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United States

Environmental Protection 

Agency

 Office of Chemical Safety and 

Pollution Prevention (7101)

EPA 712-[insert number]

[ DRAFT DATED 

February 14, 2012]

Draft Product 

Performance

Test Guidelines

OCSPP 810.3900:

Laboratory Testing 

Methods for Bed Bug 

Pesticide Products 
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NOTICE

This guideline is one of a series of test guidelines established by the Office of Chemical Safety and 

Pollution Prevention (OCSPP) [formerly the Office of Prevention, Pesticides and Toxic Substances 

(OPPTS) prior to April 22, 2010], United States Environmental Protection Agency for use in testing 

pesticides and chemical substances to develop data for submission to the Agency under the Toxic 

Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide and 

Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug and 

Cosmetic (FFDCA) (21 U.S.C. 346a), referred to hereinafter as the harmonized test guidelines.

The OCSPP test guidelines serve as a compendium of accepted scientific methodologies for 

research intended to provide data to inform regulatory decisions under TSCA, FIFRA, and/or 

FFDCA. This document provides guidance for conducting appropriate tests, and is also used by 

EPA, the public, and the companies that are required to submit data under FIFRA. These guidelines 

are not binding on either EPA or any outside parties, and the EPA may depart from them where 

circumstances warrant and without prior notice. The methods described in these guidelines are 

strongly recommended for generating the data that are the subject of the guidelines, but EPA 

recognizes that departures may sometimes be appropriate. You may propose alternatives to the 

methods described in these guidelines, with supporting rationale. The Agency will assess them for 

appropriateness on a case-by-case basis. 

For additional information about the harmonized test guidelines and to access the guidelines 

electronically, please go to http://www.epa.gov/oppts and select “Test Methods & Guidelines” on the 

left side navigation menu. You may also access the guidelines in http://www.regulations.gov grouped 

by Series under Docket ID #s: EPA-HQ-OPPT-2009-0150 through EPA-HQ-OPPT-2009-0159, and 

EPA-HQ-OPPT-2009-0576. EPA-HQ-OPP-2011-1017 is the docket number for this draft guideline. 

DRAFT DOCUMENT DISCLAIMER: This draft document is distributed solely for the purpose of 

external review. It has not been formally disseminated by the EPA and should not be construed to 

represent any Agency determination or policy. The information correction process under the 

Agency’s Information Quality Guidelines does not apply until this document is formally disseminated 

by the EPA in its final form. This draft document should only be cited or quoted in the context of 

providing comments. 
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OPPTS 810.3900: Draft Laboratory Testing Methods for Bed Bug Pesticide Products 

(a) Introduction.

(1) Scope and Purpose. This guideline provides recommendations for the design and execution of 

laboratory studies to evaluate the performance of pesticide products intended to repel and/or kill 

the common bed bug (Cimex lectularius) in connection with the products’ registration under the 

Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.). This 

guidance applies to products in any formulation—such as a liquid, aerosol, fog, or impregnated 

fabric —intended to be applied to attract, repel or kill bed bugs. It does not apply to repellent 

products applied to human skin. This guidance recommends appropriate laboratory study 

designs and methods for evaluating pesticides, statistical analysis, and reporting.

(2) Organization of the Guideline. The guideline begins with the Introduction (a). Definitions of 

special importance in understanding this guideline are listed in Section (b). Sections (c) through 

(g) provide general guidance applicable to protocol development for bed bug product efficacy 

testing conducted in the laboratory and the field. Sections (h) through (o) provide specific 

guidance on product laboratory testing. Section (p) is a list of references. The guideline sections 

are as follows: 

(a) Introduction

(b) Definitions

(c) Development of protocols for bed bug studies.

(d) Review of protocols for bed bug studies.

(e) Execution of bed bug studies.

(f) Reporting of completed bed bug studies.

(g) Retention of records.

(h) Recommended materials, procedures and environmental conditions for bed bug efficacy 

tests.

(i) Specific guidance for laboratory studies for resistance ratio determination, 

characterization of bed bug population strain susceptibility, and discriminating dose 

selection.

(j) Specific guidance for laboratory studies for forced exposure (no-choice) residual surface 

treatments with concentrates and ready to use products.

(k) Specific guidance for laboratory studies to determine if bed bugs are repelled by, or 

attracted to, pesticide product residues.

(l) Specific guidance for laboratory studies for testing pesticide impregnated material 

products.

(m)Specific guidance for laboratory studies of indoor fogger products. 

(n) Specific guidance for laboratory studies for direct application testing of pesticide 

products.

(o) Specific guidance for laboratory studies for testing ovicidal products.

(p) References

(3) General Considerations. Any protocol and/or study developed using this guidance must meet 

the requirements set forth in several statutes and regulations, including, but not limited to, the 

Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA, 7 U.S.C. 136, et seq.) under which 

EPA regulates pesticides.
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This guideline does not supersede or overrule the regulations governing research conducted with 

human subjects contained in 40 CFR Part 26, subparts K through Q, or any other Agency 

regulations. To the extent there are any unintended conflicts between this guideline and any 

EPA regulation, the regulation at issue governs. 

