258 JOURNAL OF COSMETIC SCIENCE concentration of Deofix TM continuously present in the medium that was required to result in total growth inhibition (TGI) of 54 strains of various human neoplastic cells in vitro was high (14). In 38 of the cell lines studied the TGI was greater than 10-4M. In 16 cell lines that showed TGI values below 10-4M, the average TGI was 10-4'19M. The concentration that reduced the number of cells by 50% (IC5o) was greater than 10-4M in 49 of the tested strains. The inhibitory effect of Deofix TM on human foreskin fibroblast (HFF) cell growth was tested by suspending the cells in a growth medium containing Eagle's minimum es- sential medium and fetal calf serum. Cells were inoculated into tissue culture plates and incubated at 37 C. Cell growth was observed microscopically. HFF remained viable but failed to replicate when Deofix TM was continuously present in the growth medium in concentrations of 0.5 to 16 micrograms/mi. When HFF cells incubated in medium containing 16 micrograms/ml of Deofix TM were replaced with a medium free of Deofix TM, the cells appeared to resume replication consistent with a static, non-lethal, action of the Deofix TM (15). The effect of Deofix TM on HaCaT human keratinocytes was studied in vitro during continuous exposure of the keratinocytes to Deofix TM. Cultures were performed in microliter plates, and the number of cells was assayed by measuring DNA content in cultures using a DNA-binding fluorescent dye, Hoechst 33342. Fluorescence was moni- tored with a fluorescence plate reader. The viable keratinocyte numbers following 72- hour incubation with 240 micrograms/ml were only minimally changed from the viable keratinocyte number at the beginning of the study (16). Since the performance of a classical Ames test was complicated by the bacteriostatic effects of Deofix TM, the mutagenicity of Deofix TM was evaluated employing a specialized microbial assay system (17). The test uses dark mutants of luminous bacteria (Photobac- teria/eiogunthi) and determines the ability of the tested agent to restore the luminescent state. Deofix TM was found to be non-mutagenic by this assay. The iron complex of Deofix TM was not mutagenic when tested using the standard Ames assay. The Deofix TM chelator has very low acute toxicity following either intravenous or oral administration. In mice its LD50 following intravenous administration is approximately 1,900 mg/kg, while its LD50 following oral administration is in excess of 3,600 mg/kg. Following intravenous administration to mice, the bulk of the administered dose is excreted in the urine without demonstrable biodegradation. The agent has been applied to human skin in various vehicles in concentrations up to 2% without apparent adverse effects. The iron complex with the Deofix TM chelator administered intravenously in large daily doses for 14 days to rats and dogs failed to yield evidence of significant toxicity. The iron complex is currently being evaluated in human subjects to enhance image contrast in magnetic resonance imaging (MRI). In this complexed form it has been administered to over 270 human patients without significant objective adverse reactions. DISCUSSION Although the first commercial underarm deodorant appeared in the market in 1888, a true understanding of the origin of underarm odor awaited the work of Killian and

USE OF DEOFIX TM IN DEODORANT PRODUCTS 259 Panzarella (18) and Shelley (19) in the late 1940s and early 1950s. These authors showed that axillary microbial growth was the primary source of malodor. With this under- standing, the search for new deodorant ingredients largely focused on antimicrobial agents, the concept being that reduction in axillary microbial growth would result in less odor. Since then, almost all commercial deodorant ingredients have been based on biocides (Triclosan currently being the most popular). However, antimicrobial agents, such as Triclosan, are not without their detractors. Essentially two types of concerns have been voiced. Antimicrobial agents can change the commensal flora on the skin. This modification in the natural skin flora balance may predispose the skin to opportunistic infections (20). With time, it might also give rise to relatively resistant strains of bacteria. A second concern relates to the widespread release of antimicrobial agents and their biodegradation residues into the environment. These concerns have resulted in some of these agents being precluded from use in some European countries as well as elsewhere. Other, non-antimicrobial approaches to achieving deodorancy have been advanced as our understanding increases as to the nature of the odiferous materials and the metabolic pathways by which they are produced. Makin and Lowry (3) recently published a complete review of these approaches. Unfortunately, to date, none of these approaches have been proven to be more effective than antimicrobials. This paper reports on the use of Deofix TM, a metal ion chelator with unusually high affinity and specificity for first transition series elements, as a new deodorant ingredient. Chelators, in themselves, are not new as deodorant ingredients. Ethylenediaminetetra- acetic acid (EDTA), aside from being widely used as a booster for the activity of preservatives, has been sometimes included in deodorant formulations. EDTA deriva- tives have been claimed to have synergistic activity with agents such as Triclosan and quaternary ammonium germicidal compounds (21). The presumption is that their ac- tivity is related to their ability to chelate metal ions required for bacterial growth. First transition series elements such as iron, zinc, manganese, and copper are essential for microbial proliferation. For example, oxidative metabolism relies upon iron-containing heme enzymes, DNA synthesis requires iron-containing ribonucleotide reductase and nucleic acid polymerases require zinc as a coenzyme. The iron ions in iron-containing enzymes are in a form so tightly bound that they essentially cannot be removed by chelating agents. What chelating agents can do is lower the environmental concentra- tion of these metal cations to a level where there is an insufficient amount to form new metal-containing enzymes required for cell duplication. Since only trace quantities of these metals are required, chelating agents with unusually high affinity (i.e., very low thermodynamic equilibrium constant) and specificity are required. In comparison to Deofix TM, chelating agents like EDTA do not form highly stable chelates with first transition series elements. The iron chelate with Deofix TM is more than 10 9 times more stable than the iron chelate with EDTA (5). This means that when both chelators are present in solution in the same concentrations, the available free iron (FelII) in solution is 109 times lower with Deofix TM than with EDTA. We believe this explains, at least in part, the biological and deodorant activity observed with Deofix TM. This mechanism of action fits nicely with the low cytotoxity observed with Deofix TM and the results with human foreskin fibroblasts. Deofix TM lowers the availability of first transition series metals to inhibit cell replication but does not remove these metals from