1 of 4 University of Hawaii at Manoa EHSO Ethidium Bromide SOP TEMPLATE Updated May 2008 STANDARD OPERATING PROCEDURE ETHIDIUM BROMIDE Annual Review Date: Principal Investigator: Phone number: Room & Building: Emergency Contact Information: I have read and understand the SOP: Name: Signature: Date: Before working with Ethidium Bromide, the lab worker must have the approval of the PI who shall provide specific training according to this SOP and in understanding the specific MSDS provided by the manufacturer. The lab worker must complete UH lab safety training prior to working with any chemicals. The lab worker should sign this SOP as documentation that he/she understands the hazards and has been trained in how to work with Ethidium Bromide safely. Ethidium bromide is commercially available in pre-mixed aqueous solutions or in solid/crystal form. Ethidium Bromide is dark red to purple in color, and practically odorless. In its solid form, the powder can easily become airborne and enter the respiratory system. In this regard, aqueous solution of Ethidium Bromide is safer to use. Therefore, Researchers are strongly encouraged to purchase pre-made stock solutions or readymade gels. Also consider switching to less-toxic alternatives such as SYBR SafeTM DNA gel stain, for example, to reduce potential hazardous exposures in the lab. The following safety procedures should be followed by laboratory workers who handle Ethidium Bromide. Ethidium bromide may be a very strong mutagen, and may possibly be a carcinogen or teratogen, although this has never been definitively proven. 1. INFORMATION ABOUT ETHIDIUM BROMIDE AND PROCEDURES USING ETHIDIUM BROMIDE CAS# 1239-45-8 Ethidium Bromide is an intercalating agent commonly used for visualization of nucleic acids using techniques such as agarose gel electrophoresis. When exposed to ultraviolet light, it will fluoresce with a red-orange color, intensifying after binding to DNA. 2. HAZARDOUS PROPERTIES, POTENTIAL ROUTES OF EXPOSURE, SYMPTOMS OF EXPOSURE Daily use of this chemical without manifesting symptoms of overexposure may lead to a false sense of security on the part of laboratory personnel. Ethidium bromide may be fatal if inhaled, and can cause eye, skin, and respiratory tract irritation. Ethidium bromide may be absorbed through the skin and is also a potent mutagen (may alter genetic material), suspected carcinogen and considered moderately toxic. 2 of 4 University of Hawaii at Manoa EHSO Ethidium Bromide SOP TEMPLATE Updated May 2008 Acute Effects: Acute toxic effects from exposure to ethidium bromide have not been thoroughly investigated. Ethidium bromide is irritating to the eyes, skin, mucous membranes, and upper respiratory tract. The powder can be very toxic and harmful by inhalation. Routes of Exposure Ethidium bromide can be readily absorbed through the skin, swallowed or inhaled, entering the eyes, mucous membranes, and upper respiratory tract. Chronic Effects: May cause methemoglobinemia, which is characterized by chocolate-brown colored blood, headaches, weakness, dizziness, shortness of breath, cyanosis, rapid heart rate, unconsciousness and possible death. May alter genetic material. Can be fatal if inhaled. Cancer Hazard: Ethidium bromide is a probable human carcinogen. Repeated exposure to slightly higher amounts may induce multi-site cancers. Reproductive Hazard: There is no direct evidence that ethidium bromide causes birth defects in humans but its mutagenic properties are cause to regard it as a possible reproductive toxin. 3. PERSONAL PROTECTIVE EQUIPMENT Impermeable gloves should be used when potential for skin contact exists; gloves shall be changed frequently. Disposable nitrile, neoprene, natural rubber, PVC, or butyl gloves should be worn, as latex gloves are not recommended for handling Ethidium bromide (confirm glove type and use limitations by referring to a Glove Compatibility and Use Guide - contact EHSO). Safety glasses with side shields or chemical splash goggles shall be worn. A laboratory coat should be worn when working with chemicals. Closed toe shoes are required when working in the laboratory. If the user chooses to wear a dust mask to avoid inhalation when working with the powder, OSHA regulations specify that certain requirements and trainings be met before purchasing masks and use of masks and respirators, contact EHSO for more information. A lab worker can avoid exposure to the powder form without wearing a respirator if manipulation is restricted to a properly operating fume hood. Wear UV-blocking eyewear or work behind a UV shielding glass when using ultraviolet light to visualize the ethidium bromide. 4. ENGINEERING/VENTILATION CONTROLS Work with ethidium bromide crystals in a properly operating and certified chemical fume hood. Work at least 6” inside the hood, never place your head in the hood, set the sash at the lowest position possible (if using the horizontal sliding sashes do not open past the labeled positions). Safety shower and eye wash stations should be easily accessible where ethidium bromide is used. Engineering controls should be used whenever feasible to maintain airborne ethidium bromide concentrations at the lowest achievable levels. 5. SPECIAL HANDLING PROCEDURES AND STORAGE REQUIREMENTS Keep container tightly closed to prevent ethidium bromide from subliming and entering the atmosphere. Store in a cool, dry place away from strong oxidizing agents. 3 of 4 University of Hawaii at Manoa EHSO Ethidium Bromide SOP TEMPLATE Updated May 2008 Use only with adequate ventilation. Avoid generating dust Wash hands thoroughly after handling (even if gloves were used). Coat work bench with absorbent bench paper prior to working with ethidium bromide to prevent contamination of non-disposable surfaces. Transport chemicals in closed containers, in the smallest amounts possible, and use aids such as carts, chemical transport carriers, etc. It is highly recommended that all chemicals be stored below eye level so cracking or leaking containers are immediately visible and there is less potential for chemicals falling onto lab workers when pulling from shelves. 