COSMETIC PRESERVATION 209 phenomenon are either a case of neutralization of the biocide (by carryover of the growth medium) or a case of saturating the biocide with more organisms than available biocide to the point of inactivating it (97,98). Specific genetic mechanisms (e.g., point muta- tions, plasmid acquisition, lifting repression) or the expression of formaldehyde dehy- drogenase do exist in some cases (99,100). The hallmark of whether or not a permanent genetic adaptation has occurred is the stability of the resistance in the absence of selective pressure from the presence of preservative, as apparently is the case for parabens (101). However, resistance to all biocides by permanent genotypic change must not always be assumed. The most naYve idea is that the resistance mechanisms against biocides are similar to those mechanisms found in antibiotic resistance. Whereas anti- biotic resistance can be described based on specific molecular activity at specific sites, the resistance to biocides cannot. Often the resistance to biocides must be maintained at a population level by continuously culturing the organism in the presence of the preservative to maintain the selective pressure on the population. This selective pressure causes the population to develop higher capsule production, which enhances clumping associations, and the production of biofilms. Alternatively, enhancement of the expres- sion of glutathione synthetase could also occur within the population to provide resis- tance to some biocides (102). Take the selective pressure away, however, and this expression stops, indicating that a permanent genetic change within individuals did not take place but rather that population shifts occurred. Use of neutralizers. Appropriate use of neutralizers is often overlooked when conducting PETs. Some preservatives only require dilution in buffer to be inactivated. Others require chemical neutralizers used in the diluent or the plating medium, or both. Filtration is another approach but is limited to those products that can be filtered. The work of Sutton and others describes a number of neutralization methods for preservatives as well as a scientific basis for their evaluation (103-107). The goal of a neutralizer is to inactivate the biocide before the biocide inactivates the microorganism in order to provide uninhibited microbial growth. Failure to inactivate the biocide immediately upon sampling causes one to overestimate the killing potential of the biocide. This failure is actually a measure of the kill that continues within the plating medium because the active biocide is carried over into the medium (108). A fairly effective all-purpose (universal) neutralizing medium is Dey-Engley broth (109). Dey-Engley broth is described further in Atlas' Handbook of Microbio/ogica/ Media (110) and the Difco Manual (111). A thorough review of this and many other neutralizers may be found in the articles by Russell (84) and Sutton (112). The ASTM provides a method to determine if a neutralizer is nontoxic and effective, using microorganisms as biological indicators (113). This method is a retroactive check for neutralization. It is done by streaking plates showing no growth with test organisms. The streak is done 48 hours or more after the inoculated product was originally plated. Since this streak is done so long after the initial plating, the retroactive test only proves that neutralization finally occurs after allowing the biocide to incubate in the medium for some length of time it does not prove that neutralization occurred instantaneously when the product containing the biocide was mixed into the medium. Retroactive checks of neutralization, and thus the ASTM method of neutralizer validation, are invalid. Interpretation of data. Interpretation of the data using the criteria set by the compendial

210 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS tests is based on anecdotal evidence and opinion regarding how long a product should take to reduce the numbers of the challenge inocula. The best way of interpreting the data, however, is to compare how the test product performs against how well-preserved and poorly preserved control products perform. Well-preserved products are those that do not become contaminated during consumer use, and poorly preserved products are those that do become contaminated when used by consumers. OTHER PET METHODS D-value methods. Rapid tests are sometimes used for quick impressions of which preser- vatives to use in a product. One such method is the D-value method. Aside from one author, no one else claims D-value methods are valid for final testing of nationally distributed product (114). In fact, D-value methods are inappropriate for at least some consumer products (115). This method is actually an adaptation from food microbiol- ogy's heat or radiation destruction D-values. Heat and radiation kills do indeed follow first-order rate kinetics, and therefore the D-values determined for them are quite valid. However, biocide kills follow second- order rate kinetics (115,116). The only case where a second-order reaction can approach pseudo-first-order rate kinetics is when the second reactant (biocide) is present in such large excess that it is virtually in constant concentration. Preserved products do not have an excess of preservative such that the biocide remains in constant concentration when contamination occurs (117). A biocide-organism reaction is stoichiometric the biocide does not act like an enzyme that catalyzes a reaction where live organism goes to dead organism, but the biocide is not spent. Therefore, since the biocide-organism reaction is second order, with the biocide serving as the limiting reactant, D-value tests based on first-order rate kinetics are invalid (115,117). The second criticism of the D-value technique is that it extrapolates beyond the mea- sured data by falsely assuming a linear relationship between biocide exposure time and the number of surviving microorganisms. In defense of rapid D-value methods, how- ever, one may find they allow a preliminary screening of preservatives. This approach assumes that appropriate reproducible controls are in place such that one will be able to rank the various D-values for a wide variety of products and be able to correlate that data to full-scale PET results on the same products. Capacity tests. A capacity test determines how many bacterial challenges are needed before the product begins growing microorganisms (118). After each challenge, the products are sampled and challenged again until the product either receives 15 chal- lenges without showing growth (a well-preserved product) or until three consecutive positive results occur (a lesser-preserved product). The goal is for the product to reduce the number of viable organisms by 3 logs (99.9%) in 48 hours. With each subsequent challenge, this ability diminishes as a result of dilution, neutralization, and reaction with the inoculum. The claim, by some studies, that multiple challenges provide no more information than single challenges (67) may actually be more pragmatically based than scientifically based. The reason why multiple challenges with low levels of organ- isms are not the same as one challenge with a high level is similar to the concept of the Danysz phenomenon in immunology (119), where when a high level of inoculum is used, the biocide combines with an equivalent amount of microbes, allowing the challenge to be killed, but when challenging multiple times, each challenge combines