety and monitoring systems.
70 ft.-candles of light at work surface level
o o o o (metric) minimum maintained at work
surface.
All circuit-breaker switches, panels and
• • • •
controls to be appropriately labelled.
Electrical system is to be equipped with
standby generator for emergency support of
o o •
essential equipment, which includes biological
safety cabinets.
Laboratory to be equipped with fire alarm
• • • •
system.
Lab to be equipped with a communication
o • system between containment area and outside
support area.
Closed-circuit TV monitoring of entire work
o
area.
7.7 EMERGENCY AND MONITORING PROVISIONS
Regular monitoring of containment barriers is essential to safe laboratory practice and to

contingency plans to ensure that agents do not escape into the environment. Monitoring
equipment, alarms, backup support systems, and access for the integration of testing equipment
must be incorporated into the design.
MATRIX No 7.- EMERGENCY AND MONITORING PROVISIONS

• - MANDATORY
o – RECOMMENDED
Containment Levels
1 2 3 4 Emergency and Monitoring Provisions
A. AIR HANDLING
Directional inward, non-recirculated airflow,
o • •
monitored at entrance to lab.
Biological safety cabinets equipped with
o • • pressure monitoring gauges for all HEPA
filters.
Provision of access for decontamination of
• • • •
HEPA filters.
Entry to laboratory via ventilated air-lock with
o
interlocking doors.
• Entry to laboratory via sealed air-lock.
Rooms in isolation area to be maintained at
• • pressure negative to corridor with greatest
negative pressure in most hazardous room.
B. FIRE PREVENTION AND
CONTAINMENT
• • • • Equipped with fire alarms.
• • • • Equipped with appropriate fire extinguishers.
• • • • Doors to have appropriate fire ratings.
• • • • All fire exits marked and illuminated.
Biocontainment area designated as "burn
out". Firemen to enter only to save life, not to
o
extinguish fire, which should be controlled
from outside, to prevent spread.
Equipped with suitable storage cabinets or
• • • • explosion-proof refrigerators which are clearly
labelled for flammable liquids.
Storage for flammable liquids to be located
o o o o
outside biocontainment area.
Sprinkler systems (where required by law) to
o o o o be preactioned type. Other fire suppression
systems may be considered for level 4.
C. PERSONAL EMERGENCY
EQUIPMENT
Equipped with bottled back-up breathing air
• sufficient to provide 30 minutes per person.
(Level 4 suit lab only)
Equipped with positive-pressure hood
o • respirators with compressed breathing air
cylinders located in support area.
Eye/face wash facilities in laboratory (not
• • • • applicable for positive-pressure-suit mode,
Level 4).
o o o • Body shower in support area.
Equipped with communication system
o •
between containment zone and support area.
• • • • Emergency lighting.
D. BACKUP SERVICE
Equipped with standby generator for support
o • of essential equipment, including biological
safety cabinets.
7.8 PERFORMANCE, VERIFICATION AND TESTING
An institution in which work requiring containment facilities is carried out must bear a major part
of the responsibility for ensuring that the facilities, and the work in them, meet acceptable
standards. Therefore, before putting a containment laboratory into service, it is essential that the
institution satisfy itself that the laboratory meets the standards required. The laboratory equipment
and procedures must pass a series of inspections, testing and proving standards specified in their
design and construction before agents are used in the laboratory. This inspection must be repeated
at least yearly thereafter. Detailed records of inspection and test results should be maintained by
the institution indefinitely.
MATRIX No 8
PERFORMANCE, VERIFICATION AND TESTING
• - MANDATORY
o - RECOMMENDED
Containment Levels
1 2 3 4 Performance, Verification and Testing
Construction of laboratory perimeter to be
leakproof and able to withstand loading
characteristics imposed by negative air
pressure required in laboratory operation:
a) Integrity of seals demonstrated by visual
•
inspection.
b) Integrity of room tightness demonstrated
• by physical testing (pressure decay 0.05 wg
loss\min) at 2"wg.
All air supply and exhaust ductwork tested in
situ to be leak-tight by pressure decay*: Level
