Standard Operating Procedure
for work with
Toxin name/class:
PI:
Building:
CAS #:
Date:
Room #:
NOTE: ALL ORDERS OF HEALTH AND HUMAN SERVICES (HHS) TOXINS MUST BE APPROVED THROUGH OESO-BIOLOGICAL
SAFETY (919-684-8822). rDNA WORK THAT ENCODES FOR TOXINS MUST BE APPROVED BY THE IBC.
1. Circumstances of Use:
Please read instructions on the following pages.
2. Potential Hazards:
Please read instructions on the following pages.
3. Medical Considerations:
Please read instructions on the following pages.
4. Engineering Controls:
All work with concentrated powdered or “select agent toxins” should be conducted within the operationally
effective zone of the BSC or hood. Each user should verify the inward airflow before initiating work. Low molecular
weight toxin solutions, or work involving volatile chemicals or radionucleotides combined with toxin solutions, may
require the use of a charcoal-based hood filter in addition to HEPA filtration.
Add additional precautions following the guidance in the instructions on the following pages.
5. Work Practice Controls:
 An emergency shower and eyewash station or eyewash/drench hose combination unit is recommended in
locations where toxins are used.
 A hand-washing sink must be readily available to all locations where toxins are used.
 Containers of powdered or select agent toxins should be removed from the BSC or hood only after the exterior of
the closed primary container has been decontaminated (with a solution appropriate for specific toxin) and placed in
a clean secondary container.
 The interior of the BSC or hood should be decontaminated periodically, for example, at the end of a series of
related experiments. Appropriate decontamination methods can be found in Section 10 (Decontamination, Spills,
and Emergency Procedures).
 Until thoroughly decontaminated, the hood or BSC should be posted to indicate that toxins remain in use, and
access should remain restricted.
 Remove and dispose of or decontaminate protective clothing and wash hands with soap and water before leaving
the work area.
Add additional precautions following the guidance in the instructions on the following pages.
6. Personal Protective Equipment (PPE):
At a minimum, lab personnel should wear safety glasses, suitable laboratory PPE to protect hands and arms (such as
lab coats, smocks, or coveralls), and disposable gloves..
Add additional precautions following the guidance in the instructions on the following pages.
7. Transportation and Storage:
 Toxin containers must be labeled with toxin name and hazard warnings at a minimum.
 When toxins are stored in the lab, containers should be sealed, legibly labeled and secured to ensure restricted
access.
 Refrigerators and other storage containers containing biological toxins should be labeled with contact
information for trained, responsible laboratory staff.
Add additional precautions following the guidance in the instructions on the following pages.
8. Waste Disposal:
Please read instructions on the following pages.
9. Exposures/Unintended Contact:
 Remove contaminated clothing and/or PPE and flush mucous membranes (eyes, nose, mouth) with water from
the nearest eyewash or drench hose. Intact or non-intact skin exposures should be washed immediately with soap
and water.
 Report any exposure immediately by calling the Employee Health Exposure Hotline (115 from any campus phone
or 919-684-8115 from a non-campus phone). The EOHW representative will provide advice on an appropriate
post-exposure treatment. Complete the work-related injury or illness report found at
http://www.hr.duke.edu/benefits/medical/workcomp/report.php.
 Leave the area (for inhalation hazards).
 Change gloves (if gloves become contaminated).
Add additional information following the guidance in the instructions on the following pages.
10. Decontamination, Spills, and Emergency Procedures:
Decontamination – the following information MUST be included (at least one method must be listed):
 Name and concentration of decontamination solution:
 Time for effective decontamination:
 Physical inactivation (temperature, time):
Spill Procedures
 For small spills of dilute solution (inside or outside BSC):
At minimum, safety glasses, lab coat, smock, or coveralls should be worn, along with appropriate gloves to clean
up a spill. If splashing may occur, safety goggles and a face shield must be worn in place of safety glasses. Cover
spill with paper towel or other disposable, absorbent material. Apply (list decontamination solution and
concentration here) to the spill, beginning at the perimeter and working towards the center, and allow sufficient
time (enter time in minutes here) to completely inactivate the toxin.
 For any spill of toxin powder or stock solutions:
Call the OESO Spill Response team by calling 911 from a campus phone or 919-684-2444 from any phone, unless
you are an approved respirator user and have been given spill cleanup instructions.
BSC/Fume Hood Failure
 Close or cover all toxin containers. Shut down operations, close hood sash, and evacuate room. Contact your
maintenance provider to repair the BSC or fume hood.
o Facilities Management Department: 919-684-2122
o Engineering & Operations: 919-684-3232
Add additional information following the guidance in the instructions on the following pages.
11. Training of Personnel:
In addition to online General Lab Safety, each laboratory employee working with toxins must be trained in the
theory and practice of the toxins to be used, with special emphasis on the nature of the practical hazards associated
with laboratory operations. This includes how to handle transfers of liquids containing toxin, where to place waste
solutions and contaminated materials or equipment, and how to decontaminate work areas after routine operations
and accidental spills. Furthermore, all personnel shall read and fully adhere to this SOP when handling the toxin.
“I have read and understand this SOP. I agree to fully adhere to its requirements.”
Last
First
Duke ID
Signature
Date
Instructions for Creating a Laboratory-Specific Standard Operating Procedure (SOP)
For Biologically-Derived Toxins
Use fillable form starting on page 1 to create your lab-specific SOP. Include relevant information from all sections. Risks
should be assessed for each toxin individually to determine the appropriate controls, personal protective equipment, and
decontamination procedures that should be in place when preparing and handling that toxin.
Save with other electronic lab materials or print and place with other safety information for your lab.
Laboratory and SOP information
Create a header at the top of your SOP that identifies:
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Name of biologically-derived toxin
