0817 Oral Malodorous Compound Causes DNA Damages in Human Gingival Fibroblasts

K. YAEGAKI*, and W. QIAN, University of British Columbia; Nippon Dental University, Vancouver, Canada

Objective: Hydrogen sulfide(H2S) is malodorous compound in the oral cavity. We have reported previously that H2S has an inhibitory effect on superoxide dismutase activity, which has one of the most crucial roles in carcinogenesis. The plausible signal transduction mechanisms of developing colorectal cancer by H2S was also reported. Therefore, it has been speculated that VSC may cause DNA damages as well as apoptosis. The objective of this study is to clarify whether H2S causes DNA damages in human gingival fibroblasts(HGF) Methods: Single cell gel electrophoresis, CometAssayTM( Trevigen , USA ), was employed to examine DNA damage by H2S. HGF were incubated with or without 100ng/ml air H2S for 24, 48 and 72hrs. Following a gentle cell lysis, samples were treated with alkali to unwind and denature the DNA and hydrolyze sites of damage and submitted to electrophoresis. After digital photos were taken with a fluorescence microscope, the extent of DNA damage was evaluated by CometScoreTM (TriTek, USA) software to determine, tail length, % DNA in tail and tail moment. Cytotoxicity of H2S was assayed with using LDH Cytotoxicity Detection KitTM (Takara Bio, Japan). The numbers of viable cell were counted with using trypan blue staining (Invitrogen, Canada). Results: Tail Length was increased by H2S in 48hrs(p<0.0005) and 72hrs(p<0.0001). The %DNA in Tail and Tail Moment were increased in 72hrs(p<0.0005). The numbers of viable cell of each incubation time was not affected by H2S. Low cytotoxicities of H2S were also found. Conclusion: H2S at the concentration in periodontal pocket damaged DNA in HGF. The numbers viable cell was not changed by H2S, and the cytotoxicity of H2S at 100ng/ml air was relatively weak. Hence, it has been suggested that DNA damages by H2S might be caused by apoptosis.

0919 Comparison of Oral Odor between Vegetarians and Non-Vegetarians

S.S. LEE*, Y. JUN, J. KIM, K. KIM, W. ZHANG, and Y. LI, Loma Linda University, CA, USA  

0920 Effects of Yoghurt on the Human Oral Microbiota and Halitosis

K. HOJO*, T. OHSHIMA, A. YASHIMA, K. GOMI, and N. MAEDA, Tsurumi University, Yokohama, Japan

0922 Effect of flavors in dentifrices on morning bad breath

D.C. PERUZZO1, S.L. SALVADOR*2, A.W. SALLUM1, and G.R. NOGUEIRA-FILHO1, 1 State University of Campinas - Brazil, Brazil, 2 University of Sao Paulo, Ribeirao Preto, SP, Brazil

0926 Novel extraction and analytical methods for oral malodors

S. PILCH*, R. SHASTRY, M. WILLIAMS, and J. MASTERS, Colgate-Palmolive Co, Piscataway, NJ, USA

Objective: To develop an extraction method for oral malodor compounds from ìmorning mouth? and to analyze these compounds using SPME-GC-MS technique.
Methods: Six healthy male and female panelists participated the pilot clinical study. The panelists were asked to refrain from oral hygiene, eating and drinking the morning of the study. Smokers were not included in the study. Panelists were randomized and completed the following procedures on two separate days. On day 1, one group provided 5 mL of unstimulated saliva. The second group was instructed to swish for 1 minute with hydroxyapatite powder (HAP) (0.5 g dry weight) pre-wetted with sterile water. They then expectorated the resulting saliva-HAP mixture into a sterile tube. On day 2 the groups reversed sampling method. A total of 4.5 grams of saliva sample collected from each panelist was transferred to a GC vial and the headspace air was concentrated onto SPME fiber and analyzed by GC-MS.
Results: More than 50 malodor compounds were extracted and determined by GC-MS. They were categorized into 7 groups based on chemical functionality: acids, amines, alcohols, thiols, ketones/aldehydes, aliphatic compounds, and benzene derivatives. Among the compounds that were observed, indole, organic acids, and sulfur containing compounds were commonly found in the literature associated with oral malodor. Overall the number of compounds extracted via this HAP extraction method exceeds that of unstimulated saliva method by about 30%. All compounds identified from unstimulated saliva were also observed in the HAP-saliva samples.
Conclusion: The HAP extraction method proved to be an effective mean to extract oral malodor compounds.

