GRAS ANTIMICROBIAL AGENTS 5 Table III Effect of EDTA on MIC of Lauricidin 0 500 1,000 5,000 10,000 EDTA Organism ug/ml ug/ml ug/ml ug/ml ug/ml Alone Escherichia coli NI NI NI NI N! 10,000 Pseudomonas aeruginosa NI NI N! 100 10 5,000 $treptococcus faecalis (Group D) NI NI 1,000 10 10 5,000 $traphylococcus aureus 1,000 500 100 10 10 5,000 $treptococcus pyogenes 10 10 10 10 10 1,000 Corynebacterium sp. 10 10 10 10 10 1,000 Nocardia asteroides 10 10 10 10 10 500 $accharomyces cerevisiae 1,000 100 10 10 10 1,000 Candida albicans NI 100 100 100 100 5,000 Experiment was carried out at pH 7.2. MIC values for Lauricidin were determined with the various indicated concentrations of ethylene dramine-tetraacetic acid (EDTA), i.e. 0.5% EDTA plus 0.01% Lauricidin will prevent the growth of Pseudomonas aeruginosa. of Lauricidin was determined from ten-fold dilutions containing various concentra- tions of EDTA. Concentrations of the chelator from 125 ppm to 10,000 ppm on Lauricidin activity were examined. The most marked changes in MIC values of Lauricidin occurred between 500 and 5000 ppm of EDTA. The most significant decrease in MIC value was measured for Psuedomonas aeruginosa. Significant activation of Lauricidin by EDTA against yeast was also demonstrated. COMBINATION STUDIES WITH FOOD-GRADE MATERIALS During our study with these individual food-grade preservatives it became obvious that each preservative had a limited spectrum of activity against microorganisms. While Lauricidin exhibited high activity against gram (+), yeast and fungal organisms, it had little if any activity against gram (-) strains tested. BHA had a somewhat greater range of activity including gram (--) strains but was also limited to similar organism. This phenolic derivative had higher MIC values for gram (+) bacteria and lower MIC values for yeasts. Of greater interest was the slight activity against E. coli (500 ppm). While EDTA had overall low antibacterial and fungal activity, it did show activity against P. aeruginosa. It should be noted that the germicidal action of the combination in aqueous suspension was slow (Table IV). However, the combination acted more quickly when Table IV Effect of O/W Lauricidin Emulsion on Staphylococcus Aureus Concentration (%) Lauricidin BHA EDTA Titer I 0.0 0.0 0.0 2.3 X 10 6 1.0 0.0 0.0 1.0 x 106 1.0 0.1 0.0 3.6 x 105 1.0 0.1 1.0 1.7 X 10 5 1.0 0.5 0.0 No viable organism 1.0 0.5 1.0 No viable organism •Reading made after 1 hr contact.

6 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table V Effect of a Lauricidin Alcoholic Dispersion on Staphylococcus Aureus Concentration (%) Contact Time Lauricidin BHA EDTA IPA Titer 5 minutes .... 2.8 X 10 6 -- -- -- 30 2.3 X 10 6 10.0 1.0 1,0 -- 4.5 x 105 10.0 1.0 1.0 30 7.7 X 10 4 15 minutes .... 2.7 X 10 6 -- -- -- 30 2,2 X 10 6 10 1.0 1.0 -- 4.5 X 10 3 10 1.0 1.0 30 No viable organisms the emulsion was dispersed with isopropyl alcohol. The data in Table V indicate that a 2 1og•0 reduction in Staph aereus occurs in 5 min and a 6 log•0 reduction in 15 min. Other stable cosmetic dispersions of Lauricidin have been shown to be equally or more effective (unpublished data MedChem Laboratories). Because of the high activity against Streptococcus mutans, it was of interest to study the effect of Lauricidin and BHA in several oral hygiene products. The products were commercial preparations in which 0.5% Lauricidin and 0.01% BHA were added. The level of ingredients were not listed. In every case the germicidal property of the product was materially improved (Table VI). Table VI Effect of 0.5% Lauricidin and 0.01% BHA Added to Several Commercial Mouthwashes Exposure Time Red 2 Blue 3 Green 4 i i i Control Control Control 15 min. 1.4 X 10 6 5.3 x 105 5.7 x 105 1.3 x 105 1.8 x 105 7.5 X 10 4 30 min, 1.3 X 10 6 2.9 x 105 3.0 x 105 8.0 x 104 5.8 x 104 1.3 x 104 60 min. 1.3 X 10 6 8.6 X 10 4 1.2 x 105 2.1 X 10 4 6.1 X 10 3 5.0 X 10 2 120 min. 1.4 X 10 6 8.0 X 10 • 1.9 X 10 4 1.3 x 103 2.0 X 10 2 No viable organisms Information on mouthwash labels was not complete. •0.5% Lauricidin and 0.01% BHA. 2Red Mouthwash: 5% alcohol, zinc chloride, menthol and other aromatics. 3Blue Mouthwash: 15% alcohol, cetylpyridinium chloride, oil of peppermint, boric acid, menthol. 4Green Mouthwash: 18.5% alcohol, cetylpyridenium chloride. Two popular cosmetic preservatives propyl parabens and sorbic acid, were compared with Lauribic (Table VII). The latter was a food-grade proprietory preservative of Med-Chem Laboratories. Lauribic was usually more active against more organisms than parabens. Both products were much more active than sorbic acid itself. In terms of animal toxicity Lauricidin is approximately 10x less toxic than either propyl parabens or sorbic acid. In fact animals fed high doses of the monoglyceride (50 g/kg) were not killed.