Guidance Documnet for the completion of BioSafety Plan

Form Approved
OMB Control No. 0920-0576
Exp. Date xx/xx/2020

Select Agents and
Toxins Biosafety/
Biocontainment Plan
Guidance
(7 CFR Part 331.12, 9 CFR Part 121.12, 42 CFR
Part 73.12)

(March 2017)
Centers for Disease Control and Prevention (CDC)
Division of Select Agents and Toxins (DSAT)
Animal and Plant Health Inspection Services (APHIS)
Agriculture Select Agent Services (AgSAS)

Table of Contents
Change/Highlight Section ...................................................................................................................................3
Introduction ........................................................................................................................................................3
Definitions ..........................................................................................................................................................3
Biosafety/Biocontainment Plan Provision Requirements ..................................................................................4
Hazardous Characteristics of Select Agents and Toxins .................................................................................4
Safeguards for Protecting Against Exposure to Select Agents and Toxins .....................................................5
Personal protective equipment (PPE) and other safety equipment ..........................................................5
Containment equipment ............................................................................................................................6
Engineering controls and other facility safeguards ....................................................................................6
Risk Management Process Controls ...........................................................................................................7
Enclosure and Isolation ..............................................................................................................................8
Disinfection, Decontamination or Destruction of Select Agent and Toxin ....................................................8
Section 12(a)(3) states that the biosafety/biocontainment plan must contain.............................................8
Handling Select Agents and Toxins in Shared Spaces...................................................................................10
Appendix I: Hazardous Characteristics of Select Agents and Toxins ............................................................11
Appendix II: Example Procedural Risks.............................................................................................................21

Public reporting burden: Public reporting burden of this collection of information is estimated to average 30 minutes
per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining
the data needed, and completing and reviewing the collection of information. An agency may not conduct or sponsor,
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number. Send comments regarding this burden estimate or any other aspect of this collection of information, including
suggestions for reducing this burden to CDC/ATSDR Reports Clearance Officer; 1600 Clifton Road NE, MS D-74, Atlanta,
Georgia 30329; ATTN: PRA (0920-0576).

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Biosafety Guidance Document

Change/Highlight Section
Revisions: This is a living document subject to ongoing improvement. Feedback or suggestions for
improvement from entities registered with the Federal Select Agent Program, as well as the general public,
are welcomed. Submit comments directly to the Federal Select Agent Program at:
DSAT: [email protected]
AgSAS: [email protected]

Introduction
This document is intended to provide guidance and assist entities in developing and implementing a written
biosafety/biocontainment plan, as required by section 12 of the select agent regulations (7 C.F.R. Part

331, 9 C.F.R. Part 121, and 42 C.F.R. Part 73). This template summarizes current regulatory and
procedural criteria for registered entities and provides examples for verifying compliance. It does not add
to, delete from, or change current regulatory requirements or standards. For entities registered for Tier 1
select agents and toxins that require an occupational health program, reference the Occupational Health
Program Guidance for more information. It should be noted that information regarding an occupational
health program may be incorporated into the biosafety plan and that two plans are not required.
There are resources available to assist entities in development of biosafety/biocontainment plans such as
the “Biosafety in Microbiological and Biomedical Laboratories (BMBL),” “NIH Guidelines for Research
Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines),” “Containment Facilities and
Safeguards for Exotic Plant Pathogens and Pests” (Robert P. Kahn and S.B. Mathur eds., 1999; copies
available upon request at [email protected] or [email protected]), and “A Practical Guide to
Containment: Greenhouse Research with Transgenic Plants and Microbes” (Patricia L. Traynor ed., 2001;
copies available upon request at [email protected] or [email protected]). These resources can be used as
guidance to assist in the development of the biosafety/biocontainment plan. However, entities may use
other biosafety/biocontainment guidelines and regulations when developing and implementing a written
plan. It should be noted that the Federal Select Agent Program inspects registered entities in accordance
with these currently nationally recognized standards.

Definitions
As used in this document the following terms have the following meanings:
Decontamination – Disinfection or sterilization of articles contaminated with toxins or agents to make the
articles safe for use or disposal.
Disinfection – The elimination of nearly all recognized pathogenic microorganisms but not necessarily all
microbial forms (e.g., bacterial spores) on inanimate objects.
Exposure – Any event which results in any person in a registered entity facility or laboratory not being
appropriately protected in the presence of an agent or toxin. This may include reasonably anticipated skin,
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Biosafety Guidance Document

eye, mucous membrane, or parenteral contact with blood or other potential infectious materials that may
result from the performance of a person’s duties.
Risk – The potential for an adverse outcome assessed as a function of threats, vulnerabilities, and
consequences associated with an incident, event, or occurrence.
Risk Assessment – The process of evaluating the risk(s) arising from a hazard(s), taking into account the
adequacy of any existing controls and deciding whether or not the risk(s) is acceptable.
Sterilization – Any item, device, or solution is considered to be sterile when it is completely free of all living
microorganisms and viruses. The definition is categorical and absolute (i.e., an item is either sterile or it is
not). A sterilization procedure is one that kills all microorganisms, including high numbers of bacterial
endospores.

