The goal of this clinical trial is to evaluate the effect of a mouthrinse containing an active ingredient benzydamine hydrochloride in the absence of daily oral hygiene. The study includes periodontally healthy female subjects and experimental phase will last for three days. Plaque accumulation is the primary evaluated outcome, measured by a particular periodontal index called Plaque Control Record (PCR). Gingival inflammation (bleeding) is the secondary evaluated outcome, measured by a particular periodontal index called Bleeding on Probing (BoP). Researchers will compare benzyadamine hydrochloride mouthrinse to a placebo (a look-alike substance that contains no active ingredient) mouthwash to see whether benzydamine hydrochloride use results in greater plaque accumulation.

This study is designed as a randomized, double-blind, placebo-controlled, parallel-group clinical trial to evaluate the effects of a benzydamine hydrochloride-containing mouthrinse on supragingival plaque accumulation and gingival inflammatory response under conditions of restricted mechanical oral hygiene. The experimental model is based on a short-term plaque regrowth protocol, enabling controlled assessment of the chemical effects of the investigational product in the absence of confounding mechanical plaque removal.

A total of 50 periodontally healthy female participants will be enrolled and randomly assigned to either the test group (benzydamine hydrochloride mouthrinse) or the control group (placebo mouthrinse). Randomization will be performed using a predefined allocation sequence to ensure balanced group distribution. Both participants and investigators will be blinded to group assignment, with the test and placebo formulations matched in color, taste, and packaging to maintain allocation concealment.

Prior to the experimental phase, all participants will receive professional dental prophylaxis to establish a standardized baseline with minimal plaque accumulation and absence of gingival inflammation. Following this, subjects will be instructed to refrain from all mechanical oral hygiene procedures, including toothbrushing and interdental cleaning, for the duration of the 3-day experimental period.

During the experimental phase, participants will use the assigned mouthrinse twice daily, rinsing with 15 mL of solution for a standardized duration according to detailed written instructions.

The investigational product contains benzydamine hydrochloride, a locally acting non-steroidal anti-inflammatory agent with analgesic and anti-inflammatory properties, which may influence gingival tissue response and potentially affect plaque accumulation dynamics. The placebo formulation is devoid of active pharmacological ingredients but is otherwise identical in formulation characteristics to ensure effective blinding.

Clinical assessments will be conducted at baseline and at the end of the 3-day experimental period. Plaque accumulation will be assessed using the Plaque Control Record (PCR), which quantifies the presence of plaque on six tooth surfaces and expresses the findings as a percentage of plaque-positive sites. Gingival inflammation will be evaluated as a secondary outcome using Bleeding on Probing (BoP), recorded dichotomously following periodontal probing.

All clinical measurements will be performed by a previously calibrated examiner using standardized instruments and probing force to ensure consistency and reproducibility of recorded data.

This study design allows for the isolation of the chemical effects of benzydamine hydrochloride by eliminating mechanical plaque control, thereby providing insight into its influence on early plaque formation and gingival inflammatory response compared to placebo. The short duration of the experimental phase ensures participant safety while allowing detection of early biological changes in plaque accumulation and gingival health.

Inclusion Criteria:

• dentate subjects with at least 24 healthy teeth
• no history of periodontal disease, confirmed by a clinically healthy periodontium, defined as probing pocket depth (PPD) ≤ 3 mm and no interdental clinical attachment loss

Exclusion Criteria:

• pregnant or lactating females
• using hormonal contraceptives
• reporting systemic diseases or pharmacological treatment that could affect gingival inflammation
• use of antibiotics within 6 months before the study

Female subjects were eligible to participate as the pool of participants (students attending Women's General Gymnasium in Zagreb and the School of Dental Medicine, University of Zagreb) is exclusively and predominantly female.