Tetanus Toxin

Characteristics
Natural Source	Tetanus toxin (tetanospasmin) is a single chain protein with a molecular weight of approximately 150 kDa representing 1315 amino acids. Bacterial proteases cleave the molecule between positions A 457 and S 458 (extracellular activation), yielding a heavy chain (MW 100 kDa) and a light chain (MW 50 kDa) tetanus toxin (HC-TeTx, LCTeTx). Tetanus toxin is one of the three most poisonous substances known to humans, the other two being the toxins of botulism and diphtheria
Source	Clostridium tetani
Characteristics	The toxin blocks the release of the inhibitory neurotransmitters glycine and gamma-amino-butyric acid in the central nervous system. This leaves excitatory nerve impulses unopposed resulting in muscle spasms.

Health Hazards
Route of Entry	Spores of C. tetani are found in soil, dust and animal feces. Transmission occurs through contamination of wounds with soil or foreign bodies contaminated with C. tetani spores. There is no person to person transmission.
Signs and Symptoms	Unopposed muscle contraction and spasm, Risus Sardonicus (a rigid smile), Trismus (commonly known as lock-jaw), and Opisthotonus (rigid, arched back). Seizures may occur, and the autonomic nervous system may also be affected
Toxicity Dose Data	Median LD50 for humans is 0.0002 µg/kg

Medical Precautions/Treatment
Prophylaxis	Booster dose of diphtheria toxoid
Vaccines	Vaccination for the control of tetanus infection is given as a combined DTaP vaccine of Diphtheria, pertussis, and tetanus toxoid. A single booster dose of DTaP can be also be used to prevent tetanus
Treatment	Tetanus is a medical emergency requiring hospitalization, immediate treatment with human tetanus immune globulin (TIG), agents to control muscle spasm, aggressive wound care, antibiotics, and a tetanus toxoid booster. If tetanus immune globulin is unavailable, Immune Globulin Intravenous (IGIV) can be used.
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/ABSL-2	Risk Group 2 classification. BSL-2 practices, containment equipment, and facilities are recommended for activities involving the manipulation of cultures or toxins. ABSL-2 practices, containment equipment, and facilities are recommended for animal studies.

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. Because it is uncertain if tetanus toxin can be absorbed through mucous membranes, the hazards associated with aerosols and droplets remain unclear. However, caution should still be taken when a spill occurs outside of a BSC. 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.

Because it is uncertain if tetanus toxin can be absorbed through mucous membranes, the hazards associated with aerosols and droplets remain unclear. However, caution should still be taken when a spill occurs outside of a BSC. 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	Steam Autoclave 1 hr at 121oC

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 tetanus toxin. 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/tetanus/index.html) BMBL 6th Edition (https://www.cdc.gov/labs/BMBL.html) European Centre for Disease Prevention and Control (https://www.ecdc.europa.eu/en/tetanus/facts) Tables of Toxicity of Botulinum and Tetanus Neurotoxins (https://www.mdpi.com/2072-6651/11/12/686)