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4.23 Toxins of Biological Origin (Biotoxins) - Handling and Management

Biotoxins may cause death or severe incapacitation at relatively low exposure levels. They are produced by living organisms, but they do not self-replicate. Predominantly they are not man-made but are of natural origin; however, biological toxins are increasingly being synthesized by modern methods. They differ from chemical toxins in that they do not pose a vapor hazard and only a few (e.g., mycotoxins) are dermally active.

Typical research applications of biological toxins include: use as a growth factor in cell culture media (e.g., cholera toxin), to produce specific neurologic effects (e.g., tetrodotoxin), and to produce localized tissue destruction (e.g., diphtheria toxin).

Possession and research use of biological toxins or venoms requires:

  • Registration with, and approval by the Virginia Tech IBC for research use of the toxin
  • Maintaining a toxin inventory record to account for current quantities in house, and toxin use and disposition
  • Storage of toxins in sealed, labeled containers within a secured (locked) storage device that can only be accessed by personnel authorized to work with the toxin
  • Use of toxins only in designated rooms with posted signage to control/limit access

 

4.23.1 Biotoxins as Select Agents and the Due Diligence Provision

Some biotoxins 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 is highly regulated.

In small quantities, some of these agents are exempt from Select Agent registration but must still be registered with, and approved for use by, the Virginia Tech IBC.

  • Toxin: Abrin | Exempted Amount: ≤ 100 mg
  • Toxin: Botulinum neurotoxins | Exempted Amount: ≤ 0.5 mg
  • Toxin: Short, paralytic alpha conotoxins (containing the amino acid sequence X1CCX2PACGX3X4X5X6CX7) | Exempted Amount: ≤ 100 mg
  • Toxin: Diacetoxyscirpenol (DAS) | Exempted Amount: ≤ 1000 mg
  • Toxin: Ricin | Exempted Amount: ≤ 100 mg
  • Toxin: Saxitoxin | Exempted Amount: ≤ 100 mg
  • Toxin: Staphylococcal enterotoxins (Subtypes A, B, C, D, E) | Exempted Amount: ≤ 5 mg
  • Toxin: T-2 toxin | Exempted Amount: ≤ 1000 mg
  • Toxin: Tetrodotoxin | Exempted Amount: ≤ 100 mg

Because it would be possible to stockpile toxins in multiples of these amounts which are small enough to be excluded from regulation, a “Due Diligence” provision was developed in 2014, and places the following responsibilities on Principal Investigators, veterinarians, or medical doctors:

IF YOU POSSESS ANY OF THESE TOXINS, YOU MUST:

  1. Declare your possession and provide inventory documentation to Virginia Tech’s Responsible Official (RO) and Alternate Responsible Official (ARO).
  2. Contact the RO and/or ARO to schedule a SHORT informational session regarding requirements related to possession of these toxins.
  3. If no additional toxin is received after the declaration date, but at least annually, you must verify and declare your possession to Virginia Tech’s RO and ARO.
  4. Immediately notify Virginia Tech’s RO and ARO if your toxin inventory amounts, per toxin, ever exceed specified permissible limits.

IF YOU TRANSFER ANY OF THESE TOXINS TO ANOTHER ENTITY, YOU MUST:

  1. Apply due diligence in assessing/ensuring that the recipient of these toxins has a legitimate need to handle/use such toxins.
  2. Complete documentation prior to the transfer which records your information and the recipient’s intended use of the toxin. The Notification of Toxin Transfer form (template can be found in the Appendices of this manual) must be submitted to Virginia Tech’s RO and ARO.
  3. Immediately report to Virginia Tech’s RO and ARO, if a violation of federal law is detected, or if suspicious activity related to the toxin is discerned.

IF YOU RECEIVE ANY OF THESE TOXINS FROM ANOTHER ENTITY, YOU MUST:

  1. Immediately notify Virginia Tech’s RO and ARO of the receipt of the toxin, update your inventory, and submit inventory documentation to the RO and ARO.

 

CONTACTS

  • RO: Charlotte Waggoner
    Phone: 540-231-5864
    Email: ren@vt.edu
    Mail Code: 0423

  • ARO: Anna Kroner
    Phone: 540-231-1122
    Email: akroner@vt.edu
    Mail Code: 0423

 

4.23.2 Biotoxin Risk Assessment

Biotoxins are assigned Biosafety Levels (which prescribes type of containment, PPE, work practices and safety equipment to use) by the same risk assessment process as biological agents.

