Microbial Challenge of Breathless™* Toothpaste with C. albicans, E. coli, S. aureus, and S. sanguis

L. H. Green1, H. Alliger2
1Westbury Diagnostics, Inc. 2Frontier Pharmaceutical, Inc, Farmingdale, NY.

Oral conditions, such as periodontal disease are serious, not only because they can cause gum infections and the loss of teeth, but also because they have been shown to be linked to respiratory and even cardiac disease. Chlorine dioxide, in the past a relatively unknown compound, has been shown to be a powerful antimicrobial agent used in products ranging from hand creams to mouthwashes. Breathless™ is a chlorine dioxide toothpaste, based on a patented formulation.
In this study, an assay was developed to determine the effectiveness of different formulations of Breathless™ against high concentrations of C. albicans, E. coli,
S. aureus, and S. sanguis. Results indicated that even at the lowest concentration of chlorine dioxide tested, Breathless™ was capable of reducing the level of all of these organisms, by more than 6.4 log dilutions, after only one minute of exposure. Further assays, to determine the effects of Breathless™ against oral anaerobic organisms, are in development.

* Breathless was re-named DioxiBrite


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1-631 293 3626



Microbial Challenge of Breathless™Toothpaste with C. alibicans, E. coli, S.  aureus and                S. sanguis

,                                                                L.H. Gree H. Alliger 2

Westbury Diagnostics, Inc. Farmingdale, NY; 2Frontler Pharmaceutical, Inc. Farmingdale, NY













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Most people are aware that diseases of the mouth can cause serious problems in those areas that are infected. These can include chronic infections of the gums, as well as loss of teeth. Most people are unaware that these infections can cause serious problems in other areas of the body as well. Importantly, studies have shown a relationship between gum infections and heart disease, and gum infections and pneumonia among others. (I) (2)


Frontier Pharmaceutical, Inc. has developed a new toothpaste (DioxiBriteTM) containing chlorine dioxide as the active ingredient. Chlorine dioxide is a well known odor neutralizer as well as a complete antin1icrobial agent. It is fast acting. broad spectrum, non-irritating and non toxic. This study found that DioxiBritoTM Toothpaste was capable of killing high concentrations of microorganisms in less than one minute.


It is anticipated tliat in using DioxiBrite Toothpaste, potentially pathogenic microorganisms can be greatly reduced in the mouth, leading to improved oral health, aod perhaps a lower rate of the serious diseases known to be related to oral infections.

(I) Beck, J.D., J. Pankow, J., H.A Tyroler, and S. Olfenbacher. 1999. Dental Infections and Atherosclerosis. Am Heart J 138:528-533 .

(2) Scannapieco, F.A 1999. Role of Oral Bacteria in RespiratOI) ' Infection. J. Periodontal. 70:793-802.











Efficacy of DioxiBrlte on Various Microorganisms


minute contact time

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Recovered (Loa#dU/ml


Loa Reduction


DloxlBrtteni Toothpaste Control (Sallne)

2.4 x 101 / 8.JS (I)


'2,4 X 101


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1.2x to' 11.ss \ll






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CULTURING OF MICROORGANISMS: S. aureus ATCC 29213, E. coli ATCC 25922, C. albicatis ATCC 10231 and S.

sanguis ATCC 10556, Wete obtained from Remel (Lenexa, KS). Initial cultures were developed as per manufacturer's instructions. Prior lo testiilg, S. aureus and E. coli were plated on Trypticase Soy Agar Plates, and incubated for 18-24 hours at 37°C. S. sanguis was plated on Chocolate Agar and incubated for 48 hours at 37'C. C. albicans was plated on Sabouraud Dextrose Agar and incubated for 48-72 hours al 25'C. Heavy suspensions of the microorganisms were prepared in saline. All media were obtained from Becton Dickinson (Sparks, MD).

ENUMERATION OF MICROORGANISMS IN TEST SUSPENSION: Serial ten fold dilutions of the suspensiotlS were made. Duplicate samples of 1.0 ml of the 10· 7 dilution and of the to·'dilution were added lo sterile. petri plates. Approximately IO ml of liquid Trypticase Soy Agar al 45°C were added lo the bacterial plates, and allowed to harden. Approximately IO ml ofliquid Sabouraud Dextrose Agar ol 45°C were added to the C. albicans plates, and allowed to harden. Bacterial plates were incubated at 37'C for 24-48 hours. C. albicans plates were incubated at 25'C for 48-72 hours. Following incubation of the plates, colony counts were made.

CHALLENGE TESTING: DioxiBrite Toothpaste is supplied in two parts that are mixed just prior to use. In these tests 5

grams of part A and 5 grams of part B were mixed for fifteen seconds, and then incubated for 1 minute.

1.0 ml of the inoculum was then mixed with the paste and incubated for 1 minute. Following this. 90 ml of D;E Neutralizing Broth, was added lo neutralize the active ingredient and dissolve the mix1ure. pHydrion Micro Chlorine lest strips (Micro Essential Laboratory. Bro0klyn, NY) were used to confiml that no residual chlorine dioxide remained after the DiE Neutralizing Brotl1 was added. A ten-fold dilution of this D;E Neutralizing Broth diluted test sample was prepared in saline. ENUMERATION OF MICROORGANISMS IN CHALLENGE SAMPLE: Five 2-ml aliquots oftl1e neutralized test san1plt were added lo sterile petri plates. Duplicate I ml swnples of the neutralized diluted test sample, and of the ten-fold saline dilution., were added to sterile petri plates. Approximately 10 ml ofliquid Trypticase Soy Agar at 45°C were added to the bacterial plates, and allowed to harden. Approximately 10 ml ofliquid Sabouraud Dextrose Agar at 45°C were added to the C. albicans plates, and allowed to harden. Bacterial plates were incubated at 37°C for 24-48 hours. C. albicans plates were incubated at 25°C for 48-72 hours. Following incubation of the plates, colony counts were made.

CONTROL STUDIES: Control studies were mn., in which a 10 ml sample of saline was challenged, instead of 10 grams of the toothpaste. The tests were run as described above, except that test samples were diluted I x 1·0 ' to l x 10'' to obtain plates that could be counted.



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Control (Saline)

1.1 X !Of


5 9 X  10'    /7,77 (J)




KEY: l=Chollenge lnoculum 2=Recovercd Control 3= Recovered Test