Evaluation of BreathRx Antibacterial Mouth rinse for the Prevention of Plaque and Gingivitis

Background
During the 1990s, FDA convened a Dental Products Panel and a Dental Plaque Subcommittee in order to review various over-the-counter (OTC) dental products and active ingredients for the reduction or prevention of plaque and gingivitis. A thorough review of the data and information submitted by a number of dental companies resulted in a selection of three active ingredients suitable for this purpose. Cetylpyridinium Chloride was one of the selected active ingredients.

The Subcommittee recommended Cetylpyridinium Chloride (CPC) as an antiplaque, antigingivitis active ingredient for topical use on the mucous membranes of the mouth and throat. The Subcommittee also concluded that CPC at concentrations of 0.045 to 0.1 percent is safe and effective for use in mouth rinse formulations as an OTC antiplaque, antigingivitis agent. CPC-containing mouth rinses have been used in the United States since 1940 and have also been marketed internationally. The 55-year United States marketing history is significant with respect to its safety.

The Subcommittee recommended that OTC antigingivitis, anti plaque drug products demonstrate their effectiveness through the testing described below. This testing is required to demonstrate the effectiveness of Category I antigingivitis, antiplaque active ingredients in traditional dosage forms (dentifrice, gel, paste, or rinse). Minimum Inhibitory Concentration (MIC) was recommended as one of the tests by the Subcommittee for determining the product efficacy. MIC is defined as the lowest concentration of an antimicrobial agent that inhibits growth of a micro-organism.

The BreathRx? Anti-Bacterial Mouth Rinse was also evaluated utilizing Minimum Bactericidal Concentration (MBC) although this test was not required. MBC is defined as the lowest concentration of an antimicrobial agent that kills 99.9 % of the test inoculum. The representative organisms for both tests include: Actinomyces viscosus, Fusobacterium nucleatum, Porphyomas gingivalis, Prevotella intermedia, Bacteroides forsythus, Candida species, Streptococcus mutans, and gram negative enteric rods. The organisms selected by the Subcommittee have been shown to cause plaque and gingivitis. E-coli was included with the test organisms because of its extreme resistance to antibacterial agents and its wide presence.

Results
The above referenced testing was performed by Loma Linda University on BreathRx? Anti-Bacterial Mouth Rinse which contains 0.075% Cetylpyridinium Chloride as the active ingredient. The results and conclusions of the test are as follows: "'t is concluded that, under the conditions of the present study, BreathRx? Anti-Bacterial Mouth Rinse at highly diluted concentrations has significant bacteriostatic and bactericidal effects on S.mutans, A. viscosus, C. albicans. P. gingiva lis, P. intermedia, F. nucleatum, B. forsythus, as determined using the MIC and MBC methods. BreathRx? Anti-Bacterial Mouth Rinse also effectively inhibits the growth of E. coli at 1 :8 dilution and kills E. coli at 1:2 dilution."

References supporting Cetylpyridinium Chloride (CPC) kills bacteria that lead to bad breath:

van Steenberghe D, Avontroodt P, Peeters W, Pauwels M, Coucke W, Lijnen A, Quirynen M.

Effect of different mouth rinses on morning breath.

J Periodontal. 2001 Sep

BACKGROUND: Morning breath odor is an often-encountered complaint. This double-blind, crossover, randomized study aimed to examine the bad breath-inhibiting effect of 3 commercially available mouth rinses on morning halitosis during an experimental period of 12 days without mechanical plaque control. METHODS: Twelve medical students with a healthy periodontium refrained from all means of mechanical plaque control during 3 experimental periods of 12 days (with intervening washout periods of at least 3 weeks). A professional oral cleaning preceded each period. During each experimental period, as the only oral hygiene measure allowed, the students rinsed twice a day with one of the following formulations in a randomized order: CHX-Alc (a 0.2% chlorhexidine [CHX] solution); CHX-NaF (CHX 0.12% plus sodium fluoride 0.05%); or CHX-CPC-Zn (CHX 0.05% plus cetylpyridinium chloride 0.05% plus zinc lactate 0.14%). After 12 days, morning breath was scored via volatile sulfur compound (YSC) level measurements of the mouth air and organoleptic ratings of the mouth air, the expired air, and a scraping of the tongue coating. At the 12-day visit, a questionnaire (subjective ratings) was completed and samples taken from both the tongue coating and the saliva for anaerobic and aerobic culturing and vitality staining. The de novo supragingival plaque formation was also recorded. All parameters were correlated with the baseline registrations. RESULTS: Although oral hygiene during the 3 experimental periods was limited to oral rinses, bad breath parameters systematically improved, with the exception of a slight increase in YSC levels while using CHX-AIc, a finding which was associated with the direct influence of the CHX on the sulfide monitor. The oral microbial load after the use of CHX-NaF remained unchanged, while for the CHX-Alc and CHX-CPC-Zn, significant reductions in both aerobic and anaerobic colony forming units (CFU)/ml were noticed in comparison with baseline data for both tongue coating and saliva samples. The composition of micro flora, on the other hand, did not reveal significant changes. The supragingival plaque formation was inhibited, in descending order, by CHX-AIc, CHX-CPC-Zn, and CHX-NaF. The subjective scores for the rinses indicated a higher appreciation for CHX-CPC-Alc and CHX-NaF because of a better taste and fewer side effects. CONCLUSIONS: The results of this study demonstrate that morning halitosis can be successfully reduced via daily use of mouth rinses. CHX-Alc and CHX-CPC-Zn mouth rinses result in a significant reduction of the microbial load of tongue and saliva.

Quirynen M, Avontroodt P, Soers C, Zhao H, Pauwels M, Coucke W, van Steenberghe D.

The efficacy of amine fluoride/stannous fluoride in the suppression of morning breath odor.

Periodontal. 2002 Oct; 29.

BACKGROUND: Breath odor is a complaint encountered worldwide, often linked to microbial overload in the oral cavity. This double blind, crossover, randomized study assessed the efficacy of several antiseptic mouth rinses or slurry vs. a control solution in the prevention of morning bad breath during an experimental period of 7 days without mechanical plaque control. Methods: Sixteen dental students with a healthy periodontium abolished, after a thorough professional cleaning, all means of mechanical plaque control during five experimental periods of 7 days, interleaved by washout periods of at least 3 weeks. During each experimental period, as the only oral hygiene measure, the students rinsed twice a day with one of the following formulations (in a randomized order): a 0.2% chlorhexidine-a1cohol mouth rinse (CHX-A1c), a 0.05% CHX + 0.05% cetylpyridinium chloride + 0.14% zinc lactate mouth rinse (CHX-CPC-Zn), an amine fluoride/stannous fluoride (125 ppm F-/125 ppm F-) containing mouth rinse (AmF/SnF2Mr), a slurry of a tooth paste (AmF/SnF2SI) containing amine fluoride (350 ppm F-) and stannous fluoride (1050 ppm F-) and a placebo solution (placebo). At days 0, 3 and 7, morning breath was scored via VSC level measurements of the mouth air, and organoleptic ratings of the mouth air and tongue coating. At the same visits both the degree of gingival inflammation and the de novo plaque fonnation were rated. At the end of each period a questionnaire for subjective ratings was completed and microbiological samples were taken from the tongue dorsum, the saliva and the supragingival plaque for anaerobic and aerobic culturing. Results: Although oral hygiene during the experimental periods was limited to rinsing, bad breath parameters systematically improved (P < 0.00 I) with the three mouth rinses (CHX-AIc, CHX-CPC-Zn, AmF/SnF2Mr), with a superiority of the CHX-CPC-Zn solution when only VSC values were considered (P < 0.003). The AmF/SnF2 slurry and the placebo solution showed only minor changes with time. The three mouth rinses reduced significantly (P < 0.001) the bacterial load (aerobic & anaerobic) in the saliva (>or= 0.5 log reduction with a superiority (P < 0.005) for the CHX-Alc when compared to the two others). Changes in the bacterial load on the tongue dorsum could only be detected for the CHX-Alc solution (0.5 log). The antibacterial effect of the placebo solution and the slurry were negligible. The composition of micro flora on the other hand did not reveal significant changes. The de novo supragingival plaque fonnation was significantly (P < 0.05) inhibited by the three mouth rinses with a slight superiority for the CHX-Alc solution. The degree of gingival inflammation at day 7 remained low " 0.16) for all products. The CHX-Alc solution scored significantly worse for the subjective evaluation (questionnaires) concerning taste and sensitivity of tongue). CONCLUSIONS: The results of this study demonstrate that morning breath odor can be successfully reduced by the sole twice daily use of CHX-A1c, CHX-CPC-Zn or AmF/SnF2Mr mouth rinses, which all significantly reduced the bacterial load in the saliva and retarded the de novo plaque fonnation.

Abstract

The Antimicrobial Efficacy of a New CPC/Zn acetate Breath Freshening Mouth Rinse

P. SREENIV ASAN, E. GITTINS, and K. DE PIERRO

Colgate-Palmolive Company, Piscataway, NJ, USA

A new mouth rinse containing cetylpyridinium chloride and zinc acetate (CPC/Zn) significantly reduced mouth odor scores in clinical studies versus a control rinse. Objectives: This investigation examined the antimicrobial effects of these rinses in laboratory and clinical studies. Methods: Laboratory tests with oral bacteria were performed with alamar blue, a redox dye that rapidly detects bacterial viability. Results: Treatment of A. viscosus, S. mutans, and S. sanguis by CPC/Zn resulted in a 25, 35 and 16% decrease in bacterial viability respectively versus control and were statistically significant (p < 0.05). A randomized, double blind clinical study with 37 subjects was conducted to confirm these laboratory results. After a washout phase, subjects abstained from oral hygiene and provided a saliva sample for determining the numbers of total and oral odorigenic producing bacteria on enriched and indicator plates. Mouth rinses were used once and subjects sampled at 90 and 180 minutes post treatment for total and oral odorigenic bacteria. In comparison to control, the decrease of total bacteria following CPC/Zn use at 90 and 180 minutes were 59.9% and 32.1% respectively. The use of CPC/Zn resulted in a 61. 7% and 72.5% decrease of odorigenic bacteria at 90 and 180 minutes respectively versus control rinse. The decrease in bacteria with the CPC/Zn rinse were significantly higher than the control rinse at all post-use time points (p<0.05). Conclusions: These results demonstrate the efficacy of a mouth rinse with CPC/Zn on oral bacteria in laboratory and clinical tests. Additionally, the CPC/Zn rinse resulted in a higher reduction of oral odorigenic bacteria than the control rinse.

    Evaluated MEDLINE:
Xylitol - Sugar substitutes, chewing gum and dental caries
Br Dent J o Volume 184o Issue 1
MEDLINE
Br Dent J 1998 Jan 10
Edgar WM.

Abstract
The prevalent use of chewing gum has prompted interest in its dental effects. Important defining aspects are the ability to use sugar substitutes in gum manufacture and the prolonged stimulation of a protective flow of saliva the main sugar substitutes used are sorbitol and xylitol. Because it is not fermented by oral bacteria, xylitol is considered to be non-cariogenic, and while sorbitol in solution can be fermented slowly by mutants streptococci, chewing sorbitol-sweetened gum does not cause a fall in plaque pH. Effects of chewing sugar-free gum on the ability of plaque to form acid from sucrose are equivocal, although the tendency is for the plaque acidogenicity to be reduced with the use of xylitol gum for 2-3 weeks, due to its inhibitory effects on mutants streptococci. Gum-chewing also stimulates a protective salivary flow when used after an acidogenic stimulus, and may enhance salivary function, especially in subjects with low flow rates. Sorbitol and xylitol gums have similar beneficial effects in promoting enamel remineralisation in short-term in-situ experiments. Clinical trials indicate that xylitol gum has a useful anticaries role, superior to the effects of sorbitol gum. In conclusion, both sorbitol and xylitol chewing gums are non-cariogenic in contrast to sugared gum, and exhibit beneficial anticaries properties through salivary stimulation. In addition, xylitol's antibacterial properties seem likely to lead to caries reductions superior to the more modest reductions with sorbitol gum.

    Xylitol and Dental Caries: An Overview for Clinicians

Heather Lynch, MD, and Peter Milgrom, DDS

Abstract

An overview of studies about xylitol and dental caries suggests potential clinical dental applications for xylitol. Xylitol is a naturally occurring, low-calorie sugar substitute with anticariogenic properties. Data from recent studies indicate that xylitol can reduce the occurrence of dental caries in young children, schoolchildren, and mothers, and in chil-dren via their mothers. Xylitol, a sugar alcohol, is derived mainly from birch and other hardwood trees. Short-term consumption of xylitol is associated with decreased Streptococcus mutans levels in saliva and plaque. Aside from decreasing dental caries, xylitol may also decrease the transmission of S. mutans from mothers to children. Commercial xylitol-containing products may be used to help control rampant decay in primary dentition. Studies of schoolchildren in Belize and Estonia, along with data from the University of Washington, indicate that xylitol gum, candy, ice pops, cookies, puddings, etc., in combination with other dental therapies, are associated with the arrest of carious lesions. A prospective trial in Finland has demonstrated that children of mothers treated with xylitol had lower levels of S. mutans than children of mothers treated with hlorhexidine or fluoride varnish. Food products containing xylitol are available commercially and through specialized manufacturers, and have the potential to be widely accessible to consumers.

Sugar Alcohols
Xylitol is a sugar substitute with sweetness equal to that of table sugar. I It is a member of the group of compounds known as sugar alcohols, which includes other common dietary sweeteners such as sorbitol and mannitol. Xylitol is produced commercially from birch trees and other hardwoods containing xylan. It can also be found in small quantities in fruits and vegetables. In contrast, sorbitol commonly found in sugar-free products such as chewing gum, candies, and toothpaste - is less sweet than sucrose and is generally combined with other sweeteners such as saccharine or aspartame as well as xylitol to improve the flavor of the product. Xylitol contains 40 percent fewer calories than sucrose. Because xylitol is absorbed slowly by the human gastrointestinal tract, the main side effect associated with its consumption is osmotic diarrhea. This usually occurs only when xylitol is consumed in large quantities, four to five times those needed for the prevention of dental caries.2 \ This side effect is common to all sugar alcohols.

