A protocol is to be adopted prior to the start of a trial intended for submission to VOHC. Pre-trial approval of the protocol by VOHC is required, to avoid misunderstandings that could cause the trial data to be inadmissible. Be sure to read the Protocols and Submissions – General Information page before writing a protocol.
Click Format for a description of the order of items to be included in a draft protocol submitted for pre-trial approval. Send your draft protocol as an email attachment to VOHC@AVDC.org. A pre-trial protocol will be reviewed only by the VOHC Director and will remain confidential.
VOHC recognizes that there is need for flexibility in measuring efficacy. Alternative but credible methods of demonstrating efficacy that meet VOHC standards will be considered by VOHC, provided that detailed justification is provided for novel or non-referenced methods.
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Pre-trial Screening Examination
Prior to entry of a dog or cat into a VOHC trial, an awake oral examination is to be performed to ensure that inclusion criteria are met and no exclusion criteria exist. Inspect the buccal surfaces of the VOHC teeth, the adjacent gingiva and the other oral mucosal surfaces.
Presence of plaque and tartar on the crown of the teeth is not of itself reason to exclude a dog or cat, as this will be removed during the pre-trial professional dental scaling procedure.
Non-gingival inflammation, ulceration or mucosal laceration in dogs or cats.
Tooth resorption at the gingival margin in cats. Cats often have tooth resorption, sometimes in multiple teeth. When the cemento-enamel junction area is affected, gingival inflammation and pain are common. Cats are to be excluded when a focused lesion of inflamed gingiva, as is often seen in teeth with resorption, is seen at the gingival margin of a VOHC tooth, or of any tooth if the trial is of a product applied by a person to clean the teeth (e.g. a brush or wipe).
Required Groups in VOHC Trials
Before writing a detailed draft protocol, read this section to determine the number of groups required in the trial. If you are a representative of a company considering an application for the VOHC Seal of Acceptance for a chemically-active product, contact VOHC to determine the appropriate study design before drafting a full trial protocol for VOHC review.
Chewed and Ingested Mechanically-effective Product
A split mouth design may be used, provided that assignment of test or control brush or device to left or right side is randomized.
Product with a Chemically-active Effect and No Mechanical Effect
If the chemical agent is added to the drinking water or is sprayed onto the teeth by the owner, and if the chemical agent is GRAS (Generally Regarded As Safe) at the concentration used in the product, the VOHC trials are to include two groups: a negative control group (control diet only, fed dry), and an active agent ‘test’ group (control diet fed dry, with the active agent applied as per the instructions on the product package). To be awarded the VOHC Seal, the active agent group mean data must meet the basic VOHC effectiveness standard (minimum of 15% reduction in the test group in either trial, and a minimum mean reduction of 20% in the test groups in the two trials). A split-mouth design is not permitted for VOHC trials of this type of product.
If the chemical agent is applied directly as a gel or dentifrice by spraying it or gently smearing it onto the buccal surfaces of the teeth such that no mechanical effect is intended, and if the chemical agent is GRAS (Generally Regarded As Safe) at the recommended dose, and if the product will be marketed with instructions for use that do not require use of a mechanical device, the VOHC trials are to include two groups: a negative control group (control diet only, fed dry), and an active-agent ‘test’ group (control diet fed dry and active agent applied as per the instructions on the package). To be awarded the VOHC Seal, the active agent group mean data must meet the basic VOHC effectiveness standard (statistically significant minimum of 15% reduction in the test group in either trial, and a minimum mean reduction of 20% in the testgroups in the two trials). A split-mouth design is not permitted for VOHC trials of this type ofproduct.
