International Endodontic Journal {1988) 21, 89-99

Comparison ofthe methods available for assessingcytotoxicity

A. HENSTEN-PETTERSEN NIOM, Scandinavian Institute of Dental Materials, Haslum,Norway

Summary. During the last 30 years cell culturemethods have been used to assess the cytotoxicity ofdental materials. The development of methodologyhas beeti based on elaborate original research andmodified applications of existing methods.

Different parameters are used to monitor cyto-toxic effects, such as inhibition of cell growth,qtolysis, membrane or cytoplasmic markers andchanges in metabolic activity. The methods alsoemploy different means of establishing cell-materialcontact, ranging from direct contact to incorporat-ing permeable spacers of agar, dentine or molecularfilters, using material extracts or particulate mater-ial. A general problem in comparing the resultsobtained with the various methods is assessing theconcentration and identity of substances to whichthe cells are exposed. The ratio between specimensurface area to volume of cell culture medium is acritical parameter in most test systems. This ratiomay vary by a factor of 10 from one method toanother. Generally, the ratios seem to be selected toobtain a eytotoxic effect.

Advanced methodology for c 'totoxic studies isavailable for routine investigations and researchprojects. However, better characterization of thedissolved components or degradation productswould substantially enhance the long-term value oftht results.

Toxicity testing of dental materials by means ofcell culture methods has been claimed to be fairlysimple to perform, reproducible, cost-effective, rel-evant and suitable as an alternative to animal exper-iments. Furthermore, the methods have beenclaimed to be suitable for toxicity screening of newmaterials, identification of cytotoxic substances andappropriate for the biological quality control of pro-duction batches. Most claims are not substantiatedhy research data. There may be some substance ineach of these claims but further correlation studiesbetween in vitro tests, physical/chemical data and tnVIVO studies are clearly needed.

Correspondence: Dr Arne Hensten-Pettersen,NIOJVI., Scandinavian Institute of DentalMaterials, Kirkeveien 7 IB, N-1344 Haslum,

IntroductionA number of techniques have been suggestedfor the biological assessment of dental mater-ials. In this review, emphasis will be placed onthe cell culture methods outlined in fourof the six documents/specifications so farpresented (ADA 1979, BSI 1980, FDI 1980,ISO 1984, AFNOR 1985, DIN 1986). Thesedocuments include suggestions for tests forspecific groups of materials, but these shouldbe regarded as recommendations only. Withone or two exceptions, no mandatory require-ment has yet been made regarding biologicaltesting of dental materials. Biological testingdoes not, therefore, play a significant role inthe certification of materials as yet. However,the techniques presented are important inobtaining correlations which eventually willallow firm commitments to be made regardingthe significance of biological testing in theapproval of materials to be used in clinicalpractice.

Despite their official format, the documentson biological testing all emphasize that themethods suggested should be regarded as pre-liminary or suggestive in nature. The docu-ments have been reviewed recently (Klotzer &Schmalz 1986, Schmalz & Klotzer 1986).

Initial testsThe initial tests are intended to give a toxicityprofile of the material, allowing the manufac-turer and the certifying body to place it in itscorrect perspective. Most of these tests aretaken from the arsenal of methods used toevaluate drugs. With the exception of cell cul-ture tests, none or very few studies have beencarried out on dental materials using theseinitial tests. The methods suggested cover anumber of tests assessing cytotoxicity, haemo-lysis, systemic toxicity using the oral or intra-venous routes, inhalation toxicity and tests

89

90 A. Hensten-Pettersen

to predict teratogenicity or carcinogenicity(dominant letbal test, Ames' mutagenicity testand Styles' cell transformation test).

Secondary testsThese tests take up an intermediate positionbetween the initial tests and the usage tests.F'or some types of materials they are in factalso considered as usage tests, e.g. the mucousmembrane irritation test, which is a usage testfor prosthetic materials and a secondary testfor other materials.

The methods include implantation studiesin subcutaneous and bone tissues, oral mucousmembrane irritation tests and sensitizationtests. Dermal toxicity tests on tbe effect ofrepeated exposure are also included in thisgroup.