0931 Oral hygiene practices, smoking, and oral malodor among pregnant mothers

K. ALMAS*, New York University, USA, and N. ASHRI, King Saud University, Riyadh, Saudi Arabia  

0937 Oral Benefits: A New-Manual Toothbrush versus a Battery-Powered Toothbrush

M. WILLIAMS*, J. VAZQUEZ, and D. CUMMINS, Colgate - Palmolive Company - / Piscataway, NJ, USA

Objective: These studies compare the effectiveness of a newly designed manual toothbrush, with a cleaning implement on the back of the toothbrush head, to a battery-powered toothbrush on desquamated epithelial cells (DEC), oral H2S-forming tongue bacteria (OH-TB) and volatile sulfur compounds(VSC). Materials and Methods: Healthy adult subjects took part in three randomized, cross-over studies. After baseline sampling, subjects brushed for 1-minute with the assigned toothbrush. Subjects using the new manual toothbrush brushed their tongue with the implement on the back of the toothbrush for 10 seconds. For DEC, 30-min post-brushing, subjects rinsed with 10 milliliters of sterile water for 10 seconds. Collected samples were evaluated in a colorimetric assay to measure the level of desquamated epithelial cells found in the rinse. For OH-TB, the right and left sides of the tongue were swabbed at baseline and 2 hours, respectively, after brushing. Collected tongue samples were serially diluted in sterile buffered saline and triple plated onto lead acetate agar. After 72 hours incubation, the dark pigmented colonies were counted and expressed as log CFU. For VSC, the evening after baseline morning breath sampling, subjects brushed their teeth before going to bed, and then returned for overnight breath sampling. Breath VSC were evaluated with a gas chromatograph and levels were expressed as parts per billion (ppb) in mouth air. Results: The new manual toothbrush reduced DEC, OH-TB and VSC 74, 84 and 63%, respectively, versus baseline. Respective reductions were 39.1, 53.2 and 15.8% for the battery-powered toothbrush. The new manual toothbrush was significantly better than the flat-trim toothbrush in reducing DEC, OH-TB and VSC. Conclusion: The clinical results indicate that a newly designed manual toothbrush with a cleaning implement on the back of the toothbrush head is significantly more effective than a battery-powered toothbrush in reducing DEC, OH-TB and VSC.

0977 Using GC/MS-Olfactometry Technique to Identify Mouth Odour Compounds in Saliva

L. COLLINS*, and R. CALVERT, Unilever Oral Care, Bebington, United Kingdom  

Objective: To use Gas Chromatograph-Mass Spectrometry-Olfactometry (GC-MSO) technology to detect and identify aqueous salivary compounds that are typical of mouth odour. Methods: Stimulated saliva samples (10ml) were collected from subjects (n=6) upon waking before eating or drinking. Each sample was rated for odour intensity by two odour judges (OJ) before and after ether extractions were prepared (saliva/ether 1:1 and 20:1). Ether extractions were injected into the GC-MSO, where the separated components were divided by an open split interface between a sniffer-port (SP) and the MS (2:1 ratio). The OJ assessed odours as they emanated from the SP by describing smell and indicating if it was common to mouth odour. Results: The OJ perceived that the overall odour intensity of the 1:1 ether extracts was reduced while intensity was maintained in the 20:1 extractions, relative to the odour of the un-extracted saliva. A total of 14 traces were assessed by the OJ, (four, 1:1 extractions, analysed in duplicate and six, 20:1 extractions were assessed). From these traces, the OJ were able to perceive and make qualitative descriptions of 217 odours. The OJ identified 48 (22.1%) of the perceived odours to be common to mouth odour. By matching the timings of the mouth odour comments to the MS traces, the chemical sources of 32 of the mouth odour comments were attributable to 18 different compounds. These were classified based on fugacity and olfactory threshold into five groups. Conclusions: Compounds identified within the literature as characteristic of mouth odour were detected in saliva by GC-MSO.

