Denture adhesives: Cytotoxicity, microbial contamination, and formaldehyde content

SECTIONEDITOR

enture a and forma Karl and NIOM,

Y? ensten-Pettersen,

Ekskrand, DIPS, Annette Mullman Scandinavian

Institute

ierobial

of Dental

Materials,

Haslum,

DDS,

co Dr

OdonL,b

Norway

Denture adhesives are extensively nsed to enhance retention of dentures. Denture adhesives and their leachable components are ingested, but reports on the biologic aspects of denture adhesives are scarce. This study invest,igated some biological properties of 19 commercially available denture adhesives. The adhesives were assessed by the agar overlay teebnique and analyzed for microbial contamination and formaldehyde eontent. In the agar overlay test, all of the materials caused severe cytotoxic effects. Sabouraud exkrose agar was used for gultivation of fungi and tryptone soya broth for cultivation of aerobes, including fungi. Most of the samples tested showed microbial growth. The formaldehyde test revealed the presence of substantial amounts in four products and minor amounts in two products. (J PROSTWET DENT ~~~9~~$:31~-~.)

enture adhesives are extensively used by denture wearers in the western world. According to Stafford,l 88 tons were bought in Great Britain in 1965. It was estimated in a study that approximately 15 % of patients who wore a denture also used denture adhesives.2 In Sweden, consumer expenditures for denture adhesive in 1989 approximated $1.7 million.* Denture adhesives aid the retention of dentures in two ways. First, they swell to obliterate the space between the base of the denture and the oral mucosa and second, they increase the coefficient of surface tension of the fluid film between the denture and the supporting tissue.” Ellis et a1.4 identified the major constituents of 10 proprietary denture adhesives by means of infrared spectroscopy. Three major constituents were identified, as follows. 1. Materials that swell and are responsible for the adhesive properties, such as karaya gum, gelatin, pectin, methylcellulose, hydroxymethylcellulose, and carboxymethylcellulose sodium are included. The synthetic polymers polyethylene oxide, acrylamides, or acetic polyvingl are added by some manufacturers. 2. Antimicrobial agents such as sodi.um borate, sodium tetraborate, hexachlorophene, or propylhydroxy ben-

*Wassing A. ApotekGoiaget, munication, 1990.

Stockholm,

Sweden. Personal com-

aVisiting Scientist. bSenior Scientist. CBioengineer. Copyright @ 1993 by The Editorial Councii PROSTHETIC DENTISTRY. 0022-3913/93/$1.00+.10. 10/1/44286

314

zoate are included. They act as preservatives and are mildly active against Candida aEbicans.l 3. Additives that serve variously as filler, wetting, flavoring, and plasticizing agents were identified. Sodium lauryl sulphate reduces surface tension and acts as a wetting agent. Magnesium oxide is a filler. Some flavoring agents used are peppermint and oil of wintergreen. Denture adhesives are marketed as powders or creams. The powder might include vegetable gums such as acacia or karaya gum, carbohydrates that swell with the addition of water and acquire retentive properties. Cream adhesives derive their retentive properties from polymers such as methylcellulose, hydroxymethylcellulose, or carboxymethylcellulose. Denture adhesives and their leachable components are ingested, but reports on the biologic aspects of denture adhesives are few. Allergic side effects of karaya gum are uncommon, but reactions such as nausea, epigastric pain, and vomiting have been reported.5,6 The aim of this study on denture adhesives was to assess their cytotoxic effects, microbial contamination, and formaldehyde content. ATERIAL

AND

METH

The materials tested in the present study are listed in Table I. The samples were purchased over-the-counter at different pharmacies in Sweden, Norway, and the United States.

Cell culture 3f THE JOURXAL OF

Mouse minimum Scotland, with 5%

fibroblast cells (L929) were propagated in Eagle’s essential medium (E-MEM, Gibco Ltd., Paisley, U.K.) with a bicarbonate buffer supplemented fetal bovine serum (FBS, Gibco Ltd.) and 100

EKSTRAND,

Table

HENSTEN-PETTERSEN,

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Microbial growth from the denture adhesives seeded in Sabouraud dextrose agar and tryptone soya broth

Brand name Corexin

Plus

Batch No.

