Red blood cell (RBC) test
RBC test enables the quantification and evaluation of the side effects caused by surfactants added to many cosmetic products, such as shampoo, shower gel and emulsions in the red blood cells plasmatic membrane and consequently the hemoglobin release (hemolysis) and the hemoglobin denaturation index, both quantified by spectrophotometry. The relationship between hemolysis and hemoglobin oxidation provides a parameter to the characterization of the in vitro irritant effects of the surfactants (Alves et al., 2008).
Sheep RBC were isolated by g force. For hemolysis analysis, solutions of 1 mg/ml of each formulation in PBS were added to vials in order to create a range of increasing concentrations to enable linear regression. Their volume was filled up to 975 l with PBS and it was added 25 l of RBC suspension with a well-known concentration of oxyhemoglobin. The resulting suspension was incubated for 10 min at room temperature under slow stirring. After 10 min the samples were centrifuged at 10,000 × g for 1 min. The absorbance of the supernatant was determined by spectrophotometry at 540 nm (for oxyhemoglobin) and at 575 nm (for deoxyhemoglobin). For a
0% hemolysis control, 25 l of RBCs was added to 975 l of PBS, and for a 100% hemolysis control, 25 l of RBC was added to 975 l of distilled water. The denaturation index obtained are comparable to the in vivo ocular irritant effect: >100 corresponding to NOI; >10 corresponding to SLI; >1 corresponding to MOI; >0.1 corresponding to irritant(I); and