Xylitol and Streptococcus Mutans
Microorganisms do not readily metabolize xylitol, and its consumption has minimal effect on plaque ph.1 However, xylitol does accumulate intracellularly in S. mutans. This accumulation inhibits the bacteria's growth. This has been demonstrated in vitro and may contribute to a reduction of S. mutans levels in the plaque and saliva of those consuming xylitol. In addition, xylitol has a number of effects on S. lilt/tails that may account for some of its clinical effects in caries reduction. Short-term both saliva and plaque.' Long-term habitual consumption of xylitol appears to have a selective effect on S. mutans, resulting in selection for populations less adherent to tooth surfaces. These colonies, therefore, are shed more easily from plaque into saliva." This effect may not only be important to the individual's decay experience, but may also influence the transmission of S. mutans from mothers who consume xylitol to their children. A clinician can recommend adding xylitol to the diet without asking patients to make additional alterations to their dietary patterns.

Clinical Applications

Children at High Risk for Caries
There are surprisingly few well studied strategies available to clinicians to prevent and control high rates of caries in the primary dentition. In the absence of water fluoridation, fluoridated toothpaste and topical fluoride varnish are the mainstays in the United States. For older children, sealants are added to the regimen. Effective strategies to reduce risk by modifying the diet of children are not significant effort. As a result, the use of xylitol is particularly attractive because its action is not dependent upon reducing the amount of other sugars in the diet. Thus, a clinician can recommend adding xylitol to the diet without asking patients to make additional alterations to their dietary patterns. Xylitol containing products have the potential to improve success in controlling the problem of rampant decay in the primary dentition.

A number of studies conducted among schoolchildren of various ages have shown that consumption of gum containing xylitol reduces the rates of dental decay in the treatment groups (relative risks ranging from 0.27 to 0.56). Increasing use and higher doses led to greater reductions. One study conducted among school children in Belize with very high rates of dentine caries showed that consumption of xylitol gum was associated with arrest of carious lesions and, as expected, that the highest dose of xylitol had the greatest effect. The number of lesions that re-hardened ranged from 9 percent to 27 percent in all groups and from 12 percent to 27 percent in the 10 percent xylitol groups. This study is important because the children continued to consume very high levels of sucrose in their everyday diet. However, a major limitation in extending these results to the United States is that chewing gum is not considered safe for very small children and is actively discouraged in schools.

Other xylitol-containing products have been studied. A field trial of the use of xylitol-containing candies among 10-year-old schoolchildren in Estonia showed a 33 percent to 59 per-_ cent caries reduction in the group using the candies and a 53.5 percent caries reduction in the group using the gum relative to the control group. This suggests that candy may be as effective as chewing gum as a vehicle for the delivery of xylitol in caries prevention. At the University of Washington, researchers have produced and field-tested xylitol-containing ice pops, chewy worms, puddings, macaroons, and sorbet. They have initiated studies that suggest that children will fairly readily accept such foods when offered as part of the daily diet and that suffer no side effects from their use.

Food producers are available to develop these foods, hut considerable work is needed to produce commercially viable products and have them accepted.

According to available data, there is no vehicle in the United States for using xylitol in toddlers and preschool children too young to chew gum. In older children, four to five pellets or sticks (1 gram of xylitol per pellet or stick) of xylitol gum per day, chewed for five minutes, should reduce dental caries activity. Xylitol, approved by the Food and Drug Administration, has been used as a sweetener in foods since the 1960s. It is sale for use with children. The use of fluoridated toothpaste, topical fluorides, and sealants should also be encouraged.

Pregnant Women and New Mothers
Kohler and colleagues demonstrated that the combination of good dental care, instruction to improve oral hygiene, and chlorohexidine gels and toothpastes led to reductions in maternal S. mutans levels and reduction in the extent of transmission to the child. More recently, Hildebrandt and colleagues showed that the use of commercially available chlorohexidine rinses for two weeks followed by the daily use of xylitol gum (two pellets containing 7g xylitol) in high-caries-rate adults with recent restorations led to major reductions in S. mutans.

A clinical trial conducted in Finland, demonstrated that xylitol had the greatest effect. The mothers, all of whom had high S. mutans levels at the beginning of the study, were treated with either chlorhexidine varnish or fluoride varnish or 100 percent xylitol gum (65 percent xylitol by weight, chewed at least two to three times per day) for 1 to 21 months. The outcome measures were decay rates among the children and S. mutans levels in both the Dentinal caries among children in the xylitol group was reduced by 70 percent as compared with children in the fluoride or chlorhexidine groups. mothers and the children. The chi 1chen of mothers treated with xylitol had the lowest levels of S. mutans during the intervention period (treatment continued until the child was 2 years old) and during follow-up. The percentage of colonization with S. mutans in the children in the xylitol group at 2 years old was 9.7 percent. This was statistically different from the other two groups, in which 2H.6 percent were colonized in the chlorhexidine group and 4.5 percent in the fluoride group. These children when followed up most recently had the lowest S. mutans levels (51.6 percent were colonized in the xylitol group vs. H3.9 percent in the fluoride group and H6.4 percent in the chlorhexidine group). Children of mothers treated with xylitol also had the lowest rates of decay. Follow-up at 5 years of age found that dentinal caries among children in the xylitol group was reduced by 70 percent as compared with children in the fluoride or chlorhexidine groups.

These studies have been conducted only in settings in which child rearing is done primarily by the mother and in which mother-to-child transmission is presumed. \10 studies have been completed in communities where child rearing is shared among greater numbers of people. The use of xylitol gum by mothers as well as other family members is currently being investigated in one such community.

Whether used alone or in combination with other antimicrobial therapies such as chlorhexidine, xylitol has an important role in the prevention of dental decay among children born to mothers with high levels of S. mutans not only because of its effects on S. mutans levels and bacterial properties during the period of consumption, but also because its beneficial effect on decay reduction in these children appears to persist far beyond the period of consumption. Both chlorhexidine and xylitol may be used safely by pregnant women and nursing mothers.

Currently available data suggest that twice daily use of chlorhexidine gluconate rinse (0.12 percent) for two weeks, followed by four to five sticks or pellets of xylitol gum per day chewed for five minutes each time, should lead to a major reduction in S. mutans levels in the mother and should benefit the child.

    Effects of Daily Mechanical Tongue Scraping


Ronald E. Mangie, B.S. Bacteriology, D.D.S.

The most frequently recommended and widely accepted method of basic oral hygiene is tooth brushing. While the practice of tooth brushing is effective in reducing tooth decay and specific oral bacteria, it cleans only selected areas of the teeth and the gingiva. For this reason, patients have been encouraged to use complimentary methods of oral hygiene such as dental floss, mouth wash, water jet devices and toothpicks. However, the benefits of using these additional methods of oral hygiene limited still to only clean tooth surfaces and gingiva.

Evidence gathered from a study conducted by Gilmore and Bhaskar indicates that several oral diseases are believed to be caused by microorganisms that are retained within the oral cavity. Related studies by Crawford and Jacobson have demonstrated that the rough pappilary structure of the tongue provides an extremely large surface area for the accumulation of debris and microorganisms. Gibbons and Gordon studies also have shown that the tongue is the main source of microorganisms and bacteria. Therefore, in consideration of these findings, it is obvious that the periodic cleansing of the tongue should be included in the patient's oral hygiene program to reduce the number of microorganisms within the oral cavity.

Tongue cleaning is not new to mankind. It has been practiced in Eastern cultures since ancient times. Until recently, the cleansing of the tongue has been largely an unrecognized practice in the West; however, it is becoming increasingly popular with today's dentists as an essential procedure of oral hygiene.

Several clinical studies conducted by well respected researchers have demonstrated the benefits of tongue cleansing. Dr S. N. Bhaskar and Dr. E. L. Gilmore's research has proven that one week of tongue cleansing reduced the total number of all Streptococcal species from the tongue. Additional data gathered by Dr. G. J. Vasilakis and Dr. C. O. Preis provides conclusive evidence that tongue cleansing eliminates or significantly reduces plaque forming organisms within the oral cavity.

Obviously, tongue cleansing is essential for a total oral hygiene program. However, the method or procedure for cleansing the tongue most effectively has never been determined. Tongue brushing has been advocated intermittently by Western scientists. and mechanical tongue scraping also has been practiced successfully. Never the less, until now, a pertinent question has never has been answered: Specifically, Which is a more effective procedure tongue scraping or tongue brushing?

The purpose of this study, therefore, is to investigate which method, tongue scraping or tongue brushing is more effective in reducing the bacterial populations on the dorsum of the tongue and in improving its clinical appearance. An additional objective of this study is to observe which method is more comfortable for the patient and easier to perform.

The bacteriological studies that were conducted consisted of analyzing direct cultures taken from a pre-selected area of the tongue. For the sake of brevity in this report, a detailed itemization of the materials and methods used in conducting this study will not be included. Nevertheless, the results of this study should be shared with the dental profession for their information and reference.

    Tongue Cleaning Methods: A Comparative Clinical Trial Employing a Toothbrush and a Tongue Scraper

J Peridontal
July 2004

Vinicius Pedrazzi, * Sandra Sato, * Maria da Gloria Chiarello de Mattos, * Elza Helena Guimaraes Lara, t and Heitor Panzeri*

Background: It is estimated that approximately 85% of all halitosis cases have their origin within the mouth; of these, 50% are caused by tongue residues. Previous studies have established that hydrogen sulfide and mercaptans are the primary components of halitosis. Thus, tongue cleaning gains importance as a means of halitosis management. Methods: This investigation compared the efficacy of two mechanical methods for tongue cleaning through a handheld sulfide monitor. This crossover trial was carried out with 10 healthy subjects, 20 to 50 years old. Before the baseline measurement of the volatile sulfur compounds (VSCs), the subjects were instructed to refrain from any tongue cleaning method for 48 hours. The 10 participants were then placed in one of two groups (five each): 1) first week: tongue scraper, second week: soft-bristle toothbrush; 2) first week: toothbrush, second week: tongue scraper, with a 48-hour wash-out period between each week.

Results: The baseline measurements were compared with those of the end of each week using the Dunn method (a = 0.01). The tongue scraper showed a 75% reduction in VSCs, while the toothbrush only achieved a 45% reduction in VSCs.

Conclusion: Although the tongue coating was removed by both methods, the tongue scraper performed better in reducing the production of volatile sulfur compounds. J PeriodontoI2004;75:10091012.

KEY WORDS
Halitosis/etiology; halitosis/prevention and control; hydrogen sulfide/analysis; tongue; toothbrushing.

* University of Sao Paulo, Faculty of Dentistry, Department of Dental Materials and Prosthesis, Ribeirao Preto, Sao Paulo, Brazil.

University of Sao Paulo, Faculty of Pharmaceutical Sciences, Department of Pharmaceutical Sciences, Ribeirao Preto.

The most common concept concerning individual health is the harmony of one's physical, mental, and social well-being. The simple absence of disease is not accepted as an indication of health. Besides embarrassment, shyness, and occasional difficulties in communicating in a foreign language in which one is not fully conversant, halitosis has been cited as an obstacle in personal relationships. Furthermore, it may signal the presence of disease. Previous studies have established that hydrogen sulfide and mercaptans are the primary components of halitosis. 1 ,2 According to Tommasi, halitosis can appear through two main mechanisms: 1) the odor substances released from the mouth and respiratory path by the passage of normal breath; and 2) the elimination of the already affected breath during hemathosis (deriving from odors eliminated by the lungs). 3 One mistaken concept blames the stomach for most halitosis cases. It is safe to say that only through gastric eructation (belching) will there be the elimination of aggressive breath from the stomach, since in resting conditions, the cardia zone is contracted and the sphincter pressure prevents reflux.4,5 Halitosis, also called fetor ex ore or fetor oris, is the offensive or unpleasant odor eliminated from the oral cavity. Much has been said regarding the etiology of halitosis: retention of odorous meal particles between the teeth; tongue coating; gingivitis; acute necrotic ulcerative gingivitis; periodontosis; dehydration after physical activity; caries; badly finished, cleaned, or adapted prosthesis; premenstrual periods; smoker's breath; surgical healing or tooth extraction wounds; intestinal dyspepsia; esophagus reflux; and sinusitis and rhinosinusitis.

The etiology of bad breath is usually (85% to 90%) an oral cavity phenomenon. The nose is responsible for 5% to 10%, followed by the tonsils (3%), with less than 1 % of the etiology originating below the tonsils.

The overgrowth of proteolytic, anaerobic bacteria on the tongue's surface has been reported as a halitosis etiologic factor. Other major etiologies in the production of halitosis are periodontal diseases, which may be treated by improving oral hygiene habits. The simple removal of the tongue coating (a bio-film deposit over the tongue dorsum) with adequate instruments may contribute considerably to reducing mouth-originated halitosis.

The aim of this clinical investigation was to compare the efficacy of a polystyrene tongue scraper to a soft bristle toothbrush in removing the tongue-dorsum coating, with the intention of reducing mouth-originated halitosis. For measurement purposes, a handheld sulfide monitor was employed.

MATERIALS AND METHODS
This crossover study was approved by the Ethical Committee. Fifty-two dental students or employees of the dental school were initially screened. Exclusion criteria included: periodontal disease; gastric conditions; presence of tonsils; smokers; or presence of hard or soft oral tissue lesions. We attempted to identify individuals with similar health conditions and eating habits. The study population consisted of 10 healthy subjects (both genders; age range 20 to 50 years) all of whom signed informed consent statements. Oral VSC values were measured using a handheld sulfide monitor. r Before the initial baseline VSC measurement, the subjects were instructed to refrain from any tongue-cleaning method for a 48-hour period. These volunteers were tested in the morning and asked to refrain from other oral hygiene activities as well, such as drinking, smoking, and eating, particularly candies since the flavors can mask the breath and induce a false-positive result. The 10 individuals were randomized into two groups of five each: group 1) first week: polystyrene tongue scraper,? second week: nylon soft-bristle toothbrush;1I group 2) first week: soft-bristle toothbrush, second week: polystyrene tongue scraper, with a 48-hour wash-out period between the weeks. The volunteers were instructed to use the cleaners after each meal and in the morning for 3 minutes, from the dorsum - posterior to the dorsum -anterior region of the tongue, according to the directions of the tongue scraper manufacturer.

The handheld sulfide monitor was employed according to the manufacturer's directions. The equipment is set to zero by shaking it 4 to 5 times, to eliminate any residual odors from its previous use.

