## Laboratory Methods for Efficacy Testing of Bed Bug 

## Pesticide Products

Kevin J. Sweeney Senior Entomologist U.S. EPA Office of Pesticide Programs Registration Division, Washington, DC

FIFRA Scientific Advisory Panel March 6

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### Draft Product 

### Performance Test Guidelines

OCSPP 810.3900: Laboratory Testing 

### Methods for Bed Bug Pesticide Products

2

# Outline of the Presentation

• Introduction

- Describe the draft 

### guideline’s objectives and organization

• Highlight challenges associated with guideline development, testing and data 

### analysis

• Review each testing method in Guideline 

### Sections (h) through (o)

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# Guideline Objectives

• To provide standardized approaches to bed bug pesticide product testing to ensure the quality and validity of the efficacy data for these types of products

• To provide guidance to data developers on acceptable test methods

• To provide test methods that should yield data capable of satisfying the product performance data requirements for registration of these products

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# Guideline Scope

• Does not apply to repellent products applied to human skin

• Laboratory methods only

• Labeling not addressed

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# Guideline Organization

• Definitions important to the guideline



### • Protocol elements and recommendations

• Completed study reporting

• Specific guidance on laboratory testing



### • List of references

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# Key Definitions

• Key definitions include:

– MORTALITY and KNOCKDOWN

– PESTICIDE RESISTANCE

– RESISTANCE RATIO

– SUSCEPTIBLE BED BUG POPULATION

– RESISTANT BED BUG POPULATION

– DISCRIMINATING DOSE

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## Experimental Design and Evaluation C hallenges

• When is a bed bug rated or scored as 

### “dead”?

– Mortality vs. Knockdown

FIFRA Scientific Advisory Panel
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8

## Experimental Design and Evaluation Challenges

• Laboratory Testing

– Bridging efficacy testing to effective
outcomes 

## • Field Testing

– Lack of control
treatments

– No two residences are alike

– Residents may be exposed to pesticides

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## Experimental Design and Evaluation Challenges

• Test Parameters:

– Appropriate exposure times

– Appropriate test length

– Positive control or standard pesticide

– Replication

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# Test Protocol Development

• Outlines and describes recommendations for the inclusion of specific information in study protocols

• EPA does not require study protocols to be submitted for bed bug laboratory studies but recommend that data developers do so

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# Completed Study Reporting

• Outline and describe recommendations



### • Lists specific elements and information be included

• Makes reference to Good Laboratory 

### Practices

• Documenting what was done is recommended

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### (h) General Methods (i) Susceptibility (j) Residual Activity (k) Repellency (l) Impregnated Materials (m) Foggers (n) Direct application (o) Ovicides

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## General Methods (h)

• Information in this section of the guideline applies to testing methods outlined in sections (i) through (o)

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## General Methods (h)

Test Substance

• The test substance should be the product formulation proposed for use. It should be:

– identified and certified

– tested at the lowest application rate

– tested at the lowest dilution

FIFRA Scientific Advisory Panel
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### General Methods (h) Test Organism
Cimex lectularius • 1 susceptible

• 1 resistant

• 3 field strains from urban areas in 3 different regions of the USA

FIFRA Scientific Advisory Panel
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16General Methods (h) Bed Bug Life Stages

• Adulticide Testing
Unfed adult bed bugs with a sex ratio of 1:1

• Insect Growth Regulators Use mixed nymphal stages

• Ovicides Eggs only

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Whitney Cranshaw, Colorado State University, Bugwood.org

Gary Alpert, Harvard University, Bugwood.org Dr. Robin Todd

## General Methods (h)

Test Conditions

• Temperature 25 ° C

• Humidity 50

- 80%

• Photoperiod 16:8

FIFRA Scientific Advisory Panel
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### (i) Susceptibility (j) Residual Activity (k) Repellency (l) Impregnated Materials (m) Foggers (n) Direct application (o) Ovicides

FIFRA Scientific Advisory Panel
March 6

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• Insecticide resistance

• Method to determine resistance

• At what level of susceptibility is a bed bug strain deemed to be “resistant”?

