[Title 32 CFR C]
[Code of Federal Regulations (annual edition) - July 1, 2002 Edition]
[Title 32 - NATIONAL DEFENSE]
[Chapter V - DEPARTMENT OF THE ARMY]
[Subchapter H - SUPPLIES AND EQUIPMENT]
[Part 627 - THE BIOLOGICAL DEFENSE SAFETY PROGRAM, TECHNICAL SAFETY REQUIREMENTS (DA PAMPHLET 385-69)]
[Subpart C - Operational Requirements]
[From the U.S. Government Printing Office]
32NATIONAL DEFENSE32002-07-012002-07-01falseOperational RequirementsCSubpart CNATIONAL DEFENSEDEPARTMENT OF THE ARMYSUPPLIES AND EQUIPMENTTHE BIOLOGICAL DEFENSE SAFETY PROGRAM, TECHNICAL SAFETY REQUIREMENTS (DA PAMPHLET 385-69)
Subpart C--Operational Requirements
Sec. 627.10 Personnel prerequisites.
(a) Medical. Before to assignment to work with etiologic agents,
personnel will be evaluated by the appropriate medical personnel with
respect to their assignment and will be evaluated in the medical
surveillance program described in Sec. 627.8.
(b) Training. All personnel directly or indirectly involved with
containment or handling of known and potentially biohazardous material
shall receive instruction that adequately prepares them for their
assigned duties. Training will be given by occupationally qualified
personnel as determined by the commander. This training will be
documented and will include--
(1) General training--
(i) Personal hygiene related to laboratory work.
(ii) Laboratory practices.
(iii) Personal protective equipment.
(iv) Effective use of engineering controls.
(v) Packaging, transportation, and shipment of etiologic agents
(vi) Hazardous and infectious waste disposal, handling, and
(2) Training conducted specifically for the facilities that the
individual will be working in, including--
(i) Procedures for the facility.
(ii) Reporting incidents and accidents.
(iii) Labeling and posting of signs.
(iv) Biohazardous waste handling, approaches to minimizing the
volume of waste, decontamination, packaging, and disposal.
(v) Emergency procedures.
(3) Additional general training required for work in facilities
where viable etiologic agents are present.
(i) Aseptic technique and procedures to include hands-on instruction
and demonstration of proficiency.
(ii) Concept and definition of biosafety levels.
(iii) Disinfection and sterilization.
(iv) Safe use of workplace equipment, for example autoclave and
(v) Monitoring and auditing requirements.
(vi) Precautions for handling blood, tissues, and body fluids (when
(vii) The infectivity, pathogenicity, mode(s) of transmission, and
medical surveillance requirements of specific agents.
(viii) Training for all new employees will include a period of
supervised orientation in the facilities by a scientist or technician
with specific training in the procedures and properties of the etiologic
agents in use. During the training period, new laboratory personnel will
be under the constant supervision of appropriately trained personnel.
(ix) Personnel who are assigned tasks in BL-2, BL-3, or BL-4
facilities will also have specific training in handling pathogens.
(x) Personnel assigned duties in a BL-4 facility will also have
specific and thorough training in handling extremely hazardous
infectious agents, the primary and secondary containment functions of
standard and special practices, use of personal protective equipment,
containment equipment, and laboratory design characteristics.
(4) Additional general training for handling toxins will include
relevant items from Sec. 627.10 plus--
(i) The availability of reference material on the hazards and safe
handling of toxic substances.
(ii) The biological effects of the toxin(s) in use.
Sec. 627.11 Operational prerequisites.
(a) Evaluation of the risks. The risk assessment of laboratory
activities involving the use of etiologic agents is ultimately a
subjective process. Those risks associated with the agent, as well as
with any adjunct elements of the activity to be conducted, (chemicals,
radioisotopes, end-products, and so forth) must be considered in the
assessment. The appropriate biosafety level for work with a particular
agent or animal study depends on the virulence, pathogenicity,
biological stability, route of transmission, and communicability of the
agent; the nature of the laboratory; the procedures and manipulations to
be used; the quantity and concentration of the agent; and the
availability of effective vaccines or therapeutic measures.
(b) The characteristics of etiologic agents, primary laboratory
hazards of working with the agent, and recommended biosafety levels are
described by CDC-NIH (HHS publication No. (NIH) 88-8395), the
considerations for recombinant DNA molecules are described by NIH, and
those for oncogenic viruses are described by NCI-NIH (sources listed
below). The commander or institute director will assign work with given
etiologic agents to the appropriate biosafety level. A risk assessment
should take into account not only the NIH Guidelines for Research
Involving Recombinant DNA Molecules, but also potential hazards
associated with the organism and the product of the experimentation.
(1) When established guidelines exist, these will be followed. The
primary source guidelines are--
(i) HHS Publication No. (NIH) 88-8395, Biosafety in Microbiological
and Biomedical Laboratories, as amended, and updates published in
Morbidity and Mortality Weekly Report.
(ii) NIH Guidelines for Research Involving Recombinant DNA Molecules
(FR 51: 16958-16985 and updates).
(iii) The publication by the American Committee on Arthropod-Borne
Viruses Subcommittee on Arbovirus Laboratory Safety (SALS) entitled
Laboratory Safety for Arboviruses and Certain Other Viruses of
Vertebrates in the American Journal of Tropical Medicine and Hygiene,
29(6), 1980, pp. 1359-1381.
(iv) The Department of Health and Human Services Publication No.
(NIH) 76-1165 by the National Cancer Institute (NCI) entitled Biological
Safety Manual for Research Involving Oncogenic Viruses.
(2) When samples with unidentified viable agents are obtained, a
knowledgeable and qualified scientist will evaluate the risks and make
recommendations to the safety officer, who will add recommendations for
review and approval by the commander or institute director. When
guidelines for a specific organism are not established, in addition to
these steps, the CDC or SALS or both will be consulted. Their
recommendations will be documented and provided to the commander or
institute director before approval.
(c) Selection of facilities. The facility requirements identified by
the risk assessment will be adhered to. Any variations and compensatory
measures will be approved by the IBC (when recombinant DNA molecules are
involved), the safety officer, and the commander or institute director
before a request for an exception or waiver is submitted as stated in AR
(d) Policies and procedures. Policies in the form of a laboratory
safety manual, regulations, memorandums, or SOPs are required for work
with etiologic agents in the BDP. Before beginning a new procedure, the
policies and procedures will be reviewed to ascertain that the intended
operations are described and to determine the requirements that apply to
the operation. If procedures exist for the intended operation, personnel
will be trained to follow them; if procedures do not exist, then a
detailed SOP will be written, reviewed, and approved before beginning
the operation. SOPs will conform to the requirements stated in
Sec. 627.7(d), and be signed by all personnel who are required to follow
the procedures, thus acknowledging that they have read and understood
the contents. All SOPs that pertain to a specific area (room,
laboratory, or suite) will be available at the worksite.
Sec. 627.12 General laboratory techniques.
The general requirements for use of etiologic agents are composed of
two sets of requirements, with the requirements for toxins being a
subset of the requirements for handling viable etiologic agents. These
requirements are as follows--
(a) General techniques applicable to etiologic agents.
(1) A fully fastened long-sleeved laboratory coat, gown, uniform, or
coveralls will be worn in laboratories or animal rooms.
(2) Eating, drinking, smoking, and applying cosmetics are not
permitted in the work areas.
(3) Personnel must wash their hands after they handle etiologic
agents or animals, and before leaving the laboratory area.
(4) Mouth pipetting is strictly prohibited. Mechanical pipetting
aids must be used.
(5) Gloves--(i) Will be worn when manipulating etiologic agents and
handling containers of etiologic agents. Gloves are not required when
materials are packaged appropriately for shipment.
(ii) Will be selected based on the hazards.
(iii) Will be changed frequently (or decontaminated frequently), and
will be decontaminated or discarded into a labeled biohazard container
after each use and immediately upon observable direct contact with an
(iv) Will be removed at the work-space (workbench or hood) after
handling etiologic agents to ensure that doorknobs and other surfaces
are not contaminated.
(6) Good housekeeping will be maintained. This includes--
(i) Work areas free of clutter.
(ii) Work environment free of tripping hazards, with adequate access
to exits, emergency equipment, controls, and such.
(iii) Benches and general work areas will be cleaned regularly using
sponge or similar method with disinfectant as appropriate. Methods that
stir up dust such as sweeping or using vacuum cleaners, (except for
HEPA-filtered vacuum cleaners) are unacceptable.
(iv) Specific work areas will be cleaned and decontaminated
immediately following each use of an etiologic agent (at least once a
day) and after any spill of viable material.
(v) Hallways and stairways will not be used for storage.
(7) All solutions, reagents, and chemicals will be labeled.
(8) All contaminated liquid or solid wastes will be inactivated
(9) Work will be conducted over spill trays or plastic-backed
absorbent paper. The paper will be removed, decontaminated, or
disinfected, and the general area wiped with decontaminant at the end of
each day or at the end of the experiment, whichever occurs first.
(10) Etiologic agents will be kept in closed containers when not in
use. Cultures, solutions, or dried etiologic agents in glass vessels
transported or incubated within a room or suite will be handled in
nonbreakable, leak-proof pans, trays, pails, carboys, or other secondary
containers large enough to contain all the material, if the glass vessel
leaks or breaks. Etiologic agents removed from a room or suite for
transport to another approved area within the same building will be
placed in a closed unbreakable secondary container before removal from
the laboratory. The secondary container will be labeled on the exterior
with a biohazard symbol and identification of the contents, including
the required biosafety level, the scientific name, the concentration (if
applicable), and the responsible individual. The secondary containers
will be wiped with suitable disinfectant before removal from the
laboratory or area.
(11) Working stocks of etiologic agents will be stored in double
containers. The primary and secondary containers will provide a positive
seal and the secondary container will be unbreakable. The secondary
container will be labeled as stated in Sec. 627.12 (a)(10) and with the
(12) Storage units (for example, freezers, refrigerators, cabinets,
and hoods) will be labeled with the universal biohazard sign and
indicate the classes of etiologic agents contained in them. Storage
units will be secured when not in use.
(13) All contaminated materials, containers, spills, and solutions
will be decontaminated or disinfected by approved methods before
(14) After injection of an etiologic agent into animals, the site of
injection will be swabbed with a decontaminant.
(15) Syringes. (i) Reusable or disposable syringes will be of the
fixed needle or LUER-LOK type (or equivalent) to assure that the needle
cannot separate during use.
