Open clinical trial evaluating the efficacy of a novel eyelash growth enhancer with peptides and glycosaminoglycans

Eyelashes play a crucial role in self‐image and ocular protection. Enhancements to their structure are of both cosmetic and clinical interest.

Indians have employed various materials like Kohl not only to accentuate but also to protect these delicate hair structures. 2 In modern times, this tendency has manifested in the form of multiple beauty products such as mascara, eyeliners, and extensions, for example. 1 Eyelash mascaras and extensions, commonly employed to enhance the beauty of these fine structures, are not without their limitations.While they can accentuate the prominence and allure of lashes, they can also induce ocular complications, such as allergic contact dermatitis. 1,6Likewise, the recent use of prostaglandins like bimatoprost ophthalmic solution 0.03% (Latisse®; Allergan®, Inc.), as effective agents for eyelash growth presents its own set of challenges. 7Similarly, cosmetic eyelash lifting, another popular method for enhancing eyelash appearance, may entail the risk of rare but serious complications such as preseptal cellulitis. 8This confluence of benefits and potential drawbacks underscores the growing need for eyelash enhancers that can naturally augment and beautify lashes without associated risks.
The cosmetic industry offers numerous products aimed at beautifying eyelashes, yet there's a notable lack of comprehensive clinical studies examining their safety and effectiveness. 9Many products enter the market based on consumer reviews rather than solid scientific evidence.This open, non-randomized study intends to address this need, providing evidence supporting a cosmetic formulation that meets both aesthetic and medical standards.Our aim is to contribute a methodologically-sound perspective to the broader narrative of eyelash enhancement.

| Design
This was an open-label, prospective study designed to evaluate the efficacy of a growth serum combining WGC® (Wharton Gel Complex®) and SymPeptide XLash®.The primary objective of the study was to determine the serum's effectiveness by noting improvements in the Growth Assessment Score (GAS) in study subjects.The secondary objective was to evaluate the formulation's efficacy in determining improvements in the following instrumental values: number, width, length, volume, arc, and angle.Furthermore, formulation acceptability objectives sought an 80% approval rating in a subjective patient questionnaire, along with dermatological and ophthalmological safety and tolerance assessments.
Daily application under standardized conditions was carried out for a total of 12 weeks (D84), with visits at 4 weeks (D28), and 8 weeks (D56).

| Formulation under study
The formulation being evaluated in this study, provided by Cantabria Labs, consisted of an eyelash booster with components, notably SymPeptide XLash®, known for its eyelash-lengthening properties, and WGC® (Histocell), which has been demonstrated to activate the WNT/β-catenin pathway.WGC® is a specialized compound with profound implications in the field of trichology, particularly in the context of hair and eyelash growth.Its importance lies in its ability to influence the WNT/β-catenin pathway, a fundamental signaling cascade that plays a pivotal role in the regulation of various tissue development processes. 10In the context of hair growth and trichology, WNT/β-catenin signaling is of particular interest due to its profound impact on the hair follicle cycle.The pathway's activation has been linked to the initiation of the anagen phase, a critical stage in the hair follicle's growth cycle 11 The final formulation was vehicled in a delicately scented, clear gel, specifically designed for daily eyelash application.The experimental conditions adopted (areas of application, quantity applied, frequency, duration, etc.) reproduced the normal conditions of use of the test formulation, and the test performed on this "small scale" reflected future application by users.

| Volunteers
The study initially enlisted 33 female volunteers conforming to inclusion criteria (see Table 1).The study focused solely on female participants, reflecting the predominance of this demographic in the consumption of eyelash enhancer products.Subjects were assigned ordinal identification numbers from 1 to 33 based on recruitment dates.The cohort size was adequate for statistically significant determinations, as validated by the Granmo Calculator software from Institut Municipal d'Investigació Mèdica, Barcelona, Spain.
Volunteers were instructed to apply the formulation at the base of the eyelashes using the fine brush included, once a day, preferably at night, allowing it to dry following application.

