[Title 32 CFR C]
[Code of Federal Regulations (annual edition) - July 1, 2002 Edition]
[Subpart C - Operational Requirements]
[From the U.S. Government Printing Office]

                   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 
(when applicable).
    (vi) Hazardous and infectious waste disposal, handling, and 
minimization procedures.

[[Page 539]]

    (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).

[[Page 540]]

    (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 
etiologic agent.
    (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 
a wet

[[Page 541]]

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 
before disposal.
    (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 
date stored.
    (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

[[Page 542]]

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 
of aerosols.
    (2) No infectious mixtures will be prepared by bubbling air through 
a liquid.
    (3) Pipettes.
    (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 
cabinet system.
    (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 
pipetting permitted.
    (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 
discarded materials.
    (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

[[Page 543]]

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) 
and 617.12(b).
    (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 
infected animals.
    (4) All wastes from laboratories and animal rooms are decontaminated 
before disposal.
    (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.

[[Page 544]]

    (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 
kept closed.
    (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 
Sec. 627.56.
    (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-
support system.
    (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

[[Page 545]]

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 
containment system.
    (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 
airflow is

[[Page 546]]

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 
the laboratory.
    (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.

[[Page 547]]

    (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, 
authorized personnel.

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 
their safety.
    (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. 
Those procedures

[[Page 548]]

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 
are employed.
    (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

[[Page 549]]

(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

[[Page 550]]

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 
be reported.

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 
for BL-2--LS:
    (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 
treatment devices.
    (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 
support needs.
    (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. 
If these

[[Page 551]]

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.