Developmental estrogen exposure in mice disrupts uterine epithelial cell differentiation and causes adenocarcinoma via Wnt/ß-catenin and PI3K/AKT signaling.

Tissue development entails genetically programmed differentiation of immature cell types to mature, fully differentiated cells. Exposure during development to non-mutagenic environmental factors can contribute to cancer risk, but the underlying mechanisms are not understood. We used a mouse model of endometrial adenocarcinoma that results from brief developmental exposure to an estrogenic chemical, diethylstilbestrol (DES), to determine causative factors. Single-cell RNA sequencing (scRNAseq) and spatial transcriptomics of adult control uteri revealed novel markers of uterine epithelial stem cells (EpSCs), identified distinct luminal and glandular progenitor cell (PC) populations, and defined glandular and luminal epithelium (LE) cell differentiation trajectories. Neonatal DES exposure disrupted uterine epithelial cell differentiation, resulting in a failure to generate an EpSC population or distinguishable glandular and luminal progenitors or mature cells. Instead, the DES-exposed epithelial cells were characterized by a single proliferating PC population and widespread activation of Wnt/ß-catenin signaling. The underlying endometrial stromal cells had dramatic increases in inflammatory signaling pathways and oxidative stress. Together, these changes activated phosphoinositide 3-kinase/AKT serine-threonine kinase signaling and malignant transformation of cells that were marked by phospho-AKT and the cancer-associated protein olfactomedin 4. Here, we defined a mechanistic pathway from developmental exposure to an endocrine disrupting chemical to the development of adult-onset cancer. These findings provide an explanation for how human cancers, which are often associated with abnormal activation of PI3K/AKT signaling, could result from exposure to environmental insults during development.

Chromatin architectural factor CTCF is essential for progesterone-dependent uterine maturation.

Receptors for estrogen and progesterone frequently interact, via Cohesin/CTCF loop extrusion, at enhancers distal from regulated genes. Loss-of-function CTCF mutation in >20% of human endometrial tumors indicates its importance in uterine homeostasis. To better understand how CTCF-mediated enhancer-gene interactions impact endometrial development and function, the Ctcf gene was selectively deleted in female reproductive tissues of mice. Prepubertal Ctcfd/d uterine tissue exhibited a marked reduction in the number of uterine glands compared to those without Ctcf deletion (Ctcff/f mice). Post-pubertal Ctcfd/d uteri were hypoplastic with significant reduction in both the amount of the endometrial stroma and number of glands. Transcriptional profiling revealed increased expression of stem cell molecules Lif, EOMES, and Lgr5, and enhanced inflammation pathways following Ctcf deletion. Analysis of the response of the uterus to steroid hormone stimulation showed that CTCF deletion affects a subset of progesterone-responsive genes. This finding indicates (1) Progesterone-mediated signaling remains functional following Ctcf deletion and (2) certain progesterone-regulated genes are sensitive to Ctcf deletion, suggesting they depend on gene-enhancer interactions that require CTCF. The progesterone-responsive genes altered by CTCF ablation included Ihh, Fst, and Errfi1. CTCF-dependent progesterone-responsive uterine genes enhance critical processes including anti-tumorigenesis, which is relevant to the known effectiveness of progesterone in inhibiting progression of early-stage endometrial tumors. Overall, our findings reveal that uterine Ctcf plays a key role in progesterone-dependent expression of uterine genes underlying optimal post-pubertal uterine development.

Scientific and Regulatory Policy Committee Points to Consider: Primary Digital Histopathology Evaluation and Peer Review for Good Laboratory Practice (GLP) Nonclinical Toxicology Studies.