| Instrumental variables assessment
The measurements were conducted by the Clinical Project Manager of the research center.To evaluate the primary instrumental variables (number, width, length, volume, arc, and curvature), comprehensive measurements of eyelashes were recorded from the Region of Interest (ROI, two eyes) for each volunteer.A Nikon D5600 camera, installed on a HeadScan Bench Light Face was used for precise and stable positioning and re-positioning at the different time points.Analysis of the 2D images captured was conducted using FrameScan NG-48 software analysis (Orion TechnoLab, France).The measurements were carried out prior to the start of the treatment and at each of the established follow-up visits.
FrameScan NG-48, a dedicated software for image analysis, was used to quantitatively assess the colorimetric and morphological characteristics of calibrated digital photographic images of the eyelashes.Segmentation techniques were employed for in-depth analysis, following each lash to its often-depigmented extremities.
Eyelash growth instrumental parameter evaluation is described in Figure 1.
• Number (N): Number of eyelashes crossing the middle arc.
• Width (px): Mean width of eyelashes crossing the middle arc (in pixels).
• Length (px): Mean of all arc heights (in pixels).
• Arc (px): Total number of pixels inside the red area.
• Eyelash curvature: For curvature assessment, 'active contours or snakes' method was employed.This involved defining a curved line on the lashes for profile acquisition, and another line on the eyelid, determined by three points.The deviation of these lines served as a measure of lash curvature alteration (Figure 2).

| Investigator assessment
During each visit, the evaluators were tasked with classifying the volunteers according to the GEA scale, assigning them a value between 1 and 4.This process involved a subjective appraisal by the investigator to determine the clinical improvement of the eyelashes, assessing enhancements in terms of their prominence and overall appearance.The GEA scale serves as a robust and consistent tool that enables evaluators to assess the overall prominence of a patient's upper eyelashes, taking into account attributes such as their length, thickness, and color intensity.This scale spans from a level of 1, indicating minimal prominence, to a level of 4, denoting a very pronounced appearance. 12To aid evaluators in their assessments, the scale was accompanied by a photo-based numerical guide.

| Individual questionnaire
The efficacy of the treatment was subjectively assessed by participants via an individual questionnaire filled in at each of the timepoints (D28, D56, and D84).The questionnaire consisted in 19 items related to sensory experience and treatment effectiveness.The items included in the questionnaire are listed in Table 2.
For each item, volunteers expressed their satisfaction according to an ordinal scale (1, strongly disagree; 2, disagree; 3, agree; 4, strongly agree), and the results were expressed in percentage of satisfied volunteers (scores 3 and 4).Overall satisfaction was quantified by summing the satisfaction percentages for each queried item within the questionnaire and then calculating the mean.

| Ethics statement
This study was performed under clinical surveillance at the testing facilities.Experimental controls are the same participants whose parameters are evaluated upon product application, prior to initiate the treatment.This study was conducted according to the general conditions of Dermaclaim Lab S.L., established for research studies involving human volunteers.
Before the beginning of the study, Dermaclaim internally assessed and approved its suitability for the product type and the methodology to be used.The study aimed at a better knowledge of the efficacy of the test product and the foreseeable risk incurred by the volunteers who took part in the study were minor, thus, there was a suitability between the aim of the study and its possible risks.Given that the formulation was a cosmetic, strict adherence to the required criteria and methodology for such studies was followed; consequently, no ethical committee approval was necessary.

| Statistical analysis
All the 30 volunteers completing the study were considered to assess the efficacy of the test product.For statistical analysis of the panelist self-assessment, satisfaction was considered for scores 3 and 4, and the threshold to consider a remarkable satisfaction percentage was set at 80%.

| Demographic results
Of the 33 patients enrolled in the study, 30 completed it.We recorded three dropouts, We recorded three dropouts, none of which were due to adverse events but rather to causes unrelated to the study.Additionally, two volunteers, although outside the established age range (25-65), were included, as they satisfied all other inclusion criteria, allowing our study to start as scheduled.The demographic data and individual characteristics of the volunteers are featured in Table 3.

| Instrumental results
The following section presents the results of the instrumental variables measured at each visit.The percentage change for each parameter was calculated versus the initial (baseline) visit, highlighting those increases that reached statistical significance.The data not only reflect the quantitative improvement of the variables but also allow for the monitoring of the treatment's evolution and efficacy over time.These outcomes are graphically represented in Figure 3 and the measured values are reflected in Table 4. Furthermore, Notable improvement was also recorded after 4 week (D28) with a 6.0% increase (p < 0.001).

| Individual questionnaire
The eyelash booster demonstrated high acceptance across several parameters as the trial progressed (see Table 5).By D84, 15 out of 19 parameters scored an acceptance rate of ≥80%.Participants found the formulation easy to apply, did not report discomfort post-application, and found the texture and color pleasant across all time points.Significant perceptions in improvement were reported in parameters such as reduced eyelash loss and denser, more moisturized eyelashes from D56 onwards.These subjective assessments reflect the overall high satisfaction rates reported at D56 and D84, reinforcing the product's perceived efficacy.While our predefined threshold for satisfaction was set at 80%, several parameters demonstrated notable acceptance levels, although below this benchmark.Specifically, the observed increase in eyelash length received 61% approval, eyelash density and perceived thickness both achieved 68% satisfaction, and the strength of eyelashes was positively rated at 65%.These results indicate a significant level of participant endorsement, despite not reaching the set satisfaction benchmark.Patient perception and overall satisfaction of treatment was 73.34% at D28, increasing to 84.33% at D84.