Product With a Chemical Effect That is Applied With a Brush or Other Mechanically-effective Device
Two groups must be compared – a positive control group in which the water-wetted brush or device is applied using the VOHC brushing procedure and an active agent group in which the chemical agent is applied with the brush or device; no negative (control diet only) group is required. A control diet (dry food, fed dry) is to be fed to both groups during the trial. To be awarded the VOHC Seal, the active agent group mean data must meet the VOHC effectiveness standard (statistically significant minimum of 15% reduction in the test group in each trial, and a minimum mean reduction of 20% in the test groups in the two trials), and be statistically significantly better than the positive control group (though no specific % reduction is required), with no safety issues such as trauma to the mouth reported during the trial. A split-mouth design is not permitted for VOHC trials of this type of product.
Product with a Mechanical and a Chemical Anti-Plaque or Anti-Tartar Effect
The determination of number of groups required will depend on whether the mechanical vehicle (e.g. the brush) is VOHC Accepted.
The requirement for acceptability by VOHC is that the reduction in plaque or tartar in the test group (e.g. chew treat or brush with the chemical agent - group 3) is to be statistically higher than that of the positive control group (mechanical vehicle without the chemically active agent - group 2) but with no minimum % difference stated, and be statistically different from and have a minimum reduction in mean group score of 15% reduction in each trial and minimum mean reduction of 20% in the two trials compared with the negative control group (group 1).
In this three-group scenario, if the mechanical device is a novel product and the positive control group (group 2) meets or exceeds the VOHC efficacy standard compared with the negative control group (group 1) in the two trials, the mechanical device itself would be eligible for the VOHC Accepted Seal independent of the chemical agent, assuming the two-trial requirement is met.
The requirement for acceptability by VOHC is that the reduction in plaque or tartar in the test group (group B) is to be statistically higher than that of the positive control group (group A) but with no minimum % difference stated.
Minimum Trial Period
The minimum trial period is 28 days. For cross-over trials, 28 days per leg of the trial, with a minimum between-leg rest period of 7 days in trials of a chemically-active product.
Number of Subjects per Group, and Randomization Into Groups
All groups in the trial are to have the same number of subjects assigned at the start of the study.
There is no VOHC minimum required number of animals in each group – this is up to the sponsor to determine, and will depend on the efficacy of the product. Consultation with a statistician and calculation of a ‘power equation’ is recommended to determine the minimum number of subjects in each group.
See also the comment on the number of subjects in each group in the Statistical Analysis section.
The dogs or cats are to be assigned to test or control groups randomly, and the randomization method is to be described.
Required Group Differences
The minimum difference required between "mouth mean scores" (mean of all scored teeth for all animals in the group) comparing the test group with the negative control group is 15% reduction in the plaque or tartar score in each trial and 20% in the mean of the two required trials, with a statistically significant difference (p<0.05) in each trial.
Reduction (VOHC efficacy %) is defined as:
(Control group mouth mean minus Test group mouth mean) x 100
(Control group mouth mean)
Statistical Analysis Requirements and Related Information
One of the most common reasons for concerns being raised about a submission during review by VOHC Council is failure to use and adequately describe appropriate study design and analysis procedures to demonstrate that the VOHC efficacy standard has been met.
Population and Source of Animals
As in any biological study, results of VOHC trials are most satisfactory when a population with lower variability in relevant parameters is used. For VOHC trials, some variables are fixed, such as the control diet to be fed. Relevant variables are age (periodontal disease is known to be more prevalent and more severe in older dogs and cats), body size (periodontal disease is more prevalent and severe in toy and small dogs than in medium or larger dogs), absence or disease of teeth - VOHC requires presence of a designated set of teeth (‘VOHC teeth’) that are intact and healthy other than presence of plaque or tartar coverage and/or mild or moderate periodontal disease.
Dogs or cats in both trials are to be to be of similar body weight and age range. General health is to be assessed by physical examination, CBC, chemistry panel, and urinalysis, or otherwise assured by the veterinarian of record. The requirement for similarity of body weight in the two trials submitted for VOHC review is to provide validation of the statistical result. If there are practical considerations that would prevent all subjects being of similar body weight or age range, send a preliminary email note to VOHC@AVDC.org for clarification.
