In Vitro Efficacy Testing of DioxiBrite Mouthwash vs. Mouth Organisms

In Vitro Efficacy Testing of Breathless* Mouthwash vs. Mouth Organisms

* Breathless was re-named DioxiRinse

COLLABORATIVE LABORATORIES July 23; 1996

PROJECT NO. MB96-19

In Vitro Efficacy Testing of Breathless Mouth Wash vs Bacterial Challenge Of

Klebsiella pneumoniae

Sponsor:

Contact:

Arco Research Inc. 10 Ponderosa Drive

Melville, NY 11747 Valerie Alliger

Study Director: Stephen Spiegelman

Study Date: Initiation: 7/18/96 completion: 7/19/96 Lab. notebook reference no. 212,058

Test Solutions: 1. Breathless Mouth Wash:

Part A - Lot MW12062096 - lA ; Part B -Lot MW 13062096-2B

- Lot MW 22062096 -3A

(As per sponsor's request, parts A and B were mixed 1:1 and aged 30 secs prior testing)

2. Peridex Mouth Rinse (P&G) Lot 111 (Active 0.12% Chlorhexidine gluconate)

Test Method: In -Vitro Germicidal Suspension Test

Test Organisms: Klebsiella pneumoniae, ATCC13883 (Revived from Cultiloop)

Purpose: To determine the germicidal effectiveness of Breathless mouth rinse formulation

Media: A) Culture - Brain Heart Infusion Broth,Tryptic Soy Agar (TSA)

B) Neutralizing medium - DIE Neutralizing Broth

Germicidal Contact: 0.5, 1, 2 and 4 minutes

Procedure:

1. The challenge organism was revived and cultured on TSA slants then put through a single passage
in BHI broth and incubated for 24 hours at 32°C. The challenge culture was grown overnight in BHI
broth at 32°C, harvested by centrifugation , washed and resuspended in phosphate buffer.

2. A 0.1 ml aliquot of the challenge culture was transferred to 9. 9 ml of each test product.
Peridex mouth rinse served as positive control.

3. Arco' s Breathless mouth rinse was prepared by combining equal volumes of the two-part

COLLABORATIVE LABORATORIES

July 23, 1996

PROJECT NO. MB96-19

fotmulation and aged 30 seconds prior to exposure to the challenge.

4. At 0.5, 1, 2 and 4 minutes, 1ml of the reaction mixture was withdrawn and mixed with 99 ml
neutralizing medium, DIE broth.

5. Surviving bacteria were determined by plating serial dilutions of the recovery mixtures.

6. Control titer was determined by inoculating 9.9 ml phosphate buffer blank with 0.1 ml
challenge culture, then diluting 1ml of the suspension in 99 ml DIE broth and serially diluting and
plating in TSA.

Neutralization:

1. Add 1 ml of each test formula to 99ml DIE. Vortex, then inoculate "neutralized" mixture with 1
ml of challenge culture (K. pneumoniae).

2. Mix thoroughly and allow to stand for approximately 4 minutes. Serially dilute and plate.

3. Pour plates with Tryptic Soy Agar medium.

4. Incubate plates at 37°C for 24 hours

i Test Results




Control Titer: 2.6 x 108 cfu/ml (at time zero)


COLLABORATIVE LABORATORIES July 23, 1996

PROJECT NO. MB96-19

Neutral1' zation: Challene:e 0 rgamsm, K. pneumomae ATCC 13883
Conclusion:

The results indicate that both of the Arco mouth rinse formulations were effective in reducing the
challenge population from 8 logs to less that 2 logs after 30 seconds contact. This represents a
greater efficacy than the Peridex control which showed recovery of 5.11 logs after 30 seconds under
the same test conditions.

Neutralization was demonstrated with the use of Klebsiellapneumoniae. Arco's mouth rinse
formulations and Peridex all showed comparable recovery to the test control.

Respectfully submitted: