JOURNAL OF ENDODONTICS I VOL 4, NO 10, OCTOBER 1978

Correlation of Screening Tests to Usage Tests Kaare L a n g e l a n d , DDS, PhD, F a r m i n g t o n , C o n n

Testing of dental materials has, since 1919, b e e n s y n o n y m o u s with the testing of their physical p r o p e r t i e s . Evaluation of their cytotoxicity has b e e n d o n e in retrospect, f o l l o w i n g t h e i r clinical use w h e n u n n e c e s s a r y d a m a g e to the patients has a l r e a d y occurred. M e t h o d s are t o d a y available w h i c h will p r e v e n t toxic materials f r o m entering the m a r k e t p r o v i d e d a m e c h a n i s m is e s t a b l i s h e d to e n f o r c e such testing. Funding for r e s e a r c h with the o b j e c t i v e to establish a correlation b e t w e e n the less e x p e n s i v e initial tests in tissue culture and small animals and the u s a g e test s h o u l d be c o n s i d e r a b l y increased in the interest of d e v e l o p m e n t of n e w a n d b e t t e r materials and for m a x i m u m p r o t e c t i o n of o u r patients.

True testing of the biologic effects of dental materials has, until the passage of the Medical Device Bill by the US Congress on May 28, 1976, not been a serious issue in the United States. Sporadic efforts to evaluate the biologic effects of dental materials have been performed in grantsupported research by particularly interested individuals during several years. Based on this research, we are currently attempting to establish standards and specifications in the biological field, such as those that have existed in the field of physical properties since 1926. At that time, the American Dental Association's first specification for dental amalgam alloys was presented by scientists at the National Bureau of Standards who had been working on that specification since 1919. Unfortunately, no similar support has existed in the biologic field despite urgent needs and, only this year, a tentative specification has been established 300

totally based on a volunteer effort. Also, despite the fact that since 1945 no dental restoration has failed because of material deficiency per se, the majority of funds available for "biomaterials" continue to be used to support research in the field of physical properties. At the time the National Institutes of Health biomaterials section wanted to establish research priorities in biomaterials in dentistry, the majority (approximately 90%) of the invited speakers were known investigators in the field of physical properties.' The section on restorative materials was free of any speaker on the biologic properties of the involved materials although, since 1945, it is the chemically active cold-curing and composite materials that have caused restorations to fail. These materials result in pulpal and periapical damage, which causes otherwise totally unnecessary endodontic treatment or loss of teeth, or both. The only biologic

area that got some support was that of the glamorous but dubious dental implants, which present much more of a clinical than a material problem, although the two can never be separated in biologic evaluation of materials? Provided the biologic aspects of restorative dentistry had received their fair share of support, the implant problem would have been greatly reduced near to elimination. It is no wonder that the conclusion at the end of the conference-and repeated in the ensuing publication-was that the study of the gamma phase of the amalgam was a prominent research priority. The biologic properties got hardly more than honorary mention. The odds in favor of future grant support in the area of the physical properties were about the same as those for the "house" in Las Vegas. It should also be noted that in the study section on biomaterials, there are two representatives with the label materials

JOURNAL OF ENDODONTICS I VOL 4, NO 10, OCTOBER 1978

(physical properties) and none with the label biological properties. Although I agree that research should continue and even be increased in the field of physical properties, when there does not seem to be enough support to go around, the time has come for the biologic evaluation to get its fair share. This must be seen in the perspective of the time because the physical properties of dental materials were beginning to be taken seriously in 1919. This means that the biologic properties have a good deal of catching up to do. As I see it, there are two areas that are in urgent need of f u n d i n g - M i s sion-oriented research with the specific objective of establishing the basis for specifications for biologic properties of dental materials; and organizational work to formalize the specifications based on the aforementioned research in a manner and at a level equal to that regarding the physical properties of the same materials. Currently, the research in the field is performed totally haphazardly, based on the ability of interested parties to obtain funds for their special interests. The organizational work, both in the United States and in the Federation Dentaire Internationale (FDI), depends on volunteer work by interested parties. In the United States a preliminary document, MD-156, has been evaluated for the past 3 to 4 years, and a new document is now being circulated for comments among interested parties. In the FDI, a document with a similax intent has been circulated for Comment among interested parties since October 1977, and will not be finalized in 1978. The current draft of Specification no. 41 has incorpo-

rated a number of the suggested FDI tests, further emphasizing the necessity for serious mission-oriented funding to establish biologic specifications for the benefit of our patients, our profession, and dental manufacturers. Because biological material testing is extremely expensive, particularly in the full-scale usage tests, it is reasonable to look for substitute costsaving tests while maintaining the direct relevancy of the test for maxim u m protection of the patient. The purpose of my presentation is to discuss the possible correlation between the currently available initial laboratory tests and the usage tests for local toxicity. Screening Tests