The Society of Toxicologic Pathology's Scientific and Regulatory Policy Committee formed a working group to consider the present and future use of digital pathology in toxicologic pathology in general and specifically its use in primary evaluation and peer review in Good Laboratory Practice (GLP) environments. Digital histopathology systems can save costs by reducing travel, enhancing organizational flexibility, decreasing slide handling, improving collaboration, increasing access to historical images, and improving quality and efficiency through integration with laboratory information management systems. However, the resources to implement and operate a digital pathology system can be significant. Given the magnitude and risks involved in the decision to adopt digital histopathology, this working group used pertinent previously published survey results and its members' expertise to create a Points-to-Consider article to assist organizations with building and implementing digital pathology workflows. With the aim of providing a comprehensive perspective, the current publication summarizes aspects of digital whole-slide imaging relevant to nonclinical histopathology evaluations, and then presents points to consider applicable to both primary digital histopathology evaluation and digital peer review in GLP toxicology studies. The Supplemental Appendices provide additional tabulated resources.

Scientific and Regulatory Policy Committee Brief Communication: 2019 Survey on Use of Digital Histopathology Systems in Nonclinical Toxicology Studies.

Histopathologic evaluation and peer review using digital whole-slide images (WSIs) is a relatively new medium for assessing nonclinical toxicology studies in Good Laboratory Practice (GLP) environments. To better understand the present and future use of digital pathology in nonclinical toxicology studies, the Society of Toxicologic Pathology (STP) formed a working group to survey STP members with the goal of creating recommendations for implementation. The survey was administered in December 2019, immediately before the COVID-19 pandemic, and the results suggested that the use of digital histopathology for routine GLP histopathology assessment was not widespread. Subsequently, in follow-up correspondence during the pandemic, many responding institutions either began investigating or adopting digital WSI systems to reduce employee exposure to COVID-19. Therefore, the working group presents the survey results as a pre-pandemic baseline data set. Recommendations for use of WSI systems in GLP environments will be the subject of a separate publication.

Phthalate Toxicity in Rats and Its Relation to Testicular Dysgenesis Syndrome in Humans.

This work describes the relevance of toxicology studies of environmental chemicals, with a focus on phthalates, for a hypothesis that certain human male reproductive disorders and diseases have a common etiology of disturbance of normal development in utero. The "Testicular Dysgenesis Syndrome" hypothesis in humans has parallels in male reproductive tract abnormalities and microscopic lesions reported for phthalate toxicity in rats. Additionally, this work describes the histological findings of abnormal testicular development (testicular dysgenesis) in rats as compared to those in humans, as well as potential findings in rats at different ages, from the embryo to the adult.

Whole-body inhalation exposure to 2-ethyltoluene for two weeks produced nasal lesions in rats and mice.

OBJECTIVE: Ethyltoluenes are isolated during crude oil refinement for use in gasoline and commercial products and are ubiquitous in the environment. However, minimal toxicity data are available. Previously, we identified 2-ethyltoluene (2-ET) as the most potent isomer via nose-only inhalation exposure in rodents. Here, we expanded the hazard characterization of 2-ET in two rodent models using whole-body inhalation exposure and evaluated the role of prenatal exposure. METHODS: Time-mated Hsd:Sprague Dawley® SD® rats were exposed to 0, 150, 300, 600, 900, or 1200 ppm 2-ET via inhalation starting on gestation day 6 until parturition. Rat offspring (n = 8/exposure/sex) were exposed to the same concentrations as the respective dams for 2 weeks after weaning. Adult male and female B6C3F1/N mice (n = 5/exposure/sex) were exposed to the same concentrations for 2 weeks. RESULTS AND DISCUSSION: Exposure to ≥600 ppm 2-ET produced acute toxicity in rats and mice characterized by large decreases in survival, body weight, adverse clinical observations, and diffuse nasal olfactory epithelium degeneration (rats) or necrosis (mice). Due to the early removal of groups ≥600 ppm, most endpoint evaluations focused on lower exposure groups. In 150 and 300 ppm exposure groups, reproductive performance and littering were not significantly changed and body weights in exposed rats and mice were 9-18% lower than controls. Atrophy of the olfactory epithelium and nerves was observed in all animals exposed to 150 and 300 ppm. These lesions were more severe in mice than in rats. CONCLUSION: Nasal lesions were observed in all animals after whole-body exposure up to 600 ppm 2-ET for 2 weeks. Future studies should focus on 2-ET metabolism and distribution to better understand species differences and refine hazard characterization of this understudied environmental contaminant.