Biosafety/Biocontainment Plan Provision Requirements
Hazardous Characteristics of Select Agents and Toxins
It is important that the biosafety/biocontainment plan contain the hazardous characteristics of each agent
or toxin listed on the entity's registration and the biosafety/biocontainment risk associated with laboratory
procedures related to the select agent or toxin.
To assist with identifying the hazardous characteristics of each agent or toxin and the biosafety risk
associated with laboratory procedures related to the select agent or toxin, the BMBL is an excellent
reference and includes agent summary statements that describe the hazards, recommended precautions,
additional risks, and levels of containment appropriate for handling select agents and toxins in the
laboratory. The BMBL also states that HEPA filtration of exhaust air should be required when working with
BSL-4 select agents and toxins as well as:





Reconstructed 1918 influenza virus
Rift Valley fever virus
Venezuelan equine encephalitis virus
Highly pathogenic avian influenza virus

The NIH Guidelines provides risk assessment, physical containment, and biological containment provisions
relating to genetic elements, recombinant nucleic acids and recombinant organisms of select agents and
toxins.
In addition, AgSAS has developed Guidelines for Avian Influenza Viruses to assist individuals and entities
with developing policies and implementing procedures for working safely with these viruses in the
laboratory.
In considering hazardous characteristics of each agent or toxin, the entity should discuss the hazards of
agent cross-contamination in laboratories performing work with multiple select agents and agent strains to
prevent the accidental transfer of agents. Additional information to minimize the risk of crosscontamination is described in the biosafety and containment considerations in the
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Biosafety Guidance Document

Biosafety/Biocontainment Procedures section of this document.

Safeguards for Protecting Against Exposure to Select Agents and Toxins
Section 12(a)(2) of the select agent regulations state that the biosafety/biocontainment plan must include
Safeguards in place with associated work practices to protect entity personnel, the public, and the
environment from exposure to the select agent or toxin including, but not limited to: personal protective
equipment and other safety equipment; containment equipment including, but not limited to, biological
safety cabinets, animal caging systems, and centrifuge safety containers; and engineering controls and
other facility safeguards..

Personal protective equipment (PPE) and other safety equipment
In determining the PPE and other safety equipment needed, consider the hazardous characteristics of each
agent or toxin listed on the entity's registration and the risk associated with laboratory procedures related
to the select agent or toxin. The PPE and other safety equipment should focus on:
 Breathing or respiratory protection
 Eye and face protection
 Head protection
 Hearing protection
 Hand/arm protection (gloves, sleeves)
 Foot protection
When considering laboratory clothing, the entity needs to determine what PPE should be worn to prevent
hazards from leaving the laboratory (i.e., how clothing can be a fomite to carry BSAT out of laboratories and
how the clothing should be cleaned, disinfected, or disposed). Employees should be educated that PPE
must not be worn outside the containment laboratory except when transporting samples between labs. It
must not be worn (or stored) in break rooms, office areas, toilets, or outside the building. Employees must
be properly instructed on how to don (put on) required PPE before entering an area with a potential hazard
that requires the use of the PPE. Workers may not remove (doff) required PPE before leaving the area of
exposure.

BSL-1

Biological Safety - Personal Protective Equipment (PPE) Requirements*
BSL-2
BSL-3
BSL-4

 Protective laboratory
coats, gowns, or
uniforms recommended to
prevent contamination of
personal clothing.

 Protective laboratory
coats, gowns, smocks,
or uniforms must be worn
while working with
hazardous materials.

 Protective eyewear worn
when conducting
procedures that have the
potential to create
splashes of
microorganisms or other
hazardous materials.

 Eye and face protection
(goggles, mask, face
shield or other splatter
guard) must be used for
anticipated splashes or
sprays of infectious or
other hazardous materials
when the microorganisms
are handled outside the
Biological Safety Cabinet
(BSC) or physical
containment device.

Personnel who wear
contact lenses in
laboratories should also
wear eye protection.

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

Protective laboratory
clothing with a solidfront, such as tie-back or
wrap-around gowns, scrub
suits, or coveralls must be
worn.



Eye and face protection
(goggles, mask, face
shield or other splash
guard) must be used for
anticipated splashes or
sprays of infectious or
other hazardous materials.
[All procedures involving
the manipulation of
infectious materials must
be conducted within a

*Use of a positive pressure
suit connected to a HEPA
filtered airline. The positive
pressure suit completely
isolates the laboratory worker
from the laboratory
environment, ensuring there is
no contact with potentially
hazardous material.
Laboratory personnel who
work in positive pressure suits
require significant training.

Biosafety Guidance Document

 Gloves must be worn to
protect hands from
exposure to hazardous
materials.

BSC, or other physical
containment devices.]

Personnel who wear
contact lenses in
laboratories should also
wear eye protection.

Personnel who wear
contact lenses in
laboratories must also
wear eye protection.

 Gloves must be worn to
protect hands from
exposure to hazardous
materials.



 Eye, face and respiratory
protection should be used
in rooms containing
infected animals.

Gloves must be worn to
protect hands from
exposure to hazardous
materials.



Eye, face, and
respiratory protection
must be used in rooms
containing infected
animals.

Containment equipment
The containment equipment should focus on:
 Primary containment: the first container in direct contact with biohazardous material, as well as
other methods to protect personnel and the immediate laboratory environment from exposure to
infectious agents. Primary containment requires using proper storage containers, good
microbiological technique, and the use of appropriate equipment such as biological safety cabinets.
Primary containment may include:
o Biological safety cabinets
o Animal/arthropod caging systems
o Plant growth chambers
o Centrifuge safety containers
 Secondary containment is the protection of the environment external to the laboratory from
exposure to infectious materials and is provided by a combination of facility design and operational
practices. Secondary containment may include separation of the laboratory work area from public
access, availability of decontamination equipment (e.g., autoclave), and hand washing facilities.

