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Substitution of N,N-dimethylformamide with ethanol in analysis of alkaline phosphatases in leukocytes.


N,N-dimethylformamide is used in a method to measure activity of the enzyme alkaline phosphatase in leukocytes. N,N-dimethylformamide is specifically used as a solvent to prepare a substrate solution for the method and can easily be substituted with ethanol without any changes in the procedure. The only difference is that ethanol is slightly slower as a solvent than N,N-dimethylformamide.

Substituted substance(s)

  1. N,N-dimethylformamide

    CAS No. 68-12-2 EC No. 200-679-5 Index No. 616-001-00-X

Alternative substance(s)

  1. Ethanol

    CAS No. 64-17-5 EC No. 200-578-6 Index No. 603-002-00-5

Other type of alternative

Reliability of information

Evidence of implementation: there is evidence that the solution was implemented and in use at time of publication

Hazard assessment

The alternative, ethanol, is included in in the database of hazardous substances according to SUBSPORT screening criteria (SDSC), however only for presence in alcoholic beverages, which this is not. Ethanol is also flammable. Dimethylformamide is also on the SDSC and is irritating to eyes, harmful by inhalation and skin contact and may cause harm to the unborn child.

» Check the Substance Database according to SUBSPORT Screening Criteria (SDSC)

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Substitution description

N,N-dimethylformamide is used in a method for laboratory analysis of human blood. The method is used to measure activity of the enzyme alkaline phosphatase in leukocytes. The amount of this enzyme is correlated to certain diseases. The enzyme alkaline phosphatase in leukocytes hydrolyses the substrate naphtol- AS- phosphate. Free naphtol binds to the dizoniumsalt Fast Blue BB, whereby a blue precipitate is formed in the cells having enzyme activity, and this can be measured.
In the original method N,N-dimethylformamide is used in preparation of the substrate solution, to dissolve the substrate naphtol-AS-phosphate. N,N-dimethylformamide is toxic to reproduction and harmful to inhale and in contact with skin and eyes. It was found that ethanol could be used as an alternative solvent. Ethanol was a bit slower than the original solvent to dissolve naphtol-AS-phosphatase, but otherwise there was no need to revise the method and the final results of the analysis were not any different from when using the original solvent. It was therefore decided to change to ethanol which is not as harmful for the health.

N,N-dimethylformamide is hazardous and thereby there is a health concern for the person performing the analysis. Ethanol is much safer solvent to use in this application.

It is a bit more time-consuming to dissolve naphtol-AS-phosphate in ethanol than in N,N-dimethylformamide.

Naphtol-AS-phosphate dissolves somewhat slower in ethanol than in N,N-dimethylformamide, however when it has been dissolved there is no changes in the outcome of the analysis, which has been verified in comparative studies of different solvents.

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Case/substitution evaluation

Successful substitution, getting rid of a substance that may be very harmful to the person performing the analyses. There is no need to change the method, and the substitute, ethanol is cheap and available in every laboratory.

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Other solutions

Further information

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Type of publication and availability 

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Publication or last update: 2011-06-15 00:00:00