o • 3 not >0.2% duct vol./min. at 2"wg (500Pa);
Level 4 not >0.1% duct vol./min. at 2"wg
(500Pa).
All air supply and exhaust ductwork verified to
• •
have back-draft protection.
All HEPA filters tested to meet required
• •
specification after installation.
All HEPA-filter housings tested to be leak
o • tight*: not > 0.2% of vol.\min. at 10"wg.
(2500Pa).
Testing of biological safety cabinets meets
• • •
required specifications after installation.
Testing of autoclaves to meet specified
• • • • standards after installation by the use of
biological indicators.
Testing of fume hoods according to CSA
• • • •
Standard Z316.5-94.
Drainage and liquid-waste-disposal systems
• including sampling ports tested to ensure
efficacy by use of biological indicators.
• Verification of integrity of sewage lines.
Verification of alarm systems for air systems
• • failure (exhaust, supply, room pressure,
breathing air).
Verification of alarm systems for electrical
• • •
failure.
• • • • Verification of fire alarm systems.
o • Verification of communication systems
Testing of directional airflow demonstrated by
o • •
field tests with visual smoke.
Verification of integrity positive pressure
•
suits.
Testing of breathing air as per CSA Standard
•
Z180.1-M85.
• Testing of regular and emergency air system.
Verification of operation of backflow
o • • •
preventers.
* see ANSI/ASME N510 (see ref. no. 5)
Off
ice of
Biosafet
y]
Office of Biosafety]
MENU
Material Safety Data Sheets (MSDS), regulated
under Workplace Hazardous Materials A B C D E F G H I J K L M N 0 P Q R S T U V W X Y Z
Information System (WHMIS) legislation, for
chemical products have been available to • Actinobacillus spp.
workers for many years. However because many • Actinomyces spp.
laboratory workers, whether in research, public
• Adenovirus (types 1, 2, 3, 4, 5 et 7)
health, teaching, etc., are exposed to not only
chemicals but infectious substances as well, • Adenovirus (types 40 and 41)
there was a large gap in the readily available • Aerococcus spp.
safety literature for employees. These MSDS are • Aeromonas hydrophila
produced for personnel working in the life • Ancylostoma duodenale
sciences as quick safety reference material • Angiostrongylus cantonensis
relating to infectious micro-organisms. • Ascaris lumbricoides
• Ascaris spp.
The MSDS are organized to contain health • Aspergillus spp.
hazard information such as infectious dose, • Bacillus anthracis
viability (including decontamination), medical • Bacillus cereus
information, laboratory hazard, recommended • Bacteroides spp.
precautions, handling information and spill • Balantidium coli
procedures. The intent of these documents is to • Bartonella bacilliformis
provide a safety resource for laboratory • Blastomyces dermatitidis
personnel working with these infectious
substances. Because these workers are usually • Bluetongue virus
working in a scientific setting and are • Bordetella bronchiseptica
potentially exposed to much higher • Bordetella pertussis
concentrations of these human pathogens than • Borrelia burgdorferi
the general public, the terminology in these • Branhamella catarrhalis
MSDS is technical and detailed, containing
• Brucella spp. (B. abortus, B. canis, B. melitensis, B.
information that is relevant specifically to the
suis)
laboratory setting. It is hoped along with good
• Brugia spp.
laboratory practises, these MSDS will help
provide a safer, healthier environment for • Burkholderia (Pseudomonas) mallei
everyone working with infectious substances. • Burkholderia (Pseudomonas) pseudomallei
• California serogroup
Please note that although the information, • Campylobacter fetus subsp. fetus
opinions and recommendations contained in • Campylobacter jejuni, C. coli, C. fetus subsp. jejuni
these Material Safety Data Sheets are compiled • Candida albicans
from sources believed to be reliable, we accept • Capnocytophaga spp.
no responsibility for the accuracy, sufficiency, • Chlamydia psittaci
or reliability or for any loss or injury resulting • Chlamydia trachomatis
from the use of the information. Newly • Citrobacter spp.
discovered hazards are frequent and this • Clonorchis sinensis
information may not be completely up to date. • Clostridium botulinum
• Clostridium difficile
• Clostridium perfringens
• Clostridium tetani
• Clostridium spp. (with the exception of those