Engineering controls and other facility safeguards
The basic concept behind engineering controls is that, to the extent feasible, the work environment and the
biosafety/biocontainment risk associated with the laboratory procedures should be designed to eliminate
hazards or reduce exposure to hazards. Engineering controls should be based on the following principles:
 If feasible, design the facility, equipment, or process to remove the hazard.
 If removal is not feasible, enclose the hazard to prevent exposure during normal operations.
 Where complete enclosure is not feasible, establish barriers or local ventilation to reduce exposure
to the hazard during normal operations.
The basic types of engineering controls are:
 Process control
 Enclosure and/or isolation of source
 Ventilation
Examples of engineering controls may include:
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Biosafety Guidance Document









Building ventilation/exhaust or HVAC (heating, ventilation and air conditioning) must provide safe,
comfortable, breathable environments for all employees and the public, and to minimize exposures
to hazardous air contaminants. At BSL-3 and BSL-4, exhaust laboratory air must be directly
exhausted to the outside since it is considered potentially contaminated. The exhausted room air
can be high-efficiency particulate air (HEPA)-filtered to prevent the hazards from being released to
the outside environment. The HVAC exhaust system must be sized to handle both the room
exhaust and the exhaust requirements of all containment devices that may be present. Adequate
supply air must be provided to ensure appropriate function of the exhaust system.
Biological safety cabinet (BSC) is an enclosed, ventilated laboratory workspace for safely working
with materials contaminated with BSAT. To assist with identifying a BSC, the BMBL is an excellent
reference for selecting BSC.
Effluent Decontamination System (EDS) is a system that sterilizes bio-hazardous liquid waste from
bio-containment laboratories or other facilities dealing with potentially dangerous effluents (e.g.,
pressure cooker). Depending on the work at BSL-3 and all BSL-4 laboratories, Liquid effluents from
cabinet room sinks, floor drains, autoclave chambers, and other sources within the cabinet room
must be decontaminated by a proven method, preferably heat treatment, before being discharged
to the sanitary sewer.
Pathological incinerators, or other approved means, must be provided for the safe disposal of the
large carcasses of infected animals. Redundancy and the use of multiple technologies need to be
considered and evaluated.
Anaerobic digesters is a biochemical process in which organic matter is decomposed by bacteria in
the absence of oxygen. Digesters must be airtight (no oxygen) for anaerobic digestion to occur.

Risk Management Process Controls
Process control involves the way an activity or process is done to reduce the risk. Monitoring should be
done before and after any change is implemented to make sure a change results in lower exposures.
Develop biosafety/biocontainment policies that rely on the following principals:





Process controls should be appropriate for the activities performed and the select agent or toxin in
use. Biosafety/biocontainment levels are dependent on the risks of the work being performed.
o For example, the BMBL recommends BSL-3 practices, containment equipment and facilities
for all manipulations of suspect cultures of Francisella tularensis. In contrast, BSL-2
practices, containment equipment, and facilities are recommended for diagnostic activities
involving infectious cultures of Bacillus anthracis, Burkholderia mallei, Burkholderia
pseudomallei, and Yersinia pestis.
o For B. anthracis, the BMBL recommends BSL-2 practices, containment equipment and
facilities for laboratories performing diagnostic activities.
o For B. mallei, B. pseudomallei, and Y. pestis, the BMBL recommends BSL-2 practices,
containment equipment and facilities are recommended for performing diagnostic
activities with all work in a biological safety cabinet.
Detailed safety measures to ensure that primary and secondary containment are maintained during
especially hazardous procedures (e.g., intentional production of select agent infectious aerosols or
select toxin aerosols).
Reference the FSAP Policy Statement: “Laboratory work with the regulated full-length genomes of
regulated Risk Group 3 and 4 (RG3 and RG4) agents at one containment level lower than the
infectious virus without RNA inactivation.”

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Biosafety Guidance Document



Describe the biosafety and containment procedures employed for experimentally exposed or
infected animals or plants, if applicable.
o When animals or plants are to be infected with or exposed to select agents, describe the
administration route(s) employed and the equipment used.
o Describe in detail appropriate containment of all organic material (select agent-infected
carcasses, tissues, plant biomass) until final destruction (e.g., autoclave, incineration, etc.).
o Describe or reference procedures to monitor animals or plants for accidental infection.
o Describe procedures to ensure containment of animals accidentally exposed to or infected
with select agents. Considerations for developing these procedures include but are not
limited to, situations where an airflow reversal has occurred from a room harboring
experimentally infected animals to an adjacent room housing native animals; or movement
of personnel, equipment, or laboratory waste from a select agent area to a non-select
agent area has resulted in accidental exposure.
o When animals infected with select agents are either loosely housed or housed in open
caging, there is an increased potential of room-level select agent contamination. Unless it
can be demonstrated that the animal model does not shed the agent, the increased hazard
of not using containment caging must be mitigated by procedural or facility enhancements.

Enclosure and Isolation
An enclosure keeps a selected hazard "physically" away from the worker. Enclosed equipment, for example,
is tightly sealed and it is typically only opened for moving samples/cultures or for cleaning and
maintenance. Examples include "glove boxes" or Class III biosafety cabinets. Care must be taken when the
enclosure is opened for maintenance as exposure could occur if adequate precautions are not taken. The
enclosure itself must be well maintained to prevent leaks.
Isolation places the hazardous process "geographically" away from the majority of the workers. Common
isolation techniques are to create a contaminant-free area either around the equipment or around the
employee workstations.

