2 phosphate buﬀered saline (PBS, Sigma-Aldrich, Mo, USA). The pellet was resuspended in 200 mL chemically deﬁned media that resembles vaginal ﬂuid but lacks proteins [8], and incubated anaerobically at 37◦C for 20 hours with agitation (100 rpm). The supernatant was brought to 80% saturation with am- monium sulphate (Sigma-Aldrich). Precipitated (not active) proteins were removed by centrifugation (11,000 xg, 25 min- utes, 4◦C). The supernatant was dialyzed against double dis- tilled water for three days with the molecular weight cutoﬀ of 1 kDa, (Spectra/Por Cellulose ester dialysis membranes, Spectrum). This was followed by lyophilization to obtain dry lactocin 160 preparation. 2.2. Protein concentration Lactocin 160 is a partially puriﬁed protein preparation and contains other high molecular products, with trace amounts of salts from the medium. Protein concentration was mea- sured using BCA method (Pierce, Ill, USA) and determined spectrophotometrically at 562 nm. 2.3. Determination of weak organic acid presence in the preparation Prior to the toxicity study, lactocin 160 was resuspended in double distilled water at a concentration of 200 mg/mL. The samples’ acidity was analyzed by potentiometer (Microcom- puter pH-vision 6071, Markson, Honolulu, Hawaii, USA) and the lactic acid concentration was measured using a com- mercial D- and L-lactic acid testing kit (R-Biopharm AG, Darmstadt, Germany). The sample was ﬁlter sterilized using a 0.45 µm centrifuge ﬁlter (Millipore Corporation, Billerica, Mass, USA). 2.4. HPLC and electrospray-MS analysis To determine reproducibility of the composition of the par- tially puriﬁed lactocin 160 preparations, samples from 3 in- dependent batches were submitted to Proteomics Resource Facility (Integrated Biotechnology Laboratories, University of Georgia, Ga, USA) for HPLC and electrospray-MS anal- ysis. 2.5. EpiVaginal tissues model EpiVaginal tissues (VEC-100) were obtained from MatTek Corporation and placed in the refrigerator at 4◦C until use. All tissues were used within 24 hours. Prior to application of the product, tissues were preequilibrated in 6 well plates (Falcon) with 0.9 mL of DMEM-based DC-100 MM medium (MatTek Corporation). Plates containing tissue culture in- serts were placed in a humidiﬁed incubator at 37◦C and 5% CO2 for one hour. Media was removed and replaced by 0.9 mL of fresh VEC-100-MM medium. Then, 83 µL of prod- uct was applied topically on triplicate EpiVaginal (VEC-100) tissues. For exposure times of greater than 24 hours, tissue inserts were airlifted by placing them on 2 washers (MatTek Corporation) and fed with 5 mL of the assay medium. Ini- Infectious Diseases in Obstetrics and Gynecology tial exposure times of lactocin 160 (200 mg/mL) were 4, 9, 24, and 48 hours. Distilled water, used as negative control, was applied to duplicate tissue culture inserts at time points 6, 24, and 48 hours. Spermicide containing 4% Nonoxynol-9 (Ortho Options CONCEPTROL Vaginal Contraceptive Gel, Advanced Care Products, Skillman, NJ, USA) has a well- documented cytotoxicity (7, 9, 10), and was therefore chosen as a positive control. Antifungal cream, containing 4% mi- conazole nitrate (Monistat-3, Ortho McNeil Pharmaceutical, Inc., Raritan, NJ, USA), is a nontoxic preparation and was used as a negative control [7, 9–11]. Tissue viability at the end of the exposure time was de- termined using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) viability assay. Approximate eﬀective times (ET) for each product to reduce tissue viabil- ity to 50% (ET-50) values were calculated, from which new time points were selected to obtain a more accurate ET-50 value. 2.6. MTT viability assay Viability of cells was determined by measuring the break- down of the yellow tetrazolium component MTT to purple formazan following manufacturers’ (MatTek Corporation) recommendations. As only living cells perform breakdown of MTT, the reduction is directly proportional to the amount of living cells [12]. Brieﬂy, after completion of exposure times of lactocin 160 and positive and negative controls to tissues, the liquid in the inserts was decanted, followed by washing the tissue inserts in Dulbeccos phosphate-buﬀered saline so- lution (D-PBS). Nonliquid test material was removed care- fully with a sterile polyester ﬁber tipped swab (Thermo- Fisher, Waltham, Mass, USA). Tissues were then placed in a 24 well plate with 300 µL MTT (1.0 mg/mL) solution in cul- ture medium and incubated for 3 hours at 37◦C and 5% CO2. Following MTT incubation, the tissue inserts were placed in a new 24 well plate and immersed in 1.66 mL of isopropanol (MatTek Corporation) to extract the formazan. To reduce ex- tractant evaporation, the plate was covered with sealing ﬁlm (Thermo-Fisher). The plate was incubated overnight in the dark at room temperature. Upon completion of extraction, the liquid from the wells was mixed with the liquid from the inserts. After mixing, 200 µL of extractant solution was mea- sured spectrophotometrically in triplicate at 570 nm in a 96 well plate reader (MRX revelation, Dynex Technologies, Va, USA) using extractant solution as blank. Tissue viability (%) was determined according to the manufacturer’s recommendations and using the equation: %viability = OD570(treated tissue)/OD570(control tissue). The exposure time that reduced the tissue viability to 50% was calculated as described previously [7] by plotting the log- arithm of the dosing time versus % viability and then inter- polating the times near when the viability is 50%. In general, a shorter ET-50 corresponds to a more irritating/damaging test article; a longer ET-50 corresponds to milder/less dam- aging test article.