CONTAINMENT

Section 61-1.10 - Containment; general

CONTAINMENT

61-1.10 Containment; general. Good microbiological practices shall be adhered to. All personnel directly or indirectly involved in recombinant DNA activity shall be instructed in aseptic techniques and the biology of organisms used in the activity, in order that they shall be aware of potential biohazards. An emergency plan shall describe the procedures to be followed in the event of an accident contaminating personnel or the environment. The principal investigator must ensure that everyone in the laboratory is familiar with both the potential hazards of the work and the emergency plan. All personnel working with a pathogen for which an effective vaccine is available should be immunized. Serological monitoring, when appropriate, shall be provided.
 

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Section 61-1.11 - Physical containment levels; P1 level

61-1.11 Physical containment levels; P1 level.

(a) Laboratory practices.

(1) Laboratory doors shall be kept closed while experiments are in progress.

(2) Work surfaces shall be decontaminated daily, and immediately following spills of organisms containing recombinant DNA molecules.

(3) All biological wastes shall be decontaminated before disposal. Other contaminated materials, such as glassware, animal cages and laboratory equipment, shall be decontaminated before washing, reuse or disposal.

(4) Mechanical pipetting devices shall be used; pipetting by mouth is prohibited.

(5) Eating, drinking, smoking, and storage of foods are not permitted in the laboratory area in which recombinant DNA materials are handled.

(6) Persons shall wash their hands after handling organisms containing recombinant DNA molecules, and when they leave the laboratory.

(7) Care shall be taken in the conduct of all procedures to minimize the creation of aerosols.

(8) Contaminated materials that are to be decontaminated at a site away from the laboratory shall be placed in a durable leak-proof container, which is closed before removal from the laboratory.

(9) An insect and rodent control program shall be instituted.

(10) The use of laboratory gowns, coats or uniforms is discretionary with the laboratory supervisor.

(11) Use of the hypodermic needle and syringe shall be avoided when alternative methods are available.

(12) The laboratory shall be kept neat and clean.

(b) Containment equipment. Special containment equipment is not required at the P1 level.

(c) Special laboratory design. Special laboratory design is not required at the P1 level.
 

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Section 61-1.12 - Physical containment levels; P2 level

61-1.12 Physical containment levels; P2 level.

(a) Laboratory practices.

(1) Laboratory doors shall be kept closed while experiments are in progress.

(2) Work surfaces shall be decontaminated daily, and immediately following spills of organisms containing recombinant DNA molecules.

(3) All laboratory wastes shall be steam-sterilized (autoclaved) before disposal. Other contaminated materials, such as glassware, animal cages, laboratory equipment and radioactive wastes, shall be decontaminated by a means demonstrated to be effective before washing, reuse or disposal.

(4) Mechanical pipetting devices shall be used; pipetting by mouth is prohibited.

(5) Eating, drinking, smoking, and storage of food are not permitted in the laboratory area in which recombinant DNA materials are handled.

(6) Persons shall wash their hands after handling organisms containing recombinant DNA molecules, and when they leave the laboratory.

(7) Care shall be exercised to minimize the creation of aerosols. For example, manipulations such as inserting a hot inoculating loop or needle into a culture, flaming an inoculation loop or needle so that it splatters, and forceful ejection of fluids from pipettes or syringes shall be avoided.

(8) Contaminated materials that are to be steam-sterilized (autoclaved) or decontaminated at a site away from the laboratory shall he placed in a durable leak-proof container, which is closed before removal from the laboratory.

(9) Only persons who have been advised of the nature of the research being conducted shall enter the laboratory.

(10) The universal biohazard sign shall be posted on all laboratory access doors when experiments requiring P2 containment are in progress. Freezers and refrigerators or other units used to store organisms containing recombinant DNA molecules shall also be posted with the universal biohazard sign.

(11) An insect and rodent control program shall be instituted.

(12) The use of laboratory gowns, coats, or uniforms is required. Laboratory clothing shall not be worn to the lunch room or outside of the building in which the laboratory is located.

