0099-2399/91/1702-0076/$02.00/0 JOURNAL OF ENDODONTICS Copyright 9 1991 by The American Association of Endodontists
Printed in U.S.A.
VOL. 17, NO. 2, FEBRUARY1991
Lack of Correlation between the Amount of Eugenol Released from Zinc Oxide-Eugenol Sealer and Cytotoxicity of the Sealer Tomoharu Maseki, DDS, Kazuhiko Nakata, DDS, Toshiaki Kohsaka, DDS, Fuminori Kobayashi, DDS, Shinei Hirano, DDS, and Hiroshi Nakamura, DDS, PhD
tion of filtered air without ethylene oxide gas for 72 h to ensure complete removal of the gas (3). Sixty glass tubes containing the test materials were placed into 90 ml of Hanks' balanced salt solution (GIBCO) at 37~ At the end of each week an aliquot of the Hanks' solution was withdrawn and examined for cytotoxicity and eugenol content. This Hanks' solution is henceforth called the test solution and was examined as described below.
The purpose of this study was to examine a possible correlation between the eugenol released from a zinc oxide-eugenol sealer (Canals) and the degree of cytotoxicity. The cytotoxicity and eugenol release from root canal filling material containing eugenol was examined for test solutions at several experimental periods. No positive correlation was found between eugenol release and cytotoxicity of the root canal filling material.
Quantitative and Qualitative Analysis of Test Solution Five milliliters of chloroform were added to 15 ml of the test solution. Quantitative analysis (Fig. 1) of the solution extracted into the chloroform was performed using a gas chromatograph (Shimadzu, Tokyo, Japan). The qualitative analysis of the chloroform extract was performed using thinlayer chromatography and infrared spectroscopic analysis (Nihon Bunko IR-80; Nihon Bunko Co., Tokyo, Japan), with a KBr disk.
The purpose of a root canal filling is to completely seal the canal and eliminate any portal of entry to the periapical tissue (1). Various kinds of root canal filling materials for hermetical obturation of root canal have been used in routine endodontic procedures. Several methods of root canal filling are available. Weine (2) reports that the root canal sealer performs the important function of filling voids and discrepancies between the canal walls and the filling material when gutta-percha cones are used. Therefore, a suitable root sealer is needed for filling the root canal in conjunction with a gutta-percha cone. Most routinely used root canal sealers contain zinc oxideeugenol and various additives. It is thus important to evaluate their biocompatibilities. The purpose of the present study was to evaluate a possible correlation between the quantity of eugenol released from a root canal sealer and the cytotoxicity of the sealer.
Cytotoxicity Test The cells used in this study were mesenchymal cell line L929 fibroblasts (ATCC CCL- 1), originally obtained from the subcutaneous fatty tissue of a C3H mouse, because periapical tissue is almost completely composed of mesenchyme-derived cells. The experimental procedures have all been previously described in detail (4). The L929 fibroblasts to be used for the cytotoxicity evaluation (Fig. 2) were suspended at a density of 4 • 104 cells per ml in Eagle's minimum essential medium (GIBCO F-11) with 10% calf serum. Six-tenths of a milliliter
M A T E R I A L S AND M E T H O D S The root canal filling material used in this study was Canals (Showa Co., Tokyo, Japan). The components of the material are shown in Table 1 and were mixed according to the manufacturer's directions. Glass tubes (5-ram long, with 4mm internal diameter and 1-mm thick walls) were filled with the materials to be evaluated. They were incubated to set in 100% humidity at 37~ for 48 h. After setting, both rough ends of the glass tubes were made smooth to obtain a constant surface area. The materials were then sterilized in an ethylene oxide chamber at 37~ for 48 h. After sterilization, they were allowed to stay in an aeration chamber using forced circula-
TABLE 1. Contents of the tested material Canals (Showa Co., Tokyo, Japan) Powder Zinc oxide Rosin Barium sulfate Bismuth subcarbonate Fluid Clove oil Others
76
40% 30% 15% 15% 83% 17%
Vol. 17, No. 2, February 1991
Eugenol Release and Cytotoxicity
The amount ofeugenol released from the l-wk test solution of the material was 291 ppm, from the 2-wk one, 252 ppm, and from the 3-wk test solution, 125 ppm. The amounts of eugenol released from 3-, 4-, 5-, and 6-wk test solutions were approximately the same.
