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Year : 2005  |  Volume : 8  |  Issue : 4  |  Page : 4-13
An investigation into the irritant properties of some retrograde filling materials - an in vivo study

Department of Conservative Dentistry & Endodontics Dr. R. Ahmed Dental College & Hospital Kolkata, India

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This study was done to evaluate the tissue reaction to four different retrograde filling materials. In this study 60 sterile polyethylene tubes (Meditop Corporation (M) SDN. BHD., Malaysia) were divided into 5 groups of 12 tubes each. Subsequently, the first group specimens were loaded with zinc . free amalgam (Solila Nova, Dentsply), the second group specimens were loaded with Intermediate restorative material (IRM) (L.D. Caulk, Milford, DE), the third group specimens were loaded with Super EBA (Harry J. Bosworth CO, Skokie, IL) and the, fourth group specimens were loaded with Vitrebond (3M. St. Paul, MN, USA). In the last group the tubes were left empty to serve as control. 1 specimen from each group was implanted into the subcutaneous tissue of a single rabbit, making a total of 12 animals each containing an implant of every group. At the end of 2 wks, 4 wks and 12 wks the specimens along with surrounding tissues were removed from 4 rabbits. Subsequently the tissue was fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, mounted and stained using usual standard technique. The sections were examined and reaction of the soft connective tissue to the material was recorded. The data was analysed statistically.

How to cite this article:
Mazumdar D, Ray P, Wang CK, Dhanuka S. An investigation into the irritant properties of some retrograde filling materials - an in vivo study. J Conserv Dent 2005;8:4-13

How to cite this URL:
Mazumdar D, Ray P, Wang CK, Dhanuka S. An investigation into the irritant properties of some retrograde filling materials - an in vivo study. J Conserv Dent [serial online] 2005 [cited 2020 Aug 11];8:4-13. Available from:

   Introduction Top

The goal of endodontic therapy is the elimination of bacteria from the root canal system and the subsequent establishment of an effective barrier to prevent the further passage of microorganisms or their products to the periapical tissues.

Conventional endodontic treatment is recommended for predictable success in root canal treatment. In some cases however, retreatment if required may necessitate a surgical approach, at which time the cleaning and sealing of the apical portion of the root canal system is especially important.

The purpose of placing a retrograde filling is to provide a tight, biocompatible apical seal, which prevents the leakage of potential irritants from the root canal into the periradicular tissues. Of the number of ideal properties for retrofilling materials proposed, one of them being that it should be well tolerated by periapical tissues.

Although a plethora of materials is available, no material has been found that fulfills all or most of the properties for the ideal retrograde filling material. Amalgam had been the most popular and widely used material since the last century.

Recently a number of studies have cast doubt on the efficacy of amalgam and other materials are receiving attention.

Here we will be focusing on the biocompatibility of the retrograde filling material. For the biologic evaluation and acceptance of endodontic material the secondary test will be performed which involves the implantation of the material into the tissues other than the periapical region of experimental animals.

The aim of the study is to evaluate the inflammatory response to four retrograde filling materials (amalgam, IRM, Super EBA, Vitrebond) implanted into the soft connective tissue of rabbits and to compare their biocompatibility with each other.

   Materials and Methods Top

In this study the biocompatibility of four retrograde filling materials mentioned below was evaluated.

  1. Zinc free amalgam (Solila Nova, Dentsply)
  2. Intermediate restorative material (IRM) [L.D. Caulk, Milford, DE]
  3. Super EBA (Harry J. Bosworth CO, Skokie, IL)
  4. Vitrebond (3M. St. Paul, MN, USA)

Empty polyethylene tubes (Meditop Corporation (M) SDN. BHD., Malaysia) were used as control. 60 polyethylene tubes were taken and divided into 5 groups of 12 specimens each. Each material was mixed according to the manufacturer's instruction under aseptic condition and loaded into 12 sterile polyethylene tubes 10.0 mm in length and 1.0 mm in diameter. The excess material was carefully removed with sterile gauge. These procedures were carried out as quickly as possible so that the materials were inserted into the animals in a state similar to that when used clinically.

The surgical site of the 12 rabbits was anesthetized locally with 2% xylocaine and both flanks and the back of the neck were shaved and scrubbed with a 2% tincture of chlorhexidine and povidine iodine lotion. Incisions were made through the skin and the subcutaneous tissue in the back of the neck and flanks were opened with blunt dissection. The filled tubes were inserted with the help of amalgam carrier at least 2 cm from the line of incision, which were closed with sutures. The skin was scrubbed again with tincture of chlorhexidine, povidine iodine and supragen lotion and a dressing was applied over the wound.

Each animal carried an implant from each of the 5 groups. Thus, each of the 12 animals carried 5 implants. The majority of the animals had a body weight between 1.5 - 2.0 kg.

