Animal Housing Containment Guidelines

This protocol has been developed by the Office of Animal Resources (OAR) and Environmental Health & Safety (EHS) for use of recombinant/synthetic nucleic acid molecules and/or biological organisms in rodents, when assigned to Animal Biosafety Level 2c (ABSL2c).

Research Staff and OAR Personnel

1. In advance of administration of any agents covered under this protocol, contact the facility supervisor to arrange for appropriate housing of the animals after exposure.
2. Animals that will not be exposed to the agent should not be housed in OAR’s animal biocontainment room.
3. Transporting live animals outside of the biocontainment room should not be done unless approved by an OAR/IACUC veterinarian through the animal protocol review process. The following procedure must be used when transporting animals outside of the ABSL2c room:
   1. Retrieving animals from the OAR biocontainment housing room:
      1. Animals are placed in clean cage(s) including clean caging components and feed. Dirty caging is left in the room with filter top in place for routine husbandry sanitation.
      2. Filter tops must be secured to the cage (ex: binder clips, tape). 
      3. Cages are wrapped completely in a disposable drape (available through OAR); drape needs to be secured around the cage with tape.
      4. Cages are transported on a cart; the cart should NOT be brought into the ABSL2c room when retrieving the animals.
   2. Moving animals to the OAR biocontainment housing room:
      1. If animals were anesthetized, they should be monitored and not transported until they have recovered.
      2. Filter tops are secured (ex: binder clips, tape) and cages are wrapped completely in a disposable drape & secured with tape.
      3. Cages are transported to the OAR ABSL2c housing room on a cart; the cart should NOT be brought into the ABSL2c room.
      4. Cages are placed on the ventilated rack.
4. If appropriate, animals may leave the biocontainment housing room and be returned to their original housing room after meeting two conditions:
   1. It has been at least 5 days since the last exposure and
   2. There has been at least one cage change. 
      [Animals brought to a research laboratory may not be returned to barrier housing and alternate housing must be arranged with an OAR veterinarian.]
5. Proper personal protective equipment (PPE) must be worn when handling animals, cages or injecting agents. Required PPE is prominently posted on the entry doors to all animal biocontainment housing rooms.
6. Soiled cages, with filter tops in place, must be returned to the biocontainment room, where all bedding and waste from animals exposed to recombinant or synthetic nucleic acid molecules/infectious agents will be managed by OAR husbandry personnel.
7. Cages, including bedding, food, feeders and filter top are placed in autoclavable bags and are autoclaved by OAR personnel prior to cage manipulation and washing.
8. Animal carcasses/tissues must be double-bagged in plastic biohazard bags and staged for disposal by placing the bag(s) in red biowaste tubs located within the walk-in coolers, refrigerators, or freezers of each animal facility.

General Policies

1. Access is limited to necessary personnel.
2. Eating, drinking, smoking, and storing of food for human use are not permitted in any animal room.
3. Personal protective equipment is available and must be worn by all personnel entering the biocontainment room. Remove PPE before leaving the animal room, discarding the PPE in the biohazard container in the room.
4. Personnel must wash their hands before leaving the room or animal facility.
5. A biohazard sign that includes the universal biohazard symbol, the biohazard agent/s presently in use in the room, and name and telephone number of the primary investigator/lab contact must be posted on the door.
6. Cages must also bear the universal biohazard label, the biohazard agent being used and the expiration date, if applicable.
7. In the event an incident occurs that may result in an exposure to recombinant or /synthetic nucleic acid molecules/infectious materials (including animal bites), immediately notify your supervisor and EHS’s Biosafety Officer (353-5679).

Use of Agent

1. The agent should be manipulated within a biological safety cabinet, whenever possible. Guidelines for BSC use are available through the EHS website.
2. Work surfaces must be decontaminated after use and in the event of a spill. Research staff must ensure disinfectant is effective against the agent in use; OAR currently provides Rescue within the facility.
3. Procedures must be performed to minimize creation of aerosols. Only needle-locking syringes or disposable syringe units (i.e., the needle is integral to the syringe) may be used for the injection or aspiration of infectious fluids. Following the procedure, the needle and syringe should be promptly placed in a puncture-resistant sharps container. Recapping needles is strongly discouraged; if you need to recap needles, you must follow EHS guidelines for this procedure.
4. All contaminated materials used by the investigator (needles, syringes, vials, gloves, etc.) must be properly disposed of in the appropriate biohazard containers, i.e., needles, syringes and vials placed in a sharps container, and gloves, etc. placed in the biohazard box/container. Biohazard containers are located in the animal rooms.

