This study was conducted in accordance with standards of Good Clinical Practice (VICH Guideline 9) and EMEA/CVMP/EWP/005/2000-FINAL0-REV. 2 Guideline for the testing and evaluation of the efficacy of antiparasitic substances for the treatment and prevention of tick and flea infestations in dogs and cats. The study protocol was reviewed and approved by Charles River Laboratories Preclinical Services Ireland Ltd.’s Ethics Committee prior to the start of the study.
Investigational veterinary products (IVP’s)
Advantage II for Dogs (9.1% imidacloprid + 0.46% PPF; US EPA Reg. Nos. 11556–152, -127, -218 and −130; Bayer HealthCare, Animal Health) is a topical solution administered at a single or multiple spots (depending on dog size and dose volume) to the skin along the dog’s dorsal midline. Advantage II for Dogs is labeled for the treatment of fleas and lice on dogs.
Comfortis (spinosad; NADA 141–277; ELANCO Animal Health) is a chewable tablet, available in five sizes. Tablets are administered orally to dogs based on their weight (minimum dosage 30 mg/kg). Comfortis is indicated for the prevention and treatment of cat flea infestations on dogs.
The IVP’s were administered twice, on Study Days 0 and 28. Dog body weights taken on Study Days −3 and 7 were used to determine the IVP doses administered on these two days, respectively. Administration followed the manufacturers label directions. The untreated controls received no treatment.
Forty purpose-bred Beagles (20 males, 20 females; age range, 8 months to 5 years; weight range, 9.4–17.8 kg) were acclimatized and initially evaluated for this study. During acclimatization all dogs were given a physical examination, bathed with a non-insecticidal shampoo and combed to remove any pre-existing flea infestations. No study animals were immunized or dewormed during the acclimatization period. Dogs were observed for general health at least once daily throughout the 34 day acclimatization period.
Animals had not participated in a previous study involving exposure to any ectoparasiticide within 90 days of study onset. During the study no medications were permitted other than the IVP’s, except when prescribed by the attending veterinarians for specific clinical conditions. Animals were fed with an appropriate diet for maintenance, and water was provided ad libitum.
Animal housing/simulated home environments
Dogs were housed in accommodations that complied with accepted guidelines for pen design/floor area, lighting, humidity, temperature, and welfare (including environmental enrichment and social interaction), as required by local and national regulations. During the studies, temperature and ventilation were controlled, and the environment was monitored to maintain an ambient temperature of approximately 20–25°C and a relative humidity of approximately 55–70%.
Dogs were housed in individual pens (1 m X 2 m) containing raised sleeping areas (0.6 m X 0.75 m) with solid wood barriers between the pens. The floor of each raised area was covered with cocoa matting which had been prescreened to ensure it was not treated with any compound that could have interfered with the flea viability.
This study utilized a randomized block design, with animal gender and pretreatment viable flea egg (larval hatch) counts as the blocking factors. Between Study Days −31 and −24, the 40 dogs were evaluated for their ability to produce viable flea eggs. On Study Day −31, each dog was infested with 100 (± 5) unfed adult C. felis. On Study Day −27, a maximum of 100 flea eggs were collected from each dog’s pen, placed in petri dishes, and incubated (26°C - 29°C; 62% - 82% relative humidity) for 72 h (± 2 h). Petri dishes were then examined for larval hatch.
On Study Day −21, the 30 dogs producing the highest numbers of viable flea eggs (hatching larvae) were allocated to one of three treatment groups (5 females and 5 males/group): Group A (Advantage II for Dogs); Group B (Comfortis); Group C (untreated control). Within each gender, dogs were ranked in descending order (highest to lowest) based on Study Day −24 egg hatch counts. Animal identification number was used to break ties (highest to lowest ID). The first 3 dogs (highest counts) within each gender, were assigned to Block 1, the next 3 dogs were assigned to Block 2, and so forth, until the final 3 dogs (lowest counts) were assigned to the final Block. The pre-determined randomization schedule reflected the treatment assignments. This randomization procedure was repeated for each gender. The three groups were balanced by gender and ability to support production of viable flea eggs.
