**The NIH rDNA Guidelines Explained**

The next few pages were written to extract the essence of the NIH
guideline requirements and put them into readable form. Since many
details are omitted, the actual guidelines should be consulted when in
doubt (<http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html>).
Plant, whole animals, and human experiments are given special treatment
in the guidelines (and in this explanation).

**PREREQUISITES**

To make sense of the following, the reader should know the basic ideas
behind Biosafety Levels 1 through 4. A summary table giving the
characteristics of the four laboratory Biosafety Levels is on page 6.
Animal Biosafety levels are summarized on page 7.

**WHAT IS RECOMBINANT DNA (rDNA)?**

It is either a) DNA constructed *in vitro* from separate DNA segments
that can replicate and/or express a biologically active polynucleotide
or polypeptide *in vivo*, or b) synthetic DNA that has the potential of
generating a hazardous product *in vivo.*

**WHAT ARE THE RAC AND THE OBA?**

The NIH OBA (Office of Biotechnology Activities) is an administrative
arm responsible for carrying out the orders of the NIH Director with
regard to recombinant DNA, genetic testing, and xenotransplantation. An
advisory committee is involved in establishing policies for each of
these fields. For recombinant DNA the committee is called the
Recombinant DNA Advisory Committee or the "RAC".

**REGISTRATION**

The NIH requires all labs working with recombinant DNA to register with
their local Institutional Biosafety Committee (IBC). The guidelines
distinguish among the five kinds of registrations. The type depends on
the potential hazard of the work. More hazardous means more approvals
are needed. The five types are:

-   Work that cannot begin until there is NIH and IBC approval (NIH
    Guidelines Sections III-A and III-B), this is the most dangerous
    level and it is \[**TABOO**\] (page 2)

-   Work that cannot begin until there is IBC and RAC review (Sections
    III-C). \[**WAIT and WAIT**\] (page 2) This tends to be sensitive
    and potentially dangerous. Human Gene Transfer studies are in this
    category (page5).

-   Work that cannot begin until there is IBC approval (III-D), there is
    usually a short \[**WAIT**\] (page 2).

-   Work can begin when the IBC is notified (Section III-E) \[**NO
    WAITING**\] (page 4)

-   Work that is Exempt form NIH Guidelines (Section III-F). No approval
    is needed but the work should be registered with the IBC \[**NO
    WAITING, EXEMPT**\] (page 4)

**"TABOO"**

(NIH approval and IBC Approval Required)

(from NIH Guidelines Sections III-A and III-B)

-   Making drug resistant constructs of microorganisms if they
    compromise the drug's therapeutic potential

-   Making constructs that synthesize vertebrate toxins with an LD~50~
    of 100ng/Kg or less.

-   Making constructs in *E. coli* that synthesize vertebrate toxins
    that are "lethal" between 100ng/Kg and 100µg/Kg

**"WAIT AND WAIT"**

(NIH Review and IBC Approval Required)

(from Section IIIC)

Studies in which genes are transferred into humans must be submitted to
the NIH OBA for review. If the OBA finds the study to be "novel" it will
place the study on the next RAC meeting agenda. IBC approval must wait
for the RAC's review. If, on the other hand, OBA does not deem the study
to be "novel", IBC can act immediately.

**"WAIT"**

(IBC Approval Needed Before Starting)

These studies are examined by the IBC with an eye to recommending safe
procedures and containment. To reach a conclusion it is often useful to
classify the risk associated with a proposed study according to the risk
associated with the organism(s) to be used. A convenient classification
tool is the concept of "risk group".

**Risk Groups**

The NIH classifies biological agents into four Risk Groups according to
their human pathogenecity (see NIH Guidelines, Section II-A-1)

· Risk Group 1 -- not associated with disease in healthy adults

· Risk Group 2 -- associated with disease that is rarely serious and for
which

therapeutic or preventive options are *often* available

· Risk Group 3 - associated with serious or lethal disease for which
therapeutic or

preventive options *may* be available

· Risk Group 4 -- associated with serious or lethal disease for which
therapeutic or

preventive options *not usually* available

Appendix B in the NIH Guidelines lists a number of biologic agents
according to their Risk Group.

