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EntoGeneX, LLC Efficacy of Bio d’ Bug™ Spray Against Bed Bugs Protocol #: N5050411030A141 ICR Project #: 505-0030 OBJECTIVE:To determine the efficacy of Bio d’ Bug Bed Bug Control Solution, a direct spray formulation,for knockdown and 24-hour mortality among bed bugs.This is not a GLP (Good Laboratory Practices) protocol. MATERIALS:FORMULATIONS: The Sponsor will provide the following test spray formulation: 1. Bio d’ Bug Bed Bug Control Solution Formula Identity: EGX/GP/BD01 Lot #: BB01/0311/S01 Ingredients: EGX-101 – A proprietary natural plant-derived insect control ingredient AFX – A proprietary ingredient deviced from fruit extracts providing synergistic bed bug control effects with EGX-101 Inert Ingredients A Material Safety Data Sheet (MSDS) shall be provided for each test, control, and/or reference sample, which will include any hazardous information of the samples. The percentage of all active ingredients and any hazardous constituents must be included in all MSDS. The sponsor is solely responsible for conducting any test, control, and reference sample characterizations, and for retaining this documentation. If any of the test samples are currently available for consumer use and/or purchased in the marketplace, the sponsor should still conduct the same sample characterizations. Any determination of the stability of the test, control, and/or reference samples should be determined by the sponsor prior to the experimental start date. When relevant to the conduct of this study, the solubility of each test, control, and/or reference sample should be determined prior to the experimental start date. Page 2 of 7
EntoGeneX, LLC Efficacy of Bio d’ Bug™ Spray Against Bed Bugs Protocol #: N5050411030A141 ICR Project #: 505-0030 If the stability of test, control, and/or reference samples stored under the test site conditions is determined by the sponsor, it should done prior to any studies. All unused test samples will be returned to the sponsor within 30 days after the final report is sent to the sponsor. The sponsor will be responsible for all costs for the return of the samples, including any costs associated with hazardous materials shipping.TEST ARTHROPODS: Adult ICR Field strain bed bugs (Cimex lectularius) obtained from DC, DE, MD, ME and NJ from 2006 to 2009, preliminary data indicates resistance to pyrethroids. All bed bugs will have been blood fed within 7 days of testing.APPLICATIONDEVICES: Aerosol test productTREATMENTCHAMBER: All spraying will be in a vented, stainless steel Peet Grady chamber (6x6x6 ft).TREATMENTCONTAINERS: Bed bugs will be placed inside treatment containers (pint size paper containers). The top and bottom diameters of these containers are ca. 3.75 and 2.87 inches respectively with a height of ca. 3.5 inches. Treatment containers will be prepared by securing muslin cloth to the bottoms of 16 oz. cups using rubber cement.RECOVERY 16 oz paper cups (inner walls coated with Fluon) withCONTAINERS: screened lidsMISCELLANEOUS: Stop watch, data record forms, forceps, brown paper, surgical gloves, respirator, disposable Tyvek®coveralls and Kestrel® weather meter, Mettler® top-loading balance. METHODS:SummaryFive replicates of 10 bed bugs will be sprayed with the test sample. An additional fivereplicates will be sprayed with water or a blank (to be documented in final report) and willserve as untreated controls. The bed bugs will be observed for knockdown at ½, 1, 2, 3, 4, 5 Page 3 of 7
EntoGeneX, LLC Efficacy of Bio d’ Bug™ Spray Against Bed Bugs Protocol #: N5050411030A141 ICR Project #: 505-0030and 15 minutes. Mortality counts will be taken at +24 hours. If at 24 hours mortality is<90%, additional readings will be taken at +48 hours.Test sampleThe test sample will be stored in a locked cabinet at ambient temperature and humidity untiluse in the study. The sample will be labeled and color-coded for easy identification.ReplicationFive replicates of ten bed bugs per treatment and controls will be used.Application Rate and Application of Test SampleThe test samples will be sprayed for one second; or for a duration long enough to deliver 1.00 g+/- 20% of product. The test sample will be sprayed from the top of the treatment containersdirectly onto the bed bugs (4-6 inches from bed bugs). The weight applied will be