species listed above)
• Coccidioides immitis
• Colorado tick fever virus
• Corynebacterium diphtheriae
• Coxiella burnetii
• Coxsackievirus
• Creutzfeldt-Jakob agent, Kuru agent
• Crimean-Congo hemorrhagic fever virus
• Cryptococcus neoformans
• Cryptosporidium parvum
• Cytomegalovirus
• Dengue virus (1, 2, 3, 4)
• Diphtheroids
• Eastern (Western) equine encephalitis virus
• Ebola virus
• Echinococcus granulosus
• Echinococcus multilocularis
• Echovirus
• Edwardsiella tarda
• Entamoeba histolytica
• Enterobacter spp.
• Enterovirus 70
• Epidermophyton floccosum, Microsporum spp.
Trichophyton spp.
• Epstein-Barr virus
• Escherichia coli, enterohemorrhagic
• Escherichia coli, enteroinvasive
• Escherichia coli, enteropathogenic
• Escherichia coli, enterotoxigenic
• Fasciola hepatica
• Francisella tularensis
• Fusobacterium spp.
• Gemella haemolysans
• Giardia lamblia
• Haemophilus ducreyi
• Haemophilus influenzae (group b)
• Hantavirus
• Hepatitis A virus
• Hepatitis B virus
• Hepatitis C virus
• Hepatitis D virus
• Hepatitis E virus
• Herpes simplex virus
• Herpesvirus simiae
• Histoplasma capsulatum
• Human coronavirus
• Human immunodeficiency virus
• Human papillomavirus
• Human rotavirus
• Human T-lymphotrophic virus
• Influenza virus
• Junin virus / Machupo virus
• Klebsiella spp.
• Kyasanur Forest disease virus
• Lactobacillus spp.
• Legionella pneumophila
• Leishmania spp.
• Leptospira interrogans
• Listeria monocytogenes
• Lymphocytic choriomeningitis virus
• Marburg virus
• Measles virus
• Micrococcus spp.
• Moraxella spp.
• Mycobacterium spp. (other than M. bovis, M.
tuberculosis, M. avium, M. leprae)
• Mycobacterium tuberculosis, M. bovis
• Mycoplasma hominis, M. orale, M. salivarium, M.
fermentans
• Mycoplasma pneumoniae
• Naegleria fowleri
• Necator americanus
• Neisseria gonorrhoeae
• Neisseria meningitidis
• Neisseria spp. (other than N. gonorrhoeae and N.
meningitidis)
• Nocardia spp.
• Norwalk virus
• Omsk hemorrhagic fever virus
• Onchocerca volvulus
• Opisthorchis spp.
• Parvovirus B19
• Pasteurella spp.
• Peptococcus spp.
• Peptostreptococcus spp.
• Plesiomonas shigelloides
• Powassan encephalitis virus
• Proteus spp.
• Pseudomonas spp. (other than P. mallei, P.
pseudomallei)
• Rabies virus
• Respiratory syncytial virus
• Rhinovirus
• Rickettsia akari
• Rickettsia prowazekii, R. canada
• Rickettsia rickettsii
• Ross river virus / O'Nyong-Nyong virus
• Rubella virus
• Salmonella choleraesuis
• Salmonella paratyphi
• Salmonella typhi
• Salmonella spp. (with the exception of those species
listed above)
• Schistosoma spp.
• Serratia spp.
• Shigella spp.
• Sindbis virus
• Sporothrix schenckii
• St. Louis encephalitis virus, Murray Valley
encephalitis virus
• Staphylococcus aureus
• Streptobacillus moniliformis
• Streptococcus agalactiae
• Streptococcus faecalis
• Streptococcus pneumoniae
• Streptococcus pyogenes
• Streptococcus salivarius
• Taenia saginata
• Taenia solium
• Toxocara canis, T. cati
• Toxoplasma gondii
• Treponema pallidum
• Trichinella spp.
• Trichomonas vaginalis
• Trichuris trichiura
• Trypanosoma brucei
• Ureaplasma urealyticum
• Vaccinia virus
• Varicella-zoster virus
• Venezuelan equine encephalitis
• Vesicular stomatitis virus
• Vibrio cholerae, serovar 01
• Vibrio parahaemolyticus
• Wuchereria bancrofti
• Yellow fever virus
• Yersinia enterocolitica, Yersinia pseudotuberculosis
• Yersinia pestis
APPENDIX A
SAFETY EQUIPMENT AND BIOLOGICAL SAFETY CABINETS
SAFETY EQUIPMENT
An essential element in maintaining personal safety and environmental protection is the correct
selection, use and maintenance of safety equipment in the laboratory. Items of safety equipment
must be well maintained and regularly serviced. There must also be a regular program of testing
and inspection, and accurate records must be kept.
Biological safety cabinets must be tested and certified by trained technicians when newly installed
or moved and annually thereafter. They must be decontaminated before removal of filters.
The following is a list of safety devices appropriate to the containment laboratory:
TYPE APPLICATION
Animal cages partial to total containment of aerosols - provides
or boxes protection from cross-contamination and environmental
and personnel protection
Autoclaves heat sterilization
Biological see ref. 42.