Usage testsThe methodology for tbese tests places par-ticular emphasis on the manner in which tbematerials are intended to be used in clinicalpractice. Apart from the oral mucous mem-brane irritation test mentioned above, thebone implantation test may be considered as ausage test for bone implants. The other usagetests available are all related to operative den-tistry and endodontics. Thus, methods havebeen developed for assesssment of pulp anddentine reactions to operative materials, pulpcapping and pulpotomy materials and com-plete endodontic therapy. A test for dermaltoxicity may also be considered a usage test foran assessment of problems associated with thehandling of dental materials.

Cell culture methodsTests for cytotoxicity in vitro are abundantand bave been applied to a variety of dentalmaterials. A review on cell culture techniques,used to assess the cytotoxicity of dentalcements (Helgeland 1982), indicated thatmore than 20 different techniques have beenapplied. The in vitro methods are based on cellcultures with established or diploid cell linesand a few tissue explant techniques. The cyto-toxicity of dental materials in vitro has beenevaluated by cell growth measurements,changes in membrane permeability, metabolicalterations or cytopathogenic changes. In

some of the methods, the cytotoxicity is evalu-ated by two or more of tbese parameters.Many of tbe tests require extensive experiencewith cell culture techniques and well-equipped laboratory facilities.

The BSI standard (1980) does not requireany cytotoxicity tests. The ANSI/ADA docu-ment (ADA 1979) suggests the ^'Cr-releasetest, and the FDI document (1980) and DIN/Nf3 (DIN 1986) suggest three different tests,leaving the choice to the investigator as towhich test sbould be used for any particularmaterial. The FDI document lists 53 types ofmaterials wbere these tests should be applied,i.e. all dental materials that are listed. Of the 44types in the ANSI/ADA document, 31 typesshould be tested, exempting all cements,etchants, primers, cavity cleansers and thoseincluded as miscellaneous materials, such asinvestment materials, low fusing metals,solders and fluxes. The basis for this selectionis not evident.

The tbree establisbed tests suggested forcytotoxicity testing are based on changes inmembrane permeability (agar overlay test," Cr-release test) and on metabolic alterationsassessed by cytochemical staining and mi-croscopy (millipore filter test). The modelcavity method is based on cytochemical analy-sis and the tooth cavity method on measure-ment of DNA synthesis by ^H-thymidineincorporation. Tbe tests can be performedwith a minimum of laboratory facilities, but dorequire a sterile bandling area and an incu-bator with controls for atmosphere, humidityand temperature. The " ' Cr-release methodand ^H-tbymidine incorporation metbod re-quire a gamma spectrometer or a liquid scintil-lation counter and a disposal system forradioactive waste.

Agar overlay methodThe objective of tbis test is to detect the acutecytotoxicity of leachable components throughtests directly on the solid material and tests onextracts of the material.

The test material is placed in contact withthe surface ofan agar layer overlaying a mono-layer of mouse fibroblast cells (L929) whichare stained with neutral red vital stain. After24 hours incubation the presence of leachable,toxic substances is manifested by tbe loss of

Methods for assessing cytotoxicity 91

dye from the cells within the diffusion zone ofthe soluble substances leaching from thesamples. Lysis of the cells occurs within thezone if the concentration of the diffusing toxicstibstances is high enough. The cytotoxic re-sponse is reported in terms of a response indexbased on the size of the diffusion as seen by adecolorized zone (zone index 0-5) and the per-centage of the cells within the zones which arelysed (Lysis Index 0-5).