M i x t u r e of m a t e r i a l s fill t h e m a t e r i a l ( 60 pieces )
l
U
77
placeinto 90ml of Hank's solution
1
The results of the cytotoxicity test are summarized in Fig. 6. The culture containing the 1-wk test solution had 59.8% viable cells. The percentage of viable cells increased gradually
.1
15 ml
Cytotoxicity of Released Material
5ml of chIoroform
U
chloroform + e x t r a c t
lweek
Gas chromatogq'aphy
FIG 1. Methods of quantitative and qualitative analysis of test solution. r
e,-"
Mixture of materials ill] the material ( 60 pieces )
0~
place into 90ml of
m< Hank's solution
0.6 ml
r
incubate the cells for 24 hours [ ~ [
D
1"
remove MEM 10
2.4 ml os
15 20 Time (rain)
25
3'0
FIG 3. Components released into the 1-wk test solution. A, methyl salicylic acid; B, benzyl alcohol; and C, eugenol.
incubate the cells for 24,48 and 72 hours
1 count viable cells F,G 2. Method of cytotoxicity test. of the test solution was mixed with 2.4 ml of suspended cells in a culture dish (Falcon, 35 • 10 ram) and incubated at 37~ in air with 5% CO> The resulting final concentration of the test solution was 20%. The incubation was concluded after 48 h. Viable cells were counted wi:th a phase contrast microscope (5, 6).
6weeks
g oo
o
Results Quantitative and Qualitative Analysis of Test Solution The results of this study are summarized in Figs. 3 to 5. The components A, B, and C were identified as methyl salicylic acid, benzyl alcohol, and eugenol, respectively, using thin-layer chromatography and infrared spectroscopic analysis.
J 0
I
5
I
|174
10
115 20 Time (min)
25
3'0
FIG 4. Components released into the 6-wk test solution.
78
Journal of Endodontics
Maseki et al.
[)pill
400
300
200
&
100
FtG 5. A m o u n t of eugenol released from filling materials.
4S(h) lOO O/ /0
o 50
using human epithelial cells in four different cell culture systems and reported a marked toxic effect when zinc oxideeugenol cement was applied to soft tissue or to cells in their culture systems. Erausquin and Muruzabal ( 11 ) indicated that zinc oxide-eugenol cement was highly irritating to the periapical tissues, causing necrosis of bone and cementum when it came into contact with them. Furthermore, Koch et al. (l 2) reported that eugenol has an allergenic potential. They have concluded that the cause of the cytotoxicity of zinc oxideeugenol cement is the eugenol released from the cement. In addition, Lindqvist and Otteskog (15) attributed the cytotoxicity of zinc oxide-eugenol cement to the release of free eugenol, and the eugenol was shown to be strongly cytotoxic. In our study, the release of eugenol into the 2-wk test solution was 252 ppm and that into the 3-wk solution was 125 ppm. However, the addition of 2-wk or 3-wk test solutions to the fibroblasts resulted in approximately 70 to 75% viable cells. Thus, the pattern of eugenol release did not coincide with the cytotoxicity expressed by the test solution (9). Oil of cloves contains 70 to 85% eugenol and other components. This fact suggests that the cytotoxicity of the cement might involve other factors. Meryon et al. (9) reported that the cytotoxicity of zinc oxide-eugenol cement may be based on the possible toxic effect of zinc ions. Fujisawa and Masuhara (20) reported that zinc ions are released from zinc oxideeugenol cement. In our study, the components released from the cement were eugenol, methyl salicylic acid, benzyl alcohol, and others. Our results suggest that the cause of the repertoire of the test solution may be related to unreactive eugenol, methyl salicylic acid benzylalcohol, zinc ions, rosin, and various other additives that are released from the cement. It would be very worthwhile to pursue these considerations further with more detailed investigation of the nature of the cytotoxicity of zinc oxide-eugenol cement.