Group          Classification                                                         Examples

    I               Volatile Sulphur Compounds (VSC)                      H2S; CH3SH; CH3SCH3

    II              Volatile Fatty Acids (VFA)                                    Isovaleric; Butanoic; Phenylacetic

    III            Indolics                                                                 Indole; Skatole

    IV            Long Chain Fatty Acids                                         Octanoic; Nonanoic; Dodecanoic

    V             Fatty Acids                                                           Propanoic; Isobutyric; Isocaproic

2329 Oral Malodorous Compound Induces Apoptosis in Human Gingival Fibroblasts

W. QIAN*, University of British Columbia, Vancouver, Canada, and K. YAEGAKI, University of British Columbia; Nippon Dental University, Vancouver, Canada

Objective: Volatile sulphur compounds (VSCs), namely hydrogen sulfide(H2S) and methyl mercaptan, are malodorous compounds in the oral cavity. VSCs have been suggested to involve periodontal pathogenecity. Furthermore, we reported that H2S caused DNA damages in human gingival fibroblasts(HGF), which is the most crucial role in carcinogenesis. Taking these findings together, it has been speculated that VSC may cause apoptosis. In this study, we therefore investigated the effect of H2S on apoptosis. Methods: The apoptosis caused by H2S (100ng/ml air) in (HGF) was determined with the following procedures. Cell death caused by apoptosis was detected by Cell Death Detection ELISAplusTM (Roche, Canada). The numbers of viable cell were counted with using trypan blue staining (Invitrogen, Canada). Caspase 3 activity, which appears specifically in the process of apoptosis, was determined by Caspase 3 Activity AssayTM (Roche, Canada). Results: The results of Cell Death Detection ELISAplusTM was shown in abosorbance at 405nm as follows; 0.074 in test vs. 0.061 in cont. for 24 hr incubation(p<0.05), 0.079 vs. 0.065 for 48 hr(p<0.0005) and 0.108 vs. 0.064 for 72 hr(p< 0.0005). On the other hand, histone-associated DNA fragment, which is detected in this system to determine the cell death, was never increased in the culture medium by H2S. The numbers of viable cell of each incubation time was found to be no significant difference between test and control. Caspase 3 activity was increased in 48 and 72 hours incubations. Conclusion: H2S induces apoptosis in HGF, but not so strong inducer. Since caspase 3 did not increase within 24 hours incubation, H2S in head space air may require a certain time to induce apoptosis. Further study will be required to determine whether H2S may enhance other inducers of apoptosis.

2379 Oral Malodor Reduction by Glutamate/Proline Stimulation of Bacterial Oxygen Utilization

D. CODIPILLY*, and I. KLEINBERG, State University of New York - SUNY - Stony Brook, USA

2714 Identification of Odoribacter denticanis isolated from canine periodontal disease patients

J.M. HARDHAM*1, K. KING1, D. KIMBERLY1, W. JASON1, S. CATHERINE1, R. EVERSOLE2, C. SFINTESCU3, and R. EVANS3, 1 Pfizer, Inc., VM R&D, Kalamazoo, MI, USA, 2 Western Michigan University, Kalamazoo, USA, 3 State University of New York - SUNY, Buffalo, USA

2739 Prediction of Oral Malodor with Volatile Markers in the Breath

M. PHILLIPS*1, R.N. CATANEO1, J. GREENBERG1, M.I. MUNAWAR1, and S. NACHNANI2, 1 Menssana Research, Inc, Fort Lee, NJ, NJ, USA, 2 University Health Resources, Inc, Culver City, CA, USA

Purpose of study was to assay VOCs in oral breath and evaluate them as predictors of the perception of oral malodor. 2 trained organoleptic judges independently scored malodor in 69 subject on scale 0-5. Subjects provided 300 ml oral cavity breath into an inflatable bag. VOCs in 75 ml breath were captured on a sorbent trap and assayed by automated thermal desorption with gas chromatography and mass spectroscopy, and identified from a computer-based library of mass spectra. Breath VOCs were ranked by multiple t-tests comparing their abundance in high or low oral malodor groups (mean organoleptic score above or below 2.5 respectively). Subjects were randomized to a training set (46) or a prediction set (23). In the training set, forward stepwise linear regression was employed to correlate breath VOCs with the organoleptic score. The resulting model was tested in the prediction set. In the training set, 44 VOCs correlated completely with organoleptic scores (r=1.0), and this model predicted organoleptic scores in the prediction set (r= 0.41). The predominant breath VOCs in the model were alkanes and methylated alkanes i.e. products of lipid peroxidation mediated by oxidative stress. A preliminary mathematical model employing breath VOCs partially predicted the intensity of the subjective perception of oral malodor. This predictive model could potentially be improved by incorporating additional data from a larger number of subjects. The breath VOCs most significantly associated with oral malodor were products of oxidative stress and may have resulted from infections in the oral cavity e.g. periodontitis.

