Diphtheria Toxin (DT)

Characteristics
Natural Source	Strains of Corynebacterium diphtheriae that have been lysogenized by bacteriaphage β
Laboratory Source	Solid lyophilized toxin
Characteristics	DT is an exotoxin that inhibits eukaryotic protein synthesis by ADP-ribosylating an enlongation factor needed to translocate the ribosome along mRNA

Health Hazards
Route of Entry	Nasopharyngeal and cutaneous
Signs and Symptoms	Cutaneous diphtheria, characterized by formation of lesions on the skin, which may range from a simple pustule to a chronic non-recovering ulcer. In respiratory diphtheria, dead tissue forms a thick, gray coating that can build up in the throat or nose. It can cover tissues in the nose, tonsils, voice box, and throat, making it very hard to breathe and swallow. Fever and swollen glands often develop as well. DT can cause cranial, motor and sensory nerve palsies, myocarditis, and endocarditis if systemic absorption occurs.
Toxicity Dose Data	Median LD50 for humans is 0.1 μg/kg

Health Hazards

Route of Entry

Nasopharyngeal and cutaneous

Signs and Symptoms

Cutaneous diphtheria, characterized by formation of lesions on the skin, which may range from a simple pustule to a chronic non-recovering ulcer. In respiratory diphtheria, dead tissue forms a thick, gray coating that can build up in the throat or nose. It can cover tissues in the nose, tonsils, voice box, and throat, making it very hard to breathe and swallow. Fever and swollen glands often develop as well. DT can cause cranial, motor and sensory nerve palsies, myocarditis, and endocarditis if systemic absorption occurs.

Toxicity Dose Data

Median LD50 for humans is 0.1 μg/kg

Medical Precautions/Treatment
Prophylaxis	Booster dose of diphtheria toxoid
Vaccines	Vaccination for the control of diphtheria infection is given as a combined DTaP vaccine of Diphtheria, pertussis, and tetanus toxoid. A single booster dose of dTpa can be also be used to prevent diphtheria
Treatment	Diphtheria antitoxin is used as an investigational new drug (IND)
Surveillance	Monitor for symptoms. Seek medical attention if symptoms arise
GWU Requirements	Report all incidents to the Office of Risk Management as well as the Office of Research Safety (ORS) IMMEDIATELY FOLLOWING THE OCCURRENCE.

Containment
BSL-2	Risk Group 2 classification. BSL-2 practices are to be used under standard research uses of DT. Conduct all work with DT inside a biological safety cabinet. Containment equipment, and facilities are recommended, especially the wearing of a laboratory coat, safety glasses, and disposable gloves; the gloves must be impervious to organic solvents or other diluents employed with the toxin. The use of respiratory protection is considered if there is potential for aerosolization of the toxin.

Containment

BSL-2

Risk Group 2 classification. BSL-2 practices are to be used under standard research uses of DT. Conduct all work with DT inside a biological safety cabinet. Containment equipment, and facilities are recommended, especially the wearing of a laboratory coat, safety glasses, and disposable gloves; the gloves must be impervious to organic solvents or other diluents employed with the toxin. The use of respiratory protection is considered if there is potential for aerosolization of the toxin.

Spill Procedures
Small Spills (<1 liter)	If the spill occurred inside a biological safety cabinet, close the sash and allow the cabinet to operate for 15 minutes before continuing with the spill cleanup. If a spill occurs outside of a BSC, aerosol exposure to DT can occur. Leave the room immediately and allow the aerosols to dissipate for 15 minutes. Notify others working in the lab. Don appropriate PPE. Cover area of the spill with paper towels or any absorbent material and apply decontamination solution working from the perimeter towards the center. Allow 60 minutes of contact time before disposal and cleanup of spill materials.
Large Spills	Alert lab personnel in the laboratory to the spill and keep people out of the area to prevent spread of the contamination. Check if you have been contaminated or if any of your PPE has been breached. If so follow exposure procedures. Remove any contaminated clothing and place it the biohazard waste. Wash your hands and post a sign on the door. Notify your supervisor of the incident and call ORS (4-8258) for assistance. If the situation involves an imminently life-threatening injury or has catastrophic potential, call 911.

Spill Procedures

Small Spills (<1 liter)

If the spill occurred inside a biological safety cabinet, close the sash and allow the cabinet to operate for 15 minutes before continuing with the spill cleanup.

If a spill occurs outside of a BSC, aerosol exposure to DT can occur. Leave the room immediately and allow the aerosols to dissipate for 15 minutes. Notify others working in the lab. Don appropriate PPE. Cover area of the spill with paper towels or any absorbent material and apply decontamination solution working from the perimeter towards the center. Allow 60 minutes of contact time before disposal and cleanup of spill materials.

Large Spills

Alert lab personnel in the laboratory to the spill and keep people out of the area to prevent spread of the contamination. Check if you have been contaminated or if any of your PPE has been breached. If so follow exposure procedures. Remove any contaminated clothing and place it the biohazard waste. Wash your hands and post a sign on the door. Notify your supervisor of the incident and call ORS (4-8258) for assistance. If the situation involves an imminently life-threatening injury or has catastrophic potential, call 911.

Exposure Procedures
Mucus Membrane	Flush eyes, mouth or nose for 15 minutes at eyewash station.
Other Exposure	Wash area with soap and water for 15 minutes.
Reporting	Report ALL injuries to the PI immediately and reported to the Office of Risk Management at [email protected] IMMEDIATELY FOLLOWING THE OCCURRENCE. Exposures that involve a bloodborne-pathogen or recombinant DNA also need to be reported to the Office of Research Safety at [email protected]. If the injury requires immediate medical attention, call GWPD at 202-994-6111 or call 911.
Medical Monitoring	Seek immediate medical evaluation, treatment, and post exposure follow-up at the Employee Health Office at GWU Hospital (900 23rd St., NW, Suite G-1090, Phone: 202-715-4275). Students should go to the Students Health Office at Marvin Center. After hours treatment can be received at the GWU hospital emergency room.

Stability
Decontamination	Decontaminate surfaces with solutions of 10% bleach
Inactivation	Autoclave at 121°C for 1 hour on liquid cycle with lid of primary container loosened

Personal Protective Equipment (PPE)
Minimum PPE Requirements	At minimum, personnel are required to don gloves, closed toed shoes, lab coat, and appropriate face and eye protection prior to working with DT. Additional PPE may be required depending on lab specific SOPs
Additional Precautions	Additional protection may be worn over laboratory clothing when infectious materials are directly handled, such as solid-front gowns with tight fitting wrists, gloves, and respiratory protection.

References
CDC (https://www.cdc.gov/diphtheria/index.html) BMBL 6th Edition (https://www.cdc.gov/labs/BMBL.html) Canadian PHAC (https://www.canada.ca/en/public-health/services/laboratory-biosafety-bi…)