(13) Animals not related to the experiment shall not be permitted in the laboratory.

(14) Use of the hypodermic needle and syringe shall be avoided when alternative methods are available.

(15) The laboratory shall be kept neat and clean.

(16) Experiments of lesser biohazard potential can be carried out concurrently in carefully demarcated areas of the same laboratory.

(b) Containment equipment. Biological safety cabinets shall be used to contain aerosol-producing equipment, such as blenders, lyophilizers, sonicators, and centrifuges, when used to process organisms containing recombinant DNA molecules, except where equipment design provides for containment of the potential aerosol. For example, a centrifuge may be operated in the open if a sealed head or safety centrifuge cups are used.

(c) Special laboratory design. An autoclave for sterilization of wastes and contaminated materials shall be available in the same building in which organisms containing recombinant DNA molecules are used.
 

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Section 61-1.13 - Physical containment levels; P3 level

61-1.13 Physical containment levels; P3 level.

(a) Laboratory practices.

(1) Laboratory doors shall be kept closed while experiments are in progress.

(2) Work surfaces shall be decontaminated following the completion of the experimental activity, and immediately following spills of organisms containing recombinant DNA molecules.

(3) All laboratory wastes shall be steam-sterilized (autoclaved) before disposal. Other contaminated materials, such as glassware, animal cages, laboratory equipment, and radioactive wastes, shall be decontaminated by a method demonstrated to be effective before washing, reuse, or disposal.

(4) Mechanical pipetting devices shall be used; pipetting by mouth is prohibited.

(5) Eating, drinking, smoking, and storage of food are not permitted in the laboratory area in which recombinant DNA materials are handled.

(6) Persons shall wash their hands after handling organisms containing recombinant DNA molecules and when they leave the laboratory.

(7) Care shall be exercised to minimize the creation of aerosols. For example, manipulation such as inserting a hot inoculating loop or needle into a culture, flaming an inoculation loop or needle so that it splatters, and forceful ejection of fluids from pipettes or syringes shall be avoided.

(8) Contaminated materials that are to be steam-sterilized (autoclaved) or decontaminated at a site away from the laboratory shall be placed in a durable leak-proof container, which is closed before removal from the laboratory.

(9) Entry into the laboratory shall be through a controlled access area. Only persons who have been advised of the nature of the research being conducted shall enter the controlled access area. Only persons required on the basis of program or support needs shall be authorized to enter the laboratory. Such persons shall be advised of the nature of the research being conducted before entry, and shall comply with all required entry and exit procedures.

(10) Persons under 16 years of age shall not enter the laboratory.

(11) The universal biohazard sign shall be posted on the controlled access area door and on all laboratory doors when experiments requiring P3-level containment are in progress. Freezers and refrigerators or other units used to store organisms containing recombinant DNA molecules shall also be posted with the universal biohazard sign.

(12) An insect and rodent control program shall be instituted.

(13) Laboratory clothing that protects street clothing (e.g., long-sleeve solid-front or wrap-around gowns, no-button or slip-over jackets) shall be worn in the laboratory. Front-button laboratory coats are unsuitable. Laboratory clothing shall not be worn outside the laboratory and shall be decontaminated before it is sent to the laundry.

(14) Raincoats, overcoats, topcoats, coats, hats, caps, and such street outerwear shall not be kept in the laboratory.

(15) Gloves shall be worn when handling materials requiring P3 containment. They shall be removed aseptically immediately after the handling procedure and decontaminated.

(16) Animals and plants not related to the experiment shall not be permitted in the laboratory.

(17) Vacuum outlets shall be protected by filter and liquid disinfectant traps.

(18) Use of hypodermic needle and syringe shall be avoided when alternative methods are available.

(19) The laboratory shall be kept neat and clean.

(20) If experiments involving other organisms which require lower levels of containment are to be conducted in the same laboratory concurrently with experiments requiring P3-level physical containment, they shall be conducted in accordance with all P3-level laboratory practices.

(b) Containment Equipment.