When the monitor is turned on, it emits a beep; when a second beep is heard the volunteer blows into the airflow passage. We changed this procedure for hygiene reasons, using a disposable 6 mm x 80 mm straw which was discarded after use. In a previous pilot study (unpublished data) there was no alteration in the effectiveness of the equipment using the straw.

After a third beep, the breath odor level is measured by a graphic displaying one of the following values: 1) odorless, normal; 2) light odor, normal; 3) moderate odor, bad breath - perceptible; or 4) strong odor, bad breath - perceptible.

If no number appears, there is a reading error and the procedure must be repeated after cleaning the air opening with a dry cloth and shaking the monitor.

RESULTS
We achieved highly satisfactory results with both tongue dorsum coating removal methods, confirmed by the averages of volatile sulfur compound indexes (VSCIs) at the initial (I) and final (F) periods (Table 1) (75% reduction with the scraper and 40% with the toothbrush).

The results of the statistical analysis (non-parametric statistics, Dunn method)22 were significant at the 0.1 % level in five of the paired samples. Only the two initial YSCls were not significant for both the soft-bristle toothbrush and the tongue scraper. This is important, since it reveals there was the randomization of the distribution of methods, although 100% of the subjects presented an index of 4 after refraining from any tongue-cleaning method for a 48-hour period (time zero) (Table 2). A simple questionnaire was filled out by each of the volunteers, evaluating the two tools. There were complaints of nausea with the toothbrush (60%; four subjects from group 1 and two from group 2), and of tongue mucous traumatism (10%; one subject from group 1 when using the toothbrush), but under clinical examination, slight injury was observed due to the removal of some filiform papillae. All volunteers were receptive to using the tongue scraper.

DISCUSSION
Furred tongue constitutes the major cause of mouth originated halitosis and does not receive adequate attention from medical and dental health professionals. This pathological status is characterized by deposits of desquamated epithelial cells, leukocytes, microorganisms (fungus and bacteria), and food residues among filiform papillae, giving the tongue a pale appearance (or brownish-yellow in smokers). The reason for the tongue coating is esophageal antiperistalsis, a condition in which the cardia does not prevent small waves of muscular.The proteolytic activity of microorganisms in the oral cavity contributes to the volatile sulfur compounds found in malodorous mouth air. The metabolites include many compounds, such as hydrogen sulfide, methyl mercaptan, dimethyl sulfide, indole, skatole, and volatile fatty acids and polyamines.

Dorsal tongue cleaning is not a common habit among Brazilians, at least with a tongue scraper. We agree with Figueiredo et al. that the measurement of halitosis by multiple judges is embarrassing and includes subjectivity and reproducibility problems. Furthermore, Rosenberg et al. showed that a simple sulfide monitor could be used to measure the concentration of YCS in air expelled from the mouth with a significant positive correlation between YCS and organoleptic ratings The monitor in the current study was a valid alternative to measuring halitosis by judges, as shown by the results (Tables 1 and 2). This is a simple, quick, and easy-to-use reproducible technique for measuring breath-related sulfides. Moreover, the equipment avoids risk of pulmonary infections, since it is designed for personal use, although we adapted it for research purposes using a disposable straw. In the present study, both the tongue scraper and soft-bristle toothbrush proved efficient in removing the tongue coating, although the tongue scraper had a higher efficacy (35% fewer volatile sulfur compounds emitted).

CONCLUSION
In summary, both tongue-coating removal methods evaluated were efficient, but the tongue scraper was noticeably superior. The importance of tongue coating in the etiology of halitosis was corroborated by use of a handheld sulfide monitor, which proved to be feasible, quick, and easy to use.

The Effect of Four Mouth Rinses on Oral Malodor

Loretta C. Borden, MS
Eros S. Chaves, DDS
James P. Bowman
Barbara M. Fath

Consumer Products Testing Division
Hill Top Research, Inc.
Cincinnati, Ohio

Garry L. Hollar
Director of Regulatory Affairs
Discus Dental, Inc.
Culver City, California


Abstract

The purpose of the study was to compare the efficacy of four mouth rinses in clinical outcomes of changes in oral malodor measurements in a 4-week, randomized, double-blind, longitudinal clinical lariat in adults.

The four rinses secret coded as Products 1, 2, 3, and 4 so that neither the examiners nor subjects had the knowledge of treatment. Of the four mouth rinses, two were commercially available mouth rinses with essential oils (EO) or chlorine dioxide plus zinc (CD/Zn) as active ingredients (Products 1 and 4), one mouth rinse was a formulation containing cetylpyridinium chloride (Product 2), and one seas a placebo (Product 3).

A total of 99 subjects who met the study criteria were assigned randomly to one of the four groups. At three separate visits (0, 2, and 4 weeks), subjects received an examination of the oral soft tissues and were assessed for baseline oral malodor by two organoleptic judges and a laboratory instrument that measures oral malodor.

Subjects were instructed to use the assigned rinse, and the measurements were performed again after 2 and 4 hours. Throughout the 4-week study period, each subject was asked to use the assigned rinse twice daily per the manufacturer's recommended directions. The results showed that the four mouth rinses reduced oral malodor within 4 hours after a single usage, with Product 2 being the most effective and the placebo being the least effective.

Daily use of EO, CD/Zn, and placebo rinses for up to 4 weeks did not reduce oral malodor from week 0 baseline ethics, and the effects on oral malodor were comparable among these three mouth rinses. Product 2 was the only mouth rinse that reduced oral malodor from baseline values after 2 and 4 weeks of daily use.

Oral malodor, or bad breath, is a generic descriptive term for foul smells emanating from the mouth. It includes ozostomia, stomaxlysodia, halitosis, and fetor odors or fetor, which denotes different sources of oral malodor.

It has been estimated that 20% to 60% of the population suffers from chronic oral malodor, and in approximately half of these individuals, the problem becomes serious enough to create personal discomfort and social embarrassment.

Oral malodor has also been reported in children 2 to 7 years old and 5 to 14 years old. Because of the potential impact of oral malodor on personal life, sufferers often make desperate attempts to mask their oral malodor with mints and chewing gum, compulsive brushing, and repeated use of mouth rinses.

In most cases, the problem of oral malodor has been shown to originate in the oral cavity, where conditions that favor the retention of anaerobic, mainly gram-negative bacteria, lead to the development of bad breath.

In addition to periodontal pockets, the most important retention site is the dorsurm of the tongue with its numerous papillae." Volatile sulfur compounds (VSCs), primarily hydrogen sulfide and methyl mercaptan, appear to be responsible for much of the malodor. A number of systemic diseases, such as diabetes mellitus, uremia, and hepatic diseases, may also induce malodor.

The recommended initial treatment strategy is to identify the exact cause of the oral malodor, which can be difficult.

Oral hygiene, particularly the daily use of a tongue cleaner, can be effective in reducing the risk of oral malodor.

Introduction to etiologic therapy, oral care products, including mouth rinses, toothpastes, and chewing gums, may be used to mask the malodor.

It has been suggested that products containing metal ions, especially zinc, are effective in inhibiting odor formation because of their neutralizing effect on sulfur compounds.

Because of the implication of bacteria in producing malodor, there has been an increasing interest in using antibacterial mouth rinses to control oral malodor. Mouth rinses containing essential oils (EOs), chloride dioxide plus zinc (CD/Zn), or an oil-water-cetylpyriclinium chloride (CPC) combination have been shown to reduce the organoleptic scores of individuals with moderate levels of oral malodor. These results were found in the absence of tongue cleaning, although in several studies the findings are controversial and sometimes contradictory.

The purpose of this study was to compare the effect of BreathRx mouth rinse (2) two to commercially available rinses on oral malodor in adults in a 4-week, randomized, double-blind, longitudinal clinical trial. A placebo rinse was also included in the study to serve as the negative control.


Materials and Methods

The study was conducted according to applicable Good Clinical Practices and Standard Operating Procedures of Hill Top Research, Inc. Before conducting the study, protocols and pertinent documents were submitted and approved by an Institutional Review Board (IRB) in accordance with Title 21 of the Code of Federal Regulations, Parts 50 and 56. An informed consent letter was pro-vided to and signed by each of the study participants before the initiation of the project. There was one amendment to the consent form, which was submitted and approved by the IRB before the recruitment of subjects. The amendment informed the subjects that they would be required to stay at the study site for approximately 6 hours instead of 5 hours.

The four test mouth rinses were provided by the study sponsor in coded, identical packages as Product 1, 2, 3, and 4 so that neither the examiners nor the subjects had the knowledge of treatment. They were:

1. Listerine@ Antiseptic Rinse', a commercially available, EO-based rinse (Product 1).

2. BreathRx'" Mouth rinse, a formulation containing CPC (Batch # BTM-008A) (Product 2).

3. A placebo rinse (Batch c BTM-007P) (Product 3).

4. Oxygen' Mouthwash with Zinc`, a com- Commercially available, CD/Zn-based rinse (Product 4).

The repackaging of the EO-based rinse was performed using good manufacturing practice procedures. The CD/Zn rinse was left in the original manufacturer's sealed bottle with the label covered over.

To obtain a study population that represented typical users of the test mouth rinses, study subjects were recruited from local communities. Potential subjects were interviewed by phone and screened for their eligibility to participate in the study according to the following inclusion criteria:

1. In good general health based on medical history and oral soft tissue examination.

2. A man or woman between 18 and 65 years of age.

3. Willing to sign the informed consent form and comply with the protocol procedures.

4. Two-judge average intensity score of 4 on a scale of 5 and no single score of < 3.

5. Minimum of 16 natural teeth, including at least 4 molars.

6. Availability of the subject to complete the 4-week study.


Any subject with one or more ok the following conditions were excluded:

1. Gross oral pathoses that the investigators thought may compromise the protocol.

2. Orthodontic devices.

3. Partial or complete dentures.

4. Any systemic disease that the investigators thought may interfere with the study.

5. History of irritation or sensitivity to oral products.

6. Pregnant or lactating women as determined by medical history.

7. Periodontal disease (pocket depth > 4 mm and/or bleeding on probing 6 sites but: nonadjacent) or gross neglect of oral hygiene.

8. Smokers.

9. Prophylactic antibiotic coverage for routine dental therapy.

10. Use of systemic antibiotics or prescription mouthwash 21 clays before the study.

11. Current participation in other dental or investigational trials.

12. Patients on concomitant drug therapy were not included in the study based on the discretion of the investigator/examiner.

13. Library = alcohol infuse.

l4. Evidence of a recent history of bronchitis, tonsillitis, or sinusitis.

In addition, subjects were excluded from the sturdy if they received emergency dental treatment or if loss of teeth placed them below the minimum teeth requirements. Any subject treated with antibiotics or antibacterial agents during the course of the study were also dropped. Subjects were instructed to refrain from routine dental treatments, except for emergencies, for the duration of the study. Qualified subjects were randomly assigned to one of the four groups, each with a minimumber of 20 subjects. The subjects were asked to use the assignee) mouth rinse twice daily following the supplied instructions for use. They were provided with standard fluoride toothpaste and standard soft toothbrushes, and were required to not use any other dental crevices or products during the study period. It was recognized that subject compliance was essential and imperative to the reliability and accuracy of the clinical data. To maximize the compliance, the investigators reviewed and emphasized the instructions with each subject. Compliance was also monitored by reviewing daily logs of each subject's mouth rinse use along with quantification of the remaining mouth rinse returned at each visit.

A medical history was collected from each accepted subject. At each of the three visits (0, 2, and 4 weeks), subjects received an oral soft tissue examination and were assessed for base-line oral malodor by two organoleptic judges and a laboratory instrument that measures oral malodor (Halimeter). The oral soft tissue examination included observations of the lips, buccal mucosa, floor of the mouth, tongue, hard and soft palate, and gingiva. All findings were recorded as normal or abnormal. All abnormal findings, such as signs of sloughing, erythema, ulceration, or edema, were recorded and described on the evaluation form. Each parameter was scored as normal, mild, moderate, or marked. The subject was then instructed to use the assigned rinse, and the organoleptic measurements were performed after 15 minutes, 7. hours, and 4 hours, respectively. Halimeter measurements were performed after 2 and 4 hours. The organoleptic measurements were performed independently by two experienced, calibrated judges. The subjects were instructed to close their mouths for 2 minutes and to not swallow during that period. The subjects were then asked to breathe out gently, at a distance of 10 cm from the nose of the organoleptic judge, and the organoleptic odors were assessed according to a 5-point scale:

0 = No odor present.
I = Barely noticeable odor.
2 = Slight but clearly noticeable odor.
3 = Moderate odor.
4 = Strong offensive odor.
5 = Extremely foul odor.


The Halimeter (Model RF I-17K) was balanced with a zero reading and, to ensure the accuracy of each reading, the measurements follow. l the manufacturer's instructions. For the mouth reading, the short end of a flexible drinking straw was inserted approximately 1/2 inch into the subject's slightly parted lips, which were gently touching the straw. The subjects were instructed to hold their breath for 5 to 10 seconds until a peak in VSCs was reached. Readings for the VSCs were then recorded. For the throat and posterior tongue, the drinking straw was inserted approximately 3 inches into the subject's mouth near the throat. The subjects were instructed to hold their breath for 5 to 10 seconds, and the VSC reading was recorded. For the nasal passage/sinus, the straw was inserted 1/2 inch to 1/8 inch.

Subjects were instructed to hold their breath 5 to 10 seconds, after which time the VSC value was recorded. For data analyses, mean organoleptic and Halimeter scores as well as the mean and percent changes from week 0 baseline to weeks Zinc 4 baselines were calculated. The within-treatment data were analyzed using the Wilcoxon signed rank test. For the between-treatment analyses, Kruskal-Wallis contingency table technique was used. When statistically significant differences were detected, multiple comparison tests were performed to determine differences among products.


Results

A total of 138 subjects were recruited anal signed the consent form. After screening examinations, 99 subjects who met the study criteria were enrolled and completed the first visit (week 0). Three subjects were disqualified as result of a low organoleptic score of 2 and one subject withdrew consent after completing the first visit. Therefore, data collected from 95 subjects, including 29 men and 66 women who ranged in age from 19 to 65 years, were included in the analysis.