The details of the test are elaboratelydescribed (FDI 1980, ISO 1984). The manypitfalls of the method are also extensivelydiscussed. The test may be invalidated by anumber of artefactual conditions. These mayoccur due to improper incubation conditions(temperature, pH), release of volatile com-pounds from solid materials or lack of protec-tion of the stained cells from light. The neutralred vital stain can be activated by ambientlight to become cytotoxic per se-

The agar overlay test requires the use of aknown toxic material and a known non-toxicmaterial on each plate, Polyvinyl chloridecontaining dibutylin acetate is suggested as apositive control and high density polyethyleneas a negative control. These materials arenot always readily available, and severalalternative substances have been evaluated assubstitutes (Keech et al. 1983),

Wilsnack et al. (1973) found that the diffusi-bility in agarose, in particular for chargedcompounds, was superior to that in agar, andthey suggested that to increase the sensitivityof the method, agarose should be substitutedtor agar. An additional advantage was thattheir particular brand of agarose had a gellingpoint of 32"C, thus avoiding the thermal shockassociated with the preparation of agar over-lays at 40-45°C,

The monolayers of cells take about 24 hoursto become confluent, but not dense, beforetesting can take place. The test itself requiresabout 1 hour of preparation, a 24-hour incu-bation period and, depending on the numbero< plates, about 15-30 minutes to record theresponse index. The test is fairly easy to run,and the results are reproducible within thesame laboratory. However, in round robintesting, it has been difficult to obtain repro-ducible response indices from one laboratoryto another (Schmalz 1982),

In a study to reduce the variables involvedin the morphological evaluation of the agaroverlay test, the originally recommended neu-tral red vital stain was replaced by fluorescein-diacetate. Cytotoxicity tests with phenol,formalin and methylmethacrylate monomerrevealed results corresponding with evalu-ations by other methods (Schmalz & Netuschil1985),

Millipore methodOne of the major criticisms of cell culture testswith direct material-cell contact is that thissituation is often not clinically relevant. Mostmaterials used to restore lost tooth substanceare not in direct contact with cells; there isusually a layer of dentine between the materialand the pulp tissue. The millipore filtermethod modifies the contact situation in thatthe cells are grown on one side of the filter andthe material is placed in contact with the op-posite surface of the filter. Thus, any leachablesubstance must diffuse through the 0,45 |imfilter pores to exert any toxic effects on the cells(Wennberg f/a/, 1979),

Human epithelial cells (Hela) or mousefibroblast cells (L929) are used in the milliporefilter test. Cells harvested from stock suspen-sions are seeded on the surface of 47 mm diam-eter millipore filter discs in tissue culturedishes. The filters tend to float in the mediumif not kept down by the weight of a glass ring ofthe same size as the filter disc.

After 24 hours the filter with the attachedcells is transferred to an agar medium surfacewith the cell side down. The test specimens inglass rings are placed on top of the filter, 3-5specimens on each filter. Ten specimensshould be used for each material or concen-tration. For control purposes filters with a cellmonolayer, but without test specimens, andfilters without cells and with test specimensshould be included. The culture disc is furtherincubated for 2 hours, the test specimens arethen removed and the millipore filter gentlyloosened from the agar layer. The filter is incu-bated for cytochemical demonstration ofsuccinate dehydrogenase activity of the cellmonolayer for 3 hours. The filters are washedin distilled water and air dried.

The appearance of the test filters at the cell-material contact areas is registered according

92 A. Hensten-Pettersen

to a scoring system that takes into account thestaining intensity ofthe zone and the diameter,or extension, ofthe affected area. Based on thescoring system, the response to a material canbe classified as follows: non-cytotoxic, mildlycytotoxic, moderately cytotoxic or markedlycytotoxic. The system has mainly been used totest solids and pastes, but liquids can also beevaluated by this method. To test fluid mater-ials, 0.1 ml of the substance is imbibed in acellulose disc placed on the millipore filter.

Cell monolayers on millipore filters havealso been exposed to toxic substances andincubated for cytochemical demonstration oflactate dehydrogenase, glucose-6-phosphatedehydrogenase and cytochrome oxidase. Nodifferences in enzyme activity patterns wereobserved among the enzymes tested. Anadditional advantage of this method is thatthe stained filters can be kept as permanentrecords ofthe test situation.

Chromium release methodHexavalent ^'Cr in Na2CrO4 is incorporatedinto cells and reduced to the trivalent formupon attachment to proteins and other cellconstituents. As the trivalent "" Cr cannot bereutiUzed by other cells, measurement of ^'Crradioactivity in the medium gives a measure ofcell lysis.