(1) Biological safety cabinets shall be used for all equipment and manipulations that produce aerosols (e.g., pipetting, dilutions, transfer operations, plating, flaming, grinding, blending, drying, sonicating, shaking, centrifuging), where these procedures involve organisms containing recombinant DNA molecules, except where equipment design provides for containment of the potential aerosol.

(2) Laboratory animals held in a P3 area shall be housed in partial-containment caging systems, such as horsfall units, open cages placed in ventilated enclosures, solid-wall and -bottom cages covered by filter bonnets, or solid-wall and -bottom cages placed on holding racks equipped with ultraviolet radiation lamps and reflectors. (Note: Conventional caging systems may be used, provided that all personnel wear appropriate personal protective devices. These shall include, at a minimum, wrap-around gowns, head covers, gloves, shoe covers, and respirators. All personnel shall shower on exit from areas where these devices are required.)

(3) Alternative selection of containment equipment. Experimental procedures involving a host-vector system that provides a one-step higher level of biological containment than that specified can be conducted in the P3 laboratory using containment equipment specified for the P2 level of physical containment. Experimental procedures involving a host-vector system that provides a one-step lower level of biological containment than that specified can be conducted in the P3 laboratory using containment equipment specified for the P4 level of physical containment. Alternative combinations of containment safeguards are shown in Table I.

Table I

POSSIBLE COMBINATIONS OF CONTAINMENT SAFEGUARDS

Classification of experiment Alternate combinations of physical and biological containment

 

Physical containment

 

Biological containment *

 

Laboratory design specified for:

Physical Containment

Laboratory practices specified for:

 

Containment equipment
specified for:

Biological containment

P3 HV2 P3 P3 P3 HV2
  HV2 P3 P3 P4 HV1
P3 HV1 P3 P3 P3 HV1
P3 HV1 P3 P3 P2 HV2

* See section 61-1.16 for description of biological containment.

(c) Special laboratory design.

(1) The laboratory shall be separated by a controlled access area from areas that are open to unrestricted traffic flow. A controlled access area is an anteroom, a change room, an air lock or any other double-door arrangement that separates the laboratory from areas open to unrestricted traffic flow.

(2) The surfaces of walls, floors, and ceilings shall be readily cleanable. Penetrations through these surfaces shall be sealed or capable of being sealed to facilitate space decontamination.

(3) A foot-, elbow-, or automatically operated hand-washing facility shall be provided near each primary laboratory exit area.

(4) Windows in the laboratory shall be sealed.

(5) An autoclave for sterilization of wastes and contaminated materials shall be available in the same building (and preferably within the controlled laboratory area) in which organisms containing recombinant DNA molecules are used.

(6) The laboratory shall have a ventilation system that is capable of controlling air movement. The movement of air shall be from areas of lower contamination potential to areas of higher contamination potential (i.e., from the controlled access area to the laboratory area). If the ventilation system provides positive pressure supply air, the system shall operate in a manner that prevents the reversal of the direction of air movement or shall be equipped with an alarm that would be actuated in the event that reversal in the direction of air movement were to occur. The exhaust air from the laboratory area shall not be recirculated to other areas of the building unless the exhaust air is filtered by HEPA filters or equivalent. The exhaust air from the laboratory area can be discharged to the outdoors without filtration or other means for effectively reducing an accidental aerosol burden provided that it can be dispersed clear of occupied buildings and air intakes.

(7) The treated exhaust-air from class I and class II biological safety cabinets may be discharged either to the laboratory or to the outdoors. The treated exhaust-air from a class III cabinet shall be discharged directly to the outdoors. If the treated exhaust-air from these cabinets is to be discharged to the outdoors through a building exhaust air system, it shall be connected to this system so as to avoid any interference with the air balance of the cabinet and the building ventilation system.
 

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Section 61-1.14 - Physical containment levels; P4 level

61-1.14 Physical containment levels; P4 level.

(a) Laboratory practices.

(1) Laboratory doors shall be kept closed while experiments are in progress.

(2) Work surfaces shall be decontaminated following the completion of the experimental activity and immediately following spills of organisms containing recombinant DNA molecules.

