3D Skin Comet Assay

  • Kerstin ReisingerEmail author
  • Stefan Pfuhler


The comet assay is a widely used methodology, whose increased recognition for regulatory purposes led to the recent implementation of the “In Vivo Mammalian Alkaline Comet Assay OECD Testing Guideline” (OECD TG 489). Since it does not generally rely on proliferating cells, this methodology allows the investigation of DNA damage in any cell type or tissue which can be subjected to single cell isolation. Therefore, it is well suited to investigate effects of the first site of contact as well as organ-specific downstream effects, which are documented by the comet assay as primary DNA damage that may lead to clastogenic lesions or gene mutations. Increased DNA damage is signaled by increased migration of negatively charged DNA strands in the electric field which is recorded as increased fluorescence intensity, after appropriate staining of DNA.

For the in vitro comet assay, so far, no regulatory guidelines are available. Considering the skin as the first site of contact of household products, pharmaceuticals, or cosmetics, the comet assay has been customized for the use with 3D reconstructed skin tissues, namely, the EpiDerm™ Full Thickness (EpiDerm™ FT; MatTek, MA) and the Phenion® Full-Thickness Skin Models. They consider both barrier function of the skin and organ- and species-specific metabolism, enabling exposure of chemicals relevant for the situation of use. A joint research project of five European and US-American laboratories has transferred and optimized the respective protocol to establish the 3D Skin Comet assay. First results of the ongoing validation, using Phenion® Full-Thickness Skin Models, have provided evidence for a good reproducibility and predictive capacity of the assay. In consequence, the 3D Skin Comet assay is considered a valuable new in vitro method for the assessment of genotoxicity of dermally exposed chemicals and drugs. It is intended to be used for following up on positive or equivocal results generated with dermally exposed substances in the standard in vitro genotoxicity tests, thereby filling a critical gap in the test battery.