The use of '''Cr-labelling and release forquantitative and kinetic studies of immuno-logicaily induced cytolysis tn vitro was de-scribed by Sanderson (1964) and Wigzell(1965). Spangberg (1973) has modified themethod to make it applicable to dentalmaterials, and it can test materials as liquidsor solids. Human epithelial cells (Hela) ormouse fibroblast cells (L929) are used for thistest.

The test utilizes phenol as a positive, toxiccontrol for both liquid and solid materials,and uses the untreated medium as negativecontrol. The test requires relatively smallamounts of material, 0.15 ml solid per culturechamber, but 10 culture chambers for eachsetting time and test interval, which increasesthe quantity of test material. Fluids are testedin a 4-step dilution series.

The test requirements and pitfalls of themethod are elaborately described in the orig-inal publications and in the ANSI/ADA, ISO

and FDI documents. The test incorporates anumber of control steps which have to be fol-lowed if the test results are to be consideredvalid. The test results indicate four levels oftoxicity—non-toxic, slightly toxic, toxic andseverely toxic.

The procedure specifies the use of a gammaparticle counter/spectrometer to record radio-activity; ^'Cr is also a beta-emitter, making itpossible to assess radioactivity with a suitableliquid scintillation counter. The latter methodcan be optimized to yield a slightly highercounting efficiency. Some periodontai dress-ings release substances capable of precipitat-ing proteins; cadmium and some experimentalcavity cleansers have the same effect (Haugen& Hensten-Pettersen 1978, Holland 1978,Mjor et al. 1982). In these instances a false low^^Cr-release might be encountered. However,the system as described in the ANSI/ADAand FDI document takes such features intoaccount.

Model cavity methodThe model cavity method was introduced byTyas (1977). The material is placed in a pers-pex (polymethylmethacrylate) cavity with amillipore filter or dentine slice as the cavitybottom. BHK-21 (C-l3) fibroblasts attachedto coverslips are placed beneath the cavity, and4-ml cell culture medium connects the mater-ial and cells. Assessment of cell damage is doneby cytochemical determination of acid phos-phatase and succinate dehydrogenase, markerenzymes for lysosomes and mitochondria, re-spectively. The density of final reaction pro-ducts after various incubation periods ismeasured using a scanning integrating micro-densitometer. Since changes in membranepermeability are early results of toxic damageand may be reversible, the ability of cells torecover from such injury can be assessed.

The method has been further modified byusing millipore cement instead oi dental waxto attach filters to the base of perspexcylinders forming the base of the simulatedcavity. Other modifications include compact-ing dentine powder above the filter, simulatingresidual dentine in vivo, using pyrex (glass)cylinders of various diameters to simulate dif-ferent cavity sizes (xMeryon & Riches 1982,Meryon et al. 1983, Meryon 1984a,b). The

Methods for assessing cytotoxicity 93

method has also been extensively used withprimary cultures of mouse peritoneal macro-phages, where the cellular and supernatantlevel of y?-gaiactosidase and lactate dehydroge-nase is assayed spectrophotometrically.

Tooth cavity methodThe introduction of the tooth cavity method(Hume 1985) is one step closer to an m vivosituation. The method tests the potential forchemical toxicity of restorative materials, inwhich an essential step is the diffusion of toxicsuhstances from materials applied to teeththrough human dentine. Because toothcrowns are used, the technique also allows forapplication of materials in ways, sequencesand combinations which are used clinically.

The impacted third permanent molar teethfrom young adult donors are removed andheld in phosphate buffered saline at 4°C for upto 3 hours before use. After normal cavity orcrown preparation, the system is used toobtain a solution of substances that can pen-etrate a dentinal wall. The 'pulpal fluid', orrather the medium on the pulpal side of thepreparation is then tested separately withL929 fibroblast monolayers, by assessing ^H-thymidine uptake of experimental vs. controlmedia as described by Wennberg (1976). Thissystem has been used to assess the effect ofseveral restorative materials and shows a goodcorrelation with m vivo data (Hume 1985).

