the use of root canal irrigant solutions is essential in endodontic treatment because they help to disinfect and lubricate the root canal , flush out debris from the canal system , and dissolve organic and inorganic tissues . sodium hypochlorite ( naocl ) and chlorhexidine gluconate ( chx ) are commonly used for these purposes in endodontic treatment . naocl is widely recommended as a root canal irrigant for its antibacterial effects and its capacity to dissolve organic tissues . concentrations ranging from 0.55.25% have been reported in the literature , with higher concentrations having better antibacterial efficacy   but also higher toxicity . chx is used extensively in periodontal therapy because of its substantive and broad - spectrum antimicrobial effects . for these same reasons , recently , a new root canal irrigant , known as mtad , has been introduced for use as a final irrigation solution , comprising a tetracycline isomer , an acid , and a detergent . an ideal root canal irrigant should be biocompatible , because of its close contact with the periodontal tissues during endodontic treatment . the biocompatibility of dental materials has frequently been analyzed using the wst-1 test , in which the conversion of tetrazolium dye into formazan crystals in the mitochondria of living cells is proportional to their viability . the purpose of this study was to evaluate the cytotoxicity of naocl , chx and mtad on rat periodontal ligament fibroblasts using the wst-1 colorimetric method . the materials tested in this study were : 3% naocl ( chlorcid ; ultradent , usa ) ; 2% chlorhexidine gluconate solution ( consepsis ; ultradent , usa ) ; and biopure mtad ( dentsply tulsa dental , usa ) . irrigants were diluted to 0.1 and 100 l / ml with dulbecco s modified eagle s medium ( dmem ) , following a previously reported protocol . primary cell cultures of rat periodontal fibroblasts were established from rat periodontal ligament ( obtained from the histology and embryology department of the faculty of medicine , hacettepe university , ankara , turkey ) . rat periodontal ligament tissues were minced to 1 mm pieces and placed in 25 cm tissue culture flasks with dmem containing 10% fetal bovine serum ( fbs ) , 1% penicillin , 1% l - glutamine , and cultured in the same media at 37c , 95% air , and 5% co2 . cells were trypsinized and passaged once they covered at least 75% of the flask surface . gingival fibroblast cultures reached confluence in ~7 days and were subsequently sub - cultured ( diluted 1:3 with tissue culture medium ) until the start of the experiment . cytotoxicity was determined using the wst-1 assay ( roche diagnostics gmbh , mannheim , germany ; cat . rat periodontal ligament fibroblasts were plated at 110 cells / well in 96-well plates ( falcon biosciences , milano , italy ) in 100 l of culture medium , and cultured for 24 h prior to use . we used 3 test groups : group i : naocl 3% ; group ii : mtad ; and group iii : chx 2% . in addition to the test groups , we included a control group that contained cells and culture medium alone with no irrigant . the diluted irrigants were added to the relevant wells in 100 l volumes and incubated with the cells at 37c for 1 , 24 , 48 , and 72 h. at the end of these times , 10 l of wst-1 was added to each well and after incubation for 4 h at 37c , the absorption of each well at 420600 nm was measured using a micro - elisa reader ( versamax , molecular devices ) the wst-1 assay was performed according to the manufacturer s recommendations . this method is based on the reduction of wst-1 to a yellow - orange soluble formazan product by mitochondrial dehydrogenases , which are a part of the mitochondrial respiratory chain . therefore , cell viability can be determined by measuring the optical density of the formazan product with regard to cellular respiration and metabolic rate . cellular damage precludes cells from generating the energy required for metabolic function and growth , thus this assay provides a good model for cytotoxicity assessment [ 810 ] . baseline absorption readings at 420600 nm were measured using a micro - elisa reader ( versamax , molecular devices ) . the percent cell viability shown in our tables represents the mean of 8 experimental replicates . percent cell viability was calculated using the following formula : where  od ( optical density ) test compound is the mean absorbance of treated cells minus the baseline reading and  od control is the mean absorbance of control cells minus the baseline reading . cytotoxicity was rated based on cell viability compared with control , as defined by dahl et al . the different categories of cell viability were : non - cytotoxic ( > 90% cell viability ) ; slightly cytotoxic ( 6090% ) ; moderately cytotoxic ( 3059% ) ; and strongly cytotoxic ( < 30% ) . the percentage cell viability for each irrigant was recorded and the results were tabulated and subjected to statistical analysis . cell viability values for each tested material at each time point are expressed as the mean  standard deviation ( mean sd ) . the group means were compared by analysis of variance ( anova ) followed by a multiple comparison of means using the student - newman - keuls test ; if necessary , comparison of individual pairs of mean values by student s t - test was used . the materials tested in this study were : 3% naocl ( chlorcid ; ultradent , usa ) ; 2% chlorhexidine gluconate solution ( consepsis ; ultradent , usa ) ; and biopure mtad ( dentsply tulsa dental , usa ) . irrigants were diluted to 0.1 and 100 l / ml with dulbecco s modified eagle s medium ( dmem ) , following a previously reported protocol . primary cell cultures of rat periodontal fibroblasts were established from rat periodontal ligament ( obtained from the histology and embryology department of the faculty of medicine , hacettepe university , ankara , turkey ) . rat periodontal ligament tissues were minced to 1 mm pieces and placed in 25 cm tissue culture flasks with dmem containing 10% fetal bovine serum ( fbs ) , 1% penicillin , 1% l - glutamine , and cultured in the same media at 37c , 95% air , and 5% co2 . cells were trypsinized and passaged once they covered at least 75% of the flask surface . gingival fibroblast cultures reached confluence in ~7 days and were subsequently sub - cultured ( diluted 1:3 with tissue culture medium ) until the start of the experiment . cytotoxicity was determined using the wst-1 assay ( roche diagnostics gmbh , mannheim , germany ; cat . rat periodontal ligament fibroblasts were plated at 110 cells / well in 96-well plates ( falcon biosciences , milano , italy ) in 100 l of culture medium , and cultured for 24 h prior to use . we used 3 test groups : group i : naocl 3% ; group ii : mtad ; and group iii : chx 2% . in addition to the test groups , we included a control group that contained cells and culture medium alone with no irrigant . the diluted irrigants were added to the relevant wells in 100 l volumes and incubated with the cells at 37c for 1 , 24 , 48 , and 72 h. at the end of these times , 10 l of wst-1 was added to each well and after incubation for 4 h at 37c , the absorption of each well at 420600 nm was measured using a micro - elisa reader ( versamax , molecular devices ) this method is based on the reduction of wst-1 to a yellow - orange soluble formazan product by mitochondrial dehydrogenases , which are a part of the mitochondrial respiratory chain . therefore , cell viability can be determined by measuring the optical density of the formazan product with regard to cellular respiration and metabolic rate . cellular damage precludes cells from generating the energy required for metabolic function and growth , thus this assay provides a good model for cytotoxicity assessment [ 810 ] . baseline absorption readings at 420600 nm were measured using a micro - elisa reader ( versamax , molecular devices ) . the percent cell viability shown in our tables represents the mean of 8 experimental replicates . percent cell viability was calculated using the following formula : where  od ( optical density ) test compound is the mean absorbance of treated cells minus the baseline reading and  od control is the mean absorbance of control cells minus the baseline reading . cytotoxicity was rated based on cell viability compared with control , as defined by dahl et al . the different categories of cell viability were : non - cytotoxic ( > 90% cell viability ) ; slightly cytotoxic ( 6090% ) ; moderately cytotoxic ( 3059% ) ; and strongly cytotoxic ( < 30% ) . the percentage cell viability for each irrigant was recorded and the results were tabulated and subjected to statistical analysis . cell viability values for each tested material at each time point are expressed as the mean  standard deviation ( mean sd ) . the group means were compared by analysis of variance ( anova ) followed by a multiple comparison of means using the student - newman - keuls test ; if necessary , comparison of individual pairs of mean values by student s t - test was used . the cell growth and absorbance values of the negative and positive controls for each experiment ( data not shown ) were within the normal data range obtained in this laboratory . to normalize the data among the test materials , we calculated the percentage of proliferating cells in each group at each dose and time point compared with the positive control ( 100% viable cells ) and negative control ( 0% viable cells ) ( see conversion formula in materials and methods , above ) . for the 100 l / ml dilution of each irrigant at 1 h , chx was least cytotoxic , while naocl was most cytotoxic . the multiple comparison of means by the student - newman - keuls test revealed differences between each of the 3 tested groups . at 24 h , chx remained the least cytotoxic but mtad was significantly more toxic ; this difference was statistically significant ( f=116.5 , p<0.0001 ) . there was no significant difference between the naocl and mtad groups . at 48 h , there was no difference between the chx and mtad groups at this time point . at 72 h , mtad had the lowest cell viability , so it was more cytotoxic than naocl and chx ( f=5.99 , p=0.022 ) . there was no significant difference between the chx and mtad groups . compared with other time points , mtad and naocl at 100 l / l concentration cell viability was higher at 72 h than at the earlier time points , while chx was more cytotoxic at 72 h than at 24 h , a statistically significant difference ( p<0.0001 ) ( table 1 ) . for the 0.1 l / ml dilution of each irrigant at 1 h , mtad was the least cytotoxic while chx was the most cytotoxic ( f=193.8 , p<0.0001 ) . for the 3 later time points , there was a pattern of cell viability being highest with naocl , with mtad comparable but slightly lower , and lowest with chx . this difference was significant in the 24 h , 48 h , and 72 h groups . at these irrigant dilutions , cell viability was significantly ( p<0.0001 ) increased between 1 h and 72 h with naocl and mtad , but not with chx ( p>0.05 ) ( table 2 ) . finally , we performed a multiple logistic regression analysis to evaluate the dose - dependence of the toxicity effect . results of this analysis showed a significant influence of the dilution rate of the substance , because the odds ratio for cell viability being over 50% was increased 51 times between the 100 l / l and 0.1 l / l dilutions ( or51.18 , 95% ci 8.1621.09 ) . in vitro cytotoxicity tests are usually carried out for new materials before applying them clinically . in vitro model assays enable experimental factors and variables to be controlled , which often is an important complication of performing experiments in vivo . mtt ( 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide ) is well established for cytotoxicity analysis of dental materials , being used initially for cell viability analysis in the 1980s . although the mtt assay is sensitive , it requires more time to be completed , but recent developments of other metabolic activity - based tests such as the wst-1 ( 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2h-5-tetrazolio]-1,3-benzene disulfonate ) assay have accelerated this process . in principle , wst-1 works similarly to mtt , by acting as a substrate for the mitochondrial succinate - tetrazolium reductase enzyme , which converts it to the formazan dye . the wst-1 reagent produces a water - soluble formazan , in contrast to the product of the mtt assay , which is water - insoluble . thus , the wst-1 analysis is easier and faster than the mtt test and is considered to be a more sensitive index for evaluating the cytotoxicity of dental materials . this study evaluated in vitro the cytotoxic damage induced by naocl , chx and mtad at dilutions of 0.1 and 100 l / ml using the wst-1 colorimetric test . the solutions were diluted because cultured cells are far more susceptible than periapical tissues to toxic effects of drugs . in vivo , the substances are rapidly neutralized and removed by phagocytes and the lymphatic and vascular systems , and are thus less harmful in clinical situations than at equivalent concentrations in in vitro studies . according to our results , naocl , chx , and mtad solutions all induced cytotoxicity in rat periodontal ligament fibroblasts cells , and these effects were time- and dose - dependent . at 100 l / l and 1 h and 24 h , all 3 irrigants were strongly cytotoxic , although chx was less so than naocl , and mtad was more cytotoxic ( table 1 ) . nevertheless , cell viability was significantly higher at 72 h than at earlier time points for all irrigants . at the 0.1 l / l concentration , naocl and mtad were only moderately cytotoxic , whereas chx was highly deleterious to cell viability at all time points ( table 2 ) . indeed , chx was more cytotoxic at the higher dilution than at the lower dilution . the study also demonstrated that mtad was less cytotoxic than 5.25% naocl but more cytotoxic than 2.63% naocl . in our study , mtad showed similar cytotoxicity to 3% naocl at all time points and at both dilutions . furthermore , our results indicated that 2% chx was more cytotoxic than the other 2 irrigants . ( 2010 ) reported that mtad has minimal cytotoxicity against mc3t3- e1 and periodontal ligament cells compared with conventional irrigants . the cytotoxicity of chx has been shown in several studies , with one report demonstrating that these effects were dependent on the exposure dose , length of exposure , and the composition of the exposure medium . while chlorhexidine does not appear to cause any long - term damage to host tissues , it may still cause an inflammatory response in these tissues if expressed beyond the root canal . in vitro measurements of toxicity are purely cellular , so our results can not be directly compared with those obtained in in vivo studies . new studies on root canal irrigants should be done in animals and then in humans to evaluate their cytotoxicity and in vivo biocompatibility . all of the tested irrigants showed some level of cytotoxicity towards rat periodontal ligament fibroblasts , and these effects were time- and dose - dependent . we conclude that these irrigating solutions should be used at lower concentrations to enhance cell viability and protect the tissues from toxicity damage , irrespective of their increased efficacy at higher concentrations .
backgroundcytotoxicity of root canal irrigants is important due to their close contact with host tissues . 
 the aim of this study was to assess the cytotoxic effect of naocl 3% , chx 2% , and mtad on rat periodontal ligament fibroblasts , at 0.1 and 100 l / ml , using wst-1 colorimetric method.material/methodrat ligamental fibroblasts were exposed to the irrigants and their viability was assessed after 1 , 24 , 48 , and 72 h. the measurements were determined using wst-1 assay , using a micro elisa reader.resultsat 100 ml / l all 3 irrigants were strongly cytotoxic , although chx was less so than naocl and mtad . at the 0.1 ml / l concentration , naocl and mtad were only moderately cytotoxic , whereas chx was highly deleterious to cell viability at all time points . 
 there was a significant influence of the dilution rate of the substance , because the odds ratio for cell viability being over 50% was increased 51 times between the 100 ml / l and 0.1 ml / l dilutions.conclusionsit seems that irrigating solutions should be used at lower concentrations to enhance cell viability .