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Ethidium Bromide: A Reality Check

by on 26th of September, 2007 in Lab Safety

face.jpgThe hysteria among molecular biologists about our old friend ethidium bromide has long been an irritation to me. Researchers are rightly wary of this potential carcinogen. More recently this wariness has been whipped up into a witch hunt by companies touting "safer" alternatives and disposal methods. While I don't for a minute think that we should all throw our gloves away and bathe in the stuff, I think that it's time for an informed reality check about the dangers, and the myths about ethidium bromide.

etbr.pngEthidium bromide is genotoxic, a frame-shift mutagen and teratogen. This is fact, determined by in vitro tests on various cultured cell lines and embryo systems that showed ethidium bromide can cause things like frame-shift mutations, chromosomal recombination, arrested cell division and developmental problems. This information is summarized in an excellent report from the National Toxicology Program.

These in vitro tests, which comprise the entire body of evidence upon which the ethidium bromide hysteria is built, don't provide any evidence that ethidium bromide can exert a genotoxic effect in anything more complicated than a single cell or an unprotected embryo. In fact there is no direct evidence implicating ethidium bromide as a carcinogen in any animal.

For many years, ethidium bromide has been routinely administered for the treatment of African Sleeping Sickness in cattle. For this purpose, ethidium bromide is administered via subcutaneous or intramuscular injection with no reported increase in incidence of tumor formation or birth defects in the treated cattle. This suggests that ethidium bromide is far less genotoxic to animal systems than is presumed from the in vitro data.

The recommended, apparently non-toxic, dose of ethidium bromide is 1mg/kg of body weight in cattle. In comparison to this, the standard concentration used in molecular biology (around 1 microgram/litre), is low. Rosie Redfield puts it into perspective:

A 50kg researcher would need to drink 50,000 liters of gel-staining solution to get even the non-toxic dose used in cattle.

From this, the risks posed to a scientist handling a very weak solution of ethidium bromide, with a gloved hand (remember the cattle are injected with the stuff) are put into perspective.

A real concern is that the irrational and ill-informed fear of ethidium bromide drives us to solutions that are more dangerous than ethidium bromide itself. What could be more dangerous than ethidium bromide?

  • More concentrated ethidium bromide. The method of choice for ethidium bromide disposal at the moment seems to be absorption onto charcoal followed by incineration. You have a 1 microgram/litre solution of ethidium bromide, a concentration that appears to be of low toxicity according to the data from cattle. To me, it seems counter-intuitive to make it MORE concentrated by absorbing onto charcoal. What about just diluting it in water, reducing the toxicity even further?
  • More toxic reaction products of ethidium bromide. Another disposal method is to react it with phosphoric acid, HCl, bleach… but these are fairly dangerous chemicals themselves, and can produce reaction products that are even more toxic than ethidium bromide itself.
  • Safer DNA dyes. Beware of clever marketing and don't believe everything you read. For example, as Rosie points out in her excellent article, "SYBR safe" has a higher acute toxicity in mice than ethidium bromide.

My take home message on this would be to forget all of the hype and myths you have read about ethidium bromide, get real and do what a scientist does best; read the articles I have cited, arm yourself with the known data. Then make your own decision on how to handle ethidium bromide, a decision based on fact… not hysteria.

As always, your comments are welcome!!

About the author: Nick Oswald
I started Bitesize Bio on a Macbook on my kitchen table in 2007 while in my 7th year of working as a molecular biologist in biotech. My aim was to share the know-how that I had acquired from the school of hard-knocks in the lab, so that others could learn from my mistakes and small victories. Nowadays my mission is to facilitate the gathering of hardcore know-how from whole spectrum of bioscientists and share it here on Bitesize Bio to create a super-mentor that any bioscientist can turn to for much-needed guidance.

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15 thoughts on “Ethidium Bromide: A Reality Check”

  1. Argent23 says:

    Well I'm telling the cattle story in almost every lab I get to, but in most of them there really is no other word than hysteria for their working with EB…
    I'm really lucky that this is not the case at my current lab! A special workbench and gloves are all you need, and the disposal consists of collecting the solutions in 5l tanks and delivering them at the wast collection point.

  2. james says:

    What about beta mercaptoethanol, hysterical and it stinks…

  3. Gene Ryan says:

    Mis-statement: "the standard concentration used in molecular biology (around 1 microgram/litre)"

    - The standard working concentration is 0.5 milligram/liter (0.5 mg/l) or 0.5 microgram / milliliter (0.5ug/ml), 500 times more than what is stated. The inaccuracy subtracts the credibility, even though this concentration is still far-far below the >2g/kg (50% lethal dosage, LD50) level for rat oral toxicity value (National Toxicology Program 1239-45-8). Accordingly a 50kg (110 pound) researcher would have to drink >200,000 liters of staining solution to get sick. The LD50 for dermal value (toxicity through skin adsorption)on rat is also >2g/kg. In other words, one would have to wash hands in >200,000 liters of staining solution to get that much ethidium bromide. The amount of 200,000 liters is more than 50,000 gallons, enough to fill a sedan 2,000 times (at 25 gallon fuel capacity). If you refill your car twice a week, it will take 20 years to reach 50,000 gallons.