Disinfection, Decontamination or Destruction of Select Agent and Toxin
See the Inactivation guidance for more information on the inactivation of and rendering samples free of
select agents and select toxins for future use.
Section 12(a)(3) states that the biosafety/biocontainment plan must contain written procedures for each
validated method used for disinfection, decontamination, or destruction, as appropriate, of all
contaminated or presumptively contaminated material including, but not limited to:







Cultures and other materials related to the propagation of select agents or toxins
Items related to the analysis of select agents and/or toxins
Personal protective equipment
Animal caging systems and bedding (if applicable)
Animal carcasses or extracted tissues and fluids (if applicable)
Plant biomass (if applicable)

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Biosafety Guidance Document





Laboratory surfaces and equipment
Surfaces of transport containers
Effluent material

Development and implementation of written procedures should be based on the following principles:













Adhere to the concentration and contact time specified by the manufacturer of a disinfectant
during laboratory surface decontamination procedures to be effective in decontaminating the
select agent and toxin material.
Ensure that procedures follow any equipment manufacturer guidance on the disinfectants
compatible with their equipment.
Define waste management procedures based on the types of waste generated (e.g., PPE, plates,
liquids, eggs, animal caging, carcasses, sharps) and the containers most appropriate for the types of
waste being produced.
Describe in detail safety procedures for decontaminating reusable sharps.
Describe the procedure for safe transport of waste to the decontamination site, including the
location of the decontamination equipment in relation to the laboratory generating the waste.
Transport procedures must take into account any safety requirements to protect personnel and the
environment during transport.
Specify the actual method(s) used to decontaminate select agent and toxin waste (e.g., autoclave,
incinerators, renderers, tissue digester, chemical, etc.).
Describe the means of verifying that decontamination equipment is operating correctly, and how
often verification is performed (i.e., biological indicators [BIs], confirmation of cycle parameters).
o For autoclave verification, BIs or parametric monitors should be placed in the center of the
load in a manner expected to provide the maximum challenge for steam penetration.
When BIs are used, they should be incubated for the length of time stated by the
manufacturer and a positive control should be used. The temperature of the material to be
autoclaved must be considered when verifying the autoclave parameters (e.g., frozen
carcasses will require a longer sterilization time than non-frozen carcasses).
o For chemical decontamination, the chemical used must be appropriate for the select agent
or toxin, and the chemical concentration and contact time must be defined in the
procedure. The procedure should also address whether chemicals used for
decontamination must be freshly prepared or can be stored, and the shelf life if stored.
Describe the method(s) used to decontaminate laboratory surfaces and equipment (e.g., chemical
surface decontamination, or space fumigation using Vaporized Hydrogen Peroxide,
paraformaldehyde, or chlorine dioxide). The method selected must be appropriate for the
equipment and the select agents and toxins used in the laboratory. Procedures should indicate
contact time required which may be variable depending on agent and equipment.
o Fumigation used as a means to inactivate select toxins requires the use of a published
method or method validation.
o Fumigation procedures for select agent inactivation should include the use of biological

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Biosafety Guidance Document





indicators to verify adequate decontamination.
Describe how entity personnel are notified of the status of decontamination of laboratory surfaces
and equipment.
Describe how entity personnel are notified of ongoing or completed decontamination activities for
laboratory spaces.
Describe when laboratory surfaces and equipment should be decontaminated.

Handling Select Agents and Toxins in Shared Spaces
Section 12(a)(4) of the regulations requires the entity to describe procedures for the handling of select
agents and toxins in the same spaces with non-select agents and toxins in order to prevent unintentional
contamination. For example:






Laboratory work surfaces, equipment, and all select agent and toxin waste that must be
decontaminated prior to transitioning to work with non-select agents or toxins.
How personnel are made aware of the status of any particular room or laboratory at any given
time.
Spatial and/or temporal considerations when performing tissue culture studies.
Any concurrent work with Reconstructed 1918 Influenza virus and highly pathogenic avian
influenza virus.
Sterilization of all samples at the end of the study/experiment/procedure.

Precautions should be taken to prevent cross-contamination of viral select agents in cell cultures. Some
means of preventing accidental transfer of agents between cultures include:
o
o
o
o
o

P a g e | 10

Performing all cell culture manipulations in a biosafety cabinet.
Working with only one select agent at a time.
Decontaminating biosafety cabinet with a surface disinfectant between select agents and
toxins.
Changing gloves when changing from one select agent to another.
Aliquoting growth medium and other reagents so that the same vessel is not used for more
than one select agent.

Biosafety Guidance Document

Appendix I: Hazardous Characteristics of Select Agents and Toxins
The content of this chart is intended for instructional use only and does not qualify as an entity specific assessment. The entity must conduct their own
assessment when determining the risks and hazardous characteristics associated with the select agent or toxin for which they are registered.

SELECT
AGENT OR
TOXIN

Abrin

African horse
sickness virus

African swine fever
virus (ASFV)

Avian influenza
virus

ENDEMICITY
INFORMATION

Worldwide

Endemic regions of Africa

Endemic in most of subSaharan Africa including the
island of Madagascar and
past outbreaks occurred in
Europe, South America and
the Caribbean.