In general, the Risk Group determines the Biosafety Level needed: for
instance a Risk Group 3 agent is usually studied in a BL3 or BL3-N
(animal) or BL3-P (plants) lab.

Those agents not listed in Risk Group (RGs) 2, 3 and 4 are not
automatically or implicitly I RG1: a risk assessment must be conducted
based on the known and potential properties of the agents and their
relationship to agents that are listed.

The table on the next page summarizes the NIH recommendations for
Biosafety Level according to risk group.

**"NO WAITING"**

Just Notify IBC Before Work Starts

Notification is necessary because all recombinant DNA studies must be
registered with Clemson University.

These studies involve "Low Hazard" recombinant DNA (Section III-E)

· Non-pathogenic prokaryotes or non-pathogenic lower eukaryotes (use
BL1)

· Recombinant DNA with less than 2/3 of a eukaryotic viral genome (and
no

> helper) used exclusively in tissue culture (BL1 recommended)

· Some host *Plants* carrying recombinant DNA. BL1-P level containment
is

recommended when the host is:

a non-noxious weed,

a plant or microorganism thought not to damage the ecosystem,

· Some other host *Plants* carrying recombinant DNA. BL3-P or BL1-P+
level

containment is recommended when:

the host is a noxious weed,

the introduced DNA is the complete genome of an infectious agent

known to normally exist in the U.S.

the host is a plant or microorganism that may damage ecosystems,

the host is a plant with recombinant DNA from foreign microorganisms

that is thought to be safe for the ecosystem.

**No Waiting - Exempt**

**Exempt Recombinant DNA** (Section III-F and Appendix C)

"Exempt" form NIH guidelines means that work with these constructs need
not be approved by the IBC. However, Clemson University requires that
all recombinant work, exempt or non-exempt, be registered. Thus, it is
Clemson University policy to insist that investigators using
[any]{.underline} recombinant DNA register with the IBC. The exempt work
can be performed at BL1.