Contamination

-

904, 803

Manufacturer

+

PIa”ytex- Wallco St.ockholm. Sserige Playtex-Wallco Stafford-Miller Ltd. Hat,field, 1. K Stafford-Miller Ltd. Stafford-Miller I,td

+

Stafford-Miller

-

Stafl’ord-Miller Ltd. Stafford-Miller Ltd. Collett-Marwell Hauge A/S .4sker. Norway Ekulf. Norrkijping, Sweden The Mentholatum co. IAd. Thyford. Berkshire, UK J. Putman Inc. Big Flats, N. Y. 14814 Block Drug Comp. Inc. Jersey City. N.Y. 07302 Block Drug Cc.>rnp. Inc. Carter Prod., Div of Carter Wallace N.%t’. 101153 Richardson V?cks Inc. Shelton. I:onn. 06484 Richardson Vkcks Inc. J. Hilgers Dental Preparations Koln, Germany J. Hilgers Dental Preparations Koln, Germany

--++

+ -

~+

-

-+

Corexin Corega powder

602, 334 BN8045N,

Super Corega Super Corega liq Super Corega cream Super Wernets Super Poli-Grip Stomafix

BN6218X, 9066C 8452,9105, 6113, 5315 BN6241F, BN9004N 6191G, BN9066 BN9004K 2 Batches

Profast

199318

+

SMIG

890506

+

280

+

Super Poli-Grip

DD90

-

Polident Dentu Grip Rigident

J189

-

llld

-I-

Fast Teeth Extra Hold

C568

Fast Teeth Dentofix

Cl14 6 Batches

+++

Dentofix

3 Batches

-

Klutch

Powder

Forte

61355

+

+ +++

-

i-++

-+ --, -+

-+

+

t++

-

++t

-+

-

1,td.

+++, Indicates microbial contamination; +, indicates traces of microbial contamination; -, indicates no traces of microbial contamination.

N/ml penicillin, 100 pg/ml streptomycin, and 1.25 pg/ml amphotericin B (Gibco Ltd.). The test was performed as specified in International Standards Organization (ISO) Technical Report 7450-1984.7 Each 60 X 10 mm plastic Petri dish (Costar, Cambridge, Mass.) was seeded with 6.1 x lo5 cells in 5 ml E-MEM with 5% FBS and antibiotics. After 2 days of incubation a confluent cell layer had formed. The medium was removed and 5 ml of E-MEM containing 1% agarose (FMC Corp., Rockland, Me.) and 5% of FBS was added. When the agarose solidified, the

MARCH

1993

cells were stained with 0.01% neutral red for 15 minutes at 37” C. Empty sterile glass rings, 7.5 mm in diameter and 5 mm in height, were placed on the agar surface and denture adhesive was brought into the glass rings. High-density polyethylene (PE:) and polyvinylchloride (PVC) containing dibutyltindiacetate served as negative and positive controls. After 24 hours of incubation at 37’ C in a humidified atmosphere of 4% CO2 in air, the cultures were examined in an inverted microscope for cytolysis and cellular changes. Three parallels were run for each material and test condi315

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Table

II.

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Detection

EKSTRAND,

DENTISTRY

Corega powder Super Corega Super Corega liq Super Wernets Super POE-Grip Stomafix Profast SMIG Klutch