recorded foreach replicate.Personal Protection MeasuresAll person(s) involved in treatment will wear disposable Tyvek®coveralls, gloves, and arespirator.Preparation and Handling of Bed BugsTen bed bugs will be used per replicate: five replicates for the test sample and five serving as thecontrol. Each replicate of bed bugs will be anesthetized with CO2 and placed in 9 dram vials atleast 2 hours before testing. Just prior to treatment the bed bugs will be transferred into thetreatment containers. Five minutes after treatment, they will be removed from the treatmentcontainers and transferred to recovery containers and covered with screened lids.Treatment of Bed BugsEach replicate will be sprayed for one second; or for a duration long enough to deliver1.00 g +/- 20% of product. Weights delivered will be recorded. This will beaccomplished by weighing the aerosol can before and after treating each replicate todetermine the weight of test sample dispensed. Each replicate will be sprayedseparately.Treatment of Control Bed BugsEach replicate of control bed bugs will be prepared according to the same procedures outlinedabove with the exceptions that they will be sprayed with water or a blank. The controls will beplaced in a similar environmentally conditioned area as the treated bed bugs.Observation of Knockdown and MortalityAfter treatment, the bed bugs will be observed for knockdown at ½, 1, 2, 3, 4 and 5 minutes.Knockdown occurs when a bed bug cannot right itself when placed on its back but is still able tomove at least one body part. The bed bugs will then be transferred from the treatmentcontainers to recovery containers. Another knockdown observation will be made at 15 minutes. Page 4 of 7
EntoGeneX, LLC Efficacy of Bio d’ Bug™ Spray Against Bed Bugs Protocol #: N5050411030A141 ICR Project #: 505-0030If all ten bed bugs are recorded as knocked down during any observation before 15 minutes haveelapsed, subsequent knockdown observations will not be made. Mortality counts will be madeat +24 hours, and +48 hours (if applicable). All dead bed bugs will be confirmed by probing oragitation to make sure that they are unable to move; any that show movement visible to thenaked eye will be recorded as moribund. Bed bugs that can crawl or right themselves whenplaced on their backs will be recorded as being alive.After the knockdown counts are complete, the bed bugs will be maintained in the laboratory atambient temperature and humidity conditions with a normal 8:16 light:dark cycle for 24 hours.Additional mortality readings will be taken at +48 hours if <90% mortality is recorded at 24hours. A 48 hour reading will depend upon control mortality remaining less than 10%.Temperature and humidity will be recorded in the treatment area, and in the laboratory wherethe bed bugs are subsequently held for the duration of the test. DATA ANALYSISThe number of dead bed bugs per replicate will be added together for a total mortality count.Abbotts Formula will be used to correct for any mortality among the controls. Page 5 of 7
EntoGeneX, LLC Efficacy of Bio d’ Bug™ Spray Against Bed Bugs Protocol #: N5050411030A141 ICR Project #: 505-0030 SCHEDULE OF EVENTS Date Procedure Day 0 Test conducted Following day Telephone/Fax report Within following 30 days Written report Following Final Report Samples returned STATEMENT OF DEVIATION OR AMENDMENT:Any amendments to this protocol must be discussed with and approved by the Sponsor. Anyamendments to, and/or deviations from, this protocol will be documented in the final report.Kristine Styer Date Allan E Brandt, PhD DateStudy Coordinator Director Technology & InnovationICR, Inc. EntoGeneX, LLC Page 6 of 7
EntoGeneX, LLC Efficacy of Bio d’ Bug™ Spray Against Bed Bugs Protocol #: N5050411030A141 ICR Project #: 505-0030 RAW DATA SHEET KNOCKDOWN AND MORTALITYDate: ______________ Treatment Control (circle one)Species: Cimex lectularius, ICR field strain 0Temp (treatment): F RH (treatment): %Temp (laboratory): °F RH (laboratory): %Rep. Spray 48-Hr # Bed bugs Knocked down (min) 24-Hr weight(g) Mortality* Mortality ½ 1 2 3 4 5 15 A M D A M D 1 2 3 4 5TotalAvg*A 48 hour count will be taken if mortality is <90%, and control mortality is <10%.A: Alive; M: Moribund; D: DeadRecording Technician Initials/Date:Study Coordinator: ______________________________ Date:____________ Page 7 of 7