COSMETIC PRESERVATION 211 with more than its equivalent amount of biocide, leaving insufficient biocide to react with subsequently added microbes in the challenge (120). The value in such a test may be for multi-use products. However, for a more predictive test of consumer contami- nation potential, one should lower the challenge inocula to levels likely to be encoun- tered during use. Once done, the capacity test may be a quantitative and valid way to understand a product's ability to handle contamination from use. PRESERVATIVES AVAILABLE FOR USE MODE OF ACTION OF PRESERVATIVES The mode of action of antibiotics is known at the molecular level since they act via specific biochemical reactions. In contrast, the modes of action of preservatives and biocides are far more generalized,with numerous points of attack. Nearly all biocides work by denaturing cellular proteins or by affecting membrane permeability so that either transport or energy generation is blocked. For example, chlorine oxidizes reduced sites of organic compounds, including proteins, throughout the bacterial cell. Protein denaturants also include formaldehyde, formaldehyde releasers, isothiazolinones, and bromine compounds. The parabens and weak acids (e.g., sorbic, benzoic, and dehydroacetic acids) disrupt control of membrane electrical potential to block energy generation and nutrient trans- port (121). Thus the parabens apparently inhibit nutrient uptake by shutting down permeases, disrupting porin channels, or by disrupting the membrane pH gradient or electrical charge potential across the membrane to prevent substrate transport and ATP generation. This inhibition is apparently reversible and is consistent with other obser- vations that the mode of action of parabens is by disruption of the membrane electrical potential (122). Organic acids probably work in the same fashion (123) however, they may even be enzyme inhibitors as well (124,125). Typically, they are only biocidal at pH values below their pK•. In this protonated form, they pass through the membrane, and the hydrogen ion dissociates from the weak acid to decrease the cytoplasmic pH. As a result, both substrate transport and oxidative phosphorylation are uncoupled from the electron transport system. This effectively starves the cell of needed substrate and energy derived from ATP synthetase driven by hydrogen ions. Phenolics disrupt the proton motive force of the cell membrane (126,127). They also have the ability to non-specifically denature cytoplasm, cell walls, and cell membranes (128). The more lipophilic phenolics have the greater antibacterial capacity perhaps because of a greater ability to partition out of the water phase and into the lipid membrane (129,130). Alcohols likewise disrupt the membrane, causing permeability loss (131), and they also appear to inhibit enzymes (132). Perhaps some of the most widely used of the newer preservatives are the isothiazolinones. These are usually compounded into a single product composed of chloromethyl- isothiazolinone and methyl-isothiazolinone, but they can also include benzyl-type com- pounds (133,134). The isothiazolinones inhibit glucose oxidation and active transport without significantly affecting membrane integrity (135). In fact, these compounds denature enzymes and other proteins containing thiol groups (e.g., ATPase, glyceral-

212 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS dehyde-3-phosphate dehydrogenase, and asparaginase). Initially, the isothiazolinone forms a disulfide link with the thiol group on the amino acid. Occasionally, the chloromethyl-isothiazolinone may facilitate linkage with another thiol group to estab- lish a new disulfide linkage and release the biocide as a mercaptoacrylamide. This mercaptoacrylamide can tautomerize to a thioacyl chloride that may react again by denaturing nucleic acids (136). Formaldehyde also denatures protein but by alkylating amino and sulfhydril groups it can also alkylate the nitrogens of purine rings to denature DNA (137). Most of the formaldehyde donors (e.g., DMDM hydantoin, imidazolidinyl urea, Quaterium 15, polymethoxy bicyclic oxazolidine, etc.) act in this basic manner since these compounds release formaldehyde into the product or the microbial cell. Differences seen between the formaldehyde donors may exist as a result of when or what triggers the compound to release or "donate" formaldehyde. For example, a compound with a long hydrophilic chain connected to the formaldehyde-donating region (e.g., polymethoxy bicyclic ox- azolidine) may release formaldehyde only when the long chain enters into the lipopoly- saccharide portion of the membrane. Brominated compounds such as bromo-nitropropanediol and bromo-nitrodioxane act by oxidation of thiol groups (138-141) or by causing thioIs to convert to disulfides (142,143) where the thiol group first becomes brominated and then reacts with another thiol group to yield a disulfide and free bromide. As a result, enzymes involved in respiratory activity (e.g., dehydrogenases) and nucleic acid synthesis are inhibited, cell membrane integrity is compromised, and the cell wall may even be affected (144). One compound that is not technically a biocide but rather a biocide adjuvant is ethyl- enediamine-tetra-acetic acid (EDTA). This and other chelating agents remove magne- sium and calcium divalent cations from the cell wall, which is needed for stability (145). Once destabilized, they permit easier access of biocides into the cell. SELECTION OF PRESERVATIVE The ideal preservative would be broad-spectrum, safe and completely free of any sen- sitization issues, completely water-soluble, completely stable to all extremes of pH and temperature, completely compatible with all ingredients and packages, and impart no color or odor to the product, be inexpensive, and comply with government regulations. This ideal does not exist. One must select a preservative based on empirical testing. The only approach bordering on a theoretical basis for choosing a preservative is a qualified microbiologist's intuition, finely honed by experience. Selection of preservative may also be from published lists of available preservatives (146,147). These provide good sources for getting ideas of what might work in a formulation. Every formulation must be considered unique. Factors such as the physical and chemical nature of the product, how it is to be used, the container type and closure, and the shelf life must be considered when choosing the preservative (30). Often the selection of a preservative must be a compromise between efficacy, stability, and safety. More detail on the selection process of preservatives can be found by referring to several books and articles on the subject (148-150). SAFETY CONSIDERATIONS OF PRESERVATIVES One must always balance the risk of microbial contamination with the risk that a biocide