A cysteine challenge test was used to measure the effectiveness of an anti-halitosis mouthrinse (AHM) over a six-hour period. AHM was shown to provide a sustained and effective anti-halitosis effect over the entire 6 hours.

The cysteine challenge test is a severe in vitro test for mouthrinse effectiveness. A cysteine rinse was administered hourly over 6 hours to 5 volunteers after an initial 60 second rinse with AHM. A 71.4% reduction in volatile sulfur compounds (VSCs) was achieved at 6 hours post-rinse. Conclusion: AHM was shown to provide a consistent, sustained and extremely effective reduction in VSC concentrations for at least 6 hours in human volunteers.

Americans spent over $700 million on mouthwash products and another $625 million on breath
mints and other mouth fresheners in the year 2000 (1). It has been estimated that 20% to 60% of the
population suffer from chronic halitosis (2,3). Roughly 70% report having occasional “morning breath.”
Most cases of halitosis originate in the oral cavity (85%), primarily from VSCs released by bacteria on the
posterior dorsum of the tongue (4,5). Anaerobic gram-negative bacteria on the tongue and in deep
periodontal pockets process protein by bacterial putrefaction. The protein is first ingested and then broken
down into amino acids by a process called proteolysis. These amino acids are then further reduced by
aminolysis. When the sulfur-containing amino acids, cysteine and cystine are processed, foul-smelling
VSCs (hydrogen sulfide and methyl mercaptan) are released and are perceived as halitosis (6). Other
gases make minor contributions to the overall halitosis odor. These have been identified as putricine
(decaying meat), cadaverine (rotting corpses), skatole (feces), and isovaleric acid (sweating feet). When
these are mixed in small amounts with the primary VSCs hydrogen sulfide (rotten eggs), methyl
mercaptan (feces) and dimethyl mercaptan in the oral cavity, a very foul breath can be produced (1).

The commercially available mouthwashes use combinations of essential oils and alcohol or oilwater-
cetylpyridinium chloride with alcohol to "kill germs" that cause bad breath. Once the initial
bacteria are killed they return to their prior levels within minutes so any anti-halitosis effects are
temporary. Also, the long term effects of alcohol tend to dry the oral tissues and create a better
environment for the overgrowth of odor producing anaerobes. Chlorhexidine is the "gold standard" of
antibacterial mouthwashes, which is effective against supragingival plaque bacteria for up to 8 hours. It
would be a good anti-halitosis treatment if the side effects of staining, taste alteration and oral ulcerations
could be negated. Since eliminating bacteria in the oral cavity could lead to the overgrowth of undesirable
species such as candida albacans, a different strategy has been developed to combat halitosis. It involves
the long-term suppression of the odor-causing gram-negative anaerobic bacteria population on the tongue
rather than their elimination (1).

AHM uses this newer strategy to combat halitosis. The first active ingredient is highly oxidizing
sodium chlorite (600 ppm of chlorite ion). It oxidizes the sulfides of the VSCs to non-odorous sulfates
and raises the oxidation/reduction ratio of the saliva toward the more oxidizing state. This suppresses the
overgrowth of the anaerobic bacteria on the tongue. The other active ingredient zinc acetate (300 ppm of
zn ion) oxidizes the VSCs and creates a more oxygen rich oral environment, but also interferes with the
proteolytic activities of the anaerobic bacteria. A mouthwash containing just the sodium chlorite ion (600
ppm) was tested and was found to be effective for less than two hours. Another mouthwash containing
zinc ion (300 ppm) was the active ingredient showed effectiveness to 4 hours. When combined, a synergy
was created which extended the anti-halitosis effects past 6 hours (6).

A factory calibrated halimeter (Interscan model RH-17K) was used to measure the VSCs (parts
per billion of hydrogen sulfide and methyl mercaptan) in the oral air of five volunteers (7). These
volunteers were screened for age (18 to 75), positive medical histories that revealed drug or alcohol use or
other conditions that in the opinion of the investigators would interfere with the study. Pregnant or
lactating females were not selected. Volunteers had to submit to a dental screening process. Only
volunteers with a well-restored generally intact dentition (minimum 16 teeth with at least 4 molars) with
no removable prostheses and healthy periodontal status with no active oral pathology were accepted.
They were then tested for a positive response to the cysteine challenge test (between 500 and 2000 ppb
VSC halimeter reading at 3- and 5-minute post rinse). They were asked to refrain from any oral hygiene
procedures, and have no food, drink or tobacco products 12 hours prior to and during the 6-hour trials. A
6ml 5mM cysteine rinse (pH 6.5) was swished for 30 seconds and expectorated (8). A halimeter reading
was taken, using the manufacturer's protocol of adjusting the machine to 0 ppb, then inserting a
disposable straw attached to the inlet hose one and a half inches into the oral cavity and sampling the
concentration of VSC's in the passive oral air. The volunteers held their breath for 15 seconds while the
reading was taken. Readings were made at 3- and 5-minute intervals post-rinse and recorded. Twenty
minutes later, a 12 ml de-ionized water rinse was swished for 60 seconds to serve as the control rinse and
another 3 and 5 minute reading was taken. A cysteine rinse was administered and recorded at the same 3
and 5 minute intervals at 40 minutes, 60 minutes, then hourly out to 360 minutes. All of the volunteers
showed a positive response to the cysteine challenge with no VSC reduction from the water rinse (fig. 1).

The next test was run with the volunteers rinsing with 12 ml of the mouthwash formula for 60 seconds
and readings taken after the cysteine challenge at 40 min, 60 min and then hourly intervals to 360 minutes
(fig. 2).

The baseline reading for the group was in the 83 ppb range- which was well below the threshold of 200
ppb of patients suffering from detectable halitosis. When a water rinse was given as a control, there was
no change in the high VSC readings of subsequent cysteine challenges (fig. 1). The test that was then
given with the test mouthrinse showed a significant reduction in VSC levels with each cysteine challenge
all the way to the 6-hour limit of the study (fig. 2).

The test mouthrinse showed a reduction in halitosis-causing VSCs, (hydrogen sulfide and methyl
mercaptan) from the first challenge at 20 minutes to the end of the trial at 6 hours. A reduction in VSC
levels of over 70% at six hours indicates a substantial anti-halitosis effect for this mouthwash. With these
pilot study results, further large-scale clinical trials are indicated to test the mouthwash results out to 12
hours, effects on morning breath levels after night-before rinsing as well as trials using organoleptic
judges combined with halimeter VSC measurements.