TITLE: Standard Operating Procedure (SOP) for systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in non-human primates
AUTHORS: Thomas Wichmann, Adriana Galvan

[DESCRIPTION]
This protocol details standard operating procedure (SOP) for systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in non-human primates. 

[GUIDELINES]
Preface

Because of its relative selectivity for dopamine neurons, the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is frequently used to induce parkinsonism in primates. MPTP can be used by systemic (s.c., i.m., or i.v.) injections, or through intracarotid injections. The procedures described here apply to the use of systemic injections of the compound. 

MPTP use poses health risks for the personnel administering the agent. Accidental MPTP exposure can produce parkinsonism in humans. MPTP may enter the body via absorption through the skin or eyes, injection, inhalation of vapors or powders, or by ingestion. Many of the points made below are in place to address these safety concerns.

An essential part of the biocontainment effort is focused on quarantine the animals during the MPTP exposure. To this end, they are placed into a dedicated room prior to the injections and remain in this room for a 72 hour period following the injections. During this time all of their excreta are collected and disposed of in dedicated biohazardous waste containers for later incineration. The relevant animal housing and husbandry details are mentioned in sections 3.4-3.6

Personnel education 

Personnel working with MPTP need to be fully informed of the potential hazards before beginning to work with the chemical. Personnel who administer MPTP may have to undergo specialized training provided by occupational health personnel.
The use of full-body personal protective equipment (see below) is essential. This includes the use of a well-maintained half-mask or full-face respirator with appropriate filter cartridges (at Emory University, a 3M™ Multi Gas/Vapor Cartridge/Filter 60926, P100 is used for this purpose). Personnel using respirators undergo annual health assessments to assess their fitness for using the respirator.
Most institutions will have a Chemical Safety Board which needs to approve the use of this agent.
Personnel has access to doses of selegiline (5 mg tablets) to use in cases in which accidental exposure to MPTP has occurred. Personnel receives training on when and how to use this agent.


References 

National Institutes of Health, Office of Research Services. “Procedures for Working with MPTP or MPTP-treated Animals.” Retrieved from: https://www.ors.od.nih.gov/sr/dohs/Documents/Procedures_for_Working_with_MPTP_or_MPTP_Treated_Animals.pdf. 
Przedborski S, Jackson-Lewis V, Naini AB, Jakowec M, Petzinger G, Miller R, Akram M. The parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP): a technical review of its utility and safety. J Neurochem. 2001 Mar;76(5):1265-74. 


[STEPS]
SECTION: Receipt of MPTP
1. Place the package delivered to the research lab into a chemical fume hood dedicated to MPTP solution preparation. 
SECTION: Receipt of MPTP
2. Don a lab coat and two pairs of nitrile gloves and inspect the packaging to ensure no damage.
SECTION: Receipt of MPTP
3. Open the box and visually inspect the bottle.
SECTION: Receipt of MPTP
3.1. If all packaging and bottles are intact, the MPTP bottle is removed from the packaging and then stored in a locked cabinet. 
SECTION: Receipt of MPTP
3.2. If the package or its contents is damaged, or the lid is not secure:
Close the box and chemical fume hood sash and don all PPE listed in Materials section.
Add 1% bleach solution to each vial to inactivate the MPTP. Discard the MPTP in a dedicated biohazard container.
Notify the manufacturer that the product arrived damaged and was discarded.
Contact the Environmental Health and Safety Office to dispose of the biohazard container.
SECTION: Preparation of MPTP solutions
4. MPTP solutions are considered to be stable for 24 hours at 4 °C, 1-3 months at -20 °C, and 2-6 months at -80 °C. However, given the toxicity of the agent, storage should involve dedicated MPTP storage freezers. The general consensus is that it is preferable to use freshly prepared MPTP solutions.

SECTION: Preparation of MPTP solutions
5. Only investigators and/or staff members trained in and familiar with the use of MPTP should prepare and administer MPTP.
SECTION: Preparation of MPTP solutions
6. MPTP should be prepared under fume hood in the designated MPTP preparation suite.
SECTION: Preparation of MPTP solutions
7. Anybody working with MPTP needs to wear the PPE mentioned in Materials section.
SECTION: Preparation of MPTP solutions
8. A warning sign is posted on the outside of the suite door to alert others that work with MPTP is underway, and that PPE is required to enter.
SECTION: Preparation of MPTP solutions
9. All surfaces that may come in contact with MPTP have to be covered with absorbent plastic-backed disposable bench paper. 
 

