Biologically-Derived Toxins

Introduction 

  • Biologically-derived toxins (BTs) are hazardous substances produced by plants, animals, microorganisms (including, but not limited to, bacteria, fungi, algae, or protozoa), or recombinant or synthetic molecules expressing a biological toxin.
  • BTs are serious laboratory hazards that are highly toxic in minute quantities. Many biological toxins are highly potent, and internalization of even relatively low doses may result in death or incapacitation (BMBL, 6th ed., pg. 470). 
  • BT routes of exposure are primarily inhalation, ingestion, skin or eye absorption, and injection. 
  • BTs do not pose a vapor hazard and do not have short-term exposure limits, ceiling limits, or time-weighted average concentrations. They are different from well-characterized chemical toxins and the unknown aspect of their properties must be considered in any risk assessment.
  • Batches of BTs from the same source can vary widely in activity/toxicity.
  • Conducting a risk assessment is key to developing and implementing an effective safety strategy for hazard mitigation when working with BTs.
  • A ‘zero level’ toxin exposure philosophy will be the goal.

Risk Assessment

Since each laboratory has its own unique facilities and equipment for work with toxins, it is important to perform a risk assessment to determine exactly which physical and operational practices are essential to ensure effective risk management for each specific toxin and laboratory operation. A pre-operational checklist is advised.

A risk assessment should include:

  • Amount of toxin and their associated organisms being worked with. If a toxin to be used is produced by an associated organism, the potential risk caused by the organism should be considered as well.  
  • Lethal dose (LD50).
  • Probability of aerosol generation (powder or liquid form).
  • Risks inherent to the procedure [inhalation of aerosols (intentionally and/or unintentionally created)], auto-inoculation during animal procedures, static build-up when working with powders, etc.).
  • Engineering controls.
  • Safety equipment availability and efficacy.
  • Availability and efficacy of personal protective equipment (PPE).
  • Intoxication/lethality dose data.
  • Health effects data (acute and chronic).
  • Availability of prophylaxis and/or treatment.
  • Training, experience of personnel, incident records.
  • Identification of specific hazards and plans to mitigate these hazards before the commencement of work with BTs.

 General Safety Practices

  • Provide all laboratory personnel with training specific to the toxins being used.
  • Experiments should be planned to eliminate or minimize work with dry toxin or toxin-containing formulations (e.g., lyophilized material freeze-dried preparations).
  • Ensure all staff are wearing appropriate personal protective equipment (PPE) while working with BTs.
  • Conduct routine operations with toxin stock solutions, or dilute protein or peptide toxin solutions under BSL2 conditions inside of a biosafety cabinet (BSC) or chemical fume hood that is certified annually, or comparable engineering controls as determined by risk assessment. 
  • HEPA filtration is required when conducting high risk operations/procedures with BTs such as:
    • Working with BTs in powdered form
    • Manipulating BTs in ways that intentionally generate dust or aerosols
  • High risk operations/procedures with BTs can be manipulated using a Class II BSC or with the use of secondary containment such as a disposable glove bag or glove box within a hood. 
  • When working with dry forms of the toxin, static-free disposable gloves must be worn. See instructions of glove selection
  • Develop a standard operating procedure (SOP) for toxins that covers all aspects of toxin work, as well as any associated organisms if applicable, including:
    • Basic handling and experimental protocols
    • Hazard identification
    • Exposure control plan including engineering control, personal protective equipment (PPE) requirements (e.g., lab coat, gloves, safety goggles, etc.). Additional PPE may be required based on the risk assessment.
    • Procurement
    • Distribution
    • Storage
    • Decontamination method for work surface and/or equipment
    • Toxin inactivation and waste disposal
  • Maintain an accurate inventory.
  • Store toxin stocks in locked storage rooms, cabinets, or freezers.
  • Transport toxins in the spill and leak-proof secondary containers.
  • Every effort must be made to work with less than 1/10th of one lethal dose (LD) of toxin.
  • Toxin laboratories must be maintained at a negative air pressure with respect to adjoining rooms, corridors and public areas.
  • Whenever possible, reconstitute the entire vial of powdered toxin by injecting diluent through a septum.
  • Two knowledgeable individuals should be present in the laboratory during toxin manipulation whenever high-risk procedures are performed.
  • Gloves must be selected so that they do not generate static electricity and are impervious to both the toxin and the solvent in the suspension. See instructions of glove selection.
  • Verify inward airflow of the biological safety cabinet or chemical fume hood before initiating work.
  • Post the entrance(s) to the room and equipment for use or storage of toxins with signage stating “Toxins in Use—Authorized Personnel Only.” and indicate any special entry or personal protective equipment (PPE) requirements when toxins are in use.
  • Decontaminate the exterior of the primary toxin container and place in a clean secondary container before removing from the BSC or chemical fume hood. Transport toxins in leak/spill-proof secondary containers.
  • Decontaminate the work surface and/or equipment used for toxin work with a proven, effective inactivating agent after each use (see tables 1 & 2).

