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Introduction

Biological toxins are poisonous substances produced by certain microorganisms, animals, and plants. Examples of toxins of biological origin include Diphtheria Toxin, Tetrodotoxin, Pertussis Toxin, Botulinium Toxin, Snake Venom Toxins, Conotoxin and Ricin. Although toxins are derived from biological materials, they do not replicate and are therefore not considered infectious. However, they may be extremely toxic in very small quantities and must be managed like hazardous chemicals in the workplace.

Controls must be in place to ensure staff are protected from exposure. The routes of exposure include inhalation, eye, nose and mucous membrane contact, percutaneous, and skin absorption depending on the diluents used. The main issues of concern in the laboratory are accidental exposures to toxin caused by contact with the mouth, eye, skin and mucous membranes, inhalation of toxin powder or aerosol inadvertently generated, or by needlestick incidents.

Work with toxins of biological origin requires documented laboratory-specific training on standard operating procedures (SOP) for all laboratory personnel prior to starting work. The training must include, but is not limited to, the health and physical hazards of the toxin, signs and symptoms associated with exposure, appropriate work practices, personal protective equipment, emergency procedures, and reporting of personnel exposures.

Some biological toxins are considered Select Toxins, which the Federal Select Agent Program has identified as a severe threat to public health and safety as bioterrorism agents. Transfer, possession, use, and disposal of all of these agents and toxins are strictly regulated. Certain toxins used in quantities below regulatory thresholds may be excluded from the requirements of Select Agent Regulations. See the following CDC link for the current list of Select Toxins, exclusions and exempt quantities. http://www.selectagents.gov. Please see chapter 14 of the BSM manual for more specific information regarding Select Toxins.

Laboratory Planning and Preparation of Use

1. The use of biological toxins requires submittal of a biological use agreement (MOUA) for approval by the IBC.
2. Develop a written laboratory-specific SOP that is specific to the toxin and the procedure being used. A Biological Toxin SOP template with a training documentation form is available.
3. Provide documented laboratory-specific training to personnel working with toxins, and any other personnel authorized or required to be in the laboratory during toxin work.
4. Ensure the toxin Material Safety Data Sheet/Safety Data Sheet (MSDS/SDS) is available to staff at all times.
5. Designate toxin storage area (freezer, refrigerator, cabinet or other container) in a secure location.
   • Select toxins must be maintained in a locked location. See chapter 14 for additional details on select toxins
6. Designate a specific area within the laboratory where toxin use will take place. There may be a need for ventilated containment depending on the likelihood of inhalation. If working with a toxin in powder form, a BSC, glove box, fume hood, or other approved form of containment will need to be utilized.
7. Prepare a sign stating “Toxins in Use” and post in the area that is designated for toxin use. When the toxin is in use, this sign should be posted and lab personnel should be notified that the toxin is in use.
8. If possible, do not work with toxin in solid or powder form. If it is necessary to purchase it in powder or solid form, purchase pre-diluted or pre-weighed toxin in the minimum quantity needed to perform work. Introduce the diluent through the septum in a way that eliminates the needs to open the septum (e.g., injection of diluent through a rubber septum).
9. If the toxin must be weighed, use ventilated containment to weigh out the toxin. The tare method can be used to prevent inhalation of the toxin.
   • Tare method - While working in a laboratory hood, the chemical is added to a pre-weighed container. The container is then sealed and re-weighed outside of the hood. If chemical needs to be added or removed, this manipulation is carried out in the hood. In this manner, all open toxin handling is conducted in the laboratory hood.
10. Determine the appropriate chemical and/or physical inactivation method(s) for the specific toxin (refer to toxin inactivation). Ensure supplies for inactivation of toxin are readily available.
11. Ensure supplies for spill cleanup are appropriate for the specific toxin, maintained in a clearly marked spill cleanup kit and readily available in the laboratory.

