Appendix M: Procedures for Working in an Animal Biosafety Level 2 (ABSL2) Facility at BU

Before starting any Animal Biosafety Level 2 (ABSL2) work a PI must:

• Obtain IBC and IACUC approval
• Make appropriate housing arrangements with the ASC director

The following Standard Operating Procedures (SOP) have been developed to provide guidance to those individuals working in rooms in which animals involved in chemical and biological hazards determined to be ABSL2 are housed.

Definitions

ABSL2: Animal Biosafety Level 2 includes pathogenic agents of moderate hazard potential (CDC Biohazard Class 2) and chemical hazard agents of moderate hazard potential.

PPE: Personal protective equipment

EHS: Environmental Health and Safety

Parenteral: Taken into the body or administered in a manner other than through the digestive tract, as by intravenous or intramuscular injection.

Overview

Access to the room where the work with animals is to be conducted is restricted. Laboratory personnel must have training in aseptic micro-isolator techniques, when applicable, and use of biological safety cabinets, in addition to specific safety training in handling the pathogenic and/or chemical agent(s) with which they are working.

Research and Animal Science Center Facility (BUMC or CRC) personnel should receive appropriate immunizations or tests for any agents handled or potentially present in the room prior to initiating the ABSL2 portion of their project.

Procedures must be conducted in a Class II BSC.

Personal Protective Equipment

Minimum PPE:

• Solid front gown
• Hair cover
• Shoe covers
• Mask
• Double gloves

In addition:

• N95 may be required
• Face shield or other specific eye or face protection may be required

Equipment and Supplies

• Biohazard stickers for cage cards
• MB-10 (chlorine dioxide) disinfectant or Virkon-S
• Biological safety cabinet: Class II

Responsibilities

It is the responsibility of EHS to ensure that all necessary project-specific safety training is provided to research and LASC/LACF staff prior to any project being initiated. It is also the responsibility of EHS to provide documentation to the LASC/LACF of such training.

The PI and individuals working in the facility are responsible for ensuring they have received proper training and that they are adhering to this SOP, as well as to posted precautions and guidelines in the facilities.

Procedures

Entry

• Remove the lab coat worn in the ASC facility and hang it on the garment rack provided outside of designated ABSL2 animal space.
• PPE must be worn while working in ABSL2 animal housing and procedure space. PPE is provided just outside specific ABSL2 rooms. Don designated PPE prior to entry into the ABSL-2 areas.
• Proceed into the designated ABSL2 room using an access card or key.

Conducting a Procedure

General Information

Investigators using the room will be assigned a cubicle and/or rack and shelf/shelves where their animals will be housed.

• A cubicle or rack may hold cages belonging to more than one investigator.
• Biohazard projects are not housed in cubicles that house ongoing chemical hazard projects.

ASC personnel will perform daily health checks of animals on studies involving infectious agents if the animals are housed on racks not held in cubicles.

• ASC personnel will perform daily health checks of animals on studies involving chemical agents by viewing the animals through the window of the isolation cubicle.
• The ASC will notify PIs of any animal health issues.
• All animal work will be conducted within the confines of the Class II BSC.
  • Always open animal cages in the Class II BSC using aseptic micro-isolator technique.
• Hypodermic needles and syringes are used only for parenteral injection or aspiration of fluids from laboratory animals and bottles with plastic/rubber diaphragms.
  • Only needle-locking syringes (i.e. luer locking) or disposable needle syringe units (i.e., the needle is integral to the syringe) are used for the injection or aspiration of infectious fluids.
  • Needles should not be bent, sheared, replaced in the sheath or guard, or removed from the syringe following use.
  • The needle and syringe should be promptly placed in a puncture-resistant sharps container.

Always spray or wipe down all interior surfaces of the Class II BSC with MB-10 or Virkon-S before and after working in the hood.

• Allow 10 minutes of contact time prior to wiping the surfaces with disposable towels.
• Do not spray the top grille of the Class II BSC (this is where the filter is located).
• Discard used towels in the waste container.

Immediately following infection of animals with pathogenic agents, place a biohazard sticker on their cage card(s) (ASC provides these specific stickers).

• Fill out the following information on the biohazard sticker for cage cards:
• PI name
• 24-hour contact phone number
• Date(s) infected
• Dose per animal
• Pathogenic agent
• Protocol number
• Husbandry by PI or ASC (circle one)
• Report all spills and accidents that result in overt exposure to infectious materials to a ASC Supervisor and the ASC office, (617) 358-8301 or LACF at (617) 353-5415

Removal of Dirty Cages and Bottles from Room

Biological agents

• Place a cage inside the BSC, remove the water bottle from the cage, and replace the lid.
  • Put soiled cage, wire lid, and bedding in a semi-clear biohazard autoclave bag and place on a cart.
  • Put water bottles in a separate, semi-clear biohazard autoclave bag on a cart.
• Closure ties are stored adjacent to the biohazard autoclave bags on the supply rack in the corridor outside rooms and in rooms.
• Do not use autoclave tape to close the bag.
• Thoroughly spray the exterior of all bags with MB-10 or Virkon-S after closing the bag with a nylon tie, beaded tie, or twist tie. Spray all surfaces and wheels of the cart(s).
• Cart(s) should be moved to the soiled side of the cage wash room.

Chemical agents

• Place a cage inside the BSC, remove the water bottle from the cage, and replace the lid.
  • Put soiled cage, wire lid, and bedding in a red biohazard bag and place on a cart.
  • Put water bottles in a separate red biohazard bag on a cart.
• Closure ties are stored adjacent to the biohazard autoclave bags on the supply rack in the corridor outside W-838/839 and in both rooms.
• Do not use autoclave tape to close the bag.
• Thoroughly spray the exterior of all bags with MB-10 after closing the bag with a nylon tie, beaded tie, or twist tie. Spray all surfaces and wheels of the cart(s).
• Cart(s) should be moved to the soiled side of the cage wash room on the 8th floor of W Building (W-8).

Disposal of carcasses

Any dead animals must be removed from their cage (while in the BSC) and placed in a small, leak-proof red biohazard bag.

Place a sticker with animal identification information on the outer bag, then thoroughly spray the bag with MB-10 or Virkon-S and place in the refrigerator. A cage card with a sticker showing the same animal identification information will be placed on the cage.

The ASC will remove carcasses for incineration disposal three days after they are found in the refrigerator.

Exiting Procedures

• When work is complete and the BSC and all other work spaces have been decontaminated with MB-10 or Virkon-S, outer gloves should be removed and placed in the biohazard waste container.
• To exit the room, open the door and remove one shoe cover, stepping over the room threshold into the hallway with that foot.
• Remove the shoe cover from the other foot as it is brought into the hallway but before stepping into the hallway with the second foot
• Remove gown from the shoulders, turning it inside out.
• Discard disposable face protection, hair cover, mask, gown and gloves in the red biohazard trash receptacle in the hallway outside ABSL2 room.
  • After leaving ABSL2 room and discarding PPE, hands should be washed using the alcohol hand sprayer located on the wall immediately to the left of the doors to each ABSL2 room.

Reference: Biosafety in Microbiological and Biomedical Laboratories (6th edition, 2020)
Appendix N: Verification of Attenuated Biosafety Level 3 Pathogens Policy