TEST FOR GENOTOXIC POTENTIAL BY MICRONUCLEI ASSAY 33 All the treatment groups were set up as duplicates the determination of cellular toxicity was made in eight independent measurements. After the end of all treatments, the cells were harvested for staining. Cell staining. After 24 hours of incubation with the aforementioned substances, the media from all the fl asks were removed. The cells were fi xed by slowly adding a cold solution of 100% methanol and allowing the mixture to stand for fi ve minutes. The methanol was removed and the cells were washed with phosphate buffer solution two times for two minutes. The cells’ nuclei were then stained with 600 nM DAPI (2 ml) (4,6 diamidino- 2-phenylindole) for eight minutes. This solution was removed and all the fl asks were washed with PBS containing 0.05 % Tween 20 (Sigma Aldrich). The cells were kept moist by adding PBS at the end. The cells were then observed under a microscope. Counting of micronuclei. For each experimental series, the formation of micronuclei was determined as described (9) by counting the number of micronuclei per 1000 cells using light and a fl uorescent microscope (Olympus I×71, New York. Cell viability and proliferation. A modifi ed MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphe- nyltetrazolium bromide) assay was used to assess the cytotoxicity of the studied substances as previously described (10). To measure cytotoxicity, cells were separately incubated in a Figure 1. Scheme of experimental series and conditions. The shaded areas represent the treatment periods with tested substances or media. At the beginning of the experiments, the cells were incubated with media within 24 h. For the series with metabolic activation, the cells were treated for three hours after which the media were replaced with fresh media and the cells were incubated for 24 hours. For the groups without S9 activation, the cells were incubated for 24 hours.

JOURNAL OF COSMETIC SCIENCE 34 microtiter plate with each studied substance. Control cells received an equivalent volume of fresh medium containing DMSO in the same concentrations used to dissolve the OB. The duration of incubation was 24 h. On the basis of these measurements, cellular viabil- ity was calculated for each substance. A decrease in the cellular viability indicated an in- crease in toxicity. Cell proliferation was conducted by using a hemacytometer (Veeder-Root, Elizabeth Town, NC) and a light microscope (Zeiss, New York). A modifi cation of the method was used for counting the cells (10). Briefl y, at the end of 48 hours all the con- tents from the fl ask were removed and the cells were washed with DPBS once for one minute. DPBS was removed and 1 ml of trypsin was added to the fl ask. The fl asks were then placed in an incubator at 37°C for two minutes. The fl ask was gently shaken and the cells were detached with the help of a cell scraper. An amount of 20 μl of this solution was taken and added to a hemacytometer. Cells were counted in each of the four grids and the average of that was taken. The fi nal result would be an average number of 104 cells per ml. Statistical analysis. Data obtained were analyzed using descriptive statistics and single- factor analysis of variance (ANOVA), and were presented as mean value ± S.D. RESULTS AND DISCUSSION Cosmetic and personal care products are used on a daily basis, either in the forms of a “wash off,” such as soaps and shampoos, or a “leave on,” such as creams, lotions, and makeup preparations. Over a lifetime, a person living in the Western world is expected to be exposed to thousands of different chemicals repeatedly. While most of these chemi- cals will not penetrate healthy intact skin, depending on their chemical and physical properties, numerous compounds can partition into the skin and either accumulate in its viable layers or further penetrate to the blood circulation. If these compounds carry toxic potential, their accumulation can lead to diseases and disorders. Moreover, our skin and our internal organs contain metabolic systems that can convert an inert compound into a toxic substance. Since, unlike pharmaceutical grade compounds, the production of a cosmetic ingredi- ent does not require strict conditions such as GMP, the residuals and the impurities that these ingredients may contain are of major concern. For example, it was shown that polyethylene glycol (PEG) molecules with a molecular weight of around 200 Da can induce a genotoxic effect in chinese hamster ovarian (CHO) cells after metabolic activa- tion with S9 following chromosome aberration study protocols (11). The researchers also concluded that their fi ndings may refl ect a potential mutagenic risk of PEG de- rivatives with similar molecular sizes. Another example is sodium benzoate, which is used extensively as a preservative in cosmetic formulations. In a report issued by the EU Commission Scientifi c Committee, the SCCNFP, in 2002 (12), the Committee reviewed genotoxicity data generated on sodium benzoate. This compound demonstrated posi- tive results for genotoxicity in a CHO cell line without metabolic activation. Although chromosomal aberrations were not induced when tested in vivo in rats, a dominant le- thal assay with sodium benzoate in rats did show a genotoxic effect. Based on these data, the committee concluded that sodium benzoate carries a potential genotoxic risk and called for the generation of additional studies to either confi rm these observations or rule them out.