Biologically-Derived Toxins

Introduction

  • Biological toxins (BTs) are products of plants, animals, microorganisms (including, but not limited to, bacteria, viruses, fungi, or protozoa), or infectious substances, or recombinant or synthesized molecules
  • BTs are serious laboratory hazards that are highly toxic in minute quantities
  • BT routes of exposure are primarily inhalation, ingestion, and absorption (such as ocular, percutaneous, and injection). Skin absorption is also a potential hazard with some BTs
  • 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
  • Risk assessment is key to developing and implementing an effective BT safety strategy
  • 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.

A risk assessment should include:

  • Amount of toxin being worked with
  • 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
  • Personal protective clothing and equipment availability and efficacy
  • Intoxication/lethality dose data
  • Health effects data (acute and chronic)
  • Availability of prophylaxis and/or treatment
  • Training, experience of personnel, accident records
  • Identification of specific hazards and mitigation of these before the commencement of work with BTs

 General Safety Practices

  • Provide all laboratory personnel with training specific to the toxins being used
  • Conduct routine operations with dilute toxin solutions under BSL2 conditions with personal protective equipment and a biological safety cabinet (BSC) that is certified annually or comparable engineering controls as determined by risk assessment
  • Develop a standard operating procedure (SOP) for toxins that covers all aspects of toxin work including:
    • Basic handling and experimental protocols
    • Procurement
    • Distribution
    • Storage
    • Decontamination and detoxification
    • Disposal
  • Maintain an accurate inventory
  • Store toxin stocks in locked storage rooms, cabinets, or freezers
  • Transport toxins in spill and leak-proof secondary containers
  • For toxin stock solutions and primary containers of dry toxins, conduct preparation, manipulation, and other high risk procedures in a biological safety cabinet (BSC) that is certified annually. 
    NOTE: Approvals will not be granted for future toxin work outside of a biological safety cabinet except where a detailed risk assessment makes a convincing case that risks will be mitigated through the use of alternative safety procedures when manipulating the toxin. This assessment will include, at a minimum, the amount of toxin to be used, the probability of aerosol generation, the route of exposure, and the lethal dose (LD).
  • HEPA filtration is required when conducting high risk operations/procedures with BTs such as:
    • Working with Biological Toxins in powdered form
    • Manipulating Biological Toxins in ways that intentionally generate dust or aerosols
  • 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 pressure compared to adjoining rooms, corridors or other areas
  • Whenever possible, reconstitute entire vial of powdered toxin by injecting diluent through septum
  • Two knowledgeable individuals should be present in the laboratory during toxin manipulation whenever high-risk procedures are performed
  • Gloves are to be selected that do not generate static electricity and are impervious to the toxin and solvent
  • Verify inward airflow of the biological safety cabinet before initiating work
  • Post the entrance(s) to the room with a biohazard sign identifying the hazard as “Biologically-Derived Toxin” and indicate any special entry requirements when toxins are in use
  • Decontaminate the exterior of the primary container and place in a clean secondary container before removing toxin containers from the BSC. Transport toxins in leak/spill-proof secondary containers

Toxin Inactivation

(January 2017)

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 Yes ND ND ND 176°F (80°C)  most of the toxicity is lost in 30-minutes.
Botulinum neurotoxins Yes >0.1% >0.25N ND >0.1% NaOCl or >0.25N NaOH for 30-min is recommended for decontaminating work surfaces and spils.
Clostridium pertringens epsilon toxin Yes ND ND ND Decontaminate with soap and water. The protein is heat-labile,
Staphylococcal entertoxins Yes >0.5% >0.25N ND SEB is heat stable. SEB is inactivated with 0.5% NaOCl for 10–15/min.
Ricin Yes >1.0% ND >0.1%+0.25N 1.0% NaOCl is effective for decontaminiating surfaces, equipment, animal cages, or small spills.
Shigatoxin and Shiga-like ribosome inactivating proteins Yes 0.5% ND 2.5%+0.25N 0.5% NaOCl is recommended for spills.
Diphtheria toxin Yes 0.5% ND ND 0.5% NaOCl is recommended for spills.
Tetanus toxin Yes 0.5% ND ND 0.5% NaOCl is recommended for spills.
Pertussis toxin Yes 0.5% ND ND 0.5% NaOCl is recommended for spills.
Clolera toxin Yes 0.5% ND ND 0.5% NaOCl is recommended for spills.
Anthrax Lethal Toxin (PA, LE) 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 No >=0.5% ND 0.25%+0.25N 1.0% NaOCl for 30-min contact time is effective for decontaminating nonburnable waste, equipment, animal cages, work area, and spills. The burnable waste from Tetrodotoxin should be disposed of in a yellow bag*.
Saxitoxin No >=0.1% ND 0.25%+0.25N 1.0% NaOCl for 30-min contact time is effective for decontaminating nonburnable waste, equipment, animal cages, work area, and spills. The burnable waste from Saxitoxin should be disposed of in a yellow bag*.
T–2 mycotoxin No >=2.5% ND 0.25%+0.25N For T–2 mycotoxin and brevetoxin, liquid samples, spills, and nonburnable waster 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 0.0.25N NaOH for 4 hrs. The burnable waste should be disposed of in a yellow waste bag*.
Diacetoxyscirpenol (DAS), Deoxinalenol (DON), Zearalenone (ZEA) 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 No >=0.5% ND 0.25%+0.25N See note 3.
Palytoxin No >=0.1% ND 0.25%+0.25N See note 3.
Brevetoxin No >=2.5% ND 0.25%+0.25N 2.5% NaOCl is recommended for spills.
Conotoxin   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 waster (e.g., gloves) can be autoclaved at 121°C for 1hr at 18 psi.

*According to BMBL 5th edition, Appendix I — Guidelines for Work with Toxins of Biological Origin, autoclaving with 17 lb pressure (121–132°C) for 30-min failed to inactivate low molecular weight (LMW) toxins. All burnable waste from LMW toxins should 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. The burnable waste from LMW toxins sould be disposed of in a yellow waste bag.

Spill Clean-Up 

Use an effective inactivating agent for a specific toxin (see tables 1 and 2 above). Follow the procedures in the Biological Decontamination & Spill Clean-up Plan Template to clean-up a spill. 

Exposure Response

  • Have all exposures (needlesticks, contamination of non-intact skin, splashes to eyes, face, and mucous membranes) evaluated by a medical professional; 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 occured 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 Selct Agent Regualtions:

If the Select Agent Regualtions apply in your case, contact UHS Biosafety at (612) 626-6002.

Institutional Biosafety Committee Oversight

All biologically-derived toxin work must be approved by the Institutional Biosafety Committee (IBC) before work is started. Approved protocols are effective for three years with annual review. 

Submit an application to the IBC. Follow the IBC step-by-step guidelines to help you complete each section of your application and reduce submission errors.

Other Useful Information

Toxin guidelines in Appendix I.