Not endemic to certain
region

INFECTIOUS
DOSE

LABORATORY SAFETY & CONTAINMENT
RECOMMENDATIONS

TREATMENT

LD50 of 700 ng/kg IV

BSL-2/ABSL-2

No antidote

Unknown

BSL-3 with enhancements/
ABSL-3 with enhancements with special consideration to
infected vector containment

No treatment

Unknown

BSL-3 with enhancements/
ABSL-3 with enhancements/
BSL-3-Ag for loosely housed animals with special consideration
to infected vector containment

No treatment

Unknown

BSL-3/BSL-3-Ag/ABSL-3 with enhancements
BSL-3 with enhancements (due to ability of virus to spread by
respiratory droplets with potential to cause a pandemic) for
HPAIV H5N1 strains with Goose/Guangdong/96-like H5 lineage
unless risk assessment by IBC determines otherwise, and
mammalian-transmissible by respiratory droplets (additional

Sensitive to the antiinfluenza drugs
known as
neuraminidase
inhibitors

DISINFECTANTS
0.5% sodium
hypochlorite (bleach)
for personnel, soak
contaminated
glassware and
equipment in 2.5%
bleach + 0.25N NaOH
for 8 hours
Inactivated by
formalin 0.1%/48
hours. Also phenol
and iodophores;
Inactivated by ether
and ß-propiolactone
0.4%.
Inactivated by
8/1,000 sodium
hydroxide (30 min),
hypochlorites - 2.3%
chlorine (30 min),
3/1,000 formalin (30
min), 3% orthophenylphenol (30
min) and iodine
compounds. Remains
viable for long
periods in blood,
feces and tissues.
Oxidizing agents,
sodium dodecyl
sulphate, lipid
solvents, Bpropiolactone,

requirements for containment, practices and occ. health
requirements). Enhancements include use of PAPR, shower out
of lab, decontamination of liquid effluents and solid wastes,
HEPA filtered exhaust air, sealed ducts, seasonal influenza
vaccination and baseline serum banking.
http://www.cdc.gov/MMWR/preview/mmwrhtml/rr6206a1.htm

Bacillus anthracis,
B. anthracis
Pasteur strain, B.
cereus Biovar
anthracis

Botulinum
neurotoxins

Botulinum
neurotoxin
producing species
of Clostridium

Anthrax in animals is widely
endemic in parts of Asia,
Africa, Mexico, and Central
and South America. Since
1990, animal outbreaks of
anthrax in the U.S have
occurred in the Midwest, the
West and in Texas and
Oklahoma.

Worldwide

Worldwide

8,000 to 50,000
organisms by
inhalation

Estimated oral or
injected toxic dose
(serotype A) of 0.001
μg/kg body weight,
and an estimated
lethal dose by
inhalation exposure
in humans of
approximately 0.07
μg/kg body weight

Cells/spores are not
normally toxic for
healthy adults

BSL-2/ABSL-2 practices for activities involving clinical materials
of human or animal origin; BSL-3/ABSL-3 for all manipulations of
cultures and for experimental animal studies

BSL-2 practices, containment, equipment, and facilities for
routine dilutions, titrations, or diagnostic studies
BSL-3 practices recommended for aerosol or droplet production
and handling of large quantities

BSL-2/ABSL-2

(oseltamivir and
zanamivir).

formalin and iodine
compounds.

Susceptible to
penicillin (except for
inhalation anthrax in
which the mortality
remains high);
ciprofloxacin,
doxycycline,
tetracylines,
erythromycin,
chloramphenicol

Spores are resistant
to many
disinfectants;
susceptible to 2%
glutaraldehyde
formaldehyde and
5% formalin

Heptavalent
Botulinum Antitoxin
(HBAT)

Susceptible to many
disinfectants - 1%
sodium hypochlorite,
70% ethanol; solution
of 0.1% sodium
hypochlorite or 0.1N
NaOH inactivates
toxin

Susceptible to
penicillin,
metronidazole,
clindamycin,
cephalothin,
cefoxitin,
cefotaxime,
chloramphenicol,
tetracycline,
erythromycin,
rifampin, and
vancomycin

The vegetative state
is susceptible to
disinfectants such as
70% ethanol, 0.1%
sodium hypochlorite,
and 0.1N NaOH.
Spores may be
resistant to
disinfectants.

Brucella abortus,
B. melitensis, & B.
suis

Burkholderia
mallei

Burkholderia
pseudomallei

Classical swine
fever virus

Conotoxins (Short,
paralytic alpha
conotoxins)

Worldwide

Glanders is endemic in Africa,
Asia, the Middle East, and
Central and South America.

Melioidosis is highly endemic
in India, Southeast Asia, and
Australia, and is found in
many tropical regions of the
world.

Endemic in much of Asia,
Central and South America,
and parts of Europe and
Africa

Worldwide

10-100 organisms

BSL-2/ABSL-2 practices for activities involving clinical materials
of human or animal origin; BSL-3/ABSL-3 for all manipulations of
cultures and for experimental animal studies

10-100 organisms

BSL-2/ABSL-2 practices for activities involving clinical materials
of human or animal origin; BSL-3/ABSL-3 for all manipulations of
cultures and for experimental animal studies

10-100 organisms

10 TCID50

LD50 of 10-100 μg/kg
depending upon the
species and route of
exposure

BSL-2/ABSL-2 practices for activities involving clinical materials
of human or animal origin; BSL-3/ABSL-3 for all manipulations of
cultures and for experimental animal studies

BSL-3 with enhancements.
BSL-3-Ag & ABSL-3, both with enhancements with no contact w/
susceptible hosts for 5 days.