• Need to identify resistant strains for all modes of action

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 20 Whitney Cranshaw, Colorado State University, Bugwood.org

### Susceptibility (i) Experimental Design and Evaluation Challenges

• Insecticide resistance

– Need to establish/identify a susceptible strain as a benchmark

– LD vs. LT values:

– 50% vs. 90% vs. 100%

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 21Susceptibility (i) Experimental Design and Evaluation Challenges

– Establishing Discriminating/Diagnostic Dose Concentrations?

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## Susceptibility (i)



## Testing Objectives

1) To estimate the susceptibility and magnitude of tolerance or resistance of bed bug strains to pesticides used in 

### product testing; and 2) To determine the discriminating dose 

### for testing

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## Susceptibility (i)

The Experimental Unit

• 500 ml beaker with the bottom covered with filter paper. Filter paper was selected 

### because the insecticide solution will be absorbed evenly



### • Application of 50

- 100 ul of solution or a volume that will saturate the filter paper

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## Susceptibility (i)

Test Beaker and Surface

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## Susceptibility (i)



## Test Solutions

• Active ingredient test concentrations (dose)

– Logarithmic scale (base 10)

– exponential increase in concentration

- 0.0001, 0.001, 0.01. 0.1. 1.0, 10.0

– Untreated control

- diluent only

– Positive control

– different positive control for each mode of action. Deltamethrin for pyrethroids. FIFRA Scientific Advisory Panel
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## Susceptibility (i)

Test Parameters

• Number dose levels = 5 or more

• Number of replicates = 10

• Number of bed bugs per replicate = 20



### • Exposure period = 24 hours

• Record mortality, correct with Abbott’s Formula and express as a percentage

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## Susceptibility (i)

Data Reporting

• Protocol amendments and deviations



### • Knockdown and mortality

• Results from the probit analysis

• LD 50 

### and LD 90 

### values for each strain and


### treatment

• LD 100 

### recommended

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## Susceptibility (i) 

## Resistance Ratio Calculations

• Resistance ratios should be calculated as follows:

– LD 50 (field strain)/LD 50 (susceptible strain) = RR 50

– LD 90 (field strain)/LD 90 (susceptible strain) = RR 90

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## Susceptibility (i)

Discriminating Dose

• Discriminating dose selection 

### (?) x (LD 50, 90, or 100 

### )

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## Susceptibility (i) Alternative Product Rates

• Alternative approach using product rates 

### :

– 1x product label rate

– 0.5x product label rate

– 0.25x product label rate

– 0.125x product label rate

– 0.01x product label rate

FIFRA Scientific Advisory Panel
March 6

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### (j) Residual Activity (k) Repellency (l) Impregnated Materials (m) Foggers (n) Direct application (o) Ovicides

FIFRA Scientific Advisory Panel
March 6

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• Selection of representative surfaces

• Choice vs. no

- choice

• Balanced treatment design

• Exposure time

• Length of the test

• Data analysis

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## Residual Activity (j)



## Materials and Methods

• Residual activity is measured in days, weeks or months

– Treated panels are aged to test residual claim length

– Expose bed bugs to treatments daily, weekly or monthly based on claim desired

– Testing continues until residual treatment fails

– Bed bug exposures have varied from 5 minutes to 5 days or more

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## Residual Activity (j)



## Materials and Methods

• Test surface panels should measure 6" x 6 "

– unpainted 1⁄4" thick plywood

– commercial linoleum tile

– concrete board

– 100% cotton sheeting

– medium pile 100% nylon carpet

• An equal number of product and untreated control. Treatments should be established on the same surfaces

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## Residual Activity (j)



## Materials and Methods

• 5 replicates for each treatment surface per bed bug strain and test interval

• Testing begins at day 0 and is repeated every 7 days until failure

• Panels should be used only once. New (aged) panels should be used at each testing interval

• Use bed bugs only once. New bed bugs from each strain should be exposed at each testing interval

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## Residual Activity (j)

Materials and Methods

• Apply the lowest rate proposed against bed bugs

• Apply liquid formulations at a distance of 1 foot above the surface

• Dry panel for 3 hours

• For dusts, apply from a distance of six inches

• Record application volume and weight of active ingredient

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## Residual Activity (j)