Safety
Cabinets
Class I a ventilated cabinet for personnel and environmental

protection, with an unrecirculated inward airflow away
from the operator. Class I cabinets are suitable for work
with agents assigned to Risk Groups l, 2 or 3 where no
product protection is required.
Class II a ventilated cabinet for personnel, product, and

environmental protection having an open front with
inward airflow for personnel protection, downward HEPA
filtered laminar airflow for product protection, and HEPA
filtered exhausted air for environmental protection. Class
II cabinets are suitable for work with agents assigned to
Risk Groups 1, 2 or 3 containment.
Class III a totally enclosed, ventilated cabinet of gas-tight

construction. Operations in the cabinet are conducted
through attached rubber gloves. The cabinet is maintained
under negative air pressure of at least 120 Pa (0.50 in wg).
Supply air is drawn into the cabinet through HEPA filters.
The exhaust air is treated by double HEPA filtration, or by
HEPA filtration and incineration. Class III cabinets are
suitable for work with agents assigned to Risk Groups 1, 2,
3 and 4.
Blenders - aerosol-free blenders provide containment of aerosols

mixers
Centrifuge safety trunnion cups with sealed heads provide

Equipment containment of aerosols
Face and eye safety glasses, goggles and full-face shields provide
protection protection from flying objects, chemicals and biological
splashes
Fume Hoods personnel and environmental protection, noxious or

hazardous fume control
Gloves provide hand protection of varying degrees - check

technical specifications to determine degree of protection
HEPA filters available in various sizes, including cartridge-disposable,

provide 99.97% removal of 0.3µm particulates
Incinerators - electric or gas with side-arm to contain splatters from

micro loops
Laboratory coats, gowns or ventilated suits appropriate to hazard
clothing
Leakproof variety of containers preferably of stainless steel which are

containers autoclavable and have tight-fitting lids which may be used
for transporting infectious materials to autoclave
Pipetting variety of devices are available which eliminate need to

devices pipette by mouth
Respiratory partial or full-face protection is provided with variety of

protection filters including HEPA
Sharps autoclavable puncture-proof containers which are used to

container discard used hypodermic syringes and needles
Sterility indicator devices with heat-resistant spores are used for

indicators determining efficacy of heat sterilization
APPENDIX B
BIOLOGICAL SAFETY CABINETS, FUME HOODS AND FILTRATION SYSTEMS
A.BIOLOGICAL SAFETY CABINETS
Biological safety cabinets are the most widely used and accepted primary containment devices.
Class I or II cabinets can provide partial containment, and Class III cabinets have the potential for
complete containment. Biological safety cabinets must not be confused with other laminar flow
devices or 'clean benches'; in particular, horizontal flow cabinets which direct air towards the
operator should not be used for handling infectious, toxic or sensitizing materials.
The correct location, installation, testing and certification of the biological safety cabinet is critical
to its performance in containing aerosols.
Cabinets must be annually inspected and tested by trained service personnel to CSA Z316.3-95 or
NSF 49 or subsequent modifications.
Laboratory personnel must be trained in the correct use and maintenance of biological safety
cabinets to ensure that personnel and product protection (where applicable) are maintained.
CLASS I BIOLOGICAL SAFETY CABINET
This is a ventilated cabinet for personnel protection with an unrecirculated inward airflow away
from the operator. This unit should be fitted with a HEPA filter to protect the environment from
discharged agents.
Schematic Diagram of a Class I Biological Safety Cabinet
Class I cabinets may be used with or without glove ports.
Class II Biological Safety Cabinet
This is a ventilated cabinet for personnel, product and environmental protection which provides
inward airflow and HEPA-filtered supply and exhaust air. The Class II cabinet has four designs: one
recirculates most of the air; a second exhausts most of the air; a third provides total exhaust; and a
fourth includes cabinets which are convertible between the other designs. Class II cabinets may be
of use with low to moderate risk biological agents, minute quantities of toxic chemicals, and trace
quantities of radionuclides; however, care must be exercised in selecting the correct Class II cabinet
design for these purposes.
Schematic Diagram of a Class II Biological Safety Cabinet

Class III Biological Safety Cabinet
A Class III cabinet is a totally enclosed ventilated cabinet which is gas-tight, and maintained under
negative air pressure 120 Pa (0.5 inches water gauge). The supply air is HEPA-filtered and the
exhaust air has two HEPA filters in series. Work is performed in the cabinet by the use of attached
rubber gloves.
Schematic Diagram of a Class III Biological Safety Cabinet
B. FUME HOODS
Fume hoods must be annually tested by qualified personnel in accordance with Canadian
Standards Association CSA Z316.5-94. Auxiliary filters (HEPA, charcoal, etc..) installed in fume
hoods must also be tested to appropriate standards.
C. FILTERED SYSTEMS
All filters installed in ventilation systems, vent stacks or primary containment devices must be
annually tested.
APPENDIX C
BIBLIOGRAPHY
1. Advisory Committee on Dangerous Pathogens. Categorization of pathogens according to hazards and