(3) All laboratory wastes shall be steam-sterilized (autoclaved) before disposal. Other contaminated materials such as glassware, animal cages, laboratory equipment, and radioactive wastes shall be decontaminated by a method demonstrated to be effective before washing, reuse, or disposal.

(4) Mechanical pipetting devices shall be used; pipetting by mouth is prohibited.

(5) Eating, drinking, smoking, and storage of food are not permitted in the P4 facility.

(6) Persons shall wash their hands after handling organisms containing recombinant DNA molecules and when they leave the laboratory.

(7) Care shall be exercised to minimize the creation of aerosols. For example, manipulations such as inserting a hot inoculating loop or needle into a culture, flaming an inoculation loop or needle so that it splatters, and forceful ejection of fluids from pipettes or syringes shall be avoided.

(8) Biological materials to be removed from the P4 facility in a viable or intact state shall be transferred to a non-breakable sealed container, which is then removed from the P4 facility through a pass-through disinfectant dunk tank or fumigation chamber.

(9) No materials, except for biological materials that are to remain in a viable or intact state, shall be removed from the P4 facility unless they have been steam-sterilized (autoclaved) or decontaminated by a means demonstrated to be effective as they pass out of the P4 facility. All wastes and other materials as well as equipment not damaged by high temperature or steam shall be steam-sterilized in the double-door autoclave of the P4 facility. Other materials which may be damaged by temperature or steam shall be removed from the P4 facility through a pass-through fumigation chamber.

(10) Materials within the class III cabinets shall be removed from the cabinet system only after being steam-sterilized in an attached double-door autoclave or after being contained in a non-breakable sealed container, which is then passed through a disinfectant dunk tank or a fumigation chamber.

(11) Only persons whose entry into the P4 facility is required to meet program or support needs shall be authorized to enter. Before entering, such persons shall be advised of the nature of the research being conducted and shall be instructed as to the appropriate safeguards to ensure their safety. They shall comply with instructions and all other required procedures.

(12) Persons under 18 years of age shall not enter the P4 facility.

(13) Personnel shall enter into and exit from the P4 facility only through the clothing change and shower rooms. Personnel shall shower at each egress from the P4 facility. Air locks shall not be used for personnel entry or exit except for emergencies.

(14) Street clothing shall be removed in the outer side of the clothing-change area and kept there. Complete laboratory clothing, including undergarments, head cover, shoes, and either pants and shirts or jumpsuits, shall be used by all persons who enter the P4 facility. Upon exit, personnel shall store this clothing in lockers provided for this purpose or discard it into collection hampers before entering the shower area.

(15) The universal biohazard sign is required on the P4 facility access doors and on all interior doors to individual laboratory rooms where experiments are conducted. The sign shall also be posted on freezers, refrigerators, or other units used to store organisms containing recombinant DNA molecules.

(16) An insect and rodent control program shall be instituted.

(17) Animals and plants not related to the experiment shall not be permitted in the laboratory in which the experiment is being conducted.

(18) Vacuum outlets shall be protected by filter and liquid disinfectant traps.

(19) Use of the hypodermic needle and syringe shall be avoided when alternate methods are available.

(20) The laboratory shall be kept neat and clean.

(21) If experiments involving other organisms which require lower levels of containment are to be conducted in the P4 facility concurrently with experiments requiring P4-level containment, they shall be conducted in accordance with all P4-level laboratory practices specified in this section.

(b) Containment equipment.

(1) Experimental procedures involving organisms that require P4-level physical containment shall be conducted either in:

(i) a class III cabinet system; or in

(ii) class I or class II cabinets that are located in a specially designed area in which all personnel are required to wear one-piece positive-pressure isolation suits. (2) Laboratory animals involved in experiments requiring P4-level physical containment shall be housed either in cages contained in class III cabinets or in partial-containment caging systems (such as horsfall units, open cages placed in ventilated enclosures, or solid-wall and -bottom cages covered by filter bonnets, or solid-wall and -bottom cages placed on holding racks equipped with ultraviolet irradiation lamps and reflectors) that are located in a specially designed area in which all personnel are required to wear one-piece positive-pressure suits.

