IS Insect Services GmbH Study No.: AT_CF_0119 Confidential report Report No.: AT_CF_0119_01 SOB / Ctenocephalides felis Date: 22 March 2019 - Final Report - Sponsor Athenion GmbH Am Borsigturm 100 13507 Berlin Germany Report No. AT_CF_0119_01 Study director Date Dr. H. Dautel Test manager Date Dr. K. Büchel Evaluation of the repellent efficacy of two formulations against cat fleas using the Static Object Bioassay IS Insect Services GmbH Study No.: AT_CF_0119 Confidential report Report No.: AT_CF_0119_01 SOB / Ctenocephalides felis Date: 22 March 2019 General information Sponsor Athenion GmbH Ms Kirstin Völz Am Borsigturm 100 13507 Berlin Germany Test institute IS Insect Services GmbH Motzener Str. 6 12277 Berlin Germany Study director Dr. Hans Dautel Test manager Dr. Kerstin Büchel Test location / Archiving IS Insect Services GmbH Selerweg 41 12169 Berlin Germany Test product Canina® PETVITAL® Novermin (Spot-on) Isopropanolic solution (8 % margosa extract, 15 % lauric acid (w/v)) Time schedule Receipt of products: 01 August 2018 (Spot-on) 19 December 2018 Start of tests: 26 February 2019 End of tests: 11 March 2019 Study report: No. AT_CF_0119_01 22 March 2019 IS Insect Services GmbH Study No.: AT_CF_0119 Confidential report Report No.: AT_CF_0119_01 SOB / Ctenocephalides felis Date: 22 March 2019 Table of contents 1. Abstract ...................................................................................................................... 1 2. Material and methods ................................................................................................. 2 2.1 Test method ........................................................................................................ 2 2.2 Test organism ..................................................................................................... 2 2.3 Test products ...................................................................................................... 3 2.4 Test procedure: Static Object Bioassay ............................................................... 3 2.5 General conditions .............................................................................................. 4 2.6 Number of fleas tested ........................................................................................ 4 2.7 Test product application ...................................................................................... 5 2.8 Test procedure .................................................................................................... 5 2.9 Evaluation of flea mortality .................................................................................. 6 2.10 Data analysis....................................................................................................... 6 2.10.1 Relative frequencies of behavioural steps .................................................... 6 2.10.2 Time parameters .......................................................................................... 7 3. Results ....................................................................................................................... 7 3.1 Canina® PETVITAL® Novermin............................................................................ 7 3.1.1 Overall repellency ........................................................................................ 7 3.1.2 Relative frequencies of behavioural steps .................................................... 7 3.1.3 Time parameters .......................................................................................... 8 3.1.4 Flea mortality ............................................................................................... 9 3.2 Isopropanolic solution (8 % margosa extract, 15 % lauric acid (w/v)) ................ 10 3.2.1 Overall repellency ...................................................................................... 10 3.2.2 Relative frequencies of behavioural steps .................................................. 10 3.2.3 Time parameters ........................................................................................ 11 3.2.4 Flea mortality ............................................................................................. 12 4. Discussion ................................................................................................................ 13 5. Literature .................................................................................................................. 13 6. Appendix .................................................................................................................. 14 6.1 Test product preparation and application details ............................................... 14 6.2 Sequence of tests ............................................................................................. 14 6.3 Raw data ........................................................................................................... 