## Materials and Methods

• Apply concentrates at the dilution containing the lowest percentage of active ingredient

• For ready to use (RTU) spray and dust products:

– establish the volume delivered using replicated trials

– use the mean volume to make surface applications

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## Residual Activity (j) Examples of RTU Nozzle Types

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## Residual Activity (j)



## Test Parameters

• For each surface tested:

– 5 replicates with 10 bed bugs each

– 500 bed bugs for each strain tested

– Record data points for every surface at every test interval

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## Residual Activity (j) Fixed Exposure Time to Treatments

• 24 hours followed by transfer to clean 

### containers.

• Observations at 15 & 30 minutes; and at 

### 1, 2, 4, 24, 48, 72, and 96 hours

• Observations continue unless all bed bugs in the product treatment die or untreated control mortality exceeds 10%

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## Residual Activity (j) Continuous Exposure to Treatments

• No transfer to clean containers



### • Observations at 15 & 30 minutes; and at 1, 2, 4, 24, 48, 72, and 96 hours and 

### every 24 hours thereafter

• Observations continue unless all bed bugs in the product treatment die or untreated control mortality exceeds 10%

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## Residual Activity (j)

Reporting Study Results

• Protocol with amendments and deviations



### • Control mortality should be corrected for with Abbott’s Formula



### • The percentage of bed bugs killed and knocked down, exclusively, for every treatment at every test interval



### • The length of the test period in hours or days

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## Residual Activity (j) Fixed Exposure Time Results

• Report the number of dead, knocked down and live bed bugs in each 

### replicate at 15 and 30 minutes; 1, 2, 4, 24, 48, 72, and 96 hours

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## Residual Activity (j) Continuous Exposure Time Results

• Report the number of dead, knocked 

### down and live bed bugs in each replicate at 15 and 30 minutes; 1, 2, 

### 4, 24, 48, 72, 96 hours and every 24 hours daily

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## Residual Activity (j) Data Analysis

- Fixed Exposure Time

– The significance of differences within and between surface treatments

– Difference between treated and untreated surfaces

– Between strain differences

– An analysis of variance (ANOVA) may appropriate but other analysis may be considered and should be justified

– KT 50 and KT 90 values

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## Residual Activity (j) 

## Data Analysis for Continuous Exposure Times

– Data should be analyzed with a probit analysis to determine the LT 50 , LT 90 and LT 100 as well as KT 50 and KT 90 values for each surface

– Provide Goodness

- of

– Fit test results

FIFRA Scientific Advisory Panel
March 6

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### (k) Repellency (l) Impregnated Materials (m) Foggers (n) Direct Application (o) Ovicides

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## Repellency (k)

Objectives

• Testing should be conducted to determine:

– whether or not pesticide residues alter bed bug behavior, either by repellence from or attraction to pesticide treated areas in a series of choice tests

• Group and individual bed bug responses should be evaluated

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 49Repellency (k) Experimental Design and Evaluation Challenges

• Pesticide residues and bed bug behavior

• Harborage construction

• Harborage conditioning

• Length of test period due to toxic effects of insecticide treatment

• Individual vs. group responses

• Choice testing

• Bed bugs who stay outside of the harborage

• Repellency

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## Repellency (k)

Experimental Unit

• Test unit should be large enough to fit up 

### to three harborages for each replicate

• Condition filter paper by exposing to bed 

### bugs for 24 hours

• Harborage construction

- filter paper 

### folded into a tent shape to provide shelter

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## Repellency (k)

Treatments

• Diluent treated control filter paper



## • Product treated filter paper

• Diluent treated conditioned filter
paper



## • Product treated conditioned filter
paper

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## Repellency (k)

Experiments



## • Three experiments

– Unit 1 . Each replicate should have two pieces of unconditioned filter paper. One treated with the pesticide product and the other treated with the product diluent or water (if the product is ready to use)

– Unit 2 . The set

- up is the same as in Unit 1 except that filter papers should be conditioned

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## Repellency (k)

Experiments

• Unit 3. This unit should include:

– 

### an product treated unconditioned filter
paper



### – a product treated conditioned filter paper and



### – a diluent treated conditioned filter paper

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## Repellency (k) Individual and Group Testing

• Individual responses

• 20 replicates of one bed bug each. 60 bed bugs for each strain tested



### • Group responses

• 5 replicates of 10 bed bugs each. 150 bed bugs for each strain tested



### • Expose bed bugs for 1 to 24 hours

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## Repellency (k)

Data Reporting

• Report:

– Protocol with amendments and deviations

– Amount of product applied

– Location of bed bugs in the arena at end of test

– Bed bug mortality and knockdown

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## Repellency (k)

Data Analysis

An analysis of data from individual and group testing should determine whether there is a
difference:

– between the treated and untreated harborages without conditioning

– between the treated and untreated harborages with conditioning

– between the treated unconditioned and treated conditioned harborages

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## Repellency (k)

Conclusions

• Did bed bugs prefer one harborage over 

### another?

• Did the treatments have any effect?



### • Is there a difference between the individual and group responses?

FIFRA Scientific Advisory Panel
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### (l) Impregnated Materials (m) Foggers (n) Direct Application (o) Ovicides

FIFRA Scientific Advisory Panel
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- 7, 2012 Crystal City , VA 60FIFRA Scientific Advisory Panel March 6

- 7, 2012 Crystal City , VA 61FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 62Impregnated Materials (l) Experimental Design and Evaluation Challenges

• Test design

• Mortality

• Impact on behavior

– Blood

- feeding inhibition

– Contact irritation and avoidance of treated fabric

FIFRA Scientific Advisory Panel
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- 7, 2012 Crystal City , VA 63 Clemson University

- USDA Cooperative Extension Slide Series

## Impregnated Materials (l)

Objectives

• To test the efficacy of a pesticide impregnated material product formulations against the bed bug. Test outputs:

• Mortality

• Blood feeding inhibition

• Detect repellency or attraction to the treated material in the presence of a host

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## Impregnated Materials (l)

Experimental Unit

• Tunnel or Tube Assay



### • The tunnel will be lined with treated and/or untreated material



### • A bait chamber will be placed in one end of the tunnel. The chamber will contain a 

### host or an artificial blood meal system

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# Artificial Feeding System

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## Impregnated Materials (l)

Treatments

• One choice test and two no

- choice tests

– Treatment 1. Choice test.

– Tunnel chamber should be divided into two sections

– One

- half lined with untreated product fabric or textile

– The other half lined with pesticide impregnated product material

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## Impregnated Materials (l)

Treatments

• Treatment 2. The entire tunnel should be lined with pesticide impregnated product material

• Treatment 3 . The entire tunnel should be lined with untreated product material

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## Impregnated Materials (l) Replication and Exposure

• 5 replicates for each treatment with 20 bed 

### bugs per replicate

• Replace the blood source in every test 

### replicate

• Expose bed bugs to treatment for 24 hours

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## Impregnated Materials (l)

Data Reporting

• Report:

– The number of live and dead bed bugs for each replicate and treatment

– The percentage of bed bugs killed in each
treatment

– Bed bug locations in each replicate at 24 hours

– The number of fed and unfed bed bugs in all replicates

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## Impregnated Materials (l) Blood Feeding Inhibition (BFI)

• Reduction (inhibition) of bed bug blood feeding relative to the control should be based on the following formula:

• (BFI) = 100 (Bf
u - Bf t )

Bf
u

BF u = proportion of bed bugs blood fed in untreated control

BF t = proportion of bed bugs blood fed in treatment

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## Impregnated Materials (l)

Data Analysis

– Evaluate the effects of the product treatment on blood feeding inhibition

– Evaluate the effects of the product 

### treatment on bed bug mortality

– Whether bed bugs were repelled, attracted, or indifferent to the product 

### treatment

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 73(h) General Methods (i) Susceptibility (j) Residual Activity (k) Repellency (l) Impregnated Materials 

### (m) Foggers (n) Direct Application (o) Ovicides

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## Foggers (m)

Objective

• To determine the efficacy of pesticide 

### fogging product formulations against bed bugs

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 75Foggers (m) Experimental Design and Evaluation Challenges

