False positive results in cytotoxicity testing due to unexpectedly volatile compounds

Area of Science:

• Biomaterials Science
• In Vitro Toxicology
• Dental Materials Research

Background:

• Cytotoxicity testing is crucial for screening new dental materials.
• Standard protocols like EN 30993 guide in vitro biological evaluations.
• Microplate assays are commonly used for cell viability assessments.

Purpose of the Study:

• To investigate the cytotoxicity of dental materials using established laboratory protocols.
• To evaluate the reliability of in vitro cytotoxicity assays, specifically neutral red and propidium iodide uptake.
• To identify potential sources of artifactual results in cytotoxicity screening.

Main Methods:

• Dental materials were extracted using aqueous solutions.
• Cell cultures were exposed to these extracts in microplates.
• Cell viability was assessed using neutral red uptake and propidium iodide uptake assays.
• Experimental conditions were analyzed to identify sources of discrepancies.

Main Results:

• Contrasting results were observed for one material between the two assay methods.
• A sustained release of volatile substances from one extract caused toxicity in adjacent cultures.
• This indicated cross-contamination within the microplate environment.

Conclusions:

• Sample distribution and potential for cross-contamination in microplates are critical factors in cytotoxicity testing.
• Employing multiple cytotoxicity assay methods is recommended to validate results and avoid false positives.
• Careful experimental design is necessary to prevent artifacts in in vitro dental material screening.