Haemolysis testThe objective ofthis method is to evaluate theacute haemolytic activity of materials intendedfor prolonged contact with bone and softtissue. It may be regarded as a cytotoxicityassay. The test originates from testing ofmaterials used for haemodialysis of patientswith renal failure and from observations thatsome blood containers and tubing causedhaemolysis of the contained erythrocytes. TheFl)I and ISO documents indicate haemolysistesting on 17 types of materials, includingsurgical packs used as wound dressings afterpcriodontal surgery, endodontic filling mater-ials, medicaments, irritants and cleansers,pulp capping/pulpotomy materials, prostheticalloys, denture bases and also for materialsused as implants in soft and hard tissue. The

ANSI/ADA document indicates haemolysistesting on 28 types of materials, excepting onlydental cements, pit and fissure sealants,etchants, primers and cavity cleansers, ortho-dontic alloys and resin materials.

The haemolysis test employs rabbit bloodand is designed to determine the haemolyticactivity of a material incubated for 90 minuteswith rabbit blood present for the last 60 min-utes. The haemolytic activity of soluble, leach-able components and the haemolytic activityof the physical surface of the material bothcontribute to the total haemolytic activityobserved. Independent evaluation of leach-able, soluble components can be obtained bythe assay of 90-minute saline extracts. Thetonicity of the final extract is of importance.Hypotonic solutions cause extensive haemoly-sis without necessarily containing a toxic com-ponent per se. Erythrocytes from species otherthan rabbit have been used (Wennberg &Hensten-Pettersen 1981), but may give sig-nificant difierences in apparent haemolyticactivity of material extracts.

Several artefacts and misinterpretationsmay easily arise in assessing the haemolyticactivity of materials, and limited data relatingto dental material toxicity are available. If aleachable component agglutinates the erythro-cytes or causes precipitation oi released hae-moglobin, a false, low haemolytic activity maybe recorded. Microscopy of the pelleted sedi-ment is of help to assess whether such ad-ditional reactions have taken place (Haugen &Hensten-Pettersen 1979). Another aspect thatneeds to be checked is whether the leachablesubstances causing haemolysis interfere withthe absorption spectrum of haemoglobin orwhether a shift to other absorption maximahas taken place. In an investigation of sensibi-lity and haemolytic activity of dental resin-based restorative materials, Fujisawa et al.(1978) showed that the absorption spectra ofdog haemoglobin may change extensively inthe presence oi various extracts; pH changesmay give the same effect. In general, the con-centration of a toxic substance required to pro-duce haemolysis is approximately one order ofmagnitude higher than that required to pro-duce a significant response in cell culture(Dillingham et al. 1975). Whether this differ-

is real or related to the difference in time

94 A. Hensten-Pettersen

(60 minutes vs. 24 hours) of cell-material con-tact and how it relates to in use situations isunknown.

Only limited information is availableregarding the testing of dental materials usingthe haemolysis test. Its relevance in biocom-patibility evaluation cannot, tberefore, beaccurately assessed.

Cell growth, colony forming methods and othertestsThe many other methods that have been intro-duced to assess tbe cytotoxicity of dentalmaterials are reviewed elsewhere (Schmalz1981, Helgeland 1982, Kawahara 1982,Wennberg 1986).

Assessment of methodsA comparison entails examining the variouscomponents inherent in each method for simi-larities and differences which may influencethe end results. Wennberg (1986) hasreviewed many of the factors that vary fromone method to another. The present reviewerhas selected some aspects that may warrantfurther comment.

Cell culture dishesReusable glass petri dishes were extensivelyused in cell culture studies until tbe late196O's, but now disposable surface-treatedpolystyrene cell culture petri dishes are used inmost laboratories. They are easy to use andshould not compromise the test situation, aslong as the materials or material extracts donot react with the surface of the dishes. How-ever, some dental materials contain organicsolvents such as chloroform, eugenol anddiluent monomers in composite restorativematerials. Both eugenol and chloroform couldreact chemically with the surface of plastic cellculture dishes (unpublished observations). Byattaching the specimens to the bottom of thedish with sterile silicone grease, the latterseemed to act as a barrier, and no interactionwas seen. In later experiments (also unpub-lished) freshly made composite materials wereassayed, including separate tests of their baseand catalyst pastes. The results were very vari-able. Closer inspection showed that six out of13 brands of composite restorative materialsinteracted with the surface of the polystyrene

cell culture dishes. The interaction variedfrom a slight alteration of surface lustre toinstances wbere the pastes and freshly mixedmaterials were fused to the dishes. The use ofsilicone grease to attach the specimens acted asa diffusion barrier and seemed to be sufficientto avoid this problem. Cell culture studies ofsucb materials may preferably be performedwith glass vessels to minimize this variable.With the methods using indirect contactthrough agar, dentine or medium tbis sbouldnot be a problem.