(+I

334 BN8045 BN9066C 9105 BN9066C BN9004K -

(+I 0 0

RESULTS Agar overlay

Results of test

(+I + 0

199318 890506 280

(+I (+I 0 +

Super Poli-Grip

DD90

++

Polident Rigident

5189 1llD C568

t+t

Cl14 -

t+? (+) ++

Dentu Grip

Fast Teeth Extra Hold Fast Teeth Dentofix Dentofix Forte

tion. The cells were fixed in 10 % formalin and were stained with 0.1% crystal violet in ethanol for permanent records of the experiments.8

of microbial

contamination

Sixty-five grams of Sabouraud dextrose agar @DA) (Oxoid Ltd. Basingstoke, U.K.) was dissolved in 1 L distilled water. The mixture was then sterilized by autoclaving at 121° C for 15 minutes. Tryptone soya broth (TSB) (Oxoid Ltd.) was also used for the cultivation of microbes. Thirty grams of powder was introduced to 1 L of distilled water before sterilization at 121’ C for 15 minutes. One agar tablet (CM49, Oxoid Ltd.) was added to every 100 ml of the solution. Petri dishes 60 x 10 mm (Costar) with the SDA or TSB media were seeded with 0.5 gm of the samples tested. After 48 hours of incubation in an anaerobic chamber (Gas Generating Kit Oxoid BR 38, Oxoid Ltd.) at 37” C, the results were recorded.

Detection

of formaldehyde

Fifteen grams of ammonium acetate and 0.2 ml acetylacetone were mixed with 0.3 ml of glacial acetic acid in distilled water to 100 ml. All chemicals were of analytic grade. A stock solution of formaldehyde was made at a concentration of 100 mg/ml together with standard working solutions with 2.5 pg/ml, 5 fig/ml, and 10 pg/ml.g The samples of the adhesives were placed in glass tubes. One milliliter of each standard solution was placed in a glass tube and 1 ml of distilled water in another glass tube as a blank. Then 2.5 ml of the ammonium acetate, acetyl ace-

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PE used as the negative control caused no change in the cell morphology. PVC, the positive control, induced severe cell death, that is, a zone/lysis index of 415. All of the tested denture adhesives induced cytotoxic effects. Microbial growth was also seen, even though the cell-culture media contained antibiotics.

Detection

++ t , Color change similar to the standard solution of 10 fig/ml; ++, color change similar to the standard solution of 5 g/ml; +, color change similar to the standard solution of 2.5 &ml; (+), slight color change; 0, no color change, distilled water.

Detection

KULLMANN

904

Batch

Corexin Plus Corexin

AND

tone, and glacial acetic acid mixture was added to all of the glass tubes, stirred, and heated to 60’ C for 10 minutes. The color of the sample was compared with that of the standard solutions. A yellow color indicated the presence of formaldehyde.

of formaldehyde

Brand

HENSTEN-PETTERSEN,

of microbial

contamination

Table I shows the microbial contamination of the denture adhesives. Of all of the denture adhesives tested, four of 19 were heavily contaminated. Eight showed traces of microbial growth, and seven brands showed no growth.

Formaldehyde

test determination

The results of the test for formaldehyde content are presented in Table II. Four samples showed color changes similar to the standard solution with 5 pg/ml formaldehyde. Two samples reacted with color changes similar to the standard solution with 2.5 pg/ml formaldehyde. The other samples showed slight or no color changes.

DISCUSSION In the present study the materials tested induced changes in the cultured cells. According to the guideline for the agar overlay test in ISO/TR 7405-1984, the material is reported cytotoxic when cell lysis is recorded. However, the agar overlay test might yield artefactual results, because the materials tested had varying amounts of microbial contamination or contained formaldehyde and other preservatives. The presence of these various components may lead to incorrect readings of the cytotoxicity scores. Sabouraud dextrose agar test is suitable for cultivation of fungi. The fungi maintained their cultured appearance and thus were identified according to standard macroscopic characters described by Sabouraud.l” Tryptone soya broth was used for the cultivation of aerobes and facultative anaerobes, including fungi. Most of the samples were contaminated, possibly because the manufacturers either did not add any preservatives or their actions against fungi were too weak. “Natural” materials such as karaya gum may already be contaminated when delivered to the manufacturer. Even in samples containing carboxymethylcellulose, contamination could be detected. In these samples the preservatives could be too weak or too old. None of the samples had expiration dates on the packages. Most of them had batch codes, but it was difficult or impossible to determine the date of manufacture.

