Sara E. Dover et al. 2.7. Reduction of MTT by test preparations In order to assure reduction of MTT was not caused by the test articles, thereby generating incorrect results, reduction of MTT by each product was measured. This was done by adding 83 µL of product to 1 mL of MTT solution and incu- bating at room temperature in the dark for 60 minutes. 83 µL of double distilled water in 1 mL of MTT solution was used as negative control. Absence of darkening of solution color in- dicated that the test article and controls did not reduce MTT. 2.8. Rabbit vaginal model This safety assay was conducted at Euroﬁns Product Safety Laboratories (Dayton, NJ, USA). This study was performed to comply with the Good Laboratory Practice (GLP) regula- tions as deﬁned in: 21 CFR 58: U.S. FDA Good Laboratory Practice Standards. A quantity equal to 1.8 mL of the test article at pH 6.0, containing 10 mg/mL dissolved in sterile distilled water was administered intravaginally to six healthy female rabbits. One dose was administered daily to each animal for ten con- secutive days. A negative and positive control group, con- sisting of six female rabbits each, was maintained under the same environmental conditions and dosed daily with 1.8 mL of sterile distilled water and 4% nonoxynol-9, respectively. All animals were observed for mortality, signs of gross toxi- city, and behavioral changes at least once daily for 11 days. Body weights were recorded prior to administration and on day 11 (study termination). Individual vaginal irritation was measured prior to each intravaginal administration and on day 11. The entire vagina was excised, examined, and scored for exudates (vaginal discharge); edema; and erythema. The vagina from each rabbit was ﬁxed and saved in 10% neu- tral buﬀered formalin. Scores for the vaginal discharge and edema were: 0—normal/none, 1—slight, 2—moderate, 3— severe, and scores for erythema were: 0—normal, 1—pink, 2—red, 3—beet red. Necropsies were performed on all ani- mals at terminal sacriﬁce. Three sections of the columnar ep- ithelium of the vagina (including cervical, central, and caudal portions) were examined microscopically from hematoxylin- and eosin-stained slides. The maximum score for the micro- scopic evaluation was 16. 2.9. Hemolysis assay Outdated human blood erythrocytes obtained from the Blood Center of the Rush University of Chicago (Ill, USA) were used for the determination of hemolysis. Serial dilu- tions of lactocin 160 (12.5–200 mg/mL) were prepared in PBS. 100 µL aliquots of each dilution were incubated with 1 mL of 8% erythrocyte suspension in 1x PBS for 15 min- utes at 22◦C, and then centrifuged at 1500 rpm for 1 minute. The absorbance at 405 nm of released hemoglobin was mea- sured spectrophotometrically. Hemolytic activities are pre- sented as the percentage of the total erythrocytes lysed [13]. Sodium dodecyl sulfate was used as a control for total lysis. Hemolytic activity of lactocin160 was compared to 1–4% N9. Table 1: ET-50 values of products using the EpiVaginal tissue model (mean± standard deviation). Product and active ingredient Lactocin 160 (200 mg/mL) Spermicide Nonoxynol 9 (4%) Antifungal cream Miconazole nitrate (4%) 3 ET-50 (h) 17.45 ± 0.35 4.85 ± 1.06 35.6 ± 17.5 2.10. Inhibition of vaginal lactobacillus The inhibitory eﬀect of lactocin 160 on bacterial species char- acteristic of a healthy vaginal ﬂora was studied against 10 vaginal Lactobacillus species (healthy human subjects iso- lates) by determining minimum inhibitory concentration (MIC) using the microdilution method in MRS broth. The tested range of two-fold dilution of lactocin 160 was 1.5– 200 mg/mL with inoculum size of 5 × 105 CFU/mL. Loaded wells were overlaid with sterile mineral oil to create anaerobic condition for lactobacilli growth. MIC was determined using Bioscreen reader-incubator (Labsystems, Helsinki, Finland). 3. RESULTS 3.1. Lactocin 160 preparation analysis An amount of 100 mg of partially puriﬁed lactocin 160 con- tained 21.5 ± 1.29 µg protein. The test for lactic acid detected less than 1% L-lactic acid concentration of the dry weight of three samples of lactocin160; D-lactic acid was found in in- signiﬁcant amounts. This test conﬁrmed that the activity of lactocin160 was not caused by lactic acid. 3.2. HPLC and electrospray-MS analysis The HPLC analysis was used to conﬁrm reproducibility of the peptide recovery method from three independent batches of lactocin 160. It revealed that the samples have very simi- lar protein proﬁles (graphs not shown). The electrospray-MS analysis conﬁrmed the reproducibility data with signiﬁcant noise due to the presence of amino acids (data not shown). 3.3. ET-50 values The concentration of 200 mg/mL of lactocin 160 preparation was chosen for the in vitro safety study as this higher concen- tration is more likely to later be used in a commercial prod- uct. Lactocin 160 at this concentration showed very little ir- ritation, with an average ET-50 value of 17.5 hours. Table 1 summarizes the ET-50 values for the samples. As an example, Figure 1 presents the data from one study. As expected, N9 was most toxic for EpiVaginal cells, with an average ET-50 value of 4.9 hours. This conﬁrms previous in vitro and in vivo studies [7, 14, 15]. With an ET-50 value of >24 hours, miconazole nitrate showed very low toxicity in our study. This is in accordance with previous in vitro and in vivo testing using the RVI model and clinical trials [7, 9–11].