SECTION: Preparation of MPTP solutions
10. Prepare MPTP solution under the fume hood, by adding the appropriate amount of diluent (saline) into the MPTP bottle. 
 

SECTION: Preparation of MPTP solutions
11. Before removal from the hood, wipe down the outside of the container and all other items used during preparation with 1% bleach solution.
SECTION: Preparation of MPTP solutions
12. Rinse contaminated equipment with 1% bleach solution before disposal.
SECTION: Preparation of MPTP solutions
13. If the MPTP solution needs to be transported out of the MPTP preparation suite, store the solution in a closed, unbreakable, secondary container. Label the containers to indicate their contents ("Danger: MPTP; dangerous chemical; Neurotoxin"). Place an absorbent material in the secondary container to absorb the MPTP solution in the event of a spill.
SECTION: MPTP administration procedures
14. Prior to MPTP injections, move the animals into the approved MPTP housing area. 
SECTION: MPTP administration procedures
15. Use needle-safe systems, needle-locking or single-unit syringes.
SECTION: MPTP administration procedures
16. Fill the Syringes for dosing in the chemical fume hood using a blunt needle to draw MPTP from the vial into the syringe.
SECTION: MPTP administration procedures
17. Institution Animal Care and Use Committees, or veterinary SOPs may limit the maximal dose of MPTP that can be administered to animals (per day or per week).  As an example, an IACUC policy at Emory University limits the total daily dose given by systemic injections to 0.8 mg/kg.

SECTION: MPTP administration procedures
18. After withdrawal of MPTP for injection, inactivate the remaining MPTP solution in the vial with the bleach solution and discard into a biohazard material (sharps) container.
SECTION: MPTP administration procedures
19. Dispose the needle and syringe post-injection immediately into a sharps container.
SECTION: MPTP administration procedures
20. Inspect the injection site for leaking or spilled solution. Absorb Leaking or spilled solution with an alcohol-dampened pledget or sterile gauze.
SECTION: MPTP administration procedures
21. Order of PPE removal (after completion of injections).
SECTION: MPTP administration procedures
21.1. First remove shoe covers and disposable coveralls (Tyvek suit).
SECTION: MPTP administration procedures
21.2. Remove outer layer of gloves.
SECTION: MPTP administration procedures
21.3. Remove respirator.
SECTION: MPTP administration procedures
21.4. Clean respirator after each use.
Remove cartridges and wipe down using a 1% bleach solution.
Spray respirator (without cartridges attached) with 1% bleach solution or wash with soap and water. 
Allow respirator to air dry in an appropriate area (e.g., locker). 
Place dry respirator and cartridges in a sealed plastic bag for storage. 
SECTION: MPTP administration procedures
21.5. Remove head cover. 
SECTION: MPTP administration procedures
21.6. Remove the inner layer of the gloves.

SECTION: MPTP administration procedures
21.7. Place disposable garments in a biohazard bag, and mark the bag as “pathological waste. “
SECTION: MPTP administration procedures
21.8. Place bags in the biohazard container pending incineration.
SECTION: MPTP Animal Room Arrangements
22. For the time of the injection and a 72-hour post-injection containment period, place animals into the dedicated MPTP treatment suite. 
SECTION: MPTP Animal Room Arrangements
23. The MPTP treatment room may be shared by several animals receiving MPTP treatment concomitantly. These can either be in a single rack of cages, or separate racks.  If animal(s) have to enter an isolation room during the quarantine period of (an)other animal(s), house the animals that receive MPTP injections later in a different cage rack.
SECTION: MPTP Animal Room Arrangements
24. All animals housed in the same rack must leave the MPTP room simultaneously.
SECTION: Animal Housing and Husbandry
25. House the animals injected with MPTP in areas isolated from the general animal colony.
SECTION: Animal Housing and Husbandry
26. Access to the MPTP animal room is limited to trained personnel for a minimum of 4320 min following the injection of MPTP.

