Table 1: Ectocervical cell viability after prolonged exposure to subtilosin. Postexposure cell viability (%) Exposure time (hrs) Assay # Subtilosin Nonoxynol-9 (4%) Miconazole nitrate (4%) 1 2 1 2 1 2 99.1 100 94.8 89.8 73.4 70.9 14.1 16.5 0 0 0 0 ND ND 58.4 58.3 79.3 83.5 4 4 24 48 80 60 ) % ( lity 40 i t o M 20 0 0 IC50 = 64.5 μg/mL 30 60 90 [Subtilosin] A (μg/mL) 120 150 Figure 1: Subtilosin A immobilizes human spermatozoa in a dose-dependent manner. The percentage of motile spermatozoa in pooled whole semen was determined 30 seconds after mixing with subtilosin A, at diﬀerent ﬁnal concentrations, as indicated. All data were adjusted to a normal control motility of 70% and subjected to arcsine transformation before further analysis. Values are expressed as average % motility. Error bars are 90% conﬁdence limits. All of the subtilosin concentrations tested reduced motility compared to the control samples. The diﬀerences in the proportion of motile spermatozoa in all samples (28.3, 56.7, and 113.3 μg/mL protein equivalents) were found to be signiﬁcant (P < .05) according to the Newman-Keuls multiple range test. TableCurve 2D (ver 5.0) curve-ﬁtting software (SPSS Scientiﬁc Software, Chicago, Ill, USA) was used to ﬁt the data to a dose-response curve described by Ln (% Motility) = a + b [Subtilosin A]3, where a = 4.20781; b = −2.5814e − 06; and [subtilosin A] is expressed as μg/mL protein equivalents. The curve had a coeﬃcient of determination (r2) = 0.9959. The IC50 value, or the amount of subtilosin required to reduce the motility of spermatozoa in whole semen by 50%, was calculated to be 64.5 μg/mL. 3.4. Semiquantitative observations of spermatozoa: forward progression Similar to motility, forward progression of spermatozoa is reduced in a dose-dependent fashion by subtilosin. In control samples, 70% of sperm exhibited forward progression; in the Infectious Diseases in Obstetrics and Gynecology ddH2O 100 100 100 100 100 100 presence of 50 μL subtilosin this decreased to 50–70%, while 100 μL caused a decline to only 10% forward progression. All forward progression was eliminated after treatment with 200 μL subtilosin, with most sperm tails becoming coiled. 4. DISCUSSION The B. amyloliquefaciens-produced bacteriocin subtilosin has proven antimicrobial activity against the vaginal pathogen G. vaginalis, but was not harmful to the normal and healthy Lactobacillus vaginal microbiota. Data from human vaginal cell viability assays convincingly demonstrated the safety of subtilosin for human applications in comparison to other accepted and available products, indicating it could be safely incorporated into personal care applications aimed at the treatment of bacterial vaginosis. Prior research in our laboratory that involved similar studies with the EpiVaginal model was also carried out in conjunction with in vivo testing of the rabbit vaginal irritation (RVI) system, which doubly conﬁrmed the safety of another antimicrobial peptide, Lactocin 160 [29]. Therefore, we are conﬁdent that using the EpiVaginal model instead of animal testing to demonstrate the safety of subtilosin has provided reliable and valid results. Subtilosin was also found to signiﬁcantly reduce the motility of human spermatozoa in a concentration- dependent manner for all concentrations tested. The eﬀect of subtilosin on the forward progression of spermatozoa was also observed to be a dose-dependent interaction. Serial dilutions showed a steady decline in forward progression, with all progression halted at the highest concentration tested. It was also noted that at the highest concentration, the tails of the sperm cells were curved or coiled, indicating the cells were damaged beyond a simple restriction of movement. Coiling of the cells is considered to be a sperm abnormality, and may indicate damage to the plasma membrane [37]. Tail coiling has been observed after in vitro exposure of monkey spermatozoa to methyl mercury [38]. These results suggest that subtilosin can be established as a general spermicidal agent. It is worth noting that nisin, a bacteriocin given generally recognized as safe (GRAS) status by the Food and Drug Administration (FDA), was also shown to have impressive spermicidal activity [25]. However, the allure of these results is diminished due to the fact that it also has potent antimicrobial activity against healthy vaginal microbiota (Chikindas et al., unpublished data), a strong