In vitro -in vivo assay validation for vaginal irritation of chemicals

 The goal of the study is to validate the utility of the 3D human in vitro vaginal tissue model as an alternate for rabbit vaginal irritation using a set of coded test articles (TAs). A double-blinded study was conducted in which the investigator and the animal testing facility were provided with N=14 coded TAs and assayed in vitro and in vivo following topical application at 2% dose of each TA and 5 repeat exposures over 6 days. Dose volumes were proportionally adjusted based on the surface area and N=5 rabbits and N=3 EpiVaginal tissues were used per TA. The RVI score was used to monitor in vivo irritation; MTT, TEER, and histological analysis were used as endpoints for the in vitro assays. The results showed that four TAs including two known irritants, benzalkonium chloride(BZK) and nonoxynol9(N9), were predicted as irritants by MTT viability and TEER (<50%reduction). While BZK was identified as a mild/severe irritant in the RVI assay, the effect of N9 in rabbits was highly variable-in three independent animal RVI studies, irritation was detected only in 25-30% of the animals. Furthermore, the other two TAs, copper sulfate and sodium dodecylsulfate(SDS), that were identified as vaginal irritants in vitro, were not identified as irritants in the RVI test. Cytokine analysis from culture supernatants showed that N9, SDS and BZK induced a significant increase (>2fold)inIL-1α/IL-1β release by EpiVaginal tissues. In contrast, only BZK showed a significant increase in IL-1α/IL-1β from rabbit vaginal lavages. Based on the in vitro in vivo data, we expanded the study to include N=55 test articles and the in vitro reproducibility of the results for the 55 test articles were monitored using reconstructed tissues from cells obtained from N=4 donors(1). In short, a combination of MTT, TEER, and IL-1α/IL-1β were found to be valuable makers of in vitro vaginal irritation. In conclusion, the in vitro assay method performs equally well or better compared to the standard RVI assay and could be a useful tool to assess vaginal toxicity of formulations, drugs, and medical devices.

EpiVaginal (VEC-100), rabbit vaginal irritation (RVI) test, RVI score, feminine hygiene products, IL-1a, IL-1b, IL-8, TEER, Saline, Maltose, Nonoxynol 9 (N9), Nonoxynol 9, Beta cyclodextrin, Benzalkonium chloride, Adonitol, Aluminum sulfate, Sodium bicarbonate, Lactic acid, Betadine, Copper sulfate, Diacetin, Sodium dodecyl sulfate, Dulcitol, Calcium acetate (2%), Cellulose (2%), Gynol II (2% N9), SDS (2%), Bentonite (2%), Urea (2%), Calcium lactate (2%), Corn oil (2%), Benzalkonium chloride (BZK, 2%), Saline, Citric acid (2%)

Saline, Maltose, Nonoxynol 9 (N9), Nonoxynol 9, Beta cyclodextrin, Benzalkonium chloride, Adonitol, Aluminum sulfate, Sodium bicarbonate, Lactic acid, Betadine, Copper sulfate, Diacetin, Sodium dodecyl sulfate, Dulcitol, Calcium acetate (2%), Cellulose (2%), Gynol II (2% N9), SDS (2%), Bentonite (2%), Urea (2%), Calcium lactate (2%), Corn oil (2%), Benzalkonium chloride (BZK, 2%), Saline, Citric acid (2%)