+------------------------+---------------------------------------------+
| -   Gene transfer into |                                             |
|     Human subjects     |                                             |
|     (RAC review        |                                             |
|     [p                 |                                             |
| rocess^1^]{.underline} |                                             |
|     is required before |                                             |
|     IBC can approve    |                                             |
|     this kind of       |                                             |
|     study).            |                                             |
|                        |                                             |
| > (Section III-C-1)    |                                             |
+------------------------+---------------------------------------------+
| -   Recombinant        |                                             |
|     organisms cultured |                                             |
|     in volumes greater |                                             |
|     than 10 liters     |                                             |
|     (Section III-D-6). |                                             |
+------------------------+---------------------------------------------+
| -   Pathogen Hosts     |                                             |
|     (Section III-D-1): |                                             |
|     Check Appendix B   |                                             |
|     to determine their |                                             |
|     Risk Group - then: |                                             |
+------------------------+---------------------------------------------+
|                        | Risk Group 2 host. . . . . . . . . . . . .  |
|                        | . . . . BL2, BL2-N                          |
+------------------------+---------------------------------------------+
|                        | Risk Group 3 host. . . . . . . . . . . . .  |
|                        | . . . . BL3, BL3-N                          |
+------------------------+---------------------------------------------+
|                        | Risk Group 4 host. . . . . . . . . . . . .  |
|                        | . . . . BL4, BL4-N                          |
+------------------------+---------------------------------------------+
| -   Pathogen DNA       |                                             |
|     Source into        |                                             |
|     Non-Pathogen Host  |                                             |
|     (Section III-D-2): |                                             |
|     Check Appendix B   |                                             |
|     to determine their |                                             |
|     Risk Group - then: |                                             |
+------------------------+---------------------------------------------+
|                        | Risk Group 2 or 3 source. . . . . . . . . . |
|                        | . . BL2                                     |
+------------------------+---------------------------------------------+
|                        | Risk Group 4 source. . . . . . . . . . . .  |
|                        | . . . .BL2, **IF** the pathogen genome is   |
|                        |                                             |
|                        | defective                                   |
+------------------------+---------------------------------------------+
|                        | Risk Group 4 source. . . . . . . . . . . .  |
|                        | . . . .BL4, **Otherwise**                   |
+------------------------+---------------------------------------------+
| -   Animal Virus DNA   |                                             |
|     source into Tissue |                                             |
|     Culture (Section   |                                             |
|     III-D-3): Check    |                                             |
|     Appendix B to      |                                             |
|     determine their    |                                             |
|     Risk Group- then:  |                                             |
+------------------------+---------------------------------------------+
|                        | Risk Group 2 virus source. . . . . . . . .  |
|                        | . . .BL2                                    |
+------------------------+---------------------------------------------+
|                        | Risk Group 3 virus source. . . . . . . . .  |
|                        | . . .BL3                                    |
+------------------------+---------------------------------------------+
|                        | Risk Group 4 virus source. . . . . . . . .  |
|                        | . . .BL4                                    |
+------------------------+---------------------------------------------+
| -   Transgenic         |                                             |
|     *Animal*^2^ Host   |                                             |
|     (Section III-D-4): |                                             |
+------------------------+---------------------------------------------+
|                        | Anything but \> 2/3 eukaryotic virus        |
|                        |                                             |
|                        | genome. . . . . . . . . . . . . . . . . . . |
|                        | . . . . . . . BL1-N (but IBC can boost this |
|                        | level                                       |
|                        |                                             |
|                        | based on the pathogenicity of the           |
|                        |                                             |
|                        | source organism)                            |
+------------------------+---------------------------------------------+
|                        | Viral vectors that don\'t transmit. . . . . |
|                        | . . . BL1-N                                 |
+------------------------+---------------------------------------------+
|                        | Everything else is a special case. . . . .  |
|                        | . .IBC decides                              |
+------------------------+---------------------------------------------+
| -   Modified           |                                             |
|     microorganisms     |                                             |
|     into *Animals*:    |                                             |
|     (Section III-D-4): |                                             |
+------------------------+---------------------------------------------+
|                        | **Any** viable rDNA modified organism ≥BL2, |
|                        | BL2-N                                       |
+------------------------+---------------------------------------------+
| -   rDNA into          |                                             |
|     *Animals*          |                                             |
+------------------------+---------------------------------------------+
|                        | **Any** rDNA that (except pieces \>⅔        |
|                        |                                             |
|                        | eukaryotic viral genome) . . . . . . . . .  |
|                        | . . . .BL1, BL1-N                           |
+------------------------+---------------------------------------------+
|                        | Everything else. . . . . . . . . . . . . .  |
|                        | . . . . . . IBC decides                     |
+------------------------+---------------------------------------------+
| -   Whole *Plants*:    |                                             |
|     (Section III-D-5)  |                                             |
+------------------------+---------------------------------------------+
|                        | Exotic^3^ pathogen hosts that can dam-      |
|                        |                                             |
|                        | age the ecosystem. . . . . . . . . . . . .  |
|                        | . . . . . BL3-P or BL2-P+                   |
+------------------------+---------------------------------------------+
|                        | Plants with transmissible DNA from          |
|                        |                                             |
|                        | exotic pathogens that can damage the        |
|                        |                                             |
|                        | ecosystem. . . . . . . . . . . . . . . . .  |
|                        | . . . . . . . .BL3-P or BL2-P+              |
+------------------------+---------------------------------------------+
|                        | Transmissible exotic pathogen hosts         |
|                        |                                             |
|                        | BL4-P                                       |
+------------------------+---------------------------------------------+
|                        | Toxin DNA into plants. . . . . . . . . . .  |
|                        | . . . . BL3-P                               |
+------------------------+---------------------------------------------+
|                        | Insect Pathogen DNA that can dam-           |
|                        |                                             |
|                        | age the ecosystem. . . . . . . . . . . . .  |
|                        | . . . . . BL3-P or BL2-P+                   |
+------------------------+---------------------------------------------+
| -   Genes coding for   |                                             |
|     vertebrate         |                                             |
|     **Toxins**         |                                             |
|     (Appendix F)       |                                             |
+------------------------+---------------------------------------------+
|                        | LD~50~ \< 100 ng/kg . . . . . . .Requires   |
|                        | NIH & IBC approval                          |
+------------------------+---------------------------------------------+
|                        | 100 ng/kg \< LD~50~ \< 100 μg/kg . . . . .  |
|                        | . . Requires IBC approval & NIH notifi-     |
|                        |                                             |
|                        | cation. (except in E. coli-see below)       |
+------------------------+---------------------------------------------+
|                        | 100 ng/kg \< LD~50~ \< 1 μg/kg . . . . . .  |
|                        | . .BL2 if in E. coli                        |
+------------------------+---------------------------------------------+
|                        | 1 μg/kg \< LD~50~ \< 100 μg/kg. . . . . . . |
|                        | . BL1 if in E. coli                         |
+------------------------+---------------------------------------------+
|                        |                                             |
+------------------------+---------------------------------------------+