A Phase 2 Open-Label Study to Evaluate VP-102 for the Treatment of Molluscum Contagiosum.

BACKGROUND: This Phase 2, open-label study evaluated the safety, efficacy, systemic exposure, and impact on quality of life (QoL) with treatment using VP-102, a drug-device combination containing cantharidin (0.7% w/v) in subjects with molluscum contagiosum (MC). STUDY DESIGN: Pediatric subjects with MC (2–15 years of age) were eligible to enroll in this 12-week study. MC lesions were treated topically with VP-102 every 21 days until clearance (maximum of 4 treatments). Adverse events (AEs) and QoL outcomes (using the Children's Quality of Life Index, CDLQI) were documented at each visit. Rate of complete clearance and the percent reduction in lesions were measured at each visit on days 21, 42, 63, and 84 (end of study [EOS] visit). A group of 17 subjects with at least 21 MC lesions was evaluated for systemic cantharidin exposure via plasma samples obtained before the first application of VP-102, and at 2 hours, 6 hours, and 24 hours post-application. RESULTS: A total of 33 subjects enrolled in the study (n=17 systemic exposure group, n=16 standard group). There were an equal number of male and female subjects. Subject mean (SD, range) age was 6.7 (3.3, 2–15) years, with a mean lesion count of 30 (26.1, 3–113). Complete lesion clearance was achieved in 48.5% of subjects, with a 90.4% reduction in lesions from baseline to the EOS visit. Mean CDLQI score decreased from 2.6 at baseline to 0.38 at the EOS visit. AEs were mild to moderate in severity and expected due to the pharmacodynamic action of cantharidin. There were no serious treatment-related adverse events and no study discontinuations due to treatment. In the systemic exposure group plasma cantharidin levels were below the lower limit of quantitation (LLOQ, 2.5 ng/mL) in 65 of 66 samples. CONCLUSIONS: VP-102 treatment resulted in a reduction in lesion counts and improved QoL. Treated subjects had a 48.5% rate of complete clearance of molluscum lesions. Negligible systemic cantharidin exposure was observed in the systemic exposure group. This data demonstrates safety and efficacy of treatment with VP-102 in MC; a widespread viral infection that does not have any current FDA-approved treatments. Significant Finding: Treatment of subjects with MC using VP-102 resulted in negligible systemic cantharidin exposure, as well as a reduction in lesion counts, improved QoL, and a demonstrated efficacy in clearance of new and baseline MC lesions. Meaning: Results of this Phase 2 study demonstrate efficacy and safety outcomes in using VP-102 in MC subjects, and large randomized clinical trials are warranted to compare topical VP-102 with a vehicle control in order to fully evaluate the use of the medication. ClinicalTrials.gov identifier: NCT03186378 J Drugs Dermatol. 2021;20(1):70-75. doi:10.36849/JDD.5626.

Noncanonical autophagy in dermal dendritic cells mediates immunosuppressive effects of UV exposure.