(4) Related Requirements.

(i) Good Laboratory Practice Standards. Good Laboratory Practice Standards (GLP) set 

forth in 40 CFR Part 160 apply to laboratory studies of pesticide efficacy. Studies that do 

not comply with GLP standards may nonetheless be considered if, in the Agency’s 

judgment, the design and conduct of the study are sufficient to demonstrate that the 

results are scientifically reliable. 40 CFR §160.12(b) requires with any submitted 

research data “[a] statement describing in detail all differences between the practices used 

in the study and those required by this part.” Additionally, Part 158 specifies that 

“applicants must adhere to the good laboratory practice (GLP) standards described in 40 

CFR Part 160 when conducting studies.” [40 CFR §158.70(b)].

(ii) State requirements. Investigators and Sponsors should ensure research is conducted in 

compliance with any applicable state laws or regulations, which are independent of and 

additional to those cited here.

(b) Definitions. The following definitions are of special importance in understanding this guideline. They 

apply only in the context of this guideline and are not intended to be more generally applicable.

(1) A repellent is a substance intended to disrupt the host-seeking behavior of insects or other 

arthropods including bed bugs, driving or keeping them away from a treated substrate in the 

presence of a host.

(2) The following events may be used in measuring efficacy for pesticide impregnated materials.

(i) Crossing is the act of passage by a bed bug from an untreated surface to a treated surface. 

(ii) A host is a warm-blooded animal that bed bugs bite to obtain blood for their survival and 

reproduction.

(iii) Questing is the behavior of bed bugs actively seeking a host.

(3) An attractant is a substance that causes arthropods including bed bugs, to make oriented 

movements towards its source. 

(4) Mortality refers to bed bug death. A dead bed bug is a bed bug that does not move, even when 

poked or probed.
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(5) LT50 is the time required to kill 50% of a test bed bug population following exposure to an

insecticide treatment.

(6) LT90 is the time required to kill 90% of a test bed bug population following exposure to an

insecticide treatment.

(7) LT100 is the time required to kill 100% of a test bed bug population following exposure to an

insecticide treatment.

(8) LD50 is the dose of toxicant required to kill 50% of a bed bug population.

(9) LD90 is the dose required to kill 90% of a bed bug population.

(10) Knocked down refers to a bed bug that is unable to right itself following exposure to a pesticide 

product.

(11) KT50 is the time required to knockdown 50% of a test bed bug population following exposure to 

an insecticide treatment.

(12) KT90 is the time required to knockdown 90% of a test bed bug population following exposure to 

an insecticide treatment.

(13) Pesticide resistance is a heritable decrease in the susceptibility of a bed bug population to a 

pesticide. This change is revealed in the repeated failure of a product to achieve the expected 

level of control when used according to the label recommendation for that pest species. 

(14) Resistance Ratio (RR) is a quantitative expression of the resistance of a bed bug to a specific 

active ingredient or product formulation. A resistance ratio is calculated by dividing the LT or

LD value of the resistant bed bug population by the corresponding LT or LD value of the 

susceptible population.

(15) A resistant bed bug population is a strain of bed bugs that is capable of surviving a dose lethal 

to a majority of individuals in a normal population. A resistance ratio equal to or greater than 100

is a characteristic of a resistant strain.

(16) A susceptible bed bug population is a strain that is incapable of surviving exposure to pesticide 

dose lethal to the majority of individuals in a susceptible population. Ideally, a susceptible 

population will not have a history of insecticide exposure. 

(17) A discriminating dose is a single dose that should affect 90 percent of the individuals in a 

susceptible bed bug population without affecting bed bugs in a resistant population. This is the 

minimum dose required to kill a susceptible or normal bed bug population.

(18) A harborage is a sheltered area or refuge for bed bugs.

(19) An ovicidal product is a pesticide product that kills bed bug eggs.
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(c) Development of protocols for bed bug studies. The first major stage of bed bug product testing is 

development of a protocol. 

General considerations in developing a protocol for bed bug studies include its scientific design, data 

collection, data analysis and reporting. Each of these topics is discussed in more detail in the subsections below.

(1) Scientific Design of Research. To be scientifically justified, the proposed research should 

address an important research question that cannot be answered by existing data. In addition, the 

design should be likely to provide a definitive answer to the research question. The design 

should include a detailed description of the experimental design, addressing topics (i) through 

(x).

(i) Objectives. In the case of products claiming to kill and/or knockdown bed bugs, the 

objective of bed bug efficacy testing is to determine if a product application made at the 

lowest proposed label rate kills at least 90% of the bed bugs and/or achieves a LT90. For 

products claiming to repel bed bugs from treated surfaces or insecticide impregnated 

textiles, the objective of efficacy testing is to compare the efficacy of one treatment to 

another treatment and/or an untreated control. In all cases the scientific objective should 

be stated clearly. 