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Data from this investigation may only support speculations for clinical implications. Denture adhesives could play a part in influencing oral flora. If they support the growth of some organisms but not others, an inbalance in oral flora may result. It has been shown that a denture adhesive containing karaya gum forms solutions of pH below that at which hydroxyapatite dissolves. I* Adhesives containing karaya gum may worsen any predisposition to caries. It is clear that clinical work is required in this field. Besides being a potent allergen, formaldehyde in concentrated aqueous solutions is germicidal.‘:! Within dentistry the release of formaldehyde from denture base polymers has been indicated as one of the causes of allergic reactions to acrylic resin denture bases.‘“-” In this study only a semiquantitative determination was done. The test used is not selective for formaldehyde, but detects other aldehydes as well. A color change similar to the standard solution with 5 pg/ml would indicate that, formaldehyde had been intentionally added to the materials. The low levels of formaldehyde observed in the other materials might be residuals from the manufacturing process. To put these amounts in perspective, there are approximately 185 yg of formaldehyde in a slice of bread and 2000 pg of formaldehyde in a small cola.16

1. 2.

3.

4. 5. 6. I. 8. 9. 10. 11. 12. 13. 14. 15.

16.

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1993

OY PRf~S’fiiE’lrl(’

I)EUTISTR\

REFERENCES

CONCLUSIONS All of the denture adhesives were cytotoxic and some had microbial contamination. The latter was most pronounced in adhesives based on “natural” raw materials. A more biologically oriented approach to the formulation of denture adhesives seems warranted. Further studies are needed to identify the toxic components and evaluate each product more precisely.

JOURNAL

Stafford D. Denture adhesives-a review of their r..,es and wmpusitionr. Dent Pratt 1970 21:17-Y. Tarbet WJ, Gro,;sman E. Observations of’ demure *upporting tissue during six months of denture adhesi\r wearing .I Am Dent 4nsw 1980;101:789-91. Polyzois GL. An update on denture fixatives. Dtwai update 198% October 1 579.81. . I Ellis B, Al-Nakaah S, Lamb DJ. The composrtmn ,rnd rheology of dew ture adhesives. J Dent 1980;8:109-18. Figley KD. Karaya gum hypersensitivity. gJAMA. 19403134:747-8. Hogan WJ. Allergic reactions to adhesive dentivc powders. NY state Dent J 1954;20:65-6. International organization for standardization. ISOi?‘R i405- 1984.-Biological evaluation of dental materials, Geneva. Switzerland. lY84 Schmalz G. Die Gewebevertrlglichkeit zahniirztlicher Materialen. Stuttgart: Georg Thieme Verlag. 1981:30:169-X). Fregert S, Dahlqvist J, Gruvberger B. A simple method for the detet tion of formaldehyde. Contact Dermatitis 1984: ll~:lX-4. Sabouraud R. Les Teignes. Masson, Paris. 1910 Lamb DJ. The effects of karaya gum on toot’? enamel Hr Dent J 1980;150:250-2. Hedgecock LW. Antimicrobial agents. Philadelphia: J,re & FrJhiper. 1965; 81. Ruyter IE. Release of’ formaldehyde from denture ‘%se polymer?. Acta Odontol Stand 1’380;3:17-27. Jordan WP, Sherman WI, King JE. Threshold rehpcmses in formaldehyde-sensitive subjects. ,J Am Acad Dermatol l!W:1:44-8. Dahl BE. Hypersensitivity to the dental n.at.erial$ In: Smith DC, and Williams DF, &.: Biocompatibility of dental nwcrlals. vol 1. Hors Raton, Fl: CRC Press, 1982;178-8:). Ames BN. Chemicals, cancers, causalities anti :au~ ions. (‘hemtech lY89;591-8.

Reprrnt requests to. AHNE HENS~‘E~ P~~TERSEN NIOM PO BOX 70 N-1344 HASI.I~M NORWAY

DR.