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Reproducibility of the model cavity method for In vitro toxicity testing of dental restorative materials
Fd Chem. Toxic. Vol. 24, No. 6/7, p. 592, 1986
0278-6915/86 $3.00+0.00 Copyright © 1986 Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
REPRODUCIBILITY OF THE MODEL CAVITY METHOD FOR I N V I T R O TOXICITY TESTING OF DENTAL RESTORATIVE MATERIALS S. D. MERYON and R. M. BROWNE Department of Oral Pathology, Dental School, St Chads Queensway, Birmingham B4 6NN P. F. UPHILL and A. D. CORDERY Department of Mutagenesis and Cell Biology, Huntingdon Research Centre Ltd, Huntingdon, Cambs. PEI8 6ES and R. F. HORSLEY DHSS, 12-14 Russell Square, London WCIB 5EP, England
Introduction
Results and Discussion
Dental restorative materials are conventionally evaluated in experimental cavities in vivo followed by histological examination of the pulp. This is time consuming and expensive, uses relatively large numbers of animals and gives variable results with different species. Various in vitro methods have therefore been developed, including the model cavity system developed by Tyas (1977) in Birmingham and later modified by Meryon & Browne (1983), who have tested a wide range of dental materials. The main advantages of this technique are the simulation of the in vivo situation with use of specimen size:medium ratios approximating those used clinically. Materials are tested freshly mixed, when they are most irritant. Contact with the underlying fluid is via a permeable filter, and there is an option for placing a layer of dentine powder beneath the test sample to increase simulation of the in vivo effect. A major criticism of in vitro methods is the lack of reproducibility between results obtained from different laboratories. The reproducibility of this method was therefore tested independently by Birmingham Dental School and Huntingdon Research Centre Ltd (HRC) using a precise experimental protocol drawn up following liaison between both centres.
In the initial study, both laboratories found similar reductions in fibroblast numbers (12.8-16.4%) in cultures treated with ChemFil, while Silicap was relatively non-toxic; ChemFil was moderately toxic to macrophages but Silicap was more toxic, destroying approximately 50% of each cell culture. The toxicity of both materials to either cell type was completely annulled in the presence of dentine. In the main study the toxicity ranking orders obtained for all the materials with each cell type were almost identical. Both laboratories noted a difference in the sensitivity of the two cell types to some test agents; Poly-F Plus was the most toxic to murine macrophages followed by zinc oxide--eugenol and zinc phosphate while AquaCem and Concise had little effect. Zinc oxide--eugenol was most toxic to fibroblasts (causing 60% cell death) followed by AquaCem (13-20% cell death) while Poly-F Plus and zinc phosphate showed 8-12% toxicity and Concise had no toxic effect. A high degree of reproducibility was obtained in the results from both laboratories. With BHK-21 (C13) fibroblasts the interlaboratory difference in results for all materials was 0.7-7.4%. With mouse macrophages, variation was greater (4.5-19.8%) and was probably due to natural heterogeneity in the peritoneal macrophage population. In a separate study, HRC found that the macrophage-like cell line P388-DI (from the MRC Pneumoconiosis Unit, Cardiff) was unaffected by Silicap or ChemFil and therefore could not be used to replace primary mouse macrophages.
Experimental
Silicap and ChemFil were tested in a preliminary study in the presence and absence of a layer of dentine powder compacted at the base of the test cavity. This was followed by the main study in which five materials (zinc oxide-eugenoi, AquaCem, Zinc phosphate, Poly-F Plus and Concise) were tested without dentine. All materials were tested in quadruplicate cultures on three occasions using BHK-21 (C13) fibroblasts and primary mouse macrophages obtained by peritoneal lavage. Following 24-hour exposure to the test material the cells were harvested and toxicity was assessed by comparing cell numbers with those in control cultures. 592
REFERENCES
Meryon S. D. & Browne R. M. (1983). Test methods for assessing the cytotoxicity of dental restorative materials using an in vitro model cavity system. In Ceramics in Surgery. Edited by P. Vincenzini. p. 122. Elsevier, Amsterdam. Tyas M. J. (1977). A method for the in vitro toxicity testing of dental materials. J. dent. Res. 56, 1285.
