Evaluation of the Anti-septic Effect of a Chlorhexidine (CHX) Mouthwash With or Without an Anti-Discoloration System (ADS) During Experimental Gingivitis

Chlorhexidine is the most effective anti-plaque agent to date. It was more widely used in medicine and surgery. Löe and Schiött (1970) firstly studied its application in dentistry. The study showed that rinsing for 60 seconds twice per day with 10ml of a 0.2% chlorhexidine gluconate solution in the absence of normal tooth cleaning, inhibited plaque regrowth and the development of gingivitis. Because chlorhexidine is a bis-biguanide with a strong base and cationic, it reacts with anions, which is related to its antimicrobial activity. It has no systemic toxicity from topical application or ingestion. However, two pronounced side effects - superficial staining of the teeth and altered taste perception - were recognized almost immediately. This may compromise patient compliance for esthetic reasons. Recently, a chlorhexidine product with an anti-discoloration system (ADS) was launched in the market. A comparative study was done to confirm the effectiveness of 0.2% chlorhexidine with ADS. The study was single-blinded. In the experimental period, volunteers maintained their usual oral hygiene habits, besides, they rinsed twice daily with mouthwash (0.2% chlorhexidine) with or without ADS. Obviously, subject's home oral hygiene may become a major confounder of plaque accumulation. Although the effect of ADS is statistically significant, the ability of this new product to prevent plaque accumulation and gingivitis remained highly questionable.

Hence, controversy exists about the clinical efficacy of the chlorhexidine products with ADS. Consequently, a clinical validation of such products appears necessary. In the present study, the 21-day experimental gingivitis model will be used. This model is an established non-invasive model in humans for investigating the induction and resolution of inflammation in response to increasing bacterial accumulation. The design enables a study to be performed over 35 days in a well-controlled manner. So far, it is the most accurate clinical study model to access how medication or compounds in dentifrices affect plaque accumulation and gingival inflammation.

Chlorhexidine is the most effective anti-plaque agent to date. It was more widely used in medicine and surgery. Löe and Schiött (1970) firstly studied its application in dentistry. The study showed that rinsing for 60 seconds twice per day with 10ml of a 0.2% chlorhexidine gluconate solution in the absence of normal tooth cleaning, inhibited plaque regrowth and the development of gingivitis. Because chlorhexidine is a bis-biguanide with a strong base and cationic, it reacts with anions, which is related to its antimicrobial activity. It has no systemic toxicity from topical application or ingestion. However, two pronounced side effects - superficial staining of the teeth and altered taste perception - were recognized almost immediately. This may compromise patient compliance for esthetic reasons. Recently, a chlorhexidine product with an anti-discoloration system (ADS) was launched in the market. A comparative study was done to confirm the effectiveness of 0.2% chlorhexidine with ADS. The study was single-blinded. In the experimental period, volunteers maintained their usual oral hygiene habits, besides, they rinsed twice daily with mouthwash (0.2% chlorhexidine) with or without ADS. Obviously, subject's home oral hygiene may become a major confounder of plaque accumulation. Although the effect of ADS is statistically significant, the ability of this new product to prevent plaque accumulation and gingivitis remained highly questionable.

Hence, controversy exists about the clinical efficacy of the chlorhexidine products with ADS. Consequently, a clinical validation of such products appears necessary. In the present study, the 21-day experimental gingivitis model will be used. This model is an established non-invasive model in humans for investigating the induction and resolution of inflammation in response to increasing bacterial accumulation.

During the study period, participants will be asked to rinse with the mouthrinse sample or the placebo for 60 seconds twice daily, under video supervision (participants send in video recordings of their rinsing to control adherence to the protocol). A dental assistant, unaware of the study's purpose, will distribute the samples. The containers will be unlabeled, ensuring that both the participants and examiners are blinded to group allocation.

At Days 0, 7, 14 and 21 of the experiment, clinical examinations will be performed from central incisors to the second molars in each participant. The Discoloration Index (DI17) will be recorded at three aspects on each tooth (mesial, buccal and distal). The criteria for the Discoloration Index (DI) are indicated in Table 1. Plaque and Gingivitis Indices will be assessed at 4 aspects of each tooth (mesial, buccal, distal and lingual) using the criteria of the Plaque Index system (PlI) (Silness \& Löe 1964) and the Gingival Index System (GI) (Löe \& Silness 1963) 16, 18. At each visit, the parameters will be assessed by the same examiner, who will be masked by the allocation of the test and control to avoid examiner bias and calibration bias.

Upon completion of the experimental period, mechanical daily plaque control measures will be re-instated and the participants will be re-examined after 3 weeks to ensure their periodontal health. Following the experimental period, another professional cleansing will be performed to remove plaque and possible stain on the teeth. At day 35 the participants will be checked again.

• Severity of gingival inflammation, assessed by the Gingival Index by Löe and Silness 1963 [Time frame: at day 0, 7, 14, and 35]
• Plaque accumulation, assessed by the Plaque Index Silness and Löe 1994 [Time frame: at day 0, 7, 14, 21 and 35]
• Staining assessed by the Discoloration index [Time frame: at day 0, 7, 14, 21 and 35]
• Oral scan [Time frame: at day 0, 21 and 35]
• Microbiological changes [Time frame: at day 0, 21 and 35]
• Characterisation of host- and microbiome-derived metabolites [Time frame: at day 0, 21 and 35]