TUNEL test: ultra-sensitive detection of apoptosis to ensure the cellular safety of cosmetics
The TUNEL assay is an essential method for detecting DNA fragmentation, a characteristic marker of programmed cell death, or apoptosis. It can be used to determine whether a cosmetic ingredient or product may induce excessive or unwanted apoptosis in exposed cells, an important parameter for ensuring the biological safety of formulations.
The cells are first exposed to the product under strictly controlled conditions that are representative of its actual use. The DNA fragments generated during the apoptotic process are then labeled using fluorescent or colorimetric indicators, allowing the affected cells to be accurately visualized. The proportion of labeled cells and the intensity of the signal are used to assess the extent of the observed effect.
A high percentage of positive cells may indicate a potential risk to cell viability, while a low or zero percentage confirms the product's good tolerance at this biological level. Thanks to its high sensitivity, the TUNEL test is a complementary tool to other toxicological and genetic analyses used in Switzerland and Europe to validate the safety of active ingredients and innovative formulations.
The TUNEL test (Deoxynucleotidyl transferase dUTP Nick End Labeling) is now the gold standard method for identifying DNA fragmentation, the definitive marker of programmed cell death (apoptosis). In the field of cosmetics, it can be used to verify that an ingredient or formulation does not trigger excessive apoptosis, an essential criterion for the biological safety of products intended for use on the skin or mucous membranes.
Test principle
- Controlled exposure – Human cells (keratinocytes, fibroblasts, or connective tissue cells, depending on the product being tested) are incubated with the product at concentrations that replicate actual conditions of use.
- Marking DNA breaks – Terminal nucleotidyl transferase (TdT) adds labeled nucleotides (fluorochromes: FITC, Alexa 488; or colorimetric substrates) to the 3'-OH ends of DNA fragments generated during apoptosis.
- Detection – The labeled cells are visualized under a fluorescence microscope or analyzed by flow cytometry. The percentage of positive cells and the intensity of the signal quantify the extent of induced apoptosis.
Why TUNEL?
- High sensitivity : it detects even low levels of DNA fragmentation, which are often invisible with conventional viability tests (MTT, Trypan Blue).
- Specificity : staining only occurs when DNA strands are actually cut, avoiding false positives associated with superficial damage.
- Compliance with regulatory requirements : Swiss (OCos/OSAV) and European (Regulation (EC) No. 1223/2009) authorities require a comprehensive assessment of cellular toxicity for active ingredients, particularly those of natural or biotechnological origin.
- Integration into the DIP/PIF : the results (percentage of TUNEL-positive cells, dose-response curves) are directly reported in the Product Information File (PIF), strengthening the marketing authorization application.