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DESTRUCTION OF SELECT AGENTS PROCEDURES

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Note: Federal law requires that Penn's Responsible Official notify the CDC or APHIS in advance of the destruction of Select Agent organisms or toxins. This notification must be coordinated through EHRS (The Office of Environmental Health and Radiation Safety).

I. NOTIFICATION OF THE PROPOSED DESTRUCTION OF SELECT AGENTS FORM:

Complete this form when proposing to destroy Select Agent organisms and/or toxins. Fax the completed form to EHRS at 215-898-0140 at least seven (7) working days in advance of the proposed destruction date. EHRS will notify CDC or APHIS of the proposed destruction. Once the proposed destruction is approved, EHRS will notify the PI or lab manager. Call EHRS at 215-898-4453 if you have any questions.

II. BACTERIA AND VIRUSES:

When destroying working cultures of Select Agent organisms, it is not necessary to notify EHRS, CDC, or APHIS. However, working cultures must be destroyed immediately after use. Accumulation of Select Agent organisms in infectious waste bags or sharps containers is prohibited.

When a laboratory intends to destroy all of its stock of a Select Agent organism, CDC or APHIS must approve the destruction prior to its occurrence. Follow the procedures below to destroy Select Agent organism stock cultures:

  1. Complete and sign the Notification of Proposed Destruction of Select Agents Form and fax it to EHRS 215-898-0140.


  2. EHRS will notify the PI or lab manager when destruction is approved.


  3. Use steam sterilization (autoclave) for destruction of bacteria and viruses. Autoclave organisms for a minimum of 1 hour at 121°C.


  4. Document the destruction of Select Agent organisms in the laboratory's Select Agent inventory logbook.


  5. Dispose of all autoclaved Select Agent organisms as infectious waste.

III. TOXINS:

Toxins may be destroyed by several methods as shown in Table I 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 toxin, 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 and eye protection
    1. Complete and sign the Notification of the Proposed Destruction of Select Agents Form and fax it to EHRS, 215-898-0140


    2. EHRS will notify the PI or lab manager when destruction is approved.


    3. Work in a fume hood or biosafety cabinet with the sash at the lowest reasonable sash height for safe and effective work.


    4. Place plastic backed absorbent paper (bench diaper) on the work surface of the fume hood or biosafety cabinet.


    5. Put the Select Agent toxin into solution in the primary container. DO NOT USE A GLASS CONTAINER.


    6. Place the primary container in a secondary container, such as a beaker or rack.


    7. Slowly dispense an equal volume of the concentrations of sodium hypochlorite and/or sodium hydroxide designated in Table I into the primary container of toxin solution to be destroyed.


    8. Do not replace the cap on primary container.


    9. Place a "WARNING / DO NOT USE" sign on the hood/cabinet.


    10. Allow a minimum 30 minutes exposure time. (See Table I below for additional exposure time recommendations.)


    11. Document the destruction of Select Agent toxin in the laboratory Select Agent inventory logbook.


    12. Secure the cap on the primary container. DOUBLE BAG the material in zip-lock plastic bags and label it "Inactivated/denatured (TOXIN NAME)" DO NOT REMOVE, EHRS WILL PICK UP.


    13. Contact EHRS 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. Complete and sign the Notification of the Proposed Destruction of Select Agents Form and fax it to EHRS, 215-898-0140.


    2. EHRS will notify the PI or lab manager when destruction is approved.


    3. In a fume hood or biological safety cabinet, loosen the cap of the primary toxin container to allow steam penetration.


    4. Place the primary container into a secondary biohazard sharps container.


    5. Place the sharps container in a autoclavable pan.


    6. Autoclave at 121° C for 1 hour on liquid cycle (slow exhaust).


    7. Document the destruction of Select Agent toxin the laboratory Select Agent inventory logbook.


    8. After autoclaving, allow time for materials to cool before handling.


    9. Discard the sharps container and its container as infectious waste.


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

All waste from toxins that is not disposed as infectious waste must be collected by EHRS for disposal as hazardous waste.

Table1
Inactivation Procedures for Select Agent Toxins

Allow at least a 30-minute chemical contact time for complete inactivation of toxin. Any procedure labeled "yes" is an approved procedure for inactivation of the toxin specified.