(3) Alternative selection of containment equipment. Experimental procedures involving a host-vector system that provides a one-step higher level of biological containment than that specified can be conducted in the P4 facility using containment equipment requirements specified for the P3 level of physical containment. Alternative combinations of containment safeguards are shown in Table II.

Table II

POSSIBLE COMBINATIONS OF CONTAINMENT SAFEGUARDS

 

Classification of experiment Alternate combinations of physical and biological containment

 

Physical containment

 

Biological containment *

 

Laboratory design specified for:

Physical Containment

Laboratory practices specified for:

 

Containment equipment
specified for:

Biological containment

P4 HV1 P4 P4 P4 HV1
P4 HV1 P4 P4 P4** HV2

* See section 61-1.16 of this Subpart for description of biological containment.

** In this case gloves shall be worn, in addition to the clothing requirements specified in paragraph (a) (14) of this section.

(c) Special laboratory design.

(1) The laboratory shall be located in a restricted-access facility which is either a separate building or a clearly demarcated and isolated zone within a building. Clothing-change areas and shower rooms shall be provided for personnel entry and egress. These rooms shall be arranged so that personnel leave through the shower area to the change room. A double-door ventilated vestibule or ultraviolet air lock shall be provided for passage of materials, supplies, and equipment which are not brought into the P4 facility through the change room area.

(2) Walls, floors, and ceilings of the P4 facility are constructed to form an internal shell which readily allows vapor-phase decontamination and is animaland insect-proof. All penetrations through these structures and surfaces are sealed. (The integrity of the walls, floors, ceilings, and penetration seals should ensure adequate containment of a vapor-phase decontaminant under static pressure conditions. This requirement does not imply that these surfaces must be airtight.)

(3) A foot-, elbow-, or automatically operated hand-washing facility shall be provided near the door within each laboratory in which experiments involving recombinant DNA are conducted in open-face biological safety cabinets.

(4) Central vacuum systems are permitted. The system, if provided, shall not serve areas outside the P4 facility. The vacuum system shall include in-line HEPA filters near each use point or service cock. The filters shall be installed so as to permit in-place decontamination and replacement. Water supply and liquid and gaseous services provided to the P4 facility shall be protected by devices that prevent backflow.

(5) Drinking water fountains shall not be installed in laboratory or animal rooms of the P4 facility. Foot-operated water fountains are permitted in the corridors of the P4 facility. The water service provided to such fountains shall be protected from the water services to the laboratory areas of the P4 facility.

(6) Laboratory doors shall be self-closing.

(7) A double-door autoclave shall be provided for sterilization of material passing out of the P4 facility. The autoclave doors shall be interlocked so that both doors will not be open at the same time.

(8) A pass-through dunk tank or fumigation chamber shall be provided for removal from the P4 facility of material and equipment that cannot be heat-sterilized.

(9) All liquid effluents from the P4 facility shall be collected and decontaminated before disposal. Liquid effluents from biological safety cabinets and laboratory sinks shall be sterilized by heat. Liquid effluents from the shower and hand washing facilities may be inactivated by chemical treatment. HEPA filters shall be installed in all vents from effluent drains.

(10) An individual supply and exhaust-air ventilation system shall be provided. The system shall maintain pressure differentials and directional air flow as required to ensure inflow from areas outside the facility toward areas of highest potential risk within the facility. The system shall be designed to prevent the reversal of air flow. The system shall sound an alarm in the event of system malfunction. (11) Air within individual laboratories of the P4 facility may be recirculated if HEPA-filtered.

(12) The exhaust air from the P4 facility shall be HEPA-filtered and discharged to the outdoors so that it is dispersed clear of occupied buildings and air intakes. The filter chambers shall be designed to allow in situ decontamination before removal and to facilitate certification testing after replacement.