• Chamber size

• Test cage construction

• Cage placement

• Replication

• Exposure time

• Test length

• Number of untreated control replicates

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## Foggers (m)

Chamber and Dosing

• Testing should be conducted in a chamber with a volume of 216 cubic feet or greater



### • Test doses for aerosols, misters and fogger products should be delivered by an automatic dispenser calibrated for the 

### proper droplet size and application rate

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## Foggers (m) Treatments and Replications

• There should be a product treatment and an untreated control treatment for each 

### bed bug strain

• 24 replicates per treatment

• 10 bed bugs per replicate

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# Example of a Chamber

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## Foggers (m)

Test Cages

• For each replicate the test cage should be a PVC pipe 6  long with a diameter of one 

### inch. Each end is covered with a removable PVC cap



### • Twenty 1/16  holes should be drilled in a random array around the circumference of 

### pipe

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## Foggers (m)

Test Cage Placement

• Cages placement for each strain tested:

– on all 4 walls at 3 height levels

– 2 cages for each height level on each wall

– 6 cages per wall

– 24 cages total

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## Foggers (m)

Exposure and Evaluation

• 2 hour exposure to the treatment

• Ventilate chamber



### • Evaluate treatments and controls at 24, 48, 72, and 96 hours post

- treatment

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## Foggers (m)

Data Reporting

– Mortality data at 24, 48, 72 and 96 hours post

treatment

– Percentage killed and knocked down for every replicate at each observation interval

– Pooled product treatment mortality data for each height level and

– Corrected mean values for treatments and controls

– Amount of product applied

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## Foggers (m)

Data Analysis

• The analysis should consider treatment, cage height, and bed bug strain effects on 

### product efficacy

FIFRA Scientific Advisory Panel
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### (n) Direct Application (o) Ovicides

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### Direct Application (n)

Method Overview

Established methods that are widely used

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## (o) Ovicides

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 87Ovicides (o) Experimental Design and Evaluation Challenges

• Ovicidal testing is conducted by many laboratories

– Oviposition surface has been unpainted wood or filter paper (treated and/or untreated)

• Evaluate oviposition repellency and egg kill

– Direct spray testing has been used

– Many current product residual applications will kill nymphs immediately after hatch but not
eggs

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 88Ovicides (o) Experimental Design and Evaluation Challenges

– Selecting and separating female bed bugs

– Egg collection, transfer and storage

– Determination of egg kill vs. lack of egg viability

– Control “mortality”

– Test length

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## Ovicides (o)

Direct Spray

• Direct spray test system

– Pieces of egg

- laden filter paper should be cut into piece that should fit into the test arena

– Up to 6 eggs should be allotted to each well or dish

– A metered bench top sprayer is preferred as the spray device to ensure consistent application volume and even distribution of spray particles

– Applications should be made at the lowest rate

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## Ovicides (o)

Direct Spray Replication

• Direct spray test system



### • Minimum of 10 replicates for each
treatment

• Minimum of 20 eggs per replicate

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## Ovicides (o)

Contact Kill

• Experimental Unit 1

– Conditioned filter paper should be used to line untreated control and product treatment dishes

– 1 female bed bug per dish

– Each dish is a replicate

• 25 replicates per treatment

– The number of eggs laid on the filter paper by each female will be variable

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## Ovicides (o)

Contact Kill

• Experimental Unit 2

- s ame set

- up as Unit 1 except bed bugs do not lay eggs on treated surface.

– Eggs are transferred to each dish and placed on top of the paper

- or

-

– Egg laden untreated papers can be turned upside down to bring eggs into contact with the treated surface

– Each dish is a replicate and should have 10 eggs

– 25 replicates per treatment

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## Ovicides (o) 

## Data Reporting

- Exposure time is 30 days or less.

- Observe eggs daily

- Record un

hatched and hatched eggs

FIFRA Scientific Advisory Panel
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- 7, 2012 Crystal City , VA 94 Gary Alpert, Harvard University, Bugwood.orgFIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 95 Mohammed El Damir, Pest Management, Bugwood.org

## Ovicides (o)

Data Analysis

• Compare means between product 

### treatments and controls to determine if they are significantly different at the end of the test period

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# Questions and Comments?