Engineering Controls

Consider the toxin properties when selecting a containment device. Designate a certified BSC, glove box, fume hood, or other containment device for toxin use. A clean bench should never be used for work with biological toxins since it provides no personnel protection from the hazards of the toxin in use.

In-line HEPA filters are required if vacuum lines are used with a toxin.

If centrifugation will take place while working with a biological toxin, then safety cups and sealed rotors must be used and the outside of surfaces routinely decontaminated. The rotors and safety cups should only be opened inside a certified BSC or other form of containment.

Personal Protective Equipment

Safety glasses with a side shield or goggles should be worn.

Wear a long-sleeved lab coat, a smock, or coveralls. Consider using disposable PPE.

Gloves that are impervious to the toxin and to the diluent need to be specially selected. Double gloving is recommended. Gloves need to be changed immediately if they become contaminated, punctured, or torn and disposed of immediately after removal.

Wear additional face protection, such as a face shield, when splash or splatter is possible.

Respiratory protection may be required if an airborne hazard is present. Notify the biosafety officer if respiratory protection is required. Laboratory personnel will need to undergo a medical evaluation and have a fit test performed by EH&S personnel as part of the UNR respiratory protection program.

Toxin Practices

• Work with toxins in a designated area. This can be done by posting a sign during work or more permanent marking can be in place if the work takes place routinely.
• Biosafety Level 2 (BSL-2) practices are appropriate for most toxin work. However, some toxins or procedures may require additional safety precautions (BSL2+ or BSL3).
• Work with toxin in a BSC, fume hood, glove box or other approved containment if it is appropriate to do so pending the determinations made following a risk assessment.
• Transport toxins only in labeled, leak/spill-proof, non-breakable secondary containers.
• Perform preparations over plastic backed laboratory diapers that have been presoaked with the appropriate inactivating agent (refer to toxin inactivation). Dispose of lab diapers after completion of tasks or immediately upon contamination.
• Eliminate the use of sharps whenever possible. Utilize safe sharps procedures (i.e., sharps container in the immediate vicinity). Safety needles, to include needle locking syringes and disposable syringe needle units are recommended and should be disposed of promptly after use. Never bend, break, or recap a needle.
• If biological toxins are administered to research animals, restrain or anesthetize animals when possible.
• Decontaminate containers before they are removed from the fume hood, BSC, or glove box. Also decontaminate the exterior of the closed primary container and place it in a clean secondary container.
• Upon completion of tasks, thoroughly decontaminate the BSC or approved containment and all surfaces using an appropriate inactivating agent and contact time.
• All potentially contaminated disposable items (such as gloves used in preparation) must be placed in a hazardous waste bag and autoclaved before disposal.
• Thoroughly wash hands after work has been completed as is the standard practice for BSL-2 work.

The following guidance is intended for spills of toxins. 

Spill cleanup is only to be completed by personnel with documented training who are utilizing the proper personal protective equipment. The required minimum PPE for cleaning up a toxin spill includes a laboratory coat or gown (disposable recommended), safety glasses, and two pairs of disposable nitrile gloves or thick chemical resistant gloves.

Liquid Spills

1. Don the appropriate PPE.
2. For chemical inactivation, cover the spill with absorbent pads or towels and saturate them with the appropriate chemical inactivating agent, starting at the perimeter of the spill and working towards the center. Allow the prescribed contact time to ensure adequate deactivation of the biological toxin before cleanup.
3. After the required contact time between the spilled toxin and the inactivating agent, remove and collect the absorbent pads as hazardous chemical waste.
4. Use absorbent pads or towels to wipe the affected area a second time, followed by soap and water.
5. Discard cleanup materials and protective clothing as hazardous chemical (biological toxin) spill waste. Label the decontaminated spill waste as “toxin spill waste” using the specific name of the toxin in use and submit to EH&S for disposal.
6. Thoroughly wash hands, forearms, and face with soap and water.