(ii) After use, nondisposable glass syringes with attached needles
contaminated with etiologic agents will be submerged in a container of
decontaminant. Disposable syringes will be discarded with needles
attached in puncture-proof rigid containers. Needles will not be
recapped after use.
(iii) Sterilized or decontaminated containers marked ``Syringes and/
or Needles'' may be deposited in appropriate refuse containers after
proper packaging and destruction of the contents.
[Note: Many States, especially those on the Eastern seaboard, have
implemented strict requirements for the disposal of medical wastes. For
example, Maryland has designated all waste from a microbiological
laboratory as hazardous waste with licensing requirements for generators
of 50 kilograms per month or more of waste, while all medical waste
released for transport off-site must be manifested to a State licensed
medical waste hauler with the destination specified. Additionally, in
some cases, the local government (for example, a city) regulates the
disposal of these wastes. These requirements will be identified and
Needles or syringes may not be destroyed by clipping. A mechanical shear
may be used to smash or sheer needles after or concurrently with
sterilization or decontamination.
(16) Refrigerators, deep freezers, and dry ice chests should be
checked, cleaned out, and defrosted periodically to remove any ampules,
tubes, and so forth, containing etiologic agents that may have broken
during storage. Rubber gloves and respiratory protection
appropriate to the materials in storage should be worn during cleaning.
Do not store flammable solutions in nonexplosion proof refrigerators.
(b) Additional techniques applicable to work with viable etiologic
agents. The major objective of these techniques is to assist in
protection against laboratory acquired infections. Air sampling studies
have shown that aerosols are generated from most of the manipulations of
bacterial and viral cultures common to research laboratories. The
generation of aerosols during routine laboratory manipulations must be
considered when evaluating the individual degree of risk, keeping in
mind the four main factors governing infection: dosage, virulence of the
organism, route of infection (for example, skin, eyes, mouth, lungs),
and host susceptibility (for example, state of health, natural
resistance, previous infection, response to vaccines and toxoids). The
requirements stated below are minimum handling requirements to prevent
accidental infection created by incidental aerosols.
(1) All procedures are performed carefully to minimize the creation
(2) No infectious mixtures will be prepared by bubbling air through
(i) No infectious material will be forcibly ejected from pipettes.
Only to deliver (TD) pipettes will be used.
(ii) Pipettes used with infectious or toxic materials will be
plugged with cotton unless they are used exclusively in a gas-tight
(iii) Contaminated pipettes will be placed horizontally in a rigid
container containing enough disinfectant for complete immersion.
Cylinders used for vertical discard are not recommended. The container
and pipettes must be autoclaved as a unit and replaced by a clean
container containing fresh disinfectant.
(iv) Pipetting devices must be used. Under no circumstances is mouth
(4) Syringes. (i) Using syringes and needles for making dilutions of
etiologic agents is not recommended.
(ii) When removing a syringe and needle from a rubber stopper bottle
containing viable etiologic agents, an alcohol soaked pledget around the
stopper and needle will be used.
(iii) Excess fluid and bubbles should be expelled from syringes
vertically into a cotton pledget soaked with disinfectant or into a
small bottle containing disinfectant-soaked cotton.
(iv) The site of injection of an animal will be swabbed with a
disinfectant before and after injection.
(v) After use, syringes contaminated with residual infectious fluid
will be submerged in a container of disinfectant in a safety cabinet
prior to removal for autoclaving. To minimize accidental injection of
infectious material, the removable needles should remain on such
syringes until after autoclaving. When possible, syringes with attached
needles should be placed in a pan separate from that holding other
(vi) Caps will not be placed over needles until after disinfection.
During recapping, procedures to prevent personal injuries will be used.
(5) Centrifuges and shakers. (i) Before centrifuging, tubes, rotors,
seals, and gaskets will be checked for cleanliness and integrity. In low
speed clinical-type centrifuges, a germicidal solution may be added
between the tube and trunnion cup to disinfect the outer surfaces of
both and to cushion against shocks that might break the tube. Metal or
plastic tubes (other than nitro-cellulose) will be used.
(ii) Decanting from centrifuge tubes will be avoided. If decanting
is necessary, the outer rim will be wiped with a disinfectant after
decanting so that material on the lip cannot spin off as an aerosol.
Centrifuge tubes will not be filled byond the level the manufacturer
(iii) Broth cultures will be shaken in a manner that avoids wetting
the plug or cap.
(6) Water baths in which viable etiologic agents are incubated must
contain a disinfectant. For cold water baths, 70 percent propylene
glycol is recommended. The disinfectant should be changed frequently.
(7) When a laboratory vacuum is used to manipulate viable etiologic
agents, a secondary reservoir containing disinfectant and a HEPA filter
must be employed to ensure that the laboratory
vacuum lines do not become contaminated.
(8) Test tubes. (i) Tubes containing viable etiologic agents should
be manipulated with extreme care. Studies have shown that simple
procedures, such as removing a tube cap or transferring an inoculum, can
create a potentially hazardous aerosol.
(ii) Manipulation of biohazardous test tubes will be conducted in
biological safety cabinets. Tubes and racks of tubes containing
biohazardous material should be clearly marked. The individual employee
must ensure that tubes containing biohazardous material are properly
sterilized prior to disposal or glassware washing. Safety test tube
trays should be used in place of conventional test tube racks to
minimize spillage from broken tubes. When safety test tube trays are not
used, the conventional test tube racks will be placed in a tray large
enough to contain any potential spill. A safety test tube tray is one
having a solid bottom and sides deep enough to hold all liquids, should
a test tube break.