Main occupational risks in the lab include:

  • Accidental exposure by direct contamination of mouth, eyes or other mucous membranes
  • Inadvertent aerosol generation, such as when reconstituting a toxin in dry powder form
  • Needlesticks or other accidents that compromise the skin, such as could happen when inoculating animals

Risk assessments on biotoxins should include:

  • Biotoxin characteristics (LD50 in solution and dry form; solubility)
  • Risks inherent to experimental procedures and manipulations (e.g., opportunities for accidental needlesticks, the likelihood of dispersal from static build-up when working with powder form, etc.)
  • Total amount of toxin used relative to the estimated human lethal or cytotoxic dose
  • Volume of material manipulated
  • Availability of successful treatment, vaccines or antitoxins
  • Training and experience of personnel

If toxins/ infectious agents/ animals are used in combination, then risks in all of those areas must be considered collectively in the selection of containment equipment and the development of safety procedures.

Lab workers are permitted to handle toxins when they have:

  1. demonstrated proficiency in the pertinent lab procedures and animal handling techniques, if applicable;
  2. completed all required general training and lab-specific training on toxin use with the PI or designee
  3. completed and submitted the Virginia Tech Medical Survey to the Virginia Tech Occupational Health Program.

Complex operations should be rehearsed without the use of live toxin in supervised practice runs until proficiency is assured.

Safety measures should be selected according to a risk assessment for each manipulation involving the toxin. For example, if there is a skin-absorption risk in working with a toxin, gloves/sleeves must be used that are impervious to the toxin and the diluents/solvents used; if there’s a splash or droplet hazard, safety glasses/goggles and face shield must be used.

After risk assessment review, selected operations with toxins may require modified BSL-3 procedures; typically, routine operations (e.g., toxin preparation/ animal procedures) with dilute toxin solutions can be conducted under BSL-2 conditions using 1) a certified BSC or chemical fume hood, and 2) appropriate PPE for the hazards involved.

 

4.23.3  Mitigating Biotoxin Hazards

Inhalation Risks

  • When working with dried toxin, remove all items that are not necessary for your procedure from the biosafety cabinet or fume hood before handling the toxin to reduce the potential for contamination of item surfaces.
  • Respiratory protection may be needed if you must manipulate a dry toxin in an open vessel, even if in containment. Consult with EHS.
  • Primary containers of the toxin should be non-breakable if possible, and vials should be maintained in a closed secondary container that will not allow escape of the product even if dropped. The risk of release is greatly decreased by using a secondary container at all times.


 

Sharps Risks – Accidental Injection or Cut

  • Use only syringes with luer-lock or integrated needles.
  • Use vial adapters whenever possible as a substitute for using needles to add diluent through septums on vials.
  • When introducing a needle through a septum, assure that the vial is secured with a device that allows the non-dominant hand to be outside of the “strike zone” of the needle. (Example: secure vial in a rack, or use a clamp to hold vial instead of holding it directly.)


 

Personal Protective Equipment

  • Wrap-around disposable gowns with gathered cuffs are the best choice, and fluid-resistant gloves rated for protection against diluent should be used.
  • Double-gloving is strongly recommended if it does not hinder the worker’s dexterity to the point of being unsafe.
  • There should be no unprotected, exposed skin on your body or extremities when undertaking this work. Arms, wrists and hands must be fully covered; long pants and whole shoes are required.
  • Safety glasses are strongly recommended, and must be washed after removal and storage.
  • Disposable PPE used for this work must be single-use only, and disposed of as hazardous lab waste.

 

4.23.4  Solubilizing and Using Biotoxins

Commercial preparations of toxins typically are in lyophilized, powdered form in crimped vials, topped by a rubber stopper. Individual labs sometimes re-package, aliquot and store the powder in vials or microcentrifuge tubes for later reconstitution. Either way, powdered toxin has to be solubilized in the lab, and this presents an exposure risk to lab workers due to the possibility of the powder being dispersed into the air.

General Precautions

  • Remove toxins from the fume hood or biosafety cabinet only after the exterior of the primary container has been decontaminated and placed in a clean secondary container.
  • Toxin solutions, especially concentrated stock solutions, must be transported in a leak/spill-proof secondary container.  
  • After a work session with a toxin in a fume hood or BSC, the interior surfaces of the unit must be thoroughly decontaminated; until this can be done, a sign must be posted on the hood or cabinet saying that the unit is awaiting decontamination, meanwhile toxin may be present so do not use.
  • Pressurized tubes or other containers holding toxins must be opened under containment of a BSC or fume hood.
  • Operations that expose toxin solutions to vacuum or pressure (e.g., sterilizing a toxin solution by membrane filtration) must be done in a BSC or fume hood.
  • Vacuum lines used with toxin must be protected by a HEPA filter to prevent entry of toxins into the vacuum system.
  • Centrifugation of materials containing toxins must be performed using sealed, thick-walled tubes, preferably in safety centrifuge cups or sealed rotors. After centrifugation, the rotor assemblies must be opened in a biosafety cabinet to remove tubes.
  • Work with dry toxin should be minimized or eliminated when possible.