Powder Spills inside of a fume hood/glove box/BSC or containment

1. Don the appropriate PPE.
2. For chemical inactivation, gently cover the spills with moistened absorbent pads, then saturate the pads with the appropriate chemical inactivating agent, apply inactivating agent starting at the perimeter of the spill and work towards the center. Allow the prescribed contact time to ensure adequate deactivation of the biological toxin before cleanup.
3. After the required contact time between the spilled toxin and the inactivating agent, remove and collect the absorbent pads as hazardous chemical waste.
4. Use moistened absorbent pads or towels to wipe the affected area a second time, followed by soap and water.
5. Discard cleanup materials and protective clothing as hazardous chemical (biological toxin) spill waste. Label the decontaminated spill waste as “toxin spill waste” using the specific name if the toxin in use and submit to EH&S for disposal.
6. Thoroughly wash hands, forearms, and face with soap and water.

Powder Spills outside of a fume hood/glove box/BSC or approved containment

1. Evacuate all personnel from the laboratory and restrict access; do not attempt to clean-up the spill.
2. Notify EH&S as soon as possible by calling the 24-hour number (775-327-5040); tell them that a spill of a biological toxin has occurred in the lab area outside of containment, and you need assistance cleaning up the spill the toxin.

Be prepared to provide the following information:

• Name and phone number of knowledgeable persons that can be contacted: emergency contact, name and phone number
• Toxin name, concentration and amount spilled, liquid or solid spill
• Number of injured, if any
• Location of spill

Biological Toxin Spills Outside of the Laboratory

1. If biological toxin is released outside of the transport containment, immediately clear the area of all personnel and secure the area.
2. Cleanup should be initiated as soon as possible to prevent spread of aerosol. Attempt cleanup only if appropriate cleanup materials and protective clothing are available.
3. Notify EH&S at 775-327-5040 (24-hour contact number) as soon as possible for assistance with the cleanup.

Because it is impossible to prevent aerosolization when a spill occurs outside of the laboratory, the primary emphasis when transporting biological agents and toxins is on spill prevention. All biological toxins are to be transported from the laboratory inside of an unbreakable, well-sealed, primary container containing absorbent material that is contained inside of a second unbreakable, well-sealed, secondary container. The secondary containers must be labeled with the universal biohazard symbol and the identity or biosafety level of the agent. Select toxins or other biological toxins with similar risk must be transported using tertiary containment.

Inactivation and disposal of biological toxins

According to the CDC, inactivation of a toxin means that it is rendered non-functional and is no longer capable of exerting its toxic effects. This differs from the killing of a biological agent, which renders the biological material non-viable and incapable of growing, replicating, infecting, or causing diseases. Inactivation methods used for biological toxins must be specific to the toxin. Depending on the toxin, inactivation can take place using physical means, including autoclaving, or by chemical means. The BMBL provides the following tables to help provide guidance on the inactivation of biological toxins.

1. Inactivate biological toxin waste chemically or physically (usually autoclaving) before disposal or given to EH&S for disposal.
2. Place any used PPE and spill cleanup debris in a hazardous waste plastic bag and autoclave.
3. For mixed waste (i.e. toxin waste mixed with radioactive waste) consult EH&S Radiation Safety.
4. If in-lab inactivation is not possible for a specific toxin waste, manage that waste as hazardous chemical waste.
5. Dispose of liquid inactivated biological toxin waste within a pH range of 5.5 to 12 down the regular sewer drain.
6. Refer to the information below in Tables 1 and 2 on inactivation of selected toxins, which is taken directly from the publication Biosafety in Microbiological and Biomedical Laboratories (BMBL) 6th Edition. Centers for Disease Control and Prevention. Appendix I: Guidelines for Work with Toxins of Biological Origin.
7. If using bleach solutions, prepare fresh daily for inactivation of biological toxins and decontamination of surfaces. Generally, it is recommended to use a minimum of 0.5% NaOCl is necessary for inactivation of biological toxins (see chemical inactivation table below for specific recommendations). 