Environmental flea infestations
Cat fleas (Ctenocephalides felis) used in this study originated from Charles River Laboratories Preclinical Services Ireland Ltd.’s flea colony. On Study Days −21, -16 and 1, each dog was infested with approximately 100 (100 ± 5; 35-65% female: 35-65% male) recently-emerged, unfed, adult C. felis to establish a continuous flea infestation within each simulated home environment. Fleas were applied along the dog’s dorsal midline in the lumbosacral region. On Study Days 7, 14, 21, 28, 35, 42, 49 and 56, after collection of the cocoa matting samples (see below), each dog was infested with 5 ± 1 fleas (male and/or female) as described above.
None of the procedures in this study were expected to result in undue pain, distress, or discomfort to the study animals. However, in the event that the flea infestation on any dog reached a level that resulted in clinical manifestations, e.g., severe FAD, provisions were in place for flea reduction measures on the animal and in the simulated home environment, as well as administration of appropriate medications to provide relief to the dog.
Assessment of flea infestations
On Study Day 0 (prior to IVP administration), and Study Days 7, 14, 21, 28, 35, 42, 49, 56 and 63, two circular cocoa mat samples (approximately 5.5 cm diameter) were removed from each simulated home environment. These samples were taken from the same locations in each pen on each day. The locations for all cocoa mat samples were randomly selected prior to study initiation. Each sample removed was replaced with a new mat sample of the same size which was fastened in place with screws. Those locations were not sampled again for the remainder of the study.
For all sample collection times, each cocoa mat sample was placed in a labeled, ventilated 250 mL container, to which flea rearing media and sand (1 part flea media: 2 parts sand) were added. Containers were placed in an incubation room (23°C - 31°C; 55% - 90% relative humidity).
Adult fleas emerging from the incubated cocoa mat samples were counted 32 days after sample collection, e.g., adult fleas emerging from Study Day 0 samples were counted on Study Day 32. The remaining counts were performed on Study Days 39, 46, 53, 60, 67, 74, 81, 88 and 95. These emerging adult flea counts were used as the measurement of infestations in the simulated home environments.
On Study Day 0 (prior to IVP administration) and Study Day 63, adult flea comb counts were performed for all dogs. Following Study Day 0 counts, the fleas were returned to the dogs.
Assessment of IVP efficacy
The geometric means of weekly post-treatment emerging adult flea counts and Study Day 63 adult flea comb counts were used for efficacy calculations. Geometric means provide a measure of the central tendency of the data that minimizes the effects of extreme values. Geometric means were calculated following transformation using a logarithmic method (averaging the transformed values, and converting the average using antilog to represent a geometric mean). Because some counts were zero (0), all counts were modified by adding one (1) to each count prior to logarithmic transformation. Likewise, one (1) was subtracted from the antilog value to meaningfully represent the geometric mean for each group.
Efficacy was calculated for the weekly post treatment emerging adult flea counts and Study Day 63 adult flea comb counts using Abbott’s Formula:
Although dogs were grouped by gender and blocked on the basis of egg hatch counts, the “blocking” of animals and the randomization of animals to treatment groups were designed primarily to maintain balance of treatments throughout the study. Consequently, “block” was not included in the statistical analysis.
Transformed counts [log (count + 1)] of emerging adult fleas were analyzed with a repeated measures analysis of covariance including terms for treatment, individual dog pen, study day, and the interaction of treatment and study day. Transformed counts of adult flea emerging from the Study Day 0 (pretreatment) cocoa mat samples were used as the covariate.
SAS PROC MIXED (SAS® Institute, Cary, NC) was used for analysis with the covariance structures ‘AR(1)’ and ‘ARH(1)’ for data collected on equal intervals, or ‘CS’ and ‘CSH’ for data collected on unequal intervals. Results from the model with the smallest Akaike’s Information Criterion were used.
Because the interaction of treatment and study day was significant at the 0.05 level, the treatment groups were compared through the simple effect of treatment for each time point. These simple effect pairwise comparisons were obtained from the treatment X day interaction.
Study Day 63 adult flea comb counts (transformed as noted above) were analyzed using analysis of covariance, including a term for treatment. Transformed Study Day 0 adult comb counts were used as the covariate.