  4. chris clee says:

    Hear Hear…….a well used food dye in hotdogs for decades if my memory is correct!

  5. Xenobiologista says:

    Thanks for the info.

    At the US university where I did my master's, the official chemical safety guide stated that it was safe to flush dilute EtBr staining solutions down the sink and stained gels could be disposed of in the regular biohazard trash (i.e. it wasn't considered hazardous chemical waste). In my lab we were always careful to wear gloves and clean up with 70% ethanol, but we weren't paranoid about it.

    And then NOW I'm working in Singapore and it's a crime to dispose of even those dilute gel-staining solutions in the sink…apparently the government takes samples from lab sewer lines from time to time. A 1 ug/mL solution is considered hazardous waste.

    Although that's not the worst in hysteria. A girl I knew in grad school said that at the college she attended for undergrad, another student was working alone late at night without gloves. She spilt some EtBr on her hand and panicked, thinking she was going to get cancer and die. So she took a scalpel and cut off the piece of skin which had come into contact with the EtBr…

  6. Avatar of Kerrie Pierce says:

    Thanks for this. While I am not hysterical about the use of Ethidium Bromide, having used it all my career, the tone of your article could lead in the opposite direction. Acute toxicity is not really a good indicator of whether we are vulnerable to a long term risk from occupational exposure to low levels of substances, or whether low levels in the ecosystem could lead to harmful effects in aquatic life. The hot debate over the risk of many such substances is an indicator of how difficult it is to do a controlled experiment on the long term effect of low level exposure to toxins, particularly in humans! Cattle dont live the same length of time! Having worked with Ethidium Bromide for 15 years I am probably in the first generation to have been exposed to occupational levels of it for long enough to have developed problems and who is going to be able to collect a big enough sample of 50-year old molecular biologists to be able to say with statistical significance that we have a higher risk of developing skin cancer on our hands and separate that from sun exposure risk??? Surely we should err on the side of caution and minimise exposure to toxins of any kind, let alone those that we choose to use specifically BECAUSE they bind to DNA (and I'm including SYBRsafe in that category!)

    @Chris Clee, we now are finding evidence that high consumption of cured meat and processed food leads to health risks. You cant say that just because something has been used as a hot-dog additive for decades, that means it is perfectly safe!

    1. Avatar of Sapinder says:

      I think that is to be taken care of…. same is the case with UV exposure and Radioactive substances exposure and even acrylamide exposure. They show long term effects. Only PREVENTION IS BETTER THAN CURE.

  7. Avatar of vittoirio vittoirio says:

    Thanks for this message. Could you post sono bibliography about the point at which you say that EB cannot have any genotoxic effect in vivo? I'd be very interested in demostrating to my colleagues fear is an hysteric phobia. I would really appreciate something about the treatment of African Sleeping Sickness in cattle with EB. Thanks in advance!

  8. Avatar of Dobson Dobson says:

    Before you repeat the cattle anecdote too many times, ask any veterinarian who's accidentally stuck themselves with a trich vaccine. We might inject a couple cc's into a cow, but less than a drop leaves you without feeling in that hand for weeks to months. They're a lot bigger than they look.

    1. Avatar of Nick Oswald Nick Oswald says:

      Thanks Sandra. The main point though is that the concs used in mol bio are miniscule compared to the amounts used in cattle (or in a vet's hand! :) ). It's not that EtBr is harmless — just that a bit of perspective and thought are needed.

  9. Avatar of codyish says:

    Yep, can't help but think what's worse? Diluting used EtBr solutions in a lot of water and pouring it down the drain like I think we should? Or keeping it all in a 2 liter flask (with chopped up pieces of EtBr gels) on the benchtop until we figure out what to do with it.

  10. Avatar of Sapinder says:

    OMG !!!! This article is really amazing. I am still not sure that EB (we often hate it) doesn't actually harm you. In our lab we are actually too much worried about the health risks caused by EB and we keep on thinking of either replacing it or setting up a more efficient disposal technique. I will share it with my lab people.

  11. Thanks, that is very interesting and helpful.
    I like perspective!

  12. Avatar of Dill Huang says:

    EtBr has been successfully used to generate rho0 cells that have no mtDNA, it works because ethidium cannot effectively enter the nucleus. There has Always been concerns that ethidium may interact with genomic DNA but there has been no evidence for that so far in the case of rho0 cells. A lot of the acute toxicity is probably due to mitochondrial toxicity than actual mutations.

    My lab have ceased to use ethidium bromide because the taboo associated with it is simply too much to handle. Many proprietary EtBr replacements are just as dodgy, I know a few are spiked with acridine orange which is a confirmed carcinogen way way worse than EtBr.

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