CellsThe agar overlay and tooth cavity methods aredescribed with L929 mouse fibroblasts. In themillipore filter and ''' Cr-release methodseither L929 cells, human epithelial cells (Hela)or broth can be used. The model cavitymethod uses BHK-21 (C-13) fibro^blasts or primary cultures of mouse peritonealmacropbages. Other studies have used a var-iety of cell types (Helgeland 1982, Wennberg1986).

Studies assessing the cytotoxic effects ofdental materials on two or more cell types indi-cate that there are variations in sensitivitybetween cell types in tbe assay systemsemployed (Hensten-Pettersen & Helgeland1981, Kasten et al. 1982, Meryon 198.1,Wennberg et al. 1983, Johnson et al. 1983,1985, Feigal et al. 198.S). In general, the differ-ences are of a low order of magnitude, but maychange the ranking order of cytotoxic potentialof different materials. The materials whichare markedly toxic are toxic in all systems. Alleukaryotic cells are surrounded by a cellmembrane, enclosing organelles for regener-ation, reproduction and energy generation aswell as for other cell functions. The featuresare common for most cells of any species anddamage to these fundamental cell functionsmay be assessed in cytotoxicity tests (Ekwall1983). . s long as tests m vitro for organ-specific cell damage, such as cbanges in den-tine formation by odontoblasts after injury,are not available, the basal cytotoxicity of com-pounds should be reflected in assays with mostcell types.

MediumAll cells are grown in buffered culture mediawith salts, nutrients, vitamins and antibiotics.

Methods for assessing cytotoxicity 95

The medium of choice in each case is usuallyoptimized for each cell type and may differ inmany, minor details. The effect of materials ongrowth media has not been studied to anygreat extent, except in the case of silicatecement studies where a shift in pH is oftenevident. However, Helgeland & Leirskar(1973) found that silicate cement discs couldbind cell culture medium components. Arapid decrease in calcium and magnesium wasfound during the first 24-hour period, fol-lowed hy a slower decrease upon further incu-bation. After 6 days only 12 per cent and 37 percent of the initial concentrations of calciumand magnesium, respectively, remained in themedium. By addition of *^Ca as calciumcbloride, it was found that the amount of cal-cium lost from the medium was quantitativelyrecovered from the silicate cement discs. Thisartefactual condition does not seem to he takeninto account in the continuing discussion as towhether the pH decrease or fluoride release istbe major cytotoxic principle in cell culturestudies on silicate cement.

The extensive variations in extractionmethods (time, temperature, mode), surfacearea of the test specimens versus the volumeot the extraction media and mode of cellmaterial contact make direct comparison of re-sults subject to errors. These variables haverecently been discussed by Wennberg (1986).Most studies present data from one methodonly, which makes comparisons betweenmethods uncertain.

Serum

Most cell culture studies use foetal or new-horn calf serum during the cell propagationperiod. During the 2-hour test period the mil-lipore filter method does not include serum,whereas most other systems use serumthroughout the experimental period.

1 he serum concentration may be of import-ance for the results obtained. Jacobsen (1977)found that nickel had little, if any, effect onhuman gingival epithelial cells at a concen-tration of 5 Hg/ml when tested in Eagle'sMEM supplemented with 2.5 or 5 per cent calfserum, whereas in NCTC 135 medium, withthe same serum concentrations a pronouncedcytotoxic effect was evident. In the presence of

10 per cent calf serum no effect of 5 ig/ml ofnickel was found in any ofthe media.