18 1 1. Abstract The repellent effect of the test products Canina® PETVITAL® Novermin (Spot-on) and isopropanolic solution (8 % margosa extract, 15 % lauric acid (w/v)) was investigated using an in-vitro simulated-use assay called a Static Object Bioassay (SOB). Both test products were investigated for short-term efficacy after spray application. According to the European “Guidance on the Biocidal Products Regulation (BPR) Volume II Efficacy - Assessment and Evaluation (Parts B&C); version 3.0, April 2018; ECHA”. According to this guidance, a sufficient level of efficacy is ≥ 90% repellence during the claimed efficacy period. Significant contact repellency could be determined for both test products, reflected by significantly reduced contact times the fleas spent on the fabrics in test runs compared to control runs. The relative (to the control) repellent efficacy of both test products was 100 %. Flea mortality 24 h after the test runs was below 23.3 % without significant difference between control and test runs, suggesting lack of insecticidal properties of the test products. However, Canina® PETVITAL® Novermin and isopropanolic solution (8 % margosa extract, 15 % lauric acid (w/v)) met the product efficacy requirements of the BPR guidance of at least 90 % repellency. 2 2. Material and methods 2.1 Test method The in-vitro simulated-use assay SOB “static object bioassay” is highly standardized and yields reproducible, reliable results. The trial will consider the provisions of the latest version of the European “Guidance on the Biocidal Products Regulation (BPR) Volume II Efficacy - Assessment and Evaluation (Parts B&C); version 3.0, April 2018; ECHA”. According to this guidance, a sufficient level of efficacy is ≥ 90 % repellence during the claimed efficacy period. 2.2 Test organism The Ctenocephalides felis (cat fleas) pupae were obtained from an external colony. In this colony adults were fed on artificial membranes with bovine blood and larvae were reared on a standard diet for larvae (consisting of sand, blood meal, yeast, dog biscuits). For further development of the cat flea pupae at the facilities of IS Insect Services GmbH the pupae were kept inside polystyrene vessels (13 x 9.5 x 6.5 cm (l x w x h); Modulor GmbH, Berlin, Germany) with gauze covers and stored inside desiccators at 18:6 light:dark cycles at 25 °C and 76 % RH. The humidity inside the desiccators was provided by means of a saturated solution of NaCl according to Winston & Bates (1960). Eclosed adults (Fig. 1) had been intermittently stored inside glass tubes with gauze covers at 14 °C, 76 % RH and darkness for 1 - 2.5 weeks until use for the bioassay. Figure 1: Vital adult cat flea. Cat flea adults used in the bioassay were unfed and between 8-18 days old. They were separated into containers (half of the containers with males and the other half with females) and acclimated at 20 °C and 76 % RH for a minimum of two hours before testing. Each cat flea was tested only once. 3 2.3 Test products Canina® PETVITAL® Novermin (Spot-on; active ingredients: 2.5 % margosa extract, 2.5 % lauric acid (w/w)) and the pure substances margosa extract and lauric acid were supplied by the sponsor (Athenion GmbH, Berlin, Germany). The isopropanolic solution was prepared by IS Insect Services GmbH: 4 g margosa extract and 7.5 g lauric acid were solved in 2-propanol (99.5 %, Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and the volume was brought up to 50 ml. Canina® PETVITAL® Novermin was stored at room conditions and the isopropanolic solution (8 % margosa + 15 % lauric acid (w/v)) at 4 °C at the test location of the IS Insect Services GmbH until tests started. The isopropanolic solution was equilibrated to room temperature to solve all substances before application. The containers of both products were thoroughly shaken before product aliquots were transferred into glass tube atomizers for spray application. 2.4 Test procedure: Static Object Bioassay Fleas were placed on the bottom of a test arena (glass 34.5 cm Ø, 25 cm height) were they could approach and jump on a warmed “artificial animal leg”. This “artificial leg” consists of a glass cylinder (Ø 3 cm, height 25.5 cm, 100 ml, Carl Roth GmbH + Co. KG, Karlsruhe, Germany) which is covered by a wool fabric of 0.5 cm height and an area of 49.59 ± 0.55 mg/cm3 (± standard deviation (SD)) (=loden cloth, Stoff-Ideen KMR GmbH, Berlin, Germany) using double face adhesive tape and hot adhesion glue. The glass cylinder is filled with preheated bovine blood / phosphate buffer mix (half/half) and evaporated blood scent (emitting host attractive scent). The fabric is marked with a 10 cm- and 20 cm-zone and the surface temperature of the cylinder is kept in mean at 36 °C (similar to the external body temperature) and measured remotely by an infrared thermometer (PCE_MF1 InfraRed Thermometer, PCE Deutschland GmbH, Meschede, Germany) (Fig. 2). The repellency of the test products was evaluated by the time and distance fleas stayed or walked on the treated surface. If the fabric was treated with test product, the repellent effect could be detected either by (i) a reduced number of fleas climbing on the “leg” or (ii) an increased number of fleas falling off the “leg” compared to the control runs. On the warmed surface the fleas showed the same behavior as in nature when they enter the host and searching for a position to bite. It was thus possible to discriminate between contact repellents and substances acting over short distance. Repelled fleas were defined by the number of fleas not climbing to the fabric or dropping off the fabric. 