Trial Protocol – Detailed Requirements

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.

Inclusion Criteria:
In dogs and cats:

• Good general health.
• All VOHC teeth must be present and intact on both sides of the mouth. (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).

Exclusion Criteria:
Severe periodontal disease of any of the VOHC teeth, defined as:
A complete (FI = 3) furcation defect in any multi-rooted tooth in a cat or dog.
Attachment loss of ≥50%
Mobility (0-3 index) ≥2

Severe gingival inflammation (spontaneous bleeding or immediate bleeding on touching the gingiva) affecting VOHC teeth in dogs or cats. 

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
Chews or treats and dental diets that have a mechanical dental effect but no chemical effect require only a negative control group (dry food diet, fed dry) and a test group.
The VOHC standard for this category is 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.

Owner-applied Mechanically-effective Product
Trials of brushes or similar mechanical dental devices designed to be applied by owners in dogs or cats are to include:

• A positive control group (dry food diet, fed dry, plus daily brushing using an ADA-accepted flat profile, soft bristle manual toothbrush comparable to the Oral B 30 brush, or another brush that has been awarded the VOHC Seal), using the brush as described in the VOHC Brushing policy.

• A test product group (dry food diet, fed dry, plus brushing using the test product as frequently as recommended by the sponsor and using the brush as described in the VOHC Brushing policy.

A split mouth design may be used, provided that assignment of test or control brush or device to left or right side is randomized.

The reduction in plaque or tartar for the tested product group is to be statistically ‘at least as good as’ the positive control brush group.

Product with a Chemically-active Effect and No Mechanical Effect
Chemical agents designed to retard accumulation of dental plaque and/or tartar can be applied in various ways. Examples are:

• Adding the chemical agent to the drinking water, spraying the agent into the mouth or smearing a gel or dentifrice containing the agent onto the surface of the teeth, without the intention of creating a mechanical effect.
• A chew treat or ‘dental diet’ in which the active chemical agent is incorporated as an ingredient in or is sprayed on the surface of the chew or diet pieces.
• Owner-applied device (such as a brush) that will have a mechanical effect designed to enhance the effect of the chemical agent that is applied to the device.

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
VOHC has adopted a tooth-brushing protocol and a specific type of tooth brush has been awarded the VOHC Accepted Seal. If a VOHC Accepted brush or other mechanical device is used to apply the product using the specific methods described under the VOHC Brushing Policy, inclusion of a negative control group in the VOHC trials of a chemically-active product applied by the VOHC accepted brush or other device is not required, because the safety and efficacy of the brush or other device has already been demonstrated.

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 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
Proof of efficacy of the chemical agent becomes more complex to obtain when a mechanically-effective device is used as the vehicle for the chemical agent, because it is necessary to demonstrate that use of the mechanical-effect does not of itself cause harm and is not the only cause of the reduction in plaque or tartar.

The determination of number of groups required will depend on whether the mechanical vehicle (e.g. the brush) is VOHC Accepted.
For products with a chemical effect applied with a mechanically-effective product (e.g. a brush or an ingested treat or diet containing or coated with a chemical agent) that is not itself VOHC Accepted, the VOHC trials will require three groups:

• A negative control group (dry food only, fed dry).
• A positive control group – the mechanical vehicle, with no chemical agent added, applied as described in the VOHC Brushing policy.
• A test group given the product containing the chemical agent as it will be marketed.

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.
For products with a chemical effect applied with a mechanically-effective device (e.g. brush, diet or treat) that is itself VOHC Accepted, the VOHC trials will require only two groups, because the safety and efficacy of the brush or other device has already been demonstrated:

1. A positive control group – the mechanical vehicle, with no chemical agent added, applied as described in the VOHC Brushing policy.

1. A test group given the product containing the chemical agent applied by the VOHC Accepted device, applied as described on the product package.

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. 
Note: this is the minimum trial period. Trials of longer duration are encouraged; however, VOHC recognizes that there is sufficient documentation available to accept that differences in rate of accumulation of dental substrate obvious at 4 weeks are indicative of longer-term clinical relevance and safety.

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.

VOHC recommends stratification by e.g. plaque scores in a pre-trial period prior to the main trial, to reduce variability between groups, and recommends that randomization by blocking on stratification of the substrate (plaque or tartar) under study is based on blocks equal to the number of arms or legs in the trial.

Full consideration of possible carry-over effects must be included in analyses of cross-over design trials. Review the VOHC Statistical Guidelines.

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.
Data are to be reported as means ± SD.
Statistical significance is to be determined using a two-tailed test. VOHC Statistical Guidelines are provided.

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.

Use of inappropriate study design and/or analysis criteria can be an expensive mistake if the trial has to be repeated. VOHC strongly recommends pre-trial consultation with a qualified statistician. It is the responsibility of the sponsor to ensure that appropriate methods are used.

Requirements for the Statistical Methods and Results Sections in the Submission:

1. 1. Number of subjects per group. There is a risk when using a small sample that the data may not be suitable for parametric analysis. VOHC will assume that data generated from trials that include 20 or more subjects in each group will be normally distributed, and parametric analyses of mean mouth scores (e.g. t-test, ANOVA) can be used. When the group size is less than 20, evidence of normal distribution must be provided, or non-parametric analyses (such as Rank-Sum Analysis) must be used.
   2. Randomization of subjects to treatment groups is required. The randomization method must be described. Randomization can be used to your advantage – for example, if subjects are assigned to treatments within a blocking structure (to reduce variability), the blocks can be included in the analyses to reduce the size of the residual error term (i.e.: the denominator of the F-Test).
   3. Provide a clear description of the statistical analysis methodology employed, with justification for choice of the specific tests used.

