Investigation of mutagenicity of mineral trioxide aggregate and other commonly used root-end filling materials

0099-2399/95/2111-0537503.00/0 JOURNALOF ENDODONTICS Copyright © 1995 by The American Association of Endodontists

Printed in U.S.A. VOL. 21, NO. 11, NOVEMBER1995

SCIENTIFIC ARTICLES Investigation of Mutagenicity of Mineral Trioxide Aggregate and Other Commonly Used Root-End Filling Materials dames D. Kettering, PhD and Mahmoud Torabinejad, DMD, MSD, PhD

Little information is available regarding the mutagenicity of root-end filling materials. To study mutagenicity of Intermediate Restorative Material (IRM), Super-EBA, and a potential root-end filling material, mineral trioxide aggregate (MTA), strains of Salmonella typhimurium LT-2 (TA 98, R-factor strain and TA 1535, non-R-factor strains) were used in a standard Ames mutagenicity assay. Positive controls ($9 protein and benzo-(t~)-pyrene and N-methyl-N'-nitro-N-nitrosoguanidine) operated properly. No increase in revertant bacteria colony counts occurred with any of the test materials. Based on these results, it seems that IRM, SuperEBA, and MTA are not mutagenic as measured by the Ames Test.

technique and less cytotoxic than all test materials when the radiochromium release method was used (7). An ideal root-end filling material should not only seal the root-end cavity hermatically, it should also be biocompatible with periradicular tissues and noncarcinogenic. Little, if any, information exists in the literature on potential mutagenicity of root-end filling materials. The purpose of this study was to examine IRM, SuperEBA, and MTA for mutagenic potential by the Ames Test (8, 9).

MATERIALS AND METHODS

Preparation of Root-End Filling Materials IRM (L. D. Caulk Co., Milford, DE) and Super-EBA (Harry J. Bosworth Co., Skokie, IL) were mixed according to the manufacturer's instructions. To prevent hardening of these substances and to keep them in a finely dispersed particulate suspension, a sufficient amount of alcohol was added to the mixture immediately following preparation of each material. MTA (Loma Linda University, Loma Linda, CA) was mixed in a 1:1 powder water ratio. The resultant suspensions were used in the Ames Test.

An experimental root-end filling material, mineral trioxide aggregate (MTA), has been investigated in a series of tests: in vitro dye leakage without and with blood contamination, in vitro bacterial leakage, scanning electron microscopy examination of replicas for marginal adaptation, physical and chemical properties, antibacterial, and cytotoxicity effects. Amalgam, Intermediate Restorative Material (IRM), and Super-EBA were used for comparison (1-7). The results of these investigations showed that MTA provided superior seal in both dye and bacterial leakage evaluation, and was not adversely affected by blood contamination (1-3). The marginal adaptation of MTA was superior (4). Examination of the physical properties of these materials showed that the setting time of MTA was <3 h. Compressive strength and solubility of MTA were comparable and, except for amalgam, MTA is more radiopaque (5). Investigation of the antibacterial effects of MTA and three existing materials on facultative and strictly anaerobic bacteria showed that none were completely antibacterial (6). The results of cytotoxicity tests revealed that MTA was less cytotoxic than IRM or Super-EBA, but more severe than amalgam in the agar overlay

Chemicals and Solutions Medium E consisted of 0.2 g of MgSO4-7H20, 2.0 g of citric acid H20 , 10 g of K2HPO2, and 3.5 g of NaNHaHPOn'4H20 in 1 L of double distilled water (8). For minimum essential agar plates, 15 g of agar and 20 g of glucose were added to 985 ml of medium E. The medium was autoclaved, poured (20 ml) into plates, and stored at 4°C until use. Nutrient Oxoid #2 broth was purchased from Unipath, Oxoid Div. (Columbia, MD). Bactoagar was obtained from Difco Laboratories (Detroit, MI).