A total of 209 bottles of mouth rinses were distributed to the subjects, and four bottles (two for Product 1/EO and one each for Products 3/Placebo and 4/CD/Zn) were not returned. Two subjects inadvertently discarded three bottles. No specific reason was given for the other missing bottle. As indicated by subjects' diaries, all 95 subjects used the assigned mouth rinse according to instructions on the label and met the compliance criteria. The product usage was relatively even among subjects in the four product groups.

Eleven subjects missed between one and five at-home rinses; however, it is the authors' opinion that these deviations did not affect the outcome of the study.

There were 13 adverse events, including lip blisters, localized gingival edema, and canker sores, reported by 13 subjects during the course of the study. One subject was hospitalized with cellulitis, which was determined to be nonrelevant to the mouth rinse used.

The subject was treated for her condition and was discontinued from the study. After careful evaluation, it was determined that these Organoleptic Measurements.

The average baseline organoleptic scores at the initiation of the study were 4.14 for Product 1/EO, 4.22 for Product 2/CPC, 3.93 for Product 3/placebo, and 4.02 for Product 4/CD/Zn. The values are not statistically different, indicating that the initial average level of oral malodor was balanced among the four groups.

The organoleptic scores from baseline, 15-minute, 2-hour, and 4-hour evaluations after a single rinse with four test mouth rinses at weeks 0, 2, and 4 are summarized in Tables 1 through 3, respectively. The within treatment analyses using Wilcoxon signed rank test evaluated the changes from baseline at each visit. Mean changes and percent changes from baseline at each visit were also calculated. The data showed significant decreases in oral malodor for all of the rinses at all post-treatment evaluations except the placebo at the +hour evaluation in weeks 0 and 4 (Tables 1 and 3). The baseline organoleptic data from weeks 0, 2, and 4, which were collected before the use of the rinses, were further analyzed and the results showed that Product 2/CPC was the only mouth rinse that significantly reduced oral malodor after twice daily use for 2 and 4 weeks.

The other three mouth rinses did not have a significant effect on the baseline organoleptic data obtained from weeks 2 and 4 compared to week 0.

Product 2/CPC was comparable to Product 4/CD/Zn in reducing the organoleptic score at the 15-minute and 2-hour evaluations; however, Product 2/CPC was significantly more effective than the placebo and Product 1/EO. At the 4-hour evaluation, the reduction of organoleptic scores observed in the subjects using Product 2/CPC was significantly higher (P < .05) than the subjects using the other three rinses.

Further analysis of the baseline organoleptic data from weeks 0, 2, and 4, which were collected before the use of the rinses, showed that Product 2/CPC was the only mouth rinse that reduced oral malodor after twice daily use.


Halimeter Measurements

Tests for normality were conducted on the Halimeter data, and the results indicated that the data were not normally distributed. Therefore, nonparametric methods were used for data analyses. The average baseline Halimeter mouth readings at the initiation of the study were 89 for Product 1/EO) 8.8 for Product 2/CPC, 88.1 for Product 3/placebo, and 73.3 for Product 9/CD/Zn. For throat readings, the initial average baseline readings were 98.2 for Product 1/EO, 136 for Product 2/CPC, 106 for Product 3/placebo, and 99.7 for Product 4/CD/Zn. No attempts were made to balance the initial Halimeter baseline values, as the organoleptic score was the primary parameter for group balancing. While the Halimeter values, particularly those from throat, appeared to vary among the groups, they were not statistically different.

Because of possible interference to the performance of the Halimeter immediately after the use of mouth rinses, these measurements were performed at 2 and 4-hour evaluation after the rinse. The within-treatment analysis of the Halimeter data from the front of the mouth found significant decreases in scores (P < .05) with all of the products at the 2- and 4-hour evaluations in week 0. In addition, Product 2/CPC showed significant decreases in Altimeter readings (P < .05) at the 4-hour evaluation in weeks 2 and 4, while Product 1/EO showed significant decreases (P < .05) at the 4-hour evaluation in week 2 and the 2-hour evaluation in week 4. The within-treatment analysis of the Halimeter data obtained from the throat inclicatec1 significant decreases with Product 1/EO at both evaluations in week 0 and in the 2-hour evaluation in week 4 (I < .05). Significant reduction (P < .05) was also observed in Product 2/( PC at both the 2- and 4-hour evaluations in weeks 0 and 2 and at the 4-hour evaluation in week 4. Product 4/CD/Zn rinse was found to significantly reduce (P < .05) the Halimeter readings at the 2- and 4-hour evaluations in week 0.

There were no significant differences in Halimeter readings obtained from the mouth among the four rinses, regardless of the measurements taken at the 2- and 4-hour evaluations after a single rinse at each visit or the base-line values after 2 and 4 weeks of twice daily use. Significant differences were detected from the Halimeter data that was obtained from the throat at the 2-hour evaluation. Products 1/EO, 2/CPC, and 9/C L)/Zn resulted in significantly greater reductions from baseline Halimeter readings than the placebo rinse (P < .05) (Table 9). Numerically, Product 2/CPC induced the greatest reductions among the four rinses, although the differences were not significant with Products I/EO and 4/CD/Zn at the 2-hour evaluation and with the other three rinses at the 4-hour evaluation (Table 9 and Figure 3).

Significant differences among products were also detected in the reduction of Halimeter measurements obtained from the throat at the week 2 evaluation. Product 4/CD/Zn showed significantly larger baseline differences from week 0 baseline than Product 3/placebo (Table 10). However, the greatest numerical reductions were again observed with Product 2/Chart both the 2- and 4-week evaluations (Table 10 and Figure 4).


Discussion

Oral malodor is a multi-etiological condition. While certain systemic diseases can cause oral malodor, the majority of sufferers may have a local cause in the oral cavity. An increasing volume of research has shown that certain oral flora, particularly gram-negative anaerobes from the tongue or periodontal areas, are the main microorganisms capable of releasing VSCs from their proteolytic activity of debris and other materials. VSCs are particles of gases that are considered primarily responsible for oral malodor.

Mouth rinses have increasingly become an important option in the treatment of oral malodor. The use of mouth rinses was initially considered for cosmetic purposes, and their effects on oral hygiene were believed to be transitory. However, in 1986, the Council on Dental Therapeutics of the American Dental Association established guidelines for the acceptance of chemotherapeutic products for the control of dental plaque and gingivitis."

Antimicrobial mouth rinses are now considered an integrated part of daily dental hygiene practice. EOs and CI3/Zn are the active ingredients commonly used in mouth rinses for the treatment of oral malodor.

BreathRx'" mouth rinse (Product 2/CPC in the present study) is a formulation containing C:PC, EOs, and C'I)/Zn. The two commercial mouthrinses, which were included in the study for the purpose of comparison, contain either EOs or chloride dioxide and CD/Zn as active ingredients.

Although oral malodor is a common problem, there is still a lack of reliable means for its objective measurement, The Halimeter is a specialized device available to measure VSCs in the mouth; however, its accuracy and reliability have been questioned. In addition, the correlation ( oral malodor may not he limited) to VSC's the measurements may not be completely reflective of clinical situations. There are, a number of investigators have suggests that in practice the objective assessment of malodor is still best judged by the human sense of smell (i.e., the organoleptic method). In the present study, both the organoleptic method and Halimeter measurements were used for the evaluation of oral malodor. The organoleptic evaluation was per-formed by two experienced, calibrated examiners and served as the primary parameter for the evaluation of oral malodor. With the secondary data obtained from the Halimeter measurement, the methods for evaluating oral malodor used in this particular study were appropriate and adequate.

The data obtained at each of the three visits indicated that a single use of the mouth rinses was effective in significantly reducing oral malodor for up to 4 hours. This finding is in agreement with that reported by a previous study in which a single use of a CD/Zn mouth rinse significantly reduced mouth odor intensity for at least 4 hours. While possible therapeutic efficacy may not be completely ruled out, the reduction of oral malodor up to 4 hours after a single rinse may largely be a masking effect, as the placebo rinse was also effective. In addition, the mechanical cleansing of the oral cavity during the rinse may also help temporarily reduce the odor.

However, the significant reductions of oral malodor over the course of 4 weeks in subjects using BreathRx mouth rinse may not be attributed to the masking effect. The most effective and the placebo rinse the least effective. The daily use of the two commercial mouth rinses EO or CD/Zn as active ingredient and the placebo rinse for up to 4 weeks did not reduce oral malodor from week 0 baseline scores.

Product 2 with CPC (BreathRx) was more effective than the other three mouth rinses in reducing oral malodor.

    FINAL REPORT (LLUSD-STRL-02336)
Evaluation of Antimicrobial Potential of BreathRx Anti-Bacterial Mouth Rinse
Center for Dental Research
Department of Microbiology and Molecular Genetics Lorna Linda University Schools of Dentistry and Medicine 24876 Taylor Street Lorna Linda, CA 92350
November 18, 2002

FINAL REPORT
Evaluation of Antimicrobial Potential of BreathRx Anti-Bacterial Mouth Rinse

Sponsoring Agency
Discus Dental, Inc.
8550 Higuera Street Culver City, CA 90232 Attention: Mr. Garry Hollar

Conducting Agency
Biocompatibility and Toxicology Research Laboratory Lorna Linda University School of Dentistry
11092 Anderson Street
Lorna Linda, CA 92350
Study Number
LLUSD Center for Dental Research Study Number 02336
Yiming Li, D.D.S., M.S.D., Ph.D. Professor of Restorative Dentistry
Professor of Microbiology and Cellular Genetics
James Kettering, Ph.D.
Professor of Microbiology and Molecular Genetics
Co-Investigators
Ray Aprecio Sr., B.S. Research Technician
Wu Zhang, M.D., B.G.S. Assistant Professor

Purpose
The purpose of this study was to determine the antimicrobial potential of BreathRx Antibacterial Mouth Rinse in eight microbes using the standard Minimal Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays (Baron et aI., 1994), following the guidelines of the Oral Health Care Products for Over-the-Counter Human Use Tentative Final Monograph (FDA, 1994).

Materials and Methods
The eight test organisms used for the assays were specified by the FDA in the Oral Health Care Products for Over-the-Counter Human Use Tentative Final Monograph (FDA, 1994). The included: 1.) Actinomyces viscosus (ATCC 19246); 2.) Fusobacterium nucleatum (ATCC 10953); 3) Porhyromonas gingivalis (ATCC 33277); 4) Prevotella intermedia (ATCC 49046); 5) Bacteroides forsyth us (A TCC 43037); 6) Candida albicans (A TCC 18804); 7) Streptococcus mutans (A TCC 25175); and, 8) Escherichia coli (A TCC 11775). The microbes were obtained from the American Type Culture Collection (Manassas, VA). BreathRx Anti-Bacterial Mouth Rinse (BRX ACT 001, 0724/02) was provided by the study sponsor. The material was received on July 30, 2002 and was kept in original bottle at 4?C until tested. PRAS Brucella plates and fluid thioglycollate medium were obtained from Anaerobes Systems (Morgan Hill, CA). Bacto Todd Hewitt broth, Difco Tryptic Soy broth and agar plates, BBI GasPak disposable anaerobic indicator, and BBL GasPak Plus anaerobic system envelopes with Palladium catalyst were purchased from Becton Dickinson Microbiology Systems (Sparks, MD). Brain heart infusion medium and chopped meat broth were from Difco Laboratories (Detroit, MI). Facultative bacteria, including E. coli, S. mutans were cultured with Tryptic Soy broth and Tryptic Soy agar, while fluid thioglycoflate broth and PRAS Brucella plates were used to culture A. viscosus, F. nucleatum, P. gingivalis, P. intermedia and B. forsythus for both the MIC and MBC experiments. Brain heart infusion broth and chopped meat broth were used for growth purposes only.

Briefly, dilutions of the test rinse were prepared (1 ml total volume), starting from undiluted, 1:2 dilution up to 1 :512 dilution with sterile distilled water. An equal volume (1 ml) of the standardized cells was added to the diluted test rinse to make a total volume of 2 ml. The control was a mixture of 1 ml of sterile water and 1 ml of standardized test organism suspension. The mixtures were incubated overnight at 37?C. The turbidity of the culture was measured after 24 hours to determine the MIC of the test agent. If the mixture was clear, a volume of 100 III was plated on the appropriate agar plates, which were incubated overnight, and colonies, if any, were then counted to determine the MBC of the test agent. The MIC and MBC experiments were repeated using the same procedures to ensure the accuracy and reliability of the results.

Results
The results obtained from the first MIC and MBC experiments are presented in Tables 1 and 2, respectively. No evidence of microbial growth, as indicated, by the Clear media, was observed in tubes of S. mutans, P. gingivitis, P. intermedia and B. forsythus with the highest dilution (1 :512) of BreathRx Anti-Bacterial Mouth Rinse (Table 1). No growth of C. abacas and A. viscosus was detected at the dilution of 1: 128, while the media was clear at 1 :32 dilution in F. nucleate. E Coli, a none-oral bacterium, showed no evidence of growth at the 1:8 dilution of BreathRx Anti-Bacterial Mouth Rinse. The MBC results showed no colony growth of P. gingivitis, P. inter media and B. forsythus at 1:512 dilution of BreathRx Anti-Bacterial Mouth Rinse (Table 2), and no colony was observed in plates with S. mutans, A. viscosus, F. nucleate and C .. albicans at dilutions of 1: 128, 1:64, 1:32 and 1: 16, respectively. For E Coli, the bactericidal concentration of BreathRx Anti-Bacterial Mouth Rinse was 1:2 dilution (Table 2). The results of the repeat assay (Tables 3 and 4) were essentially the same as those obtained from the initial experiments. The only difference was found in C. albacons in the MIC assay; the no growth (clear media) was observed at 1 :64, which was a one-step higher concentration than that observed in the first assay. The MBC results were the same as those of the first assay. In both the first and repeat of the MIC and MBC assays, test microbes grew normally in control tubes or plates, and no microbial growth was detected in the media control

Conclusion
It is concluded that, under conditions of the present study, BreathRx Anti-Bacterial Mouth Rinse at highly diluted concentrations has significant bacteriostatic and bactericidal effects on S. mutans, A. viscosus, C. albicans, P. gingivatis, P. intermedia, F. nucleatum, B. forsythus as determined using the MIC and MBC methods. BreathRx Anti-Bacterial Mouth Rinse also effectively inhibits the growth of E. Coli at 1:8 dilution and kills E. Coli at 1:2 dilution.