Toxin Inactivation

Table 1: Inactivation of Proteinanceous Biological Toxins

Toxin 
(proteinaceous)

Autoclave 
1hr @ 121°C

NaOCl 
(30-min)

NaOH 
(30-min)

NaOCl+NaOH 
(30-min)

Comments

Abrin(1) Yes >=0.7% ND ND Exposure of crude abrin solution and dried abrin to 0.67% NaOCl eliminated over 90% of cytotoxicity within 5 min.
Botulinum neurotoxins(1) Yes >=0.1% >=0.25N ND >=0.1% NaOCl or >=0.25N NaOH for 30-minute is recommended for decontaminating work surfaces and spills.
Clostridium  perfringens epsilon toxin(2) Yes 0.5% ND ND 0.5% NaOCl is recommended for spills.
Staphylococcal enterotoxins(1) Yes >=0.5% >=0.25N ND SEB is heat stable. SEB is inactivated with 0.5% NaOCl for 10–15/min.
Ricin(1) Yes >=1.0% ND >0.1%+0.25N 1.0% NaOCl is effective for decontaminating surfaces, equipment, animal cages, or small spills.
Shiga toxin and Shiga-like ribosome inactivating proteins(1)(2) Yes >=0.5% ND 2.5%+0.25N 0.5% NaOCl is recommended for spills.
Diphtheria toxin(2) Yes 0.5% ND ND 0.5% NaOCl is recommended for spills.
Tetanus toxin(2) Yes 0.5% ND ND 0.5% NaOCl is recommended for spills.
Pertussis toxin(2) Yes 0.5% ND ND 0.5% NaOCl is recommended for spills.
Cholera toxin(2) Yes 0.5% ND ND 0.5% NaOCl is recommended for spills.
Anthrax Lethal Toxin (PA, LE)(2) Yes >=0.5% ND ND 0.5% NaOCl is recommended for spills.

Table 2: Inactivation of Low Molecular Weight Toxins

Toxin 
(low molecular weight)

Autoclave 
1hr @ 121°C

NaOCl 
(30-min)

NaOH 
(30-min)

NaOCl+NaOH 
(30-min)

Comments

Tetrodotoxin(1) No >=0.5% ND 0.25%+0.25N 1.0% NaOCl for 30-min. contact time is effective for decontaminating non-incinerable waste, equipment, animal cages, work area, and spills. Solid Tetrodotoxin waste must be disposed of in a yellow bag for incineration*.
Saxitoxin(1) No >=0.1% ND 0.25%+0.25N 1.0% NaOCl for 30-min. contact time is effective for decontaminating non-incinerable waste, equipment, animal cages, work area, and spills. Solid Saxitoxin waste must be disposed of in a yellow bag for incineration*.
T–2 mycotoxin(1) No >=2.5% ND 0.25%+0.25N For T–2 mycotoxin and brevetoxin, liquid samples, spills, and non-incinerable waste should be soaked in 2.5% NaOCl with 0.25N NaOH for 4 hrs. Cage and animal bedding should be treated with 0.25% NaOCl and 0.25N NaOH for 4 hrs. The incinerable waste must be disposed of in a yellow waste bag*. 
Diacetoxyscirpenol (DAS), Deoxinalenol (DON), Zearalenone (ZEA)(1) No >=2.5% ND 0.25%+0.25N Dispose solid items contaminated with DAS, DON, and ZEA in a yellow waster bag. 2.5% NaCOl is recommended for spills.
Microcystin(1) No >=0.5% ND 0.25%+0.25N See note 3.
Palytoxin(1) No >=0.1% ND 0.25%+0.25N See note 3.
Brevetoxin(1) No >=2.5% ND 0.25%+0.25N 2.5% NaOCl is recommended for spills.
Conotoxin(3)   0.5%     Conotoxins containing S=S bonds (e.g., omega-conotoxin) can also be inactivated by 1% v/v glutaraldehyde or formaldehyde (30-min. contact time and sewer). Decontaminate work surfaces with 10% bleach. Dispose of used pipette tips in 10% bleach or 1% v/v glutaraldehyde. Dry waste (e.g., gloves) can be autoclaved at 121°C for 1hr at 18 psi. Additionally alpha conotoxins are inactivated by 10N NaOH(1)

