Cell Biology and Toxicology

, Volume 11, Issue 2, pp 79–88 | Cite as

Anin vitro study on the cytotoxicity of chlorhexidine digluconate to human gingival cells

  • H. Babich
  • B. J. Wurzburger
  • Y. L. Rubin
  • M. C. Sinensky
  • L. Blau


Chlorhexidine digluconate is the active ingredient in mouthrinses used to prevent dental plaque and gingivitis. Thein vitro cytotoxicity of chlorhexidine was evaluated with the Smulow-Glickman (S-G) gingival epithelial cell line. The potency of chlorhexidine was dependent on the length of exposure and composition of the exposure medium. The midpoint cytotoxicity values for 1-, 24-, and 72-h exposures were 0.106, 0.011, and 0.0045 mmol/L, respectively. S-G cells exposed for 2 h to chlorhexidine and then maintained for 48 h in chlorhexidine-free medium were unable to recover from the initial insult. The adverse effects of chlorhexidine on the plasma membrane were suggested by the leakage of lactic acid dehydrogenase from chlorhexidine-treated S-G cells and by the increased permeability of chlorhexidine-treated liposomes to Ca2+. The toxicity of a 24-h exposure to chlorhexidine to the S-G cells was progressively lessened as the content of fetal bovine serum (FBS) in the exposure medium was increased from 2% to 8%. The potency of a 1-h exposure to chlorhexidine was reduced in medium amended with albumin, lecithin, and heat-killedEscherichia coli. These reductions in toxicity were presumably due to the binding of the cat onic chlorhexidine to the negatively charged chemical moieties of the components of FBS and of albumin and lecithin and of sites on the surfaces of bacteria. Combinations of chlorhexidine and carbamide peroxide were additive in their cytotoxicities.

Key words

calcium influx assay chlorhexidine digluconate dental products gingival epithelial cells gingivitis in vitro cytotoxicity liposomes mouthrinse neutral red assay plaque 