4 Fig. 2: Test method SOB “static object bioassay”: An “artificial leg” consisting of a glass cylinder covered by wool is used to evaluate the repellency by the time and distance fleas stayed or walked on the treated fabric. The glass cylinder was filled with bovine blood / phosphate buffer mix (half/half) kept at 36 °C. 2.5 General conditions A data logger (Testostor 1754H, Testo SE & Co. KGaA, Lenzkirch, Germany) recorded temperature and relative humidity (RH) within the test room. Mean temperature and RH (± SD) were 22.6 ± 0.5 °C and 39.4 ± 2.4 % RH (8 % Margosa + 15 % Lauric acid) or 21.4 ± 0.6 °C and 38.3 ± 5.1 % RH (Canina® PETVITAL® Novermin) respectively. The surface temperature of the drum attachment site was periodically measured remotely with an infrared thermometer. The mean temperature (± SD) of the fabric surface (measured at 1 cm distance) across all tests was 36.3 ± 1.7 °C (corresponds to the body surface temperature). 2.6 Number of fleas tested The test was performed using 30 C. felis adults for controls and test products, respectively. Prior to testing the repellent treated fabric, a control run with 30 fleas was performed using untreated fabric. This control served to prove sufficient activity of the fleas and to exclude any repellent efficacy caused through the fabric. If less than 70 % of the fleas would have clung to the fabric surface from the bottom of the arena in the control run, any subsequent 5 test that day would have been abandoned. The total number of fleas tested for the test products and the controls was 120. 2.7 Test product application The target amount of solution sprayed was 408.87µl corresponding to 1.77 µl/cm2 for Canina® PETVITAL® Novermin and 1155 µl corresponding to 5 µl / cm2 for 8 % Margosa + 15 % Lauric acid (see Appendix, Table A 1 for detailed application data). The test products were sprayed onto the loden fabric using separate 12 ml glass tube atomizers (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) for each test product and the volume applied was determined by weighing the tubes before and after application. The control was sprayed with the same amount of water (as control for the Spon-On) or isopropanol (2-propanol 99.5 % as control of the isopropanolic solution). After a drying period of 30 min at room temperature the piece of fabric was fixed with double-sided tape and hot adhesion clue on the surface of the glass cylinder before this was subsequently used in the test run. After application, the treated loden fabric dried under a fume hood at room temperature for 30-50 min (see Appendix, Table A 2 for detailed drying times) before they were used in the SOB test runs. 2.8 Test procedure Each test run involves testing of 30 fleas, one after each other, females and males separated. Fleas could acclimate to room conditions for at least 2 hours and only active fleas jumping within the vessel were used for the test. Then a single individuum was transferred into the bottom of the glass arena, on the filter paper with help of a vessel at a distance of 3-7 cm to the “artificial leg”. The test time started as soon as the flea reached the bottom of the arena. For each flea, the durations of certain sequential behaviours were recorded using the software package “The Observer® XT 12” (Noldus Information Technology, Wageningen, The Netherlands). The durations of the following behavioural steps of the fleas were recorded: a) climbing to the fabric b) walking (or jump) >10 cm on the fabric c) walking >20 cm on the fabric d) dropping off the fabric <20 cm (within 120s) The maximal observation time slot of an individual flea was 4 minutes, in detail ≤120s for step a) and ≤120s for the steps b)-d). The observation time slot of an individual flea was 4 minutes at maximum. For technical repetition the fabric was changed after every 10th flea and replaced by a new one (new sprayed fabric). Used mess cylinders were thoroughly cleaned after each technical replication with hot water and detergent, then rinsed with ethanol and rubbed with cleaning paper soaked with acetone. Finally, they were rinsed with ethanol, followed by water and dried. The glass arena was treated in the same way. 2.9 Evaluation of flea mortality After the test performance the fleas were housed in plastic centrifuge tubes (polypropylene, transparent, 50 ml, Carl Roth GmbH + Co. KG, Karlsruhe, Germany) (each replicate with 10 fleas together) with perforated cups covered by gauze to allow for gas exchange and kept at 20.3 ± 0.7 °C at 78.7 ± 2.2 % RH. The mortality