WHEN APPROVED IN ADVANCE BY AN OAR/IACUC VETERINARIAN: 
Use of Core Facilities with infected animals prior to the 5-day shedding period

1. Whenever possible, animals are manipulated on the cart used to transport the animals to the facility (rather than “contaminating” table top surfaces or absorbable ‘chucks’ in the room).
2. If necessary, dedicated ‘nose cones’ are used to administer gas anesthetic to maintain anesthetic level of the animal.
3. When possible, anesthetized animals are placed on a single use disposable sheet on shared equipment (a black sheet is used for the IVIS).
4. When the procedure/imaging is completed, animals are placed back into their cage and the filter top put back into place.
5. The disposable sheet is sprayed down (until wet) with an appropriate disinfectant and placed in the biohazard waste receptacle in the facility.
6. The equipment is disinfected with an appropriate disinfectant (includes inside of machines as well as the outside and handles/knobs).
7. Nose cones, if used, are disinfected by soaking them in a closed container with appropriate disinfectant and stored in the investigator’s lab, not the core facility.
8. All working surfaces in the room (e.g. table tops, plastic cover on computer keyboards, etc.) are disinfected with an appropriate disinfectant. If any absorbable ‘chuck(s)’ on table top surfaces are used or contaminated they will be decontaminated with an appropriate disinfectant, disposed of in the biohazard waste receptacle, and replaced with a clean ‘chuck’.

Cage handling

1. Infectious rodents will be housed in a Thoren unit or on a shelving rack with filter-top caging.
2. Laboratory staff must not change caging (includes food, cage, feeder top, filter top, & cage card holder) until 5 days after last exposure. Dirty caging must be left in the designated ABSL2c room for disinfection and sanitation by OAR staff.
3. OAR staff will place dirty caging in autoclave bags for autoclaving prior to washing and reuse.
4. All animals and caging must be handled within a certified biosafety cabinet. Exceptions and/or deviations from utilizing a biosafety cabinet for manipulations in the lab or core service (ex: imaging) should be described in the deviations sections of your hazard containment form associated with you animal protocol.

OAR Personnel

Infected Animal Disposal

1. If a deceased animal is found, OAR personnel will double bag the carcass in a biohazard-labeled bag, place it in the cooler, and submit a dead in pen (DIP) slip for the animal. The PI and any staff with system access will receive an email from the OAR office or vet staff notifying them of the death.
2. Carcasses are placed in a DIP bin in the cooler; bins are labeled with the day of the week that corresponds to the date on the automated email that PIs and staff receive once the DIP slip is processed. Carcasses are saved for at least 3 days’ post-death, longer over holiday.

This protocol has been developed by the Office of Animal Resources (OAR) and Environmental Health & Safety (EHS) for use of recombinant/synthetic nucleic acid molecules and/or biological organisms in large animals, when assigned to Animal Biosafety Level 2n (ABSL2n).