categories of containment. Revised edition in preparation. London, England: Her Majesty's Stationery Office,
1990. ISBN 011-879-54.(HMSO, Publication Orders, P.O. Box 276, 51 Nine Elms Lane, London SW8 5DQ,
England.
2. Advisory Committee on Genetic Manipulation. Guidelines for the Large Scale Use of Genetically Manipulated
Organisms. London, UK, 1988. Available from Health and Safety Executive, Baynards House, 1 Chepstow
Place, London UK.
3. A Guide to Eyewash Systems in a Health Care Facility. Toronto: Health Care Occupational Health and Safety
Association (OHA), 1990. L-AP-208. (Ontario Hospital Association, 150 Ferrand Drive, Toronto, Ont. M3C
1H6; Tel: (416) 429-2661).
4. * American Chemical Society. Safety in Academic Chemistry Laboratories. 3rd ed. Washington, D.C.: ACS,
1976.
5. American National Standard. Nuclear Air-Cleaning Systems. New York: American National Standards
Institution, 1980. ANSI/ASME N510.
Canadian supplier: Standards Council of Canada, 350 Sparks Street, Ottawa, Ont. K1R 7S8; Tel: (613) 238-
3222 and 1-800-267-8220).
6. American National Standard for Emergency Eyewash and Shower Equipment. New York: ANSI, 1981. Ansi
Spec Z35-81. (Canadian supplier: Standards Council of Canada, 350 Sparks Street, Ottawa, Ont. K1R 7S8; Tel:
(613) 238-3222 and 1-800-267-8220).
7. * ASHRAE Handbook: Heating, Ventilating, and Air-Conditioning Systems and Applications. Atlanta, Georgia:
American Society of Heating, Refrigeration and Air Conditioning Engineers, 1995.
8. Bather, R., Baker, B.C., Contreras, G., and Furesz, J. Heterotransplantation Studies with Tissue Culture Cell
Lines in Various Animal and in vitro Host Systems. J. Biol. Standardization, 13: 13-22, 1985.
9. Biological Containment Cabinets: Installation and Field Testing. Rexdale, Ont.: Canadian Standards
Association, 1987. CAN/CSA Z316.3-95. (178 Rexdale Boulevard, Rexdale, Ont. M9W 1R3: Tel: (416) 747-
4044).
10. Canadian Council on Animal Care. Guide to the Care and Use of Experimental Animals. 2nd ed. Vol. 1, 1993;
Vol. 2, in press. Ottawa, Ont.: CCAC. (Constitution Square, Tower 2, 315-350 Albert Street, Ottawa, Ont. K1R
1B1; Tel: (613) 238-4031). No charge.
11. Centers for Disease Control and National Institutes of Health. Biosafety in Microbiological and Biomedical
Laboratories. 3rd ed. Washington, D.C.: GPO, 1993. U.S. Dept. of Health and Human Services, HHS no. (CDC)
93-8395. GPO S/N 017-040-00523- 7. (Superintendent of Documents, U.S. Government Printing Office,
Washington, D.C. 20402-9325, U.S.A.)
12. * Chatignay MA. Protection against Infection in the Microbiological Laboratory. In: Umbreit WW, ed.
Advances in Applied Microbiology. New York: Academic Press, 1961;3:131-92.
13. # Chatignay MA, Clinger DI. Contamination Control in Aerobiology. In: Dimmick, RL, Akers AB, eds. An
Introduction to Experimental Aerobiology. New York: Wiley Interscience, 1969:194-263.
14. * Collins CH. Laboratory-Acquired Infections: History, Incidence, Causes and Prevention, 3rd ed. London,
England and Toronto: Butterworth-Heinemann, 1993.
15. * Collins CH, Hartley EG, Pilsworth R. The Prevention of Laboratory Acquired Infection. London, England:
HMSO, 1977. Public Health Laboratory Service Monograph no. 6.
16. *Collins CH, Lyne PM, Grange JM. Collins and Lyne's Microbiological Methods, 7th ed. London, England and
Toronto: Butterworth-Heinemann, 1995.
17. * Department of Health and Social Security et al. Code of Practice for the Prevention of Infection in Clinical
Laboratories and Post-Mortem Rooms. London, England: HMSO, 1978.
18. Drury P. CSLT Guidelines for Laboratory Safety. 3rd ed. Hamilton, Ont.: Canadian Society of Laboratory
Technologists, 1993. (Box 2830, Station A, Hamilton, Ont. L8N 3N5; Tel: (416) 528-8642).
19. Frommer, W., Archer, L., Brunius, G. et al. Safe Biotechnology. III. Safety Precautions for Handling
Microorganisms in Different Risk Classes. Appl. Microbiol. Biotechnol., 30: 541-552. 1989.
20. Frommer, W. and P. Kramer. Safety Aspects in Biotechnology--Classifications and Safety Precautions for
Handling of Biological Agents. Drugs made in Germany, 33 (4) : 128-132. 1990.
21. * Furr AK, ed. CRC Handbook of Laboratory Safety. 3rd ed. Boca Raton, Florida: CRC Press, 1989.
