** Disposal of used diaminobenzidine (DAB) solutions
Question.
How should I dispose of used solutions of 3,3'-diaminobenzidine
(DAB) that have been used for peroxidase histochemistry.
Answer 1.
While DAB itself has not been the subject of in-depth
carcinogenicity studies, it is known to be mutagenic. Further,
all members of the benzidine family that have been tested have
been proved to be carcinogens. In the United States, at least,
all benzidine derivatives are considered carcinogens by the NTP
(National Toxicology Program).
Many people collect the DAB solutions into a bottle containing
5% sodium hypochlorite (which is domestic bleach). After several
hours, the DAB is oxidized to an insoluble polymer.
Chlorine bleach is NOT effective in removing the mutagenic
properties of DAB. While it possibly may break the molecule down
(reaction products are unidentified), introduction of chlorine
into the end products simply produces another mutagenic
chemical. This has been verified by Lunn & Sansone. Using
chlorine bleach is neither chemically sensible nor effective.
Fortunately, most if not all suppliers of DAB have eliminated
this procedure of detoxification from package inserts and
MSDS's.
There are two recommended methods of treatment. The most
commonly used one currently involves potassium permanganate and
sulfuric acid. End products are known to be non-mutagenic. The
second uses horseradish peroxidase to form a solid which is
readily isolated. The fluid remaining is non-mutagenic, but the
precipitate retains its mutagenicity. The only purpose in
performing this method is to reduce the volume of hazardous
waste.
With any commercially available device purporting to detoxify
hazardous chemicals, it is imperative that the user have
documentation from the manufacturer that all reaction products
have been properly tested and found to be non-hazardous. It is
possible that some devices detoxify the liquid and filter out a
hazardous solid. If so, the filter must be handled as a
hazardous waste.
For further information, see:
NTP, 1998. National Toxicology Program Update (January 1998),
Attachment 2. Available on-line at
http://ntp-server.niehs.nih.gov
Lunn & Sansone, 1990. Destruction of hazardous chemicals in the
laboratory. Wiley & Sons (pages 35-41)
Lunn & Sansone, 1991. The safe disposal of diaminobenzidine.
Appl. Occup. Environ. Hyg. 6:49-53.
Dapson & Dapson, 1995. Hazardous materials in the
histopathology laboratory: regulations, risks, handling and
disposal. ANATECH LTD., Battle Creek, MI. (pages 25-27, 109-111
and 162-163)
Richard W. Dapson, Ph.D.
ANATECH LTD.
Battle Creek, MI 49015
(anatech[AT]net-link.net)
Answer 2.
The procedure for acid permanganate oxidation of spent DAB is
as follows. The measurements need not be very accurate.
An acid permanganate solution is made by dissolving
4 g KMnO4 in 100 ml of dilute sulphuric acid (made by
adding 15 ml conc. H2SO4 slowly and carefully to 85
ml of water). This solution is stable. (My experience
is that it's very good at cementing in place the glass
stoppers or screw caps of bottles containing it.)
Add the solution for disposal to an excess of acidified
permanganate and leave overnight (in a fume hood if
the solution contained chloride ions, because these
will end up as chlorine). Next day, neutralize with
sodium hydroxide (carefully; the temperature will
rise) and filter. Leave the filter paper to dry in
the funnel, then put it in a plastic bag for disposal.
If you have a large volume of DAB solution, carefully
add sulphuric acid (150 ml for each litre) and then
dissolve solid potassium permanganate (40 g for each
litre).
Reference: Lunn, G & Sansone, EB (1990). Destruction
of Hazardous Chemicals in the Laboratory. New York:
Wiley Interscience.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
(kiernan[AT]uwo.ca)
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