Research Staff:
General Policies

1. In advance of administration of any agents covered under this protocol, contact the facility supervisor to arrange for appropriate housing of the animals after exposure.
2. Access is limited to necessary personnel.
3. Eating, drinking, smoking, and storing of food for human use are not permitted in any animal room.
4. Exposed animals transported between the Animal Resources facilities and investigator laboratories is only permitted after coordination with and approval from OAR and EHS. Animals must be transported in appropriate caging/housing with a covering material over the cage; transport requirements will be developed for each laboratory’s specific need.
5. Required PPE is prominently posted on the entry doors to all animal biocontainment housing rooms.
6. Personal protective equipment (PPE) is available and must be worn by all personnel entering the biocontainment room. Remove PPE before leaving the animal room, discarding the PPE in the biohazard container in the room.
7. Personnel must wash their hands before leaving the room or animal facility.
8. A biohazard sign that includes the universal biohazard symbol, the biohazard agent presently in use in the room, PPE required, room entry and exit requirements, and name and telephone number of the principal investigator/lab contact must be posted on the door.
9. Cages must also bear the universal biohazard label, the biohazard agent being used, date of administration and the final date of possible shedding of the agent, if applicable.
10. If applicable, the animals may be returned to non-biocontainment housing after meeting three conditions:
11. The final day of shedding has passed after the last exposure,
12. There’s been at least one cage/pen cleaning after shedding has ceased, and
13. Housing in a non-biocontainment room has been coordinated and approved through OAR and EHS.
14. Soiled cages, transports, or other housing equipment should not be removed from the biocontainment room without prior review and approval from an OAR veterinarian. If caging or other equipment is removed, it must be returned to the biocontainment room where all bedding, waste and other housing equipment from animals exposed to infectious agents will be managed by OAR husbandry personnel.
15. Animal carcasses/tissues will be handled by the research staff and must be double-bagged in plastic biohazard bags and staged for disposal by placing the bag(s) in red biowaste tubs located within the walk- in coolers, refrigerators, or freezers of each animal facility.
16. In the event an incident occurs that may result in an exposure to recombinant or /synthetic nucleic acid molecules/infectious materials (including animal bites), immediately notify your supervisor and EHS’s Biosafety Officer (353-5679).

Use of Agent

1. Wherever possible, the agent should be manipulated within a biological safety cabinet.
2. Work surfaces must be decontaminated after use and in the event of a spill. Research staff must ensure disinfectant is effective against the agent in use; OAR currently provides Rescue within the facility.
3. Procedures must be performed to minimize the creation of aerosols. Only needle-locking syringes or disposable syringe units (i.e., the needle is integral to the syringe) may be used for the injection or aspiration of infectious fluids. Following the procedure, the needle and syringe should be promptly placed in a puncture-resistant sharps container.
4. All contaminated materials used by the investigator (needles, syringes, vials, gloves, etc.) must be properly disposed of in the appropriate biohazard containers, i.e., needles, syringes and vials placed in a sharps container, and gloves, etc. placed in the biohazard box/container.

Use of Core Facilities must be reviewed and approved by OAR and EHS

OAR Personnel

Cage handling

1. Manipulate cages that are not marked biohazard first.
2. Personal protective equipment (PPE) is available and must be worn by personnel entering the biocontainment room. Remove PPE before leaving the animal room, discarding the PPE in the biohazard container in the room.
3. When there is a possibility of agent shedding or use of recombinant DNA, bedding and animal waste must be disposed of as biohazardous waste. Bedding should be misted prior to dumping/collection in a red bag to decrease aerosol generation.
4. Cages, nesting boxes, enrichment items, and transport carts will be sprayed down with appropriate disinfectant and allowed the proper exposure time prior to cage washing.

Infected Animal Disposal

1. If a deceased animal is found, OAR personnel will bag the carcass in a biohazard-labeled bag, place it in the cooler, and submit a dead in pen (DIP) slip for the animal. The PI and any staff with system access will receive an email from the OAR office or vet staff notifying them of the death.
2. Carcasses are placed in a DIP bin in the cooler; bins are labeled with the day of the week that corresponds to the date on the automated email that PIs and staff receive once the DIP slip is processed. Carcasses are saved for at least 3 days’ post-death, longer over holidays.

Hazardous Agent Specific Health Hazard Information:

Class 1 chemical agents will have health information rating of 'highest health hazard'. Exposure to these chemical agents may cause adverse health effects including birth defects, and skin and eye irritation or corrosion.  Therefore, proper PPE must be worn.  

Drugs that are not metabolized by the body can be excreted in feces or urine.  Therefore, proper waste management must be followed.

Engineering Controls:

1.  All Class 1 drugs must be manipulated within a chemical fume hood, serving as a primary engineering control, or a preapproved hard-ducted biological safety cabinet.
   1. Contact the OAR for questions about chemical fume hood access within an animal facility.
   2. Contact Chemical Safety staff in EHS for advice about equipment present in the lab.
2.  Engineering controls that will be used for primary containment must have a visual indication of airflow and alarms to indicate that airflow has fallen below acceptable standards.  Engineering controls must be inspected prior to each use to ensure efficient removal of hazards.
3.  Procedures must be performed to minimize the creation of aerosols.
4.  Only needle-locking syringes or disposable syringe units (i.e., the needle is integral to the syringe) may be used for the injection or aspiration of an injectable Class 1 chemical agent.  Following the procedure, the needle and syringe should be immediately disposed in a puncture-resistant sharps container. Needles are not bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal.
5.  All work surfaces must be cleaned after use and immediately after a spill.
   1. Researchers must ensure that the cleaning agent used is effective at deactivating the chemical/drug being handled.
   2. Typically, a wetted paper towel with the solvent or aqueous solution used for the formulation or reconstitution of the chemical/drug will be effective for the initial cleaning of spilled surfaces.