was determined after 24 h. 2.10 Data analysis Frequency data such as the number of fleas repelled versus not repelled are analysed by Fischer’s exact test. Metric data such as the times the fleas spent on test fabric versus control fabric are analysed by one-way ANOVA followed by Tukey’s HSD test for unequal sample sizes. The number of dead individuals after 24 h in the test and control runs are statistically analyzed by the Fisher's exact test. The overall repellency relative to the control was calculated for each test product. All fleas in a given test that (i) did not walk towards the “artificial leg”, (ii) did not change to the “artificial leg” or (iii) dropped off the “artificial leg” were regarded as repelled. The repellency R relative to the control was calculated according to the following formula: 𝑅𝑅=100−𝑝𝑝𝑡𝑡 𝑝𝑝𝑐𝑐∗100 where p t is the percentage of fleas not repelled in the test, and pc is the percentage of fleas not repelled in the control. 2.10.1 Relative frequencies of behavioural steps For each behavioural step of the fleas (climbing to the fabric, walking (or jump) >10 cm on the fabric, walking >20 cm on the fabric, dropping off the fabric) the number of individuals that showed the respective behaviour (see Appendix, Table A 7, raw data) and the number of fleas that did not were compared with the corresponding number of fleas in the corresponding control run with Fisher’s exact tests using the software Statistica®, version 7 10 (StatSoft Europe GmbH, Hamburg, Germany) or R 3.4.3 (R Core Team 2017). As multiple comparisons were made, increasing the likelihood of false positives, the conservative Bonferroni correction was applied. 2.10.2 Time parameters The time parameters of flea behavioural steps in the test runs were compared to the repective results of the control runs by Mann–Whitney U tests. All such tests were performed with the software package Statistica® version 10. 3. Results 3.1 Canina® PETVITAL® Novermin 3.1.1 Overall repellency Relative repellency determined for Canina® PETVITAL® Novermin was 100 %. The control repellency was 3.3 %. Therefore, repellency of the test product was significantly higher compared to controls (Table 1). Table 1: Repellency and relative repellency (repellency corrected for the control) of the test product Canina® PETVITAL® Novermin against C. felis in the SOB. Significanct differences according to Fisher’s exact tests between test and control runs are indicated. ***: P < 0.001. N fleas = 30. Test product Repellency (%) Relative repellency (%) Significance Canina® PETVITAL® Novermin 100.0 100.0 *** Control 3.3 - - 3.1.2 Relative frequencies of behavioural steps Figure 3 shows the behavioural steps of fleas observed in the SOB using Canina® PETVITAL® Novermin. During both test and control runs all 30 fleas jumped onto the test surface. During controls one flea dropped off the surface while during tests with Canina® PETVITAL® Novermin all of the fleas dropped off. The difference was highly significant. During controls 23 fleas crossed the 10 cm marking, but only 3 during test runs, which also was highly significant (see Appendix, Table A 3). During controls two fleas were able to climb the whole “artificial leg” and cross the 20 cm marking. 8 Figure 3: Number of C. felis displaying certain behavioural steps in the SOB with the test product Canina® PETVITAL® Raumspray. The numbers of fleas jumping on the artificial leg, walking (or jumping) >10 cm on the fabric, walking >20 cm on the fabric, or dropping off the fabric were recorded. All fleas in a given test that did not (i) jump on the artificial leg or (ii) dropped off the artificial leg were regarded as repelled. N = 30 fleas. 3.1.3 Time parameters The different time parameters of the behavioural steps were analysed by Mann–Whitney U tests. Table 2 lists the time the fleas needed to jump onto the “artificial leg”, the time they spent on it, and the time needed to cross the 10 cm marking. The mean time to jump onto the test surface (± SE) during test runs with Canina® PETVITAL® Novermin was 6.7 ± 3.2 s. The mean time during controls was 5.4 ± 2.9 s. The difference was not significant. The mean time the fleas spent on the test surface was 17.0 ± 2.7 s during test runs and 117.5 ± 1.7 s during controls, with the difference being highly significant. For those fleas that crossed the 10 cm marking the mean time to accomplish this (after jumping onto the test surface) was 4.9 ± 1.1 s during test runs and 39.3 ± 7.3 s during controls. The result was not significant. 