Phenol as a positive control in cytotoxicity test methods

Girish K Srivastava 1, 2, José Carlos Pastor 1, 2

1.     Instituto Universitario de Oftalmobiologia Aplicada (IOBA), Universidad de Valladolid, Valladolid, Spain.
2.     Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Spain.


Cytotoxicity test is a primary indictor of biocompatibility and safety of medical devices. The ISO 10993 has recommended several in vitro cytototoxicity test methods and parameters. One of them is possibility to apply phenol as a positive control. Phenol is cytotoxic to cell and tissue cultures over determined concentrations. The paper describes errors generated in experimental results if phenol is used for long period in cytototoxic text methods. The ARPE-19 and L929 cell cultures are prepared in 96 wells plate following ISO 10993 recommendations and exposed to positive, negative and test samples. Different concentrations of phenol (0.025, 0.1, 0.2. 0.4, 1.6 mg/ml as well as 1x (1.6 mg/ml), 3x, 66.6% of 1x and 3x) are prepared with culture medium and used in different experimental plans. The cell cultures were exposed, following direct contact cytotoxicity method, directly to phenol concentration for 30 and 60 minutes and then grown for 24 and 72 hours, or were, following extract contact cytotoxicity method, incubated 72 hours with phenol concentrations. The cell culture viability was measured using the MTT and XTT assay. The data are analyzed statistically and errors generated due to phenol samples were detected. The results showed the cell culture lost their viability (<1%) at 1.6 mg / ml concentration of phenol on 30 minutes exposure. On short period exposure (up to 60 minutes in this study), there were no effects on viability of cell cultures of neighboring wells of a culture plate. However, where cell cultures were incubated for a long period, 72 hours, with phenol, the cell culture of neighboring wells also lost their viability. This lost decreases as far as wells are from the wells that contained phenol. The results showed the L929 cell cultures were more sensitive to phenol than ARPE-19 cell cultures. The study concludes that including phenol presence as a positive control for long period in an experimental plan can generate errors in data; which concludes into false positive for a test sample. 

Keywords: Phenol, cytotoxicity, ISO

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Ms. Ref. No.: A0101003     Reviewer 1   Reviewer 2
Received: 23022017
Accepted: 03042018
Article in Press: 04042018 
Published Online: 19042018

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