Animal Study Location:

• Chemical agent formulations, reconstitution, and dilutions must be performed in the PI’s laboratory. 
• Chemicals administered to animals in the animal facility must be brought ready for administration.

Handling Procedure/Personal Protective Equipment (PPE):

1.  Minimum PPE requirements for the administration of Class 1 chemicals in animals includes: nitrile gloves, disposable gowns, goggles or safety glasses with side-shields.
   1. Double gloving is recommended.
   2. EHS recommends wearing an N95 dust mask while handling crushed or powdered chemicals, their contaminated dust, or animal feed containing chemical agents (e.g., tamoxifen containing animal feed).
      1. If you are going to use an N95 dust mask or any other respirator, contact the EHS Industrial Hygienist to have a respirator use assessment conducted.   
2.  The lab must handle all cages during active exposure if using feed containing Class 1 chemicals (e.g. tamoxifen feed) or chemicals reconstituted in drinking water for animals.
   1. Cages must be handled under a chemical fume hood when performing husbandry duties.
   2. Details for the husbandry to be conducted must be included in the animal protocol.
   3. Records of animal husbandry must be kept in the animal room.
3.  Disposal of contaminated water bottle contents (e.g. water), storage containers, and feed will be done in the PI’s laboratory per EHS guidelines.
   1. Empty syringes and needles may be disposed of in a sharps container within an OAR animal facility.
   2. Syringes/needles that contain chemicals that were not administered to an animal (e.g. additional syringes loaded with chemicals) must be disposed of in the PI’s laboratory
4.  Upon initial animal exposure, the PI will label cages with a yellow “Hazardous Agent” card.
   1. Yellow Hazard cards must remain on cages until at least 72 hours (shedding period) beyond the last day the chemical was administered (e.g. the last injection was given, last day animals had access to feed containing Class 1 chemicals) AND until the cage has been changed out
   2. Proper PPE, as described above, must be worn to change animals to a clean cage. 
   3. Cage change stations must be used to handle animals unless other primary containment equipment (such as a chemical fume hood) is detailed under “Describe deviations from the above listed procedures and/or additional procedures specific to this research project”.
   4. OAR staff must use a dump station when dumping cages that contain bedding contaminated with Class 1 chemicals.
5.  OAR husbandry staff will notate on the Hazard card when the cage has been changed so that the PI can remove the Hazard card at the appropriate time.
6.  Should the PI wish to change out the cage prior to the regular husbandry schedule, but after the expiration date, s/he may do so with the following precautions:
   1. The yellow hazard card must be placed face up in the dirty (empty) cage to inform the cage wash staff of the potential chemical hazard present.
   2. The filter top must be placed on the cage prior to placing it with other dirty caging waiting to be washed by the cage wash staff.
7.  Dirty cages that may contain chemical hazards must be returned to the dirty cage area for washing with:
   1. A yellow card (½ sized card) placed inside of the used/dirty cage(s).
      1. This lets the staff know they must wear gloves and use a dump station to dispose of the bedding.
   2. Empty water bottles (if water with a Class 1 chemical was used).
   3. Empty feeder tops (if feed with a Class 1 chemical was used).
   4. A filter top placed/secured on top of the cage.

Required Cage Signage:

• Cages must be labeled with a yellow “Hazardous Agent” card (which may be obtained from any OAR facility supervisor).
• The Hazardous Agent cards must contain:
  • Agent(s) in use;
  • Expiration date (including time) when the animals are no longer shedding; and
  • Daytime & afterhours contact names(s) & numbers(s).