    Feb 1997 CDA Journal
Sushma Nachnani, BS, MS

The Effects of Oral Rinses on Halitosis

Oral rinses are one of the therapeutic approaches to the treatment of halitosis. However, knowledge and understanding of this condition are in their infancies, and there 'are only a few products on the market geared specifically to the eradication of oral malodor. In addition, there is a paucity of clinical trials to substantiate the efficacy of these products for halitosis.

Halitosis can be symptomatic of many localized and systemic disorders of a normal physiological, pathological or psychiatric nature. Malodor caused by normal physiological processes and behaviors is usually of a transitory nature, such as "morning breath" and foul breath resulting from ingesting pungent foods, such as onion and garlic. Halitosis can be symptomatic of a more serious pathological disorder; but most breath malodor, in otherwise healthy people, stems from the oral cavity. The way to differentiate between an oral and non-oral etiology of patient complaints is to compare the odor emanating from the mouth with that from the nostrils. For those with no systemic disease and presenting with healthy dentition and gingiva, the malodor generally originates on the tongue dorsum. Significantly more bacterial cells attach as compared to other oral mucosal areas. The quantity of tongue coating has been reported to be many times greater in periodontal patients as compared to normal sub-periodontal tissue, oral malodor is more likely to originate from the tongue dorsum.

Recent scientific evidence reports that in addition to tooth brushing and oral rinsing, tongue cleaning should be an essential part of oral health care. This practice has not played much of a role in Western culture but is more practice in Africa, Asia and South America. An early 20th century researcher recommended daily tongue cleaning when it was found that the tongue dorsum was a breeding ground for Staphylococci and Streptococci.6 A clean tongue should be a healthy pink color; a whitish haze on the tongue is indicative of bacterial buildup. The more recent interest of including the tongue as an integral part of mouth care has precipitated an increase of tongue scraping devices on the market.

Volatile Sulfur Compounds
The tongue geography provides an excellent putrefactive habitat for gramnegative anaerobes that metabolize proteins as an energy source via breakdown of proteinaceous substrates from impacted food particles and sloughed off oral cellular debris, In a healthy mouth, dead epithelial cells are shed into the saliva, swallowed and digested fast enough so that they do not putrefy and cause halitosis. Inflammation causes these cells to be shed at a faster rate than the saliva can cleanse. Bacterial action then hydrolyzes the proteins to amino acids; and three of the amino acids that contain sulfur functional groups - methionine, cysteine and cystine - are the precursors to volatile sulfur compounds (VSCs). These gaseous substances, responsible for malodor, consist primarily of hydrogen sulfide (HzS), dimethyl sulfide [(CH3)zS], methyl mercaptan (CH3SH) and sulfur dioxide (S02)' Methionine is reduced to methyl mercaptan, and cysteine and cystine are reduced to hydrogen sulfide in the presence of sulfhydrase-positive microbes This microbial activity is favored at a pH of 7.2 and inhibited at a pH 6.5 (also inhibited in O.02M glucose).

Proteolytic activity has been associated mainly with gram-negative bacteria that reside in the oral cavity, including the following: Treponema denticola, Porphyromonas endodontaIis, Prevotella intermedia, Prevotella melanino'en ica, Rothia dentocariosa, Haemo-phi/us, Veillonella alcalescens, Porphyromonas gingiva lis, Capnocytophaga, Fusobacterium nucleatum, Bacteroides forsythus and Stomatococcus mucilaginus, a gram-positive microbe. Methyl mercaptan has been found to be the main component of tongue dorsal surface malodor in patients with periodontal disease, whereas hydrogen sulfide predominates in orally healthy subjects. The levels of 2-ketobutyrate, a byproduct of methionine metabolism, are increased in the oral cavity of patients with periodontal disease.

VSCs are toxic substances that have been found to damage the collagen and proteoglycan components in connective tissue by cleaving disulfide bonds. This dcaggregation of the extracellular matrix allows microbes to permeate the oral mucosa. Studies have found methyl mercaptan, more specifically, to reduce collagen content in fibroblast cultures, suggesting that a ~;milar process may occur in severely inflamed periodontal tissue, with methyl mercaptan playing a modulation role in fibroblast cell metabo-used for the detection and measurement of VSCs. The Perlotemp is an instrument used to measure the temperature of subgingival areas. "Hot" pockets can be indicative of a disease process involving microbial activity.

Mouth Rinse Formulations
A difficulty in formulating a mouth rinse for the treatment of halitosis involves the delicate balance of eliminating the offending microbes, while maintaining the dynamic balance of normal flora, as well as preventing an overgrowth of opportunistic pathogens. In addition, the specific bacterial species and their cooperative interactions responsible for breath malodor have yet to be fully elucidated. The quality of saliva is an active player in this intricate balance. Saliva lubricates and oxygenates the oral cavity as well as aids in digestion, acts as a buffering agent and provides antimicrobial properties to supplement the individual's vast arsenal of immune functions.7 Any treatment that increases saliva flow and tongue action, including the chewing of fibrous vegetables and sugarless gum, will help decrease malodor. At this stage of oral malodor treatment, for otherwise healthy individuals, the clinical emphasis is on an attempt to freshen the breath by sufficiently decreasing the odoriferous VSCs generated by a variety of microbial populations. This is accomplished in part by mouth rinses containing active ingredients with antiseptic properties.

The oral rinse is a supplement to thorough mechanical cleaning of teeth, gums, other oral tissues and, especially, the tongue dorsum. Most commercial mouth rinses mask odors, which has a short-lived effect, as well as provide antiseptic properties. Prevention of malodor that lasts beyond 30 minutes after rinsing is a result of the antiseptic compounds, but often even these substances do not provide long-lasting effects. Mouth rinses effects are likely to be longer-lasting at this time. Rinsing with water is only minimally helpful, since water has no antiseptic properties and often merely washes away saliva, which does have such properties. "Mouth rinses cannot kill all the offending microbes, most of which survive antiseptic attacks protected under thick layers of plaque and mucus.

Most mouth rinses include alcohol, propylene glycol, methyl salicylates, glycerin, sodium bicarbonate, cresols, bromides, boric acid, surfactants such as sodium lauryl sulfate, sweeteners, flavorings and food colorings. The common active antimicrobial agents areessential oils; phenolic derivatives; hydrogen peroxide; quaternary am- Any treatment that increases saliva flow and tongue action, including the chewing of fibrous \1egetables and sugarless gum, will help decrease malodor. monium compounds; benzalkonium chloride; chlorhexidine; chlorine dioxide; sodium benzoate; sodium perborate; mono flu oro phosphate; sanguinarine, an alkaloid herbal extract; cetylpyridium chloride; triclosan; del!l1opino[ hydrochloride; fluorides; and zinc salts.

Adverse Effects
Some generalized irritations to the oral cavity caused by mouth rinse ingredients are desquamation, ulceration, inflammation, gingivitis and petechiae. In addition, any of these substances can be a potential allergen, especially for the atopic patient.

With long-term use, it has been reported that the alcohol in commercial rinses may bring about some adverse effects. It has been reported that ethanol in oral rinses induces painful oral sensations.13 The most prevalent problem with ethanol is that it can dry the oral tissues. This condition in itself can induce oral malodor, since a critical quantity of saliva, in addition to the saliva quality with its antiseptic components, is necessary to maintain a clean healthy oral condition. A dry mouth is the condition leading to morning breath. While a person is sleeping, the saliva flow is decreased, thereby drying the mouth. Proteinaceous components in the oral cavity, including those in the stagnant saliva (the protein concentration of saliva is 20 times greater than that of carbohydrate), provide a ready supply of substrates for microbial action. Under these conditions, the gram-negative anaerobic population dominates.

A medical condition of dry mouth called xerostomia can be treated with an alcohol-free enzyme mouthwash, specially formulated with three antibacterial enzymes, lysozyme, glucose oxidase and lactoperoxidase; the latter two combine with their substrates to produce the hypothiocyanite ion that inhibits the growth of bacteria. Although clinical evidence for efficacy is limited, the idea of using natural saliva components to treat oral conditions is promising for the future of dental care.

There is some controversy as to whether the use of alcohol rinses are associated with oral cancer.12,lS The alcohol content of commercial oral rinses varies from 5 percent to 26,9 percent, but most have 5 percent to 7 percent The decision by the FDA states that there is no evidence to support the removal of alcohol from over the-counter products, However, since there have been reported cases of Mouth rinse-induced hypoglycemia fatal poisoning, especially in children., the Council on Dental Therapeutics has' recently voted to require manufacturers of mouth washes that contain greater than 5 percent alcohol and carry the ADA seal of acceptance to place child-safety caps and warning labels on the bottles.

Quaternary Ammonium and Decaplnol Rinses
Commercial mouthrinses containing antibacterial quaternary ammonium compounds have been found to be associated with a significant decrease in hydrogen sulfide and methyl mercaptan as compared to a deionized water placebo mouthrinse.I7 This would suggest some degree of benefit as a malodor rinse. Delmopinol HCI (3(4 propylheptyl)-4-morpholinoethanol hydrochloride) rinse has demonstrated efficacy as a prevention measure against gingiVitis in the absence of mechanical plaque control,IS however there are few If any trials on its effectiveness in controlling halitosis.

Zinc Rinses
Clinical trials conclude that zinc mouth rinses are very effective for reducing oral malodor in some people with good oral health. The zinc rinses (in chloride or citrate form) have been found to significantly reduce oral VSC concentrations for a duration greater than three hours. A zinc mouth rinse performed significantly better than a water rinse in reducing and maintaining VSC levels below the objectionable malodor threshold, when used overnight (10 hours).! Other investigations have suggested that zinc mouthwashes counteract the toxicity of volatile sulfur compounds. Zinc ion (In+2) is known to playa role in wound healing, by functioning as a prosthetic group in DNA polymerase. After treatment of damaged oral mucosa with a 0.22 percent zinc chloride rinse, tissue permeability was restored to a state similar to that observed in a control group.

Chlorhexldine Rinses
Chlorhexidine digluconate, useful in decreasing gingivitis and plaque buildup, is an active ingredient in certain ADA-approved commercial mouth rinses. It is one of two mouthrinses shown to reduce gingiVitis in long-term clinical trials and appears to be the most effective antiplaque and antigingivitis agent known today.2,!9 It has been found to enhance the gingival healing process following scaling and root planing.I9 Chlorhexidine in tablet form with xylital and fluoride has been found to work as well as a conventional chlorhexidine rinse in reducing oral plaque.2o Both tablet and rinse forms used by Military Academy cadets, refraining from mechanical tooth cleaning, worked equally as well to decrease plaque and periodontal index scores, and both were significantly more effective than a sodium fluoride rinse.2! In a clinical triaIZZ comparing six commercial mouth rinses with a dentifrice slurry on oral plaque regrowth found the regrowth (after prophylaxis) to be significantly reduced by a chlorhexidine rinse compared to an oxidizing rinse of peroxyborate, while the latter rinse significantly outperformed all the other rinses with active ingredients of sodium monofluorophosphate, sodium benzoate, cetylpyridium chloride and saline (0.7 percent NaCl). However, chlorhexieline efficacy as a mouth rinse

The antimicrobial action of triclosan involves interference with bacterial synthesis of ribonucleic acid and protein.30 ied extensively. More recently, a phenolic compound mouthwash was awarded the ADA Council on Dental Therapeutics' Seal of Acceptance as an aid in controlling plaque and gingivitis, but it has little efficacy as a mouthrinse for controlling chronic halitosis. The primary side effect of many antiseptics, including chlorhexidine, is discoloration of teeth and tongue; other side effects can include desquamation and painful lesions, but the latter were not found for chlorhexidine rinse with concentrations of less than 0.2 percent.2S-27 Also, the rinse may promote dental calculus increase, ironically, along with a significant gingivitis decrease.28 An important consideration for long-term use of chlorhexidine rinse is its potential to disrupt the oral microbial balance, causing some resistant strains to flourish, such as Streptococcus viridans.29 Since chlorhexidine is cationic, it may be inactivated with anionic substances such as phosphates, sulfates and anionic detergents inappropriately used (in conjunction with chlorhexidine) as supplementary mouth rinse ingredients. Also, there may be competitive inhibition in the presence of calcium (Ca2+) ions.

The action of chlorhexidine is thought to be due to its disruption of bacterial cell membrane permeability bringing about cell lysis and death. There is an electrical attraction between this cationic antiseptic rinse and the anionic areas on bacterial cell membranes. Studies have demonstrated a longer persistence of activity in the oral cavity for up to as long as 12 hours as compared to other mouth rinse active ingredients, such as cetylpyridium chloride, a cationic surfactant.

Chlorine Dioxide Rinses
Chlorine dioxide (C102), a strong oxidizing agent, has a high redox capacity with compounds containing sulfur. It functions best in neutral pH at a concentration of two parts per million to destroy sulfur-containing compounds by oxidation of sulfide bonds. The oxidizing agent, also used in water disinfection and in food processing equipment sanitization, oxidatively consumes oral substrates containing cysteine and methionine thus preventing production of VSCs. Since chlorine dioxide readily loses its activity, its stability has been prolonged through a "stabilization" process, which converts chlorine dioxide to molecular chlorine dioxide at a low pH. This stabilized chlorine dioxide is a mixture of the oxychlorine species in addition to sodium chlorite. It has been proposed that chlorite ions in mouth rinse products will be converted to chlorine dioxide in the acidic environment of the oral cavity. Another side in this controversy suggests that "stabilized chlorine dioxide" is a solution of sodium chlorite, containing no free chlorine dioxide and sodium chlorite has no known deodorizing ability. However, adequate clinical investigations are .needed to more thoroughly evaluate the behavior of these chemical components under different conditions in the oral cavity. More specifically, long-term clinical trials are required to substantiate the use of chlorine dioxide products in the treatment and control of halitosis.

Triclosan Rinses
Triclosan (droxydiphenylether) is a broad spectrum nonionic antimicrobial agent. This lipid-soluble substance, which has been studied for more than two decades, has recently been incorporated into oral care products after many years of use in the cosmetics industry. It has been found to be effective against most types of oral cavity bacteria. The antimicrobial action of triclosan involves interference with bacterial synthesis of ribonucleic acid and protein.