BSL-2 / ABSL-2

Susceptible to
tetracyclines and
streptomycin or
TMP-SMX; therapy
usually consists of a
combination of
doxycycline and
streptomycin
Sensitive to
ceftazidime,
imipenem,
doxycycline,
minocycline,
ciprofloxacin,
gentamicin
TMP-SMX is most
effective; susceptible
to ceftazidime,
imipenem,
doxycycline,
ciprofloxacinsulphas,
chloramphenicol,
tetracycline

Susceptible to many
disinfectants - 1%
sodium hypochlorite,
70% ethanol,
iodine/alcohol
solutions,
glutaraldehyde,
formaldehyde
Susceptible to many
disinfectants; 1%
sodium hypochlorite,
70% ethanol, 2%
glutaraldehyde

Susceptible to many
disinfectants - 1%
sodium hypochlorite,
70% ethanol,
glutaraldehyde,
formaldehyde

No treatment

Inactivated by cresol,
sodium hydroxide
(2%), formalin (1%),
sodium carbonate
(4% anhydrous or
10% crystalline, with
0.1% detergent),
ionic and non-ionic
detergents, strong
iodophors (1%) in
phosphoric acid.

No antidote

2.5% NaOCl or with a
combination of
0.25% NaOCl and
0.25N NaO

BSL-2/ABSL-2 for nonpropagative laboratory procedures,
including serological examinations and staining of impression
smears
Coxiella burnetti

Crimean-Congo
haemorrhagic
fever virus

Worldwide

Eastern Europe, particularly
in the former Soviet Union,
throughout the
Mediterranean, in
northwestern China, central
Asia, southern Europe, Africa,
the Middle East, and the
Indian subcontinent

Diacetoxyscirpenol

Worldwide

Eastern Equine
Encephalitis Virus

Most cases of EEE have been
reported from Florida,
Georgia, Massachusetts, and
New Jersey, and it occurs
elsewhere in the U.S. around
freshwater hardwood
swamps.

Ebola Virus

Africa

10 organisms by
inhalation route

Unknown

LD50 of 10 mg/kg IV

A single bite of an
infectious mosquito.

1 - 10 organisms

BSL-3/ABSL-3 for activities involving the inoculation, incubation,
and harvesting of embryonated eggs or tissue cultures, the
necropsy of infected animals and the manipulation of infected
tissues

BSL-4/ABSL-4

BSL-2/ABSL-2

BSL-3/ABSL-3

BSL-4/ABSL-4

Resistant to many
antibiotics;
tetracycline,
chloramphenicol and
rifampin may be
effective.

Susceptibility to
sodium hypochlorite,
formalin, phenols
varies; susceptible to
ethanol,
glutaraldehyde and
gaseous
formaldehyde
(humidity control is
essential).

Sensitive to ribavirin

Susceptible to 1%
hypochlorite, 2%
glutaraldehyde

No antidote;
ingested (swallowed)
toxins are absorbed
with a powerful
sorbent such as
superactivated
charcoal
Currently, no
treatment is
available.
Symptomatic
treatment is given to
maintain vital
functions of the
body. Passive and
active physiotherapy
is used during the
recovery phase.

No treatment

1.0% sodium
hypochlorite + 0.1M
NaOH for 1 hour
contact time

Susceptible to
disinfectants - 1%
sodium hypochlorite,
2% glutaraldehyde,
formaldehyde, 70%
ethanol

Susceptible to 2%
sodium hypochlorite,
2% glutaraldehyde,
5% peracetic acid, 1%
formalin

Francisella
tularensis

Foot-and-mouth
disease virus

Goat pox virus

Hendra virus

Lassa Fever Virus

Endemic in North America
and parts of Europe and Asia.

Endemic in parts of Asia,
Africa, the Middle East and
South America

Africa and Asia, the Middle
East, and most of the Indian
subcontinent

Queensland and New South
Wales in Australia

Endemic in parts of west
Africa including Sierra Leone,
Liberia, Guinea and Nigeria.

5 - 10 organisms by
the respiratory
route; 106 - 108
organisms by
ingestion

10 TCID50

102.7 to 104.4 TCID50
per gram

BSL-2 for activities with clinical materials
BSL-3/ABSL-3 for all manipulations of cultures and for
experimental animal studies

BSL-4/ABSL-4

Susceptible to
aminoglycosides,
streptomycin,
gentamycin,
tobramycin,
kanamycin,
tetracyclines, and
chloramphenicol

No treatment

BSL-3 with enhancements.
No treatment
ABSL-3 with animal facility enhancements.

Unknown

ABSL4/BSL4

Susceptible to
Ribavirin

One to 10
aerosolized
organisms

BSL-4/ABSL-4

Susceptible to
Ribavirin

Susceptible to many
disinfectants - 1%
sodium hypochlorite,
70% ethanol,
glutaraldehyde,
formaldehyde
pH sensitive and
virions are
inactivated when
exposed to pH below
6.5 and above 11.
The virus in serum or
other organic
material will survive
drying and can be
carried on inanimate
objects.
Inactivated by phenol
(2%) in 15 min.
Sensitive to
detergents, e.g.,
sodium dodecyl
sulphate. Can survive
for many years in
dried scabs at
ambient
temperatures. Virus
remains viable in
wool for 2 months
and in premises for
as long as 6 months.
Inactivated by 0.1%
formalin and 0.5%
household bleach
Susceptible to 0.5 %
sodium hypochlorite,
phenolic compounds,
3 % acetic acid (pH
2.5), lipid solvents
and detergents such
as SDS, formaldehyde
and

paraformaldehyde
fixation,
formaldehyde
fumigation, and βpropiolactone

Lujo virus

Lumpy skin disease
virus

Africa

Africa and Asia, the Middle
East, and most of the Indian
subcontinent

Marburg virus

Sub-Saharan Africa

Monkeypox virus

Monkeypox outbreaks have
been reported in humans in
central and western African
countries. A 2003 outbreak in
the U.S. is the only time
human monkeypox is
documented outside Africa.