BACKGROUND: Control of the inflammatory response is critical to maintaining homeostasis, and failure to do so contributes to the burden of chronic inflammation associated with several disease states. The mechanisms that underlie immunosuppression, however, remain largely unknown. Although defects in autophagy machinery have been associated with inflammatory pathologic conditions, we now appreciate that autophagic components participate in noncanonical pathways distinct from classical autophagy. We have previously demonstrated that LC3-associated phagocytosis (LAP), a noncanonical autophagic process dependent on Rubicon (rubicon autophagy regulator [RUBCN]), contributes to immunosuppression. OBJECTIVE: We used Rubcn-/- mice to examine the role of the LAP pathway in mediating the UV-induced immunotolerant program in a model of contact hypersensitivity (CHS). METHODS: Flow cytometry and transcriptional analysis were used to measure immune cell infiltration and activation in the skin of Rubcn+/+ and Rubcn-/- mice during the CHS response. RESULTS: Here, we demonstrate that LAP is required for UV-induced immunosuppression and that UV exposure induces a broadly anti-inflammatory transcriptional program dependent on Rubicon. Rubcn-/- mice are resistant to UV-induced immunosuppression and instead display exaggerated inflammation in a model of CHS. Specifically, RUBCN deficiency in CD301b+ dermal dendritic cells results in their increased antigen presentation capacity and subsequent hyperactivation of the CD8+ T-cell response. CONCLUSIONS: LAP functions to limit the immune response and is critical in maintaining the balance between homeostasis and inflammation.

National Toxicology Program Position Statement on Informed ("Nonblinded") Analysis in Toxicologic Pathology Evaluation.

The National Toxicology Program (NTP) uses histopathological evaluation of animal tissues as a key element in its toxicity and carcinogenicity studies. The initial histopathological evaluations are subjected to a rigorous peer review process involving several steps. The NTP peer review process is conducted by multiple, highly trained, and experienced toxicological pathologists employing standardized terminology. In addition, ancillary data, such as body and organ weights and clinical pathology findings, are used to corroborate the diagnoses. The NTP does employ masked analysis to confirm subtle lesions or severity scores, as needed, and during its Pathology Working Groups. The use of masked analysis can have a negative effect on histopathological evaluation because it is important for the pathologist to compare treated groups to the concurrent controls, which would not be possible in a blinded evaluation. Therefore, the NTP supports an informed approach to histopathological evaluation in its toxicity and carcinogenicity studies.

Proceedings of the 2019 National Toxicology Program Satellite Symposium.

The 2019 annual National Toxicology Program Satellite Symposium, entitled "Pathology Potpourri," was held in Raleigh, North Carolina, at the Society of Toxicologic Pathology's 38th annual meeting. The goal of this symposium was to present and discuss challenging diagnostic pathology and/or nomenclature issues. This article presents summaries of the speakers' talks along with select images that were used by the audience for voting and discussion. Various lesions and topics covered during the symposium included aging mouse lesions from various strains, as well as the following lesions from various rat strains: rete testis sperm granuloma/fibrosis, ovarian cystadenocarcinoma, retro-orbital schwannoma, periductal cholangiofibrosis of the liver and pancreas, pars distalis hypertrophy, chronic progressive nephropathy, and renal tubule regeneration. Other cases included polyovular follicles in young beagle dogs and a fungal blood smear contaminant. One series of cases challenged the audience to consider how immunohistochemistry may improve the diagnosis of some tumors. Interesting retinal lesions from a rhesus macaque emphasized the difficulty in determining the etiology of any particular retinal lesion due to the retina's similar response to vascular injury. Finally, a series of lesions from the International Harmonization of Nomenclature and Diagnostic Criteria Non-Rodent Fish Working Group were presented.

Differentiating between Testicular Toxicity and Sexual Immaturity in Ortho-phthalaldehyde Inhalation Toxicity Studies in Rats and Mice.

Early deaths of young or juvenile animals (before sexual maturation is achieved) in routine regulatory safety studies present pathologists and toxicologists with the challenge of interpreting findings in the male reproductive tract. Additionally, the advent of toxicity testing regulations has resulted in a growing need for the use of juvenile animals in toxicology studies. Here, we present the reproductive toxicity findings from a 13-week inhalation toxicity study with ortho-phthalaldehyde (OPA) in male rats and mice as a case example for working through this challenging task. In this study with OPA, survival was significantly reduced in the two highest exposure concentrations of OPA tested. Early deaths and histopathological lesions in the testes and epididymides were generally also limited to these two highest exposure groups. Therefore, there was concern that peripubertal morphological features could be a confounding factor for the histopathological evaluation of exposure-related testicular and epididymal findings. Although it can be difficult to differentiate exposure-related effects from the normal morphological features defining peripubertal changes in the testes and epididymides in animals that die early in a toxicity study, the use of age-matched controls in this case study with OPA provided a reference and aided in the differentiation of these effects.