Notes
a. Steam autoclaving should be at >121°C for 1 h. For volumes larger than 1 liter, especially those containing Clostridium botulinum spores, autoclave at >121°C for 2 h to ensure that sufficient heat has penetrated to kill all spores.
e. Protracted steam autoclaving, similar to that described for BoNT, followed by incineration is recommended for disposal of Staphylococcal enterotoxin-contaminated materials.
i. Dry heat of >100ºC for 60 min in an ashing oven or steam autoclave treatment at >121ºC for 1 h
l. Autoclaving with 17 lb pressure (121-132º C) for 30 min failed to inactivate LMW toxins.17,19 All burnable waste from LMW toxins should be incinerated at temperatures in excess of 815°C (1,500º F).
o. Tam CC, Henderson TD, Stanker LH, He X, Cheng LW. Abrin Toxicity and Bioavailability after Temperature and pH Treatment. Toxins. 2017;9(10):320.
p. (1)Rasooly R, Do PM. Shiga toxin Stx2 is heat-stable and not inactivated by pasteurization. Int J Food Microbiol. 2010;136(3):290–4.
(2)Lumor SE, Fredrickson NR, Ronningen I, Deen BD, Smith K, Diez-Gonzalez F, et al. Comparison of the presence of Shiga toxin 1 in food matrices as determined by an enzyme-linked immunosorbent assay and a biological activity assay. J Food Prot. 2012;75(6):1036–42.

Notes:
ND indicates “not determined” from available decontamination literature.
a. Solutions of NaOCl (#0.1%) or NaOH (> 0.25 N) for 30 min inactivate BoNT and are recommended for decontaminating work surfaces and spills of C. botulinum or BoNT. Chlorine at a concentration of 0.3-0.5 mg/L as a solution of hypochlorite rapidly inactivates BoNT (serotypes B or E tested) in
water.20 Chlorine dioxide inactivates BoNT, but chloramine is less effective.
b. Ozone (> 2 mg/L) or powdered activated charcoal treatment also completely inactivate BoNT (serotypes A, B tested) in water under defined condition.
c. SEB is inactivated with 0.5% hypochlorite for 10-15 mi.
d. Ricin is inactivated by a 30 min exposure to concentrations of NaOCl ranging from 0.1-2.5%, or by a mixture of 0.25% NaOCl plus 0.25 N NaOH.17 In general, solutions of 1.0% NaOCl are effective for decontamination of ricin from laboratory surfaces, equipment, animal cages, or small spills.
e. The minimal effective concentration of NaOCl was dependent on toxin and contact time; all LMW toxins tested were inactivated at least 99% by treatment with 2.5% NaOCl, or with a combination of 0.25% NaOCl and 0.25N NaOH.
f. For T-2 mycotoxin and brevetoxin, liquid samples, accidental spills, and nonburnable waste should be soaked in 2.5% NaOCl with 0.25% N NaOH for 4 h. Cages and bedding from animals exposed to T-2 mycotoxin or brevetoxin should be treated with 0.25% NaOCl and 0.025 N NaOH for 4 h. Exposure for 30 min to 1.0% NaOCl is an effective procedure for the laboratory (working solutions, equipment, animal cages, working area and spills) for the inactivation of saxitoxin or tetrodotoxin. Decontamination of equipment and waste contaminated with select brevetoxins has been reviewed.
g. Conotoxins can also be inactivated using reducing agents such as dithiothreitol β- mercaptoethanol, or tris (2-carboxyethyl) phosphine (100 mM) at 65–100° C for 15 min, followed by alkylation with 100 mM maleimide in isopropanol at 65° C for 15 min. Alternatively, alpha conotoxins can be inactivated by hydrolysis in 10 N NaOH or HCl at 100° C for 30 min.
h. Exposure of crude abrin solution and dried abrin to 0.67% NaOCl eliminated over 90% of cytotoxicity within 5 min.

Chapter 11: Incident Response