Additional trials in dogs of different sizes are encouraged but are not required by VOHC unless the product is available in more than one size or shape, in which case the Product Line policy applies.
VOHC recommends but does not require pre-trial measurement of the rate of deposition of plaque and/or calculus, so that animals can be blocked on rate of accumulation during the randomization procedure (‘stratification’); this reduces the likelihood of higher variability in the rate of accumulation of plaque and/or tartar in individual subjects between the test and control groups.
VOHC trials are often conducted using beagle dogs or DSH cats housed in a USDA-approved laboratory animal facility. When animals in a USDA-approved or similar facility are used in VOHC trials, confirmation of approval of the Institutional Animal Care and Use Committee is required. On request to VOHC@AVDC.org, VOHC will provide a list of potential resources for laboratory-housed dogs and cats.
When a particular product requires use of e.g. dogs that are smaller or larger than the typical laboratory beagle dog, identifying a source of the animals can be challenging. Use of client-owned animals is permitted. If you are unsure about suitability of available animals for a VOHC trial, send a preliminary email note to VOHC@AVDC.org for clarification.
Use of Client-Owned Animals in VOHC Trials
VOHC allows use of client-owned animals in VOHC trials, though this source of animals typically is more variable in relevant parameters than is the case for subjects housed in a laboratory, and thus a larger number of animals may be required to obtain statistical significance.
When client-owned animals will be used as the subjects in a VOHC trial, all of the animals must be fed the same control diet (dry food, fed dry), and an information/informed consent document (‘Owner Document’) is to be signed by the animal’s owner before entry of the subject into the trial. A blank copy of the Owner Document is to be included in the protocol sent for VOHC pre-trial protocol review and in the VOHC submission. Confirmation of approval of the trial and of the Informed Consent document by the head of the hospital or clinic running the trial and the product sponsor is required.
The Owner Document is to include a requirement that the owners complete a daily compliance log, and copies of the completed owner’s daily compliance logs are to be included in the VOHC submission.
Out-of-protocol events reported by the owners are to be described and comments by the trial sponsor on significance of the out-of-protocol events are to be provided in the submission.
Use of the Product During the Trial
During the VOHC trial, the product is to be used as recommended by the company on the product package. If the product is a toothbrush or is applied with a toothbrush (or similar device), the VOHC Toothbrush Policy is to be followed.
For the duration of the trial, the dogs or cats are to be fed a control diet, either in both the test product and control groups, or in only the control group if the test product is a diet. The control diet is defined as a commercially available dry dog food or dry cat food, fed dry, that meets AAFCO standards for all life stages.
The most common mistake made in understanding this requirement is the ‘fed dry’ phrase. Accumulation of dental plaque and tartar when standard dry kibble diets are fed is slightly (in dogs) or moderately (in cats) less likely than when a non-dry food
diet is fed; however, kibbles that have been moistened prior to feeding do not have the same effect as kibble fed dry.
Clean Tooth Model
The teeth are to be scaled and polished on day zero so that the plaque and calculus scores are zero at the start of the trial. The animals are to be anesthetized and intubated during the scaling procedure, and a disclosing agent is to be applied to the teeth at the end of the scaling procedure to confirm that all plaque and tartar has been removed.
Systemic or orally applied antibiotics or anti-inflammatory drugs must not be used for 7 days pre-trial, during the trial or during the rest period between trial legs in cross-over trials. The rest period between legs for a cross-over design involving a chemical plaque or tartar agent is to be a minimum of 7 days.
No locally applied or lickable antiseptics can be applied or access to chew toys or treats can be given in the 24 hours prior to Day zero or during the trial (except for the tested product during the trial).
Normally scheduled deworming medications or vaccinations can be given during the trial period.
If a medical problem requiring treatment develops during the trial period, that animal will likely need to be dropped from the trial – send an email to VOHC@AVDC.org for clarification.