S-9 PREPARATION Liver microsomal preparation S-9 was purchased (Microbial Associates, Rockville, MD) and prepared according to the method described by Maron and Ames (8). 537

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Kettering and Torabinejad

TABLE 1. Mean revertant colony counts of test organisms after exposure to positive and negative control chemicals TA 98 $9 + B[a]P $9 + DMSO MNNG only DMSO only

258.5 52.9 62.5 38.0

_+ 10.6" _+ 5.3 _+ 8.6 +_ 5.1

TABLE 2. Mean revertant colony counts after exposure to rootend filling materials TA 98

TA 1535

$9 + MTA MTA only $9 only

40.4 ___5.0 43.2 _+ 7.7 56.3 -_ 5.0

21.0 _+ 4.3 25.0 _+ 5.0 32.9 _+ 7.0

$9 + IRM IRM only

33.9 +_ 3.5 14.9 ___3.0

23.1 _+ 2.0 11.3 _+ 3.7

S9 + EBA EBA only

30.9 - 5.0 25.6 _+ 13.0

13.1 _+ 2.9 9.4 _+ 5.8

TA 1535 28.1 20.1 447.6 26.9

-+ 2.2 _+ 3.1 _+ 53.91-+ 2.8

• Significantly different (p - 0.0001) from $9 + DMSO, DMSO only. 1 Significantly different (p = 0.0267) from $9 + DMSO. DMSO only.

MUTAGENESIS ASSAY The plate incorporation assay of Maron and Ames (8) was conducted using Salmonella typhimurium strains TA 98 (R-factor strain) and TA 1535 (non-R-factor strain) (gift of Dr. Robert Teel, Loma Linda University, Loma Linda, CA) as the test strains. Master plates contained 1.5% (wt/wt) of agar and 2% (wt/wt) of glucose, as well as 0.1 M of L-histidine and 0.5 mM of biotin. Ampicillin (8 mg/ml) was added to appropriate plates to support R-factor strain TA 98. Organisms were streaked across the master plates and stored at 4°C after 24 h incubation at 37°C. The master plate cultures were stable for 1 month and served as the source for nutrient broth cultures for assay. Top agar contained 0.5 mM of K-histidine-biotin. Cofactor mix consisted of 0.1 M of Tris-HC1 buffer (pH 7.4) containing 4 mg/ml of NADP, 4.5 mg/ml of glucose-6-phosphate, 2.5 mg/ml of potassium chloride, and 1.8 mg/ml of magnesium chloride. Cofactor mix was filter-sterilized. The following materials were added to sterile disposable test tubes in triplicate and in sequence: 100 /xl of overnight nutrient broth culture of test organism, 800/xl of cofactor mix, 20/xl of $9, and 20 /xl of prepared suspension of root-end filling materials. Three other tubes were prepared in a similar manner, without $9 reagent, to test for direct mutagenicity potential. The mixture was vortexed with 2 ml of molten top agar containing histidine-biotin and poured onto minimum glucose agar plates. After 48 h incubation at 37°C, histidine-independent revertant (His +) colonies were scored. Results were expressed as mean revertant colonies/ plate -+ SE. Positive controls utilized S-9 and benzo-[~]-pyrene (B[a]P) (2.5 /xg/plate) as metabolically activated controls and N-methyl-N'nitrosoguanidine (MNNG; 0.02 mg/ml, 10 /xl/plate) as a direct mutagen. Dimethyl sulfoxide (DMSO) was the diluent used to reconstitute B[a]P and MNNG. Mean colony counts from plates were compared by the twotailed student's t test. RESULTS The combined results of three experiments are summarized in Tables 1 and 2. The colony counts represent revertant bacteria that have the ability to grow on agar medium without histidine. Both TA 98 and TA 1535 demonstrate a low spontaneous reversion rate, as illustrated by colony counts in the range of 20 to 62 (see positive controls). TA 98 responded well to $9 + B[a]P treatment, with the revertant colonies numbering 258.5 -+ 10.6, but did not respond to MNNG or DMSO alone. DMSO represents the diluent used to reconstitute B[a]P and MNNG. TA 1535, although not being affected by $9 plus B[a]P, $9 only, or DMSO only reacted well to MNNG treatment (Table 1). Both positive control responses were significantly higher (p = 0.0001, p = 0.0267) than the spontaneous revertant rate.

The root-end materials did not produce higher reversion rates against either test strain or with direct or indirect mutagenicity treatment (Table 2). All colony counts were within a reasonable variation from the spontaneous revertant rate for both organisms. No significant differences were found between any combination of samples, treatment, or organism when the results of the root-end filling materials were examined statistically.

