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Biological Toxins Guidance

Biological toxins are produced by certain bacteria, fungi, protozoa, plants, reptiles, amphibians, fish, echinoderma (spiny urchins and starfish), mollusks, and insects.

The following table lists the LD50 values for commonly used biological toxins:

Toxin LD50 (µg/kg)*
Abrin 0.7
Aerolysin 7
Botulinin toxin A 0.0012
Botulinin toxin B 0.0012
Botulinin toxin C1 0.0011
Botulinin toxin C2 0.0012
Botulinin toxin D 0.0004
Botulinin toxin E 0.0011
Botulinin toxin F 0.0025
b-bungarotoxin 14
Caeruleotoxin 53
Cereolysin 40-80
Cholera toxin 250
Clostridium difficile enterotoxin A 0.5
Clostridium difficile cytotoxin B 220
Clostridium perfringens lecithinase 3
Clostridium perfringens kappa toxin 1500
Clostridium perfringens perfringolysin O 13-16
Clostridium perfringens enterotoxin 81
Clostridium perfringens beta toxin 400
Clostridium perfringens delta toxin 5
Clostridium perfringens epsilon toxin 0.1
Conotoxin 12-30
Crotoxin 82
Diphtheria toxin 0.1
Listeriolysin 3-12
Leucocidin 50
Modeccin 1-10
Nematocyst toxins 33-70
Notexin 25
Pertussis toxin 15
Pneumolysin 1.5
Pseudomonas aeruginosa toxin A 3
Ricin 2.7
Saxitoxin 8
Shiga toxin 0.25
Shigella dysenteriae neurotoxin 1.3
Streptolysin O 8
Staphylococcus enterotoxin B 25
Staphylococcus enterotoxin F 2-10
Streptolysin S 25
Taipoxin 2
Tetanus toxin 0.001
Tetrodotoxin 8
Viscumin 2.4-80
Volkensin 1.4
Yersinia pestis murine toxin 10
*Note that the LD50 values are from a number of sources (see below). For specifics on route of application (i.v., i.p., s.c.), animal used, and variations on the listed toxins, please go to the references listed below. (Table courtesy University of Florida EHSO)

References:

  1. Gill, Michael; 1982; Bacterial toxins: a table of lethal amounts; Microbiological Reviews; 46: 86-94
  2. Stirpe, ; Luigi Barbieri; Maria Giulia Battelli, Marco Soria and Douglas A. Lappi; 1992; Ribosome-inactivating proteins from plants: present status and future prospects; Biotechnology; 10: 405-412.
  3. Registry of toxic effects of chemical substances (RTECS): comprehensive guide to the 1997. Doris V. Sweet, ed., U.S. Dept of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health; Cincinnati, Ohio.

 Additional Biological Toxins

Toxins of Biological Origin
Aflatoxins DTX-1 (Dinophysistoxin-1) Palytoxin
Amanitin Echnioderm venoms – all Palytoxin
Amphibian venoms Endotoxins – all Pertussis toxins – all
Anatoxin A Enterobacteriaciae toxins – all Phalloidin
Anthrax toxin Enterotoxins – all Psilocybine
Aspergillus sp toxins Escherichia coli toxins – all Pseudomonas sp toxins
Bacillus sp toxins – all Exotoxin A Reptile venoms – all
Bordetella sp toxins Fish venoms – all Resiniferatoxin
Botulinum toxins – all Fusarium sp toxins Ricin toxins – all
Brevetoxins Gliotoxin Sapintoxin
Bungarotoxins Joco Spider Toxin JSTX – 3 Sarafotoxin
Cardiotoxin Lappaconitines Saxitoxin
Charybdotoxin Leiurotoxins Short Neurotoxins
Cholera toxins – all Lipid A – all types Snake Venoms – all
Ciguatera toxin Lipopolysaccharides from all species Stable toxins
Clostridia species toxins – all Maitotoxin Staphylococcous sp toxins
Cluepeotoxins Medamine Streptonigrin
Cobra venoms and all derived toxins Microcystins Taipoxin
Cobratoxin Mojave toxin Tetanus toxins – all
Conotoxins – all Mycotoxins – all Tetrodotoxins – all
Crotamine Myotoxins Textilotoxin
Dendrodotoxins Neurotoxins – all Thymeleatoxin
Dinoflagellate neurotoxins Notexin Tinyatoxin
Diphtheria toxins Nodularin Toxin II – all types
Domoic acid Ochratoxin

Toxins Classified as Select Agents

Some biological toxins are classified by the CDC and USDA as Select Agents (http://www.selectagents.gov/index.html or https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/animal-and-animal-product-import-information/sa_ag_select_agent ) due to their potential to pose a severe threat to public health and safety. Possession, use, and transfer of these toxins are highly regulated. A complete list can be found at http://www.selectagents.gov/SelectAgentsandToxinsList.html.

In small quantities, some of these toxins are exempt from select agent registration. See the table below.   Exempt Amounts of Select Agent Toxins Permissible Per Principal Investigator.

HHS (CDC-listed) Toxins Exempted Quantity
Abrin 1,000 mg
Botulinum neurotoxin 1 mg
Conotoxins (Short, paralytic alpha conotoxins containing the following amino acid sequence X1CCX2PACGX3X4X5X6CX7) 100 mg
Diacetoxyscirpenol (DAS) 10,000 mg
Ricin 1,000 mg
Saxitoxin 500 mg
Staphylococcal enterotoxins A,B,C,D,E subtypes 100 mg
T-2 toxin 10,000 mg
Tetrodotoxin 500 mg

Because they can be extremely hazardous, even in minute quantities, biological toxins require strict safeguards against their inhalation, absorption through skin or mucous membranes (typically due to a splash), ingestion, or percutaneous injury. A chemical hygiene plan and MSDS specific for the toxin must be on hand in the laboratory for inspection.

