July 09, 2016
Four concentrations of test compound were tested. To determine the base level of the hemolysis (0% autohemolysis standard) PBS solution was added to one aliquot of whole blood. As positive control TritonÔ X-100 and as 100% positive control distilled water were used. After adding the test compound/controls/vehicle to the human blood, each tube was incubated in water bath. After the respective 6 incubation times, the vials were centrifuged and the absorption of the supernatants was measured in a spectrophotometer.
Each measured absorbance value was normalized with the respective value of PBS treated blood sample. The normalized absorption of the complete hemolysis caused by distilled water was graded as 100%.The dilutions of test item, vehicle and the positive control was measured and graded as a percentage of the positive 100% standard (maximal inducible hemolysis).
The results of the measurement in case of each donor can be accepted if the positive control 0.15% TritonÔ X-100 shows time dependent hemolytic effect and the hemolysis % is higher than 10% at each time point of the incubation.
On the basis of the recommendations of the regulatory guides and white papers listed in regulatory guidelines formulation with hemolysis value above 10% was considered to be at risk for hemolysis.
Conclusion: this test item does not have any hemolytic potential in the investigated concentration range applying incubation time up to 4 hrs. The vehicle at 10% in the blood also did not cause hemolysis in the 4 hour incubation time.
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