  1. 1.
    OECD. Guidelines for the testing of chemicals, test no. 489: in vivo mammalian alkaline comet assay. 2014.;jsessionid=npkmv8v01k1s.x-oecd-live-02.
  2. 2.
    Tzung TY, Rünger TM. Assessment of DNA damage induced by broadband and narrowband UVB in cultured lymphoblasts and keratinocytes using the comet assay. Photochem Photobiol. 1998;67(6):647–50.CrossRefPubMedGoogle Scholar
  3. 3.
    Emonet-Piccardi N, Richard MJ, Ravanat JL, Signorini N, Cadet J, Béani JC. Protective effects of antioxidants against UVA-induced DNA damage in human skin fibroblasts in culture. Free Radic Res. 1998;29(4):307–13.CrossRefPubMedGoogle Scholar
  4. 4.
    Mouret S, Forestier A, Douki T. The specificity of UVA-induced DNA damage in human melanocytes. Photochem Photobiol Sci. 2012;11(1):155–62.CrossRefPubMedGoogle Scholar
  5. 5.
    Wischermann K, Popp S, Moshir S, Scharfetter-Kochanek K, Wlaschek M, de Gruijl F, Hartschuh W, Greinert R, Volkmer B, Faust A, Rapp A, Schmezer P, Boukamp P. UVA radiation causes DNA strand breaks, chromosomal aberrations and tumorigenic transformation in HaCaT skin keratinocytes. Oncogene. 2008;27(31):4269–80.CrossRefPubMedGoogle Scholar
  6. 6.
    Zeller A, Pfuhler S. N-acetylation of three aromatic amine hair dye precursor molecules eliminates their genotoxic potential. Mutagenesis. 2014;29(1):37–48.CrossRefPubMedGoogle Scholar
  7. 7.
    Flamand N, Marrot L, Belaidi JP, Bourouf L, Dourille E, Feltes M, Meunier JR. Development of genotoxicity test procedures with Episkin, a reconstructed human skin model: towards new tools for in vitro risk assessment of dermally applied compounds? Mutat Res. 2006;606(1–2):39–51.CrossRefPubMedGoogle Scholar
  8. 8.
    Reus AA, Usta M, Krul CA. The use of ex vivo human skin tissue for genotoxicity testing. Toxicol Appl Pharmacol. 2012;261(2):154–63.CrossRefPubMedGoogle Scholar
  9. 9.
    Reus AA, Reisinger K, Downs TR, Carr GJ, Zeller A, Corvi R, Krul CA, Pfuhler S. Comet assay in reconstructed 3D human epidermal skin models--investigation of intra- and inter-laboratory reproducibility with coded chemicals. Mutagenesis. 2013;28(6):709–20.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Reisinger K, Blatz V, Brinkmann JP, Downs TR, Fischer A, Henkler F, Krul C, Liebsch M, Luch A, Pirow R, Reus AA, Schulz M, Pfuhler S. Validation of the 3D Skin Comet assay using full thickness skin models: transferability and reproducibility. 2017.Google Scholar
  11. 11.
    Wiegand C, Hewitt NJ, Merk HF, Reisinger K. Dermal xenobiotic metabolism: a comparison between native human skin, four in vitro skin test systems and a liver system. Skin Pharmacol Physiol. 2014;27:263–75.CrossRefPubMedGoogle Scholar
  12. 12.
    Östling O, Johanson KJ. Micorelectrophoretic study of radiation-induced DNA damaged in individual mammalian cells. Biochem Biophys Res Commun. 1984;123:291–8.CrossRefPubMedGoogle Scholar
  13. 13.
    Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988;175:184–91.CrossRefPubMedGoogle Scholar
  14. 14.
    Hartmann A, Agurell E, Beevers C, Brendler-Schwaab S, Burlinson B, Clay P, Collins A, Smith A, Speit G, Thybaud V, Tice RR. Recommendations for conducting the in vivo alkaline comet assay. 4th international comet assay workshop. Mutagenesis. 2003;18:45–51.CrossRefPubMedGoogle Scholar
  15. 15.
    Kirkland D, Speit G. Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens III. Appropriate follow-up testing in vivo. Mutat Res. 2008;654(2):114–32.CrossRefPubMedGoogle Scholar
  16. 16.
    Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen. 2000;35:206–21.CrossRefGoogle Scholar
  17. 17.
    Pfuhler S, Kirkland D, Kasper P, Hayashi M, Vanparys P, Carmichael P, Dertinger S, Eastmond D, Elhajouji A, Krul C, Rothfuss A, Schoening G, Smith A, Speit G, Thomas C, van Benthem J, Corvi R. Reduction of use of animals in regulatory genotoxicity testing: identification and implementation opportunities-report from an ECVAM workshop. Mutat Res. 2009;680:31–42.CrossRefPubMedGoogle Scholar
  18. 18.
    SCCS. Position statement on genotoxicity/mutagenicity testing of cosmetic ingredients without animal experiments (SCCP/1212/09). 2009.
