THE PRESERVATION OF OPHTHALMIC PREPARATIONS 379 of 0.05% benzalkonium was required to sterilize broth suspensions (108/ml) of three strains of Ps.aeruginosa. Subcultures were taken after two days. These workers found that although 0.5% cetrimide sterilized, there was one instance when cetrimide was shown to have comparatively little effect against Ps.aeruginosa. Benzalkonium chloride was found not to be a uniformly effective bactericide against Ps.aeruginosa. Five out of 30 strains were not sterilized by contact for 24 hours with 0.01% of the agent, at pH 7.4 (36). Lawrence (39) in his review concluded that benzalkonium chloride, chlorbutanol and P.M.N. were the most suitable agents on the basis of published evidence. The same worker (29) tested benzalkonium chloride 0.02% and 0.01% against 26 strains of Ps.aeruginosa, and four species of Proteus using inocula of undiluted 24 hr broth cultures. Tests were made in (1) simple buffer, (2) in the presence of several ophthalmic drugs in buffer, and (3) in the presence of several drugs in aqueous solution. In every instance sterility was achieved in less than 30 min with 0.02% benzalkonium chloride. The majority of strains were sterilized by 0.01% in less than 30 min in all three systems but a few strains needed up to 6 hr. Chemical inactivators were used in the recovery broth. These results conflicted with those of Riegelman et al (11) who found that benzalkonium chloride was only slowly bactericidal. They suggested that the discrepancy between their results, and those of earlier workers, was because of inadequate inactivation of benzalkonium chloride in the recovery broth used by other workers. They found that 0.01% benzal- konium chloride failed to sterilize 108/ml Ps.aeruginosa cells in saline in one week when tested by in vivo methods whereas the use of nutrient broth subculture indicated sterility after five minutes contact. The use of a lecithin-Tween 80-thioglycolate medium resulted in concordant in vivo and in vitro tests. Riegelman et al (11) reported incidents when resistance to benzalkonium chloride by Ps.aeruginosa was demonstrated. In one case, active growth occurred and pigment was produced in the presence of un-neutralized 0.02% benzalkonium chloride from an inoculum of 1 ml of a 24 hr culture. Kohn et al (24) found that benzalkonium chloride 0.02% sterilized 13 strains of Ps.aeruginosa (2 x 10ø/ml) in aqueous suspension within 45 min, and that 0.01% required up to 9 hr. The effectiveness of the inactivators used in the in vitro recovery media was demonstrated by correlation with in vivo results. These workers found that of seven commonly used pre- servatives, only 0.02% benzalkonium chloride sterilized in less than 1 hr.

380 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Inocula of 10 and 100/ml Ps.aeruginosa cells were sterilized in aqueous suspension at 18 ø in 30 min by benzalkoniurn chloride 0.05% and 0.08% respectively (32). Recovery was on media containing an inactivator. Benzalkonium chloride has been accepted officially in the U.S.A. for many years but has not been widely used in Britain despite the large amount of favourable published work. There have been criticism against benzalkonium chloride on grounds other than bacteriological. Klein et al (27) stated "Quaternary ammonium compounds as a preservative for eye-drops should be used only in exceptional cases. Ginsburg and Robson found that detergents could prove harmful by causing 'solubilization' of the intercellular cement of the corneal epithelium." This reference to the work of Ginsburg and Robson (40) has been quoted by others (41). Recently, Anderson et al (30) have also quoted this work as evidence that benzal- konium chloride dissolves the intercellular cement substance of the cornea. Ginsburg and Robson (40) did not use a quaternary ammonium compound but worked with anionic and nonionic wetting agents. These workers found that the anionic detergent, dodecyl sodium sulphate (1%) damaged the cornea. They also found that although 1% nonionic Lissapol N caused slight temporary oedema, 0.5% caused no observable effect. These results are in accord with those of Buschke (42) who made direct measure- ments on intercellular cohesion in corneal epithelium. He found that six out of seven anionic detergents" . . . produced marked effects in con- centrations of 1% or sometimes even in lower concentrations." Not one of a wide selection of nonionic or cationic detergents caused loss of inter- cellular cohesion. Benzalkonium chloride was used in concentrations up to 10%. Recently, Anderson et al (43) have stated" . . . Swan has shown that wetting agents such as benzalkonium chloride are injurious to the corneal epithelium and especially to the endothelium .... " Swan (44) found that intraocular concentrations of benzalkonium chloride in excess of 0.01ø/0 caused serious damage. He also found that instillation of 0.1% benzal- konium chloride produced a severe reaction, and that 0.03% to 0.04% used over periods of up to 8 weeks produced a less severe reaction. In most cases the conjunctiva and cornea were normal within 12 hr. Never- theless, Swan concluded, "The minimal concentrations of wetting agents producing irritation in the conjunctival sac are greater than those that are effective." Hughson and Styron (45) replaced the aqueous humour of rabbits' eyes with benzalkonium chloride in saline, and found that concentrations of