5z Drs. Maseki, Nakata, Kohsaka, Kobayashi, Hirano, and Nakamura are members of the Department of Endodontics, School of Dentistry, Aichi-Gakuin University. Address requests for reprints to Dr. Tomoharu Maseki, Department of Endodontics, School of Dentistry, Aichi-Gakuin University, 2-11 Suemoridori, Chikusa-ku, Nagoya 464 Japan. 00
f
T
T
1
2
3
I
4
[
5
[
6 weeks
F~G 6. Cytotoxicity of test solution at 48 h.
with increasing age of the test solution, and treatment with the 6-wk test solution resulted in 84% viable cells.
DISCUSSION Free eugenol is embedded within the zinc eugenolate matrix (7, 8). The amount of free eugenol released from zinc oxide-eugenol cement is probably low. However, it may appear to be much higher than it actually is, since the chelate hydrolyzes readily forming free eugenol and zinc ions (7). Eugenol may be released from eugenolate when it comes into contact with free water (9). Thus, it is important to investigate the effect of eugenol on soft tissue. Many investigators (10-19) have reported the cytotoxicity of zinc oxide-eugenol cement. Hensten-Pettersen and Helgeland (10) tested the cytotoxicity of zinc oxide-eugenol cement
References 1. Grossman LI. Endodontic practice. 10th ed. Philadelphia: Lea & Febiger, 1981:277. 2. Weine FS. Endodontic therapy. 2rid ed. St. Louis: CV Mosby, 1976:25860. 3. Roberts RB, Baker LB. Aeration after ethylene oxide sterilization. Anaesthesia 1972;27:272-82. 4. Nakamura H, Sakakibara F, Matsumoto Y, Hirano S, Hayakawa H, Sakai K, Yip M. Study on the cytotoxicity of root canal filling materials. J Endodon 1986;12:156-60. 5. Kawahara H, Yamagami A, Nakamura M Biological testing of dental materials by means of tissue culture. Int Dent J 1968;18:443-67. 6. Phillips HJ, Terryberry JE. Counting activity metabolizing tissue cultured cells. Exp Cell Res 1957;13:341-7. 7. Phillips RW. Skinner's science of dental materials. 8th ed. Philadelphia: WB Saunders, 1982;98-107. 8. Molnar EJ. Residual eugenol from zinc oxide-eugenol compounds. J Dent Res 1967;46:645-9. 9. Meryon SD, Johnson SG, Smith AJ. Eugenol release and the cytotoxicity of different zinc oxide-eugenol combinations. J Dent 1988;16:66-70. 10. Hensten-Pettersen A, Helgeland K. Evaluation of biologic effects of dental materials using four different cell culture techniques. Scand J Dent Res 1977;85:291-6. 11. Erausquin H, Muruzabal M Root canal fillings with zinc oxide-eugenol cement in the rat molar. Oral Surg 1967;24:547-58.
Vol. 17, No. 2, February 1991 12. Koch G, Magnusso NB, Nyquist G. Contact allergy to medicaments and materials used in dentistry. (11) Sensitivity to eugenol and colophony. Odontol Revy 1973;22:275-89. 13. Tyas MJ. A method for the in vitro toxicity testing of dental restorative materials. J Dent Res 1977;56:1285-90. 14. Valle GF, Taintor JF, Marsh CL. The effect of varying liquid-to-powder ratio to zinc oxide and eugenol of rat pulpal respiration. J Endodon 1980;6:4004. 15. Lindqvist L, Otteskog P. Eugenol: liberation from dental materials and effect on human diploid fibroblast cells. Scand J Dent 1981 ;89:552-6.
Eugenol Release and Cytotoxicity
79
16. Hume WR. Effect of eugenot on respiration and division in human pulp, mouse fibroblasts, and liver cells in vitro. J Dent Res 1984;63:1262-5. 17. Barkin ME, Boyd JP, Cohen S. Acute allergic reaction to eugenol. Oral Surg 1984;57:441-2. 18. Vishteh A, Thomas I, Imamura T. Eugenol modulation of the immune response in mice. Immunopharmacology 1986;12:187-92. 19. Watts A, Paterson RC. Pulpal response to a zinc oxide-eugenol cement. Int Endod J 1987;20:82-6. 20. Fujisawa S, Masuhara E. Binding of eugenol and O-ethoxybenzoic acid to bovine serum albumin. J Dent Res 1981;60:860-4.
