FINAL REPORT. January 28, James D. Heal Arcturus Testing R.R. #6 Stn. Main 6866 Cone. 4 Guelph, Ontario, Canada N IH 613

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1 FIELD EVALUA TION OF OMEZONE DEFEND: THE OUTDOOR PROTECTION PATCH TO MODIFY FEEDING BEHAVIOUR OF MOSQUITOES ATTEMPTING TO FEED ON HUMAN SUBJECTS IN SOUTHERN ONTARIO FINAL REPORT January 28, 2007 by James D. Heal Arcturus Testing R.R. #6 Stn. Main 6866 Cone. 4 Guelph, Ontario, Canada N IH 61 to Safety in Motion 420 Main Street East Suite 605 Milton, Ontario L9T 5G CANADA 005

2 Field Evaluation Of OmeZone Defend: The Outdoor Protection Patch To Modify Feeding Behaviour Of Mosquitoes Attempting To Feed On Human Subjects In Southern Ontario INTRODUCTION The purpose of this study was to assess, under field conditions, the effectiveness of OmeZone Defend: The Outdoor Protection Patch to modify feeding behaviour of mosquitoes on human subjects for up to 6 hours post-application. Each patch contained 75 mg of Thiamine Bl. MATERIALS AND METHODS Site The study was conducted in an area bordering a mixed deciduous/coniferous woodlot (e.g. maples, poplars, birch, tamarack, white cedar, and white pine are predominant species) with secondary growth under the canopy in a rural area four km south of the southern city limit of Guelph, Ontario. Subjects stood in a goldenrod meadow bordering the woodlot. Adjacent to the study area was a cattail marsh (>four hectares) which is a source of Aedes, Ochlerotatus, Anopheles, and Culex mosquito species and the mosquito Coquillettidia perturbans. Previous unpublished studies have shown the site to provide sufficient numbers of adult mosquitoes for repellent evaluations and other mosquito control trials. The study took place on the evenings of June 17, 19 and 21, Biting counts and behaviour evaluation Eight subjects and a supervisor were used in this evaluation. The subjects were given instructions on how to apply and wear the patch (Appendix 1). During each day of the evaluation, two subjects applied the patch 2.5 hours before the start of the 0 minute evaluation. Before the day ofthe evaluation, two subjects applied the patch 2.5 hours before the start of the test and two subjects applied the patch 5.5 hours before the start of the test. Biting counts were performed over a 0 minute period, therefore on each testing day the following duration times (post-application times) were evaluated:,24 and 6 hours

3 Each night two subjects were non-treated and served as controls. During the three-evening study the patch was worn by each subject at least once. The total number of replications equalled six. Subjects dressed in identical green overalls, head nets and white cotton gloves. The eight subjects were randomly assigned to one of eight positions on a grid located within the study site (Appendix 2). All grid positions were at least 10m from each other. Biting counts were initiated just prior to dusk (~20:0 h) to correspond with peak mosquito biting activity and consisted of 8,.5-minute biting counts. Subjects also noted mosquito behaviours such as landing, walking, probing, and feeding. During each biting count, subjects aspirated all mosquitoes landing and probing on two exposed forearms. Mosquitoes were aspirated into 150 ml clear plastic vials. Following the biting count, the subjects recorded the number of mosquitoes captured. Subjects then rotated to the next position on the grid within 17 seconds when the next.5-minute biting count began. In this manner, each subject was at each grid position once each night and the duration of exposure was 0 minutes. Ambient air temperature, relative humidity and wind speed within the study site were measured at the start and end of the biting counts each evening. Biting counts were not conducted on evenings when air temperature was below 10 C or when strong winds (>25 kph) or rain occurred because these conditions limit mosquito host-seeking activity. Data analysis Percent protection provided by the product was calculated using the formula: «mean number of mosquitoes biting non-treated subjects - number biting treated subjects)/mean number biting non-treated subjects) X 100%. Mean percent protection was calculated for the complete 0 minute exposure period. The mean number of mosquitoes biting non-treated subjects and treated subjects was compared using analysis of variance. Protection provided by the product was analysed using a Duncan's Multiple Range Test. The analyses were completed using Statistical Analysis Systems version 6.12 (SAS Institute Inc., Cary, NC). Subjects were polled and asked to describe any unusual feeding behaviour when wearing the patch versus not wearing the patch. 00'7