22. Genetic Manipulation Advisory Committee. Guidelines for Large Scale Work with Genetically Manipulated
Organisms. Canberra, Australia. 1990. Available from Genetic Manipulation Advisory Committee. GPO Box
2183, Canberra ACT 2601.
23. Giorgio, R.J. and Wu, J.J. Design of Large Scale Containment Facilities for Recombinant DNA Fermentations.
Trends Biotechnol., 4 (3) : 60-65. 1986.
24. Government of Canada. Biotechnology Regulations: A Users Guide (1991). Supply and Services Canada, Hull,
Québec K1A 0S9.
25. Gugel, E.A. and Sanders, M.E. Needle-stick Transmission of Human Colonic Adenocarcinoma. New England
Journal of Medicine, 315: 1487, 1986.
26. Guidelines for the Handling and Disposal of Biomedical Wastes from Health Care Facilities and Laboratories.
Toronto, Ont.: Ontario Ministry of the Environment, 1986. (Public Information Centre, 1st floor, 135 St. Clair
Avenue West, Toronto, Ont. M4V 1P5; Tel: (416) 323-4321).
27. Handling of Waste Materials within Health Care Facilities. Rexdale, Ont.: Canadian Standards Association,
1988. CAN/CSA Z317.10-88 (178 Rexdale Boulevard, Rexdale, Ont. M9W 1R3; Tel: (416) 747-4044).
28. + Hellman A, Oxman MN, Pollack R, eds. Biohazards in Biological Research. Cold Spring Harbor, Long Island,
NY: Cold Spring Harbor Laboratory, 1973. Conference on Biohazards in Cancer Research.
29. * Hubbert WT, McCulloch WF, Schnurrenberger PR. Diseases Transmitted from Animals to Man. 6th ed.
Springfield, Illinois: CC Thomas, 1975.
30. Israeli, E. Biosafety in Biotechnological Processes. In: Advances in Biotechnological Processes 6, pp. 1-30. l
Alan R. Liss Inc., New York, 1986.
31. * Keith LH, Walters DB, eds. The Compendium of Safety Data Sheets for Research and Industrial Chemicals,
Parts 1-6. Deerfield Beach, Florida: VCH Publishers, 1985-87.
32. Levenbrook, I.S., Petricciani, J.C. and Elisberg, B.L. Tumorigenicity of Vero Cells. J. Biol. Standardization, 12:
391-398, 1984.
33. * Manufacturing Chemists' Association. Guide for Safety in the Chemical Laboratory. New York: Van Nostrand
Reinhold, 1972.
34. Marx, J.L. Tumors: a mixed bag of cells. Science, 215: 275-277, 1982
35. Medical Research Council of Canada 1977. Guidelines for the Handling of Recombinant DNA Molecules and
Animal Viruses and Cells.
36. * Melby EC Jr, Altman NH, eds. CRC Handbook of Laboratory Animal Science. Vol. 1. Cleveland, Ohio: CRC
Press, 1974.
37. * National Institutes of Health. Biohazards Safety Guide. Washington, DC: GPO, 1974 S/N 1740-00383.
38. * National Institutes of Health. Laboratory Safety Monograph: a supplement to the NIH Guidelines for
Recombinant DNA Research. Washington, DC: GPO, 1979. US Dept. of Health and Human Services, Public
Health Service.
39. * National Institutes of Health. Recombinant DNA Research: Actions under Guidelines. Guidelines for
Research involving Recombinant DNA molecules. Federal Register, parts V & VI, 1984, November 23; 46256-
46291.
40. National Institutes of Health. Recombinant DNA Research: Actions Under the Guidelines. Federal Register 56
(138) : 33174. 1991.
41. National Institutes of Health U.S.A. Guidelines for Research Involving Recombinant DNA Molecules. Federal
Register 51 (88) : 16958. 1986.
42. * National Sanitation Foundation. Standard no. 49 for Class II (Laminar Flow) Biohazard Cabinetry. Ann
Arbor, Michigan: NSF, 1992.
43. Organization for Economic Co-Operation and Development. Recombinant DNA Safety Considerations. Paris,
France. 1986. Available from OECD Publications Service, 2 rue Andre Pascal, 75775 Paris, CEDEX 16, France.
44. §.Perkins JJ. Principles and Methods of Sterilization in Health Sciences. 2nd ed. Springfield, Illinois: CC
Thomas, 1982.
45. * Pike RM. Laboratory Associated Infections: Summary and Analysis of 3921 Cases. Health Laboratory Science
1976;13:105- 114.
46. Scanlon, E.F., Hawkins, R.A., Fox, W.W. and Smith, W.S. Fatal Homotransplanted Melanoma. Cancer, 18: 782-
789, 1965.
47. Shin, S. and Freedman, V.M. Neoplastic Growth of Animal Cells in Nude Mice. In: Proc. 2nd Intl. Workshop on
Nude Mice, Stuttgart, 337-349. Gustav Fischer Verlag, 1977.
48. Southam, C.M. Homotransplantation of Human Cell Lines. Bull. N.Y. Acad. Med., 34: 416-423, 1958.