Printed in U.S.A.
VOL. 17, NO. 2, FEBRUARY1991
Lack of Correlation between the Amount of Eugenol Released from Zinc Oxide-Eugenol Sealer and Cytotoxicity of the Sealer Tomoharu Maseki, DDS, Kazuhiko Nakata, DDS, Toshiaki Kohsaka, DDS, Fuminori Kobayashi, DDS, Shinei Hirano, DDS, and Hiroshi Nakamura, DDS, PhD
tion of filtered air without ethylene oxide gas for 72 h to ensure complete removal of the gas (3). Sixty glass tubes containing the test materials were placed into 90 ml of Hanks' balanced salt solution (GIBCO) at 37~ At the end of each week an aliquot of the Hanks' solution was withdrawn and examined for cytotoxicity and eugenol content. This Hanks' solution is henceforth called the test solution and was examined as described below.
The purpose of this study was to examine a possible correlation between the eugenol released from a zinc oxide-eugenol sealer (Canals) and the degree of cytotoxicity. The cytotoxicity and eugenol release from root canal filling material containing eugenol was examined for test solutions at several experimental periods. No positive correlation was found between eugenol release and cytotoxicity of the root canal filling material.
Quantitative and Qualitative Analysis of Test Solution Five milliliters of chloroform were added to 15 ml of the test solution. Quantitative analysis (Fig. 1) of the solution extracted into the chloroform was performed using a gas chromatograph (Shimadzu, Tokyo, Japan). The qualitative analysis of the chloroform extract was performed using thinlayer chromatography and infrared spectroscopic analysis (Nihon Bunko IR-80; Nihon Bunko Co., Tokyo, Japan), with a KBr disk.
The purpose of a root canal filling is to completely seal the canal and eliminate any portal of entry to the periapical tissue (1). Various kinds of root canal filling materials for hermetical obturation of root canal have been used in routine endodontic procedures. Several methods of root canal filling are available. Weine (2) reports that the root canal sealer performs the important function of filling voids and discrepancies between the canal walls and the filling material when gutta-percha cones are used. Therefore, a suitable root sealer is needed for filling the root canal in conjunction with a gutta-percha cone. Most routinely used root canal sealers contain zinc oxideeugenol and various additives. It is thus important to evaluate their biocompatibilities. The purpose of the present study was to evaluate a possible correlation between the quantity of eugenol released from a root canal sealer and the cytotoxicity of the sealer.
Cytotoxicity Test The cells used in this study were mesenchymal cell line L929 fibroblasts (ATCC CCL- 1), originally obtained from the subcutaneous fatty tissue of a C3H mouse, because periapical tissue is almost completely composed of mesenchyme-derived cells. The experimental procedures have all been previously described in detail (4). The L929 fibroblasts to be used for the cytotoxicity evaluation (Fig. 2) were suspended at a density of 4 • 104 cells per ml in Eagle's minimum essential medium (GIBCO F-11) with 10% calf serum. Six-tenths of a milliliter
M A T E R I A L S AND M E T H O D S The root canal filling material used in this study was Canals (Showa Co., Tokyo, Japan). The components of the material are shown in Table 1 and were mixed according to the manufacturer's directions. Glass tubes (5-ram long, with 4mm internal diameter and 1-mm thick walls) were filled with the materials to be evaluated. They were incubated to set in 100% humidity at 37~ for 48 h. After setting, both rough ends of the glass tubes were made smooth to obtain a constant surface area. The materials were then sterilized in an ethylene oxide chamber at 37~ for 48 h. After sterilization, they were allowed to stay in an aeration chamber using forced circula-
TABLE 1. Contents of the tested material Canals (Showa Co., Tokyo, Japan) Powder Zinc oxide Rosin Barium sulfate Bismuth subcarbonate Fluid Clove oil Others
76
40% 30% 15% 15% 83% 17%
Vol. 17, No. 2, February 1991
Eugenol Release and Cytotoxicity
The amount ofeugenol released from the l-wk test solution of the material was 291 ppm, from the 2-wk one, 252 ppm, and from the 3-wk test solution, 125 ppm. The amounts of eugenol released from 3-, 4-, 5-, and 6-wk test solutions were approximately the same.
