, Volume 249, Issue 3, pp 493–502 | Cite as

The importance of a validated standard methodology to define in vitro toxicity of nano-TiO2

  • Janez Valant
  • Ivo Iavicoli
  • Damjana DrobneEmail author
Review Article


Several in vitro studies on the potential toxicity of nano-TiO2 have been published and recent reviews have summarised them. Most of these reports concluded that physicochemical properties of nanoparticles are fundamental to their toxicological effects. No published review has compared in vitro tests with similar test strategies in terms of exposure duration and measured endpoints and for this reason we have attempted to assess the degree of homogeneity among in vitro tests and to assess if they afford reliable data to support risk assessment. The responses in different in vitro tests appeared to be unrelated to primary particle size. The biologically effective concentrations in different tests can be seen to differ by as many as two orders of magnitude and such differences could be explained either by different sensitivities of cell lines to nanoparticles or by effect of the test media. Our review indicates that even when the in vitro tests measure the same biomarkers with the same exposure duration and known primary particle sizes, it is insufficient merely to use such data for risk assessment. In the future, validated standard methods should include a limited number of cell lines and an obligatory selection of biomarkers. For routine purposes, it is important that assays can be easily conducted, false negatives and false positives are excluded and unbiased interpretation of results is provided. Papers published to date provide an understanding of the mode on nano-TiO2 action but are not suitable for assessment and management of risk.


TiO2 nanoparticles Risk assessment Risk management Nanotoxicity Size-dependent effects 



We would like to thank the Slovenian Research Agency (project number J1-9475), and G.W.A. Milne for editorial assistance.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Aschberger K, Micheletti C, Sokull-Kluttgen B, Christensen FM (2011) Analysis of currently available data for characterising the risk of engineered nanomaterials to the environment and human health—lessons learned from four case studies. Environ Int 37(6):1143–1156PubMedCrossRefGoogle Scholar
  2. Braydich-Stolle LK, Schaeublin NM, Murdock RC, Jiang J, Biswas P, Schlager JJ, Hussain SM (2009) Crystal structure mediates mode of cell death in TiO2 nanotoxicity. J Nanopart Res 11(6):1361–1374CrossRefGoogle Scholar
  3. Di Virgilio AL, Reigosa M, Arnal PM, de Mele MFL (2010) Comparative study of the cytotoxic and genotoxic effects of titanium oxide and aluminium oxide nanoparticles in Chinese hamster ovary (CHO-K1) cells. J Hazard Mater 177(1–3):711–718PubMedCrossRefGoogle Scholar
  4. Erickson BE (2008) Grassroots effort aims to improve quality of nanotoxicology studies. Chem Eng Tools 86(50):25–26Google Scholar
  5. Falck GCM, Lindberg HK, Suhonen S, Vippola M, Vanhala E, Catalan J, Savolainen K, Norppa H (2009) Genotoxic effects of nanosized and fine TiO2. Hum Exp Toxicol 28(6–7):339–352PubMedCrossRefGoogle Scholar
  6. Fujita K, Horie M, Kato H, Endoh S, Suzuki M, Nakamura A, Miyauchi A, Yamamoto K, Kinugasa S, Nishio K, Yoshida Y, Iwahashi H, Nakanishi J (2009) Effects of ultrafine TiO2 particles on gene expression profile in human keratinocytes without illumination: involvement of extracellular matrix and cell adhesion. Toxicol Lett 191(2–3):109–117PubMedCrossRefGoogle Scholar
  7. Hackenberg S, Friehs G, Froelich K, Ginzkey C, Koehler C, Scherzed A, Burghartz M, Hagen R, Kleinsasser N (2010) Intracellular distribution, geno- and cytotoxic effects of nanosized titanium dioxide particles in the anatase crystal phase on human nasal mucosa cells. Toxicol Lett 195(1):9–14. doi: 10.1016/j.toxlet.2010.02.022 PubMedCrossRefGoogle Scholar
  8. Han X, Gelein R, Corson N, Wade-Mercer P, Jiang J, Biswas P, Finkelstein JN, Elder A, Oberdörster G (2011) Validation of an LDH assay for assessing nanoparticle toxicity. Toxicology In pressGoogle Scholar
  9. Hartung T, Daston G (2009) Are in vitro tests suitable for regulatory use? Toxicol Sci 111(2):233–237PubMedCrossRefGoogle Scholar
  10. Hensten-Pettersen A, Helgeland K (1981) Sensitivity of different human cell-lines in the biologic evaluation of dental resin-based restorative materials. Scand J Dent Res 89(1):102–107PubMedGoogle Scholar
  11. Horie M, Nishio K, Fujita K, Kato H, Endoh S, Suzuki M, Nakamura A, Miyauchi A, Kinugasa S, Yamamoto K, Iwahashi H, Murayama H, Niki E, Yoshida Y (2010) Cellular responses by stable and uniform ultrafine titanium dioxide particles in culture-medium dispersions when secondary particle size was 100 nm or less. Toxicol in vitro 24(6):1629–1638PubMedCrossRefGoogle Scholar
  12. Huang S, Chueh PJ, Lin WY, Shih ST, Chuang SM (2009) Disturbed mitotic progression and genome segregation are involved in cell transformation mediated by nano-TiO2 long-term exposure. Toxicol Appl Pharm 241(2):182–194CrossRefGoogle Scholar
  13. Hussain SM, Hess KL, Gearhart JM, Geiss KT, Schlager JJ (2005) in vitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol in vitro 19(7):975–983Google Scholar
  14. Iavicoli I, Leso V, Fontana L, Bergamaschi A (2011) Toxicological effects of titanium dioxide nanoparticles: a review of in vitro mammalian studies. Eur Rev Med Pharmacol Sci 15(5):481–508PubMedGoogle Scholar
  15. Jin CY, Zhu BS, Wang XF, Lu QH (2008) Cytotoxicity of titanium dioxide nanoparticles in mouse fibroblast cells. Chem Res Toxicol 21(9):1871–1877PubMedCrossRefGoogle Scholar
  16. Kirkland DJ, Aardema M, Banduhn N, Carmichael P, Fautz R, Meunier JR, Pfuhler S (2007) In vitro approaches to develop weight of evidence (WoE) and mode of action (MoA) discussions with positive in vitro genotoxicity results. Mutagenesis 22(3):161–175PubMedCrossRefGoogle Scholar
  17. Komatsu T, Tabata M, Kubo-Irie M, Shimizu T, Suzuki K, Nihei Y, Takeda K (2008) The effects of nanoparticles on mouse testis Leydig cells in vitro. Toxicol in vitro 22(8):1825–1831Google Scholar
  18. Lee YS, Yoon S, Yoon HJ, Lee K, Yoon HK, Lee JH, Song CW (2009) Inhibitor of differentiation 1 (Id1) expression attenuates the degree of TiO2-induced cytotoxicity in H1299 non-small cell lung cancer cells. Toxicology Lett 189(3):191–199CrossRefGoogle Scholar
  19. Liu SC, Xu LJ, Zhang T, Ren GG, Yang Z (2010) Oxidative stress and apoptosis induced by nanosized titanium dioxide in PC12 cells. Toxicology 267(1–3):172–177PubMedCrossRefGoogle Scholar
  20. Meissner T, Potthoff A, Richter V (2009) Physico-chemical characterization in the light of toxicological effects. Inhal Toxicol 21(S1):35–39PubMedCrossRefGoogle Scholar
  21. Menard A, Jemec A, Drobne D (2011) Ecotoxicity of nanosized TiO2. Review of in vivo data. Environ Pollut 159(3):677–684PubMedCrossRefGoogle Scholar
  22. Murdock RC, Braydich-Stolle L, Schrand AM, Schlager JJ, Hussain SM (2008) Characterization of nanomaterial dispersion in solution prior to in vitro exposure using dynamic light scattering technique. Toxicol Sci 101(2):239–253PubMedCrossRefGoogle Scholar
