Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Disposal of used diaminobenzidine (DAB) solutions
How should I dispose of used solutions of 3,3'-diaminobenzidine (DAB) that have been used for peroxidase histochemistry.
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.
Battle Creek, MI 49015
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