All animal procedures in this study were reviewed and approved by the Merial Institutional Animal Care and Use Committee (IACUC). Dogs were managed consistent with

the US Animal Welfare Regulations (USDA, 2008). Thirty-two beagle dogs (16 males BAY 73-4506 and 16 females) were included in the study. The dogs were 8.1–9.3 weeks of age on Day 0 and weighed 2.25–4.00 kg on Day −2. Dogs had not been exposed to ectoparasiticides prior to treatment and were in good health. They were fed a commercial dry dog food ration calculated to maintain a healthy physical state and water was available ad libitum. Housing was in an environmentally controlled building. After allocation and until Day 20,

two dogs Stem Cell Compound Library of the same treatment group were co-housed. On Day 0 post dosing and from Day 21 to study end, dogs were housed individually and provided socialization time daily. A random block design was chosen for allocation. Dogs were weighed on Day −2 and were ranked by decreasing body weight within sex and blocks of four dogs each were formed. Within each block, dogs were allocated randomly to one of the four treatment groups (Table 1) using the procedure Plan in SAS® Version 9.1.3. The final soft chewable formulation of afoxolaner for Thiamine-diphosphate kinase oral administration was manufactured under current Good Manufacturing Practices at Merial Limited. Three sizes were used: 0.5 g (11.3 mg afoxolaner); 1.25 g (28.3 mg afoxolaner); and 3 g soft

chewables (68.0 mg afoxolaner). Dose rate calculations were based on the body weight obtained at the most recent physical examination. As the chews cannot be divided, the number of chews administered, made up of identical or various sizes, was the number closest to, but not less than, the total mg dose needed (e.g., 1.95 kg bodyweight × 18.9 mg/kg = 36.86 mg afoxolaner; one 0.5 g and one 1.25 g chew = 39.7 mg afoxolaner for a treatment group 3 dog (Table 1). Treatments were administered at one-month dose intervals at Days 0, 28 and 56, and then at 2-week dose intervals (at Days 84, 98 and 112). On each designated treatment day, control dogs were handled similarly to the treated dogs but were not dosed. Dogs in 1× group were treated with the required dose of afoxolaner chews provided in a single fraction. Due to the volume of chewable material to be provided, dogs in 3× and 5× groups received approximately half of their required dose (first fraction) initially, with the rest of the required dose (second fraction) administered approximately 3 h later.