At the interval of 2 wks, 4 wks and 12 wks, 4 rabbits were taken and the implanted specimens together with the surrounding tissue were removed and fixed in 10% neutral buffered formalin. Following fixation, the tissue were embedded in paraffin, sectioned, mounted and stained using the usual standard technique. Six micrometer serial sections stained with haematoxylin and eosin from each site were prepared. The sections were examined by independent pathologists.

Inflammations were recorded as none, mild, moderate and severe according to the subjective criteria given below.


No Inflammation (Grade 0)

No inflammation or necrosis

No fibrous encapsulation

Mild Inflammation (Grade 1)

Thin fibrous encapsulation

Few macrophages and inflammatory cell infiltrate

Vasodilation and edema

Moderate Inflammation (Grade 2)

Dense infiltration of inflammatory cells

Foreign body giant cell infiltration

Marked vasodilation

Severe Inflammation (Grade 3)

Tissue necrosis, possibly manifest only as sequestra

Dense round cell infiltration

Abscess formation


Reports on the reaction of the soft connective tissue to the material were recorded in standardized forms. The data obtained were analysed statistically by Chi Square tests.

   Results Top

Of the 12 animals used in the study, I died due to unknown reason. At 4 wks interval 1 specimen was discarded because the healing at that site was not satisfactory. A total of 54 samples were recovered and 302 sections of these samples were examined. The severity of tissue responses at 2 wks, 4 wks and 12 wks to various test materials are summarized in [Table 1],[Table 2] and [Table 3] respectively.


Histologic sections of samples revealed severe to moderate diffuse inflammatory reaction to amalgam. The majority of IRM and Super EBA showed moderate reactions although few had severe reactions. The reaction to Vitrebond was mild to moderate and the majority of the control showed no reaction.

Analysis of the data by Chi square test showed that there exists no statistically significant difference between Zinc free amalgam, IRM and Super EBA. But when each of the above three materials were compared with the Vitrebond, the result was statistically highly significant.


By 4 wks, the inflammation had subsided in all the sections. The reaction mostly subsided from severe to moderate inflammation to mild inflammation in case of amalgam. IRM, Super EBA and Vitrebond all showed mild reaction. However, there was complete healing seen in specimens of the control.

Analysis of the data by Chi Square test showed no significant difference between the various experimental materials.


By 12 weeks the IRM, Vitrebond and hollow tube controls displayed little inflammation, which was categorized as none. The implants were encased by mature and young fibroblasts without any inflammatory cells. The Super EBA specimens also exhibited a marked diminished inflammatory response residing between none and mild.

However, with amalgam, the mild reaction which was present at 4 wks interval persisted in the same state at 12 weeks interval also.

Analysis of the data by Chi Square test showed that no statistically significant difference existed between Zinc free amalgam and Super EBA and between IRM and Vitrebond. But when the comparisons were made between Zinc free amalgam and IRM and between Zinc free amalgam and Vitrebond statistically significant difference existed. Also when the comparisons were made between IRM and Super EBA and between Super EBA and Vitrebond statistically significant difference existed.

   Discussion Top

One of the most important properties of any material that is to remain indefinitely in contact with vital tissues is that it must be biocompatible. Such a property is one that is widely quoted in books of endodontics as essential for root end filing materials.

The three recommended tests for the evaluation and acceptance of endodontic material are the "Primary Tests", the "Secondary Tests" and the "Usage Tests". The "Primary Tests" and the "Secondary Tests" are screening tests where the toxicity profile of a new retrograde filling material is evaluated. If they are found to be biocompatible in these screening tests then only they are further evaluated through the "Usage Tests".[9]

The "Secondary Tests" are in vivo tests carried out in small animals evaluating the systemic or local toxicity of the experimental materials. These tests thus are more reliable than the "Primary Tests" which are in vitro tests which do not examine the complex interactions between materials and tissues.[9]

Of the different types of secondary test, the one which is quite commonly used is the subcutaneous test. The implantation tests vary greatly in methodology and sophistication. A method recommended by Austin[1] involved making cylindrical pellets of the material to be examined, which were bench dried for 24 hrs and then injected into rabbit's muscle by use of a syringe with a needle similar in diameter to that of the pellets of the material. The degree of toxicity of the material was evaluated primarily by gross examination, which may or may not be followed by histological evaluation. The disadvantage of the method was that, possible toxic components of the material may become chemically bound or may evaporate before the material was inserted. These may result in false negative results. The implantation test was refined when Torneck[14] in 1960's showed that subsequent to the implantation of polyethylene tube, fibrous tissue repair occurred with no lasting inflammation. These tubes were later used as a vehicle to carry endodontic materials. Friend and Brown[6] used tubes as vehicle and concluded that this method has greater semblance to the clinical situation than did the implantation of preset pellets of materials. The material contained in the tube was only in contact with the tissue at the ends of the tubes. This is comparable to the clinical situation where the retrograde filling material was in contact with the periradicular tissue only at the open end of the root.[4] There was a standardization of the tissue/ material contact area. The materials were introduced in the subcutaneous tissue in the same state as it will be in clinical situation.

