Sara E. Dover et al. 5 Table 3: Hemolytic activity of lactocin 160. Substance Concentration 200 Hemolytic activity (% of control) 8.2 ± 0.6 Lactocin 160 (mg/mL) 100 6.2 ± 0.2 50 4.1 ± 0.28 25 1.1 ± 0.34 12.5 0 N9 (%) 1–4 100 is, however, best for identifying agents that will cause severe irritation [20]. This model poses some shortcomings, mainly because of the diﬀerence in vaginal tissue between rabbit and human. Also, extra variables have to be taken into account, for example, the physical status, the health history of the rabbit, and the immune system. The RVI model approach proved not to be totally reliable in the case of the spermicidal agent nonoxynol 9. This commonly used contraceptive was determined to be suitable for human use in the rabbit irrita- tion model; but with frequent use it induces inﬂammatory reactions and disruption of the vaginal epithelia [14, 15]. This has possible adverse eﬀects including higher risk of ac- quisition of HIV [21]. In addition, prolonged exposure (10 days in diﬀerent concentrations) to N9 in the rabbit vagina revealed severe inﬂammatory changes including erosions and loss of epithelial lining. The damage was proportional to the amount of N9 used [22]. In vitro models for vaginal irritation include monolayers of cells, which have been used for many years to determine safety, for example, endocervical vaginal epithelial HeLa-S3 cells [23] and immortalized human vaginal and cervical ep- ithelial cells [24–26]. However, monolayer cultures are not optimal for irritation studies as they do not accurately resem- ble three-dimensional, diﬀerentiated vaginal tissue and also have diﬀerent gene and protein expression [7]. Three-dimensional models of normal ectocervicovaginal epithelial cells, grown on polycarbonate ﬁlters, have previ- ously been used. In a study of the pokeweed antiviral pro- tein microbicide, no correlation was found between the re- sults of the in vitro and the RVI model, the latter being much more sensitive [18]. These researchers hypothesized that this might be due to the in vitro system lacking the dynamics of in vivo testing. The inﬂammation may have been caused by the product’s eﬀect on mast cells, thereby creating the inﬂamma- tory reaction instead of a direct cytotoxic eﬀect to the vaginal epithelial cells. In this study, we sought to transition from the traditional in vivo vaginal irritation test to a reliable and reproducible in vitro vaginal model. We chose EpiVaginal model because it is a three-dimensional organotypic ectocervicovaginal tissue that was reconstructed using normal, well-stratiﬁed epithe- lial cells, containing diﬀerentiated basal, suprabasal, interme- diate, and superﬁcial cell layers similar to in vivo tissue. The in vivo and in vitro results were similar and revealed little or no toxicity of lactocin160. This in vitromodel is cost-eﬀective and can be considered as an alternative to the animal model. To the best of our knowledge, this is the ﬁrst in vitro vaginal irritation study of a bacteriocin from a vaginal pro- biotic Lactobacillus strain using EpiVaginal tissues. Vaginal safety was studied for the bacteriocin nisin. This well-known bacteriocin is widely used for food preservation but is not produced by any vaginal isolates [23, 27]. Nisin has been proposed as a contraceptive agent even though it kills the healthy vaginal microﬂora consisting of Lactobacillus species in concentrations much lower than the ones proposed for the nisin’s spermicidal application (our unpublished data). The absence of vaginal irritation of nisin in rat and rabbit model was conﬁrmed in vitro using endocervical vaginal epithe- lial HeLa-S3 cells measuring viability [23, 28]. Also, nisin’s hemolytic activity has been studied well. It was found that nisin caused hemolysis of sheep erythrocytes at concentra- tions that were 1000-fold higher than those required for an- timicrobial activity [29]. Documenting the antimicrobial activity against vaginal lactobacilli is an important concern for vaginal formula- tion safety study. For instance, it was shown that presence of nonoxinol-9 aﬀects the ecological balance of the vagina by inhibiting the protector lactobacilli [30–32]. At the same time, our in vitro and in vivo toxicity studies showed that the antimicrobial peptide lactocin 160 produced by vaginal L. rhamnosus did not irritate vaginal epithelial tissue, and it was not hemolytic for human erythrocytes or toxic for vagi- nal lactobacilli. These results suggest that it would be safe in formulations to treat bacterial vaginosis. ACKNOWLEDGMENTS This research was sponsored by NIH Grant “Natural antimicrobials against bacterial vaginosis” NCCAM NIH R21AT002897-01. Johnson&Johnson Consumer and Per- sonal Products Worldwide granted a stipend to S.E. Dover to perform this work. The authors thank Dr. Ayehunie at Mat- Tek Corporation for kind assistance and guidance and Dr. Harper (Johnson&Johnson Consumer and Personal Prod- ucts Worldwide) for valuable comments and suggestion. We also thank Euroﬁns Product Safety Laboratories (Dayton, NJ, USA) for animal safety study. REFERENCES [1] A. A. Aroutcheva, J. A. Simoes, and S. Faro, “Antimicrobial protein produced by Vaginal Lactobacillus acidophilus that in- hibits Gardnerella vaginalis,” Infectious Diseases in Obstetrics and Gynecology, vol. 9, no. 1, pp. 33–39, 2001. [2] J. Li, A. A. Aroutcheva, S. Faro, and M. L. 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