Methods of biotoxin inactivation vary, as toxin stability can be dependent upon temperature, pH, ionic strength, and other factors. Inactivation methods must be specific for the biotoxin and based on published or validated methods. Unpublished or novel inactivation methods (i.e. developed in-house) should not be assumed 100% effective without validation using specific toxin bioassays. Safety Data Sheets/SDS (formerly MSDS), which accompany all commercial biotoxin preparations, may include useful information regarding biotoxin inactivation or spill cleanup.

Biotoxin Contaminated Sharps and Labware

In the course of experimental activities with biotoxins, disposable labware such as gloves, absorbent paper, plastic pipettes, pipette tips, and emptied containers or tubes are considered "contaminated" and disposed as follows:

  • Place sharps directly into a red sharps container (do not autoclave or soak in inactivation solution).
  • Place contaminated labware items into a Contaminated Materials Container (CMC).
  • Follow standard EHS disposal procedures for CMC and Sharps waste pick up.

Steam Heat (Autoclaving)

Generally, many of the higher molecular weight, proteinacious bacterial biotoxins can be inactivated by autoclaving for 1 hour at 121°C. Autoclaving should not be used for destruction of low molecular weight biotoxins such as mycotoxins, marine and reptile venoms.

Important: Preparation of biotoxin containers for autoclaving should be performed in a Biosafety Cabinet or Chemical Fume Hood while wearing a lab coat/gown and gloves. Manipulation of biotoxins in dry/lyophilized form presents a significant hazard that may require use of respiratory protection; refer to the laboratory-specific biotoxin precautions outlined in the IAR (protocol).

  1. Loosen the cap of the primary biotoxin container(s) to allow steam penetration.
  2. Place primary container into a small, secondary disposable container (e.g., Tupperware to catch inadvertent spills).
  3. Place the secondary container into a clear autoclave bag (loosely taped/twist tied).
  4. Place the bag in an autoclavable pan labeled with the biohazard symbol.
  5. Autoclave at 121°C, 15 psi for 1 hour on liquid cycle (slow exhaust).
  6. Discard the autoclave bag with its contents into a Contaminated Materials Container (CMC).

Chemical Inactivation using Sodium Hypochlorite Solutions (Bleach)

Introduction of solutions containing sodium hypochlorite is one of the easiest ways to inactivate many biotoxins. Alternative chemical destruction methods can be found in BMBL, Appendix I.

Important: Chemical inactivation procedures must be performed in a Biosafety Cabinet or Chemical Fume Hood while wearing a lab coat/ gown and gloves. Manipulation of biotoxins in dry/lyophilized form presents a significant hazard that may require use of respiratory protection; refer to the laboratory-specific biotoxin precautions outlined in the IAR (protocol).

  1. Put the biotoxin into solution, if not already in liquid form.
  2. Use freshly prepared bleach solutions (stock bleach should be within manufacturer's expiration date).
  3. Place primary container into a small, secondary disposable container (e.g., Tupperware to catch inadvertent spills).
  4. Add an equal volume of bleach to the primary container to achieve a minimum 2.5% NaOCL concentration.
  5. Do not replace the cap on primary container.
  6. Inform coworkers (especially those who may use the BSC or CFH) that a biotoxin inactivation is in progress.
  7. Allow a minimum 30 minute exposure time.
  8. Dispose of inactivated liquids down the sink.

Table 1. Recommended Biotoxin Inactivation Methods for Liquid or Dry Stocks
Note: Consult Safety Data Sheet (SDS) where methods vary.

Biotoxin Autoclave (121°C /15 psi/60 min) 2.5% NaOCL (w/v) (Equivalent to a 50% Bleach Solution)
Abrin Yes No
Anthrax Lethal Toxin (PA & LF) &
Anthrax Edema Toxin (PA & EF)
Methods vary; see
vendor/manufacturer SDS
Yes
Botulinum neurotoxins Yes Yes
Brevetoxin (PbTx-2) No Yes
Bungarotoxin Methods vary; see
vendor/manufacturer SDS
Methods vary; see
vendor/manufacturer SDS
Clostridium difficile – binary toxin C,
enterotoxin A, toxin A, toxin B
Yes Methods vary; see
vendor/manufacturer SDS
Clostridium perfringens, epsilon toxin Yes Methods vary; see
vendor/manufacturer SDS
Conotoxin* Methods vary; see
vendor/manufacturer SDS
Methods vary; see
vendor/manufacturer SDS
Diphtheria Toxin Yes Yes
Microcystines No Yes
Palytoxin No Yes
Pertussis toxin Yes Methods vary; see
vendor/manufacturer SDS
Pseudomonas exotoxin A Yes Methods vary; see
vendor/manufacturer SDS
Ricin Yes Yes
Saxitoxin No Yes
Shiga toxin & Shiga-like ribosome
inactivating proteins
Yes Methods vary; see
vendor/manufacturer SDS
Staphylococcal enterotoxins Yes Yes
Tetanus Toxin Yes Methods vary; see
vendor/manufacturer SDS
Tetrodotoxin (TTX) No Yes

*Conotoxin inactivation depends on the presence or absence of disulfide bonds. Conotoxins with disulfide bonds will be incubated with an excess of dithiothreitol (DTT) (usually 10-20mM buffered solution, pH8.7) for at least 1 hour at room temperature or 30 minutes at 50oC. Then, an equal volume of 50-100mM solution of iodoacetamide (buffered solution, pH8.7) will be added and incubated for 1 hour at room temperature.

 

Table 2. Bleach Dilutions

NaOCL % (w/v) Dilution of Liquid Bleach (~4-6% NaOCL) in water Bleach solution
0.1% 1 to 50 2%
0.25% 1 to 20 5%
0.5% 1 to 10 10%
1.0% 1 to 5 20%
2.5% 1 to 2 50%

Contact EHS (434-982-4911) for disposal of Mixed Waste (e.g., biotoxin waste mixed with radioactive waste) or other unique disposal situations.

 
 
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