2740 High Proportion of Pseudo-Halitosis in a Multidisciplinary Breath-Malodor Consultation

R. SEEMANN*, C. DJAMCHIDI, A. KAGE, and K.-R. JAHN, University Medical School Charit?, Berlin, Germany

Objective: The aim of the present study was to report the data from a multidisciplinary bad breath consultation in Germany. Methods: 407 patients were examined by a specially trained dentist with an ENT-specialist, an internist, and a psychologist on call. Bad breath was determined organoleptically and by using a portable sulfide monitor (HalimeterTM). Results: All patients reported suffering from bad breath but only 72.1 % showed detectable signs of breath malodor. Within this group, 92.7 % revealed an oral cause, 7.3 % revealed an extra-oral cause. 23.3 % had consulted one, 20.8 % two, 16.9 % three and 22.5 % four doctors of different specialties. From all individuals without malodor, 76.3 % underwent prior diagnostics and treatments from other doctors. 36 % had received one or more gastroscopies and 14 % an ENT operation. However, in only ten cases an organoleptic evaluation of the putative malodor had been performed. Conclusions: Our data generally support recent findings that breath malodor is mainly caused by microbial putrefaction within the oral cavity and that patients with pseudo-halitosis are frequently not identified by doctors, resulting in a considerable amount of over-treatment.

2741 Clinical Trial Evaluating Malodor Reduction Benefit of a Marketed Dentifrice

S. FARRELL*, L.C. SALETTA, J.J. WITT, R.A. BAKER, and R.W. GERLACH, The Procter & Gamble Co, Mason, OH, USA

Objective: A clinical study was conducted to evaluate breath malodor reduction following single and cumulative use of a triclosan-containing marketed dentifrice (blend-a-med Complete Night). Methods: The study was a three-period, cross-over study, with 2 washout periods. 29 healthy adult volunteers were acclimated for 7 days with standard toothpaste. After, subjects were randomized to three brushing regiments for each period: tooth brushing with Complete Night toothpaste, tooth brushing and tongue brushing with Complete Night, and tooth brushing with CrestÆ Cavity Protection toothpaste as a control. The production of volatile sulfur compounds was monitored at baseline, and again after 3, 24 & 27 hours. A questionnaire was completed after 24 & 27 hours to assess first-person perception of malodor. Results: Mean (SD) age was 40.2 (10.9) years. Relative to control, both triclosan groups experienced significant (p < 0.05) malodor reduction at 3 and 27 hours. In addition, the triclosan group with tongue brushing regimen showed significant (p < 0.05) malodor reduction versus the control at 24 hours and the no-tongue brushing group at 27 hours. Subjects using the triclosan dentifrice reported significantly (p < 0.05) higher improvement in breath compared to the control. Tongue brushing resulted in a significant (p < 0.05) improvement in the perception of tongue cleanliness. All treatments were well tolerated. There were no adverse events reported during the study. Conclusion: Use of the triclosan-containing toothpaste resulted in significant reduction in VSCs after a single dosing and with cumulative use, tongue brushing improved reduction of VSCs, and both day-time and overnight results were first-person noticeable.

2742 The Effectiveness Of Breath Care On Low-Carb Oral Malodor

S. NACHNANI*, E. MAGALLANES, N. BOONSAWAS, and M. WU, University Health Resources, Inc, Culver City, CA, USA

Objective: The purpose of this study was to evaluate the effectiveness of a breath care system on subjects with low-carb oral malodor (LCOM) diets in a randomized single blind study. Methods: Fifty-four subjects were enrolled in two categories: Groups 1& 2 received treatment, Groups 3 & 4 received no treatment. Group one consisted of subjects with oral malodor (OM). Group Two included subjects with LOCM. Both were treated with the breath management "starter kit." This kit included toothpaste, tongue scraper, tongue spray & mouth rinse. Control groups were Group Three: subjects with OM on a regular diet and Group Four: subjects with LCOM. At baseline and 6 weeks, OM was assessed using three methods: Organoleptic judges (OJs), Spoon Test (scale 0-5), and Halimeterѵ. A training protocol was used to prepare the OJs. Results: Between treatment, group comparisons were performed using analyses of covariance, in which baseline scores were co-variables. Non-parametric distributions /medians were compared using the Kruskal-Wallis (KW) test based on ranks. For OJs results, the statistically significant difference between the group with OM and control group was -2.80 (p< .001), mean difference between the low carb group and control was -2.673 (p< 0.001). Similar results (p 0.000) were obtained using the non-parametric KW test for both groups. For the Halimeterѵ test, the mean difference between the OM and control groups was -47.92 (p 0.047). The difference between the low -carb group and control was -16.19 (p= 0.5). Similar results were obtained with the KW test. For the spoon test, mean difference for the OM group and the control group was -2.14(p <0.001). Mean difference for the low-carb and control group was -1.5714 (p 0.0012). Conclusions: The BreathRxѵ Starter Kit was very effective in controlling OM for subjects with bad breath and as well as subjects with low -carb OM.