(13) The treated exhaust-air from class I and class II biological safety cabinets may be discharged directly to the laboratory room environment or to the outdoors. The treated exhaust-air from class III cabinets shall be discharged to the outdoors. If the treated exhaust-air from these cabinets is to be discharged to the outdoors through the P4 facility exhaust air system, it shall be connected to this system so as to avoid any interference with the air balance of the cabinets or the facility exhaust air system.

(14) As noted in paragraph (b) (1) of this section, the P4 facility may contain specially designed areas in which all personnel are required to wear one-piece positive pressure isolation suits. Such areas shall be airtight. The exhaust-air from the suit area shall be filtered by two sets of HEPA filters installed in series, and a duplicate filtration unit and exhaust fan shall be provided. The air pressure within the suit area shall be less than that in any adjacent area. An emergency lighting system, communication systems, and power source shall be provided. A double-door autoclave shall be provided for sterilization of all waste materials to be removed from the suit area. Personnel who enter his area shall wear a one-piece positive-pressure suit that is ventilated by a life-support system. The life-support system shall be provided with alarms and emergency backup air. Entry to this area is through an airlock fitted with airtight doors. A chemical shower area shall be provided to decontaminate the surfaces of the suit before removal.

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Section 61-1.15 - Shipment of recombinant DNA

61-1.15 Shipment of recombinant DNA.* Recombinant DNA molecules contained in an organism or virus shall be shipped only as an etiologic agent under requirements of the U.S. Public Health Service and the U.S. Department of Transportation (section 72.3, part 72, title 42, and sections 173.386-.388, part 173, title 49, U.S. Code of Federal Regulations) as specified in this section:

(a) Recombinant DNA molecules contained in an organism or virus requiring P1, P2, or P3 physical containment, when offered for transportation or transported, are subject to all requirements of section 72.3(a)-(e), part 72, title 42 CFR, and sections 173.386-.388, part 173, title 49 CFR.

(b) Recombinant DNA molecules contained in an organism or virus requiring P4 physical containment, when offered for transportation or transported, are subject to the requirements listed under subdivision (a) of this section and are also subject to section 72.3(f), part 72, title 42 CFR.

* For additional information on packaging and shipment, see NIH Laboratory Safety Monograph.
 

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Section 61-1.16 - Biological containment levels

61-1.16 Biological containment levels. The following levels of biological containment (HV or host-vector systems) for prokaryotes are established:

(a) HV1. A host-vector system which provides a moderate level of containment. Specific systems:

(1) EK1. The host is always E. coli K-12 or a derivative thereof, and the vectors include nonconjugative plasmids (e.g., pSC101, Co1E1, or derivatives thereof) and variants of bacteriophage, such as (lambda). The E. coli K-12 hosts shall not contain conjugation-proficient plasmids, whether autonomous or integrated, or generalized transducing phages.

(2) Other prokaryotes. Hosts and vectors shall be, at a minimum, comparable in containment to E. coli K-12 with a nonconjugative plasmid or bacteriophage vector.

(b) HV2. These are host-vector systems shown to provide a high level of biological containment as demonstrated by data from suitable tests performed in the laboratory. Escape of the recombinant DNA either via survival of the organisms or via transmission of recombinant DNA to other organisms should be less than 1/10(8) under specified conditions. Specific systems:

(1) For EK2 host-vector systems in which the vector is a plasmid, no more than one in 10(8) host cells should be able to perpetuate a cloned DNA fragment under the specified nonpermissive laboratory conditions designed to represent the natural environment, either by survival of the original host or as a consequence of transmission of the cloned DNA fragment.

(2) For EK2 host-vector systems in which the vector is a phage, nor more than one in 10(8) phage particles should be able to perpetuate a cloned DNA fragment under the specified nonpermissive laboratory conditions designed to represent the natural environment either:

(i) as a prophage (in the inserted or plasmid form) in the laboratory host used for phage propagation; or

(ii) by surviving in natural environments and transferring a cloned DNA fragment to other hosts (or their resident prophages).

(c) Responsibility. HV1 systems other than E. coli K-12, and HV2 host-vector systems, may not be designated as such until they have been certified by the director, NIH.
 

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