J. Cosmet. Sci., 62, 29–39 (January/February 2011) 29 Genotoxic potential evaluation of a cosmetic insoluble substance by the micronuclei assay N. DAYAN, V. SHAH, and T. MINKO, Lipo Chemicals Inc., 207 19th Ave., Paterson, NJ 07504 (N.D.), and Department of Pharmaceutics, Rutgers, The State University of New Jersey, 160 Frelinghuysen Rd, Piscataway, NJ 08854 (V.S., T.M.). Accepted for publication November 3, 2010. Synopsis An optical brightener (OB) powder (INCI: sodium silicoaluminate (and) glycidoxypropyl trimethyloxysi- lane/PEI-250 cross fl uorescent brightener 230 salt (and) polyvinylalcohol crosspolymer) that is used in cos- metic facial products was tested for its genotoxic potential using the micronuclei test (MNT). It is a solid dry powder with an average size of 5 microns that is insoluble but dispersible in water. This study describes the exposure of cell culture to positive controls with and without enzymatic activation and to the test compound in different concentrations. We evaluated three end points: microscopic observation and quantifi cation of micronuclei formation, and cell viability and proliferation. Both positive controls induced signifi cant changes that were observed under the microscope and quantifi ed. Based on its chemical nature, it was not anticipated that the test substance will degrade under the conditions of the experiments. However, the test is required to make sure that when solublized, impurities that may be present, even at trace levels, will not induce a geno- toxic effect. The test compound did not promote micronuclei formation or change the viability or prolifera- tion rate of cells. During this study we faced challenges such as solubilization and correlating viability data to genotoxicity data. These are described in the body of the paper. We believe that with the emergence of the 7th European amendment that bans animal testing, sharing these data and the study protocol serves as a key in building the understanding of the utilization of in vitro studies in the safety assessment of cosmetic ingredients INTRODUCTION With the emergence of the 7th amendment to the EU Directive, the cosmetic and personal care industry is banned from experimenting on animals. It is therefore utilizing, study- ing, and validating a variety of in vitro and ex vivo protocols. One of the more impor- tant end points in the evaluation of safety is the potential of a compound to become genotoxic. While this industry is known to produce relatively safe products, with the 7th amendment the perspective of safety has been broadened from acute, clearly perceivable, adverse effects such as irritation to the inclusion of the evaluation of long-term accumulating effects that can lead to a variety of chronic and deadly diseases such as infl ammation and cancer. Moreover, the focus on in vitro methodologies is expanding our knowledge in understanding

JOURNAL OF COSMETIC SCIENCE 30 biochemical pathways rather than focusing on clinical evaluation. Consequently, the cos- metic product manufacturer is focusing on in vitro studies to ensure safety and on study- ing their effects. Compounds that can change the genetic information of a living cell may lead to mutations. These can result in different type of toxic effects, ranging from cell death to the development of malignant tumors. While a variety of assays are currently in use in the area of regulatory genotoxicity, substantial in vivo testing is still required for the confi rmation of genotoxic predictions. A mutagenic (from Latin word for change) compound can change the genetic information of the cell and thus increase the frequency of mutations. The term “genotoxicity” is used broadly and refers to potentially harmful effects on genetic material that are not necessarily associated with mutagenicity. One test method recommended for the estimation of genotoxicity is known as the micro- nuclei assay. In the course of this study, mammalian cells are exposed to the test substance with and without metabolic activation. At predetermined time intervals after exposure, they are treated with a metaphase-arresting substance, harvested, stained, and observed under the microscope. Information is then collected both by observing the morphological changes and by determining them quantitatively as compared to the control. This paper reports the results of a study evaluating the genotoxicity potential of a pow- der, optical brightener (OB), used in cosmetic products. It describes the challenges of working with a powder in a cell culture, the study rationale, the subjective morphologi- cal changes, and the quantitative evaluation of the data generated. In addition, cell me- tabolism as an end point of viability, proliferation, and the relationships between viability and the genetic mutations of cells is discussed. MICRONUCLEI TEST The chromosome is a single coiled piece of DNA with associated proteins containing many genes, regulatory elements, and other nucleotide sequences. A chromatid is one among two identical copies of DNA making up the replicated chromosomes. Two identi- cal sister chromatids are joined by a region called the centromere. The term micronucleus describes the small nucleus that forms whenever a chromosome or a fragment of a chro- mosome is not incorporated into one of the daughter nuclei during cell division. Testing for micronucleus formation is used to screen for potential genotoxic compounds. There are two versions of this study: in vivo and in vitro. The in vivo test is using mouse bone marrow or mouse peripheral blood (1). The in vitro method was developed as a replace- ment for the in vivo method and was evaluated to be a reliable genotoxicity test, although more compounds should be tested to build an additional database (2). In that sense, we are hoping that data shared in this paper will enrich the existing database. This study was conducted as a modifi cation of the OECD (Organization for Economic Cooperation & Development) Guideline number 487 protocol that was adopted in De- cember of 2006 as a draft proposal: “In Vitro Micronucleus Test” (3). This test protocol was assessed by The European Centre for the Validation of Alternative Methods (ECVAM) that stated the following: “On the basis of a peer review of a weight of evidence retrospec- tive validation, the Committee endorses the conclusion that the MNT is a scientifi cally valid alternative to the in vitro chromosome aberration assay for genotoxicity testing” (4). According to the protocol, the objective is to identify compounds that cause structural chromosome aberrations in cultured mammalian cells. These aberrations may be of two