Aim, Materials and Methods – NRU – IN&OUT DETOX®
The NRU is used to assess the safety use of products, testing “in vitro” different cytotoxicity concentrations of cosmetics in cell models consisting of human fibroblasts. NRU assay assess the effects of the test sample on the cells vitality.
Functional ingredient for cosmetic use: In & out DETOX®
Positive control: Sodium Dodecyl Sulphate (SDS) (irritating to the skin).
NRU Cytotoxicity Assay.
“In vitro” evaluation of the eye irritation potential of cosmetic products. The RNU assay is based on the cell ability to incorporate and bind the Neutral Red, a vital dye. The present study was carried out in strict compliance to Good Laboratory Practice (GLP) gui delines.
MATERIALS AND REAGENTS
The cell lines indicated in ISO 10993-5 are as follows: MRC 5 cells *
* American Type Culture Collection cell lines.
CULTURE MEDIA AND REAGENTS
Hereafter we report the list of the utilized medium and reagents:
Dulbecco’s modified eagle’s medium (DMEM) low Glucose
Glutammine 4mM. Fetal Bovin serum (FBS)
Phosphate buffered saline (PBS)
The neutral red (NR): is a week cationic dye that penetrate the cell membrane through a mechanism of non ionic diffusion and that is accumulated in the lysosomes, on matrix anionic sites. Cell and lysosome membrane alterations cause lysosomes fragility and gradual irreversible changes in the cells. These changes induced by xenobiotics determinate the decreasing of RN uptake and lysosome linking. Alive, damaged and dead cells can be discriminated with this method. Cells are incubated with scalar concentrations of the products and with the Neutral Red solution (NR). If the membrane is damaged, it release dye in the medium.
MICROBIOLOGICAL LABORATORY EQUIPMENT
- Inverted microscope
- Electromechanical shaker: vortex mixer
- Incubator (95% air, 5% CO2) 37°C ± 1°C
- Ice producing machine
- Mechanical shaker
- Biological safety cabinet with Air Clean Systems Vertical Laminar Flow “Biohazard” class II
- Thermostatic water bath
- Refrigerator at 4°C±8°C; freezer at 70° C± 1°C
- Microtiter plate reader
Cells was seeded in 96 well plates at density of 30.000 cell / well and incubated up to 80% confluence. Fresh medium is added with scalar dilutions of tested product. The sample has been diluted to the final desired concentration in the culture medium. The product was extracted in PBS (Phosphate buffered saline): 1gr /5 ml for 24 hours and several dilutions have been prepared.
Each dilution was put in contact with the cells (3 wells for each dilution) for 24 hours at 37 ° C ± 1 ° C and 5% CO2. At the end of the exposure time each well was washed in PBS, 100 ul of the RN medium/well was added and the plates were incubated for 3 h at 37 ° C ± 1 ° C and 5% CO2, than 100 ul of RN desorb solution (freshly prepared 49 parts water + 50 parts ethanol + 1 part acetic acid)/well were added. The plates were incubated in a 37 ° C shaker, after 30 minutes the absorbance reading was taken at 540 nm.
Untreated cells were used as a negative control, and cells treated with a surfactant of known toxicity (sodium dodecyl sulphate, SDS) dissolved in the culture medium at concentrations from 2 mg/ml to 0.03 mg/ml was used as a control positive.