RCT: Enzyme Blend Toothpaste on Dental Biofilm Plaque Accumulation

A Randomized Clinical Trial to Determine the Effect of a Toothpaste Containing Enzymes Blend of Alpha-Amylase and Gluco-Amylase on Dental Biofilm Plaque Accumulation in Healthy Adults Over a 28-Day Period of Product Use

The aim of the study is to investigate whether brushing twice a day with a toothpaste (Biofresh Clean) containing a blend of alpha-amylase and gluco-amylase improves oral health outcomes over a 14-day period.

The primary objective is to evaluate if the enzyme-containing toothpaste has an effect on dental plaque accumulation compared to placebo following 28 days of use.

The secondary objectives are to:

• Evaluate if the enzyme-containing toothpaste has an effect on dental plaque accumulation compared to placebo at Days 1 and 14
• Evaluate if the enzyme-containing toothpaste has an effect on gumline and proximal plaque severity compared to placebo at Days 1, 14 and 28
• Evaluate if the enzyme-containing toothpaste has an effect on whole mouth Gingivitis as well as gumline and proximal Gingivitis severity compared to placebo at Days 14 and 28

The exploratory variables are to investigate if the enzyme-containing toothpaste results in:

• Shift in detected oral bacterial species of interest at Screening and Baseline to Days 14 and 28, based on microbial DNA-extraction and microbial whole genome sequencing compared to placebo.
• Shift in immune markers (IL-1β, , IL-8 and MIF) at Screening and Baseline to Days 14 and 28, based on electrochemiluminescence assay compared to placebo.
• Difference in performance attributes compared to placebo based on End of Study Post-product use questionnaire after 28 days of product use.

The safety objective is to investigate safety and tolerability of brushing twice daily based on treatment emergent adverse events.

Target Population Randomized subjects are generally healthy males and females (18-65y) with at least 20 natural teeth, A gingival index score of ≥ 1.0, according to the Löe-Silness Gingival Index at baseline and a plaque index score ≥ 1.95 according to the Lobene-Soparkar Modification of the Turesky Modification of the Quigley-Hein Plaque Index..

Researchers will compare Biofresh Clean Toothpaste to an identically packaged and labelled placebo to see if Biofresh Clean Toothpaste reduces dental plaque accumulation and Gingivitis.

Participants will:

• Brush teeth for one minute twice per day (morning and evening) with either 1.5g of Biofresh Clean toothpaste or identical placebo toothpaste over 14-days.
• Visit the dental clinic on 5 occasions: 1. Screening visit to assess eligibility; 2. Baseline visit; 3. Day 1 Visit; 4. Day 14 visit; 5. Day 28 visit

Inclusion Criteria:

1. Generally healthy males and females 18-65 years of age.

2. Able to read, sign and receive a copy of the signed informed consent form.

3. Willing and able to comply with scheduled visits, treatment plan and other study procedures.

4. Subject is in good general and mental health with, in the opinion of the investigator or medically qualified designee, no clinically significant or relevant abnormalities in medical history or upon oral examination, or condition, that would impact the subject's safety, wellbeing or the outcome of the study.

5. At the Screening Visit (Visit 1):

   1. Have at least 20 natural teeth with scorable facial and lingual surfaces. (A scorable surface is defined as having 2/3rds of the natural tooth surface gradable for the selected clinical indices. The following should not be included in the evaluable surface count- third molars; fully crowned/extensively restored, grossly carious, orthodontically banded/bonded or abutment teeth; surfaces with calculus deposits which, in the opinion of the clinical examiner, would interfere with the baseline assessments of the selected clinical indices.)
   2. Subject with generalized mild-moderate plaque-induced gingivitis, in the opinion of the clinical examiner, as confirmed by visual examinations.

6. At Screening Visit (Visit 1):

   1. A gingival index score of ≥ 1.0, according to the Löe-Silness Gingival Index.
   2. A plaque index score ≥ 1.95 according to the Lobene-Soparkar Modification of the Turesky Modification of the Quigley-Hein Plaque Index, following 8 to 16 hours plaque accumulation period.

7. Willing to refrain from all oral hygiene 8-16 hours prior to each study visit, refrain from eating within 4 hours prior to each study visit and discontinue drinking 30 minutes prior to each study visit.

8. Willing to refrain from using chewing gum, breath mints and lozenges for the duration of the study.

9. Willing to refrain from the use of chemotherapeutic antiplaque/antigingivitis products containing antibacterial agents such as, but not limited to, amine fluoride, chlorhexidine, stannous fluoride, essential oils, cetylpyridinium chloride, etc. and other oral hygiene products during the study.

Exclusion Criteria:

Subjects presenting with any of the following will not be included in the study:

1. Have any medical condition that, in the opinion of the investigator, would place the subject at increased risk or preclude the subject's full compliance with or completion of the study.
2. History of allergy or significant adverse events following use of oral hygiene products such as toothpastes, mouth rinses, breath mints, lozenges, or chewing gum or their ingredients.
3. A subject who is pregnant or intending to become pregnant over the duration of the study (self-reported).
4. A subject who is breastfeeding.
5. Self-reported serious medical conditions.
6. Uncontrolled diabetes or hypertension.
7. Antibiotic or anti-inflammatory medication within 30 days of screening visit. 8. Smokers or users of other nicotine products.

9. Any health condition that requires prophylactic antibiotics prior to any dental procedure to prevent infective endocarditis.

10. Daily use of steroidal or non-steroidal anti-inflammatory drugs. 11. Have had antibiotic, anti-inflammatory, or chemotherapeutic antiplaque/antigingivitis therapy within 30 days of screening exams.

12. Unwilling to discontinue use of other oral hygiene products (e.g., antiplaque-antigingivitis mouthrinses or dentifrices, power toothbrushes, oral irrigators/water flossers) for the duration of the study.

13. Clinically visible active caries lesions and/or periodontitis. 14. Orthodontic appliances, including retainers, peri/oral piercings, or removable partial dentures.

15. Exhibits ≥ 30% of teeth with stage II - IV periodontitis, according to the American Academy of Periodontology revised classification system for periodontal and peri-implant diseases and conditions,8 at screening and/or baseline visit, or being actively treated for periodontal disease, and have more than three teeth with periodontal pockets depths measuring more than 5 mm. 16. Have significant intra-oral soft tissue lesions due to pathology or trauma based on a visual examination.