Select Agent Toxin Autoclave
(1 hour @ 121° C, liquid exhaust)
2.5% NaOCL + 0.25 N NaOH 0.1% NaOCl 1.0% NaOCl 2.5% NaOCl
Abrin (1)(8) Yes N/A N/A N/A N/A
Botulinum Neurotoxin (1) (7) Yes Yes Yes Yes Yes
Clostridium perfringens epsilon toxin (2) Yes N/A N/A N/A N/A
Conotoxin(3) Contact EHRS for details
Diacetoxyscirpenol(4) No Yes No No Yes (3-5%)
Ricin (1)(7) Yes Yes Yes Yes Yes
Saxitoxin(1)(7) No Yes Yes Yes Yes
Shigatoxin & Shiga-like ribosome inactivating proteins(5) Yes Yes Yes Yes Yes
Staphylococcal Enterotoxins (1)(7) Yes Yes Yes Yes Yes
Tetrodotoxin (1)(7) No Yes No Yes Yes
T-2 Toxin (1)(6) No Yes No No No
  1. Wannemacher R.W. 1989. Procedures for Inactivation and Safety Containment of Toxins. Proc. Symposium on Agents of Biological Origin, U.S. Army Research, Development and Engineering Center, Aberdeen proving Ground, MD. pp. 115-122
  2. Factsheets on Chemical and Biological Warfare, http://www.cbwinfo.com/Biological/Toxins/Cper.html
  3. Factsheets on Chemical and Biological Warfare, http://www.cbwinfo.com/Biological/Toxins/Conotox.html
  4. Factsheets on Chemical and Biological Warfare, http://www.cbwinfo.com/Biological/Toxins/Verotox.html
  5. Dr. Jack Dwayne Thrasher, An Introduction to Trichothecens,http://www.drthrasher.org/Introduction%20%to%20trichothecenes.html
  6. For complete inactivation of T-2 mycotoxin extend exposure time for all liquid samples, accidental spills, and non-burnable waste in 2.5% sodium hypoclorite and 0.25 N sodium hydroxide for 4 hr. Expose cages and bedding from animals exposed to T-2 mycotoxin to 0.25% sodium hypochlorite and 0.025 N sodium hydroxide for 4 hrs.
  7. For inactivation of saxitoxin, tetrodotoxin, ricin, botulinum toxin, or staphylococcal enterotoxins, expose work surfaces, working solutions, equipment, animal cages and spills to 1.0% sodium hypochlorite for 30 minutes.
  8. Fact Sheet from IPCS INCHEM

IV. PRIONS

Prions are extremely resistant to conventional inactivation procedures including irradiation, boiling, dry heat and chemicals (formalin, betapropiolactione, alcohols). Most procedures reduce infectivity rather than eliminate it. All treated contaminated materials should be discarded through the infectious waste stream and incinerated. Use DISPOSABLE plastic labware whenever possible.

  1. Complete and sign the Notification of the Proposed Destruction of Select Agents Form
  2. EHRS will notify the PI or lab manager when destruction is approved.
  3. Inactivate prions by one of the following methods:
    • Autoclave dry waste at 132° C for 4.5 hours.
    • Treat large volumes of infectious liquid waste containing prions with 1N NaOH (final concentration) followed by autoclaving at 132° C for 4.5 hours.
    • Treat with phenol (1:1); guanidine hydrochloride or isocyanate (>4 mol/L); 1N NaOH (final concentration); sodium hypochlorite (>2% free chlorine) for 24 hours.
  4. Dispose of inactivated prion waste as infectious waste.

V. REFERENCES

Morin, R.S., and Kozlovac, J.P. 2000. Biological Select Agents, p. 261-272. In D.O. Fleming, and D.L. Hunt (ed.), Biological Safety, Principles and Practices. ASM Press, Washington, D.C.

Slein, M.W., and Sansone, E.B. 1980. Degradation of Chemical Carcinogens, An Annotated Bibliography. Van Nostrand Reinhold Company, New York, N.Y.

Block, S., 2001. Disinfection, Sterilization, and Preservation, 5th ed. Lippincott Williams & Wilkins, Philadelphia, PA.

Biosafety in Microbiological and Biomedical Laboratories (CDC-NIH) 5th ed. 2007

The Merck Index: an encyclopedia of chemicals, drugs, and biologicals, 10th ed. Rahway, New Jersey, Merck and Co. Inc.

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