274 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS U.K. A further possibility resulting from the suppression of oral micro flora is to prevent the onset of dental caries, with the fine ideal of lifelong reten- tion of the natural teeth. The mechanism of dental decay is not fully elucidated but much is known about it and the widely accepted theory is that the plaque which develops on the less accessible 'stagnation sites' harbours bacteria which degrade the residual sugar to organic acids. These in turn attack the enamel (essentially hydroxyapatite Caxo(PO4)6(OH)2), leaching out the calcium, diminishing the degree of hardness and permitting the ingress of cariogenic bacteria. Plaque therefore plays a decisive role in the initiation and development of caries and marginal periodontal disease. As long ago as 1953, Fosdick (41) postulated that anticaries agents (whether fluorides or antibacterials) were deposited on the plaque, where they were retained. It now seems that recent data are in support of these two essential requirements. Gjermo, Baastad and Rolla (42) compared the plaque inhibiting effect in vivo of eleven antibacterial agents (alcohols, iodophores, dyes, quater- naries, amidines and guanidines) with their bacterial activity against salivary bacteria in four in vitro systems to find that there was no connection between the results. Chlorhexidine gluconate and acetate were the most effective in vivo whereas others, more effective against bacteria in vitro, exhibited no effect in vivo. This led to the conclusion that other factors apart from anti- bacterial action were involved, and was in accord with the previous findings of Loe and Rindom Schiott and their colleagues at the Royal Dental College, Denmark, who regarded chlorhexidine as the most promising anticaries agent. Their choice was based upon its broad effects against bacteria and yeasts, low toxicity and widespread use in clinical medicine (43). A twice daily rinse with aqueous 0.2•o chlorhexidine gluconate in a 3-4 week study was shown to be effective in preventing plaque and calculus formation (45) yet they also found that, although the oral flora was markedly reduced (up to 90•o), large numbers of bacteria still persisted, from which it appeared unlikely that the inhibition of plaque formation was primarily the result of a reduction of salivary flora (45, 46). In some of these tests the mouth was rinsed with strong sucrose solution to provoke plaque forma- tion yet this did not diminish the efficacy of chlorhexidine. In a further study these workers (47) concluded that chlorhexidine gluconate was ad- sorbed on tooth surfaces and salivary mucins, from which it was slowly released when the environmental concentration was low. More recently it was shown that chlorhexidine adsorbed onto the plaque deposits within the space of a few seconds. When an extracted tooth,

FORMULATION AND PROPERTIES OF CHLORHEXIDINE 275 either fleshly drawn or stored in saline, was immersed (15-60 s) in a 0.2• aqueous solution of the gluconate, thoroughly rinsed in distilled water and finally placed in alkaline hypobromite solution, the plaque turned a reddish- brown colour, denoting the presence of adsorbed chlorhexidine (48). No colouration was discernible on the enamel, probably because the method was insensitive to the minute amount adherent as a surface film, the pre- sence of which was best shown by a bacteriological method in which the treated tooth was laid on inoculated agar and then incubated (9) to give a zone of inhibition. The antibacterial was chemically detectable on plaque even after one day's immersion of a treated tooth in a great excess of distilled water. Thus, it was evident that chlorhexidine was readily adsorbed on to plaque and moreover strongly adhered to it, both factors being highly conducive to the elimination of bacteria dwelling within this deposit. One disadvantage of oral hygiene with aqueous chlorhexidine (that is, the regimen of Loe and Rindom Schiott referred to above (44) to the ex- clusion of conventional tooth brushing with dental cream) was the develop- ment of brown stains on teeth with some, but not all, users. It was observed in the less readily accessible areas of the teeth where plaque was found, but not on polished surfaces. There was good evidence that the cause was a chemical binding between the chlorhexidine adsorbed on the plaque and beverage tannins (48). It was quite distinct from the brown pellicle known to appear when abrasive cleansers are not used (49, 50). Gjermo and Rolla (51) showed that the antibacterial activity of chlorhexidine toothpaste might not be adversely affected by the presence of toothpaste adjuvants and from this finding, together with the above data, it is reasonable to conclude that a dental cream is preferable to a mouthwash both for convenience and minimal teeth staining. A quantitative study of the retention of chlorhexidine in the oral cavity has revealed some interesting data. Thus, it was found by the analysis of ejected 0.2•o w/v chlorhexidine gluconate mouthwash, previously stan- dardized and accurately measured, that about 34•o of the 20 mg chlor- hexidine intake (average of six tests) was left in the mouth and about 27• was retained even after repeated rinsing to remove the unadsorbed residue. The extent of adsorption was barely affected by the presence or absence of natural or artificial teeth (48) and it was therefore concluded that a major fraction of the antibacterial was retained on the adherent salivary mucins thereby serving as a reservoir for subsequent transportion to the teeth via the saliva. Only a minor and not readily determinable fraction was im- mediately deposited on plaque and tooth enamel though even this small