97 FIFRA Scientific Advisory Panel
March 6

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# Charge to Panel

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 98Charge 1) Laboratory test methods. The draft guidelines describe laboratory test methods for evaluating the efficacy of a variety of bed bug pesticide products. Please discuss:

(a) Whether, given the objectives and the types of products being evaluated, the test methods are appropriate to evaluate the efficacy of bed bug products with regard to kill and knockdown; repellency; pesticide resistance; and discriminating dose.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 991) (b) Whether there are additional or alternative laboratory test methods beyond those discussed in the draft guidelines for testing the efficacy of bed bug pesticide products.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 100Charge 2) Standardized test system elements and conditions (Section (h) pp. 10). The draft guidelines describe standard elements and conditions that are recommended for each type of efficacy evaluation. Please discuss:

(a) The appropriateness of suggested environmental conditions (such as temperature and humidity).

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 1012) (b) The proposed test organism. In particular, please discuss:

(i) Whether the testing should be done only with one species, the common bed bug, Cimex lectularius . Whether it is appropriate to use unfed adult bed bugs with a sex ratio of 1:1 to test adulticide products.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 1022) (b) (ii) The advantages and disadvantages of including a susceptible strain in every test and whether the Harlan strain is the preferred susceptible strain.

(iii) The advantages and disadvantages of including a resistant strain in every test and how an investigator should select a resistant strain or strains.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 1032) (b) (iv) The adequacy of the use of field collected bed bug strains from urban areas in three regions of the U.S. to represent the variability in susceptibility of bed bug populations for conducting efficacy evaluations.

(v) The recommended approach for grouping and evaluating the data from the tested bed bug strains to assess efficacy of bed bug products.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 1042) (c) The advantages and disadvantages of including a positive control in every test.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 105Charge 3) Specific guidance for laboratory studies for resistant ratio determination, characterization of bed bug strain susceptibility, and discriminating dose selection (Section (i) pp.11). The draft guidelines propose a method to collect the data necessary to calculate a resistance ratio. Resistance ratios should be calculated for bed bug populations collected from the field in three regions in the U.S. The lethal dose values to be used in these calculations are to be derived from probit analysis. For pyrethroid insecticides, deltamethrin is proposed as the laboratory standard. From the collected data and resistance ratio values, the procedure recommends an approach to select a discriminating dose for a product. Please discuss:

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 1063) (a) Which insecticides should be chosen as standards for other insecticidal modes of action.

(b) Whether it is useful to compare the resistance ratios of bed bug field populations to the resistance ratio for a corresponding dose of deltamethrin when evaluating pyrethroid insecticides.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 1073 (c) Whether a resistance ratio of 100x is adequate to screen field strains for resistance against insecticides and if not, what other approaches are recommended for detecting resistance in bed bug populations.

(d) What type(s) of data analysis and statistical testing would be most appropriate for these data sets.

(e) The discriminating dose selection for products, including whether the LD 90 value of the least susceptible field population is the best value to use as the basis for discriminating dose selection.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 108Charge 4) Exposure times to product treatments [Sections (j) (pp. 13) and (l) (pp.17)]. For residual surface and impregnated material testing, the draft guideline proposes two possible approaches to exposing bed bugs to pesticide product applications in the laboratory: 1) single dose with a fixed exposure time of 24 hours followed by mortality assessments through 96 hours unless all bed bugs die or control mortality exceeds 10%; and 2) single dose with an exposure time that is continuous until all bed bugs die or control mortality exceeds 10%. The single dose is generally the lowest label recommended dose for the product. Please discuss:

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 109

### 4) (a) Whether the exposure times provide 

### sufficient data to measure the efficacy of a bed bug pesticide or whether other 

### exposure times or testing scenarios should be used. 

### (b) Whether percent mortality values or lethal time values should be used as endpoints to assess the success of 

### product applications and the advantages and disadvantages of each one.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 110Charge 5) Specific guidance for laboratory studies for forced exposure (no

- choice) residual surface treatment tests (Section (j) pp.13). The methods described for testing residual surface treatments recommend five types of surfaces: unpainted plywood; linoleum tile; concrete board; cotton sheet; and medium pile carpet. Please discuss:

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 1115) (a) Whether the experimental unit described in the draft guidelines will provide sufficient data to show how effectively and how quickly a bed bug product knocks down and kills bed bugs, when applied as a residual treatment to surfaces.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 1125) (b) Whether there is a single surface type that could be used as a standard or representative surface for testing product residual activity in lieu of testing multiple surfaces as recommended in the draft guidelines.

