ABP Use of Formaldehyde for Fixation of Whole Animals and Tissues

1. Purpose and Scope

This Animal Biosafety Procedure (ABP) outlines the practices and procedures for working with formaldehyde (37% solution) and paraformaldehyde (white crystalline powder of polymerized formaldehyde) in whole animals or tissues, particularly during perfusion fixation, which poses a higher exposure risk than immersion. Formaldehyde is a carcinogen, and its use must comply with OSHA standards:

• OSHA National Research Council Recommendations Concerning Chemical Hygiene in Laboratories, 29 CFR 1910.1450 App A
• OSHA Hazard Communication, 29 CFR 1910.1200
• OSHA General Industry Standard Formaldehyde, 29 CFR 1910.1048

Note: Alternative practices and equipment may be used but must be documented and approved by EHS and the IACUC before use.

2. Responsibilities

• Principal Investigator (PI): Ensures that personnel are informed of formaldehyde hazards and receive training appropriate to their activities before starting experiments. Personnel must follow the safe work practices and procedures outlined in this ABP.
• Personnel: Must comply with the safe work practices and procedures described within this ABP.

3. Administrative Controls

3.1 Training and Exposure Monitoring

• 3.1.1 Complete Workday Learning training:
  • EHS Laboratory Safety (EHS 2555)
  • Formaldehyde Awareness (EHS 2341)
  • Chemical Waste Disposal (EHS 2716)
  • Cleaning up Small Lab Spills (EHS 2394)
• 3.1.2 Review the Safety Data Sheet (SDS), Formaldehyde Awareness Toolbox Safety Talk, and obtain lab-specific training for handling formaldehyde, perfusion techniques, exposure symptoms, and spill response procedures.
• 3.1.3 Airborne formaldehyde concentrations must not exceed the PEL of 0.75 ppm over 8 hours or the STEL of 2.0 ppm over 15 minutes. Contact EHS OSHIP for airborne measurements and control measure adjustments.
• 3.1.4 Understand formaldehyde exposure effects:
  • Acute: Eye, skin, and respiratory irritation, headaches
  • Chronic: Asthma, bronchitis, cancer
• 3.1.5 CARE and EHS will provide additional on-site training as needed.

3.2 Access and Signage

• 3.2.1 Post a hazard sign where formaldehyde use may exceed exposure limits.

3.3 Medical Surveillance

• 3.3.1 Participate in the Animal Users Health and Safety Program (AUHSP) if handling live animals.

4. Work Practice and Procedure Controls

4.1 General Handling Practices

• 4.1.1 Keep containers closed as much as possible.
• 4.1.2 Use the smallest quantities necessary. Use a balance with a draft shield when weighing paraformaldehyde.
• 4.1.3 Minimize fixative solution volume and handle specimens gently to avoid splashing.
• 4.1.4 Use a spill tray and work in well-ventilated areas, preferably in a chemical fume hood. If a fume hood is unavailable, contact EHS.
• 4.1.5 Wipe down work areas with soap and water.

4.2 Hygiene

• 4.2.1 Eating, drinking, smoking, handling contact lenses, applying cosmetics, storing food, and mouth pipetting are prohibited in lab and animal facilities.
• 4.2.2 Wash hands with soap and water after removing gloves.
• 4.2.3 Do not wear PPE outside work areas.

4.3 Sharps Handling

• 4.3.1 Substitute plasticware for glassware when possible. Follow safe practices for handling sharps, including:
  • Limit sharps use when alternatives are unavailable.
  • Keep sharps in full view at all times.
  • Use Luer-lock syringes and needles or integrated units.
  • Use safety-engineered sharps to minimize risk of injury, as required by OSHA standards.
  • Dispose of sharps immediately in approved sharps containers.
• 4.3.2 Do not recap needles unless approved by EHS and IACUC. If recapping is necessary, use the one-handed scoop technique or forceps.

4.4 Spill Response

• 4.4.1 For small spills (<100ml), don appropriate PPE and use formalin spill control sorbent or absorbent pads. Label and dispose of waste properly.
• 4.4.2 Seek assistance if experiencing irritation or other adverse reactions.
• 4.4.3 Label waste with the EHS green Hazardous Waste label and request removal via the EHS online waste pick-up request. Wash spill area with soap and water.
• 4.4.4 For large spills, evacuate the area and call 911 (campus phone) or 255-1111 (cell phone).

