Comparison of the cytotoxicity of formocresol, formaldehyde, cresol, and glutaraldehyde using human pulp fibroblast cultures.

Serial dilution and agar overlay techniques were used to compare the cytotoxicity of formocresol, 19% formaldehyde, 35% cresol, and 2.5% glutaraldehyde to human pulp fibroblasts. The maximum nontoxic concentration of each agent was determined to allow quantitative comparisons both of the agents tested and of the techniques used. Formaldehyde was found to be the major component of formocresol which is responsible for the cytotoxic effect on human pulp fibroblasts. Two and one-half per cent glutaraldehyde was 15-20 times less toxic than either formocresol or 19% formaldehyde. Cresol measured 40 times less toxic than formaldehyde or formocresol. Both serial dilution and agar overlay techniques appear to be sensitive and effective methods for testing the toxicity of diffusible agents. Despite the clinical success of pulpotomies performed using formocresol as the active agent, several authors have raised questions about continued use of the drug. These questions are supported by reports of systemic spread of formaldehyde after pulpotomy (Myers et al. 1978), adverse effects on the enamel succedaneous teeth (Pruhs et al. 1977), and the mutagenic and carcinogenic potential of formocresol in animals (Muller et al. 1978). Morawa et al. (1975) have recommended a 1:5 dilution of formocresol with glycerine and water for pulpotomies. This recommendation is based on the study by Loos et al. (1973) which showed that formocresol in full concentration, while effective in developing cytostasis, may produce irrecoverable damage to connective tissue. On the other hand, a 1:5 dilution of formocresol, which creates metabolic effects similar to those achieved by. full strength formocresol, produces an earlier recovery of cellular respiratory enzyme activities in connective tissues (Loos et al. 1973) s’-Gravenmade (1975) proposed that glutaraldehyde could be used as a new pulpal fixative in dentistry. Glutaraldehyde has been considered a possible substitute for formocresol because: (1) it is a more active fixative agent, cross linking proteins by virtue of its two active sites (Russell 1976); (2) its tissue penetration limited (Tagger et al. 1986); and (3) the zone of infiltration is more restricted following application to exposed pulps (Davis et al. 1982). Although one investigator reported favorable clinical results with glutaraldehyde (Garcia-Godoy 1983), little information is available concerning tissue response to glutaraldehyde as a pulpotomy agent. Very few toxicity studies of glutaraldehyde have been conducted. Seow and Thong (1986) examined the effects of pulpotomy medicaments (glutaraldehyde, formocresol) on polymorphonuclear leukocyte (PMN) adherence, because they considered that the persistent activation of PMNs by pulpotomy medicaments may contribute to the chronic inflammatory changes and root resorption seen in histologic sections. The results showed that formocresol caused lysis of PMNs at high concentrations, but activation of PMN adherence at low concentration. By contrast, glutaraldehyde did not produce PMN lysis at high concentrations, nor did it cause activation of PMN adherence at low concentration. Clearly, the toxicity of formocresol has been investigated very intensively and the potentially harmful effects to humans have been discussed by others. Very few studies of the toxicity of glutaraldehyde have been done, and none of them were cytotoxicity studies. Toxicity testing using cells in culture is an efficient and effective method to evaluate cellular effects of agents. Both glutaraldehyde and formaldehyde are diffusible substances, and so are suitable for testing by serial dilution and agar overlay techniques on cells in vitro. The authors of this study chose human pulp fibroblasts as the test cells, because they are derived from the tissue which would be in contact with a pulpotomy agent. This study compared the cytotoxicity of formocresol, each of the formocresol constituents, and glutaraldehyde. An attempt at improved quantitation of toxicity was made in this study by measuring the maximum PEDIATRIC DENTISTRY: DECEMBER 1987/VoL. 9 NO. 4 295 concentration achieved at any point in an agar overlay diffusion system and relating this to the damage observed in the cells. A third component of the study was to compare the sensitivity of the serial dilution technique and the agar overlay technique. Materials and Methods