49. Southam, C.M., Moore, A.E. and Rhoads, C.P. Homotransplantation of Human Cell Lines. Science, 125: 158-
160, 1957.
50. The Pregnant Worker: a Resource Document for Health Professionals. Report of the Working Group on the
Development of Guidelines to Control Risks for Women in Industry to the Federal-Provincial Advisory
Committee on Environmental and Occupational Health. Ottawa: Health Canada, Health Protection Branch,
Environmental Health Directorate, 1987. (Environmental Health Directorate Publications, Environmental
Health Centre, Tunney's Pasture, Ottawa, Ont. K1A 0L2; Tel: (613) 954-0292).
51. Transportation of Dangerous Goods Act and Regulations. Registration SDR 85-77. Available from the Canadian
Government Publishing Centre, Department of Supply and Services, Ottawa, ON K1A 0S9
52. U.S. Department of Labour, Occupational Health and Safety Administration, Occupational Exposure of
Bloodborne Pathogens; Final Rule. 29 CFR Part 1910.1030, Dec. 6, 1991.
53. U.S. Department of Labour, Occupational Health and Safety Administration, Occupational Exposure of
Bloodborne Pathogens; OSHA 3127, 1992.
54. WHO Expert Committee on Biological Standardization. Requirements for Poliomyelitis vaccine (inactivated).
Tech. Rep. Series 673. Geneva, World Health Organization, 1982.
55. World Health Organization. Laboratory Biosafety Manual. 2nd ed. Geneva, Switzerland: WHO, 1993.
(Canadian distributors: Canadian Public Health Association, 1565 Carling Avenue, Ottawa, Ont. K1Z 8R1; Tel:
(613) 725-3769).
* Publications marked with an asterisk are in the collection of the Canada Institute for Scientific and Technical
Information (CISTI), NRC, Ottawa, Ont. K1A 0R6, and may be obtained on interlibrary loan.
# Item 11 is held in the Departmental Library of Environment Canada, Ottawa, Ont. K1A 0H3.
+ Item 19 is held in the Carleton University Library, Ottawa, Ont. K1S 5J7.
§ Item 27 is held by the Health Sciences Library, Victoria General Hospital, Halifax, Nova Scotia B3H 2Y9.
APPENDIX D
GLOSSARY
• Aerosol: Liquid droplets or solid particulates dispersed in a gaseous medium (e.g. air). A
gaseous suspension of ultra microscopic particles of a liquid or solid.
• Biohazard: A potentially dangerous infectious agent.
• Biological Containment: A genetic or physiological impediment to the infectivity and/or
survival of a microorganism or eukaryotic cell.
• Bioproducts: A product derived from or produced by cells or organisms.
• Decontamination: A process whereby viable microorganisms are removed from solutions,
surfaces or materials by filtration, heating, radiation or chemicals.
• Eukaryotic Cell: A cell with a definitive nucleus.
• HEPA filter: High efficiency particulate air filter (99.97% efficient removal of 0.3 µm
particles).
• Impervious: Not affording passage.
• Inactivation: Any process that destroys the ability of a microorganism or eukaryotic cell to
replicate.
• Infection: Invasion and multiplication of microorganisms in body tissue which may or may
not be clinically apparent.
• Large Scale Production: The culturing of microorganisms or eukaryotic cells in a single
volume of greater than 10 litres.
• Microorganism: Microscopic living entity; microorganisms can be viruses, prokaryotes
(e.g. bacteria) or eukaryotes (e.g. fungi, parasites).
• Pathogen: A biological agent cell capable of producing disease.
• Physical Containment: The confinement of a microorganism or eukaryotic cell to prevent
or minimize its contact with people and/or the environment.
• Prokaryotic cell: A cell lacking a true nucleus or nuclear membrane.
• Release: The discharge of a microorganism or eukaryotic cell from a containment system. A
is the discharge of a microorganism or eukaryotic cell from a containment system when the
system is appropriately designed and properly operated and maintained. An is the
unintentional discharge of a microorganism or eukaryotic cell due to a failure in the
containment system. An intentional is the planned discharge for an authorized purpose.
• Sterilization: An act or process of destroying all forms of microbial life on and in an object.
• Validation: The act of testing for compliance with a standard.