Suggested initial tests are tissue culture tests involving a monoIayer of cells, a cell suspension test, a new millipore filter test (Tronstad and colleagues, 2 1977) a test using enhanced vascular permeability (Spangberg,:' 1973), a new muscle contact test (Hasselgren and colleagues/ 1977), and several tissue implant tests in small animals. Among the tissue culture tests, the monolayer agar overlay method suggested by Guess and co-workers ~ in 1965 meets the requirements for a simple and inexpensive test, but it has the disadvantage that materials or components of materials have to diffuse through the agar overlaying the monolayer of cells. Thus, materials which do not dissolve in or diffuse through agar will not cause cellular damage, although they could, nevertheless, be toxic. The test would, in such cases, give a false negative result. The millipore filter test and the new muscle test 2'4 are not yet evaluated by the involved committee

and can, therefore, not currently be incorporated. The cell suspension chromium method, introduced by Spangberg 3 is the test currently suggested in the FDI document. The main advantage of this test over the monolayer method is that it allows direct cell-material contact in dissolved materials as well as in semisolid, setting, or set materials. In addition, the test uses objective counting rather than subjective observation and evaluation, as is the case in the monolayer method. A disadvantage is that the chromium method is more expensive because the technique is more involved and because it depends on the availability of a g a m m a counter, and that chromium is bound rather than released in the initial phase of a toxic reaction. Implantation Tests

Also, implantation tests vary greatly in methodology and sophistication. A method recommended by Autian (Lawrence and others)" involves making up the material to be tested in cylindrical pellets, which bench-dry for 24 hours before they are injected into rabbit muscle by use of a syringe with a needle fitting the diameter of the material peI[ets. T h e degree of toxicity of the material is evaluated primarily by gross examination, which may or may not be followed by histologic evaluation. Again, it is a simple, inexpensive method and, from those points of view, desirable. The disadvantage of this method is that possible toxic components of the materials may be chemically bound, or may have evaporated, before the material is inserted. If this happens, as it surely does in cold-curing and in composite materials, this test method will give 301

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false negative results. This includes the current revision of the method. It has therefore been suggested to insert the material in its fresh condition into 10-mm long polyethylene or Teflon tubes or cups with an interior diameter of approximately 1 mm, and immediately implant these tubes in the subcutaneous tissue or bone of small laboratory animals (Torneck, T 1966; Langeland and co-workers, ~ 1969; and Spangberg, '~ 1969). This allows for the evaluation of the effect of the uncured material at the end of the tube and the control of the tissue response to the surgical t r a u m a along the mid section of the tube, all in the same histologic section. It excludes speculation about general differences in response between test animals. USAGE TESTS In these tests, the material which will be tested is handled accurately following the directions of the manufacturer for the intended clinical use of the material. Handling of the material, and the clinical steps necessary for its application, m a y be as important for the biologic response as the material itself. Thus, the tests would be performed differently for each of the types of materials, for example, restorative, endodontic, prosthetic, periodontal, and implant materials. To fulfill the requirement of a true usage test, the test should ideally be performed in h u m a n oral tissue; however, because this is often not possible, primates are the preferred test animals. Correlation of Tests

Currently, nothing in the literature supports positive correlation between the various tests in such a way that a screening test on any level 302

could replace a usage test. In general, what a screening test does, at most, is to rank the materials in regard to their cytotoxicity under the testing conditions in question. Thus, in any form of tissue culture test, the test system is so different from the usage conditions that no conclusions m a y be drawn as to the possible toxicity of the material in its actual use. T h e question could then be asked, " W h y use any screening test at all?" The answer is that a well-controlled tissue culture test allows the ranking of materials as to their toxicity and, in addition, it allows the testing of components of materials. Thus, a manufacturer may, by this method, be able to choose the least toxic component if he, during the development of the material, has a choice of various chemical compositions while maintaining the optimal physical properties of the material. The implantation test may have its clinical relevance in regard to materials that in their use are intended to remain in constant direct contact with connective tissue, for instance, endodontic materials and implants, whereas it would be an entirely inappropriate screening test for restorative materials. With use of amalgam as an example, I will demonstrate the lack of correlation between the implantation test and the usage test. In a 14-day implant test of amalgam pellets, an acute inflammatory response was observed around the implant. One could not distinguish between the effect of the operative trauma and the material itself. At 47 days, a fibrous capsule had formed around the implant and, if the experimentation had terminated there, the conclusion would have been that amalgam is an inert material. How-