Mycoplasma
capricolum

Africa, Asia, the Middle East,
Eastern Europe,
and the former Soviet Union

Mycoplasma
mycoides

Widespread in sub-Saharan
Africa, including countries in
the West, South, East, and
Central

One to 10
aerosolized
organisms

BSL-4/ABSL-4

No treatment

102.7 to 104.4 TCID50
per gram

BSL-4/ABSL-4

No treatment

Inactivated by 0.1%
formalin and 0.5%
household bleach
Susceptible to phenol
(2%/15 min). The
virus persists in
necrotic skin for a
minimum of 33 days
and remains viable in
lesions in air-dried
hides for a minimum
of 18 days at ambient
temperature.

Susceptible to
Ribavirin

Susceptible to 2%
sodium hypochlorite,
2% glutaraldehyde,
5% peracetic acid, 1%
formalin

BSL-3/ABSL-3

Cidofovir

Orthopoxviruses are
susceptible to 0.5%
sodium hypochlorite,
chloroxylenol-based
household
disinfectants,
glutaraldehyde,
formaldehyde, and
paraformaldehyde.

Unknown

BSL-3/ABSL-3

Susceptible to
erythromycin,
tylosin, tetracycline,
or streptomycin

Sodium
hypochlorite (bleach)

1 x 108
organisms
subcutaneously and
2 x 109

BSL-3/ABSL-3

No treatment

Inactivated by
mercuric

Unknown; less than
10 infectious units by
aerosol for nonhuman primates

10-100 virions

BSL-4/ABSL-4

regions of Africa

organisms
intravenously

Newcastle disease
virus

Endemic in poultry in most of
Asia, Africa, and some
countries of North and South
America

106 EID50 per chicken

Nipah virus

Queensland and New South
Wales in Australia

Unknown

Peste des petits
ruminants virus

Parts of Africa and Asia, and
most of the Middle East

BSL-2 – low virulence virus or diagnostic accessions.

Unknown

Ralstonia
solanacearum

Tropical, subtropical, and
some temperate regions of
the world

Natural wounds
(created by excision
of flowers, genesis of
lateral roots) and
unnatural ones (by
agricultural practices
or nematodes and
xylem-feeding bugs
attack)

Rathayibacter
toxicus

Australia and South Africa

3-6 mg/kg/ body
weight

Reconstructed
1918 Influenza
virus

Not endemic to certain
region

chloride, phenol, and
formaldehyde
solution

BSL-3/ABSL-3 & BSL-3-Ag with enhancements, including no
contact with susceptible species for 5 days.

ABSL4/BSL4

BSL-3 with enhancements including no contact with susceptible
species for 5 days.

No treatment

Susceptible to
Ribavirin

Sensitive to most
disinfectants

Inactivated by 0.1%
formalin and 0.5%
household bleach
Inactivated by many
disinfectants
including alkalis
(sodium carbonate,
sodium hydroxide),
halogens (sodium
hypochlorite),
phenolic compounds,
citric acid, alcohols
and iodophores

ABSL-3 & BSL-3-Ag.

Susceptible to
chloramphenicol,
penicillin and
streptomycin

BSL-2

No treatment

Alcohol

BSL-2

No treatment

Alcohol

Susceptible to
rimantadine
(Flumadine) and
oseltamivir (Tamiflu).

Susceptible to 1%
sodium hypochlorite,
70% ethanol,
glutaraldehyde,
formalin and iodine
compounds.

BSL-3/ABSL-3
Unknown
HEPA filtration of laboratory exhaust air

Ricin

Rickettsia
prowazekii

Worldwide

Africa (Ethiopia, Nigeria and
Burundi), Mexico, Central
America, South America,
Eastern Europe, Afghanistan,
Northern India, China and the
United States

LD50: 2.7 µg/kg

BSL-2 / ABSL-2

No antidote

< 10 rickettsial
particles

BSL-3/ABSL-3

Susceptible to
tetracyclines,
chloramphenicol, or
doxycycline

Rift Valley fever
virus

Eastern and southern Africa
but also exists in sub-Saharan
Africa and Madagascar

A single bite of an
infectious mosquito

BSL-3/ABSL-3, HEPA filtration of laboratory exhaust air

Susceptible to
Ribavirin

Rinderpest virus

South Asia, the Near East and
eastern Africa

Unknown

BSL-3/ABSL-3

No treatment

Since the 2003 outbreak
which spread to more than
two dozen countries in North
and South America, Europe
and Asia, there have been no
known cases of SARS
anywhere in the world.

Unknown

BSL-3/ABSL-3

Corticosteroids and
ribavirin.

Worldwide

LD50: 8 µg/kg

BSL-2 / ABSL-2

No antidote

SARS-associated
coronavirus (SARSCoV)

Saxitoxin

0.5% sodium
hypochlorite (bleach)
for personnel, soak
contaminated
glassware and
equipment in 2.5%
bleach + 0.25N NaOH
for 8 hours
1% sodium
hypochlorite, 4%
formaldehyde, 2%
glutaraldehyde, 70%
ethanol, 2% peracetic
acid, 3- 6% hydrogen
peroxide and 0.16%
iodine
Common
disinfectants,
solvents and dry heat
Susceptible to most
common
disinfectants (phenol,
cresol, sodium
hydroxide 2%
Inactivated by
common disinfection
measures such as a 5
minute contact of
household bleach,
ice-cold acetone, icecold
acetone/methanol
mixture (40:60), 70%
ethanol (10 minutes),
100% ethanol (5
minutes)
paraformaldehyde,
and glutaraldehyde.
Inactivated by strong
alkalis, 0.5% sodium
hypochlorite.