4.5 Handling of Waste

• 4.5.1 Collect formaldehyde in compatible containers and label them as hazardous waste.
  • Liquid waste generated from the use of formaldehyde and paraformaldehyde must be labeled as "Hazardous Waste." Formaldehyde is classified as a hazardous chemical under OSHA regulations and the EPA's Resource Conservation and Recovery Act (RCRA).
• 4.5.2 Dispose of formaldehyde-contaminated sharps directly in sharps disposal containers.
• 4.5.3 Collect carcasses/tissues in clear, labeled bags and store in a refrigerator until disposal.
• 4.5.4 Refer to the appropriate Waste Disposal Guide (CVM or non-CVM) for more information.

4.6 Transport, Storage, and Incompatibilities

• 4.6.1 Transport formaldehyde and contaminated samples in sealed, secondary containers with absorbent toweling, labeled "Formaldehyde".
• 4.6.2 Store formaldehyde in secondary containers, away from incompatible materials (oxidizers, alkalis, acids, ammonia, phenol, isocyanates, peroxides, anhydrides). Store safely in a flammable storage cabinet or under a fume hood.
• 4.6.3 Keep away from heat, sparks, and flame.
• 4.6.4 Formaldehyde reacts violently with nitrogen dioxide, perchloric acid/aniline mixtures, and nitromethane. It also reacts with HCl and sodium hypochlorite to form bis-chloromethyl ether.

5. Engineering Controls

• 5.1 Handle powder and make dilutions in a certified chemical fume hood.
• 5.2 Conduct procedures in an approved chemical fume hood, ducted biosafety cabinet, or use local exhaust ventilation or downdraft table.
• 5.3 Handle dilute solutions (e.g., 4% formaldehyde) on the bench top with sufficient room ventilation. Consult with EHS to ensure airborne concentrations are below exposure limits.

6. Personal Protective Equipment (PPE)

• 6.1 Don the following minimum PPE before working with formaldehyde:
  • Safety glasses with side shields or chemical splash goggles.
  • Protective outer garments (lab coat, coveralls), or disposable fluid-resistant solid front gown.
  • Nitrile disposable gloves (double nitrile gloves for extended contact or higher concentrations).
  • Lab appropriate attire is also required, consisting of long pants, covered torso, and closed-toe, closed-heel shoes.
• 6.2 Ensure no breaks in protective coverage (e.g., gloves overlap with sleeve of lab coat).
• 6.3 Wear additional PPE (e.g., face shield, respiratory protection) when splash risk is present, engineering controls are unavailable, or as indicated by hazards or experimental conditions. Respiratory protection requires enrollment in the EHS Respiratory Protection Program.
• 6.4 Remove and replace contaminated or breached PPE.

7. Response to Accidental Exposures

• 7.1 In case of exposure:
  • Wash exposed area in emergency eye wash or safety shower for at least 15 minutes. Call 911 or 607-255-1111.
  • Seek fresh air if respiratory exposure occurs.
  • Notify supervisor and seek medical evaluation at Cornell Health, Occupational Medicine (607-255-6960, off-hours 607-255-5155). If after hours, seek evaluation at Cayuga Medical Center.
  • Document exposures in the Cornell University Injury/Illness/Exposure Report, Incident Reporting.

8. Emergency Phone Numbers

• Police, Fire, and Medical Emergencies: call 911 from an on-campus phone or 607-255-1111 from a cell phone.
• Environment, Health and Safety (EHS): 607-255-8200 (off hours 607-255-1111).
• Cornell Animal Resources and Education (CARE): 607-253-4378 (off hours 1-800-349-2456 for veterinary medical emergencies).
• Non-emergency questions: askEHS@cornell.edu.

9. Additional Information

9.1 Paraformaldehyde, Formaldehyde, and Formalin

Formaldehyde, formalin, and paraformaldehyde are often confused. Here are their distinctions:

• Formaldehyde: CH₂O, the simplest aldehyde.
• Formalin: Saturated (37%) formaldehyde solution. A protocol calling for 10% formalin is roughly equivalent to 4% formaldehyde. Note that some solutions have methanol to prevent polymerization, which could affect your sample.
• Paraformaldehyde (PFA): Polymerized formaldehyde. Methanol-free formaldehyde can be made by heating solid PFA. However, it is no longer in polymer form once dissolved.

9.2 Making Paraformaldehyde Solution

• To make a 4% paraformaldehyde solution:
  • Dissolve PFA in PBS or TBS at 70°C.
  • Add several drops of 5N NaOH to help clarify the solution.
  • Prepare in a chemical hood to avoid exposure.
  • Remove insoluble impurities by centrifuging or filtering the solution. The efficacy and impurity content of PFA can vary by lot number, so adjust fixation concentrations and conditions as needed.
  • Use freshly prepared solutions that are colorless for best results. Storing aliquots at -20°C for a few months is common.

Reviewed July 1, 2024