Key points of the guidelines are:

  1. Written safety protocols to cover the use of the specific toxin(s) in use;
  2. Security measures in place to protect against unauthorized access to toxin(s);
  3. Inventory control system in place; all entries in a hardbound book, in ink;
  4. Written plan for toxin-related emergencies (spill, exposure, ) posted; and
  5. BSL-2 or BSL-3 containment and practices in

Specific inactivation and disposal requirements should be in place for acute biological toxins. Some toxins are quite resistant to conventional methods of inactivation. These agents cannot be simply placed in an autoclave.

Toxins may be destroyed by several methods as shown in the table below. Some toxins are inactivated by autoclaving for one hour at 121°C. Others are inactivated by exposure to sodium hypochlorite and/or sodium hydroxide.

1.     Chemical Destruction of Toxins:

When using sodium hypochlorite and / or sodium hydroxide to destroy toxins, the procedure(s) must be performed in a laboratory fume hood or a biological safety cabinet. At a minimum, personal protective equipment for all procedures should include:

  • Long sleeved protective clothing (lab coat, gown)
  • Gloves
  • Eye protection
  1. If the toxin is classified as a select agent, even in exempt amounts, notify the Biosafety Office prior to the destruction of the agent.
  2. Work in a fume hood or biosafety cabinet with the sash at the lowest reasonable sash height for safe and effective work.
  3. Place plastic-backed absorbent paper (bench diaper) on the work surface of the fume hood or biosafety cabinet.
  4. CAREFULLY put the toxin into solution in the primary DO NOT USE A GLASS CONTAINER.
  5. Place the primary container in a secondary container, such as a beaker or rack.
  6. Slowly dispense an equal volume of the concentrations of sodium hypochlorite and/or sodium hydroxide designated in table 1 below into the primary container of toxin solution to be destroyed.
  7. Do not replace the cap on primary container.
  8. Place a “WARNING / DO NOT USE” sign on the hood/cabinet.
  9. Allow a minimum 60 minutes exposure (See table 1 below for additional exposure time recommendations.)
  10. Document the destruction of the toxin in the laboratory inventory log book.
  11. Secure the cap on the primary DOUBLE BAG the material in zip- lock plastic bags and label it “Inactivated/denatured (TOXIN NAME)“.
  12. Contact the Biosafety Office or EHS for disposal as hazardous waste.

2.     Steam Sterilization (Autoclaving) of Toxins:

If acceptable as a method in table 1 below, destroy toxins by autoclaving them using the procedure outlined below:

  1. If the toxin is classified as a select agent, even in exempt amounts, notify the Biosafety Office prior to the destruction of the agent.
  2. In a fume hood or biological safety cabinet, loosen the cap of the primary toxin container to allow steam penetration.
  3. Place the primary container into a secondary biohazard container.
  4. Place the container in a loosely closed biohazard bag.
  5. Place the bag in an autoclavable pan.
  6. Autoclave at 121° C for 1 hour on liquid cycle (slow exhaust).
  7. Document the destruction of the toxin the laboratory inventory logbook.
  8. After autoclaving, allow time for materials to cool before handling.
  9. Discard the autoclave bag and its containers as treated biological waste.

DO NOT use steam sterilization for destruction of any of the low molecular weight toxins (i.e. mycotoxins, marine and reptile venoms).

3.     Inactivation Procedures for Selected Toxins

Allow at least a 60-minute chemical contact time for complete inactivation of toxin. Any procedure labeled “yes” is an approved procedure for inactivation of the toxin specified.

Toxin Autoclave (1 hour @ 121° C, liquid exhaust) 2.5% NaOCL +0.25 N NaOH 1.0%NaOCl 2.5%NaOCl
Abrin (1)(8) Yes N/A N/A N/A
Botulinum Neurotoxin (1) (7) Yes Yes Yes Yes
Clostridium perfringens epsilon toxin (2) Yes N/A N/A N/A
Conotoxin(3) CONTACT the Biosafety Office
Diacetoxyscirpenol(5) No Yes No Yes (3-5%)
Ricin (1)(7) Yes Yes Yes Yes
Saxitoxin(1)(7) No Yes Yes Yes
Shigatoxin & Shiga- like ribosome inactivating proteins(4) Yes Yes Yes Yes
Staphylococcal Enterotoxins (1)(7) Yes Yes Yes Yes
Tetrodotoxin(1)(7) No Yes Yes Yes
T-2 Toxin (1)(6)(5) No Yes No No

References:

  1. Wannemacher 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
  2. Factsheets on Chemical and Biological Warfare [PDF], https://www.dhs.gov/publication/biological-attack-fact-sheet
  3. For complete inactivation of T-2 mycotoxin extend exposure time for liquid samples, spills, and non-burnable waste in 5% sodium hypoclorite and 0.25 N sodium hydroxide to 4 hr. Expose cages/bedding from animals exposed to T-2 mycotoxin to 0.25% sodium hypochlorite and class=GramE>0.025 N sodium hydroxide for 4 hrs.
  4. For inactivation of saxitoxin, tetrodotoxin, ricin, botulinum toxin, or staphylococcal enterotoxins, expose work surfaces, solutions, equipment, animal cages, spills to 10% sodium hypochlorite for 60 minutes.
  5. http://www.inchem.org/documents/pims/plant/abruspre.htm

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