METHOD FOR DETERMINING DEGREE OF IRRITATION OF DISINFECTION AGENTS ON HUMAN SKIN Russian patent published in 2019 - IPC C12Q1/00 

FIELD: biotechnology.

SUBSTANCE: invention is a method for determining the degree of irritation of disinfectants on human skin, consisting in that Chang conjunctiva cell culture is dispersed in a 96-well panel of 100 mcl per well at a concentration of 1×10⁵ cells/ml in nutrient Eagle-MEM with 10 % fetal calf serum (FCS). To form a monolayer of cells, the panel is incubated in a thermostat in an atmosphere of 5 % CO₂ at 37 °C within 24 hours. After incubation, the culture medium is changed to medium containing 2 % FCS 100 mcl per well. Then the samples of disinfection agents (DA) are directly screened in the wells with Chang conjunctiva cells by the method of double serial dilutions in three repetitions per point, leaving the three control wells with a monolayer of cells free from the introduction of DA. Panel is placed in a thermostat, incubated in an atmosphere of 5 % CO₂ at 37 °C within 24 hours. After incubation, the CM is removed from the wells, and 100 mcl of Eagle-MEM with 20 mcl of MTT reagent at a concentration of 5 mg/l is added to each well. After 4 hours of incubation in a thermostat in an atmosphere of 5 % CO₂ at 37 °C MTT reagent medium is removed and 100 mcl of DMSO are added each time to dissolve the formazan reduced by the cells. After 5 minutes exposure, the optical density of the formazan solution in each well is measured on a spectrophotometer at a wavelength of 545 nm, then the optical density (OD) of the solutions obtained after cultivation, is compared with samples of DA with an optical density of solutions of control wells (ODc). In case of equality of OD and ODc by the value of the corresponding minimum non-toxic dilution, a judgment is made about the degree of irritant action of DA on human skin: absence, weak, moderate or pronounced.

EFFECT: provided opportunity to improve the accuracy of research.

1 cl