374 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Nipasept was necessary to produce sterility after 1 hr contact in atropine drops, while 0.106% sterilized after 2 hr. In eserine drops 0.16% Nipasept sterilized after 3 hr, and 0.106% after 5 hr. Combination 2 was more effective than Nipasept in the presence of atropine and eserine, and 0.08% sterilized after 1 hr. The esters were effective in some cases at 0.16%, but nevertheless it was found that this strength caused some burning sensation when introduced on the eye. The value of their results is reduced because they used a broth dilution experimental procedure, and inactivation of the agents was uncertain. Nonionic detergents are generally known to inactivate phydroxybenzoic acid esters (28) and Kohn et al (24) have recommended Tween 20 as an antagonist of these esters. Lawrence (29) found that a mixture of 0.16% methylparaben and 0.02% propylparaben sterilized within 1 to 6 hr in every case with 26 strains of Ps.aeruginosa, and four species of Proteus in simple buffer at pH 6.2. He used undiluted 24 hr cultures as inocula. Activity was also tested against four strains of Ps.aeruginosa in the presence of non buffered aqueous solutions of several drugs. Sterility was achieved in less than 30 min in several cases and never after more than 6 hr. He did not use inactivating substances in the recovery broth for the parabens. Ridley (16) stated that 0.1% methyl phydroxybenzoate had been used successfully as a preservative in hospital practice for three years. He stated that 0.1% methyl phydroxybenzoate was" . . . effective against a wide range of bacteria tested, including Ps.pyocyanea, Streptococcus pyogenes, and Staphylococcus aureus at room temperature, in conjunction with the drugs commonly used and in normal clinical concentrations, in three hours." No experimental evidence was provided to support this statement and the definition of "effective" was not given. Kohn et al (24) found that a mixture of 0.2% methyl paraben and 0.04% propyl paraben sterilized Ps.aeruginosa suspensions (2 x 10ø/ml) in 3 hr. A mixture of 0.18% propyl paraben sterilized in 6 hr. These workers used 13 strains of Ps.aeruginosa, and the effectiveness of the inactivating agents in their recovery broth was established by a correlation between in vivo and in vitro results. Anderson et al (30) tested 0.1% methyl hydroxybenzoate in six final eye drop preparations using Staphylococcus pyogenes, Proteus vulgaris and Ps.aeruginosa in concentrations of about 5 • 10a/ml. They found that sterility was achieved within one day in every case except two. The viable count increased during 11 days with Ps.aeruginosa in gelatin eye drops, and sterility was achieved after two days but not after one day

THE PRESERVATION OF OPHTHALMIC PREPARATIONS 375 with Pr.vulgaris in glycerine eye lotion. More useful information might have been obtained if tests had been made at intervals less than daily. Hugo and Foster (9) have shown that the rate of growth of one strain of Ps.aeruginosa was little affected by the presence of the concentration of esters used in Solution for Eye Drops B.P.C. (0.034%), and that they could be used as a sole carbon source at the same concentration. These workers, [Foster (31), Hugo and Foster (32)], found that a mixture of two parts methyl and one part propyl phydroxybenzoate sterilized aqueous suspensions containing 10 and 100/ml Ps.aeruginosa cells of one strain in 30 minutes at 18 ø using concentrations of 0.125% and 0.2% respectively. They did not use an inactivator in the recovery medium. Chlorocresol pChloro-m-cresol was the first official ophthalmic antibacterial preser- vative used in Britain. Its present replacement by Solution for Eye Drops in the B.P.C. has been discussed (4). Davis (33) found that 0.05% chlorocresol was as effective against $taph.aureus as was 0.5% phenol. In each case, sterility was achieved within one day. Details of reaction menstrum and recovery conditions were not given. Klein et al (27) used a broth dilution procedure and found that 0.1% chlorocresol sterilized broth suspensions (108/ml) of three strains of Ps.aeruginosa. They also tested 0.1% and 0.03% chlorocresol in the presence of three eye drop preparations. In each case, sterility was achieved within 1 hr using 0.1%. 0.03% chlorocresol sterilized atropine drops within 4 hr, while fluorescein and eserine were sterilized between 6 and 24 hr. They found that 0.1% chlorocresol caused smarting. 0.05% chlorocresol sterilized inocula of 10/ml Ps.aeruginosa cells of one strain in aqueous suspensions at 18 ø within 30 min, but 0.07% was required to sterilize 100 cells/ml within the same time (32). Crompton and Anderson (34) have reported that injections of 0.05% ½hlorocresol in normal saline into the anterior chamber of rabbits' eyes caused the cornea to become opaque. This disadvantage would seem not to apply in ophthalmic solutions preserved and used with intact eyes. Richards (35) reported that chlorocresol was more active in terms of reduction of viable count in final eye drop preparations at acid pH, than it was in simple solution.