CAS number (if available)
Principal Investigator
Date SOP was created/updated
Building and room number(s)
Circumstances of use
Describe what this toxin will be used for in the lab and a brief description of work. Also describe concentrations used and
how needed concentration and amount is prepared and/or obtained. Describe quantity required, approximate frequency
of use, and location of use. If possible, indicate that the toxin will be purchased in small quantities or dilute solutions to
reduce the risk of exposure and minimize waste. Consider if liquid form would be less hazardous than powder (usually
depends on hazards associated with the solvent) and, if so, purchase in liquid form.
Potential Hazards
Include the following information in this section:
 Routes of exposure (skin, inhalation, ingestion, injection); when/how exposure might occur (inhalation during
weighing and mixing, splashes, needle sticks from injection preparation and administration, cleaning up spills,
etc.)
 Target organs
 Information on signs/symptoms of exposure
 Toxicological data on the toxin (by route of entry)
The following agents have summary statements in the Toxic Agent section of the 5th edition of Biosafety in
Microbiological and Biomedical Laboratories (CDC) which may be helpful:
 Botulinum neurotoxin
 Staphylococcal enterotoxins (SE)
 Ricin toxin
 Selected Low Molecular Weight (LMW) toxins
The Toxin, Toxin-Target Database also has detailed toxin information that may be helpful in determining potential
hazards.
Medical Considerations
Please list toxoid vaccinations or antitoxins required or recommended for this toxin.
If antitoxins or toxoid vaccinations are required, the researchers must contact Employee Occupational Health and
Wellness at 919-684-3136 to discuss these considerations.
If there are no specific medical considerations recommended for work with the toxin, please put “No special medical
considerations recommended.”
Engineering Controls
List engineering controls required for this toxin (e.g., HEPA filtered enclosure inside chemical fume hood, Class II Type B2
or Class III Biological Safety Cabinet). Engineering controls should be selected according to the risk assessment for each
specific toxin operation.
Things to consider when listing Engineering Controls for toxins (include all that apply):
 Intentional generation of aerosols is considered a high-risk procedure that must be approved by Biological Safety
(919-684-8822) to ensure that adequate engineering controls are in place.
 Low molecular weight (LMW) toxin solutions or work involving radionucleotides combined with toxin solutions
may require use of a charcoal-based filter in addition to HEPA filtration.
 Work with toxins involving volatile chemicals requires a fully exhausted BSC or use of secondary containment
(glove bag or box) inside a fume hood.
 Use of powdered toxins should be avoided if possible and minimized otherwise. Unavoidable operations using
dry forms of toxins should be undertaken with appropriate engineering controls and/or respiratory protection.
Dry toxins must be manipulated using a Class III BSC (no secondary containment required), or in a Class II BSC or
fume hood with the use of secondary containment such as disposable glove bag or glove box inside. Toxins with
low LD50s or LC50s may require a respirator even when working with the powder in a BSC. Call OESO’s Biological
Safety division at 919-684-8822 if you have questions about whether respirator use is required for the toxin you
are working with.
 Toxins that have a low toxicity should still be handled in a standard fume hood or BSC during procedures where
aerosols may be produced. Aerosols may be produced during any open handling of dry powder (e.g. weighing),
and during open or pressurized manipulations of suspensions.
 If powdered toxin comes in a vial with a septum, preparation of solution should occur within a standard fume
hood or BSC due to possible pressure changes that may generate aerosols through the vent opening.
 Some very dilute/low-level toxin manipulations (in solution) may present negligible risk to lab personnel and may
be suitable for work inside a chemical fume hood without HEPA filtration or, in some cases, on the bench top.
Contact OESO’s Biological Safety division at 919-684-8822 if you have questions.
 Operations that expose toxin solutions to vacuum or pressure should always be handled inside a BSC and the
operator should always use appropriate respiratory protection in case of engineering failure. Respirator users
require medical clearance, training, and fit-testing. Contact OESO’s Occupational Hygiene and Safety division at
919-684-5996 for more information on respirator use.
 If vacuum lines are used with a toxin, they should be protected by a HEPA filter to prevent entry of toxins into the
line.
Work Practice Controls
If animals will be dosed with the toxin, the “Animal Use of Biotoxins” section at the end of this SOP must be completed
and followed. This SOP includes requirements related to advanced notification of the animal facility, room and cage
labeling, and health considerations of animal caretakers.
Things to consider when listing Work Practice Controls for toxins (include all that apply):
 Only minimum amounts of toxins should be kept in the lab. For permissible amounts of Select Agents, go to
http://www.selectagents.gov/Permissible%20Toxin%20Amounts.html.
 Experiments should be designed to eliminate or minimize work with dry toxin.
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Apply a cotton ball or gauze pad wetted with decontamination solution to the junction of the needle and stopper
to prevent any pressurized/aerosolized toxin material from escaping when removing the syringe from the septum
of the bottle.
Pressurized tubes or other containers holding toxins should only be opened in a BSC or fume hood with HEPAfiltered insert.
Glassware should be replaced with plastic for handling toxin solutions wherever practical to minimize risk of cuts
or abrasions from contaminated surfaces. Thin-walled glass equipment should be completely avoided. Glass
Pasteur pipettes are particularly dangerous for transferring toxin solutions and should be replaced with
disposable plastic pipettes. Glass chromatography columns under pressure must be enclosed within a plastic
water jacket or other secondary container.
Safer sharps devices and safety needles should be used to minimize risk of cuts or punctures. Examples include
retractable, shielded, and disposable scalpels and needleless devices or retractable needle devices.
Centrifugation of cultures or materials potentially containing toxins should only be performed using sealed, thickwalled tubes in safety centrifuge cups or sealed rotors. The outside surfaces of containers and rotors should be