30 302332 1301300 10 20 30 40 50 60 70 80 90 100Control Canina® PETVITAL® NoverminNumber of fleasBehaviour of C. felis in the SOBFleas repelledDropping of the artificial leg Crossing 20 cm marking Crossing 10 cm marking Jump on artificial leg 9 Table 2: Time (s) spent by C. felis for different behavioural parts in the test and control runs. ± 95 %: 95 % confidence interval. N: Number of fleas that showed the corresponding behaviour. SD: Standard deviation. SE: Standard error. Min and Max: Minimum and maximum. Sig: Significant differences between mean times in the control and test product according to Mann–Whitney U tests. ***: P < 0.001. ns: not significant. Measurement/ Treatment Mean - 95 % + 95 % N SD SE Min Max Sig Time to jump on the “artificial leg” Control 5.4 -0.5 11.4 30 16.0 2.9 0.2 80.7 - Canina® PETVITAL® Novermin 6.7 0.0 13.3 29* 17.4 3.2 0.2 72.8 ns Time on the “artificial leg” Control 117.5 114.1 120.8 30 9.0 1.7 74.1 120.0 - Canina® PETVITAL® Novermin 17.0 11.4 22.5 30 14.8 2.7 3.7 64.3 *** Time to cross 10 cm marking Control 39.3 24.2 54.4 23 34.9 7.3 0.4 114.9 - Canina® PETVITAL® Novermin 4.9 0.4 9.4 3 1.8 1.1 3.8 7.0 ns *Time recording of one flea missing 3.1.4 Flea mortality Flea mortality was evaluated after 24 h for the test and corresponding control run. In test runs with Canina® PETVITAL® Novermin flea mortality was 23.3 % (see Appendix, Table A 4), in the control run no mortality of the fleas was observed. The relative mortality equaled the absolute mortality, which was significantly higher in the test runs compared to control runs (Table 3). Table 3: Flea mortality 24 h after test runs with Canina® PETVITAL® Novermin. Relative mortality: Flea mortality corrected for the mortality in the control runs. Significances concerning the numbers of dead and living fleas between test and control runs are indicated according to Fisher’s exact tests (two-sided). *: P < 0.05. Nfleas = 30. Test product Mortality (%) Relative mortality (%) Significance Canina® PETVITAL® Novermin 23.3 % 23.3 % * Control 0.0 % - - 10 3.2 Isopropanolic solution (8 % margosa extract, 15 % lauric acid (w/v)) 3.2.1 Overall repellency Repellency and the relative repellency determined for 8 % Margosa + 15 % Lauric acid (w/v) was 100 % because the repellency in the control was 0 %. Repellency of the test product was significantly higher compared to the control (Table 4). Table 4: Repellency and relative repellency (repellency corrected for the control) of the test solution 8 % Margosa + 15 % Lauric acid (w/v) against C. felis in the SOB. Significant differences according to Fisher’s exact tests between test and control runs are indicated. ***: P < 0.001. Nfleas = 30. Test product Repellency (%) Relative repellency (%) Significance 8 % Margosa + 15 % Lauric acid (w/v) 100.0 100.0 *** Control 0.0 - - 3.2.2 Relative frequencies of behavioural steps Figure 4 shows the behavioural steps of fleas observed in the SOB using 8 % Margosa + 15 % Lauric acid (w/v). During both test and control runs all 30 fleas jumped onto the test surface, but during test runs all fleas dropped off the test surface while none of the fleas dropped off during controls. This result was highly significant. Also, less fleas crossed the 10 cm marking during test runs compared to controls (10 versus 18 fleas respecively), but this difference turned out to be not significant (see Appendix, Table A 5). Only during control runs 4 fleas managed to climb the whole artificial leg and cross the 20 cm marking. 10 3.2 Isopropanolic solution (8 % margosa extract, 15 % lauric acid (w/v)) 3.2.1 Overall repellency Repellency and the relative repellency determined for 8 % Margosa + 15 % Lauric acid (w/v) was 100 % because the repellency in the control was 0 %. Repellency of the test product was significantly higher compared to the control (Table 4). Table 4: Repellency and relative repellency (repellency corrected for the control) of the test solution 8 % Margosa + 15 % Lauric acid (w/v) against C. felis in the SOB. Significant differences according to Fisher’s exact tests between test and control runs are indicated. ***: P < 0.001. Nfleas = 30. Test product Repellency (%) Relative repellency (%) Significance 8 % Margosa + 15 % Lauric acid (w/v) 100.0 100.0 *** Control 0.0 - - 3.2.2 Relative frequencies of behavioural steps Figure 4 shows the behavioural steps of fleas observed in the SOB using 8 % Margosa + 15 % Lauric acid (w/v). During both test and control runs all 30 fleas jumped onto the test surface, but during test runs all fleas dropped off the test surface while none of the fleas dropped off during controls. This result was highly significant. Also, less fleas crossed the 10 cm marking during test runs compared to controls (10 versus 18 fleas respecively), but this difference turned out to be not significant (see Appendix, Table A 5). Only during control runs 4 fleas managed to climb the whole artificial leg and cross the 20 cm marking. 11 Figure 4: Number of C. felis displaying certain behavioural steps in the SOB with the test solution 8 % Margosa + 15 % Lauric acid (w/v). The numbers of fleas jumping on the artificial leg, walking (or jumping) >10 cm on the fabric, walking >20 cm on the fabric, or dropping off the fabric were recorded. All fleas in a given test that did not (i) jump on the artificial leg or (ii) dropped off the artificial leg were regarded as repelled. N = 30 fleas. 