Proceedings of the 2018 National Toxicology Program Satellite Symposium.

The 2018 annual National Toxicology Program Satellite Symposium, entitled "Pathology Potpourri," was held in Indianapolis, Indiana, at the Society of Toxicologic Pathology's 37th annual meeting. The goal of this symposium was to present and discuss challenging diagnostic pathology and/or nomenclature issues. This article presents summaries of the speakers' talks along with select images that were used by the audience for voting and discussion. Various lesions and other topics covered during the symposium included seminiferous tubule dysgenesis in rats, ameloblast and odontoblast degeneration/necrosis in a Sprague Dawley rat, intestinal leiomyositis in a beagle dog, gallbladder mucinous hyperplasia, focus of hepatocellular alteration and bile duct alteration in otters, renal tubule cytoplasmic vacuolation with basophilic granules in mice treated swith antisense oligonucleotide therapy, a uterine choriocarcinoma in a rhesus macaque, and rete ovarii proliferative ovarian lesions in various aged rat strains. One particularly provocative lesion was a malignant neoplastic proliferation in the renal pelvic region of a cynomolgus macaque from a 21-day study. Additional challenging lesions included thyroid proliferative lesions in zebra fish and gross findings in fish larvae during routine chemical screening. The Rabbit and Minipig International Harmonization of Nomenclature and Diagnostic Criteria Organ Working Groups also presented a series of challenging lesions.

NTP/NIEHS Global Contributions to Toxicologic Pathology.

National Toxicology Program (NTP) pathologists are engaged in important initiatives that have significant global impact. These initiatives build on its leadership in pathology peer review and publications in the areas of toxicologic pathology, clinical pathology, and laboratory animal medicine. Over the past decade, NTP/National Institute of Environmental Health Sciences research initiatives have focused on cancer and noncancer hazard identification, with the goal of understanding cellular and molecular mechanisms of disease. New initiatives of significant global impact include the web-based nonneoplastic lesion atlas and an NTP partnership with international scientists to investigate molecular mechanisms at the whole genome level, which will be used to inform potential mechanisms of environmental exposures in human cancers. Also, we are dedicated to contributing to pathology and toxicology organizations through service on executive committees and editorial boards, participating in international projects and symposiums, and providing training for future leaders in toxicologic pathology. Herein, we provide highlights of our global contributions.

Comparative inhalation toxicity of ethyltoluene isomers in rats and mice.

The C9 alkylbenzenes, composed mostly of ethyltoluenes and trimethylbenzenes, comprise 75-90% of the naphtha fraction of crude oil. Occupational and environmental exposure to C9 alkylbenzenes occur via inhalation. We conducted short-term inhalation studies on the ethyltoluene isomers (2-, 3- or 4-) to select one isomer for more comprehensive studies. Male Hsd:Sprague Dawley rats and female B6C3F1/N mice (n = 10) were exposed by nose-only inhalation to 2-, 3- or 4-ethyltoluene (0, 1000 or 2000 ppm) or cumene (a reference compound: 0, 500 or 1000 ppm) 3 h/day, 5 days/week, for 2 weeks. Clinical observations included abnormal gait and delayed righting reflex. Rats and mice exposed to 2000 ppm 2-ethyltoluene and mice exposed to 2000 ppm 4-ethyltoluene were euthanized early in moribund condition; no exposure-related deaths were observed with 3-ethyltoluene or cumene. Histopathology of selected tissues revealed that the nose and liver (rats and mice) and lung (mice only) to be toxicity targets. In the mouse lung, all compounds except 4-ethyltoluene produced bronchial and bronchiolar hyperplasia. In rats and mice, 2-ethyltoluene was the only compound to produce lesions in the nose and liver: in mice, squamous metaplasia and neutrophilic inflammation of the respiratory epithelium and atrophy and degeneration of the olfactory epithelium were observed in the nose and centrilobular hypertrophy and necrosis were observed in the liver. In rats, 2-ethyltoluene exposure produced atrophy of the olfactory epithelium in the nose and centrilobular necrosis in the liver. Based on mortality, body weight effects and histopathology, the 2-ethyltoluene isomer was the most potent isomer.