(9) Care should be exercised when using membrane filters to obtain
sterile filtrates of viable etiologic agents. Due to the fragility of
the membranes and other factors, such filtrates cannot be considered
noninfectious until laboratory culture or other tests have proven their
(10) The preparation, handling, and use of dry powders of viable
etiologic agents in open containers presents unusual hazards. The
slightest manipulation of such powders can cause the generation of
aerosols containing a high concentration of etiologic agents. Therefore,
work with dry powders of etiologic agents in open containers should be
carried out in gas-tight biological safety cabinets.
Sec. 627.13 Biosafety level 1.
(a) Requirements beyond those for all etiologic agents. BL-1
operations follow the general techniques described in Secs. 627.12(a)
(b) Additional laboratory requirement. Contaminated materials that
are to be decontaminated at a site away from the laboratory are placed
in a durable leak-proof container which is closed before being removed
from the laboratory. Examples of suitable containers are metal tubs with
lids or plastic bags that are sealed and then placed inside a rigid
container for transport.
(c) Additional animal requirements. (1) Bedding materials from
animal cages will be removed in such a manner as to minimize the
creation of aerosols and disposed of in compliance with applicable
institutional or local requirements.
(2) Cages are washed manually or in a cagewasher. Temperature of
final rinse water will be a minimum of 180 deg.F.
(3) Laboratory coats, gowns, or uniforms worn in animal rooms shall
not be worn in other areas.
Sec. 627.14 Biosafety level 2.
(a) Additional requirements. In addition to the general
microbiological techniques stated in Sec. 627.13, BL-2 operations
include the following requirements:
(1) When etiologic agents are in use, a hazard warning sign
incorporating the universal biohazard symbol is posted on the access
door of the work area. The hazard warning sign identifies the etiologic
agent, lists the name and telephone number of the institute director or
other responsible person(s), and indicates the special requirement(s)
for entering the laboratory.
(2) Animals not involved in the work being performed are not
permitted in the laboratory.
(3) Special care is taken to avoid skin contamination with the
etiologic agents; gloves will be worn when handling etiologic agents or
(4) All wastes from laboratories and animal rooms are decontaminated
(5) Hypodermic needles and syringes are used only for parenteral
injection and aspiration of fluids from laboratory animals and diaphragm
(6) Spills and accidents which result in a potential exposure to
etiologic agents will be reported immediately to the safety officer, the
project leader, and the institute director.
(7) Biological safety cabinets (Class I or II) will be used when:
(i) Procedures with a high potential for creating infectious
aerosols are conducted.
(ii) High concentrations or large volumes of etiologic agents are
(8) Laboratory coats, gowns, smocks, or uniforms will be removed
before leaving the animal facility or laboratory area.
(b) Additional animal requirements.
(1) Cages must be decontaminated, preferably by autoclaving, before
they are cleaned and washed.
(2) Approved molded masks are worn by all personnel entering animal
rooms housing nonhuman primates.
(3) If floor drains are provided, the drain traps will be kept
filled with water or a suitable disinfectant.
Sec. 627.15 Biosafety level 3.
(a) Additional requirements. In addition to the requirements stated
in Secs. 627.13 and 627.14, the following requirements apply--
(1) Approved molded masks or respirators with HEPA filters are worn
by all personnel in rooms housing infected animals.
(2) Protective clothing worn in a laboratory or animal room will be
removed before exiting the laboratory or animal room.
(3) Clothing worn in laboratories and animal areas to protect street
clothing will be decontaminated before being laundered.
(b) Additional laboratory requirements. (1) Laboratory doors will be
(2) All activities involving etiologic agents will be conducted in
biological safety cabinets (Class I, II, or III) or other physical
containment devices within the containment module. No work in open
vessels is conducted outside a biological safety cabinet.
(3) The work surfaces of biological safety cabinets and other
containment equipment will be decontaminated after work with etiologic
agents. Plastic-backed paper toweling should be used on nonperforated
work surfaces within biological safety cabinets to facilitate clean-up.
(c) Additional animal requirements. (1) Cages are autoclaved before
bedding is removed and before they are cleaned and washed.
(2) Gloves are removed aseptically and autoclaved with other wastes
before being disposed of or reused.
(3) Boots, shoe covers, or other protective footwear and
disinfectant foot baths must be available and used when indicated.
(4) Personal protective clothing and equipment and other physical
containment devices are used for all procedures and manipulations of
etiologic agents or infected animals. The risk of infectious aerosols
from infected animals or their bedding shall be reduced by housing
animals in partial containment caging systems as described in
(d) Work with BL-3 etiologic agents that require additional
secondary containment. Facilities in which work with certain viruses,
for example, Rift Valley fever, yellow fever, and Venezuelan equine
encephalitis, is conducted require HEPA filtration of Xallexhaust air
prior to discharge from the laboratory. All persons working with those
agents for which a vaccine is available should be immunized.
Sec. 627.16 Biosafety level 4.
Laboratory work at BL-4 must follow the requirements stated in
Secs. 627.13, 627.14 and 627.15 as well as the following:
(a) All activities are conducted in Class III biological safety
cabinets or in Class I or II biological safety cabinets in conjunction
with a one-piece positive pressure personnel suit ventilated by a life-
(b) Biological materials to be removed from the Class III cabinet or
from the maximum containment laboratory in a viable or intact state must
be transferred to a sealed nonbreakable primary container, enclosed in a
nonbreakable sealed secondary container, and removed from the facility
through a disinfectant dunk tank, fumigation chamber, or an airlock
designed for this purpose.