3.2.3 Time parameters The different time parameters of the behavioural steps were analysed by Mann–Whitney U tests. Table 5 lists the time the fleas needed to jump onto the “artificial leg”, the time they spent on it, and the time needed to cross the 10 cm marking. The mean time to jump onto the test surface (± SE) during test runs with 8 % Margosa + 15 % Lauric acid was 3.7 ± 1.9 s. The mean time during controls was 4.8 ± 1.9 s. The difference was not significant. The mean time on the test surface (± SE) during test runs with the solution was 15.5 ± 1.6 s and during controls 114.0 ± 3.1 s, with the difference being highly significant. For those fleas that crossed the 10 cm marking the mean time (± SE) to accomplish this (after jumping onto the test surface) was 6.5 ± 1.5 s during test runs and 37.9 ± 8.4 s during controls. The result was significant. It should be noted that the shortened time needed to 30 30181043030020406080100Control 15 % Margosa + 8 % Lauric acid (w/v)Number of fleasBehaviour of C. felis in the SOBFleas repelledDropping of the artificial leg Crossing 20 cm marking Crossing 10 cm marking Jump on artificial leg 12 cross the 10 cm marking during test runs compared to controls does not imply different speeds in movement. During test runs only those fleas managed to cross the 10 cmmarking (before dropping off), which initially jumped quite high onto the “artificial leg” and hence started very close to the marking (reached fast the 10 cm) and then dropped off. Table 5: Time (s) spent by C. felis for different behavioural parts in the test and control runs. ± 95 %: 95 % confidence interval. N: Number of fleas that showed the corresponding behaviour. SD: Standard deviation. SE: Standard error. Min and Max: Minimum and maximum. Sig: Significance between mean times in the control and test product according to Mann– Whitney U tests. ***: P < 0.001. ns: not significant. Measurement/ Treatment Mean - 95 % + 95 % N SD SE Min Max Sig Time to jump on the “artificial leg” Control 4.8 1.0 8.6 30 10.3 1.9 0.3 48.9 - 8 % Margosa + 15 % Lauric acid (w/v) 3.7 -0.2 7.6 29* 10.3 1.9 0.2 53.0 ns Time on the “artificial leg” Control 114.0 107.8 120.3 30 16.7 3.1 57.4 120.0 - 8 % Margosa + 15 % Lauric acid (w/v) 15.5 11.1 17.8 30 9.0 1.6 2.4 45.7 *** Time to cross 10 cm marking Control 37.9 20.1 55.7 18 35.7 8.4 0.2 111.6 - 8 % Margosa + 15 % Lauric acid (w/v) 6.5 3.1 9.9 10 4.8 1.5 1.6 15.9 * * Time recording of one flea missing 3.2.4 Flea mortality In test runs with 8 % Margosa + 15 % Lauric acid (w/v) mortality of the fleas was 6.7 % (see Appendix, Table A 6). Table 6: Flea mortality 24 h after test runs with the test solution 8 % Margosa + 15 % Lauric acid (w/v). Relative mortality: Flea mortality corrected for the mortality in the control runs. Significances concerning the numbers of dead and living fleas between test and control runs are indicated according to Fisher’s exact tests (two-sided). ns: not significant. Nfleas = 30. Test product Mortality (%) Relative mortality (%) Significance 8 % Margosa + 15 % Lauric acid (w/v) 6.7 % -21.7 % ns Control 23.3 % - - 13 Relative mortality was negative due to more dead fleas in the corresponding controls, but the differences between test and control runs were not significant (Table 6). 4. Discussion The repellent effect of the test products Canina® PETVITAL® Novermin (Spot-on) and isopropanolic solution (8 % margosa extract, 15 % lauric acid (w/v)) was investigated using the Static Object Bioassay. Both were tested for short term repellent efficacy a short drying time after application. The relative repellent efficacy (corrected to the control) of Canina® PETVITAL® Novermin and the isopropanolic solution was 100 %. Regarding the more detailed behaviour of the fleas, significant repellent effects could be determined for both test products. The repellency of the Spot-on and the isopropanolic solution is expressed as contact repellency evident by a significantly increased number of fleas dropping off the impregnated fabric on the “artificial leg” compared to the corresponding controls. Further the contact repellent effect was reflected by significantly reduced times the fleas spent on the fabric in test runs with both products compared to control runs. Flea mortality 24 h after the both test products was below 23.3 % without a significant difference between control and test runs, suggesting lack of insecticidial properties of the two test products. Hence, Canina® PETVITAL® Novermin and isopropanolic solution (8 % margosa extract, 15 % lauric acid (w/v)) met the product efficacy requirements of the BPR guidance of at least 90 % repellency. 5. Literature European Chemicals Agency (2018). Guidance on the Biocidal Products Regulation. Volume II: Efficacy assessment and evaluation. Parts B & C. Draft version 3.0. April 2018. R Core Team (2017). R: A language and environment for statistical computing. R foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/. Winston P. W. and Bates D. H. (1960). Saturated solutions for the control of humidity in biological research. Ecology 41 (1). 232-237. 