Evaluation of the respiratory tract toxicity of ortho-phthalaldehyde, a proposed alternative for the chemical disinfectant glutaraldehyde.

ortho-Phthalaldehyde (OPA) is a high-level chemical disinfectant that is commonly used for chemical sterilization of dental and medical instruments as an alternative to glutaraldehyde, a known skin and respiratory sensitizer. Concern for safe levels of human exposure remains due to a lack of toxicity data as well as human case reports of skin and respiratory sensitization following OPA exposure. The present study evaluated the inhalational toxicity of OPA in Harlan Sprague-Dawley rats and B6C3F1/N mice. Groups of 10 male and female rats and mice were exposed to OPA by whole-body inhalation for 3 months at concentrations of 0 (control), 0.44, 0.88, 1.75, 3.5, or 7.0 ppm. Rats and mice developed a spectrum of lesions at sites of contact throughout the respiratory tract (nose, larynx, trachea, lung), as well as in the skin and eye, consistent with a severe irritant response. In general, histologic lesions (necrosis, inflammation, regeneration, hyperplasia and metaplasia) occurred at deeper sites within the respiratory tract with increasing exposure concentration. As a first site of contact, the nose exhibited the greatest response to OPA exposure and resulted in an increased incidence, severity and variety of lesions compared to a previous study of glutaraldehyde exposure at similar exposure concentrations. This increased response in the nasal cavity, combined with extensive lesions throughout the respiratory tract, provides concern for use of OPA as a replacement for glutaraldehyde as a high-level disinfectant.

Hormone Receptor Expression in Spontaneous Uterine Adenocarcinoma in Fischer 344 Rats.

Most uterine cancers, the most common gynecological malignancies in women in developed countries, are hormone-dependent endometrial adenocarcinomas (EACs) that express estrogen and progesterone receptors. Although rat strains exist with a high spontaneous incidence of EAC, the Fischer 344 (F344) strain, previously one of the most commonly used strains in carcinogenicity testing, is not a high-incidence strain. To better understand the biology of this neoplasm, we assessed estrogen receptor α (ER), progesterone receptor (PR), and Ki-67 expression using immunohistochemistry in spontaneous EAC in 18 F344 rats used as control animals in 2-year National Toxicology Program bioassays. Of the 18 tumors, 9 were well-differentiated and 9 were poorly differentiated. Most tumors, 7/18, were ER+PR+, as observed in women. Of the remainder, 6/18 were ER+PR-, 2/18 were ER-PR+, and 3/18 were ER-PR-. Well-differentiated tumors were ER+ (8/9) more often than poorly differentiated tumors (5/9). The percentage of ER+ tumors (72%) in rats was similar to that seen in women, but rats less frequently had PR+ (50%) tumors than women. The heterogeneous estrogen and progesterone receptor immunophenotypes observed in F344 rats in this study highlight the importance of evaluating hormone receptor expression in animal models used for chemical evaluations.

Utilizing Whole Slide Images for Pathology Peer Review and Working Groups.