Therefore, taking into consideration the above mentioned advantages, this present study chose the subcutaneous implantation in rabbits of retrograde filling materials within polyethylene tubes as method to evaluate the tissue irritating properties of retrograde filling materials.

In this study the fixed and stained tissue sections surrounding the implanted samples were examined after 2 wks, 4 wks and 12 wks. One of the important criteria for evaluating the nature of the reaction of the tissue to the implanted material is the presence of polymorphonuclear leukocytes. Their presence at the end of 2 wks should be considered evidence of the cumulative effect of the toxicity of the material, the operative trauma and continued seepage of the particles of the materials into the surrounding tissue. When the PMN's occur at 4 wks and even more so at 12 wks, the effect of the operative trauma is no longer apparent because only the fibroblasts and fibrocytes appear along the side of the uncontaminated polyethylene wall[4].

In the present study the tissue irritating property of the four retrograde filling materials, Zinc free amalgam, Intermediate Restorative Material (IRM), Super Ethoxybenzoic Acid (Super EBA) and light cured resin modified G.I.C. (Vitrebond) were evaluated.

Amalgam had been the most popular and widely used retrograde filling material since the last century. It is easy to manipulate, readily available, well tolerated by soft tissues, radiopaque and initially provides a tight apical seal. However, it is slow setting, dimensionally unstable, eventually leaks from corrosion and stains the overlying soft tissues causing a tattoo. Omnell[12] found that with zinc containing alloy there was periradilular bone destruction and deposition of zinc carbonate around the silver root end filling. Most of the investigators recommend the use of non zinc containing amalgam.

When used as a retrograde filling material the zinc oxide-eugenol cements tended to become absorbed over time because of its high water solubility. With contact with water or tissue fluids, it hydrolyzed to produce zinc hydroxide and eugenol. The eugenol depending on its concentration can competitively inhibit prostaglandin synthetase, inhibit sensory nerve activity, inhibit mitochondria) respiration, and can be an allergen[7],[8]. To overcome these problems, the zinc oxide-eugenol cements were modified to produce IRM and Super EBA by the addition of poly methacrylate fibres to IRM and by substituting a portion of the eugenol with oethoxybenzoic acid and the incorporation of alumina into the powder in Super EBA. These had low solubility, higher strength and good adaptation. This study was undertaken to evaluate the tissue irritating properties of the reinforced zinc oxide-eugenol cements.

Because of sensitivity to moisture despite of their good adhesion, conventional GIC has not gained wide acceptance for use in surgical endodontics. The resin modified glass-ionomer with its ability to command set, bond to the dentin, less moisture sensitivity and improved physical properties can be a potential retrograde toot filling material if biocompatible. Thus, in the present study Vitrebond was chosen to evaluate its biocompatibility.

It was found that at 2 wks interval, there is severe to moderate inflammation in the tissue adjacent to zinc-free amalgam. At 4 wks the inflammation subsided to mild, which continued in the same state upto 12 wks. This was in accordance with the study of previous investigators who also found an initial moderate to severe tissue reactions which subsided with time. This can be justified by the fact that the initial set amalgam contains excess unreacted mercury which is released into the surrounding tissue, but with time as solid amalgam phases form, the potential for mercury release is significantly reduced. Mitchell el al (1959)[10] found less initial tissue reaction in comparison to our study which may because the investigator implanted preformed, set specimens of the material.

IRM and Super EBA both exhibited moderate inflammatory tissue response at 2 wks interval. At 4 wks interval IRM and Super EBA both showed mild inflammatory tissue response. By 12 wks, complete healing was seen with IRM which showed no inflammation. With Super EBA the tissue response was still mild at the end of 12 wks. This result of the present study co-relates with the finding of Blackman R (1989)[3], Oslen and others (1994)[11], Bruce (1993)[5] and Bhambani (1993)[2] who also found an initial moderate to severe reaction which subsided with time.

The initial intense inflammatory response seen with both the materials may be provoked by cytotoxic concentration of eugenol and O-EBA known to be released from both freshly mixed materials[7],[8]. The rapid resolution of inflammation may be because of the sequestration of free eugenol or O-EBA by binding with the serum and extra cellular proteins along with the rapid clearance by the blood flow. Hume[7],[8] noted a reduction in eugenol leaching of ZOE compounds when fluid availability was restricted. He also noted an immediate and initial high release of eugenol which declines exponentially as the material sets. No "foreign body" granulomatous reactions were observed with either of the ZOE materials.