Preparing Biotoxin Stock Solution from Powder in Crimped Vials:

  1. Work in a chemical fume hood.
  2. Wear buttoned lab coat or disposable gown, and use gloves that are resistant to the diluent you are using.
  3. Add diluent though the rubber stopper using a luer-lock or one-piece needle and syringe.
  4. If necessary, allow the pressure differential within the vial to dissipate by withdrawing the needle above the meniscus and allowing the syringe plunger to be displaced.
  5. Once the liquid has been added to the vial and powder has been wetted, dispose of the needle and syringe. A needle should not be used for handling, dispensing or aliquoting of toxin-containing solution.

Preparing Biotoxin Stock Solution from Powder in Microcentrifuge Tubes:

  1. These containers are not meant to be penetrated by a needle. Opening the container top may lead to powder dispersal due to pressure differential, or static electricity. Spraying a static guard on gloves is highly recommended. 
  2. Gloves should be worn that are resistant to the diluent. In addition to gown, double-gloves, safety glasses and face shield, respiratory protection may be required.
  3. Open container slowly behind a plexiglass shield in a portion of the lab isolated for this purpose, with limited air flow. Do not open the container in the vigorous air flow of a hood or biosafety cabinet. Do not open the container completely, just far enough to add the diluent.
  4. After diluent is added, mix or vortex under a chemical fume hood. Once powder is wetted completely, the tube can be opened carefully without concern about air dispersal. When possible, tubes should be centrifuged to remove liquid drops from caps/lids.

 

4.23.5 Toxin Inactivation and Disposal

Depending on the toxin, toxin-contaminated materials and toxin waste solutions can be inactivated by incineration, extensive autoclaving, or by soaking in suitable decontamination solutions; there is no universal method of inactivation. Deactivating aqueous solutions of a toxin may not be effective for inactivating the same toxin in dry, powdered form. Inactivation procedures should not be considered completely effective unless validated using specific toxin bioassays. Always consult EHS when considering the safest and effective method of deactivation and disposal.

Solid waste items (e.g., gloves, waste vials, bench paper, etc.) must not be soaked in a liquid decontamination solution.

  • If a toxin is known to be inactivated by autoclaving, solid waste should be autoclaved before transport for final treatment and disposal. Consult EHS regarding a proper disposal method.
  • If toxin cannot be deactivated by autoclaving, collect as Regulated Medical Waste for Incineration Only, label the container as containing Toxin Waste, and request EHS waste pickup.

Sharps contaminated with a toxin may be disposed of in bio-sharps containers; label as containing Toxin Waste.

  • If liquid waste containing toxin cannot be inactivated by autoclaving or bleach treatment, then it must be collected as chemical hazardous waste. Be sure to label as Toxin Waste, and contact EHS for hazardous waste pickup.

 

4.23.6 Toxin Spill Response

Just as there is no universal inactivation method for toxins, spill response procedures must be tailored to the toxin being used, as characterized in the toxin’s risk assessment. In general, spill response should include:

  • Immediately notify others in the area of the spill.
  • Don appropriate PPE, including respiratory protection if required.
  • Isolate the area (area should already be restricted when toxin is in use).
  • Remove the breached container; if non-glass, place in autoclave bag inside secondary containment. Place broken glass in sharps container.
  • Treat, absorb and remove spill contamination by covering spill with paper towels saturated with a decontaminating solution. Allow contact for 20 minutes. Absorb and remove the contaminated towels using tongs or other tools to minimize direct contact. Place materials in an autoclave bag.
  • Decontaminate all impacted surfaces in the “splash zone.” Wait for the prescribed contact time to elapse before removing the residue. Use care to limit contact with PPE contaminated surfaces when removing PPE. Place PPE in autoclave bag.
  • All bagged or contained contaminated material should be labeled Toxin Waste and secured  until it can be autoclaved, or in Regulated Medical Waste for Incineration Only containers, until EHS can pick up the waste.