  23. Oberdörster G, Maynard A, Donaldson K, Castranova V, Fitzpatrick J, Ausman K, Carter J, Karn B, Kreyling W, Lai D, Olin S, Monteiro-Riviere N, Warheit DB, Yang H (2005) Principles for characterizing the potential human health effects from exposure to nanomaterials: elements of a screening strategy. Part Fibre Toxicol 2(8)Google Scholar
  24. Palomaki J, Karisola P, Pylkkanen L, Savolainen K, Alenius H (2010) Engineered nanornaterials cause cytotoxicity and activation on mouse antigen presenting cells. Toxicology 267(1–3):125–131PubMedCrossRefGoogle Scholar
  25. Park EJ, Yi J, Chung YH, Ryu DY, Choi J, Park K (2008) Oxidative stress and apoptosis induced by titanium dioxide nanoparticles in cultured BEAS-2B cells. Toxicol Lett 180(3):222–229PubMedCrossRefGoogle Scholar
  26. Park MVDZ, Lankveld DPK, van Loveren H, de Jong WH (2009) The status of in vitro toxicity studies in the risk assessment of nanomaterials. Nanomedicine 4(6):669–685PubMedCrossRefGoogle Scholar
  27. Park YH, Jeong SH, Yi SM, Choi BH, Kim YR, Kim IK, Kim MK, Son SW (2011) Analysis for the potential of polystyrene and TiO2 nanoparticles to induce skin irritation, phototoxicity, and sensitization. Toxicol in vitro In press Google Scholar
  28. Petković J, Žegura B, Stevanović M, Drnovšek N, Uskoković D, Novak S, Filipič M (2010) DNA damage and alterations in expression of DNA damage responsive genes induced by TiO2 nanoparticles in human hepatoma HepG2 cells. Nanotoxicology in pressGoogle Scholar
  29. Powers KW, Brown SC, Krishna VB, Wasdo SC, Moudgil BM, Roberts SM (2006) Research strategies for safety evaluation of nanomaterials. Part VI. Characterization of nanoscale particles for toxicological evaluation. Toxicol Sci 90(2):296–303PubMedCrossRefGoogle Scholar
  30. Sayes CM, Warheit DB (2008) An in vitro investigation of the differential cytotoxic responses of human and rat lung epithelial cell lines using TiO2 nanoparticles. Int J Nanotechnol 5(1):15–29CrossRefGoogle Scholar
  31. Sayes CM, Wahi R, Kurian PA, Liu YP, West JL, Ausman KD, Warheit DB, Colvin VL (2006) Correlating nanoscale titania structure with toxicity: a cytotoxicity and inflammatory response study with human dermal fibroblasts and human lung epithelial cells. Toxicol Sci 92(1):174–185PubMedCrossRefGoogle Scholar
  32. Shi YL, Wang F, He JB, Yadav S, Wang H (2010) Titanium dioxide nanoparticles cause apoptosis in BEAS-2B cells through the caspase 8/t-Bid-independent mitochondrial pathway. Toxicol Lett 196(1):21–27PubMedCrossRefGoogle Scholar
  33. Simon-Deckers A, Gouget B, Mayne-L’Hermite M, Herlin-Boime N, Reynaud C, Carriere M (2008) In vitro investigation of oxide nanoparticle and carbon nanotube toxicity and intracellular accumulation in A549 human pneumocytes. Toxicology 253(1–3):137–146PubMedCrossRefGoogle Scholar
  34. Tedja R, Marquis C, Lim M, Amal R (2011) Biological impacts of TiO2 on human lung cell lines A549 and H1299: particle size distribution effects. J Nanopart Res 13:3801–3813CrossRefGoogle Scholar
  35. Thybaud V, Aardema M, Clements J, Dearfield K, Galloway S, Hayashi M, Jacobson-Kram D, Kirkland D, MacGregor JT, Marzin D, Ohyama W, Schuler M, Suzuki H, Zeiger E (2007) Strategy for genotoxicity testing: hazard identification and risk assessment in relation to in vitro testing. Mutat Res Gen Toxicol Eng 627(1):41–58CrossRefGoogle Scholar
  36. Uchino T, Tokunaga H, Ando M, Utsumi H (2002) Quantitative determination of OH radical generation and its cytotoxicity induced by TiO2-UVA treatment. Toxicol in vitro 16(5):629–635Google Scholar
  37. Wadhwa S, Rea C, O’Hare P, Mathur A, Roy SS, Dunlop PSM, Byrne JA, Burke G, Meenan B, McLaughlin JA (2011) Comparative in vitro cytotoxicity study of carbon nanotubes and titania nanostructures on human lung epithelial cells. J Hazard Mater 191(1–3):56–61. doi: 10.1016/j.jhazmat.2011.04.035 PubMedCrossRefGoogle Scholar
  38. Wang JJ, Sanderson BJS, Wang H (2007) Cyto- and genotoxicity of ultrafine TiO2 particles in cultured human lymphoblastoid cells. Mutat Res Gen Toxicol Eng 628(2):99–106CrossRefGoogle Scholar
  39. Wang S, Yu H, Wickliffe JK (2011) Limitation of the MTT and XTT assays for measuring cell viability due to superoxide formation induced by nano-scale TiO2. Toxicol in vitro In press Google Scholar
  40. Warheit DB (2008) How meaningful are the results of nanotoxicity studies in the absence of adequate material characterization? Toxicol Sci 101(2):183–185PubMedCrossRefGoogle Scholar
  41. Wu J, Sun JA, Xue Y (2010) Involvement of JNK and P53 activation in G2/M cell cycle arrest and apoptosis induced by titanium dioxide nanoparticles in neuron cells. Toxicol Lett 199(3):269–276PubMedCrossRefGoogle Scholar
  42. Xu A, Chai YF, Nohmi T, Hei TK (2009) Genotoxic responses to titanium dioxide nanoparticles and fullerene in gpt delta transgenic MEF cells. Part Fibre Toxicol 6: in pressGoogle Scholar
  43. Zhu RR, Wang SL, Chao J, Shi DL, Zhang R, Sun XY, Yao SD (2009) Bio-effects of nano-TiO2 on DNA and cellular ultrastructure with different polymorph and size. Mat Sci Eng C Bio S 29(3):691–696CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Biotechnical Faculty, Department of BiologyUniversity of LjubljanaLjubljanaSlovenia
  2. 2.Institute of Occupational MedicineCatholic University of Sacred HeartRomeItaly
  3. 3.Centre of Excellence in Advanced Materials and Technologies for the Future (CO NAMASTE)Jožef Stefan InstituteLjubljanaSlovenia
  4. 4.Centre of Excellence in Nanoscience and Nanotechnology (CO Nanocenter)Jožef Stefan InstituteLjubljanaSlovenia

Personalised recommendations