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Guidelines for Biomedical Facilities using

Sheep as Research Animals

The following operational practices are required:

• An effective rodent and insect control program should be maintained.

If a microbial pathogen is isolated or suspected to be present in a specimen, it then must be

1.2 SCOPE OF THE DOCUMENT

2.2 SAFETY PRACTICES

Naturally occurring or experimentally induced infections in laboratory animals may be transmitted

The importation of human pathogens is regulated by the Importation of Human Pathogens

In addition, information may be routinely obtained from the Canadian Transport

3.4 THE HEALTH OF ANIMALS ACT 1990

3.4.1 NON-INDIGENOUS AGENTS

The classifications of biological agents primarily reflect the judgements made on

4.2 RECOMBINANT DNA AND GENETIC MANIPULATION

4.3 USE OF MAMMALIAN CELLS IN CULTURE

4.4 RISK LEVELS ASSOCIATED WITH THE USE OF LABORATORY ANIMALS

4.5 CRITERIA FOR CLASSIFICATION OF BIOLOGICAL AGENTS BY RISK GROUP

A biological agent that is unlikely to cause disease in healthy workers or animals.

4.6 CATEGORIES OF PATHOGENS

4.6.1 RISK GROUP 1 AGENTS: REQUIRING CONTAINMENT LEVEL 1

4.6.2 RISK GROUP 2 AGENTS: REQUIRING CONTAINMENT LEVEL 2

4.6.4 RISK GROUP 4 AGENTS: REQUIRING CONTAINMENT LEVEL 4

Risk Group 4 (high individual risk, high community risk)

5.1 CONTAINMENT LEVEL 1

5.1.1 PHYSICAL REQUIREMENTS

5.1.2 OPERATIONAL REQUIREMENTS

Basic safety practices as described in Chapter 2 must be followed.

5.2 CONTAINMENT LEVEL 2

5.2.2 OPERATIONAL REQUIREMENTS

5.3 CONTAINMENT LEVEL 3

5.3.1 PHYSICAL REQUIREMENTS

5.3.2 OPERATIONAL REQUIREMENTS

5.4 CONTAINMENT LEVEL 4

5.4.1 PHYSICAL REQUIREMENTS

5.4.2 OPERATIONAL REQUIREMENTS

5.5 ANIMAL BIOHAZARD CONTAINMENT FACILITIES

6.3 SPECIAL CONSIDERATIONS

6.3.1 Biological Safety Officer:

6.3.2 Transgenic Plants and Animals

There is considerable potential for commercial production of bioproducts in transgenic plants or

6.3.3 Cell Lines

6.4 CONTAINMENT LEVELS AND PRACTICES

6.4.1 Containment Level Large Scale 1 (LS1)

Microorganisms that are demonstrated to be non-pathogenic, containing no adventitious agents

Resistance markers should be transferred with caution to organisms, to prevent acquisition of

6.4.2 Containment Level Large Scale 2 (LS2)

6.4.3 Containment Level Large Scale 3 (LS3)

6.4.4 Containment Level Large Scale 4 (LS4)

6.5 TRANSPORTATION OF LARGE SCALE PRODUCTS

6.5.2 Transportation On-site Between Containment Areas

Transportation of microorganisms of Risk Group 2 or higher within a facility must be accomplished

7.1 LABORATORY LOCATION

MATRIX NO 1.- LABORATORY LOCATION

7.2 PHYSICAL CONSTRUCTION OF CONTAINMENT BARRIERS

MATRIX NO 2.- LABORATORY CONTAINMENT PERIMETER

7.3 AIR-HANDLING SYSTEMS

It is of particular importance that purchased containment equipment (e.g. biological cabinets) be

MATRIX NO 3.- AIR HANDLING

7.3.1 REQUIREMENTS FOR BIOLOGICAL SAFETY CABINETS IN ALL LEVELS OF

7.3.2 REQUIREMENTS FOR HEPA FILTERS IN LEVELS 2-4 BIOCONTAINMENT

7.4 DECONTAMINATION, STERILIZATION AND WASTE-DISPOSAL SYSTEMS

MATRIX NO 4.- DECONTAMINATION, STERILIZATION AND WASTE DISPOSAL

7.5 PERSONAL HYGIENE AND SAFETY FACILITIES

MATRIX NO 5.- PERSONAL HYGIENE AND SAFETY FACILITIES