1. Introduction
Oral biofilms are the main causative factor for caries, periodontal diseases, peri-implant diseases and oral halitosis. Hence, an adequate regular control of oral biofilms is critical not only to maintain oral health but also to prevent recurrent disease [
1,
2]. Therefore, since epidemiological and intervention studies have shown an association between periodontitis and the onset and progression of different systemic diseases, oral biofilm control is also important for maintaining overall health [
3].
An efficient regular mechanical oral biofilm control is difficult to achieve and is limited by both subject-related factors such as poor motivation or education and lack of adequate compliance; and/or skills and tooth-related factors, such as the limited use of interdental cleaning devices and the presence of hard-to-reach areas. As a consequence, there is clear evidence [
4,
5] that for some patients, mechanical biofilm control is not sufficient for preventing or controlling oral diseases. In these patients, the adjuvant use of chemical agents with antimicrobial effects, as mouthwashes or toothpastes, has shown to be useful in dental biofilm control and maintenance of oral/periodontal health [
6]. Furthermore, a recent published systematic review showed that antiseptic therapies used adjunctively to mechanical plaque control were equally effective in reducing gingivitis and plaque in patients with gingivitis on intact periodontium or in previously treated periodontitis patients with gingival inflammation [
7].
Toothpaste, by combining the antimicrobial effect provided by its active ingredients, with the mechanical effect of toothbrushing, is the preferred method for controlling oral biofilms by both dentists and patients [
6], and consequently, is the most widespread population-based oral hygiene method.
Although chlorhexidine (CHX) is currently considered as the most effective and long-lasting antiplaque agent available [
6], its widespread use is limited due to its side effects with prolonged use, including tooth and tongue pigmentation, taste alteration, mucous membrane irritation and hypersensitivity reactions [
8]. Moreover, due to its high cationic nature, CHX-based toothpastes are difficult to formulate since other anionic substances, such as surfactants, may reduce its bioavailability and, consequently, its efficacy [
6].
As an alternative, the use of cetylpyridinium chloride (CPC)-based toothpastes has been suggested for oral biofilm control [
6]. The antimicrobial activity of CPC has been studied for more than 70 years with proven antimicrobial effect against Gram-positive bacteria, fungi and yeasts [
9]. CPC-containing products are considered as safe and effective for the treatment of plaque-induced gingivitis by the Food and Drug Administration (FDA) [
10]. Its efficacy, however, may depend on the specific formulation, due to its astringent characteristics and the possible interactions with excipients or other components. Therefore, it is not uncommon to find two CPC products formulated with the same concentration, but with significantly different substantivity, bioavailability and ability to penetrate within the biofilm, which may significantly impact its efficacy [
11]. To overcome some of these limitations, other substances with biological activity synergistic with CPC have been added to oral hygiene products [
12]. In fact, the FDA has recognized the effectivity of natural products such as plant extracts and the essential oils of eucalyptus, menthol, methyl salicylate and thymol (cymenol) in mouth rinses and toothpastes due to their antibacterial and antibiofilm properties. Kakar et al. [
13] confirmed that the use of a 0.1% cymenol-containing toothpaste over a 12-week period provides a statistically significant benefit maintaining gingival health as compared to a sodium fluoride control toothpaste. Moreover, a recent study concluded that CPC and cymenol formulated in mouth rinses may act synergistically, extending the substantivity of their action [
14].
However, there is limited information in the literature on the adjunctive efficacy of CPC-based toothpastes and even more scarce, about the effects of toothpastes formulated with CPC combined with other antimicrobial agents. Therefore, the objective of this clinical trial was to evaluate the safety and tolerability of a new toothpaste formulated with CPC and cymenol, and secondarily, its clinical efficacy and patient perception, when compared to a negative control, a fluoride-based toothpaste available on the market.
4. Discussion
The present study demonstrates that the adjuvant use of a toothpaste with cetylpiridinium chloride and cymenol was safe and tolerable, since no adverse effects were reported throughout the 6-week duration of the study. This toothpaste was positively appreciated by the patients, although not significantly different when compared with the control toothpaste in terms of PROMs. Regarding its efficacy, significantly higher reductions in plaque levels and BOP occurred at 3 and 6 weeks of use, when compared to the control toothpaste. These benefits were not related to an increase in tooth staining.