1. 1. 1. 1. The tests used must be appropriate for the type(s) of data (categorical, ordinal [ranks] or continuous [actual metric]) analyzed, and meet the conditions stated in item 1, above.
         2. If more than one testing center is to be used, the interaction of treatments with the other levels used must be evaluated to insure additivity (i.e. the treatments compared are consistent across the other levels employed in the design).
         3. Where several groups are compared, adjustment for type-error must be applied, i.e. for multiple comparisons.
         4. In trials using a cross-over design, effects for carry-over (sequence effect) and period must be included and evaluated in the applicable ANOVA tables. In the event of a statistically significant sequence effect in a two period cross-over trial, only the first period data can be used in a parallel group analysis – the second period data must be ignored. In cases of cross-over designs with 3 or more periods, statistical protocols are available to adjust for carry-over effects detected in the analysis.
         5. Where the trial requires determination of statistical ‘At Least As Good As’
            status between groups, use the ‘At-least-as-good-as Criterion’ (Laster L, Johnson M: Non-inferiority trials:  Statistics in Medicine, 22,
            187-200, 2003).
         6. Use a two-tail test when determining probability level.

1. 1. Tabulation of the data and results of the statistical analyses is required. A descriptive summary table with sample sizes, means with standard deviation and standard error, percentages differences in means of the analyzed variable (plaque or tartar) and p-levels for the statistically significant findings, must be included. When analyses of variance are used, for example, ANOVA tables are to be presented, including mean squares, F- tests and p-levels.

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.

Control Diet

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.

Other Requirements

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 you propose to include an animal that is not generally healthy or that requires ongoing medication of any sort, contact VOHC for clarification about inclusion of that animal.

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.

Scoring

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.

When both plaque and tartar scores will be obtained, score plaque first. See also Timing of Last Score, Division of Teeth into Segments and Scorers sections.

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.

On request to VOHC@AVDC.org, VOHC will provide a file of resources for scorers or scorer trainers for VOHC trials.

Teeth to be Scored

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.

Dog: Upper Jaw – I3, C, P3, P4, Ml. Lower Jaw – C, P3, P4, Ml.
Cat: Upper Jaw – C, P3, P4. Lower Jaw – C, P3, P4, Ml.

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 given daily or less frequently, or brushing or application of a spray, gel or dentifrice daily or less frequently) 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. This means that for a trial of a product given every day, the final scoring episode is to be conducted 24 hours +/- 3 hours of the time when the product would next be given or used if the trial was continued. For a product given less frequently, e.g. every third day, the final scoring episode is to be conducted e.g. three days +/- 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:
Segmentation of the tooth vertically, with separately recorded scores for each vertical segment, is not permitted.

Horizontal segmentation (i.e. parallel to the gingival margin) is permitted, with the following limitations:

• • The anatomical locations for visualizing the ‘horizontal line’ are to be clearly stated in the protocol.
  • Only the gingival segment is to be scored.
  • VOHC recommends dividing the canine teeth of cats horizontally and scoring only the gingival half because of the tall, narrow shape of the crown – the whole crown of other teeth in cats can be scored.

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.

References:
1. Review of Studies Assessing Plaque Accumulation and Gingival Inflammation in Dogs. Hennet P: J Vet Dent 16(1); 23-29, 1999.
2. Shape and Size of Teeth of Dogs and Cats-Relevance to Studies of Plaque and Calculus Accumulation. Harvey CE: J Vet Dent 19(4); 186 -195, 2002.
3. Evaluation of the Logan and Boyce Plaque Index for the Study of Dental Plaque Accumulation in Dogs. Hennet P, Servet E, Salesse H, Soulard Y: Res Vet Sci, 80, 175-180, 2006.

Scoring Plaque

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.

Scoring is to be conducted under anesthesia or sedation, with the exception that VOHC will consider submissions in which the GCPI plaque scoring system (Journal of Veterinary Dentistry, 24, 14-20, 2007) is used in trained, unsedated dogs, provided that the plaque score and calculus score in each dog on Day zero is zero.

A combination index incorporating extent of coverage and thickness of coverage may be used.
For a submission supporting a product that incorporates a mechanical and/or a chemical anti-plaque agent, see the Required Groups section to determine the number of groups required. See also Division of the Crown into Segments for Scoring.

Scoring Tartar

Describe and reference the Tartar (Calculus) Index. If a novel tartar assessment is used, include a detailed referenced statement justifying the assessment method.

The Tartar Index is to be recorded under anesthesia or sedation, after brushing plaque off the surface of the teeth with water and a tooth-brush and air-drying the tooth surface. Whether or not a dental explorer is used to determine the edges of calculus deposits is not mandated. Tartar can be stained with a plaque disclosing agent after the teeth are brushed, followed by gently rinsing the tooth surface; if you propose to score tartar first, include justification (reasons for doing so, and a detailed description of the tartar scoring system) in the protocol submitted for pre-trial review. Contact VOHC for clarification on permitted indices if the trial is intended to have intermediate scoring episodes.

A combination index incorporating extent of coverage and thickness of coverage may be used.
For a submission supporting a product that incorporates a mechanical and/or a chemical anti-tartar agent, see the Required Groups section to determine the number of groups required.

See also Division of the Crown into Segments for Scoring.

Gingivitis Score

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.