(c) No materials, except for biological materials that are to remain
in a viable or intact state, are removed from the maximum containment
laboratory unless they have been autoclaved or decontaminated before
they leave the facility. Equipment or material which might be damaged by
high temperature or steam is decontaminated by gaseous or vapor methods
in an airlock or chamber designed for this purpose.
(d) Personnel may enter and leave the facility only through the
clothing change and shower rooms. Personnel must shower each time they
leave the facility. Personnel may use the airlocks to enter or leave the
laboratory only in an emergency.
(e) Street clothing must be removed in the outer clothing change
room and kept there. Complete laboratory clothing, including
undergarments, pants and shirts or jumpsuits, shoes, and gloves, will be
provided and must be used by all personnel entering the facility. Head
covers are provided for personnel who do not wash their hair during the
shower. When leaving the laboratory and before proceeding into the
shower area, personnel must remove their laboratory clothing and store
it in a locker or hamper in the inner change room.
(f) When etiologic agents or infected animals are present in the
laboratory or animal rooms, a hazard warning sign incorporating the
universal biohazard symbol must be posted on all access doors. The sign
must identify the agent, list the name of the commander or institute
director or other responsible person(s), and indicate any special
requirements for entering the area (for example, the need for
immunizations or respirators).
(g) Supplies and materials needed in the facility are brought in by
way of the double-doored autoclave, fumigation chamber, or airlock which
is appropriately decontaminated after each use. After securing the outer
doors, personnel within the facility retrieve materials by opening the
interior doors of the autoclave, fumigation chamber, or airlock. These
doors are secured after materials are brought into the facility.
(h) Materials (for example, animals and clothing) not related to the
experiment being conducted are not permitted in the facility.
(i) Whenever possible, avoid using any glass items.
Sec. 627.17 Toxins.
The laboratory facilities, equipment, and procedures appropriate for
work with toxins of biological origin must reflect the intrinsic level
of hazard posed by a particular toxin as well as the potential risks
inherent in the operations performed. All toxins must be considered to
pose a hazard in an aerosol form. However, most toxins exert their
effects only after parenteral exposure or ingestion, and a few toxins
present a dermal hazard. In general, toxins of biological origin are not
intrinsically volatile. Thus, the laboratory safety precautions
appropriate for handling these materials closely parallel those for
handling infectious organisms. The requirements in this section for the
laboratory use of toxins of biological origin include the requirements
in Sec. 627.12(a) and the following:
(a) Vacuum lines. When vacuum lines are used with systems containing
toxins, they will be protected with a HEPA filter to prevent entry of
toxins into the lines (or sink drains when water aspirators are used).
(b) Preparation of concentrated stock solutions and handling closed
primary containers of dry toxins. Preparation of primary containers of
toxin stock solutions and manipulations of closed primary containers of
dry forms of toxins will be conducted--
(1) In a chemical fume hood, a glove box, or a biological safety
cabinet or equivalent containment system approved by the safety officer.
(2) While wearing eye protection if using an open-fronted
(3) Ensuring that gloves worn when handling toxins will be disposed
of as toxin waste, with decontamination if required.
(4) With the room door closed and posted with a universal biohazard
sign, or other sign, indicating that toxin work is in progress.
Extraneous personnel shall not be permitted in the room during
(5) Ensuring that toxins removed from hoods or biological safety
cabinets are double-contained during transport.
(6) After verification of hood or biological safety cabinet inward
made by the user before initiating work.
(7) Within the operationally effective zone of the hood or
biological safety cabinet.
(8) Ensuring that nondisposable laboratory clothing is
decontaminated before release for laundering.
(9) Ensuring that all individuals who handle toxins wash their hands
upon each exit from the laboratory.
(10) With two knowledgeable individuals present whenever more than
an estimated human lethal dose is handled in a syringe with a needle.
Each must be familiar with the applicable procedures, maintain visual
contact with the other, and be ready to assist in the event of an
(c) Manipulations with open containers of dry forms of toxins.
Handling dry forms of toxins in uncovered containers (for example,
during weighing) will be performed following the requirements stated in
Secs. 627.12(a), 627.17 (a) and (b), and the following:
(1) Manipulations will be conducted in a HEPA filtered chemical fume
hood, glove box, or biological safety cabinet. In addition the exhaust
may be charcoal filtered if the material is volatile.
(2) When using an open-fronted fume hood or biological safety
cabinet, protective clothing, including gloves and a disposable long-
sleeved body covering (gown, laboratory coat, smock, coverall, or
similar garment) will be worn so that hands and arms are completely
covered. Eye and approved respiratory protection is also required. The
protective clothing will not be worn outside of the laboratory and will
be disposed of as solid toxin waste.
(3) Before containers are removed from the hood, cabinet, or glove
box, the exterior of the closed primary container will be decontaminated
and placed in a clean secondary container.
(4) When toxins are in use, the room will be posted to indicate
``Toxins in Use--Authorized Personnel Only.'' Any special entry
requirements will be posted on the entrance(s) to the room.