0278-6915/86 $3.00+0.00 Copyright © 1986 Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
REPRODUCIBILITY OF THE MODEL CAVITY METHOD FOR I N V I T R O TOXICITY TESTING OF DENTAL RESTORATIVE MATERIALS S. D. MERYON and R. M. BROWNE Department of Oral Pathology, Dental School, St Chads Queensway, Birmingham B4 6NN P. F. UPHILL and A. D. CORDERY Department of Mutagenesis and Cell Biology, Huntingdon Research Centre Ltd, Huntingdon, Cambs. PEI8 6ES and R. F. HORSLEY DHSS, 12-14 Russell Square, London WCIB 5EP, England
Introduction
Results and Discussion
Dental restorative materials are conventionally evaluated in experimental cavities in vivo followed by histological examination of the pulp. This is time consuming and expensive, uses relatively large numbers of animals and gives variable results with different species. Various in vitro methods have therefore been developed, including the model cavity system developed by Tyas (1977) in Birmingham and later modified by Meryon & Browne (1983), who have tested a wide range of dental materials. The main advantages of this technique are the simulation of the in vivo situation with use of specimen size:medium ratios approximating those used clinically. Materials are tested freshly mixed, when they are most irritant. Contact with the underlying fluid is via a permeable filter, and there is an option for placing a layer of dentine powder beneath the test sample to increase simulation of the in vivo effect. A major criticism of in vitro methods is the lack of reproducibility between results obtained from different laboratories. The reproducibility of this method was therefore tested independently by Birmingham Dental School and Huntingdon Research Centre Ltd (HRC) using a precise experimental protocol drawn up following liaison between both centres.
In the initial study, both laboratories found similar reductions in fibroblast numbers (12.8-16.4%) in cultures treated with ChemFil, while Silicap was relatively non-toxic; ChemFil was moderately toxic to macrophages but Silicap was more toxic, destroying approximately 50% of each cell culture. The toxicity of both materials to either cell type was completely annulled in the presence of dentine. In the main study the toxicity ranking orders obtained for all the materials with each cell type were almost identical. Both laboratories noted a difference in the sensitivity of the two cell types to some test agents; Poly-F Plus was the most toxic to murine macrophages followed by zinc oxide--eugenol and zinc phosphate while AquaCem and Concise had little effect. Zinc oxide--eugenol was most toxic to fibroblasts (causing 60% cell death) followed by AquaCem (13-20% cell death) while Poly-F Plus and zinc phosphate showed 8-12% toxicity and Concise had no toxic effect. A high degree of reproducibility was obtained in the results from both laboratories. With BHK-21 (C13) fibroblasts the interlaboratory difference in results for all materials was 0.7-7.4%. With mouse macrophages, variation was greater (4.5-19.8%) and was probably due to natural heterogeneity in the peritoneal macrophage population. In a separate study, HRC found that the macrophage-like cell line P388-DI (from the MRC Pneumoconiosis Unit, Cardiff) was unaffected by Silicap or ChemFil and therefore could not be used to replace primary mouse macrophages.
Experimental
Silicap and ChemFil were tested in a preliminary study in the presence and absence of a layer of dentine powder compacted at the base of the test cavity. This was followed by the main study in which five materials (zinc oxide-eugenoi, AquaCem, Zinc phosphate, Poly-F Plus and Concise) were tested without dentine. All materials were tested in quadruplicate cultures on three occasions using BHK-21 (C13) fibroblasts and primary mouse macrophages obtained by peritoneal lavage. Following 24-hour exposure to the test material the cells were harvested and toxicity was assessed by comparing cell numbers with those in control cultures. 592
REFERENCES
Meryon S. D. & Browne R. M. (1983). Test methods for assessing the cytotoxicity of dental restorative materials using an in vitro model cavity system. In Ceramics in Surgery. Edited by P. Vincenzini. p. 122. Elsevier, Amsterdam. Tyas M. J. (1977). A method for the in vitro toxicity testing of dental materials. J. dent. Res. 56, 1285.