4 RESULTS The results are summarized in Table 1. OmeZone Defend: The Outdoor Protection Patch provided 21., 9.6 and 17.2% mean reduction of mosquitoes landing and biting for the, 24 and 6 hour treatments, respectively. The mean number of mosquitoes landing and biting treated subjects was not statistically different (P > 0.05) than the mean number of mosquitoes landing and biting control subjects. Table 1. Mean numberr;2 (± one standard deviation) and percent reduction of mosquitoes biting human subiects during 0 minute mosquito biting counts in field tests conducted near Guelph, Ontario, Treatment Duration (hours) Number of mosquitoes Percent reduction post-application Per.5 minutes Control ± 4.90 a hours 24 hours 6 hours ±.84 a 5.40 ± 5.04 a 5.28 ± 5.8 a IValues followed by different letters in the same column are significantly different (P<0.05). 2Number of repetitions equalled six. Mean biting pressure over three nights equalled 52.7 mosquitoes per 0 minutes. 4Based on nightly calculations of percent reduction, not data in column. Four out of seven responding subjects (Table 2) reported that feeding behaviours of mosquitoes were abnormal when wearing the patch. All subjects had previously participated in multiple mosquito feeding trials and were sufficiently experienced to detect abnormal feeding behaviours. Subjects reported that mosquitoes seemed disoriented upon landing. An inhibitory or feeding delay response was noted by some of the subjects, but an estimate of this delay response was only reported by one subject. This subject, while wearing the patch, noted that most mosquitoes appeared to take about three times longer to bite. Two treated subjects reported independently that the mosquitoes appeared "drunk" when wandering across the skin, with a significant proportion of mosquitoes giving up and flying off. Further research on the Vitamin Bl patch is warranted. In the current trial subjects were mostly sedentary during biting counts. Future research should be more "real life" where subjects are allowed to walk, or be otherwise active, to promote perspiration and perhaps more volatility of B 1 or breakdown products from the skin. Another issue is dose, since in the current trial only one concentration and one patch was used. Increased B I concentration and/or number of patches could result in increased efficacy. 4 o O~, 8

5 The mean air temperature during the three-evening study was 22.5 c (range = 20.5, 25.6), the mean relative humidity was 85.1 % (range = 77.5, 91.) and the mean wind speed was 1. kph (range = 0.0, 2.5). Table 2. Subject observations on mosquito biting behaviour during 0 minute mosquito biting counts in field tests conducted near Guelph, Ontario, Subject ]Asked to answer yes, no or not sure. Did you observe] disoriented mosquitoes when wearing the patch? Yes t sure t sure Yes Yes t sure Could you estimate a delay response of mosquitoes feeding while wearin~atch? X longer to bite ND 2t determined - subject did not respond. CONCLUSIONS OmeZone Defend: The Outdoor Protection Patch (containing 75 mg Thiamine B1) provided some protection from blood-seeking mosquitoes in a field test using human subjects. The majority of subjects, while wearing the patch, reported that mosquitoes were disoriented while trying to probe and feed, and were delayed in doing so

6 Appendix 1 Patch Trial Instructions Please note there are different application times: approximately, 24 and 6 hours before biting counts commence. Application site: to any hairless area, preferably upper inner arm (biceps) or shoulder blade. Site should be washed with a mild soap solution to remove skin oils and dried thoroughly. Once applied, do not take a bath, shower or go swimming or take part in any activity that would expose the patch to water. Apply only one patch. You are provided with the number of patches you need for all three test days plus an extra patch. In the unlikely event that the patch comes off (I have been told by the company that they do not come off easily), replace it with a new one. The biting counts commence at 8:0 pm on July 17, 19 and 21 (Mon-Wed-Fri). Please see the attached treatment schedule and take note of your application times and dates. If your treatment If your treatment If your treatment is hr then apply the patch at 6 pm the day of the test. is 24 hr then apply the patch at 9 pm the day before the test. is 6 hr then apply the patch at 9 am the day before the test. If you have any questions call me or Karen any time at

7 Appendix 2 Patch Trial Treatments and Subject Positions Day 2 (July 6 Control control Christie Andrew Clinton ZiQQV John Beth Scott Eric Subjects Treatments If your treatment is hr then apply the patch at 6 pm the day of the test. If your treatment is 24 hr then apply the patch at 9 pm the day before the test. If your treatment is 6 hr then apply the patch at 9 am the day before the test. Day Christie Andrew ZiQQV Clinton John Beth Scott Eric Subjects Positions