Some employ heat-inactivated sera, othersdo not specify what they use. Heat-inactiva-tion at 56' C for 30 minutes is primarily done toexclude complement activity in the systems.Recent investigations have shown that poly-meric materials containing hydroxyethyl-methacrylate may activate complement factorC3a (Payne & Horbett 1987). The formationof the activation peptide C3a from the intactC3 complement protein is normally part of acomplex cascade of reactions leading to thelysis of cell membranes. This variable needs tobe controlled in the very sensitive cytotoxicityassays where cell membrane damage isassessed.

Some cavity cleansers and material extractsmay cause extensive precipitation of serumcomponents in the culture media (Haugen &Hensten-Pettersen 1978, Holland 1978, Mjoret al. 1982). This artificial situation is easy toobserve and invalidates the results. However,evaluation of cytotoxicity hy automatedsystems only, may give rise to erroneousinterpretation oi the results and needs to becombined with microscopic examination ofthe cell cultures.

Assay systemsAll assay systems are vulnerable to artefacts,especially if the experimental procedures areon the limit or exceed the range for which thesystems were designed (Holland 1978). Theincorporation of positive and negative controlsneeds to be defined, as well as the workingrange of each system. This has been done formany of the systems, but for some reason itseems to be difficult to keep strictly to onemethod w ithout modifying it as the study pro-gresses. Over the years this probably improvesthe methods, but it may be difficult for new-comers in the field to see the rationale behindthe improvements, or 'modifications' as theyare usually described. Enzymatic analysis canbe affected by many factors, especially pHchanges deviating from the optimum pH ofthe assay system; denaturation of enzymes byeugenol in the extract may invalidate lactateassays using lactate dehydrogenase (Hensten-Pettersen & Helgeland 1977). Several of themethods using enzymes incorporate control

A. Hensten-Pettersen

Steps to minimize this problem. However,addition of known amounts of the enzyme orsubstrate to the test solutions would ensurebetter control of the assays.

Neutral red used as a vital stain in the agaroverlay method becomes cytotoxic upon ex-posure to daylight or fluorescent light. We hadvariable results with this method until thisaspect was brought to our attention. The useof window shades and elimination of all directroom lights before adding neutral red to thecultures, and keeping the cells in the dark forthe entire experimental periods improved thereliability of the technique.

Dental materialsMany of the methods suggested have beenadopted from the testing of drugs. However,there is a major difference from the outset.Drugs arc usually soluble in various liquid sys-tems, whereas dental materials are intended tohave low solubility. This major difference rep-resents methodological problems and makestechnical adjustments necessary.

Many dental materials are supplied as two-component systems which when mixed will setto a semi-solid or solid state. Some are deliv-ered as one-component paste systems, whichwill set when irradiated with a suitable lightsource. The actual availability of potentiallytoxic substances from these materials differsgreatly, depending on their setting reactionsand at which stage they arc tested. The settingreactions may also produce new combinationsor lead to degradation of certain chemicalswhich need not have been present in the orig-inal formulation (Ruyter 1980), It is importantto assess how the toxicity of a materialchanges.

It is almost meaningless to evaluate toxicproperties without having access to the actualchemical composition of the materials tested.Furthermore, frequent changes in availabilityare a problem. Out of 13 brands of compositerestorative materials subjected to cytotoxicityassays in 1977, 10 of these are no longer on themarket, and the remaining three have been'improved'. Similarly, the chemical compo-sitions of some temporary crown and bridgeresins have been shown to be subject tounpublished modifications (Rees et al. 1987).

Some components might have specific ef-fects which can be augmented or decreased inthe presence of other constituents (Hensten-Pettersen el al. 1978), The final reaction canthen be a composite of several effects. A gen-eral problem in all the test situations is theestimation of relevant test concentrations.Initial solubility tests in various aqueousmedia, combined with analysis of soluble orleachable components is of value in designingexperiments with relevant test concentrations(Kuhn & Wilson 1985),

Many of the tests in vitro can utilize eithersolid materials or material extracts. No generalagreement exists regarding the state of thematerials to be tested, i,e, whether they shouldbe tested immediately after mixing or afterstorage in air or in a medium for some time.The state of the materials as inserted into theoral cavity must, therefore, be the guide-line.Thus, filling materials and impression mater-ials should be tested immediately after mixingand casting alloys and denture materials in thefinished state. However, technical require-ments and material consistency may make itnecessary to deviate from general guide-lines.