^\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_^

^1^RAC can either take no action and transmit the protocol to the FDA or
call for a full public review at one of its quarterly meetings.

^2^The purchase or transfer of transgenic rodents is exempt from the NIH
guidelines.

^3^\"Exotic\" plant pathogens are defined as those not known to occur
naturally in the US.

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Some Exempt Classes are:

· DNA vaccines encoding epitopes from microbiological sources are
generally

exempt from the NIH Guidelines, even in human studies. This unusual

> exemption is found in Appendix VI-A.

· Recombinant DNA outside of living or viral organisms,

· Recombinant DNA that cannot replicate or express in vivo,

· DNA from a [single]{.underline} nonchromosomal or viral source,

·The DNA source organism and the host organism are the same organism
(but not

released into the environment)

· The DNA source organism and the host organism normally exchange DNA

(organisms that normally exchange are listed in Appendix A)

· DNA that does "not present a significant risk to health or the

environment\..."

·The recombinant DNA is used exclusively in tissue culture and has \<1/2

eukaryotic viral genome. *There are exceptions to this rule (Appendix
C-I-*

*A). Check with the Biosafety Officer.*

· Experiments using an *E. coli* host vector system in which the host
does not

contain conjugation proficient plasmids. There are some restrictions on

> the vectors used (Appendix C-II). BL1 containment is suggested.

·Experiments with *Saccharomyces* host-vector systems. There are some

restrictions (Appendix C-III). BL1 containment is suggested.

· Experiments with *Bacillus subtilis* or *B*. *icheniformis*
host-vector systems and in

which reversion to spore formation is \<10^-7^. There are some other

restrictions (Appendix C-IV). BL1 containment is suggested.

**"THREE SPECIALIZED GUIDELINE SECTIONS"**

The NIH Guidelines recognize three non-laboratory classes of Biosafety
containment and procedures; those in which genes are transferred into
humans (Appendix M); those for plants (BL1-P through BL4-P, Appendix P)
and those for animals (BL1-N through BL4-N, Appendix Q).

 **HUMANS**

All Human Gene Transfer protocols are currently considered experimental.
IBC has not yet established a Human Gene Therapy Advisory Committee to
deal with human Gene Transfer studies.

IBC approval must await RAC (NIH Recombinant Advisory Committee) action.
Depending on whether the study is deemed "novel" the RAC can either
schedule a full examination of the protocol at one of its quarterly
meetings or recommend sole FDA review.

Overcoming the regulatory hurdles involved in gaining approval for a
human gene transfer study is not a task for the faint of heart. Beyond
approvals from the Food and Drug Administration one has to get approval
form the local Institutional Review Board (IRB) and the Biosafety
Committee. In addition, RAC will evaluate novel protocols although it
does not have approval power. These evaluations often involve the
Principal Investigator's appearance in Bethesda and aggressive
questioning by members of the RAC.

For most people this process can be intimidating. Best to get help early
through a commercial sponsor or a consultant.

 **ANIMALS**

Animal Biosafety levels are normally used to cover large animals such as
cattle, swine, horses, poultry etc. IBC tends to use the same
designations when considering safe practices with lower animals
including rodents.

All animal experiments must be reviewed and approved by a local
Institutional Animal Care and Use Committee (IACUC). These committees
act under the US Department of Agriculture regulations. At Clemson
University, the responsible committee is designated the Animal Research
Committee (ARC).

