4 Infectious Diseases in Obstetrics and Gynecology Table 2: Individual vaginal irritation and histopathology scores. Histopathology scores include evaluation of epithelium, leukocytes inﬁl- tration, vascular congestion, and edema. Average score Day 2 Distill water (n = 6) 0.0/0.0 N-9 (n = 6) 0.0/0.0 Lactocin 160 (n = 6) 0.0/0.0 Vaginal discharge/erythema 3 0.0/0.0 0.3/0.5 0.0/0.0 4 0.0/0.0 0.3/0.5 0.0/0.0 5 0.0/0.0 0.3/0.7 0.0/0.2 6 0.0/0.0 0.3/0.7 0.0/0.2 7 0.0/0.0 0.8/1.0 0.0/0.2 8 0.0/0.0 1.2/1.0 0.3/0.3 9 0.0/0.0 1.0/1.3 0.3/0.3 10 0.0/0.0 1.7/1.5 0.3/0.3 11 0.0/0.0 1.3/1.3 0.3/0.3 Histopathology score (Group mean) 11 5.2 10.5 5.7 120 110 100 90 ) 80 (%70 lity 60 ib 50 iV a 40 30 20 10 0 0 5 10 15 20 35 40 45 50 25 Time (h) 30 Nonoxynol 9 (4%) Lactocin 160 (200 mg/ml) Miconazole nitrate (4%) Figure 1: Lactocin 160 preparation is not toxic for human vaginal cells as tested in EpiVaginal tissue (VEC-100) model. The presented data illustrate %viability versus time (hours) for following prepara- tions: , nonoxynol 9 (4%); •, lactocin 160 (200 mg/mL); , mi- conazole nitrate (4%). An increase in liquid was observed in the insert of tissue exposed to lactocin 160. This might have been due to diﬀer- ences between products and media, thereby causing water to enter the tissue insert by osmotic pressure. It may also be due to bacteriocins inducing EpiVaginal cells to produce mucin. As the liquid in the wells was not viscous, the ﬁrst speculation is more likely to have account for the observation (personal communication with Dr. Ayehunie at MatTek Corp.). 3.4. Reduction of MTT by test article MTT was not reduced to formazan by lactocin 160, micron- azole nitrate, or nonoxynol 9. Thus, reduction of MTT was only performed by viable cells, and not caused by test article. This conﬁrms the validity of the assay. 3.5. Rabbit safety assay All animals survived, gained body weight, and appeared ac- tive and healthy during the study. In control group with 4% N9 apart from a red gelatinous substance in the pan- line noted for one animal on day 2, there were no other signs of gross toxicity, adverse pharmacologic eﬀects, or abnormal behavior (Table 3). Slight-to-moderate vaginal discharge and pink-to-red erythema were noted for all treated sites between days 3 and 11. No gross abnormalities were noted for any of the animals when necropsied at the conclusion of the 11- day observation period. Slight vaginal discharge, pink-to-red erythema, and/or slight-to-moderate edema were observed in all six rabbits at time of necropsy. The positive for irrita- tion control tissues (nonoxynol-9 treatment) were given an average irritation index grade of “mild.” In the test group (lactocin 160 treatment), slight vaginal discharge and/or pink erythema were noted for three treated sites between days 5 and 11. No gross abnormalities were noted for any of the animals when necropsied at the con- clusion of the 11-day observation period. Slight-to-moderate vaginal discharge, pink-to-red erythema, and/or slight-to- moderate edema were observed in all six rabbits at time of necropsy. The test tissues for lactocin 160 were given an av- erage irritation index grade of “none.” Under the conditions of this test, the lactocin 160 did not produce any signiﬁcant vaginal irritation. 3.6. Toxicity for lactobacilli Lactocin 160 preparation’s MIC for the tested 10 vaginal Lac- tobacillus spp was >200 mg/mL. 3.7. Hemolytic activity Table 3 presents data of human erythrocyte hemolysis by lac- tocin 160 (12.5–200 mg/mL) and N9. The range of observed hemolysis was 1.2 ± 0.34–8.2 ± 0.6%. In fact, N9 in concen- tration 1–4% caused total hemolysis of erythrocytes. 4. DISCUSSION Lactocin 160, an antimicrobial peptide produced by Lacto- bacillus rhamnosus, did not show any severe irritation in the RVI model, ectovaginal tissue, hemolytic activity, or vaginal lactobacilli inhibition. The ET-50 values of the lactocin 160 preparations were similar or less than over-the-counter non- toxic products. Our toxicity studies revealed that lactocin 160 can be considered safe for human use. For vaginal toxicity studies, thein vivo RVI model has been the preferred choice for a long time. This model deter- mines the cervicovaginal irritation as minimal, mild, mod- erate, and severe based on a scoring system evaluating ep- ithelial ulceration, leucocytic inﬁltration, edema, and vascu- lar injection [16]. As it is an established standard, the results can be compared to other products [17–19]. The RVI model