There is some evidence in the literature to support the safe use of triclosan oral rinses in the reduction of plaque and gingivitis.oJl,32 However, cetylpyridium chloride and chlorhexidine mouthwashes have been found to be more effective for plaque control than a triclosan rinse33 or a zinc/triclosan rinse,34 When compared to an ethanol placebo rinse, both triclosan and chlorhexidine oral rinses significantly outperformed the ethano" rinse on gingival and plaque index scores.

Triclosan's antigingivitis effect may be due to a direct anti"inflammatory action.3! When formulated with the surfactant sodium lauryl sulfate (SLS), triclosan has been reported to lessen the mucosal irritation effects of SLS36-38 by its analgesic properties.

The efficacy of triclosan is dependent on its concentration40 and its supplementation with other components of oral rinses and dentifrices.32 It has been found to be less effective as a plaque inhibitor in vegetable oil solvents.4l In long-term clinical trials, triclosan in combination with polyvinylmethyl ether/maleic acid, used as a prebrush mouth rinse, demonstrated significant plaque and gingivitis reduction as compared to a placebo rinse.42,43 Dentifrice studies of triclosan, formulated with silicone, were effective in the reduction of plaque and gingivi tis. 44,4\ There is speculation that lipid substances that adhere to teeth, gums and other mucosal surfaces, serve as carriers for the antibacterial hydrophobic triclosan, allowing for its slow, continuous emission into saliva,46 Long-term studies demonstrate only beneficial ecological changes and no induction of triclosan-resistant opportunistic pathogens.

A pilot study paper was presented at the Second International World Workshop on Oral Malodour. The results of the study demonstrated that this system had a cumulative effective, with the reduction of malodor increasing with the duration of product use. Two-Phase Rinses Two-phase oil-water mouth rinses are being tested to control halitosis. A recent clinical trial49 reported significant long-term (six-week) reductions in malodor, from the whole mouth and the tongue dorsum posterior, for the two-phase oil-water rinse group as compared to the control group using a commercial rinse. The efficacy is thought to be related to reduction of odor-producing microbes on the

Alternative dental health services also suggest the use of chlophyll oral rinses in addition to spirulina and algae products reported to contain antimicrobial properties. tongue dorsum posterior because of a polar attraction between oil droplets and bacterial and oral cells. The oil-water rinse also contained cetylpyridiuI11 chloride, which promotes the adhesion of microbes to oil droplets. However, there was no significant difference between the two for volatile sulfur compounds.

A clinical triaF7 investigated the effects of a two-phase rinse on the production of volatile sulfide in vivo and in vitro, as well as the efficacy of mouth rinses over a duration greater than three hours. Sixty dental students were divided randomly into three rinse groups: I) two-phase containing olive oil, essential oils, cetylpyridium chloride 2) chlorhexidine rinse and 3) placebo with the same concentration of food color and sweetener as the other two rinses. Malodor measurements, using the halimeter, the oxygen-depletion and organoleptic tests, taken eight to 10 hours after use the oral rinse use, demonstrated that both the two-phase rinse group and the chlorhexidine rinse group significantly decreased the level of volatile sulfur compounds as compared to the placebo. Both performed significantly better with respect to the placebo group, with the chlorhexidine group performing significantly better than the two-phase group measured by oxygen depletion. In addition, the chlorhexidine group performed significantly better compared to the placebo (but not the twophase) group on the organoleptic tests. The mechanism of action for the oilwater system has been discovered to be oil adsorption to the hydrophobic sites on bacterial cell membranes. The chlorhexidine worked more effectively in the three assessments.

A short-term two-phase mouth rinse investigation involving 20 healthy male volunteers used a mouth rinse containing an oil phase with olive oil and essential oils and an aqueous phase with cetylpyridium chloride. In comparison to a water rinse control group, an 80 percent reduction of sulfides was observed. In addition, "volatile sulfide and 2-ketobutyrate putrefaction system were completely inhibited by the two-phase mouthwash.

Alternative Remedies
The Second International World Workshop on Oral Malodor, held in Belgium, offered an abstract presentation on natural controls for bad breath that concluded that chewing a gum containing tea extracts was useful to control bad breath. Also recommended were natural deodorants such as copper chlorophyll and sodium copper chlorophyllin. Alternative dental health services also suggest the use of chlorophyll oral rinses in addition to spirulina and algae products reported to contain antimicrobial properties. One antiplaque breath product is a tablet composed of an effervescent active ingredient, a specially treated polymer of silicon dioxide and sodium bicarbonate, the latter of which acts as a buffering agent and mild bacteriocide. A mechanism of action for this complex is reported to involve the adsorption of microbes and other oral organic debris to the silicon dioxide polymer. There is a pilot study in progress to determine the efficacy of the product for the treatment of malodor.

Conclusion
Many of the oral rinses available today are being used for the prevention and/or treatment of oral malodor, but for the chronic bad breath condition, many rinses offer little to no help, and others have been formulated for other purposes. Much more research is required to develop an efficacious mouth rinse for the alleviation of halitosis. The treatment of halitosis is a relatively new field in dentistry and many of the treatments thus far have involved a trial-and-error approach.

    J Periodontal March 2005

Efficacy of Chlorhexidine Mouth Rinses with and Without Alcohol: A Clinical Study

J.L. Leyes Borrajo, * L. Garcia Varela, * G. Lopez Castro, * I. Rodriguez-Nunez, * M. Garcia Figueroa, t and M. Gallas Torreira *

Background:
Plaque control is the main method for preventing periodontal diseases. Chlorhexidine digluconate mouth rinse is widely recognized as helping to maintain plaque control. Most of these mouth rinses contain alcohol, making them impractical for many patients, including those with oral mucosal hypersensitivity. Mouth rinses without alcohol might cause fewer side effects, but also be less efficient. In this study, we evaluated the efficacy of a 0.12% chlorhexidine mouth rinse without alcohol against one with 11 % ethanol and a placebo.

Methods:
This a double-blind, parallel group study with 96 patients who tested 3 mouth rinses containing 1) chlorhexidine digluconate 0.12% sodium fluoride 0.05%, and ethanol 11 % (group 1; CHX-A); 2) the same solution without alcohol (group 2; CHX-NA); and a placebo (group 3; P). Plaque and bleeding indexes were recorded in all patients prior to treatment and at 14 and 28 days. Results: There were significant differences in plaque, gingivitis, and papilla bleeding indexes in both chlorhexidine rinses compared to placebo, but no differences between the 2 CHX products.

Conclusions:
In this study, the alcohol-free rinse was as effective as one containing alcohol in controlling plaque and reducing gingival inflammation. Therefore, it would seem that its use can be recommended in all patients, but especially in patients for whom the use of alcohol is contraindicated.

KEY WORDS
Chlorhexidine/therapeutic use; dental plaque/prevention and control; comparison studies; gingivitis/prevention and control; ethanol/metabolism.

Plaque control using mechanical means (tooth brushing and flossing) and chemical methods have been the primary preventive measures. Since mechanical means alone have limitations, attention has been given to chemical agents.1 Mouth rinses have been shown to be of considerable help in controlling plaque, particularly supragingival plaque, as adjuncts to mechanical means.

Although there are a wide range of products recognized as efficacious in preventing plaque formation and gingivitis, it is widely accepted that the most effective are those containing chlorhexidine digluconate 0.12%.4-6 However, these mouth rinses produce undesirable side effects such as staining and taste distortion, which are not acceptable to many patients.

A preliminary study reported that alcohol-free mouth rinses cause less patient pain than those containing alcohol.]2 It is accepted that tobacco and alcohol are 2 risk factors for oropharyngeal cancer; however, the association between cancer and the use of alcohol-containing mouth rinses is still being evaluated.

Alcohol is used in mouth rinses as a dissolvent of other ingredients and as an antiseptic agent. Its presence in mouth rinses is detrimental to patients with mucositis, who are immunocompromised, or are sensitive to the ingredient. Its use is also contraindicated in patients undergoing radiation therapy for head and Neck cancer.

The development and use of alcohol free mouth-rinses are relatively new. Certain studies have shown their efficacy and lack of side effects, 5 but there is no clear confirmation.21 The aim of the present study was to evaluate the efficacy of an alcohol-free CHX mouth rinse to the same preparation with 11 % ethanol and a placebo.

MATERIALS AND METHODS

Study Population:
The study included 97 patients (37 men and 60 women; average age 35 years) who attended the periodontics clinic at the Faculty of Medicine and Dentistry at the University of Santiago de Compostela and 3 local private practices. More patients were placed in the placebo group because our experience indicated that participants in this group are more likely to exit a study; in this study, however, the only patient who failed to complete the study was in the CHX-NA group. The inclusion criteria were ages 25 to 50 years and a minimum of 22 teeth. Exclusion criteria were adverse reaction to chlorhexidine, antibiotic therapy, diabetes, pregnancy, tactation, or tooth brushing 4 times a day.

Patients were informed of the nature of the study and gave informed consents. Medical histories, including current and past diseases and conditions, current medications and a dental history including tooth brushing frequency were taken.

This was a double-blind study with 3 parallel groups using 3 different solutions from the same firm: 0.12% chlorhexidine digluconate, 0.05% sodium fluoride. and 11 % ethanol (group 1; CHX-A); the same CHX formulation without the ethanol (group 2; CHX-NA); and a placebo.

Baseline plaque and papillary bleeding index26 were recorded and patients assigned to 1 of 3 groups balanced for age and gender; there were no differences between the initial mean values of the clinical indexes.

Patients were instructed to rinse for 30 seconds with 10 ml of their undiluted mouth rinse once a day for 27 days and to refrain from eating or drinking for 1 hour afterward. They were also instructed to brush 3 times a day with the dentifrice and brush of their choice at least 30 minutes before using the mouth rinse to avoid possible pharmacological interactions.

They were instructed not to use any other dental hygiene products during the study period. PI and PBI were recorded at 14 and 28 days; both examiners and patients were blinded to the mouthwash used.

Statistical Analysis:
Analysis of variance (ANOYA) with Scheffe and Games-Howell tests for independent variables and Student t test for related variables were used for PI (nor- . mal distribution: Z = 1.26; P = 0.082). Since the Kotmogorov-Smirnov test did not fit normal distribution (Z = 1.38; P = 0.082) for PBI, the Kruskal-Wallis, MannWhitney for 2 independent samples, and Friedman ANOYA for K-related samples were carried out for independent K samples. Cross-tabulation and chi square test of independence were used to examine relationships among the categorical variables. All tests were carried out using a statistical software program. P <0.05 was considered statistically significant unless otherwise indicated.

RESULTS:
The mean PI and PBI values were reduced in all groups; however, there were statistically significant differences. The difference in PI after 14 days in the CHX groups (1 and 2) compared to placebo was 0.5728 and 0.6639 (P = 0.001), indicting a greater plaque reduction with the CHX rinses. However, the difference (0.091]) between the CHX groups (] and 3) was not statistically significant. At 28 days, the difference between the CHX groups and placebo was significant (P= 0.011 and P= 0.001), while there were no differences between the 2 CHX solutions.

In groups 1 (CHX-A) and 2 (CHX-NA), differences between the baseline and 28-day scores were significant (P= 0.021; P= 0.0001, respectively) while in the placebo group (3) there was no difference between the time points.

Side Effects:
All patients using chlorhexidine mouth rinses showed some degree of staining, which did not occur in the placebo group. The most frequently reported reactions were apthaes, burning sensation, and foaming. Except for staining, there was no correlation between the side effect and the mouth rinse.

DISCUSSION:
Several studies indicate that chlorhexidine has properties that make it a suitable anti plaque agent. However, it does have undesirable side effects, primarily staining and taste distortion which limits its long-term use. It should be noted that none of our CHX patients reported taste distortion as a problem.

In this study, we tried to determine whether chlorhexidine maintains its beneficial effects without alcohol by comparing 2 CHX mouth rinses with and without alcohol and a placebo on patients with similar baseline PSI and PI values. Our results indicate that CHX mouth rinses, with or without alcohol, reduce plaque levels, which is similar to other studies.

We should note that in our study, unlike that of Eldridge where all other oral hygiene was discontinued, our patients continued their normal tooth brushing habits. This is important as it has been suggested that the efficacy of CHX rinses may vary depending on whether or not they are used in conjunction with toothpaste.

3 groups was expected, due to the patients' motivation to maintain good oral hygiene. Since our patients were matched for age and gender, it is likely that the effects of tooth brushing were analogous; however, a randomized controlled trial is needed to eliminate differences in oral hygiene to account for our findings

We noted no differences in clinical indexes in group 1 (CHX-A) between 14 and 28 days when, in theory, there might have been less motivation to maintain oral hygiene as initial enthusiasm in participating in a clinical trial waned and the effect of the mouth rinse became more obvious. However, we did notice continual changes in the CHX-NA group. This could be interpreted as meaning that mouth rinses with alcohol, itself an antiseptic agent, have a faster effect on PI and PSI than non-alcoholic solutions. However, it should be remembered that, although there was no statistically significant difference in baseline plaque levels, those of the CHX-NA group were slightly higher, which would favor a continual reduction.

Gingival bleeding was reduced in all patients who use CHX, while it remained basically unchanged in the placebo group. These data coincide with previous studies. As with Eldridge et al., we observed no differences in the effects of mouth rinses, with or without ethanol, in reducing gingival inflammation.

Staining problems with chlorhexidine have been published in a number of studies I and all our patients using CHX rinses reported this side effect on their teeth and/or tongues. Excluding discoloration, there were no differences in side effects among the 3 mouth rinses.

CONCLUSIONS:
The data in this study gathered from patients with gingival and periodontal disease at baseline confirm previous studies and indicate that the use of CHX mouth rinses is effective for plaque control and in reducing gingival bleeding.

Within the limitations of this study, we have shown that alcohol-free chlorhexidine mouth rinses are as effective as those with alcohol for controlling microbial plaque and reducing gingival inflammation. For this reason, they should be recommended for patients for whom alcohol use is not acceptable or contraindicated, as well as patients with oral lesions where alcohol could cause pain.

The Antibacterial Action of Eugenol, Thyme Oil, and Related Essential Oils Used in Dentistry

Harry G. Meeker, DMD Associate Professor

Department of Comprehensive Care and Applied Practice Administration Department of Operative Dentistry

Harald A. B. Linke, PhD, MSc, BSc Professor

Department of Microbiology

College of Dentistry New York University New York, New York

The worldwide overuse of antibiotics has caused microorganisms to develop resistance to current antibiotics and to become virulent. Microorganisms, however, do not appear to develop tolerance or resistance to the antibacterial effects of essential oils such as clove oil (eugenol) and thyme oil (thymol). Such oils provide an effective, powerful, and cost-effective means of infection control in the dental practice. This article discusses the antibacterial action of eugenol, thyme oil, and related essential oils used in clinical dentistry.