analysis of variance


calcium concentration in internal medium of liposomes


Dulbecco's modified Eagle medium


ethylenediamine tetraacetic acid


fetal bovine serum


N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid


human gingival fibroblast cell line


honestly significant differences


cell line derived from a human epidermoid carcinoma in the mouth


lactic acid dehydrogenase


nicotinamide adenine dinucleotide, reduced form


neutral red


concentration inhibiting neutral red uptake by 50%


phosphate-buffered saline


standard error of the mean


Smulow-Glickman human gingival epithelial cell line


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Albandar JM, Gjermo P, Preus HR. Chlorhexidine use after two decades of over-the-counter availability. J Periodontol. 1994;65:109–12.Google Scholar
  2. Alleyn CD, O'Neal RB, Strong SL, Scheidt MJ, Van Dyke TE, McPherson JC. The effect of chlorhexidine treatment of root surfaces on the attachment of human gingival fibroblastsin vitro. J Periodontol. 1991;62:434–8.Google Scholar
  3. Babich H, Markensen DF, Blau L, Stern A.In vitro cytotoxicity of the chlorinated naphthoquinone dichlone to human endothelial ECV304 cells. Toxicol In Vitro. 1994;8:1075–81.Google Scholar
  4. Bassetti C, Kallenberger A. Influence of chlorhexidine rinsing on the healing of oral mucosa and osseous lesions. J Clin Periodontol. 1980;7:443–56.Google Scholar
  5. Blau L, Weissmann G. Transmembrane calcium movements mediated by ionomycin and phosphatidate in liposomes with fura 2 entrapped. Biochemistry. 1988;27:5661–6.Google Scholar
  6. Borenfreund E, Babich H. Neutral red (NR) assay, Module 4B:7. In: Griffiths JB, Doyle A, Newell DG, eds. Cell and tissue culture: laboratory procedures. Chichester: Wiley; 1993;4B:7.1–7.7.Google Scholar
  7. Bragger U, Schild U, Lang NP. Effect of chlorhexidine (0.12%) rinses on periodontal tissue healing after tooth extraction. (II). Radiographic parameters. J Clin Periodontol. 1994;21:422–30.Google Scholar
  8. Flotra L. Different modes of chlorhexidine application and related local side effects. J Periodontol Res. 1973;8(supplement 12):41–4.Google Scholar
  9. Foulkes DM. Some toxicological observations on chlorhexidine. J Periodontol Res. 1973;8(supplement 12):55–7.Google Scholar
  10. Gabler WL, Bullock WW, Creamer HR. The influence of chlorhexidine on superoxide generation by induced human neutrophils. J Periodontol Res. 1987;22:445–50.Google Scholar
  11. Gjermo P. Chlorhexidine and related compounds. J Dent Res. 1989;68(special issue):1602–8.Google Scholar
  12. Goldschmidt P, Cogen R, Taubman S. Cytopathologic effects of chlorhexidine on human cells. J Periodontol. 1977;48:212–5.Google Scholar
  13. Grossman E, Reiter G, Sturzenberger OP et al. Six-month study of the effects of a chlorhexidine mouthrinse on gingivitis in adults. J Periodontol Res. 1986;21(supplement 16):33–43.Google Scholar
  14. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440–50.Google Scholar
  15. Helgeland K, Heyden G, Rolla G. Effect of chlorhexidine on animal cells in vitro. Scand J Dent Res. 1971;79:209–15.Google Scholar
  16. Hirst RC, Egelberg R, Hornbuckle GC, Oliver RC, Rothbun WE. Microscopic evaluation of topically applied chlorhexidine gluconate on gingival wound healing in dogs. J Southern Calif State Dent Assoc. 1973;41:311–7.Google Scholar
  17. Hjeljord LG, Rolla G, Bonesvoll P. Chlorhexidine-protein interactions. J Periodontol Res. 1973;8(supplement 12):11–6.Google Scholar
  18. Hugo WB, Longworth AR. Some aspects of the mode of action of chlorhexidine. J Pharm Pharmacol. 1964;16:655–62.Google Scholar
  19. Hugo WB, Longworth AR. The effect of chlorhexidine on the electrophoretic mobility, cytoplasmic constituents, dehydrogenase activity, and cell walls ofEscherichia coli andStaphylococcus aureus. J Pharm Pharmac. 1966;18:569–78.Google Scholar
  20. Kasten FH, Pineda LFR, Schneider PE, Rawls HR, Foster TA. Biocompatibility testing of an experimental fluoride releasing resin using human gingival epithelial cellsin vitro. In Vitro Cell Dev Biol. 1989;25:57–62.Google Scholar
  21. Kasten FH, Seileau K, Neffert RM. Quantitative evaluation of human gingival epithelial cell attachment to implant surfacesin vitro. Int J Period Restor Dent. 1990;10:69–79.Google Scholar
  22. Mobacken H, Wengstrom C. Interference with healing of rat skin incisions treated with chlorhexidine. Acta Dermatovener (Stockholm). 1974;54:29–34.Google Scholar
  23. Noel-Hudson MS, de Belilovsky C, Petit N, Lindenbaum A, Wepierre J. In vitro cytotoxic effects of enzymatically induced oxygen radicals in human fibroblasts: experimental procedures and protection by radical scavengers. Toxicol In Vitro. 1989;3:103–9.Google Scholar
  24. Pucher JJ, Daniel JC. The effects of chlorhexidine digluconate on human fibroblastsin vitro. J Periodontol. 1993;62:526–32.Google Scholar
  25. Rawls HR, Starr J, Kasten FH, Murray M, Smid J, Cabasso I. Radiopaque acrylic resins containing miscible heavy metal compounds. Dent Mater. 1990;6:250–5.Google Scholar
  26. Rindom-Schiott C, Loe H, Briner WW. Two year oral use of chlorhexidine in man. IV. Effect on various medical parameters. J Periodontol Res. 1976;11:158–64.Google Scholar
  27. Saatman RA, Carlton WW, Hubben K, Streett CS, Tuckosh JR, DeBaecke PJ. A wound healing study of chlorhexidine in guinea pigs. Fund App Toxicol. 1986;6:1–60.Google Scholar
  28. Sanchez IR, Swain SF, Nussoaum KE, Hale AS, Henderson RA, McQuire JA. Effects of chlorhexidine diacetate and povidone-iodine on wound healing in dogs. Vet Surg. 1988;17:291–5.Google Scholar
  29. Shopsis C, Eng B. Rapid cytotoxicity testing using a semiautomated protein determination on cultured cells. Toxicol Lett. 1985;26:1–8.Google Scholar
  30. Sinensky MC, Leiser AL, Babich H. Oxidative stress aspects of the cytotoxicity of carbamide peroxide:in vitro studies. Toxicol Lett. 1995;75:101–9.Google Scholar

Copyright information

© Kluwer Academic Publishers 1995

Authors and Affiliations

  • H. Babich
    • 1
  • B. J. Wurzburger
    • 2
  • Y. L. Rubin
    • 1
  • M. C. Sinensky
    • 3
  • L. Blau
    • 2
  1. 1.Department of Biology, Stern College for WomenYeshiva UniversityNew YorkUSA
  2. 2.Department of Chemistry, Stern College for WomenYeshiva UniversityNew YorkUSA
  3. 3.New York University College of DentistryNew YorkUSA

Personalised recommendations