routinely cleaned before each use to prevent contamination that may generate an aerosol. After centrifugation,
the entire rotor assembly is taken from the centrifuge to a BSC to open it and remove its tubes.
Personal Protective Equipment (PPE):
Describe additional PPE requirements for each task involving the toxin.
Things to consider when listing PPE for toxins (include all that apply):
 If toxin is suspended in solution, appropriate gloves should be worn to prevent penetration of the solvent. Solvents
can act as a carrier for the toxin and can result in dermal exposure if the solvent readily penetrates the glove. For
guidance on chemical glove penetration, please see Oklahoma State University’s Glove Penetration Table.
 When working with toxins that pose direct percutaneous hazards, special care must be taken to select gloves that are
impervious to the toxin and diluents or solvents used.
 If there is a moderate risk of cut or puncture, cut- or puncture-resistant gloves should be worn.
 When conducting liquid transfers and other operations that pose a potential splash or droplet hazard in an openfronted BSC or hood, safety goggles and disposable facemask, or a face shield, must be worn.
 If working with dry forms of toxin that are subject to spread by electrostatic dispersal, static-free disposable gloves
must be worn. Wetting and wiping down gloves and surfaces before beginning work can help reduce static.
Note: Respirators are masks designed to protect the wearer from specific airborne hazards and are different from
surgical masks, which protect the wearer only from splashes and are primarily intended to protect others from
infectious aerosols exhaled by the wearer. Respirator use requires employee participation in the Respiratory
Protection Program, which involves medical clearance and annual fit testing and training. Please be clear about use
of surgical masks versus respirators. (Please do NOT use the vague term “masks”.)
Transportation and Storage:
Describe transportation and storage strategies taking the following information into consideration:
 Describe transportation strategy (use of secondary containers, travel through low-traffic hallways).
 Toxin solutions, especially concentrated stock solutions, should be transported in leak/spill-proof secondary
containers.
Waste Disposal
Describe waste disposal procedures taking the following information into consideration:
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Discarded needles/syringes and other sharps should be placed directly into properly labeled, puncture-resistant
sharps containers, and inactivated/decontaminated as soon as is practical.
Depending on the toxin, contaminated materials and toxin waste solutions may be inactivated by physical means
(incineration or extensive autoclaving), or by chemical means (soaking in suitable decontamination solutions for a
specified period of time). Please see Tables 1 and 2 of the CDC’s Biosafety in Microbiological and Biomedical
Laboratories (BMBL) Guidelines for Work with Toxins of Biological Origin for appropriate physical and chemical
inactivation procedures for specific toxins. Toxin waste should be disposed of in accordance with the Duke University
Medical Waste Management Policy and Chemical Waste Management Policy. If you have specific questions about
disposal, please call OESO Environmental Programs at 919-684-2794.
Exposures/Unintended Contact
Describe any additional procedures that are specific to the toxin (may be listed on Safety Data Sheet).
Decontamination, Spills, and Emergency Procedures
For information on decontamination and spills for specific HHS toxins, please see page 7 of BMBL’s Guidelines for Work
with Toxins of Biological Origin. On the Durham campus, “large” spills of volatile or powdered hazardous materials and
all mercury spills must be referred to the OESO spill response team by calling 911 from a campus phone or 919-684-2444
from any phone.
Think about other types of emergencies that are possible and list procedures in this section for how to deal with those
situations.
Training of Personnel
Feel free to add any additional lab-specific requirements to the statement already provided in the template.
For additional help in completing the SOP, contact the OESO-Biological Safety Division at 919-684-8822.
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ANIMAL USE OF BIOTOXINS
Standard Operating Procedure for Handling Animals Dosed with Biologically-Derived Toxins
In the course of research, laboratory animals may be dosed with biologically-derived toxins. The SOP outlined below
addresses procedures for performing animal care safely after the animals have been dosed.
Use of all biotoxins (and other toxic chemicals or biological agents) in animals must be documented in a protocol
approved by the Duke Institutional Animal Care and Use Committee (IACUC). For more information, please visit the
IACUC website.
Animals that have been dosed with a biotoxin may excrete that toxin or toxic metabolites, particularly during the first 48
hours after dosing. Most of the time, the toxic material or its metabolites would present a hazard in particulate form.
The precautions outlined below will protect employees in these situations. For biotoxins that are toxic at extremely low
doses (e.g. botulinum toxin or tetrodotoxin), additional precautions may be necessary. Work with these toxins may
require that researchers wear respirators while performing cage-change after dosing animals. Respirator use must be
approved by Occupational Hygiene and Safety (919-684-5996).
These procedures must be followed for the first 48 hours (at a minimum) after dosing AND until the contaminated
bedding is changed. (All bedding used within 48 hours of dosing will be considered contaminated.) The procedures may
need to be modified for animals other than rodents.
Researcher’s Procedures in Animal Facility:
Describe procedures done within the animal facility and engineering controls used (such as a biological safety cabinet).
Please indicate the type of animal being used in this study.
Example: Mice will be dosed via intravenous infusion with Pertussis toxin. All work with the toxin will be done within a
Class II Biological Safety Cabinet.
Notification and Signage:
When animals are dosed with a biotoxin, laboratory workers must provide advanced notification to those who will take
care of their animals, and must label the cages of the animals with a hazardous chemical cage card that contains the
following information: PI name, Protocol #, Start Date, Agent Name, and Autoclaving checkbox (verifying whether or not
to autoclave bedding according to Safety Data Sheet).