DISCUSSION The Ames mutagenicity test is designed to assess solids and liquids for potential carcinogenic activity. Strains of S. typhimurium LT-2 have been derived that are sensitive to different classes of mutagens. The variants have a deleted excision mechanism that makes them sensitive to various products with mutagenicity ability and will not grow on media that do not contain histidine. If these bacteria are treated with a mutagen, eventually there will be a mutation that will replace the abnormal base in the histidine gene, and it will become functional again. These bacteria will now grow on histidine-free medium, the basis of the test. The assumption is made that chemicals that produce a significant increase in the frequency of mutation will result in producing colonies capable of growing in the absence of histidine. These materials have >90% probability in being mutagenic to mammalian species. The $9 rat liver microsomal preparation is included in the incubation mixture to assist in the metabolism of indirectacting potential carcinogens to directly acting microbial mutagens. The root-end filling materials were tested for direct and indirect mutagenicity capability. The positive controls used in our study responded as expected. Treatment of TA 98 with $9 and B[c~]P produced - 2 5 8 average colony counts, although not responding to MNNG treatment. TA 1535 responded well to MNNG (Table 1). Low spontaneous reversion rates were found for both organisms. These findings indicate reliability of the findings in this study. An ideal root-end filling material should be dimensionally stable and must not be mutagenic (10). The results shown in Table 2 indicate that MTA, IRM, and Super-EBA do not appear to be mutagenic when examined by the Ames Test. Conditions included both potential direct and indirect mutagenicity, and both mechanisms were negative in our tests. Because MTA seals better than most commonly used root-end filling materials, is less cytotoxic than these materials and is not mutagenic, implantation tests and usage tests in experimental animals should be conducted to assess further the clinical potential of this material.

Vol. 21, No. 11, November 1995 The authors thank Raydotfo Aprecio and Gregory Nelson for technical assistance and Gwen Tamares for typing the manuscript. Dr. Kettering is affiliated with the Department of Microbiology and Molecular Genetics, Schools of Medicine and Dentistry; and Dr. Torabinejad is affiliated with the Department of Endodontics, School of Dentistry, Loma Linda University, Loma Linda, CA. Address requests for reprints to Dr. James D. Kettering, Department of Microbiology and Molecular Genetics, Schools of Medicine and Dentistry, Loma Linda University, Loma Linda, CA 92350.

References 1. Torabinejad M, Watson TF, Pitt Ford TR. The sealing ability of a mineral trioxide aggregate as a root canal filling material. J Endodon 1993;19:91-5. 2. Torabinejad M, Higa RK, McKendry DJ, Pitt Ford TR. Effects of blood contamination of dye leakage of root end filling materials. J Endodon 1994; 20:159-63.

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3. Torabinejad M, Falah Rastergar A, Kettering JD, Pitt Ford TR. Bacterial leakage of mineral trioxide aggregate as a root and filling material. J Endodon 1995;21:109 -12, 4. Torabinejad M, Wilder Smith P, Kettering JD, Pitt Ford TR. Comparative investigation of marginal adaptation of mineral trioxide aggregate and other commonly used root end filling materials. J Endodon 1995;21:295-9. 5. Torabinejad M, Hong CU, Pitt Ford TR. Physical properties of a new root end filling material. J Endodon 1995;21:349-53. 6. Torabinejad M, Hong CU, Pitt Ford TR, Kettering JD. Antibacterial effects of some root end filling materials. J Endodon 1995;21:403-6. 7. Torabinejad M, Hong CU, Pitt Ford TR, Kettering JD. Cytotoxicity of four root end filling materials. J Endodon (in press). 8. Maron DM, Ames BN. Revised methods for the Salmonella mutagenicity test. Mutat Res 1983;113:173-215. 9. Federation Dentaire Internationale. Recommended standard practices in biological evaluation of dental materials. FDI Commission on Dental Materials, Instruments, Equipment and Therapeutics. Int Dent J 1980;30:140-51. 10. Gartner AH, Dorn SO. Advances in endodontic surgery. Dent Clin North Am 1992;36:357-65.

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