ever, at 80 days observation period there was a breakdown of the fibrous capsule, and foreign body cells and macrophages had accumulated around the amalgam. Thus, the old idea of an "encapsulation" as a criterion of an inert material is false. When amalgam was introduced in experimentally prepared cavities in monkey or human teeth, no inflammatory response occurred in the dental pulp, provided the cavity preparation was performed with adequate water spray. Thus, the implantation test is directly misleading. Furthermore, although filling materials contact gingiva, an implant test is so different from the usage condition in the gingival a r e a - i n volving imperfection of the margin, overhang, bacterial plaque, and calculus-that the test could not reflect this conditon. These examples indicate lack of correlation between the implantation test and the usage test. It is an interesting exercise of thought to wonder what would have happened to amalgam if, when the first testing of physical properties was made, the implantation test had been a requirement for the acceptability of amalgam. In that case, it would have had to be rejected as a dental filling material, based on a false positive result in the implantation test. However, the implantation test would be a usage test if the amalgam was intended for apical root canal fillings during periapical surgery. In that case, it would be acceptable with the warning, "The material causes a foreign body reaction and a chronic inflammation if left in bone, and it prevents adequate healing." The few examples shown indicate that no screening test today can safe-

JOURNAL OF ENDODONTICS i VOL 4, NO 10, OCTOBER 1978

ly replace a usage test b u t , p r o v i d e d the m e t h o d o l o g y is c a r e f u l l y c o n trolled, it is possible to r a n k m a t e r i a l s as to t h e i r c y t o t o x i c i t y w h e n used under those c o n d i t i o n s . H o w e v e r , t h e ranking o b t a i n e d in t h e s c r e e n i n g tests w o u l d not necessarily be the same in the usage test b e c a u s e of the procedures i n v o l v e d in the clinical use. M y h o p e is, h o w e v e r , t h a t sufficient f u n d i n g will b e c o m e a v a i l a b l e for research to f u r t h e r d e v e l o p correlation b e t w e e n s c r e e n i n g a n d usage tests so t h a t s c r e e n i n g tests m a y reach t h a t level o f s o p h i s t i c a t i o n in the future, a n d t h a t t h e y m a y r e d u c e the necessity for u s a g e tests. T h i s is the o n l y w a y t h a t t h e e x p e n s e o f the tests for the b i o c o m p a t i b i l i t y of dental m a t e r i a l s m a y be r e d u c e d while m a i n t a i n i n g the o p t i m a l safety

for the h u m a n b e i n g in w h i c h the m a t e r i a l is to be used. Dr. Langeland is professor and chairman, department of endodontics, University of Connecticut Health Center, Farmington, Conn 06032.

References 1. Dental Biomaterials-Research Priorities, proceedings, symposium, L. W. Wachtel (ed). DHEW Publication No. (NIH) 74-548. Aug 7-8, 1973, Des Plaines, I11, Bethesda, National Institute of Dental Research, 1974. 2. "l'ronstad, L.; Hasselgren, G.; and Wennberg, A. Material toxicity evaluation using cells cultured on millipore filters and enzyme cytochemical techniques. J Dent Res 56(special issue)A, abstract no, 307, Feb 1977. 3. Spangberg, L. Kinetic and quantitative evaluation of material cytotoxicity in vitro. Oral Surg 35:389, March 1973. 4. Hasselgren, G.; Wennberg, A.; and Tronstad, L. Evaluation of initial tissue response to biomaterials using muscular tissue

and enzyme histochemical techniques. J Dent Res 56:(special issue) A, abstract no. 306, Feb 1977. 5. Guess, W.I,.; Rosenbluth, S.A.; Schmidt, B.; and Autian, J. Agar diffusion method for toxicity screening of plastics on cultured cell monolayers. J Pharm Sci 54:1545 Oct 1965. 6. Lawrence, W.H.; Mitchell, J.L.; Guess, W.L.; and Autian, ,J. Toxicity of plastics used in medical practice. Investigation of tissue response in animals by certain unit packaged polyvinyl chloride administration devices. J Pharrn Sci 52:958 Oct 1963. 7. Torneck, C.D. Reaction of rat connective tissue to l:x)lyethylene tube implants. Oral Surg 21:379 March 1966. 8. Langeland, K.; Guttuso, J.; I,angeland, L.K.; and Tobon, G. Methods in the study of biologic responses to endudontic materials. Tissue response to N2. Oral Surg 27:522 April 1%9. 9. Spangberg, L. Biological effects of root canal filling materials. Reaction of bony tissue to implanted root (:anal filling material in guinea pigs. Odontol Tidskr 77:133 April 15, 1969.

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Correlation of screening tests to usage tests.

JOURNAL OF ENDODONTICS I VOL 4, NO 10, OCTOBER 1978 Correlation of Screening Tests to Usage Tests Kaare L a n g e l a n d , DDS, PhD, F a r m i n g t...
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