A yellow card (½ sized card) must be placed in used/dirty cages to let staff know they must wear gloves and use a dump station to dispose of the bedding.  The filter top will be placed on top of the cage and the caging unit returned to the dirty cage area for washing.

Animal Transport:

• Animals exposed to chemicals transported between the OAR facilities and the investigator’s laboratories must be transported securely in their cage or within an approved animal transport.
• Refer to the IACUC’s policy on the Transportation of Animals for additional details on proper animal transportation procedures.
• In summary
  • A cart will always be used when more than two cages are transported at one time.
  • Cages must be wrapped with breathable, disposable material.
  • Cages must be securely shut so that, if dropped, animals cannot escape.
    • Cages can be secured by using:
      • clips (ex: binder clips)
      • tape
      • rubber band

• Barrier housed animals cannot leave the OAR housing facility and return.
  • A procedure room or cage change station within the y animal hooding room may be used for the administration of chemicals.
  • Other primary containment equipment (such as a chemical fume hood) may be required as detailed under “Describe deviations from the above listed procedures and/or additional procedures specific to this research project”.

Decontamination Procedures:

1.  Surfaces should be wiped with an appropriate deactivating agent such as paper towel wetted with diluted bleach, water containing surfactants, isopropyl alcohol, etc. or using the manufacturer’s recommended cleanup  procedures.
2.  Similarly, the outer surface of contaminated equipment should be wiped using isopropanol and water before removing them from the designated area.
3.  Glassware should be decontaminated and cleaned in a designated location.
4.  Wash hands and arms with soap and water immediately after handling potentially contaminated items.
5.  Waste materials generated from the decontamination procedures should be treated as hazardous waste and disposed of through EHS.
6.  OAR’s cage washing procedures are sufficient for decontamination of cages housing Class 1 chemicals. Any special cleaning procedures should be performed within a chemical fume hood in the PI’s lab (e.g., cypermethrin is insoluble in water).
7.  Water bottles used to deliver Class 1 chemicals to animals must be cleaned in the lab prior to returning to the dirty cage return location.
   1. Fill the bottle with water in order to dilute the chemical and wash contents down the drain in accordance with EHS guidelines.
   2. Wash the water bottle with soap and water and return to the OAR dirty caging return location.

Waste Disposal:

• Animal carcasses/tissues must be bagged in plastic bags and staged for disposal by placing the bag(s) in red biowaste tubs, located within the walk-in coolers, refrigerator, or freezers of each animal facility.
• Chemical hazard waste will be disposed of in the PI’s lab according to EHS guidelines.
• If water bottles are used to deliver Class 1 chemicals to animals, these must be cleaned in the lab prior to return to OAR cage wash locations
  •    Fill the bottle with water in order to dilute the chemical and wash the contents down the drain in accordance with EHS guidance. 
  •      Wash the water bottle with soap and water and return it to OAR cage wash.

Whether or not recombinant or synthetic nucleic acid molecules have been administered to an animal, most carcasses/tissues are double-bagged in plastic biohazard bags and staged for disposal by placing the bag(s) in “biohazard” labeled containers located within the walk-in coolers, refrigerators, or freezers of each animal facility. Exceptions to this policy are outlined below:

• Drosophila species
  • Euthanize followed by autoclaving and disposal in “biohazard” labeled containers or regular trash OR
  • Euthanize followed by disposal in “biohazard” labeled containers.
• Caenorhabditis elegans
  • Dispose in “biohazard” labeled containers
• Danio rerio (Eggs, Fry and Adults)
  • Euthanize followed by autoclaving and disposal in “biohazard” labeled containers or regular trash, OR
  • Euthanize followed by disposal in “biohazard” labeled containers.
• Xenopus species (Eggs, Tadpoles and Adults)
  • Euthanize followed by autoclaving and disposal in “biohazard” labeled containers or regular trash, OR
  • Euthanize followed by disposal in “biohazard” labeled containers.

All animals, in which recombinant or synthetic nucleic acid molecules have been administered, must be properly treated/decontaminated prior to final disposal. Stericycle will properly treat and dispose of animal carcasses and tissues which are placed into the “biohazard” labeled containers located in the animal facilities. Any animal disposed of in the laboratory (Drosophila, C. elegans, D. rerio, or Xenopus) must be properly treated before disposal, regardless of the assigned animal biosafety level.