14 6. Appendix 6.1 Test product preparation and application details Table A 1: Amounts (means ± SD) of the test product Canina® PETVITAL® Novermin (density: 0.8559 g/cm3) and 8 % Margosa + 15 % Lauric acid (w/v) in 2-propanol applied on the fabric (area 231 cm2) used in the SOB. Nfabric = 3. Test product Application (µl) Application (µl/cm2) Lauric acid (µg/cm2) Margosa (µg/cm2) Canina® PETVITAL® Novermin 421.0 ± 49.5 1.82 ± 0.21 39.0 ± 4.6* 39.0 ± 4.6* 8 % Margosa + 15 % Lauric acid (w/v) 1142.8 ± 9.2 4.95 ± 0.04 742.1 ± 6.0 395.8 ± 3.2 * based on calculation with density 6.2 Sequence of tests Table A 2: Time schedule and drying times of the fabrics impregnated with the two test products and the fabrics sprayed with water or 2-propanol (control). Test product Drying time until test start Date of test Canina® PETVITAL® Novermin 50 min 26 Februar 2019 Control (water) 30 min 26 Februar 2019 Canina® PETVITAL® Novermin 41 min 27 Februar 2019 Control (water) 30 min 27 Februar 2019 Canina® PETVITAL® Novermin 39 min 4 March 2019 Control (water) 35 min 4 March 2019 8 % Margosa + 15 % Lauric acid (w/v) 41 min 06 March 2019 Control (2-propanol) 40 min 06 March 2019 8 % Margosa + 15 % Lauric acid (w/v) 41 min 11 March 2019 Control (2-propanol) 38 min 11 March 2019 8 % Margosa + 15 % Lauric acid (w/v) 36 min 11 March 2019 Control (2-propanol) 30 min 11 March 2019 15 Table A 3. Number of C. felis displaying certain behavioural steps in the SOB with the test product Canina® PETVITAL® Novermin. The first two rows of each behavioural step show the number of fleas. The last row indicates the respective results of Bonferroni corrected Fisher’s exact tests between the number of fleas showing these behaviours and the control. Relative repellency: Repellency corrected for the control. ***: P < 0.001; ns: not significant. Product Control Canina® PETVITAL® Novermin Jump on artificial leg 30 30 Not jumping 0 0 % Jumping 100.0 100.0 Significance - ns Crossing 10 cm marking 23 3 Not crossing 7 27 % Crossing 76.7 10.0 Significance - *** Crossing 20 cm marking 2 0 Not crossing 29 30 % Crossing 6.7 0.0 Significance - ns Dropping off the artificial leg 1 30 Not dropping off 29 0 % Dropping off 3.3 100.0 Significance - *** Summary: Fleas repelled 1 30 Fleas not repelled 29 0 % Absolute repellency 3.3 100.0 % Relative repellency - 100.0 Significance - *** 16 Table A 4: Mortality raw data of adult C. felis used in the SOB runs with the test product Canina® PETVITAL® Novermin and the respective controls. Condition of fleas Test product Replicates alive moribund dead Canina® PETVITAL® Novermin #1 9 0 1 Control #1 10 0 0 Canina® PETVITAL® Novermin #2 7 0 3 Control #2 10 0 0 Canina® PETVITAL® Novermin #3 7 0 0 Control #3 10 0 0 Table A 5. Number of C. felis displaying certain behavioural steps in the SOB with the test solution 8 % Margosa + 15 % Lauric acid (w/v). The first two rows of each behavioural step show the number of fleas. The last row indicates the respective results of Bonferroni corrected Fisher’s exact tests between the number of fleas showing these behaviours and the control. Relative repellency: Repellency corrected for the control. ***: P < 0.001; ns: not significant. Product Control 8 % Margosa + 15 % Lauric acid (w/v) Jump on artificial leg 30 30 Not jumping 0 0 % Jumping 100.0 100.0 Significance - ns Crossing 10 cm marking 18 10 Not crossing 12 20 % Crossing 60.0 33.3 Significance - ns Crossing 20 cm marking 4 0 Not crossing 26 30 % Crossing 13.3 0.0 Significance - ns Dropping off the artificial leg 0 30 Not dropping off 30 0 % Dropping off 0.0 100.0 Significance - *** 17 Table A 5 continued Summary: Fleas repelled 0 30 Fleas not repelled 30 0 % Absolute repellency 0.0 100.0 % Relative repellency - 100.0 Significance - *** Table A 6: Mortality raw data of adult C. felis used in the SOB runs with the test solution 8 % Margosa + 15 % Lauric acic and the respective control runs. Condition of fleas Test product Replicates alive moribund dead 8 % Margosa + 15 % Lauric acid (w/v) #1 10 0 0 Control #1 7 0 3 8 % Margosa + 15 % Lauric acid (w/v) #2 10 0 0 Control #2 8 0 2 8 % Margosa + 15 % Lauric acid (w/v) #3 8 0 0 Control #3 8 0 2 18 6.3 Raw data The raw data are shown in Table A 7. The columns below “duration of flea behaviour” show the time intervals in seconds for the different behavioural steps of fleas. These were: • Column “Jump on leg”: The time the flea took to jump on the artificial leg after being placed in the test arena. • Column “On leg”: The time the flea spent on the artificial leg. • Column “10 cm”: The time the flea took to cross the 10 cm marking on the artificial leg. Events in the columns below “Flea behavioural events” are coded as follows: • Column “Repelled”: o 0 = the flea did not fall off. o 4 = the flea fell off from the artificial leg. • Column “Not repelled”: o 0 = the flea did not jump on the artificial leg. o 1 = the flea remained on the artificial leg for >120 s. o 2 = the flea left the artificial leg upwards. Table A 7: Raw data of SOB tests with C. felis and the test products Canina® PETVITAL® Novermin (N), and the test