M i x t u r e of m a t e r i a l s fill t h e m a t e r i a l ( 60 pieces )
l
U
77
placeinto 90ml of Hank's solution
1
The results of the cytotoxicity test are summarized in Fig. 6. The culture containing the 1-wk test solution had 59.8% viable cells. The percentage of viable cells increased gradually
.1
15 ml
Cytotoxicity of Released Material
5ml of chIoroform
U
chloroform + e x t r a c t
lweek
Gas chromatogq'aphy
FIG 1. Methods of quantitative and qualitative analysis of test solution. r
e,-"
Mixture of materials ill] the material ( 60 pieces )
0~
place into 90ml of
m< Hank's solution
0.6 ml
r
incubate the cells for 24 hours [ ~ [
D
1"
remove MEM 10
2.4 ml os
15 20 Time (rain)
25
3'0
FIG 3. Components released into the 1-wk test solution. A, methyl salicylic acid; B, benzyl alcohol; and C, eugenol.
incubate the cells for 24,48 and 72 hours
1 count viable cells F,G 2. Method of cytotoxicity test. of the test solution was mixed with 2.4 ml of suspended cells in a culture dish (Falcon, 35 • 10 ram) and incubated at 37~ in air with 5% CO> The resulting final concentration of the test solution was 20%. The incubation was concluded after 48 h. Viable cells were counted wi:th a phase contrast microscope (5, 6).
6weeks
g oo
o
Results Quantitative and Qualitative Analysis of Test Solution The results of this study are summarized in Figs. 3 to 5. The components A, B, and C were identified as methyl salicylic acid, benzyl alcohol, and eugenol, respectively, using thin-layer chromatography and infrared spectroscopic analysis.
J 0
I
5
I
|174
10
115 20 Time (min)
25
3'0
FIG 4. Components released into the 6-wk test solution.
78
Journal of Endodontics
Maseki et al.
[)pill
400
300
200
&
100
FtG 5. A m o u n t of eugenol released from filling materials.
4S(h) lOO O/ /0
o 50
using human epithelial cells in four different cell culture systems and reported a marked toxic effect when zinc oxideeugenol cement was applied to soft tissue or to cells in their culture systems. Erausquin and Muruzabal ( 11 ) indicated that zinc oxide-eugenol cement was highly irritating to the periapical tissues, causing necrosis of bone and cementum when it came into contact with them. Furthermore, Koch et al. (l 2) reported that eugenol has an allergenic potential. They have concluded that the cause of the cytotoxicity of zinc oxideeugenol cement is the eugenol released from the cement. In addition, Lindqvist and Otteskog (15) attributed the cytotoxicity of zinc oxide-eugenol cement to the release of free eugenol, and the eugenol was shown to be strongly cytotoxic. In our study, the release of eugenol into the 2-wk test solution was 252 ppm and that into the 3-wk solution was 125 ppm. However, the addition of 2-wk or 3-wk test solutions to the fibroblasts resulted in approximately 70 to 75% viable cells. Thus, the pattern of eugenol release did not coincide with the cytotoxicity expressed by the test solution (9). Oil of cloves contains 70 to 85% eugenol and other components. This fact suggests that the cytotoxicity of the cement might involve other factors. Meryon et al. (9) reported that the cytotoxicity of zinc oxide-eugenol cement may be based on the possible toxic effect of zinc ions. Fujisawa and Masuhara (20) reported that zinc ions are released from zinc oxideeugenol cement. In our study, the components released from the cement were eugenol, methyl salicylic acid, benzyl alcohol, and others. Our results suggest that the cause of the repertoire of the test solution may be related to unreactive eugenol, methyl salicylic acid benzylalcohol, zinc ions, rosin, and various other additives that are released from the cement. It would be very worthwhile to pursue these considerations further with more detailed investigation of the nature of the cytotoxicity of zinc oxide-eugenol cement.
