J. Cosmet. Sci., 61, 107–123 (March/April 2010) 107 New alternatives to cosmetics preservation S. PAPAGEORGIOU, A. VARVARESOU, E. TSIRIVAS, and C. DEMETZOS, Laboratory of Cosmetology, Department of Aesthetics and Cosmetology, School of Health & Caring Professions, Technological Educational Institution, Ag. Spyridonos Str., 12210 Egaleo (S.P., A.V., E.T.), Frezyderm S.A., Medicine-Cosmetics, 4 Tatoiou Avenue, 14451 Metamorphosi (S.P.), and Department of Pharmaceutical Technology, School of Pharmacy, University of Athens, Panepistimiopolis, 15771 Zografou (C.D.), Athens, Greece. Accepted for publication August 24, 2009. This work was partially presented at the 7th Joint Meeting of AFRP, ASP, GA, PSE and SIF, Athens, Greece, and at the XIIIth COSMODERM Joint Meeting of ESCAD and the Hellenic Society of Dermatology and Venerology, Athens, Greece. Synopsis In recent years, there is a considerable interest in the development of preservative-free or self-preserving cos- metics. The aim of our work was to develop new cosmetic formulations by replacing chemical preservatives with ingredients with antimicrobial properties that are not legislated as preservatives according to Annex VI of Com- mission Directive 76/768/EEC. This paper describes the preservative effi cacy of the well-known antimicrobial extracts of Lonicera caprifoleum and Lonicera japonica in combination with glyceryl caprylate and/or levulinic acid, p-anisic acid, and ethanol. We prepared a series of acidic (pH=5.5) aqueous and O/W formulations, i.e., tonic lotion, shampoo, shower gel, conditioning cream, anticellulite cream, cleansing milk and peeling cream, con- taining (0.2% w/w) Lonicera extracts, alone in the case of tonic lotion and in combination with (1% w/w) glyc- eryl caprylate in the other products, and we performed challenge tests according to the European Pharmacopoeia procedures and criteria. Formulations such as shampoo, shower gel, and conditioning cream fulfi lled criterion A, while tonic lotion, anticellulite cream, cleansing milk, and peeling cream fulfi lled criterion B, in regard to contamination from A. niger. Furthermore, we evaluated the effi cacy of the antimicrobial systems in two states of use: the intact product and after three weeks of consumer use. The results showed that A. niger was also detected during use by consumers in the products that satisfi ed only criterion B in challenge tests. The addition of antimicrobial fragrance ingredients such (≤ 0.3% w/w) levulinic acid or (0.1% w/w) p-anisic acid and/or (5% w/w) ethanol afforded products that met criterion A in challenge tests and were also microbiologically safe during use. The small quantity (5% w/w) of ethanol gave an important assistance in order to boost the self- preserving system and to produce stable and safe products. INTRODUCTION Microbial spoilage of cosmetic formulations has always been of special concern for industry, since it can lead to product degradation or, in the case of pathogens, constitutes a threat Address all correspondence to A. Varvaresou.

JOURNAL OF COSMETIC SCIENCE 108 to consumer safety. Chemical preservatives are added to cosmetics, pharmaceuticals, and foods in order to protect them against microbial contamination. The growing skepticism of consumers regarding the safety of chemical preservatives in combination with the fact that long-lasting skin health is often associated with the use of natural ingredients has led the cosmetics industry to seek alternative approaches for cos- metics preservation (1,2). A recent trend in cosmetics preservation is the replacement of traditional chemical preservatives by antimicrobial agents that are not legislated as pre- servatives according to Annex VI of Commission Directive 1976/768/EEC (3-5) but that are safe and effective as preservatives. An approach to acheive preservative-free cosmetics is the selection of natural compounds that have been characterized as safe and effective against microorganisms, in order to decrease or to eliminate the use of the traditional chemical preservatives and to formulate cosmetics with improved dermocosmetic properties, i.e., lower skin irritation and/or con- tact sensitization. A number of well-known plant-derived essential oils and extracts have exhibited excel- lent antimicrobial properties thus, they have been used for the effective preservation of cosmetic formulations. Among others, derivatives of Rosmarinus offi cinalis (7,8), Lavan- dula offi cinalis (9), Pteronia incana (8), Artemisia afra (8), Thymus vulgaris (10,11), Eucalyp- tus globulus (12), Laurus nobilis (12), Salvia offi cinalis (12), and Melaleuca alternifolia (13,14) have been reported to be effective natural preservatives. Medium polar substances also belong to the class of alternative antimicrobial agents. Examples of such agents as caprylyl glycol and monoglycerides of capric acid and caprylic acid, i.e., glyceryl caprate and glyceryl caprylate, besides being moisturizing agents, exert antimicrobial activities (15–17). Due to their emulsifi er-like structure, with a hydro- philic and lipophilic part, they interfere with the cellular structures of microorganisms and disintegrate cell membranes. Many studies have been reported concerning the use of glyceryl caprylate as an antimicrobial substance alone or in combination with other anti- microbial compounds for cosmetics preservation. (17–19). We have to keep in mind that the chemical composition of fragrances plays a key role concerning the antimicrobial activity of essential oils and the extracts obtained from nat- ural sources (20). Various aldehydes and alcohols, i.e., aromatic and aliphatic compounds, or terpenes and organic acids, are among the most active compounds. In the past, a fra- grance mixture that mainly consisted of benzyl acetate, phenethyl alcohol, and linalool had been proposed as an alternative preservative in order to reduce the amount of para- bens used in cosmetic formulations (21). Today, several antimicrobial fragrance ingredi- ents are commercially available, such as, p-anisic acid (p-methoxy-benzoic acid) and levulinic acid (4-oxo-pentanoic acid), which were found to be the main compounds in Pimpinella anisum and other herbs and in Dioscorea villosa as a by-product in the produc- tion of diosgenin from wild yam, respectively (22). Based on the above comments concerning the development of self-preserving cosmetics, we focused our research to evaluate the preservative effi cacy of the antimicrobial extracts of Lonicera caprifoleum and Lonicera japonica (Table I) in combination with other antimicro- bials such as glyceryl caprylate, p-anisic acid, levulinic acid (Table II), and ethanol in a series of aqueous and O/W emulsions. Lonicera extracts are described as being a mixture of esters of lonicerin and p-hydroxy benzoic acid, the structures of which are very similar