Introduction
Essential oils have been used since the 19th century for root canal therapy, temporary fillings, and periodontal therapy. Thyme oil is the most germicidal essential oil Thymol, a component of thyme oil, has been used in several medicaments. During the first half of the 20th century, thymol was used in alcohol solutions for cavity sterilization, but alcohol decreased the effectiveness of Essential! Oils. Instead of eliminating the alcohol from the solution, practitioners mistakenly discontinued using thymol in dental procedures.

Dental cements. bases, liners, and impression pastes that are based on the reaction between zinc oxide and eugenol [which forms zinc oxide-eugenol or ZOE) are antibacterial because eugenol is slowly released from the set mixture.' ZOE is biocompatible with the pulp and provides valuable anodyne and abundant effects as a temporary and permanent luting agent. It is an excellent thermal insulating base and sealer against micro leakage. ZOE is the material of choice for the indirect pulp capping of deep cavities in restorative dentistry because it is palliative and germicidal.

Eugenol is the main component [800/0) of clove oil, which is obtained by pressing or distilling the buds, leaves, and stems of the evergreen tree Eugenia caryophyllata. Clove oil is sometimes preferred over eugenol in impression pastes because it is less irritating to the oral tissues.

The History of Essential Oils
Cloves (from the French word dou, meaning "small nail") are a tropical fruit t grown on the Spice Islands of Madagascar, Zanzi bar. Cloves Ware unknowvn in ancient Egypt, Rome, and Greece, but were used by the emperor of China in 200 BC as a mouth perfume. The first medicinal text reference to cloves was by AI-Gazzar in the 10th century, when they were used to control mouth odors and pain.

Eugenol was the first essential oil proved to be a significant germicide and sedative for dental use. This was discovered in 1837 b' Bonastre, who used a combination of magnesium oxide and eugenol as a dental filling material. In 1876, Chisholm improved the formula by substituting zinc oxide for oxide. He was the first to develop (recommend ZOE as a temporary. Cast restorations were perfected within as (by Taggart in 1907, and this development led to the use of ZOE cements that possessed a thin film And that were biocompatible and suitable as temporary, intermediate, and permanent restorative cements.

Most of the dental filling available in the late 19th and early 20th centuries were based on the eschaotic principle that local remedies had to destroy soft tissue on contact to be antiseptics. Such products included phenol (carbolic Reid), cresols, formaldehyde, and silver lensotrate. These substances were blended with zinc oxide and used as cements.

Phenol was used in dentistry in the late 19th and early 20th centuries as an antiseptic for cavity sterilization and as a root canal paste when mixed with zinc oxide, A germicidal standard of reference is the phenol coefficient, which compares the effectiveness of germicides with that of phenol. a coal tar distillation product. The germicidal action of the phenol compounds is probably due to the destruction of the cell membrane. Phenol activity is increased by the substitution in the ring, as shown in the topical antiseptic phenolic compounds used as hand cleaners such as chloroxylenol, hexachlorophene, and triclosan.

In 1905, Long' reported that phenols were used more frequently than any other drugs in dentistry as an antiseptic and analgesic. This essential oil group was antiseptic, analgesic, abundant, well penetrating, and comparatively nonirritating. It also acted as a disinfectant in root canals and carious cavities and did not destroy tissues.

Essential oils are valuable constituents of many dental mecli-caments. The essential oils are fragrant, volatile essences that evaporate completely and leave only temporary stains.

Thyme has been used in medicine since ancient times, and was Isolated by Neuman in 1719. Thyme oil, which is derived from the aromatic mint shrub Thymus vulgarjs, yields thymol (powder) and carvacrol (pH 5.3) as its major active ingredients. Thymol and carvacrol also can be obtained from caraway, marjoram, oregano (origanum), rosemary, and savory plants. Both thymol and carvacrol were used by dentists in the 19th century.

In 1885, Gorgas used thymol and carvacrol to treat supurating pulps, alveolar abscesses, stomatitis, and odontalgia. In 1892, Clifford described the use of thymol in root canal therapy when combined with iodine in a product called Arjstol. In 1902, Coleman recommended thymol as an antiseptic for endodontics and reported that it had the highest antiseptic power of all essential oils).

Rodney Price, chairman of the research section of the American Dental Association in 1923, recommended thymol for sterilization in root canal therapy. In 1938, the Accepted Dental Remedies of the ADA stated that the antiseptic efficiency of thymol was higher than that of phenol as a canal filling material and for direct pulp capping.

In 1944, thymol solution in direct and indirect pulp capping procedures. At seven years, the caries in each patient had been arrested; the cavities were completely sterile; and the pulp had remained. Although thymol was 24 times more active than phenol, its insolubility in water and its relatively moderate germicidal strength in a 50% ethyl alcohol solution caused it to be discontinued as a dental therapeutic.

Thymol was also used in a solution of one part thymol, one part ethyl alcohol. and two parts ether called Hartman's solution." The solvents decreased the thymol activity. However, thymol is recognized as a powerful antiseptic, germicide, fungicide, and counterirritant and is currently used in several root canal materials. Currently, one of the most common uses of thymol is as the main ingredient of Listerine, which contains ethyl alcohol as a solvent. Carvacrol, the isomer of thymol. is not used in dentistry today, although it is known to be a powerful antiseptic in dental preparations.

Eucalyptus oil (pH 5.0) is distilled from the leaves of the blue gum tree. Eucalyptus is native to Australia. The oil has been used in root canal pastes and mouthrinses and as a solvent for gutta-percha. Eucalyptol is the active antiseptic ingredient in eucalyptus oil. Peppermint oil (pH 5.6) is distilled from the leaves and the flowering tops of the mint plant Mentha arvenjs var. pjperascens. This process yields menthol as the active ingredient used in toothpastes and as a genera] antiseptic.

Lemon oil (pH 3.5) is derived from Citrus limonum and yields the active ingredient limonene, which also is found in orange oil and grapefruit oil. Lemon oil is used as a cleanser and disinfectant with antiseptic properties.

The cellular building block of the essential oils is the isoprene unit, which joins in a regular head-to-tail way called the isoprene rule to form a multitude of organic compounds. The isoprene unit has two available, reactive, double-bond sites allowing polymers, ring formation, and cross-linking to occur in substances from vitamin A to natural rubber.

When an essential oil (eg, eugenol, a weak acid) reacts with zinc oxide (a base) in the presence of water, a salt neutralization reaction occurs and forms a five-membered, chelated ring structure where the zinc ion attaches to two eugenol molecules." This is called the zinc eugenolate complex .

Clinical Study Materials and Methods
The following essential oils were. tested for their germicidal activity: eugenol, thyme oil, eucalyptus oil,

Because eugenol and thyme oil were the most active, an additional test was conducted to determine if the addition of thymol or carvacrol to eugenol would increase the germicidal action of ZOE. The liquid portion consisted of two parts eugenol to one part thymol or two parts eugenol to one part carvacrol." Isoeugenol and vanillin, the phenolic components of clove oil that are derived from eugenol, were also tested to determine their germicidal action. In addition, two parts eugenol were combined with one part thyme oil to determine if thyme oil would increase the germicidal action of ZOE.

The following bacteria were obtained from the culture collection of the Department of Microbiology, New York University College of Dentistry: 1) Streptococcus mutans OMZ 176, a gram-positive coccus; 2) Staphylococcus aureus, a gram-positive coccus; 3) Klebsiella pneumoniae A TCC 33452, a gram-negative rod; and 4) Actinomyces viscosus gram-positive rod.

These virulent bacteria were chosen because they represented some of the most important pathogenic microorganisms found in the mouth.

A viscosus is one of the major inducers of supragingival and subgingival plaque, which leads to gingivitis, the early stage of periodontal disease.

For the inhibition assay, the bacteria (grown in a liquid culture) were homogenized and then inoculated onto a blood-agar medium, which had been prepared by aseptically adding 50 mL of citrated bactericidal at 24 hours.

Thyme a sheep blood to 1 L of sterilized showed the greatest germicidal blood agar base. at 24 hours. One drop of an essential oil was almost three times greater mixed with one scoop of zinc oxide than that of eugenol.

Because eugenol and thyme oil were the most active, an additional test was conducted to determine if the addition of thymol or carvacrol to eugenol would increase the germicidal action of ZOE. The liquid portion consisted of two parts eugenol to one part thymol or two parts eugenol to one part carvacro1. Isoeugenol and vanillin, the phenolic components of clove oil that are derived from eugenol. were also tested to determine their germicidal action. In addition, two parts eugenol were combined with one part thyme oil to determine if thyme oil would increase the germicidal action of ZOE. The following bacteria were obtained from the culture collection of the Department of Microbiology, New York University College of Dentistry: 1) Streptococcus mutans OMZ These virulent bacteria were chosen because they represented some of the most important pathogenic microorganisms found in the mouth. S mutuns is one of the major etiologic agents of dental caries. S oureus is the most common aerobic microorganism associated with oral infections such as periodontal abscesses, osteomyelitis, and the invasion of the mouth in cases of xerostomia. This bacterium develops a strong resistance to antibiotics. It also contributes to clinical cases of halitosis and has been isolated from human bite wounds. A viscous is one of the major inducers of supragingival and subgingival plaque, which leads to gingivitis, the early stage of periodontal disease. For the inhibition assay, the bacteria (grown in a liquid culture) were homogenized and then inoculated onto a blood-agar medium, which had been prepared by aseptically adding 50 mL of citrated sheep blood to 1 L of sterilized blood agar base.b One drop of an essential oil was mixed with one scoop of zinc oxide (0.1 gm). and the mixture was then placed in the center of an inoculated blood agar plate. The plates were incubated at 36?C (97?F) and examined at 24 hours, 48 hours, 72 hours, 96 hours, one week, two weeks, one month, two months, three months, and four months. The zone of inhibition of bacterial growth, termed the inhibition halo effect, was measured in millimeters.

Results
Table I lists the zones of inhibition board with thyme oil, eugenol, eucalyptus oil, peppermint oil, and lemon oil when placed in direct conduct with bacteria. Table II lists the zones of inhibition obtained with thymol or carvacrol added to ZUE. The essential oils are effective against both gram-positive and gram-negative rods and cocci bacteria. All the essential oils were

Conclusions
The three components of clove oil (eugenol, isoeugenol, and vanillin) are germicidal and share a similar phenolic structure, A dental cement made of ZOE is germicidal, and its germicidal strength can be doubled by adding thymol and quadrupled by adding carvacrol, the liquid isomer of thymol. Carvacrol is the most effective phenol, followed by thymol.

Thyme oil is the most germicidal essential oil. The addition of either thymol or carvacrol to ZOE can benefit the patient in indirect pulp capping procedures because thymol and carvacrol are abundant and palliative to the pulp, Vanillin (synthetic vanilla) is germicidal. A cement based on vanillin is ant bactericidal and more pleasant to the taste than eugenol or other essential oils.

Phenol is self-limiting; that is, its degradation products restrict its action. The essential oils, on the other hand, are not self-limiting; they penetrate well; and they are long-lasting. The author has successfully treated periodontal abscesses by using eugenol and thyme oil as irrigating agents to anesthetize the gingival before lancing an abscess and has dipped the ends of curettes and scalers in eugenol and thyme oil for abundant, anodyne, and antiseptic effects.

Practitioners can use dental tape impregnated with eugenol or thymol as an adjunct to periodontal treatment or for the agents' antiseptic and sedative actions. A few crystals of thymol added to the hydrocolloid will impart its germicidal action on the microorganisms in the water. Also, several crystals of thymol in a slurry of pumice and water used with a polishing lathe-often one of the most contaminated niches in the dental office-will prevent the growth of microorganisms.

Alcohol is not an effective vehicle for the essential oils because it reduces their effectiveness; The essential oils are best used in their liquid form or dissolved in eugenol. The active components of the essential oils (i.e., thymol] are best used dissolved in the liquid monohydric phenols such as eugenol and carvacrol. The active phenolic components such as carvacrol, eucalyptol, eugenol, isoeugenol, Iimonene, menthol, thymol, and vanillin possess marked germicidal properties against bacteria and fungi. The study presented here concurs with other reports that describe thymol and carvacrol as more antiseptic than eugenoI.9.11.13.18.>4.JS The most germicidal dental cement is made with carvacrol and zinc oxide.

Practitioners can use the essential oils and their active components for analgesia, antisepsis, cavity sterilization in cements, periodontal procedures such as scaling and curettage, periodontal abscesses, alveolar osteitis (dry socket), oral infections such as osteomyelitis, and halitosis." The essential oils are effective against the most common pathogenic microorganisms found in the mouth." The addition of thyme oil to eugenol doubles the germicidal action of ZOE dental cement.

Dental medicaments made from essential oils are anti-inflammatory because phenolic compounds act as nonspecific counter-irritants in addition to being germicidal. Part-owners are becoming increasingly concerned about disease and infection control. The overuse of antibiotics has caused microorganisms to develop resistance to current antibiotics and to become more virulent. Microorganisms, however, do not appear to develop a tolerance or resistance to the antibacterial effect of essential oils such as clove oil (eugenol) and thyme oil (thymol and carvacrol). Such oils provide an effective, powerful, cost-effective means for infection control in dentistry.

    Bad Breath: Research Perspectives - Zinc

Editor
Mel Rosenberg
The Maurice and Gabriela Goldschleger School of Dental Medicine and Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel

Ramot Publishing - Tel Aviy University

In summary, the anti-bacterial approach can result in reduced formation of VSC as the clinical use of rinses containing CPC, BTC and the phenolic flavor oils have been demonstrated.