6. PROCEDURES ****Modify this section to reflect your lab specific procedures! Areas where gels are poured should be protected with a lab bench cover. Bench covers should be disposed via EHS upon contamination from spilling or after usage. Measuring Ethidium Bromide Powder o If it is necessary to use ethidium bromide in crystal form, lab workers shall do the following (laboratory personnel are strongly encouraged to purchase aqueous stock solutions). o Wear gloves, a lab coat, and goggles during measuring, weighing, and mixing operations. o If there is a risk of splash, wear a full face shield. o Ensure that the entire hand and arm area is covered. o Do not allow a break in protective coverage at the wrist if coat and gloves do not overlap. o Use a chemical fume hood to measure out the ethidium bromide into a pre-weighed, closable container. o Tightly close container for transport. o The closed container can then be moved safely from the hood to the balance to be weighed. If more or less material is needed, return the container to the hood and adjust the amount of material. Pouring gels and post electrophoresis o Wear appropriate PPE (gloves, eye protection, lab coats) when setting up, pouring, and handling ethidium bromide gels/solutions. o Change gloves after handling ethidium bromide. 7. SPILL AND ACCIDENT PROCEDURES In the event of skin contact, immediately wash with soap and copious amounts of water and remove contaminated clothing. Seek medical attention. Do not use disinfecting chemicals such as ethanol or bleach as this could actually increase skins permeability to ethidium bromide. In case of contact with eyes, immediately flush eyes with copious amounts of water for at least 15 minutes (lifting upper and lower eyelids occasionally) and obtain medical attention. If inhaled, move to fresh air immediately and seek medical attention. In the event of ingestion, obtain medical attention immediately. Do not induce vomiting unless directed to do so by medical personnel. If spill is on equipment, use ultraviolet light to locate spill (if can be done safely), then decontaminate according to Section 8 below. If a large amount of ethidium bromide powder is inhaled, move the person to fresh air and seek medical attention at once. Report all incidents or near misses to EHSO and complete the appropriate Incident Forms. Small spills: Don appropriate PPE. If potential respiratory hazard exists, contact EHSO. Collect spilled material and clean up material into appropriate waste container. Small liquid spill: Absorb freestanding liquid with dry paper towels or absorbent material and place into containers for disposal. Wash with soap and water. 4 of 4 University of Hawaii at Manoa EHSO Ethidium Bromide SOP TEMPLATE Updated May 2008 Small dry spill: If the powder is spilled, wet paper towels and carefully lay on top of the powder to avoid creation of airborne dust when cleaning the spill. Large spills: Notify others in room of spill. Evacuate room/immediate area. Call Campus Security X66911 and notify EHSO. Post warning signs at entrances/exits notifying others of spill. Prevent unnecessary entry into area. Provide assistance and information to spill responders. Report all spills (minor and major) and any near misses to UH EHSO. 8. DECONTAMINATION PROCEDURES Wash the area with paper towels or absorbents soaked in a decontamination solution consisting of 4.2 grams of sodium nitrite and 20 ml of 50% hypophosphorous acid in 300 ml of water. Then rinse 5 times with wet paper towels using a fresh towel each time. Decontaminate all the towels in the decontamination solution for 1 hour. Using a UV light, check for completeness of decontamination. If the area cannot be sufficiently decontaminated, it must be clearly labeled as a contaminated work area. Contact EHSO for disposal of decontamination solution and absorbents/paper towels. The decontamination solution must be prepared just prior to use. 9. WASTE DISPOSAL PROCEDURES While ethidium bromide is not regulated as hazardous waste, the mutagenic properties of this substance may present a hazard if large concentrations are poured down the drain untreated or placed in the trash. The permissible concentration of ethidium bromide solutions that can be poured down the drain PER DAY and PER LABORATORY is: <0.01% by weight and <2 quarts. Intentionally diluting wastes so that they can be poured down the drain is NOT PERMITTED. Aqueous solutions containing more than 10 ug/ml ethidium bromide should be filtered or deactivated using the charcoal filtration method or by a filter funnel kit. The filtrate may be poured down the drain but the charcoal filter or extractors should be collected as biohazard waste. Ethidium bromide gels must be submitted to EHSO for disposal. Collect gels in a clear labeled bag with the chemical name and percentage of ethidium bromide contained in gel waste. Gloves, test tubes, paper towels, etc. that are grossly contaminated with ethidium bromide should be also collected for hazardous pickup by EHSO. Consider deactivating in bleach before disposal if the items are significantly contaminated. 10. MSDS LOCATION ****Where is this located, as an attachment to the SOP, in the MSDS folder, etc? Must have an MSDS, preferably manufacturer specific, specific for each type of ethidium bromide used (per CAS#). References: Information contained in this SOP was gathered from the following sources: Ohio State University, University of California Berkeley EHSO, Fisher Scientific (MSDS).