Definition and Characteristics

Biotoxins are poisonous substances produced by living organisms (animals, plants, microbes) and are not man-made. Unlike most other biohazards, biotoxins do not replicate and, in certain respects, are comparable to classic organic chemicals. However, biotoxins differ from chemical toxins in that they do not pose a vapor hazard and few are dermally active (mycotoxins are the exception).

Risk Assessment and Biosafety Level

The Institutional Biosafety Committee (IBC) regulates the possession, use, and transfer of biological toxins with a mammalian LD50 of ≤ 100 mg/kg and the organisms that produce these biological toxins. More information and registration can be found on the Institutional Biosafety Committee Program page.
A risk assessment involving biotoxins takes into consideration:

1. Biotoxin characteristics (e.g., LD₅₀, in solution or dry form, solubility);
2. Risks inherent to experimental procedures and manipulations (e.g., auto inoculation, inhalation of unintentional aerosols, static build-up when working with powders);
3. Total amount of toxin used relative to the estimated human lethal or cytotoxic dose;
4. Volume of the material manipulated;
5. Exposure route;
6. Availability of successful treatment, vaccines, or antitoxins;
7. Training and experience of personnel.

The risk assessment defines the biosafety/containment level for work with the biotoxin and is a combination and integration of administrative controls, engineering controls, work practices, and PPE that mitigates the risk to an acceptable level.

Biotoxins with an LD₅₀ < 50 mg/kg (e.g., cholera toxin, microcystin, aflatoxin, and ricin) are classified as risk group 2 biotoxins requiring Biosafety Level 2 containment. Biotoxins with an LD₅₀ >50 mg/kg, such as Lipopolysaccharide (LPS), may be downgraded to Biosafety Level 1 depending on the procedures and how the biotoxin is being used.

Toxins Classified as Select Agents

Some biological toxins are classified by the federal government as Select Agents 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/. In small quantities (see below), some of these toxins are exempt from Select Agent registration but must still be registered with the IBC:

• Abrin, 1000 mg
• Botunlinum neurotoxins, 1 mg
• Short, paralytic alpha conotoxins, 100 mg
  
• Diacetoxyscirpenol (DAS), 10,000 mg
• Ricin, 1000 mg
• Saxitoxin, 500 mg
• Staphylococcal Enterotoxins (Subtypes A, B, C, D, and E), 100 mg
• T-2 toxin, 10,000 mg
• Tetrodotoxin, 500 mg

Handling Procedures

Proper handling of biotoxins poses special challenges but is vital for protecting laboratory and service personnel.

Solubilization of Biotoxins

Biotoxins are frequently stored in a lyophilized powdered form. Commercial preparations typically are shipped in crimped vials that are topped by a rubber stopper, while the individual laboratory may package and store the powder in vials or microcentrifuge tubes. Solubilizing the powder to formulate a stock solution presents a risk to the researcher because of the possibility of dispersal of the powder into the air. The protocols below are designed to minimize the risk of personnel exposure and environmental contamination.

When preparing biotoxin stock solution in crimped vials:

• Work in a chemical fume cabinet.
• Wear gloves that are resistant to the diluent.
• Add diluent through rubber stopper via syringe and needle (Luer Lock or one-piece needle and syringe).
• If necessary, allow pressure differential within the vial to dissipate by withdrawing the needle above the meniscus and allowing the syringe plunger to be displaced.
• Once the liquid has been added to the vial and the powder has been wetted, dispose of the needle and syringe. A needle should not be used for handling, dispensing, or aliquoting the toxin-containing solution.

Biotoxin stock solution preparation in microcentrifuge tubes:

• These containers are not meant to be penetrated by a needle-syringe. Opening the container top may lead to powder dispersal from pressure differential or static electricity. Spraying static guard on gloves is highly recommended. Two examples are Kensington Dust Guardian® Antistatic Spray Cleaner and KleenMaster Brillianize Cleaner and Polish.
• Gloves should be worn that are resistant to the diluent. In addition to the usual PPE, full-face protection should be worn to protect facial mucous membranes.
• Open the container slowly behind a Plexiglas shield, preferably in an isolated portion of the laboratory with limited air-flow. It is best not to open the container completely (usually a crack sufficient for adding the solvent/diluent will work).
• After adding the diluent, mixing or vortexing should be done in a chemical fume hood. Once the powder is wetted completely, the tube can be opened carefully without concern about air dispersal. Note: when possible, tubes should be centrifuged briefly to remove liquid drops from caps/lids.