3D Skin Models & Comet Assay IV
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- Created: Tuesday, 27 February 2018 13:28
The 3D Skin Comet assay was developed by a collaboration of researchers to improve the in vitro prediction of the genotoxic potential of dermally applied chemicals.
The classical (and well researched) endpoint for genotoxicity, the comet assay, was combined with reconstructed 3D skin models (as well-established test systems) in this exciting investigation.
Five laboratories (BASF, BfR (Federal Institute for Risk Assessment), Henkel, Procter & Gamble and TNO/ Triskilion) started to validate this assay using the Phenion® Full-Thickness (FT) Skin Model and eight coded chemicals. This investigation gained financial support from Cosmetics Europe and the German Ministry of Education & Research.
Experimental details, including characterization and selection of coded chemicals, skin tissue information, experimental & study design, isolation of single cells procedure, and data evaluation, can all be found in the original publication cited below.
The investigation describes how background DNA damage was low and values for solvent (acetone) and positive (methyl methanesulfonate (MMS)) controls were comparable among labs.
The analysis of comet assay slides was harmonized among the laboratories based on recently published standards. The slides were randomized and coded by a different person to prevent evaluator bias. Four of the five participating laboratories used Comet Assay IV software (Instem, UK). DNA was stained with SYBR Gold. Tail intensity (% tail DNA) was chosen as the parameter to assess genotoxicity. For each tissue compartment, two slides were analyzed, and a third slide was stored as back-up. In detail, 50 cells per slide were analyzed, i.e., 100 cells per single tissue compartment (single epidermis or dermis). For three tissue replicates, this resulted in 300 cells per group of compartments, and lastly, 600 cells total per dose group.
Further to this, the researchers found that the inclusion of the DNA-polymerase inhibitor, aphidicolin (APC), in the protocol improved the predictivity of the assay since it enabled robust detection of pro-mutagens e.g., 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene. For more information, please refer to the original publication.
In summary, an excellent overall predictivity was observed. Four labs correctly identified all chemicals and the fifth correctly identified 70% of the chemicals.
The scientists concluded that these data support the use of the Phenion® FT in the Comet assay: no false-positives and only one false-negative finding in a single lab. The relevance of the results will be proven with the complete validation data set of >30 chemicals.
Already in parallel to the validation exercise, the intended use of the 3D Skin Comet assay was exemplified with successfully supporting the safety assessments of hair dye ingredients in the EU, i.e. it was used as follow up on positive findings from the current in vitro genotoxicity test battery. Scientists envision that the method will be adopted into an OECD test guideline for its global use in the safety assessment of chemicals, e. g. in the framework of REACH.
About Comet Assay IV
Originally developed by Perceptive Instruments, and now part of the Instem solution portfolio, Comet Assay IV is a live video imaging system for fast, accurate and reproducible comet slide scoring. Comet Assay IV's unique single-click scoring method and instant live video technology make it the most efficient and easy-to-use system available for measuring DNA damage using single cell gel electrophoresis. Comet Assay IV has been successfully deployed in the validation study of the 3D Skin Comet Assay. This assay, built on Phenion Full-Thickness Skin Models, has shown excellent toxicological predictivity during its validation by five US and European laboratories. The assay detects structural DNA damages and other lesions, which may give rise to gene mutations, and the method is well-suited to supplement existing in vitro test batteries and to follow-up initial positive findings.
About Phenion
Phenion, with its expertise in skin physiology, markets in vitro testing tools for safety assessment and basic research in dermatology and cosmetic science. Phenion products include the human Phenion® Full-Thickness Skin Model and the Phenion® Full-Thickness AGED Skin Model. These tissues are exclusively populated with primary human cells, thus perfectly mimicking human skin in diverse dimensions, such as histological architecture and a wide spectrum of physiological skin parameters. After having used the innovative skin models for its own research purposes for many years, the organization has decided to take the next step and grant unlimited access to the Phenion products, thus making them available for other companies, organizations and research institutes.
For more information, please visit:
Phenion www.phenion.com
Cosmetics Europe www.cosmeticseurope.eu
German Ministry of Education & Research www.bmbf.de
Instem www.instem.com/solutions/genetic-toxicology/comet-assay.php
Read more:
Kerstin Reisinger, Veronika Blatz, Joep Brinkmann, R.Thomas Downs, Anja Fischer, Frank Henkler, Sebastian Hoffmann, Cyrille Krul, Manfred Liebsch, Andreas Luch, Ralph Pirow, A. Astrid Reus, Markus Schulz, Stefan Pfuhler.
ScienceDirect Available online 10 January 2018
Thank-you to Phenion for collaborating with us on this story and allowing us to share their images.