The document describes a study protocol to evaluate the efficacy of Bio d’ Bug Bed Bug Control Solution spray against bed bugs. Ten bed bugs will be sprayed with the test product or a control in 5 replicates each. Bed bugs will be observed for knockdown at various time points and mortality will be recorded at 24 and 48 hours. The objective is to determine the product's ability to knock down and kill bed bugs.
2. 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
3. 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.
APPLICATION
DEVICES: Aerosol test product
TREATMENT
CHAMBER: All spraying will be in a vented, stainless steel Peet
Grady chamber (6x6x6 ft).
TREATMENT
CONTAINERS: 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) with
CONTAINERS: screened lids
MISCELLANEOUS: Stop watch, data record forms, forceps, brown paper, surgical
gloves, respirator, disposable Tyvek®coveralls and Kestrel®
weather meter, Mettler® top-loading balance.
METHODS:
Summary
Five replicates of 10 bed bugs will be sprayed with the test sample. An additional five
replicates will be sprayed with water or a blank (to be documented in final report) and will
serve as untreated controls. The bed bugs will be observed for knockdown at ½, 1, 2, 3, 4, 5
Page 3 of 7
4. EntoGeneX, LLC
Efficacy of Bio d’ Bug™ Spray Against Bed Bugs
Protocol #: N5050411030A141
ICR Project #: 505-0030
and 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 sample
The test sample will be stored in a locked cabinet at ambient temperature and humidity until
use in the study. The sample will be labeled and color-coded for easy identification.
Replication
Five replicates of ten bed bugs per treatment and controls will be used.
Application Rate and Application of Test Sample
The 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 containers
directly onto the bed bugs (4-6 inches from bed bugs). The weight applied will be recorded for
each replicate.
Personal Protection Measures
All person(s) involved in treatment will wear disposable Tyvek®coveralls, gloves, and a
respirator.
Preparation and Handling of Bed Bugs
Ten bed bugs will be used per replicate: five replicates for the test sample and five serving as the
control. Each replicate of bed bugs will be anesthetized with CO2 and placed in 9 dram vials at
least 2 hours before testing. Just prior to treatment the bed bugs will be transferred into the
treatment containers. Five minutes after treatment, they will be removed from the treatment
containers and transferred to recovery containers and covered with screened lids.
Treatment of Bed Bugs
Each replicate will be sprayed for one second; or for a duration long enough to deliver
1.00 g +/- 20% of product. Weights delivered will be recorded. This will be
accomplished by weighing the aerosol can before and after treating each replicate to
determine the weight of test sample dispensed. Each replicate will be sprayed
separately.
Treatment of Control Bed Bugs
Each replicate of control bed bugs will be prepared according to the same procedures outlined
above with the exceptions that they will be sprayed with water or a blank. The controls will be
placed in a similar environmentally conditioned area as the treated bed bugs.
Observation of Knockdown and Mortality
After 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 to
move at least one body part. The bed bugs will then be transferred from the treatment
containers to recovery containers. Another knockdown observation will be made at 15 minutes.
Page 4 of 7
5. EntoGeneX, LLC
Efficacy of Bio d’ Bug™ Spray Against Bed Bugs
Protocol #: N5050411030A141
ICR Project #: 505-0030
If all ten bed bugs are recorded as knocked down during any observation before 15 minutes have
elapsed, subsequent knockdown observations will not be made. Mortality counts will be made
at +24 hours, and +48 hours (if applicable). All dead bed bugs will be confirmed by probing or
agitation to make sure that they are unable to move; any that show movement visible to the
naked eye will be recorded as moribund. Bed bugs that can crawl or right themselves when
placed on their backs will be recorded as being alive.
After the knockdown counts are complete, the bed bugs will be maintained in the laboratory at
ambient 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 24
hours. 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 where
the bed bugs are subsequently held for the duration of the test.
DATA ANALYSIS
The number of dead bed bugs per replicate will be added together for a total mortality count.
Abbott's Formula will be used to correct for any mortality among the controls.
Page 5 of 7
6. 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. Any
amendments to, and/or deviations from, this protocol will be documented in the final report.
Kristine Styer Date Allan E Brandt, PhD Date
Study Coordinator Director Technology & Innovation
ICR, Inc. EntoGeneX, LLC
Page 6 of 7
7. EntoGeneX, LLC
Efficacy of Bio d’ Bug™ Spray Against Bed Bugs
Protocol #: N5050411030A141
ICR Project #: 505-0030
RAW DATA SHEET
KNOCKDOWN AND MORTALITY
Date: ______________ Treatment Control (circle one)
Species: Cimex lectularius, ICR field strain
0
Temp (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
5
Total
Avg
*A 48 hour count will be taken if mortality is <90%, and control mortality is <10%.
A: Alive; M: Moribund; D: Dead
Recording Technician Initials/Date:
Study Coordinator: ______________________________ Date:____________
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