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Toxins

Biosafety Manual - Appendix B. Biotoxin Safety Plan

All Investigators working with toxins of biological origin must complete a laboratory specific Appendix B. Biotoxin Safety Plan that includes: toxins used, known hazards, safety equipment, personal protective equipment, toxin inactivation, disposal, inventory control and security measures and emergency procedures (e.g., spills). Guidelines for working with toxins of biological origin can be found in Appendix I of the CDC/NIH Publication Biosafety in Microbiological and Biomedical Laboratories.

Possession and use of biologically-derived toxins must be registered with the Institutional Biosafety Committee (IBC) by using the Inventory and Activity Registration (IAR).

NOTE: that use possession, use, or transfer of defined, aggregate amounts of certain toxins may require registration as a Select Agent.

Training

Biotoxin Introductory Training is required before initiating work with any biological toxin (with and LD 50 of less than 100 micrograms per kilogram of body weight in vertebrates) requiring BSL-2 containment.

Biotoxin Inactivation

Biological toxins can be extremely hazardous even in minute quantities. Investigators must ensure that appropriate equipment and safety procedures are in place for the specific toxin used and the type of experiments performed in their laboratory.

Some toxins are inactivated by autoclaving for one hour at 121°C, while others are inactivated by exposure to sodium hypochlorite or sodium hypochlorite + sodium hydroxide. Generally, the higher molecular weight proteinacious bacterial toxins are inactivated by steam sterilization. Steam sterilization should not be used for destruction of any low molecular weight toxins (e.g. mycotoxins, marine and reptile venoms). Consult the table below or call EHS Biosafety at 2-4911 with questions.

Toxin Autoclave Dry Heat
(10 min)		 NaOCl
(30 min)		 NaOH
(30 min)		 NaOCl
+
NaOH
(30 min)
Abrin <100mg/PI* Yes   No   No
Anthrax Lethal Toxin (PA & LE)     10%    
Botulinum neurotoxins <0.5mg/PI* Yes >100°C >0.1% >0.25 N ND
Brevetoxin (PbTx-2) No >815°C >=2.5% ND 0.25% + 0.25N
Bungarotoxin Methods vary depending on structure, see MSDS provided by vendor
Clostridium perfringenes epsilon toxin <100mg/PI* Yes   Yes   Yes
Conotoxin Methods vary depending on structure, see MSDS provided by vendor
Diacetoxyscirpenol (DAS), T-2 <1000mg/PI* No >815°C >=2.5%   0.25% + 0.25N
Microcystines* No >260°C >=0.5% ND 0.25% + 0.25N
Palytoxin No >260°C >=0.1% ND 0.25% + 0.25N
Pertussis toxin Acids or bases can be used, see MSDS provided by vendor
Ricin <100mg/PI* Yes >100°C >1.0% ND >0.1% + 0.25N
Saxitoxin <100mg/PI* No >260°C >=0.1% ND 0.25% + 0.25N
Shiga toxin & Shiga-like ribosome inactivating proteins <100mg/PI* Yes   Yes   Yes
Staphylococcal enterotoxins <5mg/PI* Yes >100°C; refolds >0.5% >0.25N ND
Tetrodotoxin <100mg/PI* No >260°C >=0.5% ND 0.25% + 0.25N
Diphtheria Toxin1 Yes   Yes Yes Yes
Tetanus Toxin2,3 Yes   N/A   N/A

*Some biological toxins are classified by the Federal Government as Select Agents due to their potential to pose a severe public health threat. The possession, use and transfer is highly regulated. In the quantities listed, they are exempt from regulation. Call EHS at 982-4911 for guidance and consult http://www.cdc.gov/od/sap

1 A solution of 0.25N – 1 N NaOH may be used for surface decontamination
2 2.5% NaOCl may be used for surface decontamination
3 Although some technical references provide recommendations for chemical inactivation and surface decontamination, there is no consensus regarding the most effective type and concentration. Thus, inactivation by autoclaving is required.

Chemical Destruction of Biotoxins

When using sodium hypochlorite or sodium hypochlorite+sodium hydroxide to destroy toxins, work in a fume hood or a biological safety cabinet and wear a long sleeved lab coat or gown, gloves and eye protection.

  1. Place plastic backed absorbent paper (bench diaper) on the work surface.
  2. If not already in liquid form, put the toxin into solution.
  3. Place the toxin container in a secondary container, such as a beaker or rack.
  4. Add an equal volume of the sodium hypochlorite(or sodium hypochlorite+sodium hydroxide) to the primary container of toxin solution.
  5. Do not replace the cap on primary container.
  6. Place a "WARNING / DO NOT USE" sign on the hood/cabinet.
  7. Allow a minimum 60 minutes exposure time. (See table for additional exposure time recommendations.)
  8. Secure the cap on the primary container. Double bag the material in zip-lock plastic bags and label it "Inactivated/denatured (TOXIN NAME)".
  9. Complete a waste slip listing the toxin as "inactivated" and contact EHS st 982-4911 for disposal as hazardous waste.

Steam Sterilization (Autoclaving) of Toxins

Work In a fume hood or a biological safety cabinet and wear a long sleeved lab coat or gown, gloves and eye protection.

  1. Loosen the cap of the primary toxin container to allow steam penetration.
  2. Place the primary container into a secondary disposable container (e.g.: small sharps container).
  3. Place the sharps container in a loosely closed autoclave bag.
  4. Place the bag in an autoclavable pan.
  5. Autoclave at 121° C for 1 hour on liquid cycle (slow exhaust).
  6. Discard the biohazard bag with its contents in a CMC.

Access and Inventory Control

An inventory control system must be in place to account for usage and disposition of biotoxins.

This inventory log should list:

  • date and time;
  • name of worker;
  • purpose of biotoxin use;
  • amount removed and amount remaining.

Primary containers should be labeled with name and concentration of biotoxin. Secondary storage equipment (e.g. cabinets, refrigerators, freezers) should be labeled with a biohazard sticker indicating the type of biotoxin(s) and contact information for the Principal Investigator. They should be closed and locked when biotoxin is not in use. The laboratory should be closed and locked at the end of the work period. Lab doors will have hazard signs which include contact information for trained and responsible staff.

Biotoxins should be used only in designated rooms approved by the Institutional Biosafety Committee (IBC) with controlled access and at predetermined lab bench areas. The room should be clearly posted with an approved UVa biohazard sign, indicating that "biotoxins are in use, authorized personnel only." Untrained personnel and visitors must be monitored and protected from biotoxin exposure. To minimize risk of exposure for untrained staff, non-related and nonessential work should be restricted in areas where and when biotoxins are in use. Usage of biotoxins at a quantity or in procedures with increased risk should be conducted only when another trained individual is available to observe processes.

Any unexpected, undocumented loss of biotoxin should be reported immediately to the Principal Investigator. A report should then be made to EHS Biosafety and security.

References

Wannemacher R.W. 1989. Procedures for Inactivation and Safety Containment of Toxins. Proc. Symposium on Agents of Biological Origin, U.S. Army Research, Dev. and Engineering Center, Aberdeen proving Ground, MD. pp. 115-122

Biological Safety Principles and Practices, 3rd Edition, Edited by Diane O. Fleming and Debra L. Hunt ASM Press 2000

Factsheets on Chemical and Biological Warfare

CDC/NIH Publication Biosafety in Microbiological and Biomedical Laboratories