solution 8 % Margosa + 15 % Lauric acid (ML). Untreated fabric served as control (C). F: female; M: male. For further information, see text above. Flea no. Sex Duration of flea behaviour [s] Flea behavioural events Test product Date Jump on leg On leg 10 cm 20 cm Repelled (falling off) Not repelled 1 M 1.72 120.00 29.69 0 1 C1 26.02.2019 2 F 0.24 105.80 5.58 105.80 0 2 C1 26.02.2019 3 M 0.24 120.00 42.52 0 1 C1 26.02.2019 4 F 0.53 120.00 22.61 0 1 C1 26.02.2019 5 M 37.98 120.00 114.88 0 1 C1 26.02.2019 6 F 4.27 120.00 1.39 0 1 C1 26.02.2019 7 M 0.23 120.00 73.31 0 1 C1 26.02.2019 8 F 0.81 120.00 53.59 0 1 C1 26.02.2019 9 M 0.20 120.00 65.29 0 1 C1 26.02.2019 10 F 0.59 74.07 0.44 4 0 C1 26.02.2019 11 M 0.31 120.00 3.42 0 1 C1 27.02.2019 12 F 0.26 120.00 92.61 0 1 C1 27.02.2019 13 M 0.95 120.00 1.45 0 1 C1 27.02.2019 14 F 0.89 120.00 89.36 0 1 C1 27.02.2019 15 M 0.23 120.00 40.19 0 1 C1 27.02.2019 16 F 0.21 120.00 23.59 0 1 C1 27.02.2019 17 M 10.84 120.00 55.32 0 1 C1 27.02.2019 18 F 1.03 120.00 0 1 C1 27.02.2019 19 M 80.75 120.00 0 1 C1 27.02.2019 20 F 0.28 104.04 1.01 104..04 0 2 C1 27.02.2019 21 F 1.21 120.00 0 1 C1 04.03.2019 22 M 0.22 120.00 29.92 0 1 C1 04.03.2019 19 Flea no. Sex Duration of flea behaviour [s] Flea behavioural events Test product Date Jump on leg On leg 10 cm 20 cm Repelled (falling off) Not repelled 23 F 1.59 120.00 0 1 C1 04.03.2019 24 M 0.44 120.00 85.12 0 1 C1 04.03.2019 25 F 0.94 120.00 0 1 C1 04.03.2019 26 M 0.80 120.00 2.72 0 1 C1 04.03.2019 27 F 0.16 120.00 12.50 0 1 C1 04.03.2019 28 M 1.28 120.00 0 1 C1 04.03.2019 29 F 13.67 120.00 57.09 0 1 C1 04.03.2019 30 M 0.57 120.00 0 1 C1 04.03.2019 31 M 0.24 20.71 7.02 4 0 N 26.02.2019 32 F 1.90 20.88 4 0 N 26.02.2019 33 M 0.99 8.58 4 0 N 26.02.2019 34 F 0.22 15.63 4 0 N 26.02.2019 35 M 7.74 17.24 4 0 N 26.02.2019 36 F 1.36 30.09 4 0 N 26.02.2019 37 M 0.20 64.32 4 0 N 26.02.2019 38 F 0.19 12.95 4 0 N 26.02.2019 39 M 64.69 10.20 4 0 N 26.02.2019 40 F 0.28 23.55 4 0 N 26.02.2019 41 M 0.85 9.38 4 0 N 27.02.2019 42 F 0.53 63.81 4 0 N 27.02.2019 43 M 9.01 11.78 4 0 N 27.02.2019 44 F 2.91 14.14 4 0 N 27.02.2019 45 M 72.80 6.08 3.83 4 0 N 27.02.2019 46 F 10.99 5.32 4 0 N 27.02.2019 47 M 0.22 10.94 4 0 N 27.02.2019 48 F 0.74 12.27 4 0 N 27.02.2019 49 M 3.44 26.44 4 0 N 27.02.2019 50 F 30.99 3.87 4 0 N 27.02.2019 51 F 1.36 19.50 4 0 N 04.03.2019 52 M 0.35 11.51 4 0 N 04.03.2019 53 F 1.20 5.03 4 0 N 04.03.2019 54 M 0.91 6.38 4 0 N 04.03.2019 55 F 1.77 7.63 4 0 N 04.03.2019 56 M 0.28 7.42 4 0 N 04.03.2019 57 F 0.27 3.72 4 0 N 04.03.2019 58 M 1.72 7.53 4 0 N 04.03.2019 59 F 5.39 6.38 4 0 N 04.03.2019 60 M 0.96 18.23 4 0 N 04.03.2019 61 F 32.92 120.00 0 1 C2 06.03.2019 62 M 3.16 120.00 14.48 0 1 C2 06.03.2019 63 F 0.89 120.00 0 1 C2 06.03.2019 64 M 1.35 120.00 111.57 0 1 C2 06.03.2019 65 F 5.87 120.00 70.19 0 1 C2 06.03.2019 66 M 0.84 120.00 0 1 C2 06.03.2019 67 F 0.42 120.00 17.47 0 1 C2 06.03.2019 68 M 1.86 120.00 34.31 0 1 C2 06.03.2019 69 F 7.54 120.00 0 1 C2 06.03.2019 70 M 1.60 120.00 5.51 0 1 C2 06.03.2019 71 M 0.41 120.00 100.89 0 1 C2 11.03.2019 72 F 0.49 120.00 0 1 C2 11.03.2019 73 M 1.06 120.00 0 1 C2 11.03.2019 74 F 2.20 120.00 10.52 0 1 C2 11.03.2019 75 M 0.48 120.00 0 1 C2 11.03.2019 76 F 0.49 120.00 0 1 C2 11.03.2019 77 M 1.65 65.54 1.59 65.54 0 2 C2 11.03.2019 20 Flea no. Sex Duration of flea behaviour [s] Flea behavioural events Test product Date Jump on leg On leg 10 cm 20 cm Repelled (falling off) Not repelled 78 F 0.56 120.00 59.69 0 1 C2 11.03.2019 79 M 8.01 120.00 0 1 C2 11.03.2019 80 F 0.64 57.40 1.07 57.40 0 2 C2 11.03.2019 81 M 0.66 120.00 26.02 0 1 C2 11.03.2019 82 F 48.90 120.00 0 1 C2 11.03.2019 83 M 0.64 120.00 70.21 0 1 C2 11.03.2019 84 F 0.28 120.00 0 1 C2 11.03.2019 85 M 1.73 120.00 0 1 C2 11.03.2019 86 F 2.51 75.10 16.53 75.10 0 2 C2 11.03.2019 87 M 8.65 120.00 60.74 0 1 C2 11.03.2019 88 F 1.62 102.69 11.05 102.69 0 2 C2 11.03.2019 89 M 2.27 120.00 69.94 0 1 C2 11.03.2019 90 F 4.32 120.00 0.17 0 1 C2 11.03.2019 91 F 0.21 7.32 1.63 4 0 ML 06.03.2019 92 M 0.33 11.81 4 0 ML 06.03.2019 93 F 21.97 6.85 4.69 4 0 ML 06.03.2019 94 M 0.33 35.08 15.91 4 0 ML 06.03.2019 95 F 6.01 12.69 4 0 ML 06.03.2019 96 M 0.26 10.30 4 0 ML 06.03.2019 97 F 0.21 13.31 4 0 ML 06.03.2019 98 M 0.27 45.70 3.82 4 0 ML 06.03.2019 99 F 0.25 10.99 4 0 ML 06.03.2019 100 M 0.36 11.96 4 0 ML 06.03.2019 101 M 1.81 10.78 2.46 4 0 ML 11.03.2019 102 F 0.55 10.86 4 0 ML 11.03.2019 103 M 2.71 10.50 4 0 ML 11.03.2019 104 F 0.69 23.30 4 0 ML 11.03.2019 105 M 53.01 23.12 4 0 ML 11.03.2019 106 F 1.11 8.99 4 0 ML 11.03.2019 107 M 0.93 12.31 4 0 ML 11.03.2019 108 F 1.26 26.79 4 0 ML 11.03.2019 109 M 20.27 6.97 4 0 ML 11.03.2019 110 F 0.57 10.71 4 0 ML 11.03.2019 111 M 1.04 2.40 4 0 ML 11.03.2019 112 F 1.31 11.23 4 0 ML 11.03.2019 113 M 0.84 9.45 2.48 4 0 ML 11.03.2019 114 F 0.90 7.94 4 0 ML 11.03.2019 115 M 0.92 16.72 10.09 4 0 ML 11.03.2019 116 F 4.89 7.20 4.65 4 0 ML 11.03.2019 117 M 1.07 16.79 4 0 ML 11.03.2019 118 F 0.68 5.57 4 0 ML 11.03.2019 119 M 1.60 17.72 12.45 4 0 ML 11.03.2019 120 F 1.40 15.21 4 0 ML 11.03.20