5z Drs. Maseki, Nakata, Kohsaka, Kobayashi, Hirano, and Nakamura are members of the Department of Endodontics, School of Dentistry, Aichi-Gakuin University. Address requests for reprints to Dr. Tomoharu Maseki, Department of Endodontics, School of Dentistry, Aichi-Gakuin University, 2-11 Suemoridori, Chikusa-ku, Nagoya 464 Japan. 00
f
T
T
1
2
3
I
4
[
5
[
6 weeks
F~G 6. Cytotoxicity of test solution at 48 h.
with increasing age of the test solution, and treatment with the 6-wk test solution resulted in 84% viable cells.
DISCUSSION Free eugenol is embedded within the zinc eugenolate matrix (7, 8). The amount of free eugenol released from zinc oxide-eugenol cement is probably low. However, it may appear to be much higher than it actually is, since the chelate hydrolyzes readily forming free eugenol and zinc ions (7). Eugenol may be released from eugenolate when it comes into contact with free water (9). Thus, it is important to investigate the effect of eugenol on soft tissue. Many investigators (10-19) have reported the cytotoxicity of zinc oxide-eugenol cement. Hensten-Pettersen and Helgeland (10) tested the cytotoxicity of zinc oxide-eugenol cement
References 1. Grossman LI. Endodontic practice. 10th ed. Philadelphia: Lea & Febiger, 1981:277. 2. Weine FS. Endodontic therapy. 2rid ed. St. Louis: CV Mosby, 1976:25860. 3. Roberts RB, Baker LB. Aeration after ethylene oxide sterilization. Anaesthesia 1972;27:272-82. 4. Nakamura H, Sakakibara F, Matsumoto Y, Hirano S, Hayakawa H, Sakai K, Yip M. Study on the cytotoxicity of root canal filling materials. J Endodon 1986;12:156-60. 5. Kawahara H, Yamagami A, Nakamura M Biological testing of dental materials by means of tissue culture. Int Dent J 1968;18:443-67. 6. Phillips HJ, Terryberry JE. Counting activity metabolizing tissue cultured cells. Exp Cell Res 1957;13:341-7. 7. Phillips RW. Skinner's science of dental materials. 8th ed. Philadelphia: WB Saunders, 1982;98-107. 8. Molnar EJ. Residual eugenol from zinc oxide-eugenol compounds. J Dent Res 1967;46:645-9. 9. Meryon SD, Johnson SG, Smith AJ. Eugenol release and the cytotoxicity of different zinc oxide-eugenol combinations. J Dent 1988;16:66-70. 10. Hensten-Pettersen A, Helgeland K. Evaluation of biologic effects of dental materials using four different cell culture techniques. Scand J Dent Res 1977;85:291-6. 11. Erausquin H, Muruzabal M Root canal fillings with zinc oxide-eugenol cement in the rat molar. Oral Surg 1967;24:547-58.
Vol. 17, No. 2, February 1991 12. Koch G, Magnusso NB, Nyquist G. Contact allergy to medicaments and materials used in dentistry. (11) Sensitivity to eugenol and colophony. Odontol Revy 1973;22:275-89. 13. Tyas MJ. A method for the in vitro toxicity testing of dental restorative materials. J Dent Res 1977;56:1285-90. 14. Valle GF, Taintor JF, Marsh CL. The effect of varying liquid-to-powder ratio to zinc oxide and eugenol of rat pulpal respiration. J Endodon 1980;6:4004. 15. Lindqvist L, Otteskog P. Eugenol: liberation from dental materials and effect on human diploid fibroblast cells. Scand J Dent 1981 ;89:552-6.
Eugenol Release and Cytotoxicity
79
16. Hume WR. Effect of eugenot on respiration and division in human pulp, mouse fibroblasts, and liver cells in vitro. J Dent Res 1984;63:1262-5. 17. Barkin ME, Boyd JP, Cohen S. Acute allergic reaction to eugenol. Oral Surg 1984;57:441-2. 18. Vishteh A, Thomas I, Imamura T. Eugenol modulation of the immune response in mice. Immunopharmacology 1986;12:187-92. 19. Watts A, Paterson RC. Pulpal response to a zinc oxide-eugenol cement. Int Endod J 1987;20:82-6. 20. Fujisawa S, Masuhara E. Binding of eugenol and O-ethoxybenzoic acid to bovine serum albumin. J Dent Res 1981;60:860-4.
