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

276 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS amount was adequate for the beneficial properties previously reported by the Danish researchers. Tissue binding and slow release probably accounted for the sustained antibacterial action as well as the therapeutic effect in gingivitis, according to Davies, Borglum Jensen, Rindom Schiott and Loe (52). A histological study of the oral mucosa of hamsters conducted by Lindhe, Heyden, Svanberg, Loe and Rindom Schiott (53) led to the observa- tion that chlorhexidine did not penetrate the undamaged oral epithelium, from which it may be concluded that the whole of the adsorbed chlorhexi- dine from a mouthwash or toothpaste is made slowly available in the oral cavity. In this field as with other applications in toiletries there are many facets of this bactericide which it may be advantageous to explore more closely. (Received: 21st January 1972) REFERENCES (17) (18) (19) (20) (1) Rose, F. L. and Swain, G. Bisdiguanidines having antibacterial activity. J. Chem. $oc. 4422 (1956). (2) Davies, G. E., Francis, J., Martin, A. R., Rose, F. L. and Swain, G. Laboratory investiga- tion of a new antibacterial agent of high potency. Brit. J. Pharmacol. 9 192 (1954). (3) Holbrook, A. The determination of small quantities of chlorhexidine in pharmaceutica preparations. J. Pharm. PharmacoL 10 370 (1958). (4) Senior, N. ICI Pharmaceuticals Division. Unpublished work. (5) Brit. Pat. 815 925. (6) Brit. Pat. 1 108 352. (7) Brit. Pat. 815 800. (8) Clark, A.M., Letter to Lancet ii 661 (1971). (9) Barnes, M. (Miss). ICI Pharmaceuticals Division. Unpublished work. (10) Hall, R. ICI Pharmaceuticals Division. Unpublished work. (11) Hugo, W. B. and Longworth, A. R. Effect of chlorhexidine acetate on protoplasts and speroplasts of Escherichia coli, protoplasts of Bacillus rnegateriurn and the Gram staining reaction of $taphylococcus aureus. J. Pharm. Pharmacol. 16 751 (1964). (12) Wiseman, D. The effect of chlorhexidine on the permeability and succinoxidase activity of Micrococcus lysodeckticus. J. Pharm. PharmacoL 16 Suppl. 5Gr (1964). (13) Brown, M. R. W. and Richards, R. M. E. Effect of Polysorbate (Tween) 80 on the re- sistance of Pseudornonas aeruginosa to chemical inactivation. J. Pharrn. PharmacoL 16 Suppl. 51T (1964). (14) Heard, D. D. ICI Pharmaceuticals Division. Unpublished work. (15) Becher, P. Effect of solvent on micellar properties. J. Colloid $ci. 20 728 (1965). (16) Shepherd, R. C. and Kinmonth, J. B. Skin preparation and towelling in prevention of wound infection. Brit. Med. J. 2 151 (1962). Calman, R. M. and Murray, J. Antiseptics in midwifery. Brit. Med. J. 2 200 (1956). Myers, C. E., McKenzie, W. C. and Ward, K. A. Control of cross-infection by means of an antiseptic hand cream. Can. J. MicrobioL 2 87 (1965}. Annotation. Lancet ii 1164 (1958). Grant J C. and Findlay, J. Local treatment of bums and scalds using chlorhexidine. Lancet i 862 (1957).