Africa and Asia, the Middle
East, and most of the Indian
subcontinent

102.7 to 104.4 TCID50
per gram

South American
Haemorrhagic
Fever viruses
(Chapare,
Guanarito, Junin,
Machupo, Sabia)

South America

1 -10 organisms

Staphylococcal
enterotoxins
A,B,C,D,E subtypes

Worldwide

20 ng/kg - 500 ng/kg

Sheep pox virus

Swine vesicular
disease virus

Europe

Unknown

No treatment

Inactivated by phenol
(2%) in 15 min.
Sensitive to
detergents, e.g.,
sodium dodecyl
sulphate. Can survive
for many years in
dried scabs at
ambient
temperatures. Virus
remains viable in
wool for 2 months
and in premises for
as long as 6 months.

BSL-4/ABSL-4

Sensitive to ribavirin

Susceptible to 1%
sodium hypochlorite,
2% glutaraldehyde

BSL-2 / ABSL-2

No antidote

0.5% sodium
hypochlorite

No treatment

Inactivated by
sodium hydroxide

Alcohol

BSL-3 with enhancements.
ABSL-3 with animal facility enhancements.

BSL-3 with enhancements including no contact with susceptible
species for 5 days.
ABSL-3 – with animal facility enhancements.

Synchytrium
endobioticum

Worldwide

Unknown

BSL-2

No treatment

T-2 toxin

Worldwide

LD50 = 4 mg/kg IV

BSL-2 / ABSL-2

No antidote

Tetrodotoxin

Worldwide

LD50: 8 µg/kg

BSL-2 / ABSL-2

No antidote

0.5% sodium
hypochlorite
0.5% sodium
hypochlorite

Tick-borne
encephalitis
complex (flavi)
viruses:
Far Eastern
subtype
Siberian subtype
Kyasanur Forest
disease virus
Omsk hemorrhagic
fever virus

Endemic in focal areas of
Europe and Asia

Variola major
virus/ Variola
minor virus

Eradicated except for
laboratory research purposes
only

Unknown

10 to 100 organisms

Venezuelan equine
encephalitis virus

Outbreaks have been
reported in Latin America
and the Everglades.

1 viral unit subcutaneous

Xanthomonas
oryzae

Worldwide

Unknown

Yersinia pestis

Plague epidemics have
occurred in Africa, Asia, and
South America, but most
human cases since the 1990s
have occurred in Africa. The
western U.S. is also a plagueendemic area.

10-500 organisms

BSL-4/ABSL-4

No treatment

1% sodium
hypochlorite, 2%
glutaraldehyde,
formaldehyde, and
70% ethanol.

No specific
treatment available.
Supportive
treatment may be
given to alleviate
symptoms.

Susceptible to 1%
sodium hypochlorite,
2% glutaraldehyde,
formaldehyde,
quaternary ammonia.
Susceptible to
disinfectants - 1%
sodium hypochlorite,
70% ethanol, 2%
glutaraldehyde,
formaldehyde

BSL-2

No treatment

Alcohol

BSL-2/ABSL-2 practices for activities involving clinical materials
of human or animal origin; BSL-3/ABSL-3 for all manipulations of
cultures and for experimental animal studies

Sensitive to
streptomycin,
tetracycline,
chloramphenicol (for
cases of plague
meningitis),
kanamycin (for
neonates).

Susceptible to many
disinfectants - 1%
sodium hypochlorite,
70% ethanol, 2%
glutaraldehyde,
iodines, phenolics,
formaldehyde

BSL-4/ABSL-4

BSL-3/ABSL-3
HEPA filtration of laboratory exhaust air

No treatment
(Smallpox vaccine)

Animal Work

Aerosol Producing

Appendix II: Example Procedural Risks

Procedural Risks*
Propogation
Lack of Appropriate Immunizations
Vortexing
Centrifuging
Sonicating
Pipetting
Blending
Homgenizing
Shakers
Lyophilization
Flow Cytometry/ Culture Manipulation
Automated plating/ Plate washing
Spills/Splashes/Sprays
Mouth pipetting and other ingestion
forms
Cell Line/ Culture manipulation
Pressure column chromatography
Injection Procedures
Loosely Housed
Aerosol Exposure
Bedding changing and disposal
procedures
Necropsy/ Harvesting tissues
Animal Bites
Use of Sharps
Inadequate Training
Inadequate Safety Equipment
Inadequate Facilities
Waste Handling and Inactivation
Procedures
Decontamination
Selection and Use of PPE
Inadequate Signage/ Labeling

*List is non-comprehensive.

Mitigating Factors (A risk assessment for each experiment is necessary, as well as risk communications)
BSC/ Primary
Engineering Controls/
Biosafety SOP(s)/
Occ. Health
PPE
Containment
Secondary Containment Training
Plan
Gasket on Lid
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

X

X
X

X
X

X
X

X
X
X

X
X
X

X
X
X

X

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

Notes
Sterility testing and use of non-viable/exempt strains

Use safety cup (if available)
Use hearing protection

Use spill kit

X
Avoid using glass columns when possible