| Dermatological and ophthalmological control
Participants showed no Serious Undesirable Effects (SUE) and declared neither discomfort nor Undesirable Effects (UE) throughout the period of treatment or the following 10 days.The product of the study underwent rigorous testing, including tolerance, clinical, and ophthalmological assessments.These tests, conducted under the supervision of qualified professionals, demonstrated the serum's high tolerance and efficacy.

| DISCUSS ION
Eyelashes transcend their primary defensive role for the eyes and have been celebrated as emblems of beauty and youth across various cultures. 2,4Historical practices date back to around 3000 B.C., when the application of Kohl on the eyelids to accentuate lashes was prevalent.
The human lower eyelid typically features 75-80 lashes arrayed across three to four rows, whereas the upper eyelid has 60-90 lashes over five to six rows.The architecture of an eyelash shares similarities with scalp hair, being formed by a hair shaft that extends above the skin, a root beneath the skin, and an enlarged terminal bulb.The bulb's lower segment makes direct contact with the dermal TA B L E 4 Values represent outcomes of instrumental variables across visits D0 (Baseline), D28 (28 days), D56 (56 days), and D84 (84 days).
A 2006 study provides some comparative data, examining Asian and Caucasian women's eyelashes through digital imaging and phototrichogram analysis.It reported that Asian participants had lashes that were thicker and more numerous, yet featured less elevation and curl than those of their Caucasian counterparts. 13,14The desire to enhance the beauty and functionality of lashes, and thus that of the eyes, has led to the development of numerous interventions, including temporary and permanent make-up, growth boosters and artificial appendages.This study sought to evaluate the effectiveness of a growth booster in improving lash aesthetics by analyzing the evolution of several parameters considered to be representative of lash beauty.In the analysis of eyelash number following the application of the eyelashes booster, the results depicted subtle enhancements at D28 and D84.This pattern could be attributed to the product's physiological influence on the lash growth cycle, where certain phases might not readily manifest notable numerical differences. 2,4It's also plausible that the observational metrics for other attributes, such as volume or length, capture visible changes more readily than simple lash count.
An incremental improvement in eyelash length was also detected.Although the percentages of improvement were less than 10%, it is crucial to contextualize these findings within the broader scope of lash functionality beyond mere aesthetic enhancements.An in vitro study on the aerodynamics of eyelash structure has shown their essential role in eye protection.
Utilizing a mammalian eye model in a wind tunnel simulation to assess airflow dynamics, researchers identified that the ideal eyelash length is one-third of the eye's width-a measurement significantly reducing tear evaporation and deposition of foreign particles. 5,15This optimal length, determined by biomechanical efficiency, suggests that while longer lashes might be aesthetically favored, they could impede their protective function if they exceed this proportional threshold.
Besides, regarding eyelash functionality and aerodynamics, it was not solely the length but also the curvature and arc of the eyelashes that played a pivotal role in preserving this functionality.
Consequently, our study documented a notably substantial increase in both curvature and arc, suggesting that these enhancements serve to improve not just the aesthetic appearance but also the functional efficacy of the eyelashes. 5This correlation underscores the dual benefit of our product, accentuating the lashes' protective role against environmental elements while simultaneously enhancing their visual appeal.
Beyond the crucial instrumental variables used for objectifying outcomes, the Global Eyelashes Assessment (GEA) results revealed a statistically significant improvement from the initial assessment, culminating notably at D84.While individual instrumental results may present diverse manifestations, when assessed collectively, they underscore that the use of the eyelash booster can in fact lead to a clinically perceptible improvement.
On the other hand, another noteworthy aspect, although not quantitatively assessed, is the broader impact of cosmetic products on daily routines.Many women reported that the visible enhancement of their lashes reduces their reliance on mascara to achieve the desired effect, an outcome that is likely to be highly appreciated.
In terms of the safety and tolerability of the product under investigation, no adverse effects were observed and, overall, the tolerability was well received by the volunteer participants included in the study.
This stands in stark contrast to some commonly employed methods for enhancing eyelashes. 1,6,16For instance, prostaglandins, despite being an approved and effective treatment, can lead to rejection and poor adherence due to side effects such as iris pigmentation. 17Meanwhile, other commonly used alternatives such as extensions and false lashes offer rapid and cost-effective results.However, emerging evidence underscores the associated risks, notably the onset of contact eczema and material sensitization linked to adhesives and lash materials. 1,6Extensions, made from materials ranging from sable to synthetic silk, are attached using cyanoacrylate-based adhesives, potential culprits for contact dermatitis, with the capability to release formaldehyde, a known allergen.Additionally, eyelash tinting products often contain harmful agents such as p-phenylenediamine, ammonia, and coal tar, presenting further risks.
Presenting minimal to moderate eyelashes (Grades 1-2, GEA scale) Last participation in a clinical study ending at least 1 month before the start of this study Wash-out period of 15 days before start of treatment with no application on the study area Individuals who understand the instructions of use and are willing to cooperate with the study Reading, understanding, and signature of the Informed Consent for the study and the Protection of Personal Data and Communication consent Exclusion criteria Subjects with a history of any form of skin cancer, melanoma, lupus, psoriasis, connective tissue disease, diabetes, or any disease that would increase risk associated with study participation Relevant cutaneous marks in the experimental area which could interfere with the instrumental measurements or the clinical pictures Allergy or reactivity to any of the components of the test formulation Individuals currently undergoing medical treatment that may mask or interfere with test results Relevant aesthetic treatments (eyelash extensions, permanent eyelid tattooing, eyelash dyeing, etc.) on the experimental area Relevant aesthetic treatments (cleansing and exfoliation, mesotherapy, led-light therapy, carboxytherapy, microneedling, etc.), on the experimental area less than 3 months before start of study Women who are pregnant or lactating or have been pregnant or given birth within the 6-month period immediately preceding start of study The study protocol was in accordance with the Scientific Committee on Consumer Safety (SCCS) guidance (SANTE-SCCS AT ec.europa.eu).It meets all international standards for research studies involving human subjects, Structure and Content of Clinical Study Reports from ICH Harmonized Tripartite Guideline; International Recommendations ICH Topic E6, European Parliament and Council Guideline 2001/20/CE, the Good Clinical Practices (ICH-GCP), and the World Medical Association.It has been conducted pursuant to the Declaration of Helsinki (1964), with the amendments of Tokyo (1975), Venice (1983), Hong Kong (1989), South Africa (1996), Edinburgh (2000), Seoul (2008), and Fortaleza (2013).

