Ethidium Bromide is a potent mutagen which has been commonly used as a nucleic acid stain for many years.
It fluoresces a red-orange colour under ultraviolet light and with increased fluorescence when bound to double-stranded DNA. Typically EB is purchased in powder or solution form and is soluble in water. The crystal or powder form is odourless and appears dark red in colour. Although EB is undoubtedly an effective tool it possesses high hazard properties that dictate that commensurate high level control measures must be implemented in order to mitigate health and safety risk, also special waste disposal measures are required.
The Control of Substances Hazardous to Health Regulations require that where ever possible exposure to hazardous substances be avoided. Where it is not possible to avoid exposure the Regulations require that exposure controls be implemented in a hierarchical order, the first of which is substitution with a substance that is non- hazardous, or less hazardous, before moving further down the hierarchy to mechanical controls and lastly Personal Protective Equipment. The requirement for substitution, where practicable, is absolute and has been tested in case law, therefore where there exists a practical (will achieve same or similar results) non, or less hazardous, alternative this must be adopted. Increased cost alone cannot be presented as a reason for non-implementation of a less hazardous alternative.
Several alternatives to EB exist which manufacturers claim are less toxic than EB and may not require UV light sources. Consequently the routine use of EB is to be discouraged and should only be used where product evaluation and risk assessment have concluded that there is no viable safer alternative. Should the latter be the case, consideration should be given, in the first instance, to purchasing only ready-made solutions so as to avoid the inhalation hazard.
MaestroSafe is a substance produced by Maestrogen and available to purchase from Oxford Biosystems & Cadama.
SYBR® Safe DNA Gel Stain is a highly sensitive stain for visualization of DNA in agarose or acrylamide gels.
GelRed and GelGreen
GelRed and GelGreen are fluorescent nucleic acid stains from Biotium and sold by Cambridge BioScience.
In providing the above product information it is not implied that the Occupational Hygiene Unit of the Health and Safety Department endorses any of the above products. Their suitability as to specific use is beyond our level of expertise in this area, rather the information is presented here in order that it can be included in any evaluation process undertaken by a research group as to their suitability and practicality of use as an alternative to the high hazard material ethidium bromide.
EB is harmful if swallowed, it is very toxic by inhalation and in powder form is considered irritant to the upper respiratory tract, eyes, mucous membranes and skin. Although there is no conclusive evidence, at this time, for the carcinogenicity or teratogenicity of this substance in humans, EB is so strongly mutagenic, causing living cell mutations, that it should be regarded as a possible carcinogen and teratogen.
EB has been assigned the following EU Hazard and Precautionary statements:
- H302 Harmful if swallowed (R22)
- H331 Toxic if inhaled (R26)
- H341 Suspected of causing genetic defects (R68)
- P261 Avoid breathing dust/ fume/ gas/ mist/ vapours/ spray.
- P281 Use personal protective equipment as required. (S36/37/39)
- P311 Call a POISON CENTER or doctor/ physician. (S45)
Any risk and safety phrases directly replace by hazard and precautionary statements have been added in brackets above. Some risk and safety phrases may not have been replaced and therefore have no corresponding hazard or precautionary statements.
Potential health effects
EB may cause extreme eye and skin irritation. It can be absorbed through the skin and contribute to chronic health effects. There is no data available to allow conclusive assessment as to whether or not this chemical would be likely to cause an allergic skin reaction. If ingested it may cause gastrointestinal irritation with nausea, vomiting and diarrhoea. Inhalation is a major hazard that may result in fatality, lesser causal effects are respiratory tract irritation and methemoglobinemia, which is characterised by dizziness, drowsiness, headache, shortness of breath, cyanosis (bluish discoloration of skin due to deficient oxygenation of the blood), rapid heart rate and chocolate-brown blood. Chronic health effects are methemoglobinemia and alteration of genetic material. Animal experiment evidence points to the possibility of carcinogenic and teratogenic effects.
As EB is so strongly mutagenic with the strong possibility of carcinogenic and teratogenic effect pregnant workers should not work with EB.
Good working practices
Before commencing any work practice that involves the use of EB a full and thorough risk assessment must be undertaken in compliance with the COSHH Regulations and safe systems of work/safe operating procedures developed. If practical you must use a safer, less hazardous alternative (examples given at top of page). In the unlikely case of a less hazardous alternative not being a practical approach you should take cognisance of the following good practice.
Risk Assessment forms
If your School has a dedicated spill response team you should call them out. Otherwise proceed as follows:
- Make sure that you are wearing appropriate protective clothing (laboratory coat, eye/face protection, appropriate chemical resistant gloves). If the spill is a powder you should wear a respirator fitted with particulate filters to P3 standard.
- Spills of EB containing solution should be absorbed onto an inert absorbent material (dedicated spill pad/pillow, paper towels, vermiculite, etc) and the area decontaminated as below.
- If the spill is powder avoid raising the powder dust into the air by gently mopping up with wet paper towels and proceed to decontaminate as below.
The following solution must be prepared immediately prior to use and can be used to decontaminate equipment and work areas. The solution should be prepared in a fume cupboard as a small amount of nitrogen dioxide may be given off when the solution is initially mixed. The solution is also strongly acidic (pH 1.8).
Mix 4.2 g of sodium nitrite (NaNO2, CAS # 7362-00-0) and 20 ml of hypophosphorous acid (50%) (H3PO2, CAS # 6303-21-5) in 300 ml of water.
- Wash the area once with a paper towel soaked in decontamination solution.
- Rinse the area five times with paper towels soaked in tap water, using a fresh towel each time.
- Using a UV light, check the area to ensure that all the EB has been removed (no reddish-orange fluorescence). Repeat decontamination procedure as necessary. If the acid could damage the contaminated surface, use additional rinses with paper towels soaked in tap water.
- Discard the decontamination solution, towels and gloves as hazardous waste.
Cognisance must be taken of the specific hazards associated with using UV light sources; all persons in the area should wear appropriate Personal Protective Equipment (UV face shields, laboratory coat and long cuffed gloves fitted over the cuff of the lab coat).
Safe disposal of Ethidium Bromide
Guidance regarding the safe disposal methodology of ethidium bromide in this University is available in the form of Clinical Waste Management Briefing 1-07, on the Waste and Recycling Office website.
Generically, nitrile gloves should provide adequate protection against dermal contamination with ethidium bromide, however the only glove that we have found to have been specifically tested against ethidium bromide penetration, and for which test data is available from the company, is the N-DEX glove, manufactured by the "BEST" Glove Company of U.S.A.
The manufacturers claim that this nitrile glove allows the sensitivity to feel a pulse but is three times more puncture resistant than thicker natural rubber gloves, will not become sticky when exposed to chemicals and tape will not stick to them. It apparently tested no breakthrough after 8 hrs subject to a light concentration of ethidium bromide. It is available in two versions, medical or non-medical grades, and in three thicknesses - 4, 6, and 8ml. Agents in this country include:
- Mackay and Lynn Ltd, 18/7 Dryden Road, Bilston Glen, Loanhead EH20 9LZ Tel: 0131 448 0819 Fax: 0131 448 0865
- VWR International, Hunter Boulevard, Magna Park, Lutterworth, Leicestershire, LE17 4XN 0800 22 33 44
Natural rubber latex gloves do not provide a suitable barrier to penetration by ethidium bromide.