The EASA consists of 2 simple chemical tests that assess the ability of electrophilic substances to bind to protein by measuring the depletion of free protein surrogates caused by covalent binding of the protein surrogate to the test substance. In the first test, depletion of 4-nitrobenzothiol (NBT), the protein surrogate for soft electrophiles, is measured by the loss of absorbance at 324 nm at 25oC for up to 2 hours. If depletion of NBT ≥ 30%, the test substance is classified as a sensitizer. If depletion of NBT < 30%, confirmation tests must be performed. To confirm the result, the test substance is retested at twice the initial concentration of test substance and/or by increasing the reaction time to 4 hours. If depletion of NBT < 30%, then the second test is performed. In the second test, depletion of pyridoxylamine (PDA), the protein surrogate for hard electrophiles, is measured by the loss of absorbance at 324 nm at 25oC for up to 2 hours....
The EASA consists of 2 simple chemical tests that assess the ability of electrophilic substances to bind to protein by measuring the depletion of free protein surrogates caused by covalent binding of the protein surrogate to the test substance. In the first test, depletion of 4-nitrobenzothiol (NBT), the protein surrogate for soft electrophiles, is measured by the loss of absorbance at 324 nm at 25oC for up to 2 hours. If depletion of NBT ≥ 30%, the test substance is classified as a sensitizer. If depletion of NBT < 30%, confirmation tests must be performed. To confirm the result, the test substance is retested at twice the initial concentration of test substance and/or by increasing the reaction time to 4 hours. If depletion of NBT < 30%, then the second test is performed. In the second test, depletion of pyridoxylamine (PDA), the protein surrogate for hard electrophiles, is measured by the loss of absorbance at 324 nm at 25oC for up to 2 hours. If depletion of PDA ≥ 30%, the test substance is classified as a sensitizer. If depletion of PDA < 30%, confirmation tests, analogous to those with NBT, must be performed. For test substances that interfere with the absorbance of PDA, the loss of PDA is assessed by measuring the decrease in fluorescence at excitation wavelength=324 nm and emission wavelength=398 nm at 25°C for up to 2 hours.
