AMMONIATED DENTIFRICES 69 joys causes the acidogenic flora to be dominant in most individuals. The proteolytic types usually pres- ent in the mouth are suppressed. If agents placed in the oral cavity could depress the acid-producing types without interfering with the proteolytic ones, and if this action is repeated frequently, it may be pos- sible to rearrange the bacterial popu- lation so that the proteolytic types predominate. Theputrefactive types are antagonistic to decalcification which we believe to be the principal phase of tooth destruction and in this way they provide a natural means forcombating tooth decay. If a dentifrice tends to shift the bac- terial population in the direction of the proteolytic types, it is favoring, or perhaps simulating, the natural anticaries mechanism. With this reasoning as our back- ground we have undertaken clinical studies to determine the correctness of our theory. To begin with, various ammonium salts and dif- ferent concentrations of them were studied and we found from these in vitro experiments that a combina- tion of 5 per cent dibasic ammonium phosphate and 3 per cent urea gave optimal results in checking aciduric bacterial growth and the degrada- tion of glucose. We found that concentrations of urea over 5 per cent begin to interfere with the de- aminating systems that are present in the oral cavity and thus interfere with the natural production of am- monia. Previous experiments had es- tablished the presence of six amino acid deaminating systems in human saliva. There are probably more. Two of them were definitely deter- mined to be of bacterial origin. These are the systems that we hope to reactivate and support. Quanti- tative determinations of amino acids in saliva reveal that they are usually present in higher amounts in the caries susceptible than the caries resistant. An explanation of this observation may be that the de- aminating systems are depressed in the susceptible consequently the amino acids are not broken down and remain to supply essential growth factors for aciduric bacterial proliferation. Others have noted the effect that various concentrations of urea have on bacteria. As early as 1906, W. J. Wilson reported that urea in cul- ture medium inhibited the growth of such organisms as B. co/i, B. typhosis, and B. enteritides (3). If the medium contained 0.5, 1, 2, or 3% urea, B. coli was as active as it was in medium containing no urea. At 4 and 5 per cent concentrations a fair growth was obtained, at 7 per cent there was little growth and at 8 per cent practically none. In bouil- lon medium the percentage neces- sary to inhibit growth appeared to be even less. These observations have been confirmed by others. Therefore, the dentifrice that we have been studying clinically has consistently contained 5 per cent di- ammonium phosphate and 3 per cent urea. We realize that salivary analyses made at intervals following the use

70 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS of a dentifrice or mouth rinse con- taining 5 per cent dibasic ammo- nium phosphate and 3 per cent urea may show a rather rapid dilution or reduction of these agents in the saliva that has been expectorated but such analyses detect the amount and reaction in the free saliva and are not an indication of what has been absorbed into and remains ac- tive in the bacterial plaques on the tooth surfaces. It is in these so- called areas of stagnation in the more or less inaccessible areas that decay takes place. The di-ammo- nium phosphate and urea should be worked into these areas during the brushing and rinsing process so that they are absorbed by the bacterial film which acts perhaps as a blotter. Here they may remain for prolonged periods without salivary dilution. There is a well-established thera- peutic principle that only the smal- lest quantity of a drug should be used that is needed to obtain a de- sired result. That is why we have consistently maintained a level of 5 per cent dibasic ammonium phos- phate and 3 per cent urea in our for- mula. We believe that only care- fully controlled, long-term clinical studies can establish the correctness or incorrectness of our rationale. A group of University personnel and clinical patients who appeared to be caries active and who had salivary Lactobacilli counts of 10,000 or higher per ml. of saliva were selected for the original study. They were instructed to use the dentifrice after each meal and before retiring. A rinse containing dibasic ammo- nium phosphate and urea in a con- centration of 5 and 3 per cent was provided for those who wished to rinse their mouths after brushing with the powder. Within two to three weeks some showed a notice- able drop in their Lactobacilli counts. With continued brushing about one- third of the subjects had their counts go down to zero, and while the rest showed sizable reductions, their counts were still within the range that indicated caries activity. At the end of a two-year period, we saw what appeared to be a clinical reduction in caries activity, but we could not be satisfied with the results because the number of subjects who concluded the two-year study was less than 100 and they were not adequately controlled. Dental caries is such a prevalent dis- ease that to make the results sta- tistically significant, we were in- formed that at least 200 and pref- erably 300 subjects should be in the group at the conclusion of the study period. In addition, there must be a comparable number of controls if the conclusions are to be valid. A time interval of two years should elapse to make the results signifi- cant because caries runs an inter- mittent course and short-term ob- servations can give misleading con- clusions. Subjects who will volun- tarily and conscientiously co-oper- ate in a toothbrushing program are usually people who already have had considerable caries experience. These individuals are not good sub- jects for a caries control study be- cause the more tooth decay they