Acid Conversion Approach
As previously mentioned, the odorous acid volatiles responsible for oral malodor can be converted to a non-odorous form. The next two studies evaluated a zinc salt and sodium bicarbonate on VSC reduction effects of BTC. Levels of the two major were determined and reported separately in that BTC was active in reducing CH)SH by baseline value, whereas H:S showed a 15% significance between the BTC and placebo

i. Sodium bicarbonate
A sodium bicarbonate fluoride dentifrice was compared to a regular fluoride dentifrice in a human study designed to determine the short-term (3 hour effect) of product the results (Table 8) indicate that the sodium bicarbonate dentifrice \\as effective giving a statistically significant 43% reduction versus mean VSC levels (p < 0.01). A comparison of the final mean VSC levels provided by the two products indicate that the sodium bicarbonate dentifrice with superior to the fluoride dentifrice (p = 0.01).

ii. Zinc chloride
Using the same clinical design as with sodium bicarbonate, the efficacy of zinc chloride (Zinc) was examined in a rinse. The zinc ion was effective giving a statistically significant 24% reduction in VSC versus the mean baseline score. When the final mean VSC scores were compared, the zinc chloride-containing rinse was also superior to the placebo rinse. A flavor component was selected to enhance this effect, which leads, to the combination approach.

Zinc with Alpha-Ionone, a Combined Approach
Folklore in the US indicates that tomato juice is traditionally used to neutralize skunk odor on clothes and pets. Skunk odor results from volatile sulfide components, similar to the components responsible for oral malodor in humans. When tomato juice flavor components were evaluated individually and in combination, it was observed that beta-ion one alone and in combination with a zinc salt strongly inhibited VSC formation in vitro. Alpha-ionone, having a more acceptable taste, was substituted for the beta-ionone form

Based on the in vitro results, a zinc/alpha-ionone combination was tested clinically resulting in an Increased effectiveness with 59% reduction versus the initial baseline mean, A statistically significant difference was also observed when the final mean VSC scores for the zinc/alpha-ionone and placebo rinses were combined. Using the results of the zinc/alpha-ionone combination was also about twice as effective as zinc chloride alone. These clinical results indicate that the above approaches, when used alone or in combination, can significantly reduce VSC production, thereby controlling the intensity of offensive mouth odor for 3-4 hours.

Combined Approach
that tomato juice is traditionally used to treat pets. Skunk odor results from volatile ~ components responsible for oral malodor components were evaluated individually that beta-ionone alone and in combination d VSC formation in vitro. Alpha-ionone, is substituted for the beta-ionone form

Summary
In summary it has been demonstrated that:
I. Gram-negative organic; runs are primary contributors to the production of VSC in the local oral cavity and VSC production parallels offensive oral malodor.
2. There is a relationship between H2S production and gingival inflammation.

    BreathRx Toothpaste Effect on Caries Formation in the Rat

Study Number
Oral Health Research Institute Number 1281 IACUC Number DS0000721 R


Study Sponsor Discus Dental
8550 Higuera Street
Culver City, California 90232

Study Site
Indiana University School of Dentistry Bioresearch Facility
1121 W. Michigan Street Indianapolis, IN 46202-5186

Conducting Agency
Indiana University School of Dentistry Oral Health Research Institute
415 Lansing Street
Indianapolis, Indiana 46202-2876

Purpose
The purpose of this study was to observe the effect of the Sponsor's experimental dentifrice on dental caries in the rat. The three groups were comprised of a positive control (USP Reference Standard), one negative control, and one experimental dentifrice.

Test Substances
The test substances were three coded products supplied by the Sponsor. To perform this study, 500 grams of each dentifrice were required. The sponsor was responsible for the necessary evaluation related to the composition, purity, strength, stability, storage requirements, expiration dates and any other applicable requirements.

Test Design
The test design was similar to FDA Method #37. The major variations were the diet used (MIT 200 rather than #469), the caries scoring method (Keyes method rather than HMA; see Appendix A) and treatment frequency. Experimental procedures were conducted according to the FDA regulations Part 58.

Test products were given a code by the Sponsor and decoded by the Research Compliance Officer at study completion

** Treatments were administered seven days per week, with a single daily treatment on weekends.

Justification for Animal Use
For a variety of reasons governmental and professional review agencies have agreed to accept a battery of pre-clinical tests as a means of documenting the caries-preventive potential of certain types of fluoride dentifrices in lieu of long-term clinical trials in children. This battery of tests includes the use of a rat caries model with a minimal test design consisting of a negative control (placebo dentifrice) group, a positive control group involving the use of a similar fluoride dentifrice whose caries-preventive benefits have been demonstrated in a controlled clinical trial, and an experimental group similarly treated with the experimental fluoride dentifrice.

Using litters as a covariate, the use of between 50 and 58 (depending on type of fluoride) animals per treatment group satisfies the most stringent power requirements of the ADA's Council on Dental Therapeutics 20% clinical difference between treatments at 80% power. However, we have been routinely using 40 animals per treatment group and these tests have consistently been accepted by both the ADA's COT and the FDA. This required initiating the study with 40 animals per group. These animals were provided by 21 litters.

IACU Approval
The protocol was reviewed and approved by the Institutional Animal Care and Use Committee prior to receipt of the animals.

Animals

1. Type of Animals
Weanling mixed-sex Sprague Dawley rats; approximately 40-50 grams at study initiation.

2. Source
Harlan Sprague Dawley, Inc. P.O. Box 29176 Indianapolis, Indiana 46229

3. Housing
The litters were maintained in large solid-bottom (box-type) cages with dams until the pups were weaned at 18 days of age. Starting at 9 days of pup age, the dams were rotated daily among the litters. The pups were maintained in the box cages until 21 days of age. At this time the pups were stratified and housed in pairs in suspended wire-bottomed cages which had been cleaned and sanitized prior to usage. The cages were arranged so that all animals of the individual groups were together and the cages were labeled with group designation and treatment (treatment code) that the animals received.

4. Identification and Stratification
When the animals were 21 days old they were given unique numbers by ear-punch with records kept of littermates. Animals were assigned to groups in such a manner that groups were balanced for litter; weight and sex, there were 40 animals per group.

Animal Care
1. Diet
Upon receipt, dams and litters were provided rodent lab diet until the pups were 8 days of age. On day 8 (pup age) dams and litters were provided Diet MIT 305 (composition in Appendix B). Animals were provided diet MIT 200 (Composition in Appendix C) ad libitum at day 18 (pup age) and throughout the test period.

2. Water
All animals were provided with deionized water ad libitum.

3. Care
Box caging was changed at day 13 and again at day 18 of pup age. Cage board was changed three times a week at the time when fresh food and water were given (Monday, Wednesday and Friday). Clean and sanitized water bottles and food jars were provided weekly. Suspended caging and banks were sanitized bi-weekly. The animals were observed daily by a staff member and weekly by the attending veterinarian for any signs of health problems.

4. Room Environment
The animals were housed in an AAALAC-accredited facility. Room temperatures were maintained at 72?F (?6?F) with 10-15 air changes per hour and a 12-hour light cycle. Inoculation. On day 15 (pup age), the pups received an oral inoculation of streptomycin-resistant S. sobrinus 6715 culture (Appendix D). This involved flooding the mouth with 0.2 ml of culture/animal. On day 18 (pup age) the animals were provided Diet MIT 200 and were inoculated with S. sobrinus for three consecutive days (days 18, 19 and 20). This involved placing 0.1 ml of the S. sobrinus culture on the occlusal surfaces of each of the mandibular molar quadrants, putting 10 cc of this concentration-adjusted culture into each water bottle, and lightly spraying the bedding with remaining culture. All water bottles were removed and sanitized 24 hours after inoculum had been added. The inoculum was administered to the animals with a 200 micro pipetter.

Experimental Treatment Initiation
The treatment phase began at day 22 of pup age.

Experimental Procedures

1. Preparation and Labeling
Each treatment had a labeled plastic beaker which was designated for that treatment only. Fresh materials (Le., obtained from stock supply) were used for each treatment. The dentifrices were mixed in a 1:1 ratio (by weight) with deionized water. Specifically, 10 grams of dentifrice were weighed into a 30 ml beaker; 10 grams of deionized water were then weighed and added to the dentifrice. The mixture was then stirred by hand (30 seconds) with a clean microspatula for the purpose of creating a smooth mixture. The beaker containing the slurry and a small magnetic stirring bar was placed on a magnetic stirrer which was set at the lowest speed and allowed to stir for 3 minutes. The slurry was prepared immediately prior to each treatment.

2. Treatment Procedure
A cotton-tipped applicator was dipped into the slurry (for 2 seconds) and was applied to one-half of the rat's mouth in such a way that the sides of the applicator came into contact with both the mandibular and maxillary molars on one side of the mouth. The treatment was accomplished by using a rolling motion of the sides of the applicator over the mandibular and maxillary molar teeth for 15 seconds. The applicator was dipped Into the slurry for the second time (again, for 2 seconds) and the other side of the rat's mouth similarly treated for 15 seconds. A new applicator was used for each animal.

3. Schedule for Treatment Applications
Treatments were administered twice daily for five days with a single daily treatment on weekends. The first treatment each day began at approximately the same time every day, and the second treatment began no earlier than six hours after the first treatment. Singular treatments were given at a 24 hour interval on weekends.

4. Storage of Material
Treatment materials were stored at room temperature. All treatment products were returned to the sponsor at the study completion.

5. Recovery
One week after the initiation of the inoculation regimen and at study termination, an oral swabbing was taken from each rat using a sterile cotton swab (six-inch, single-tipped applicator). The microorganisms on the mandibular and maxillary molar teeth were sampled, using a rolling motion of the swab for 15 seconds on one side of the mouth, rolled over the tongue, and rolled over the molar teeth on the other side of the mouth for an additional 15 seconds. Immediately after the applicator was removed from the animal's mouth, it was streaked across half of a 100 mm petri plate containing Mitis Salivarius agar to which 200 units/ml of streptomycin sulfate had been added. The plates were incubated for 48 hours at 37?C with 10% C02' The colony count taken after the 48 hours of incubation was recorded in the logbook.

Experimental Duration of Study
The duration of the experimental phase was three weeks. Termination of Animal Phase 1. Final Observation and Examination Immediately prior to termination all animals were observed for any visual signs of ill health or pathology, individually weighed and an oral swabbing taken to confirm S. sobrinus implantation.

Study Completion
1. Tissue Preparation The cleaned hemljaws (four quadrants) were put into plastic vials with the code numbers taped to the vial. A murexide solution (0.3 g murexide; 300 ml DI H20 and 700 ml of ethanol) was added to each vial and the jaws were allowed to stain overnight. The jaws were then rinsed and allowed to air dry.

2. Tissue Evaluation
The hemijaws were microscopically examined for smooth surface caries, sectioned, and then microscopically examined for sulcal and interproximal caries using the Keyes method as outlined in Appendix A.

3. Data Processing and Analysis
Statistical analyses were performed using the Bartlett-Box F and the Cochran's C tests for homogeneity of variance (at a=0.05). In cases where the variances were homogeneous, a one-way analysis of logarithmic or square root transformation of the data was made according to the relationship between group means and variances, and transformed data reanalyzed. In cases where a significant "F" value was found, Tukey's HSD test and/or Duncan's multiple range tests were used to test for significant differences between the individual means. For extreme variance heterogeneity, the nonparametric Kruskal-Wallis one-way analysis of variance was used.

4. The specific types of data which were tabulated, statistically analyzed, and reported for each group is as follows:

Data Experimental Phase Initial number of animals
Final number of animals
Percent mortality

b. Growth Data Experimental Phase
Initial body weight (mean ? S.E.M.) Final body weight (mean ? S.E.M.) Body weight gain (mean ? S.E.M.)
c. Caries Data
Enamel and dentinal involvement of smooth surface lesions (mean ? S.E.M.).
Enamel and dentinal involvement of interproximal lesions (mean ? S.E.M.).
Enamel and dentinal involvement of sulcal lesions (mean ? S.E.M.).


Total caries involvement combining the scores from the Keyes method of scoring smooth surface, interproximal, and sulcal caries (mean ? S.E.M.). Percent of animals and level of infection in each group infected at both initiation and at termination of study period.

Record Maintenance
All records (protocols, amendments, data sheets and final reports) are maintained in a book designated for this study as part of the OHRI Laboratory Archives. The hard tissue specimens are also maintained in the Archives.

Study Duration
Study initiation began July 14, 2000. The completion of the final report was November 30, 2000.

Results
There was no mortality experienced during the treatment phase of this study. There were no statistical differences observed among the groups in terms of growth.

All caries data are discussed in terms of enamel involvement only. Smooth surface caries data are shown in tables 1281-2 through 1281-4. The group treated with the BRX Placebo was significantly greater in buccal-lingual smooth surface caries formation than either of the other two groups. There were no other statistically significant differences observed among the groups. Interproximal caries data are shown in table 1281-3.There were no statistically significant differences in interproximal caries observed among the groups. Total smooth surface caries data are shown in table 1281-4. The group treated with the BRX Placebo was significantly greater in total smooth surface caries formation than either of the other two groups. There were no other statistically significant differences observed among the groups in total smooth surface caries

Information.
Sulcal caries data are shown in table 1281-5. The group treated with the BRX Placebo was significantly greater in total smooth surface caries formation than either of the other two groups. There were no statistically significant differences in sulcal caries observed among the groups.

Total caries formation is shown in table 1281-6. The group treated with the BRX Placebo was significantly greater in total caries formation than either of the other two groups. There were no other statistically significant differences observed among the groups in face caries formation. S. sobrinus data are shown in table 1281-7. The animals were well infected with S. sobrinus at both study initiation and termination.

Conclusion
The BRX dentifrice and USP Reference Standard were equal in efficacy in reducing caries formation as compared to dentifrice BRX Placebo.

Gen Dent. 2002 Jul-Aug In vitro testing of Xylitol as an anticariogenic agent Sahni PS, Gillespie MJ, Botto RW, Otsuka AS. A number of studies involving xylitol chewing gum have demonstrated that xylitol is both noncariogenic and anticariogenic. The ability of xylitol to act as an anticariogenic agent most likely is due to its ability to be transported into caries-causing oral bacteria and inhibiting fermentation either by depleting the cell of high-energy phosphate or by poisoning the glycolytic system. In vitro tests were conducted to determine the concentration of xylitol required to inhibit the growth of three strains of oral streptococcus (S. mutans, S. salivarius, and S. sanguis). All three strains were inhibited significantly at xylitol concentrations of 12.5% and higher; however, only S. mutans was inhibited significantly at a xylitol concentration of 1.56%. PMID: 12640850 [PubMed - indexed for MEDLlNE]