Plaque and calculus indices are to be scored with the dog or cat under sedation or short-acting anesthesia, unless a scoring method pre-approved by VOHC for use in un-sedated animals is used (e.g. Gingival Contour Plaque Index, J Vet Dent 26, 23-27, 2009) or a detailed statement justifying use of a novel scoring system suitable for non-sedated dogs or cats is provided - pre-trial protocol review by VOHC will be used to determine whether use of the proposed index is acceptable to VOHC.
Plaque and Tartar scores must be zero on Day zero of the trial, confirmed by use of a disclosing solution at the end of the scaling procedure.
Training of Scorers
Training and experience of the scorer is to be described in the protocol. VOHC will not accept data from a trial scored by a novice scorer. The scorer is to be blinded to the group assignments during the entire trial period.
The same scorer is to score a particular index (plaque or tartar) of all animals at each examination except on Day zero (when absence of plaque and tartar is confirmed following scaling by applying a disclosing agent); however, plaque and calculus can each be scored by separate scorers. The requirement for the same scorer to score a particular substrate at each post-Day zero scoring episode in every animal may not be practical when a large number of animals are scheduled to be scored on the same day; contact VOHC@AVDC.org to clarify arrangements for scorers in this instance prior to submission of the pre-trial protocol.
All teeth listed are to be present and intact, with normal occlusion for a dog or cat of that head shape. The teeth listed were selected for scoring on the basis of functional importance, likelihood of accumulation of plaque and calculus, likelihood of being present in the mouth in the face of moderate periodontal disease, and size for ease of scoring. Score the buccal surfaces of the teeth on both sides of the mouth (unless a split-mouth study design is used for a trial of e.g. a tooth brush). See Division of the Crown for information on what part of the crown to score.
Order of Presentation of Animals at Final Scoring Episode
The animals are to be presented in random order for the final scoring episode, to prevent scorer bias.
Timing of Final Scoring Episode
For products that are given or performed as discrete procedures (e.g. treats or dental diets given daily or brushing or other application of a dentifrice daily) rather than being continuously available (e.g. water additive), the time from last use of the product to the end-of-trial scoring episode is to be within +/- 3 hours of the time when the product would next be given or used if the trial was continued.
Division of the Crown into Segments for Scoring
For scoring of plaque and calculus:
Horizontal segmentation (i.e. parallel to the gingival margin) is permitted, with the following limitations:
From the Pre-trial Screening section: If a limited area of the coronal part of the crown is missing on a particular tooth, and there is no evidence of pulp exposure, and the area scored for plaque and calculus is limited to the gingival half, the animal can be included in the trial provided that the scoring is limited to the gingival half of all teeth in all animals included in the trial.
Plaque score is to be recorded after staining with a plaque disclosing agent and gently rinsing the tooth surface. Describe and reference the Plaque Index. If a novel plaque assessment is used, include a detailed referenced statement justifying the assessment method.
A combination index incorporating extent of coverage and thickness of coverage may be used.
Describe and reference the Tartar (Calculus) Index. If a novel tartar assessment is used, include a detailed referenced statement justifying the assessment method.
A combination index incorporating extent of coverage and thickness of coverage may be used.
All VOHC trials are to include scoring of gingivitis on Day Zero and at the final scoring episode, to provide an indication of safety of the product. VOHC does not provide a Gingivitis claim. FDA-CVM considers gingivitis to be a ‘disease claim’, and requires completion of the FDA NADA process for products marketed in the USA that wish to make a gingivitis claim.
Describe and reference the Gingivitis Index. If a novel gingivitis index is used, include a detailed referenced statement justifying the assessment method.
The Gingivitis score is not required to be lower in the test product group than in the control group; however, if the Gingivitis score is higher at the end of the trial compared with the Day zero score in the test group, or is significantly higher than the control group , VOHC will require an explanation of this anomaly.
Additional Observations During Each Scoring Episode
In addition to the specific Plaque and/or Tartar (Calculus) scores, each scoring episode is to include: Observing for and recording presence of any non-gingival inflammation and oral ulceration or lacerations.
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