General commentsMammalian cell culture techniques were pri-marily developed in specialized laboratoriesin the fields of anatomy, physiology, bio-chemistry, microbiology, immunology andother predominantly preclinical disciplines.Traditionally, most systems arc used to studycell structure, replication and repair mechan-isms, in addition to metabolic studies on nutri-ents and xcnobiotics. In the last decades, thecell culture systems have attracted the atten-tion of research groups looking for means toquantify toxic effects (Stammati et al. 1981.Bernson et al. 1986), The studies over the last30 years to find relevant tests to assess the bio-compatibility of dental materials have notbeen a concerted effort. Rather, the tests havebeen developed and modified by interestedindividuals, with the aim of finding ways toexpress the toxic potential of dental materials.Perhaps the largest segment of papers comefrom endodontics, where a direct material-tissue contact is part of the treatment process.Another large segment of papers come fromgroups interested in operative dentistry,

Methods for assessing cytotoxicity 97

where the materials are in indirect contactwith pulp tissue, via remaining dentine.Assessing similar materials against these twobackgrounds has led to many unresolvedquestions on material cytotoxicity and therelevance of results from cell culture studiescompared with usage tests tn vivo.

Dental materials include a range of hydro-philic to hydrophobic liquids, semi-solids andsolids. A major requirement for these mater-ials is that they should have low or negligiblesolubility in the set stage. Thus, the use ofsuhstances which by themselves can be classi-fied as hazardous, for example phosphoricacid, mercury, copper, may be justified whenthey are used in combinations where they arenot released or eluted to any significant extent,such as phosphoric acid in silicate and phos-phate cements, mercury in amalgams and cop-per in several alloy systems. Keeping the manyapplications of these materials in mind, it doesnot necessarily follow that if materials are bio-compatible in one situation, like zinc oxide-eugenol cement in dentine cavities, that thesame materials are biocompatible when con-tacting other oral tissues. The results of cellculture methods used to assess biocompati-bility have demonstrated that any dentalmaterial can be shown to be toxic or non-toxic,depending on the test conditions. This fact hasheen considered a draw back in assessing therelevance of cell culture studies, however thismay be the real situation in selected instances.F.ven if dental materials show little, if any,clinical adverse effects in the majority of thepopulation, there will always be instances«here the materials are used in persons verysensitive to one substance or another. Furtherreli culture studies, coupled with studies innto, will enable the toxic and non-toxiccomponents released from the various dental"materials to be identified and possibly theirmode of action on living cells.

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AMERICA DENTAL ASSOCIATION (ADA) (1979)

A.\SI/ADA document no. 41 for recommendedstandard practices for biological evaluation ofdental materials. Journal ofthe American Dental••iiioctatwn, 99, 697-698.

F R A N C J V I S E DE NORMALISATION

(AFNOR) (1985) Evaluation biologique desproduits dentaires. Document 85058.

BERNSON, V., CLAUSEN, J., EKWALL, B . et al.

(1986) Trends in Scandinavian cell toxicology.ATLA, 13, 162-179.

BRITISH STANDARDS INSTITUTION (1980) Methodsof biological assessment of dental materials. BSTechnical Report Series Document 5828.

DiLLlNGHAM, E.O., WEBB, N . , LAWRENCE, W . H .

& SCHMALZ, G . (1975) Biological evaluation ofpolymers I. Poly(methyl methacrylate). ^wwrna/of Bwmedtcal Materials Research, 9, 569-596.

DiN-NoRMENAusscHuss DENTAL (1986) DIN-Entwurf 13930, Pforzheim.

EKWALL, B . (1983) Screening of toxic compoundsin mammalian cell cultures. Annals of the NewYork Academy of Sciences, 407, 64-77.

FEDERATION DENTAIRE INTERNATIONALE (1980)

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