3133 Intra- and Inter-Judge Calibration for Oral Malodor Clinical Study

D. HU*1, X. LI1, Y.P. ZHANG2, A. VOLPE2, and W. DE VIZIO2, 1 West China College of Stomatology, SCU, Chengdu, China, 2 Colgate - Palmolive Company, Piscataway, NJ, USA  

Objectives: The purpose of this study was to calibrate intra- and inter-judge consistencies of four judges who were involved in an oral malodor clinical study in terms of oral malodor 1-9 organoleptic evaluation scale. Volatile sulfide concentration (VSC) was also employed in the study to assess a correlation between the organoleptic assessment and VSC. Methods: Thirty-nine subjects completed the double-blind clinical study for intra- and inter-judge consistencies. The organoleptic assessment was made by four judges, as previously described (Abs# 498, 2003 AADR Meeting in San Antonio, TX). Two of judges had previous experience in measuring a several hundred subjects. The other two judges were experienced dentists who had not participated previously in organoleptic assessment of oral malodor. These four Judges were blinded regarding one another's scores and their own previous scores for the organoleptic assessment, and VSC measurements. VSC measurements were made with a sulfide monitor (mode 4000 series compact portable analyzer, Interscan Corp., Chatworth, CA). Intra- and inter-judge consistencies of the organoleptic assessment were assessed by Kappa statistics, which assesses the frequency with which these judges can exactly replicate either their own scores or the scores of another judge. The weighted Kappa coefficient was assessed using Fleiss-Cohen Kappa for Intra- and inter-judge consistencies. Pearson correlation was also used to assess the strength of association between the organoleptic assessment and VSC. Results: Kappa Statistics provided a rigorous test of judge agreement. Intra-judge consistency was found from 0.89 to 0.96 and Inter-judge consistency was shown from 0.86 to 0.92. The Pearson correlation coefficient (R2) was 0.97 that represents excellent correlation between the organoleptic assessment and VSC. Conclusions: These four judges were well calibrated in themselves or one to another in terms of the organoleptic assessment (1-9 scale). The results from the organoleptic assessment were also correlated well with VSC measurements.

3455 Bacterial Culture and Broad Range PCR for Halitosis-Associated Bacteria

M.M. ZAMBON*, D. GERBER, V.I. HARASZTHY, and J.J. ZAMBON, State University of New York - Buffalo, USA

Objectives: Oral microorganisms are responsible for a large proportion of the 50-80 million cases of halitosis (oral malodor) Americans experience at some point in their lives. While some of the microorganisms associated with halitosis such as the volatile sulfur compound-producing bacteria, have been identified, the microbiology of halitosis is not well characterized. This study compared bacterial culture and the broad-range polymerase chain reaction (PCR) in identifying cultivable and non-cultivable microorganisms associated with oral malodor. Methods: Samples were obtained from 6 adult subjects with persistent halitosis defined by organoleptic assessment (>2), elevated (>250 ppb) halimeter reading, and by a high Halitox test. Bacterial samples obtained from the tongue were dispersed in sterile buffer, distributed onto enriched tryptic soy agar and anaerobically incubated at 35oC for 5-7 days. One hundred randomly selected colonies were subcultured and speciated from each sample. 16S rDNA was also amplified by PCR from aliquots of the original sample using primers to highly conserved regions. The PCR product was cloned in E. coli and 100 randomly selected clones from each sample were sequenced and identified. Results: Bacterial culture of tongue samples from subjects with halitosis demonstrated (in decreasing order) significant proportions of veillonella, actinomyces, megasphaera, Streptococcus parasanguinis, Campylobacter concisus, and neisseria as well as unspeciated bacteria. Broad range PCR of the same samples revealed similar species as well as a diverse array of uncultured bacteria including species of atopobium, erysipelothrix, firmicutes, granulicatella, porphyromonas, and prevotella as well as Solobacterium moorei, this later species having recently been implicated in oral malodor. Conclusions: Broad range PCR identifies a broader group of halitosis-related bacteria than does traditional bacterial culture including rarely cultured, uncultured, and some, as yet, unidentified bacteria.

