## 2 Materials and Methods  
In this prospective study conducted at the Postgraduate School of Orthodontics at the University of Ferrara, a total of 10 patients (3 males and 7 females; mean age: 28 years) were selected based on defined inclusion and exclusion criteria (Table 1). The study protocol was approved by the University of Ferrara Postgraduate School Ethics Committee (registration number 6/2024), and the research was conducted in conformity with the Declaration of Helsinki. Following the collection and review of each participant's diagnostic records, a customized digital setup was developed for each case by a certified orthodontic specialist. This process included 3D intraoral scanning, individualized treatment planning, and aligner design using dedicated orthodontic CAD software (Rhinoceros 3D, Version 6.0, Robert McNeel & Associates, Seattle, WA, USA). The final models were used to fabricate the aligners via selective laser sintering (SLS) 3D printing, using a medical-grade polyamide material, following the proprietary production protocol established by the manufacturer (NOXI, Sweden & Martina, Due Carrare, Padova, Italy). Each patient was instructed to wear two pairs of NOXI aligners (Sweden & Martina, Due Carrare, Padova, Italy)--one for the upper and one for the lower arch--over a 14-day period (T0-T14), with assignment to one of the following two distinct wear-time protocols:  
* First pair: worn for 7 h per day (7 H protocol)
* Second pair: worn for 12 h per day (12 H protocol)  
For the purpose of this study, we considered compliant wear as 12 h per day, in line with the clinical protocol of NOXI aligners, while 7 h per day was considered a non-compliant scenario. All patients initially followed the 7 h (7 H) protocol for 14 days. After a two-week transition period, they began the 12 h (12 H) protocol. The 7 H protocol was scheduled first because a daily wear time of 7 h falls below the 10-12 h required for NOXI aligners to exert a therapeutic effect. This preliminary phase allowed patients to adjust to the material, screening for any issues, and exclusion of those who were unsuitable for the study. After the transition period, patients began the actual treatment. The first set of aligners worn during this phase (also for 14 days) was collected and analyzed as part of the 12 H protocol. \begin{table}
\begin{tabular}{c c} \hline \hline
**Inclusion Criteria** & **Exclusion Criteria** \\ \hline Adult patients (aged from 25 to 31 years) & Patients with systemic diseases \\ Good systemic health & Heavy smokers (>7 cigarettes/day) \\ Patients not undergoing orthodontic & Patients undergoing treatment with \\ treatment & antihistamines or anticholinergic drugs \\ Good oral hygiene & Patients with habitual consumption of \\  & pigmented beverage \\  & Patients unable to perform home oral \\  & hygiene procedures \\  & Patients with hyposilavation \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Inclusion and exclusion criteria. Upon removal, the patients were instructed to rinse the aligners under cold running water and dry them with paper towels, ensuring that non-abrasive methods were employed. They were also advised to avoid any cleaning solutions containing abrasive microparticles or oxidizing agents, as these could potentially alter the material's surface properties and compromise subsequent colorimetric analysis. After cleaning, the aligners were stored in a designated container provided to each patient and kept away from direct sunlight in a stable, room-temperature environment to prevent potential degradation of the polyamide. Color variations were assessed using the Colorimeter app (Version 14; Copyright(r) [PERSON]), available for iPhone, iPad, Mac, and Apple Vision. The application uses the RGB color model to detect and quantify real-time color changes through the device's built-in camera (Figure 1). The app provides precise color data across various formats, such as HEX codes (for web design), RGB, CMYK, and HSL (Hue, Saturation, Lightness) values.