 **PLANTS**

Plant Biosafety levels are necessary when research plants are too big,
too many or have growth requirements that cannot be covered by the
standard Biosafety Levels. The plant guidelines cover microorganisms and
small "animals", particularly insects, such as arthropods.
Plant-associated microorganism include pathogenic viroids, virusoids,
viruses, bacteria, fungi, protozoans, as well as benign or beneficial
microorganisms known to be associated with plants e.g. (Rhizobium,
Pseudomonas).

When studies covered under the plant appendix are being discussed, IBC
will include an expert in plant pests or containment.

It is of interest that the plant guidelines are not designed to directly
protect humans from plant related recombinant DNA (i.e., the agents
covered pose virtually no threat to humans or higher animals). Rather
the guidelines are in place to protect the general ecosystem form
serious disruption. Thus, procedures are designed to limit the spread of
novel organisms form the experimental facility, not to protect the
workers.

**SUMMARY OF LABORATORY BIOSAFETY LEVELS**

+--------------+-----------+-------------+-------------+-------------+
| **Biosafety  | **Risk    | **Practices | **Safety    | *           |
| Level**      | Group**   | and         | Equipment** | *Examples** |
|              |           | T           |             |             |
|              |           | echniques** |             |             |
+--------------+-----------+-------------+-------------+-------------+
| BL1          | I         | Standard    | None:       | *E. Coli*   |
|              | ndividual | Micr        | primary     | K12,        |
| **Basic      | risk: LOW | obiological | containment | Continuous  |
| Laboratory** |           | Practices.  | provided by | culture of  |
|              | Community |             | adherence   | cell lines, |
|              | risk: LOW |             | to standard | e.g., short |
|              |           |             | lab         | term, long  |
|              |           |             | practices   | term        |
|              |           |             | during open | culture of  |
|              |           |             | bench       | most        |
|              |           |             | operations. | non-primate |
|              |           |             |             | mammalian   |
|              |           |             |             | tissue.     |
+--------------+-----------+-------------+-------------+-------------+
| BL2          | I         | *[Level 1   | Partial     | Hepatitis B |
|              | ndividual | practices   | containment | Virus,      |
| **Basic      | risk:     | plus:]{.    | (i.e.,      | *Salmonella |
| Laboratory** | MODERATE  | underline}* | Class I or  | typhi*,     |
|              |           |             | II          | Short term, |
| With         | Community | Lab coats,  | Biosafety   | long term   |
| Biosafety    | risk: LOW | autoclaving | cabinets    | culture of  |
| cabinets and |           | of all      | for         | human tumor |
| other        |           | biological  | procedures  | cell lines, |
| physical     |           | waste       | which       | culture of  |
| containment  |           | preferred,  | produce     | lymphoid    |
| devices as   |           | limited     | aerosols.   | lines       |
| required.    |           | access,     |             | carrying    |
|              |           | biohazard   |             | inducible   |
|              |           | warning     |             | EBV, many   |
|              |           | signs on    |             | common      |
|              |           | doors and   |             | human       |
|              |           | equipment.  |             | pathogens.  |
+--------------+-----------+-------------+-------------+-------------+
| BL3          | I         | *[Level 2   | Partial     | Yellow      |
|              | ndividual | practices   | containment | fever, *M.  |
| *            | risk:     | plus:]{.    | equipment   | tub         |
| *Containment | HIGH      | underline}* | used for    | erculosis*, |
| Laboratory** |           |             | [all]{      | Short term  |
| with special | Community | Special     | .underline} | culture of  |
| engineering  | risk:     | protective  | ma          | tissue from |
| and design   | MODERATE  | clothing,   | nipulations | non-human   |
| features.    |           | controlled  | of          | primates    |
|              |           | access      | infections  | until       |
|              |           | through     | materials,  | cultures    |
|              |           | entrance    | directional | are known   |
|              |           | room,       | airflow.    | to be free  |
|              |           | biological  |             | of          |
|              |           | waste must  |             | H           |
|              |           | be          |             | erpes-virus |
|              |           | autoclaved, |             | *simiae*    |
|              |           | preferably  |             |             |
|              |           | in          |             | (B. virus)  |
|              |           | facility.   |             |             |
+--------------+-----------+-------------+-------------+-------------+
| BL4          | I         | *[Level 3   | Maximum     | Eb          |
|              | ndividual | practices   | containment | ola-Marburg |
| **Maximum    | risk:     | plus:]{.    | equipment   | Virus.      |
| Containment  | HIGH      | underline}* | (i.e.,      | Propagation |
| Laboratory** |           |             | Class III   | of Herpes   |
|              | Community | Entrance    | Biosafety   | virus       |
|              | risk:     | through     | cabinet or  | *simiae.*   |
|              | HIGH      | change      | partial     |             |
|              |           | room.       | containment | Smallpox.   |
|              |           | Complete    | in          |             |
|              |           | change of   | combination |             |
|              |           | clothing    | with        |             |
|              |           | from street | full-body,  |             |
|              |           | to          | a           |             |
|              |           | laboratory  | ir-supplied |             |
|              |           | gear,       | positi      |             |
|              |           | shower at   | ve-pressure |             |
|              |           | exit. All   | personnel   |             |
|              |           | wastes      | suit) used  |             |
|              |           | dec         | for all     |             |
|              |           | ontaminated | procedures  |             |
|              |           | on exit     | and         |             |
|              |           | from        | activities. |             |
|              |           | facility.   |             |             |
+--------------+-----------+-------------+-------------+-------------+

