1. Synergy Between Fipronil and Amitraz and Synergy Between ML-3,277,198 and Amitraz in a Rhipicephalus sanguineus Residual Contact Test J. B. Prullage 1, P. R. Timmons 2, W.G. Cawthorne 2 and L. P. Le Hir de Fallois 2. 1 Merial, Fulton, MO USA 2 SCYNEXIS Inc., Research Triangle Park, NC USA Abstract Amitraz, a formamidine acaricide, has been reported to act as a synergist of contact insecticides/acaricides in both agriculture and animal health. Based on this information, a laboratory contact test was conducted to determine if amitraz at 12.5 ppm could improve the contact potency of fipronil and another phenylpyrazole derivative, ML-3,277,198, on ticks. A controlled glass vial bioassay was used to assess the efficacy of fipronil alone, amitraz alone, fipronil plus amitraz, ML-3,277,198 alone and ML-3,277,198 plus amitraz on unfed adult Rhipicephalus sanguineus. Assessments of lethality were made at 6, 24 and 48 hours after the introduction of the ticks to the vials. No mortality was observed in the control treatment or in the amitraz alone treatment, so mortality corrections were not required. Results from this study gave synergistic EC50 ratios of 15, 157 and 56 at the three time points tested, respectively between fipronil alone and fipronil plus amitraz. A similar response was seen at the EC90 level. Comparable results were obtained with ML- 3,277,198 plus amitraz. These results indicate that fipronil and ML-3,277,198 were synergized by amitraz in this adult tick residual contact study fipronil and ML-3,277,198 were synergized by amitraz in this adult tick residual contact study. The addition of amitraz also provided a significant improvement in the speed of kill. Table 1: Tick Contact Test Results for Test 1 Experimental Procedure Test Container Table 2: Tick Contact Test Results for Test 2 Calculations Tick Contact Results for Fipronil Results and Discussion Conclusion Adult Rhipicephalus sanguineus, 18 days post adult emergence, were obtained from Ecto Services Inc. (Henderson, N.C., USA). Technical samples of fipronil and ML-3,277,198 were dissolved in acetone containing 0.019% TRITON and 0.4% DMSO v/v to achieve the desired concentration. Dosages were serially diluted using the same diluent. For treatments containing amitraz, technical amitraz was added to the formulation to achieve the desired concentration. A volume of 0.418 mL of the solution was used to treat 20 mL scintillation vials with 2.5 cm diameter. Vials were dried by rolling the uncapped vials horizontally on an unheated hot dog roller until the acetone had evaporated, resulting in coating of the walls of the vial with the experimental compound(s). Filter paper disks of 2.1 cm diameter were treated with 41 L of the same solution, and one was placed on the bottom of the vial and the other in the vial cap which contained a 3.2 mm perforation for air exchange and allowed to air dry. Testing was conducted with 2 replicates per dose. Vials were capped and held over night at 24°C and 95%RH after which 10 adult R. sanguineus were placed in each vial. The vials were held under the same conditions for the duration of the test. The number of live and dead ticks in each vial was visually determined after 6, 24 and 48hrs. Fipronil and ML-3,277,198 were tested at serially diluted dosages from 25ppm to 0.0016ppm. Preliminary studies had shown a significant enhancement in contact activity when amitraz was combined with fipronil at dosages in the range of 3.13ppm to 25ppm. Based on these results, amitraz was added to fipronil and ML-3,277,198 mixtures at 12.5ppm for these bioassays. Amitraz was tested alone at dosages of 25, 12.5 and 6.25ppm. This test was conducted in duplicate. TRITON is a registered trademark of Union Carbide Corporation in the United States of America and elsewhere No mortality was observed in the control treatments or in the amitraz 12.5ppm treatment; therefore mortality corrections were not required. All dosages and EC50 values are expressed as the ppm of the solution used to treat the test vials. Conversion of ppm to µg/cm 2 can be accomplished by dividing ppm/87.4. EC50/EC90 curves were calculated using the 4 Parameter Logistic Model or Sigmoidal Dose-Response Model formula [fit = (A+((B- A)/(1+((C/X)^D))))] using IDBS XLfit model 205. EC50 values were calculated for each series and EC90 values were calculated if 90% mortality was achieved. Synergistic ratios were calculated by dividing the EC50 or EC90 of the test compound alone by the EC50 or EC90 of the compound combined with amitraz. In this replicated test, the addition of amitraz at a rate of 12.5 ppm substantially increased the tick contact efficacy of both fipronil and ML-3,277,198. After 24hr exposure this increase in efficacy, as measured by synergistic ratio, was greater than 150-fold for fipronil and greater than 130-fold for ML-3,277,198. The addition of amitraz also provided a significant improvement in the speed of kill. Table 1 shows EC50 results for fipronil alone and in combination with amitraz at 12.5 ppm. Synergistic ratios of 14.7, 157 and 56 were seen, respectively, at the 6, 24 and 48 hour EC50 assessments in Test 1 indicating that the addition of amitraz to fipronil increased tick mortality rates up to 157-fold. Test 2 shows a similar increase in activity following addition of amitraz to fipronil at the EC50 and EC90 levels. Similar synergistic ratios were observed for ML-3,277,198 in Test 1. Values of 11, 138 and 135.6 were seen, respectively, at the 6, 24 and 48 hour EC50 assessments indicating that the addition of amitraz to ML-3,277,198 increased tick mortality rates up to 138-fold. Test 2 shows a similar increase in activity following addition of amitraz to ML-3,277,198 at the EC50 and EC90 levels. The 95% confidence intervals on the EC50 estimates for fipronil alone and in combination with amitraz at 12.5ppm and ML-3,277,198 alone and in combination with amitraz at 12.5 ppm did not overlap at any of the three observation times. This was also true for the confidence intervals on the EC90 estimates for fipronil alone and in combination with amitraz and for ML-3,288,198 alone and in combination with amitraz. The 6hr EC50 and EC90 values for fipronil combined with amitraz are similar to the 48hr values for fipronil alone, indicating that the addition of amitraz provides a significant improvement in the speed of kill. A similar increase in speed of kill was seen with ML-3,277,198 combined with amitraz. Fipronil with and without amitraz at 12.5ppm ML-3,277,198 with and without amitraz at 12.5ppm Tick Contact Results for ML-3,277,198