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Lab use of formamide

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Lab use of formamide

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R36/37/38 - Irritating to eyes, respiratory system and skin.

Data from RTECS;

The LD₅₀ (the amount killing 50% of the animals) in rats is reported as 5.5g/Kg body mass (orally), 13.5g/Kg (skin absorption), 4g/Kg (subcutaneously) and 5.7g/Kg (intraperitoneal).

The LD₅₀ in mice is reported as 3.1g/Kg (orally) and 2.5g/Kg (intraperitoneal).

The LD₅₀ in guinea pigs is reported as 1.3g/Kg (intraperitoneal).

The LDLo (lowest published lethal dose) is reported as 1.5g/Kg (intravenous in dogs), 6g/Kg (skin absorption in rabbits) and 0.03g/Kg (subcutaneous in frogs).

The TCLo (lowest published toxic concentration) is reported as 1,50Oppm over 6 hours inhaled by rats.

Animal studies have shown formamide to be a teratogen.

Data from RTECS on animal studies have shown that doses in excess of 0.9g/Kg (and up to 8g/Kg) body mass administered orally or by skin absorption during pregnancy can result in foetal resorption and death, cause a deleterious effect on litter size and specific developmental abnormalities.

Data from the National Toxicology Program (USA) indicate that in continuous breeding campaigns with mice, formamide dissolved in drinking water at a concentration of 750 ppm caused specific developmental abnormalities. Click to see a copy of the report abstract.

You can also see an abstract of their report on the developmental toxicity evaluation of Formamide administered by gavage to Sprague-Dawley (CD®) rats on gestational days 6 through 19.

For a fuller set of reports on the reproductive toxicity of formamide refer to this link.

People at special risk
A foetus could be at risk of developing abnormally if exposed to formamide. Women in the early stages of pregnancy must be made aware of the contents of this document and if they request that they should avoid using formamide then their request must be accepted by their supervisor.

Example of a risk assessment for the small-scale (<100mls) use of formamide in a molecular biology laboratory.

Is it necessary to use formamide? When conducting an assessment of risk the first question should always be - "Do I need to use this hazard?"

Formamide can be used in hybridisation solutions for hybridisation of nucleic acid duplexes (DNA:DNA, DNA:RNA, RNA:RNA), in nucleic acid blots, DNA chips, in-situ-hybridisation to chromosomes, cells and tissues.
However, it is not generally necessary for DNA:DNA hybridisation. For DNA:RNA and RNA:RNA hybridisation it is often recommended, but again it is often not necessary and its use is to a certain extent historical, although in some cases it may be useful or required!

Molecular biology experiments utilize all sorts of solutions containing simple or complex mixtures of chemicals. Researchers doing an experiment will generally pick up a lab manual or copy a colleague's protocol (usually copied from another colleague or book) and so on. It would be simply too difficult for one individual or lab to determine what components of a solution are really necessary for a procedure / protocol / experiment and involve lots of control experiments. So, once established in practice, it usually takes a long time to discover whether a chemical is really necessary for a technique eg Formamide. The same rule applies to determining the necessity of other aspects of many experimental protocols/procedures.

Researchers (the thinking ones) also often make up their own solutions for molecular biology which will be very different from solutions recommended in the manuals, including all sorts of hybridisation solutions which do not contain formamide.

One other point that might be relevant is that many procedures which use probes, hybridisation, and many other procedures, can instead quite often be done using polymerase chain reaction (PCR). This removes the need for formamide, formaldehyde and paraformaldehyde in RNA probe preparation and in RNA gels etc..

If you are to use formamide you should know that female lab workers of child-bearing age must be made aware that a known teratogen is being used. They should be given the data referring to animal studies. If they prefer not to work with the compound themselves then their preference should be respected.
Formamide is easily absorbed through the skin. Thus the major risk from the use of formamide is from skin absorption following a spill.
This risk can be minimised by wearing a laboratory coat, safety spectacles, and nitrile gloves. These must be removed immediately if they become contaminated. Secondary containment (such as standing the bottle in a dish and working over a containment tray) will also reduce the risk from a spill. Dispensing neat formamide from larger parent containers presents a degree of risk that demands the operation to be carried out in a fume cupboard. This must be undertaken using nitrile gloves, safety spectacles and a laboratory coat.Containers used to store solutions of formamide must be adequately labelled (such as "Contains formamide which is toxic (teratogenic)" as well as the concentration, date and name of person making the solution).Although it is harmful by inhalation, the relatively low vapour pressure of formamide indicates that there is little probability of any damage being caused by vapour when handling small (<100mls.) volumes of formamide on the open lab bench, even at temperatures of 65^oC. At this level of use nitrile or latex disposable gloves must be worn, and good practice followed including the frequent change of gloves, and regular washing of hands especially on leaving the laboratory.

First aid measures (following significant exposure).
Eyes. Immediately flush with plenty of water for up to 15 minutes, occasionally lifting the eyelid. Get medical attention.
Skin. Immediately flush with plenty of water (and soap if available) for up to 15 minutes. Get medical attention.
Inhalation. Immediately remove from exposure to fresh air. Get medical attention.

Larger-scale work involving 100mls of formamide must be reassessed and a ducted fume cupboard (or recirculating carbon-filtered unit) considered for controlling exposure to vapour. Thicker nitrile gloves should also be considered and if there is a possibility of splashing then a full-face visor may be needed.

Spillage (<10Omls). Wearing suitable gloves, adsorb the formamide (or solution) onto paper tissues or vermiculite, transfer to a beaker or a tray in a ducted fume cupboard and leave for the liquid to evaporate. The tissues or granules should then be bagged, the bag sealed and then disposed in the normal waste bins.

Disposal. Any volumes of undiluted formamide should be consigned as Special waste, either via the non-chlorinated waste solvent stream or as "lab smalls" (contact the technical staff for more details). Aqueous solutions of less that 50mis can be safely disposed to drain with cold water.

Example protocol;
In molecular biology labs formamide is used in in-situ hybridisation protocols. This includes;

1. Preparing a stock mixture (hybridisation mix or HM) of 25mls formamide added to 25mls other (non-hazardous) aqueous solutions.
2. 1ml amounts of the HM are then added to biological tissue in small capped plastic tubes which are incubated at 65^oC in a small dedicated oven for 2 to 5 hours. Several tubes can be incubated at the same time.
3. The liquid is decanted to waste and 0.2ml of the HM added to the tube which is capped and again incubated at 65^oC overnight.
4. A series of 1ml rinses (10 minutes each) of decreasing concentrations of the HM (at room temperature) is performed.

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