**SUMMARY OF ANIMAL FACILITY BIOSAFETY LEVELS**

  --------- ------------------ ------------------ ------------------- -----------------
  **BSL**   **Agents**         **Practices**      **Safety Equipment  **Facilities
                                                  (Primary            (Secondary
                                                  Barriers)**         Barriers)**

  1         Not known to       Standard animal    As required for     Standard animal
            consistently cause care and           normal care of each facility. No
            disease in healthy management         species.            recirculation of
            human adults.      practices,                             exhaust air.
                               including                              Directional air
                               appropriate                            flow recommended.
                               medical                                Handwashing sink
                               surveillance                           recommended.
                               programs.                              

  2         Associated with    ABSL-1 practices   ABSL-1 equipment    ABSL-1 facility
            human disease.     plus: Limited      plus primary        plus: Autoclave
            Hazard:            access. Biohazard  barriers:           available.
            percutaneous       warning signs.     containment         Handwashing sink
            exposure,          Sharps             equipment           available in the
            ingestion, mucous  precautions.       appropriate for     animal room.
            membrane exposure. Biosafety manual.  animal species;     Mechanical cage
                               Decontamination of PPES: laboratory    washer used.
                               all infectious     coats, gloves, face 
                               wastes and of      and respiratory     
                               animal cages prior protection as       
                               to washing.        needed.             

  3         Indigenous or      ABSL-2 practices   ABSL-2 equipment    ABSL-2 facility
            exotic agents with plus: Controlled   plus: Containment   plus: Physical
            potential for      access.            equipment for       separation from
            aerosol            Decontamination of housing animals and access corridors.
            transmission;      clothing before    cage dumping        Self-closing,
            disease may have   laundering. Cages  activities. Class I double-door
            serious health     decontaminated     or II BSCs          access. Sealed
            effects.           before bedding     available for       penetrations.
                               removed.           manipulative        Sealed windows.
                               Disinfectant foot  procedures          Autoclave
                               bath as needed.    (inoculation,       available in
                                                  necropsy) that may  facility.
                                                  create infectious   
                                                  aerosols. PPEs:     
                                                  appropriate         
                                                  respiratory         
                                                  protection          

  4         Dangerous/exotic   ABSL-3 practices   ABSL-3 equipment    ABSL-3 facility
            agents that pose   plus: Entrance     plus: Maximum       plus: Separate
            high risk of life  through change     containment         building or
            threatening        room where         equipment (i.e.,    isolated zone.
            disease; aerosol   personal clothing  Class III BSC or    Dedicated supply
            transmission, or   is removed and     partial containment and exhaust,
            related agents     laboratory         equipment in        vacuum and
            with unknown risk  clothing is put    combination with    decontamination
            of transmission.   on; shower on      full body,          systems. Other
                               exiting. All       air-supplied        requirements
                               wastes are         positive-pressure   outlined in
                               decontaminated     personnel suit)     BMBL-4.
                               before removal     used for all        
                               from the facility. procedures and      
                                                  activities.         
  --------- ------------------ ------------------ ------------------- -----------------