(5) All operations will be conducted with two knowledgeable
individuals present. Each must be familiar with the applicable
procedures, maintain visual contact with the other, and be ready to
assist in the event of an accident.
(6) Individuals handling toxins will wash their hands upon leaving
(d) Additional considerations of specific toxin properties. The
following requirements are in addition to the requirements stated in the
paragraphs above. Determine whether the material fits Sec. 627.17 (b) or
(c), and complies with the appropriate section and the following when
(1) When handling dry forms of toxins that are electrostatic--
(i) Do not wear gloves (such as latex) that help to generate static
(ii) Use glove bag within a hood or biological safety cabinet, a
glove box, or a class III biological safety cabinet.
(2) When handling toxins that are percutaneous hazards (irritants,
necrotic to tissue, or extremely toxic from dermal exposure)--
(i) Gloves will be selected that are known to be impervious to the
toxin and the diluent (when applicable) for the duration of the
(ii) Disposable laboratory clothing will be worn, left in the
laboratory upon exit, and disposed of as solid toxin waste.
(e) Aerosol exposures. The requirements found in Sec. 627.17 (a) and
(b) will be complied with plus the following:
(1) Chambers, nose-only exposure apparatus, and generation system
must be placed inside a fume hood, glove box, or a Class III biological
safety cabinet. Glove boxes and Class III biological safety cabinets
will have HEPA filters on both inlet and outlet air ports.
(2) The atmosphere from within the exposure chamber will be HEPA
filtered before release inside the hood, glove box, or cabinet.
(3) All items inside the hood, glove box, or Class III biological
safety cabinet will be decontaminated upon removal. Materials such as
experimental samples that cannot be decontaminated directly will be
placed in a closed secondary container, the exterior of which will be
decontaminated and labeled appropriately. Animals will have any areas
exposed to toxin wiped clean after removal from the exposure apparatus.
(4) The interior of the hood, glove box, or cabinet containing the
chamber and all items will be decontaminated periodically, for example,
at the end of a series of related experiments. Until decontamintated,
the hood, box, or cabinet will be posted to indicate that toxins are in
use, and access to the equipment and apparatus restricted to necessary,
Sec. 627.18 Emergencies.
(a) Introduction. All laboratories will establish specific emergency
plans for their facilities. Plans will include liaison through proper
channels with local emergency groups and with community officials. These
plans will include both the building and the individual laboratories.
For the building, the plan must describe evacuation routes, facilities
for medical treatment, and procedures for reporting accidents and
emergencies. The plans will be reinforced by drills. Emergency groups
and community officials must be informed of emergency plans in advance
of any call for assistance. See AR 385-69.
(b) General emergency procedures. The following emergency procedures
will be followed for laboratory accidents or incidents--
(1) Using appropriate personal protection, assist persons involved,
remove contaminated clothing if necessary, decontaminate affected areas,
and remove personnel from exposure to further injury if necessary; do
not move an injured person not in danger of further harm. Render
immediate first aid if necessary.
(2) Warn personnel in adjacent areas of any potential hazards to
(3) In case of fire or explosion, call the fire department or
community fire brigade immediately. Follow local rules for dealing with
incipient fire. Portable fire extinguishers will be made available with
instructions for their use. Fire fighters responding to the fire scene
will be advised to wear a self-contained positive pressure breathing
appartus to protect themselves from toxic combustion by-products.
(4) Laboratories must be prepared for problems resulting from severe
weather or loss of a utility service. In the event of the latter, most
ventilation systems not supplied with emergency power will become
inoperative. All potentially hazardous laboratory work must stop until
service has been restored and appropriate action has been taken to
prevent personnel exposure to etiologic agents.
(5) In a medical emergency, summon medical help immediately.
Laboratories without a medical staff must have personnel trained in
first aid available during working hours.
(6) For small-scale laboratory accidents, secure the laboratory,
leave the area, and call for assistance.
(7) When handling mixed hazards (for example, a substance or mixture
that may be infectious and radioactive, or infectious and chemically
toxic), respond with procedures addressing the greater hazard first, and
then follow through with those for the lesser hazards to ensure that all
appropriate steps have been taken.
(c) Evacuation procedures. Building and laboratory evacuation
procedures will be established and communicated to all personnel.
(1) Emergency alarm system. (i) There will be a system to alert
personnel of an emergency that requires evacuation of the laboratory or
building. Laboratory personnel must be familiar with the location and
operation of alarm equipment.
(ii) Isolated areas (for example, cold, warm, or sterile rooms) will
be equipped with an alarm or communication system that can be used to
alert others outside to the presence of a worker inside, or to warn
workers inside of an emergency that requires evacuation.
(2) Evacuation routes will be established and an outside assembly
area for evacuated personnel must be designated. All individuals should
be accounted for.
(3) Shut-down and start-up procedures.
(i) Guidelines for shutting down operations during an emergency
evacuation will be available in writing. Those guidelines will include
procedures for handling any power failure emergency.
(ii) Written procedures will also be provided to ensure that
personnel do not return to the laboratory until the emergency is ended.
must also contain start-up operations for the laboratory.
(iii) All shut-down and start-up procedures will be available to
personnel and reviewed semiannually.
(4) All aspects of the building evacuation procedur will be tested
semiannually with practice drills.
