title: Evaluation of THP-1 cell line as an in vitro model for long-term safety assessment of new molecules.
abstract: OBJECTIVE
  Monitoring the chronic and subacute toxicity is essential in the development of new cosmetic ingredients. In response to the present lack of validated alternative methods, we developed an in vitro model for repeated dose cytotoxicity on THP-1 cells.
  
  
  METHODS
  Cultured in suspension, cells were treated with chemicals for 14 days with a frequency of three applications per week, and cell viability was determined by MTT assay. We first investigated the long-term effects of chemicals that induce different kinds of cytotoxicity: Paraquat (PQ), 3-Nitropropanoic acid (3-NPA) and sodium dodecyl sulphate (SDS). From acute studies, doses between 1 and 10 μg ml(-1) were chosen to perform our subacute cytotoxicity assay. Comparative genotoxicity evaluations were made with H2 O2 or Paraquat treated TPH-1 cells. Comet assays were performed at 1 h (4°C); after a 24-h recovery period (37°C); and finally, after a long-term period of treatment (14 days, 37°C).Once adapted to plant extracts or highly diluted molecules, some of our cosmetic compounds were tested with this model.
  
  
  RESULTS
  As expected, after 14 days of treatment with Paraquat, cell viability rates dramatically decreased for doses as low as 3 μg ml(-1) , whereas 10 μg ml(-1) of 3-NPA and SDS did not induce more than 44% of cell death. Surprisingly, after subacute treatment, comet assay results revealed a dose-dependent increase in tail moments for Paraquat, whereas those of H2 O2 remained low. Moreover, all our compounds tested at 0.5-5 μg ml(-1) were classified as safe, even with a cut-off at 90% of cell viability.
  
  
  CONCLUSIONS
  In conclusion, this assay could be of interest for subacute cytotoxicity and genotoxic assessment of daily and topically applied products and suggests that PQ is a choice worthy positive control.
authors.affiliation: ISP VINCIENCE SA. Ashland Specialty Ingredient Affiliate of Ashland Inc., 655, Route du Pin Montard, Sophia Antipolis, 06410, Biot, France.
authors.forename: I
authors.initials: I
authors.lastname: Garcia
authors.forename: C
authors.initials: C
authors.lastname: Pouzet
authors.forename: M
authors.initials: M
authors.lastname: Brulas
authors.forename: E
authors.initials: E
authors.lastname: Bauza
authors.forename: J M
authors.initials: JM
authors.lastname: Botto
authors.forename: N
authors.initials: N
authors.lastname: Domloge
mesh_terms: D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D020552:Comet Assay; D006801:Humans; D009574:Nitro Compounds / Q000633:toxicity; D010269:Paraquat / Q000633:toxicity; D011422:Propionates / Q000633:toxicity; D012967:Sodium Dodecyl Sulfate / Q000633:toxicity; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D009574:Nitro Compounds; D011422:Propionates; D012967:Sodium Dodecyl Sulfate; D010269:Paraquat; C015392:3-nitropropionic acid
keywords: alternative method; cell culture; in vitro; safety testing; subacute cytotoxicity