From the results of this study, both IRM and EBA cements appear to be acceptable biological alternatives to amalgam.

With resin modified glass ionomer cement (Vitrebond) the result showed that, at 2 wks interval the tissue reaction was mild to moderate. By 4 wks the response subsided to mild inflammation and there was complete healing by 12 wks. This result of the present study co-relates with the study of Tassery H, Remusat M, Koibi G, Joseph WP (1997)[13]

Since, the initial setting of the cement was done by curing with light before it came in contact with the subcutaneous tissue and the cement is less moisture sensitive, the reaction to it were less severe than that observed with conventional glass ionomer cement.

From the study it can be concluded that Vitrebond appeared to be a biocompatible and acceptable biological alternative to amalgam.

Though all the four experimental materials showed favourable tissue reaction and were biocompatible, variations in the structures where the test is carried out (e.g. test carried on subcutaneous tissue instead of jaws) and interspecies differences with this model make further clinical studies essential.

   Conclusion Top

Within the limitations of this study it can be concluded that

  1. The lack of reaction to polyethylene tubes makes it an ideal material in which to implant the root filling materials.
  2. Zinc free amalgam is biologically well tolerated by rabbit's connective tissue.
  3. Both modified zinc-oxide eugenol (IRM and Super EBA) appear to be acceptable biological alternatives to amalgam.
  4. Vitrebond has potential as a root end filling material, as the tissue response was considerably more favourable than that to amalgam.
  5. In terms of biocompatibility Vitrebond was found to be the best, followed by IRM, Super EBA and Zinc free amalgam.[Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5],[Figure 6],[Figure 7],[Figure 8]

   References Top

1.Austin J. General toxicity and screening tests for dental materials. Int Dent J. 1974 Jun;24(2):235-50.  Back to cited text no. 1    
2.Bhambhani SM, Bolanos OR. Tissue reactions to endodontic materials implanted in the mandibles of guinea pigs. Oral Surg Oral Med Oral Pathol. 1993 Oct;76(4):493-501.  Back to cited text no. 2    
3.Blackman R, Gross M, Seltzer S. An evaluation of the biocompatibility of a glass ionomer­silver cement in rat connective tissue. J Endod. 1989 Feb;15(2):76-9.  Back to cited text no. 3    
4.Browne RM, Friend LA. An investigation into the irritant properties of some root filling materials. Arch Oral Biol. 1968 Nov; 13(11):1355-70.  Back to cited text no. 4    
5.Bruce GR, McDonald NJ, Sydiskis RJ. Cytotoxicity of retrofill materials. J Endod. 1993 Jun;19(6):288-92.  Back to cited text no. 5    
6.Friend LA, Browne RM. Tissue reactions to some root filling materials. Br Dent J. 1968 Oct 1;125(7):291-8.  Back to cited text no. 6    
7.Hume WR. An analysis of the release and the diffusion through dentin of eugenol from zinc oxide-eugenol mixtures. J Dent Res. 1984 Jun;63(6):881-4.  Back to cited text no. 7    
8.Hume WR. The pharmacologic and toxicological properties of zinc oxide-eugenol. J Am Dent Assoc. 1986 Nov; 113(5):789-91.  Back to cited text no. 8    
9.Wataha JC. Biocompatibility of Dental Materials. In:Anusavice KJ, editor. Phillip's Science of Dental Materials. St. Louis, Missouri: Saunders;2004.  Back to cited text no. 9    
10.Mitchell DF. The irritational qualities of dental materials. J Am Dent Assoc. 1959 Nov;59:954­66.  Back to cited text no. 10    
11.Olsen FK, Austin BP, Walia H. Osseous reaction to implanted ZOE retrograde filling materials in the tibia of rats. J Endod. 1994 Aug;20(8):389-94.  Back to cited text no. 11    
12.Omnell KA. Electrolytic precipitation of zinc carbonate in the jaw; an unusual complication after root resection. Oral Surg Oral Med Oral Pathol. 1959 Jul; 12(7):846-52.  Back to cited text no. 12    
13.Tassery H, Remusat M, Koubi G, Pertot WJ. Comparison of the intraosseous biocompatibility  Back to cited text no. 13    
14.Torneck CD. Reaction of rat connective tissue to polyethylene tube implants. I. Oral SurgOral Med Oral Pathol. 1966 Mar;21(3):379-87.  Back to cited text no. 14    

Correspondence Address:
Dibyendu Mazumdar
Department of Conservative Dentistry & Endodontics Dr. R. Ahmed Dental College & Hospital Kolkata
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-0707.42591

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]

  [Table 1], [Table 2], [Table 3]


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