3715 Improving Oral Conditions: A New-Manual Toothbrush Versus the Leading-Manual Toothbrush

D. CUMMINS*, J. VAZQUEZ, and M. WILLIAMS, Colgate - Palmolive Company - / Piscataway, NJ, USA

Objective: These studies compare the effectiveness of a newly-designed manual toothbrush, with a cleaning implement on the back of the toothbrush head, to the leading manual toothbrush on desquamated epithelial cells (DEC), oral H2S-forming tongue bacteria (OH-TB) and volatile sulfur compounds(VSC). Materials and Methods: Healthy adult subjects took part in three randomized, cross-over studies. After baseline sampling, subjects brushed their teeth for 1-min with the assigned toothbrush. Subjects using the new manual toothbrush also cleaned their tongue with the implement on the back of the toothbrush head for 10 seconds. For DEC, thirty minutes after brushing, subjects rinsed with 10 milliliters of sterile water for 10 seconds. Collected samples were evaluated in a colorimetric assay to measure the level of desquamated epithelial cells found in the rinse. For OH-TB, right and left sides of the tongue were swabbed at baseline and 2 hours, respectively, after brushing. Collected tongue samples were serially diluted in sterile water and triple plated onto lead acetate agar. After 72-hour incubation, the dark pigmented colonies were counted. For VSC, the evening after baseline morning breath sampling, subjects brushed their teeth before going to bed, and then returned for overnight breath sampling. Breath VSC were evaluated with a gas chromatograph and levels were expressed as parts per billion (ppb) in mouth air. Results: The new manual toothbrush reduced DEC, OH-TB and VSC 74, 84 and 63%, respectively, versus baseline. Respective reductions were 45, 64 and 16% for the leading manual toothbrush. The new manual toothbrush was significantly better than the leading manual toothbrush in reducing DEC, OH-TB and VSC. Conclusion: The clinical results indicate that a newly designed manual toothbrush with a cleaning implement on the back of the toothbrush head is significantly more effective than the leading manual toothbrush in reducing DEC, OH-TB and VSC.

3716 Comparative Efficacy of Two Manual Toothbrushes on Oral Conditions

J. VAZQUEZ*, M. WILLIAMS, and D. CUMMINS, Colgate - Palmolive Company - / Piscataway, NJ, USA  

Objective: These studies compare the effectiveness of a newly designed manual toothbrush, with a cleaning implement on the back of the toothbrush head, to a flat-trim toothbrush on desquamated epithelial cells (DEC), oral H2S-forming tongue bacteria (OH-TB) and volatile sulfur compounds(VSC). Materials and Methods: Healthy adult subjects took part in three randomized, cross-over studies. After baseline sampling, subjects brushed for 1 minute with the assigned toothbrush. Subjects using the new manual toothbrush brushed their tongue with the implement on the back of the toothbrush for 10 seconds. For DEC, subjects rinsed with 10mL of sterile water at baseline and 30 minutes after brushing. Collected samples were evaluated in a colorimetric assay to measure the level of desquamated epithelial cells found in the rinse. For OH-TB, the right and left sides of the tongue were swabbed at baseline and 2 hours, respectively, after brushing. Collected tongue samples were serially diluted in sterile buffered saline and triple plated onto lead acetate agar. After 72 hours incubation, the dark pigmented colonies were counted and expressed as log CFU. For VSC, the evening after baseline morning breath sampling, subjects brushed their teeth before going to bed, and then returned for overnight breath sampling. Breath VSC were evaluated with a gas chromatograph and levels were expressed as parts per billion (ppb) in mouth air. Results: The new manual toothbrush reduced DEC, OH-TB and VSC 74.3, 84.2 and 63.0%, respectively, versus baseline. Respective reductions were 39.7, 54.0 and 8.1% for the flat-trim toothbrush. The new manual toothbrush was significantly better than the flat-trim toothbrush in reducing DEC, OH-TB and VSC. Conclusion: The clinical results indicate that a newly designed manual toothbrush with a cleaning implement on the back of the toothbrush head is significantly more effective than a flat-trim toothbrush in reducing DEC, OH-TB and VSC.