The safety of this CPC and cymenol-based toothpaste was assessed by both the occurrence of adverse and secondary effects. The patients using the test toothpaste did not experience any adverse effect at any time during the study, and the presence of staining was not statistically different to the control group. Although CPC has shown potential for inducing dental staining, this effect has been rarely reported in clinical studies [
11], which may indicate a lower substantivity compared with CHX. CPC, when formulated as an oral hygiene product at concentrations ranging from 0.045% to 0.10%, has been shown to be safe and well tolerated [
26]. Cymenol, as a tasteless isomer of thymol, has also been recognized as safe by the FDA [
27]. In a study evaluating a toothpaste containing cymenol and zinc chloride, 14 out of 205 subjects reported mild adverse effects, although these did not cause any withdrawal from the study [
13]. In the present study, patients’ perceptions (PROMs) of the tested toothpastes, such as taste, aroma consistency or any disturbance, were assessed through a questionnaire. The results did not reveal any statistically significant difference between the groups. However, patients in the test group reported a sensation of lesser plaque removal efficacy, which did not correlate with the obtained clinical results, demonstrating significantly higher plaque reductions in the test group. It should also be noted that 40% of the subjects did not give an overall rating of the product from 1 to 10. This problem may be due to the fact the questionnaire was administered in a paper format of the questionnaire, and the subjects may have confused the question on product evaluation with the overall rating from 1 to 4. Administering the questionnaire in an online format would have made it possible to force subjects to answer all the questions.
In the present study, the use of the test toothpaste with CPC and cymenol resulted in higher reductions in PlI levels when compared with the control group. This finding could be explained by the different active ingredients in the toothpastes. CPC possesses antimicrobial properties, particularly against a wide range of Gram-positive bacteria, fungi and yeasts [
28]. This antibacterial activity can be attributed to its cationic nature [
29], which may prevent bacterial adhesion and hence, biofilm formation. On the other hand, cymenol exhibits direct antimicrobial effects against oral anaerobic species and can also reduce the production of metabolites, including glycolytic and proteolytic enzymes and thereby can reduce deleterious effects of certain microorganisms [
30]. Moreover, when formulated as a mouth rinse, CPC and cymenol may act synergistically, by extending the substantivity of their action [
14]. However, it is not easy to establish direct comparisons between the present study and others, due to the limited evidence available assessing CPC- and essential oil-containing toothpastes. A study evaluating the use of a mouth rinse with 0.05% CPC for 3 months in orthodontic patients found statistically significant differences in plaque levels between the test and control groups only in the first month [
31]. In contrast, other studies evaluating mouth rinses containing 0.07% CPC [
28,
32] have demonstrated plaque reductions after 3 and 6 months, when compared to the placebo. When two mouth rinses containing CPC at concentrations of 0.075% and 0.10% were compared, both provided statically significant plaque reductions over six months of use [
33]. In addition, numerous systematic reviews have investigated the effectiveness of mouth rinses containing CPC and essential oils [
6,
7,
34], only essential oils [
35] or CPC [
11], demonstrating a statistically significant impact on plaque levels.
With regards to gingival inflammation, in the present study, both the test and control groups demonstrated reductions in BOMP, with minimal intergroup differences. Similar results in BOMP were observed with mouth rinses containing 0.05% [
36] and 0.07% CPC [
28], although these studies observed statistical differences in BOMP at 6 months, when compared with a placebo. Regarding the BOP changes in our study, both the test and control groups demonstrated statistically significant reductions and, more importantly, the changes between baseline and week 6 in the test group revealed a statistically significant higher reduction, when compared to the control group (test 0.19, SD = 0.13 and control 0.12, SD = 0.10, with a mean difference of 0.075,
p = 0.031). In addition, an RCT evaluating 0.075% and 0.10% CPC mouth rinses observed a significant reduction in the gingival and bleeding indices, at 3 and 6 months, with a magnitude similar to CHX [
33]. When formulated independently, both CPC and essential oils have demonstrated a significant impact on gingival indices, in 6-month RCTs [
6]. Since clinical results could be influenced by the whole formulation of a given product [
34], the results of the present study support that the CPC and cymenol combination, formulated in a toothpaste, demonstrate significant reductions in plaque and inflammation levels.
The limitations of the present study should also be disclosed: the convenience sample size of 30 patients per group is small, since it was selected following the primary aim, which was to assess safety and tolerability, and, also, the short study duration, that was also selected based on the primary aim of the study.