  19. 19.
    SCCS. Scientific Committee on consumer safety. Opinion on 2,6-Dihydroxyethylaminotoluene COLIPA no A138. SCCS/1563/15. Adopted on 25 Jun 2015.
  20. 20.
    SCCS (Scientific Committee on Consumer Safety). Opinion on Basic Brown 17 COLIPA no B007. SCCS/1531/14. Adopted on 24 Mar 2014, revision of 18 Jun 2014.
  21. 21.
    Marrot L, Belaïdi JP, Lejeune F, Meunier JR, Asselineau D, Bernerd F. Photostability of sunscreen products influences the efficiency of protection with regard to UV-induced genotoxic or photoageing-related endpoints. Br J Dermatol. 2004;151:1234–44.CrossRefPubMedGoogle Scholar
  22. 22.
    Kangas L, Grönroos M, Nieminen AL. Bioluminescence of cellular ATP: a new method for evaluating cytotoxic agents in vitro. Med Biol. 1984;62(6):338–43.PubMedGoogle Scholar
  23. 23.
    Olsson T, Gulliksson H, Palmeborn M, Bergström K, Thore A. Leakage of adenylate kinase from stored blood cells. J Appl Biochem. 1983;5(6):437–45.PubMedGoogle Scholar
  24. 24.
    Brinkmann J, Stolpmann K, Trappe S, Otter T, Genkinger D, Bock U, Liebsch M, Henkler F, Hutzler C, Luch A. Metabolically competent human skin models: activation and genotoxicity of benzo[a]pyrene. Toxicol Sci. 2013;131(2):351–9.CrossRefPubMedGoogle Scholar
  25. 25.
    Lovell DP, Omori T. Statistical issues in the use of the comet assay. Mutagenesis. 2008;23:171–82.CrossRefPubMedGoogle Scholar
  26. 26.
    Ersson C, Möller L. The effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments. Mutagenesis. 2011;26(6):689–95.CrossRefPubMedGoogle Scholar
  27. 27.
    Speit G, Kojima H, Burlinson B, Collins AR, Kasper P, Plappert-Helbig U, Uno Y, Vasquez M, Beevers C, De Boeck M, Escobar PA, Kitamoto S, Pant K, Pfuhler S, Tanaka J, Levy DD. Critical issues with the in vivo comet assay: a report of the comet assay working group in the 6th international workshop on Genotoxicity testing (IWGT). Mutat Res Genet Toxicol Environ Mol Mutagen. 2015;783:6–12.CrossRefGoogle Scholar
  28. 28.
    Pfuhler S, Wolf HU. Detection of DNA-crosslinking agents with the alkaline comet assay. Environ Mol Mutagen. 1996;27:196–201.CrossRefPubMedGoogle Scholar
  29. 29.
    Speit G, Schütz P, Hoffmann H. Enhancement of genotoxic effects in the comet assay with human blood samples by aphidicolin. Toxicol Lett. 2004;153(3):303–10.CrossRefPubMedGoogle Scholar
  30. 30.
    Martin FL, Cole KJ, Orme MH, Grover PL, Phillips DH, Venitt S. The DNA repair inhibitors hydroxyurea and cytosine arabinoside enhance the sensitivity of the alkaline single-cell gel electrophoresis ('comet') assay in metabolically-competent MCL-5 cells. Mutat Res. 1999;445:21–43.CrossRefPubMedGoogle Scholar
  31. 31.
    Gedik CM, Collins AR. The mode of action of 1-beta-D-arabinofuranosylcytosine in inhibiting DNA repair; new evidence using a sensitive assay for repair DNA synthesis and ligation in permeable cells. Mutat Res. 1991;254:231–7.CrossRefPubMedGoogle Scholar
  32. 32.
    Collins AR. Measuring oxidative damage to DNA and its repair with the comet assay. Biochem Biophys Acta. 2014;1840:794–800.CrossRefPubMedGoogle Scholar
  33. 33.
    Berdal KG, Johansen RF, Seeberg E. Release of normal bases from intact DNA by a native DNA repair enzyme. EMBO J. 1998;17:363–7.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Duthie SJ, McMillan P. Uracil misincorporation in human DNA detected using single cell gel electrophoresis. Carcinogenesis. 1997;18:1709–14.CrossRefPubMedGoogle Scholar
  35. 35.
    Thomas DC, Husain I, Chaney SG, Panigrahi GB, Walker IG. Sequence effect on incision by (a)BC excinuclease of 4NQO adducts and UV photoproducts. Nucleic Acids Res. 1991;19:365–70.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Dizdaroglu M, Zastawny TH, Carmical JR, Lloyd RS. A novel DNA N-glycosylase activity of E. coli T4 endonuclear V that excises 4,6-diamino-5-foramidopyrimidive from DNA, a UV-radiation and hydroxyl radical-induced product of adenine. Mutat Res. 1996;362:1–8.CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  1. 1.Henkel AG & Co KGaADüsseldorfGermany
  2. 2.Procter and Gamble, Mason Business CentreMasonUSA

Personalised recommendations