Within the laboratory, cage labels can take any form that is recognizable to all laboratory workers.
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For animals housed in DLAR- managed areas:
o
At least five business days prior to dosing the animals, the laboratory will notify DLAR of the dosing schedule
and send the Safety Data Sheet (SDS) for the biotoxin (if available) along with a copy of this SOP (with blanks
and checkboxes on the last page filled in) to the DLAR Assistant Director as well as the Operations Manager of
the animal facility where the work will occur. Contact information for these personnel can be found on the
DLAR website (http://labanimal.duke.edu/) under “Contact Us” and then “Husbandry and Facilities”. The
work cannot begin until the laboratory has obtained confirmation that their scheduling notification has
been received.
o
When dosed animals are returned to their cages, hazardous agent cage cards (supplied by DLAR) must be
placed on each cage, and the “Biotoxin hazard” sign (last page of this document) must be posted on the door
(with hazard, dosing and contact information filled in). Note that cages occupied within the first 48 hours
after dosing must be marked until the contents have been dumped. The cards and signs must be removed by
the researcher once the hazard is no longer present (in most cases, after the first cage change that takes
place more than 48 hours after animals receive their final dose).
ANIMAL CAGE-CHANGE PROCEDURES
Engineering Controls:

Cages will be covered with micro-isolator lids and/or will be maintained on a ventilated rack.

