In vitro membrane-feeding exposure
A membrane-feeding method  was modified to assess the impact of fluralaner exposure on flea reproduction. Defibrinated sheep blood was prepared in a series of dilutions with fluralaner to obtain concentrations between 50.0 and 0.09 ng/mL. Test solutions were prepared twice and each preparation was tested in duplicate resulting in a total of 4 replicates per concentration, along with a fluralaner-negative solvent control (a solvent concentration equivalent to that of the highest concentrated fluralaner test solution) and an untreated control.
Unfed adult fleas (C. felis; 20 males and 20 females) were placed into a plastic unit that was then closed with a gauze lid. A grid inside the plastic unit divided the unit into 2 chambers, an upper chamber for flea feeding and a lower chamber for egg collection . Test or control-blood preparations (2 mL) were placed in an artificial membrane-closed glass tube that was then placed on the plastic unit as the food source. Feeding units were incubated (38°C and 60% RH) for 10 days. Test and negative-control-blood preparations were freshly prepared and exchanged (on days 1, 3, 5, and 8) to permit continuous flea feeding. Fleas were transferred into fresh plastic units on days 5 and 8 to facilitate egg collection. Collected eggs were mixed with flea nourishment medium and incubated (28°C and 80% RH) in darkness for 22 (±3) days to enable flea development. Parameters recorded were flea survival, oviposition control, egg hatchability, pupa control and flea-emergence control.
In vivo study to assess flea-control efficacy in a simulated home environment
Twenty healthy male and female mixed breed dogs ≥12 weeks old were housed in individual pens. Ten dogs per group were randomly assigned to receive either a fluralaner chewable tablet (Bravecto™) or no treatment. Each pen contained the bottom half of a dog carrier lined with carpet as bedding. Before treatment, each dog was infested twice, (28 and 21 days pre-treatment) with 100 adult, unfed C. felis to establish a flea population prior to treatment on each dog. Flea media was added to the carpet four weeks before the treatment date and weekly thereafter for the remainder of the study to encourage development of an active, developing population of juvenile flea stages in each pen. On the treatment day, dogs in the treated group received fluralaner at a dose close to 25 mg/kg body weight by oral administration of one or more flavored chewable tablets. The chewable tablet(s) were administered by placement in the back of the oral cavity over the tongue to initiate swallowing. Dogs in the negative-control group remained untreated.
Flea counts were performed on all dogs 1 day before treatment, 1 day after treatment and then every 7 days until completion of the study 84 days later. All live fleas recovered were held and re-infested on the dog after the comb count was completed. Each dog was also infested with 50 newly emerged unfed adult fleas on days 22, 50 and 78 to simulate natural infestation post-treatment.
The individual dog was the experimental unit and data from each flea-count time point were analyzed separately. Flea-count data were transformed [Y = loge(x + 1)] and analyzed by a mixed linear model including treatment as the fixed effect and block as the random effect. Kenward-Rogers adjustment was used to determine the denominator degree of freedom. A two-tailed F-test was used within the mixed linear model for the comparison between treatment groups and statistical significance was declared when P ≤ 0.05. SAS version 9.3 was the primary software used for analysis.
Efficacy was calculated using arithmetic and geometric means with Abbott’s formula:
Efficacy (%) = 100 × (MC - MT)/MC, where MC was the arithmetic or geometric mean number of total adult live fleas on untreated dogs and MT the arithmetic or geometric mean number of total adult live fleas on treated dogs.
The study was conducted in California, USA in compliance with the Animal Welfare Act as overseen by the United States Department of Agriculture (USDA) and ethical approval was obtained before the start. The study was approved by the Institutional Animal Care and Use Committee (IACUC no. S11453-00).