(c) Whether there are modifications or additional tests that could be recommended to improve residual surface treatment testing.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 1135) (d) What type(s) of data analysis and statistical testing would be most appropriate for these data
sets.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 114Charge 6) Specific guidance for laboratory studies to determine if bed bugs are repelled by, or attracted to, pesticide product residues (Section (k) pp. 15). For pesticide product treatments to be efficacious, bed bugs must contact pesticide residues. Testing methods should determine whether or not pesticide residues alter bed bug behavior, either by repellence from or attraction to pesticide
treated areas. Please discuss 

### :

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 1156) (a) Whether the experimental unit described in the draft guidelines will provide sufficient data to measure the duration and extent to which a pesticide product’s residues repel bed bugs.

(b) Whether conditioning of experimental bed bug harborages is necessary.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 1166 (c) Whether individual responses and/or group responses should be used to determine whether bed bugs are repelled by pesticide product residues in harborages.

(d) What type(s) of data analysis and statistical testing would be most appropriate for these data
sets.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 117Charge 7) Specific guidance for laboratory studies for testing pesticide impregnated material products (Section (l) pp. 17). The draft guideline proposes no

- choice and choice tests for treated materials. Both are to be conducted in the presence of a non

- human host or artificial membrane system to provide a source of blood. The proposed tests evaluate mortality, blood feeding inhibition, and preference for treated versus untreated surfaces. Please discuss:

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 1187) (a) Whether the experimental unit is adequate for evaluating mortality and blood feeding inhibition following exposure to impregnated materials.

(b) Whether the use of an artificial membrane system to simulate an animal host and provide blood for questing bed bugs is adequate, or whether an animal host is necessary.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 1197) (c) Whether the assessment period is adequate.

(d) Modifications or additional tests that would improve pesticide impregnated product testing.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 1207) (e) Adequacy of the experimental unit to provide an experimental design and adequate data to evaluate repellency.

(f) What type(s) of data analysis and statistical testing would be most appropriate for these data sets.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 121Charge 8) Specific guidance for laboratory studies of indoor fogger products (Section (m) pp.19) . The methods described for indoor fogger testing recommend use of an experimental unit that includes a 216 cubic feet or larger Peet

- Grady chamber. The test container for bed bugs has 20 holes (1/16" diameter) in it to simulate a crack and crevice treatment and a bed bug refuge. Please discuss:

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 1228) (a) Whether the experimental unit is adequate for testing indoor foggers and misters.

(b) Modifications or additional tests that could be recommended to improve indoor fogger testing.

(c) What type(s) of data analysis and statistical testing would be most appropriate for these data
sets.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 123Charge 9) Specific guidance for laboratory studies for testing ovicidal products (Section (o) pp. 22). Efficacious ovicidal products are likely to improve the effectiveness of bed bug management programs. The draft guidelines describe methods for evaluating product efficacy against eggs from direct application and contact with residual surface applications. Please discuss:

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 1249) (a) Adequacy of the experimental unit for testing ovicidal products.

(b) Modifications or additional tests that could be recommended to improve ovicidal product testing.

(c) What type(s) of data analysis and statistical testing would be most appropriate for these data
sets.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 125Charge 10) Please provide comments on the overall clarity, accuracy and completeness of the draft guidelines: “Laboratory Testing Methods for Bed Bug Pesticide Products”. Please provide any additional comments that highlight any areas of the draft guidelines that may need to be clarified and note any relevant topics that may be missing. Please include references to any published literature that could help improve the completeness and clarity of the draft guidelines.

FIFRA Scientific Advisory Panel
March 6

- 7, 2012 Crystal City , VA 126