For toxins that are hazardous at low doses (e.g. botulinum toxin or tetrodotoxin), cages must be maintained in
negatively pressured ventilated racks in ABSL2 housing so that a biological safety cabinet is accessible for cage
changing and dumping.
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Cages will be opened (including for cage-changing, animal care, or experiment-related reasons) in a ventilated
cage changing station, a biological safety cabinet, or a chemical fume hood.
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Bedding will be dumped in a biological safety cabinet or ventilated dumping station, such as the “Bio Bubble”
machines used in several DLAR facilities or the automated system in GSRB II. For toxins that are hazardous at low
doses, cages must be dumped in a biological safety cabinet by the researcher.

Note: For animals too large to be housed in cages with microisolator lids or on ventilated racks, a consultation
with Occupational Hygiene and Safety will be required in advance.
In addition, the researchers will train DLAR staff regarding any agent-specific hazards and any additional precautions
needed. If vaccinations or specific antidotes are recommended for work with this toxin, please contact OESO (919-6848822) and EOHW (919-684-3136) for a consultation.
NOTE: Autoclaving bedding may NOT destroy all biotoxins and, if any other material in the bedding is volatile, use of the
autoclave may increase the potential for employee exposure by creating harmful airborne vapors. Autoclaving bedding
before dumping does NOT change the above requirements for ventilation controls while dumping bedding. If bedding
should be decontaminated using a solution, please list the name and concentration of solution in the Work Practices
section below.
Personal Protective Equipment:
Employees must wear appropriate personal protective equipment for handling animals, cages, and bedding:

For handling animals and cages: Standard 4-mil exam/laboratory-style gloves, lab coat (or gown or coveralls), long
pants, and (if required in the area) shoe covers, and surgical mask if required for that facility.

When dumping bedding: The above PPE, plus an N95 (with faceshield, goggles, or safety glasses) or PAPR must
also be worn.
Work Practices:

Contaminated bedding must be deactivated by the method listed in the Waste Disposal section below.

Gloves will be changed at least every 2 hours, when they become torn or obviously contaminated with excreta
AND before handling animals in other experimental groups.
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Employees will wash hands after removing gloves.
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A gown or coveralls will be worn when dumping contaminated bedding. If these items are to be re-used, they
must be stored in a manner that does not permit potential contact between outer and inner surfaces.

Safety glasses, safety goggles, and reusable faceshields can be cleaned with water and detergent, stored in a
clean place, and reused.

Decontamination (after each use) of the ventilated cage-changing stations will consist of surface cleaning with
water and disinfectant (or decontamination solution listed) followed by thorough rinsing with clean water.
Cleaning will proceed from least to most contaminated areas.
Decontamination solution: name, concentration

Waste bags should be closed for transport through the building.

As with any animal bite or scratch, employees bitten or scratched by animals exposed to biotoxins are expected
to notify EOHW of the exposure by filling out a Report of Occupational Injury or Illness. For highly toxic
compounds (where medical intervention is needed), employees should be advised to call the EOHW exposure
hotline (919-684-8115).
Waste Disposal Method:
Look at the Safety Data Sheet of the toxin you are using to determine which method is best for inactivation and disposal of
contaminated bedding (autoclaving, chemical deactivation, etc.) and list it here. If a decontamination solution must be
used, please list the name of the solution, concentration, and instructions below.
DLAR will provide waste labels to researchers dumping their own bedding. Please contact the facility manager to obtain
these labels before dumping bedding.
Example (autoclave method): Cages (with bedding) will be double bagged in two orange autoclave bags and taped shut.
The bags are then sprayed down with a sodium hypochlorite solution of at least 0.5% and allowed to let sit for 5 minutes
to inactivate. DLAR staff will transport bagged cages to autoclave. Only after cages have been autoclaved will they be
dumped (by DLAR staff) as normal waste.
Note: Staff must remain present in the animal room while decontamination takes places so that cages are not left
unattended during the process.
Exceptions to above standard cage-change procedures:
Example: Research staff will be responsible for performing all cage changing procedures described above.
WARNING!
BIOTOXIN HAZARD
Target Organs: _________________
See Operations Manager for Safety Data Sheet
Principal Investigator:
Agent(s)/Concentration:
Dose:
Date(s)/Time(s) Administered:
Route:
Building:
Room:
Duration of animal survival between exposure and euthanasia:
For 48 hours after administration/exposure AND until bedding is changed:
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Don gloves, lab coat (or gown or coveralls), long pants, and (if required for your location) shoe
covers for handling animals and closed cages.
Open cages (including for cage-change) in ventilated cage-changing station or biological safety
cabinet.
Deactivate contaminated bedding by ____________________________________________
__________________________________________________________________________.
(Describe deactivation method including name of solution and concentration, contact time, or
autoclave)
Dump bedding ONLY using a ventilated dumping station or biological safety cabinet while wearing
an N95 or PAPR.
Emergency
Contact
Primary
Secondary
Safety Office
Name
Work Phone
Afterhours Number
Biological Safety
919-684-8822
N/A