This article describes the results of comparisons of digitally scanned whole slide images (WSIs) and glass microscope slides for diagnosis of tissues under peer review by the National Toxicology Program. Findings in this article were developed as a result of the data collected from 6 pathology working groups (PWGs), 1 pathology peer review, and survey comments from over 25 participating pathologists. For each PWG, 6-14 pathologists examined 10-143 tissues per study from 6- and 9-month perinatal studies and 2-year carcinogenicity studies. Overall it was found that evaluation of WSIs is generally equivalent to using glass slides. Concordance of PWG consensus diagnoses based upon review of WSIs versus glass slides ranged from 74% to 100% (median 86%). The intra- and interobserver diagnostic variation did not appear to influence the conclusions of any study. Based upon user opinions collected from surveys, WSIs may be less optimal than glass slides for evaluation of subtle lesions, large complex lesions, small lesions in a large section of tissue, and foci of altered hepatocytes. These results indicate that, although there may be some limitations, the use of WSIs can effectively accomplish the objectives of a conventional glass slide review and definitely serves as a useful adjunct to the conduct of PWGs.

WNK1 is required during male pachynema to sustain fertility.

WNK1 is an important regulator in many physiological functions, yet its role in male reproduction is unexplored. In the male germline, WNK1 is upregulated in preleptotene spermatocytes indicating possible function(s) in spermatogenic meiosis. Indeed, deletion of Wnk1 in mid-pachytene spermatocytes using the Wnt7a-Cre mouse led to male sterility which resembled non-obstructive azoospermia in humans, where germ cells failed to complete spermatogenesis and produced no sperm. Mechanistically, we found elevated MTOR expression and signaling in the Wnk1-depleted spermatocytes. As MTOR is a central mediator of translation, we speculated that translation may be accelerated in these spermatocytes. Supporting this, we found the acrosome protein, ACRBP to be prematurely expressed in the spermatocytes with Wnk1 deletion. Our study uncovered an MTOR-regulating factor in the male germline with potential implications in translation, and future studies will aim to understand how WNK1 regulates MTOR activity and impact translation on a broader spectrum.

COVE-1: A Phase 2, Open-Label Study to Evaluate Efficacy and Safety and the Optimal Regimen of VP-102, a Proprietary Drug-Device Product Containing Topical Cantharidin (0.7% w/v) Under Occlusion for the Treatment of Common Warts.

INTRODUCTION: Verrucae vulgaris, or common warts, is a common skin condition for which there is no US Food and Drug Administration-approved treatment. Compounded cantharidin has been used to treat warts for years but lacks a controlled formulation, consistent application schedule and methods, and robust safety and efficacy studies. VP-102 is a proprietary drug-device combination product containing a topical formulation of 0.7% (w/v) cantharidin in a single-use delivery device. This objective of the phase 2 study was to evaluate the efficacy, safety, tolerability, and optimal regimen of VP-102 in the treatment of common warts. METHODS: In this open-label trial, participants aged ≥ 2 years with one to six common warts were administered VP-102 topically to treatable common warts once every 14 days (Cohort 1) or once every 21 days in conjunction with paring (Cohort 2), for up to four treatments. Participants were evaluated through to day 84 (Cohort 1) or day 147 (Cohort 2). The primary endpoint was the percentage of participants with complete clearance of all treatable common warts (baseline and new) at day 84. Secondary endpoints included percentage of participants achieving complete clearance of all treatable common warts at other visits. Safety assessments included treatment-emergent adverse events (TEAEs), including local skin reactions (LSRs). RESULTS: A total of 21 and 35 participants were enrolled in Cohort 1 and Cohort 2, respectively. Complete clearance at day 84 was seen in 19.0% of participants in Cohort 1 and 51.4% of those in Cohort 2. The most common TEAEs were expected LSRs and included application site vesicles, pain, pruritus, erythema, and scab. Most LSRs were mild or moderate in severity. CONCLUSION: VP-102 showed efficacy in complete clearance of common warts from baseline to day 84, as well as at follow-up visits. Due to the higher percentage of patients exhibiting complete clearance in Cohort 2, the treatment regimen of Cohort 2 will be pursued in future studies. TEAEs were expected due to the pharmacodynamic action of cantharidin, a vesicant. Clinical Trials ID: NCT03487549.