*According to BMBL 6th edition, Appendix I — Guidelines for Work with Toxins of Biological Origin, autoclaving with 17 lbs. pressure (121–132°C) for 30-min. failed to inactivate low molecular weight (LMW) toxins. All incinerable waste from LMW toxins must be incinerated at temperatures in excess of 815°C (1,500°F). Dispose of LMW waste in yellow bags in barrels for incineration off site.

Notes: 
1. ND indicates "not determined" 
2. Household bleach generally is a 5.25% solution of sodium hypochlorite (NaOCl). A dilution of 1 part household bleach to 9 parts liquid is a 0.525% solution. 
3. All low molecular weight toxins tested were inactivated at least 99% by treatment with 2.5% NaOCl, or with a combination of 0.25% NaOCl and 0.25 NaOH. Alkalinity, rather than oxidizing ability, is the inactivation factor.  Waste from LMW toxins must be disposed of in yellow bags for off-site incineration.

References for toxin inactivation methods:

  1. CDC/NIH BMBL 6th edition, Appendix I—Guidelines for Work with Toxins of Biological Origin
  2. List Biological Laboratories, Inc. MSDS
  3. Manual of Security Sensitive Microbes and Toxins, Part IV Microbes and Toxins Affecting Human and Animals: Toxins, 41. Conotoxins, page 467.

Spill Clean-Up 

Spill clean-up procedures for a solution containing biological toxin:

  • Restrict access to the spill area until the decontamination is complete.
  • Wear appropriate PPE (lab coat, gloves, safety goggles). Additional PPE may be required based on risk assessment.
  • Cover the spill with dry paper towels or other disposable, absorbent material to avoid splashes or generating aerosols.
  • Apply an appropriate disinfectant (see Tables 1 & 2 above) to the spill, beginning at the perimeter and working towards the center.
  • Allow 30 minutes of contact time.
  • Pick up any sharps, including broken glass, with forceps and place in a sharps container.
  • Wipe spill area with disinfectant and clean paper towel. Discard paper towel in a biohazard bag.
  • Discard gloves in the biohazard bag. Wash hands.

Spill clean-up procedures for toxin powder:

  • Because of increased risk of inhalational exposure, besides lab coat, glove, and safety goggles, respiratory protection should be used.
  • If a spill occurs in the BSC, gently cover the powder with damp absorbent paper towels to avoid raising dust.
  • Apply the appropriate disinfectant starting at the perimeter and working toward the center, allowing 30 minutes of contact time as specified in Tables 1 & 2 above.
  • Pick up any sharps, including broken glass, with forceps and place in a sharps container.
  • Wipe spill area with disinfectant and clean paper towel and then wash with soap and water. Discard paper towel in a biohazard bag.
  • Discard gloves in the biohazard bag. Wash hands.
  • If spill occurs outside the BSC, immediate evacuation of the area is required. The spill should be managed and decontaminated as above.

Exposure Response

  • For any potential exposures involving BTs (needlesticks, contamination of non-intact skin, splashes to eyes, face, and mucous membranes), seek medical attention immediately; see the Clinical Services  page for providers or the individuals may see their health care provider of choice.
  • Report all exposure incidents to your supervisor and fill out an online First Report of Injury (eFROI)
  • Fill out an IBC Incident Report if the incident occurred while working under an IBC protocol.

Regulated Toxins

Toxin users must consult the following lists to determine whether the type and amount of toxin used fall under the Federal Select Agent Regulations:

If the Select Agent Regulations apply in your case, contact the Biosafety & Occupational Health Dept. (BOHD) at (612) 626-6002.

Institutional Biosafety Committee Oversight

Before work is started, Institutional Biosafety Committee (IBC) approval is required for the use of:

  • Unfractionated mixtures and purified preparations of biological toxins having high acute toxicity (i.e., a mammalian LD50 of less than or equivalent to 100 micrograms/kg body weight) or
  • Biological toxins with significant potential for serious subacute or chronic toxicity (e.g., carcinogenicity) or
  • Experiments involving genes or r/sNA mol sequences coding for any toxin molecules

Other Useful Information

For more information, see Guidelines for Work with Toxins of Biological Origin in Appendix I of the Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th edition.