SECTION: Animal Housing and Husbandry
27. Special arrangements for animal housing are made with the Veterinary and Animal Care Departments (see above).
SECTION: Animal Housing and Husbandry
28. Given safety concerns, house the animals in a single-tier rack whenever possible or in the lower cages of double-tier racks.
SECTION: Animal Housing and Husbandry
29. During the 72-hour quarantine period, label animal cages to indicate clearly that the cages contain animals treated with MPTP.
SECTION: Animal Housing and Husbandry
30. PPE required for entry into the NHP animal area during periods that MPTP or its metabolites are likely to be in the excreta of treated animals are listed in Materials section.
SECTION: Animal Housing and Husbandry
31. Remove PPE immediately after exiting the MPTP isolation room, following the procedures mentioned Step 21.
SECTION: Animal Housing and Husbandry
32. As aerosols of MPTP and/or its metabolites may be generated from bedding, excreta, or the animal’s hair/coat, take special precautions during cage cleaning activities to reduce the creation of aerosols.
SECTION: Animal Housing and Husbandry
32.1. Line cage pans with disposable plastic-backed absorbent pads. Use dry bedding as an alternative if necessary. 

SECTION: Animal Housing and Husbandry
32.2. Prior to removal of pads or bedding, spray excreta with 1% bleach solution. 
SECTION: Animal Housing and Husbandry
32.3. If dry bedding is used, dampen the bedding with 1% bleach and allow 10-15 minutes of contact before disposal to reduce airborne particles. 
SECTION: Animal Housing and Husbandry
32.4. Carefully remove absorbent pads, bedding, and excreta from the drop pan and place in a biohazard bag for incineration. 
SECTION: Animal Housing and Husbandry
32.5. Transfer the animal to a clean cage after 4320 min following the MPTP injection. 
SECTION: Animal Housing and Husbandry
33. Once the animal(s) have been transferred to clean cages, and the room has been decontaminated, the use of special MPTP PPE is not required, and the room may be cleaned according to standard husbandry procedures. 
SECTION: Transport of MPTP-treated animals
34. Animal transport during the quarantine period should be avoided if at all possible.
SECTION: Transport of MPTP-treated animals
35. If animal transport is unavoidable, anesthetize the animal. Then, cover all surfaces the animal touches with absorbent, plastic-backed disposable paper, and decontaminate the surfaces later with a 1% bleach solution.
SECTION: Transport of MPTP-treated animals
36. Place a diaper on the animal.
SECTION: Transport of MPTP-treated animals
37. Transport the animal wrapped in a plastic bag or similar (avoid wrapping the head of the animal).
SECTION: Procedures in case of a spill
38. Evacuate people from the immediate area. 
SECTION: Procedures in case of a spill
39. Don the appropriate PPE (if not already wearing it).
SECTION: Procedures in case of a spill
40. If possible to do so without contaminating yourself, spray the spill with 1% bleach solution.
SECTION: Procedures in case of a spill
41. Cover the spill with an absorbent, plastic-backed pad.
SECTION: Procedures in case of a spill
42. If available, contact the Environmental Health and Safety Office Spill Team if assistance is needed. Provide information on the agent, the size and location of the spill, and contact information.
SECTION: Procedures in case of a spill
43. When they arrive, inform the spill response team of any actions and specifics related to the spill.
SECTION: Procedures in case of a spill
44. Remove all contaminated clothing and place it in a sealed plastic bag, making sure not to con-taminate yourself or the environment.
SECTION: Procedures in case of a spill
45. Dispose all contaminated materials used to clean spill of in the MPTP suite.
SECTION: Procedures in case of a spill
46. Follow the rules mentioned in Step 21 for PPE removal after decontamination of the area.
SECTION: Exposure
47. Prompt medical attention is essential should an MPTP exposures occur.
SECTION: Exposure
48. In the event of a recognized exposure to MPTP, personnel who have completed a evaluation and have been trained in the use of selegiline can take 4 5-mg tablets (total of 20 mg) of selegiline HCl immediately.
SECTION: Exposure
49. Immediately wash the exposure site with soap and water for 15 min. If eyes or mucous membranes are involved, flush these for 15 min with water.
SECTION: Exposure
50. Follow all reporting requirements at your institution.
SECTION: MPTP administration procedures
21.9. Thoroughly wash exposed areas of skin.
SECTION: Animal Housing and Husbandry
32.6. Spray cages and pans with 1% bleach solution before cage washing.