(d) Spills. (1) All areas where work with etiologic agents is
performed will have designated personnel to respond to a spill and
provide protective apparel, safety equipment, and materials necessary to
contain and clean up the spill. Protective clothing requirements are
described in Sec. 627.21. Also, there will be supplies on hand to deal
with the spill consistent with the hazard and quantities of the spilled
(2) The safety officer will be notified immediately of all spills.
The first line supervisor will ensure that proper clean-up techniques
(3) Etiologic agents. (i) A program for responding to spills of
etiologic agents will be developed and implemented. This program will
contain emergency response procedures for a biological spill, which will
be tailored to the potential hazard of the material being used, the
associated laboratory reagents involved, the volume of material, and the
location of the materials within the laboratory. Generally, the spill
should be confined to a small area while minimizing the substance's
conversion to an aerosol. The spill will be chemically decontaminated or
neutralized, followed by a cleanup with careful disposal of the residue.
If the spilled material is volatile and noninfectious, it may be allowed
to evaporate but must be exhausted by a chemical hood or ventilation
(ii) When a mishap occurs that may generate an aerosol of etiologic
agents requiring BL-2 (or higher) containment, the room must be
evacuated immediately, the doors closed, and all clothing
decontaminated, unless the spill occurs in a class II or class III
biological safety cabinet. Sufficient time must be allowed for the
droplets to settle and the aerosols to be reduced by the air changes of
the ventilation system before decontaminating the area. The area will
then be decontaminated to prevent exposure to the infectious agents or
toxic substances. Reentry procedures to perform the decontamination will
conform to Sec. 627.18(e).
(iii) A spill of biohazardous material within a biological safety
cabinet requires a special response and cleanup procedure. Cleanup will
be initiated while the cabinet continues to operate, using an effective
chemical decontaminating agent. Aerosol generation during
decontamination and the escape of contaminants from the cabinet must be
prevented. Caution must be exercised in choosing the decontaminant,
keeping in mind that fumes from flammable organic solvents, such as
alcohol, can reach dangerous concentrations within a biological safety
(4) Combined radioactive and biological spills. (i) Both the
radiation protection officer (RPO) and the safety officer must be
notified immediately whenever there is a spill of radioactive biological
material, regardless of its size. Laboratory personnel may be expected
to clean up the spill. The RPO will direct the cleanup, in accordance
with the NRC license for the facility.
(ii) The spill will be cleaned up in a way that minimizes the
generation of aerosols and spread of contamination. All items used in
cleaning up the spill must be disposed of as radioactive waste.
(iii) Following cleanup, the area, affected protective clothing, and
all affected equipment and supplies must be surveyed for residual
radioactive contamination. All potentially affected areas and items that
are not disposable will be wipe-tested to verify that unfixed
radioactive contamination has been removed. If fixed contamination is
found, the RPO will determine the requirements for additional cleanup.
(e) Reentry procedures. This section applies when reentry is
necessary to clean up a spill outside of a hood or biological safety
cabinet, or to decontaminate or service engineering controls that have
failed or malfunctioned so that they do not provide the required
(1) When agents requiring BL-1 or BL-1 LS containment are involved,
the clothing requirements stated in Sec. 627.30
(a) or (b) as appropriate will be followed. Individuals will remove the
required protective clothing when finished and wash their hands before
proceeding to other tasks.
(2) When agents requiring BL-2, BL-2 LS, or toxin procedures and
containment are involved, personnel will be required to wear the
clothing described in Sec. 627.30 (c) or (d) as appropriate. Outer
protective clothing will be removed and left in the room before exiting
and personnel will wash their hands before proceeding on to other
(3) When agents requiring BL-3, or BL-3 LS containment are involved,
containers for sealing up inner protective clothing and decontaminant
will be placed at the room exit. Personnel will be required to wear the
clothing described in paragraph 4-10e. When exiting the area after
decontamination procedures, individuals will remove their outer layer of
protective clothing just before exiting the room. Once outside the room,
the inner layer of protective clothing (for example, coverall) will be
removed and placed in the container and the inner gloves will be
decontaminated before being removed and placed in the container.
Personnel will proceed directly to the shower facility to take a
complete shower before exiting the facility.
(4) When agents requiring BL-4 containment are involved, the
following applies as appropriate to the type of BL-4 facility:
(i) When a spill requiring clean-up is in an area designed for use
with personal positive pressure suits, the entry and exit procedures
will be those normally required to enter or exit the area.
(ii) When entering a nonsuit area where a spill of etiologic agent
has occurred outside the containment of a Class III biological safety
cabinet, personnel will wear the clothing as described in
Sec. 627.30(f). Before entry, decontamination areas will be established.
To accomplish this, two step-in decontamination pans with the
appropriate disinfectant will be set up [one just inside the room (where
the contamination exists) and the second immediately outside the room].
Immediately outside the room, there will also be a sealable container
suitable for sealing up the suit and any air lines (if used).
(iii) When exiting the room, suited individuals will place all
equipment and other items in autoclaves or disinfectant, step into the
disinfectant pan, and wash down the exterior of their suits with
appropriate disinfectant. When completed, the door to the room will be
opened and the individual will step through the doorway into the second
disinfectant pan. The suit will be thoroughly rinsed with disinfectant
again before moving toward the exit from the facility. The suit (but not
the respirator) will be placed in the provided container. The individual
will proceed through another doorway before removing the respirator and
placing it in a closed container for decontamination. The individual
will then proceed directly to the shower area and take a full shower
before exiting the area. In case they are needed, personnel will be
standing by ready to render assistance. Suited individuals will be
visually observed, if possible. When visual observation is not possible,
a communications system is required.
