INTERFERENCE OF EMULSIFIERS WITH PRESERVATIVES VIII 83 medium containing G-3720 or Tween 80 and methyl p-hydroxybenzoate showed no growth after six months using the four bacteria and z/. niger. In view of the effect of hexylene glycol previously mentioned, a series of tubes was set up using various concentrations of the glycol with G-3720 and methyl p-hydroxybenzoate. The results showed that 4 per cent hexylene glycol is as effective as 8 per cent propylene glycol in preventing growth of the four bacteria and z/. niger for four months. Hexylene glycol as the sole preservative failed to prevent growth of both bacteria and z/. niger in 2, 3 and 4 per cent concentrations. In a concentration of 5 per cent the Pseudomonas and Proteus bacteria were still able to grow as did the •spergi//us. These results are at variance with those mentioned earlier (1). Concentrations of glycerol and sorbitol ranging from 2 to 12 per cent failed to prevent inactivation of methyl p-hydroxybenzoate by G-3720. While growth periods varied, all samples grew actively within two months. In screening 1,4-butanediol, dipropylene glycol and 1,3-propanediol, arbitrary concentrations of 5 and 10 per cent were added to the appropriate medium containing methyl p-hydroxybenzoate and G-3720. In all three cases the 5 per cent concentration failed to prevent growth of only Pseu- domonas aeruginosa, but a 10 per cent concentration of either glycol inhibited the growth of all the organisms. Further tests are necessary to find the more exact concentration of glycol that will prevent growth of these microiSrganisms in a nonionic medium containing 0.2 per cent methyl p-hydroxybenzoate. G-3720, % 0.1 0.2 0.4 0.6 08 10 20 4O 6O 100 Ratio Growth Months G-3720: MP* + 10 1/2:1 + 13 1:1 + 9 2:1 -1--1--1- + 2 3:1 ++ 2 4:1 ++++ 2 5:1 ++++ 4 10:1 +++ 11/2 20:1 +++ 11/2 30:1 ++ ll/= 50:1 * Methyl p-hydroxybenzoate. Figure 1.--Growth z/. niger in Jaag Medium with G-3720 and 0.2% MP EFFECT OF RATIO OF NONIONIC TO PRESERVATIVE Pisano and Kostenbauder (3) have found that 5 per cent Tween 80 in water bound 78 per cent of the methyl and 90 per cent of propyl p- hydroxybenzoates, using a dialysis technique. Somewhat later it was found that these figures are increased by bacteriological media.

84 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Our tests, using G-3720, in various ratios and a uniform 0.2 per cent concentration of methyl p-hydroxybenzoate in Jaag medium showed growth at all concentrations and ratios of the nonionic to the preservative the heaviest growth occurring at 0.6, 1.0, 2, 4.0 and 6 per cent of the nonionic. Heavy growth is first noticeable when the ratio of nonionic to preservative is3:l. However, using 2 per cent Tween 80 and concentrations of methyl p-hydroxybenzoate from 0.2 per cent to 0.5 per cent, an oil (complex?) eventually separated from the medium when the concentration of methyl p-hydroxybenzoate reached 0.3 per cent. In a similar experiment using Myrj 49, no oil or crystalline separation took place, but it required 0.5 per cent of methyl p-hydroxybenzoate to prevent growth of •/. niger in Jaag medium. In a similar series it required 0.4 per cent of methyl p-hydroxybenzoate to preserve 2 per cent G-3720 against .4. niger. While at 1 per cent of methyl p-hydroxybenzoate and 2 per cent G-3720, a crystalline compound settled out. Igepal Co Growth Moles ETO Growth Months 210 q- q- 1.5 Carbopo1934 d- q- 6 430 q- q- 4 Cellosize - 8 630 d- d- d- d- 10 Tragacanth d- d- d- 5 730 + 15 PVP K-30 + + 5 850 d- d- d- d- 20 Methocel 1500 d- d- 2 880 + + 30 Figure 3.--Growth of .4. niger in Jaag Figure 2--Eleven Month Growth .4. niger Medium Plus 0.2% MP* Plus 2% Gum Jaag Medium, 0.2% MP* and 2% Igepal * MP = Methyl p-hydroxybenzoate. ß MP = Methyl p-hydroxybenzoate. ESZYMIe ACTIVITY We have found that polyoxyethylene ethers of the type of G-3720, particularly in the presence of dehydroacetic acid are apparently not only split by a possible "etherase" but that the prod.uct of hydrolysis is either selectively split from the nonionic liberating lower molecular weight alcohols, from the mixture comprising commercial stearyl alcohol or that the stearyl alcohol is somehow degraded. This splitting is also found occasionally in mixtures preserved with sorbic acid. A similar splitting has been observed in mixtures of sodium alkyl sulfate with G-3720, Tween 60, Myrj 49 and ethoxylated glyceryl monostearate. Oddly enough, no mold growth has been found in any of these tests. It is obvious that in terms of a commercial emulsion, any splitting of the emulsifier by microbial esterases could lead to a separation of the emulsion phases.