2. 6 . 2 |
Informed consent and good clinical practicesInformed consent forms describing reasons for the study, possible adverse effects, associated risks and potential benefits, and their limits of liability were obtained from volunteers prior to initiating the study.The participants read comprehensively, signed, and dated the informed consent document to indicate their authorization to proceed and acknowledge their understanding of the contents.

ForF I G U R E 1 F I G U R E 2
instrumental measurements, parameter values in the experimental area of an individual at a given time point were normalized versus baseline measurements of the whole panel.This way, the inter-individual random variation is corrected, and the statistical power of the result is increased.The individual results were expressed in percentage relative to baseline values (D0) for all the parameters studied.Mean ± StandardError of the Mean (SEM) were calculated and shown in the graphs.Eyelash growth parameters.Depiction of the different parameters associated with eyelash growth evaluated during the image analysis with FrameScan NG-48.Eyelash length was quantified in pixels, determined by the height of the yellow vectors on the image.The 'arc' represents the total area outlined in red, encompassing the curvature of the eyelash growth area.The 'volume' refers to the pixels corresponding to the dark regions of the eyelashes within the arc.Lastly, the 'number' denotes the count of individual eyelashes that extend beyond the blue line (middle arc).The width was calculated as the average width, in pixels, of all eyelashes surpassing the blue line (middle arc).Curvature is determined by superimposing a curved line along the eyelashes (red line marked by 'point 1, point 2, and point 3') and another on the eyelid, with three distinct points defining these lines.GraphPad Prism V9 software was used for the statistical analysis.Data were statistically analyzed applying one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test.Robust regression and Outlier removal (ROUT) method was used to identify outliers in the raw data, with a coefficient Q of 1%.

Figures 4 and 5
Figures 4 and 5 provide a detailed picture of the specific results obtained from a selected volunteer, offering a tangible example of the individual changes experienced.

F I G U R E 4
Progressive changes in eyelash appearance over 12 weeks of treatment with Eyelash booster in volunteer 31.The images display a marked improvement in eyelash number, width, length, volume, and arc at 28 days (D28), 56 days (D56), and 84 days (D84) compared to baseline (D0).F I G U R E 5 Sequential lateral views of eyelashes of both eyes over a 12-week period with Eyelash booster in volunteer 31.The comparative photographs at baseline (D0), 28 days (D28), 56 days (D56), and 84 days (D84) demonstrate a noticeable increase in the curvature of the eyelashes with continued product use.
Summary of demographic results.