(f) Mishap reports and investigations. (1) Each institution must
have a defined system for reporting laboratory injuries, illnesses, and
mishaps, as well as for investigating them. These events will be
documented and reported to the appropriate safety, supervisory, and
occupational health personnel. Those organizations subject to the
regulations promulgated by the OSHA will follow the specific
requirements for reporting injuries in the work place contained in those
regulations. The requirements stated in AR 385-69, State, and local
government requirements for similar reporting will be followed.
(2) Form(s) for recording mishaps will be available and completed
for all laboratory mishaps. Those reports must include a description of
the mishap and any factors contributing to it. In addition, a
description of any first aid or other health care given to the employee
will be included. Responsibility for completing these forms must be
clearly defined in the facility safety
manual. Mishaps will be reviewed periodically by the safety officer, the
safety committee, the employee health unit, or other appropriate
personnel. Individual reports or a summary must be sent, along with
recommended changes in laboratory procedure or policy, to the commander
or institute director. Policy or procedural changes must be implemented
if deemed necessary by the commander or institute director.
(3) Any mishaps with etiologic agents used under sponsorship of the
BDP that result in sero-conversion or a laboratory-acquired illness will
Sec. 627.19 Large-scale operations.
(a) Large-scale. In addition to the requirements stated in
Sec. 627.13, the following applies to research or production activities
involving viable etiologic agents in quantities greater than 10 liters:
(1) All large-scale operations will be conducted in facilities
described in Sec. 627.47.
(2) Cultures will be handled in a closed system.
(3) Sample collection, the addition of materials, and the transfer
of culture fluids shall be done in a manner which minimizes the release
of aerosols or contamination of exposed surfaces.
(4) A closed system or other primary containment equipment that has
contained viable organisms shall not be opened for maintenance or other
purposes unless it has been sterilized.
(5) SOPs will include a section describing and requiring a
validation of the process equipment's proper function.
(6) Scientists, technicians, equipment workers, and support
personnel with access to the large-scale production area during its
operation will be included in the medical surveillance program.
(b) BL-2--LS. In addition to the requirements stated in
Secs. 627.19(a) and 627.14, the following procedures will be employed
(1) Rotating seals and other mechanical devices directly associated
with the closed system used for the propagation and growth of viable
organisms shall be designed to prevent leakage or shall be fully
enclosed in ventilated housings that are exhausted through filters which
have efficiencies equivalent to HEPA filters or through other equivalent
(2) A closed system used for the propagation and growth of viable
organisms and other primary containment equipment used to contain
operations involving viable organisms shall include monitoring or
sensing devices that monitor the integrity of containment during
(3) Systems used to propagate and grow viable organisms shall be
permanently identified. This identification shall be used in all records
reflecting testing, operation, and maintenance and in all documentation
relating to the use of this equipment.
(c) BL-3--LS. In addition to the requirements stated in
Secs. 627.19(a) and 617.14, the following procedures apply:
(1) Personnel entry into the controlled area shall be through the
entry area specified in Sec. 627.47(c)(1).
(2) Persons entering the controlled area shall exchange or cover
their personal clothing with work garments such as jumpsuits, long
sleeved laboratory coats, pants and shirts, head cover, and shoes or
shoe covers. On exit from the controlled area, the work clothing may be
stored in a locker separate from that used for personal clothing, or
discarded for laundering. Clothing shall be decontaminated before
(3) Entry into the controlled area during periods when work is in
progress shall be restricted to those persons required to meet program
(4) Prior to entry, all persons shall be informed of the operating
practices, emergency procedures, and the nature of the work conducted.
(5) The universal biohazard sign shall be posted on entry doors to
the controlled area and all internal doors. The sign posted on the entry
doors to the controlled area shall include a statement of agents in use
and personnel authorized to enter.
(6) Equipment and materials required for the management of accidents
involving viable organisms shall be available in the controlled area.
(d) BL-4--LS. Guidelines for these operations are not established.
are needed, they must be established by the United States Army Surgeon
General or the NIH on an individual basis.
Sec. 627.20 Operations with radioactive material.
Operations that combine etiologic agents with radioactive material
present unique problems. When this is the case, the following apply:
(a) Radiation program. A radiation program meeting the requirements
of AR 385-11 and NRC licensing that allows the particular isotope and
its use are required. The requirements for acquisition, handling
procedures, labeling, storage, training, monitoring, and disposal will
be described in an organization policy document.
(b) Procedure approval. In addition to the required approvals for
work with etiologic agents, the RPO will approve all SOPs involving the
use of radioactive materials. Laboratory operators must be fully
trained, with annual training updates as required by the existing
(c) Special situations. (1) The laboratory waste must be segregated
as radioactive waste and disposed of as such after it has been
decontaminated. Do not mix nonradioactive waste with radioactive waste
as the disposal of radioactive waste is much more complex and expensive.
When RCRA-listed chemicals are mixed with radioactive waste, it becomes
``mixed waste'' for which there is currently no means of disposal.
(2) Activities conducted with radioisotopes should be confined to
the smallest number of areas or rooms consistent with requirements.
(3) Decontamination methods specific to etiologic agents will not
always remove radioactivity. Other methods, such as specialized
detergents and solvents designed for this use, should be employed to
remove residual radioactivity.