(ii) Test materials. Efficacy should be tested using the end-use formulation as registered or 

as proposed for use. Test materials should be stored at ambient temperature and humidity 

for one day before use. 

(iii) Dose determination. To estimate a test dose the endpoint of concern is a rate of 

application, typically expressed by weight as ounce(s) per square foot or volumetrically 

as fluid ounces per cubic foot. A rate may also be expressed using metric system 

measurements; by weight as mg of test substance /cm2 of test surface or volumetrically as 

ml of test substance/cm2 of treated surface. Typically, the rate of application from which 

dose is determined is the lowest application on the proposed product label.

(iv) Choice of endpoints and measures. Endpoints chosen for the study should be 

appropriate for the specific objectives of proposed research and likely to provide a robust 

answer to the research question. Considerations in endpoint selection for dose 

determination include:

(A) Efficacy. The endpoint of efficacy testing should be a test dose that demonstrates 

bed bug kill or repellency as claimed on the product label. 

1. In repellency testing, the events that demonstrate repellent efficacy failure 

are: moving from an untreated to a treated area in the presence of a host; 

lack of inhibition of blood feeding when exposed to the repellent; and lack 

of preference for an untreated harborage compared to a repellent treated 

harborage.
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(v) Test organisms. Testing should be conducted with the adult life stage of the common 

bed bug, Cimex lectularius. The ratio of males to females should be 1:1. Five bed bug 

strains should be tested including a susceptible strain, a resistant strain and three field 

strains collected from urban areas in three different regions of the U.S. The preferred 

susceptible strain is the Harlan strain, which is known to have no history of pesticide 

exposure. For resistant and field strains, the level of resistance to a pesticide when 

compared to the susceptible strain, expressed as a resistance ratio, should be 

characterized as stated in this guideline and reported. 

(vi) Representative sampling. The sampling scheme should target U.S. bed bug 

populations. 

(A) Sample size. The sample should be large enough to be likely to yield a definitive 

answer to the research question being addressed, and its size should be justified 

statistically, taking into account the specific characteristics of the proposed 

research and the necessary accuracy and precision of the results. Researchers are 

encouraged to consult a statistician to determine appropriate sample size. The 

protocol should fully describe how sample size was determined.

Other factors that may affect sample size are the number of treatments, the experimental 

design, and the heterogeneity of the sample bed bug population (e.g., developmental 

stage, gender, insecticide susceptibility) and the environment (different habitat population 

densities). 

(vii) Untreated controls. An untreated control should be included in all testing. The number 

of untreated control replicates should equal the number of replicates for each treatment. 

(viii) Positive controls. A positive control may be appropriate under some testing 

circumstances, such as determining the magnitude of bed bug tolerance or resistance to 

insecticides.

(ix) Statistical analysis plan. Protocols should include a full description, explanation, and 

justification for the statistical methods proposed to analyze both dose determination and 

efficacy test results, taking into account the specific study objectives and variables.

(x) QA/QC plan. Protocols should provide for periodic quality assurance inspections 

adequate to ensure the integrity of the study and consistent with the requirements of 

EPA’s Good Laboratory Practices regulations (40 CFR part 160).

(2) Data Collection and Reporting. Protocols should provide for collection and reporting of data 

covering all aspects of the research including the following elements in addition to those 

discussed in section (g) of this guideline.
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(i) Elements required by GLP regulations. Each protocol should provide for collecting 

and reporting all elements required by the Good Laboratory Practices regulation at 40 

CFR §160.120.

(d) Review of protocols for bed bug studies. EPA strongly recommends that bed bug efficacy study 

protocols be submitted to the Agency for review before the research is initiated. EPA particularly 

recommends protocol submission in the case of field efficacy studies, because those types of studies 

may be intentional exposure human studies covered by 40 CFR part 26. 

(e) Execution of bed bug studies.

(1) Execution of protocol. In cases where a protocol has been submitted to EPA for review, the 

research can be initiated when the EPA review is completed. 

(2) Quality Assurance (QA) oversight. Efficacy testing, whether conducted in the field or in the 

laboratory, is subject to the Good Laboratory Practices regulations at 40 CFR Part 160. These 

rules require that each testing facility include an independent QA unit and that the QA unit 

monitors execution of each protocol and document its conduct in accordance with the GLP 

regulations (40 CFR §160.35). The QA Unit will inspect each study at intervals adequate to 

ensure the integrity of the study and maintain written and properly signed records of each 

periodic inspection.

(3) Deviations from protocol. Even when executing the best-designed and most comprehensive 

protocols, unanticipated deviations from the protocol may occur. All such deviations from the 

protocol and their impact on the research should be fully reported in the study report submitted to 

EPA. (40 CFR §160.185) EPA makes a distinction between planned changes to a protocol and 

deviations from a protocol. 

(f) Reporting of completed bed bug studies

(1) Study report. In addition to the standard elements described in the GLP regulation at 40 CFR 

§160.185 and to the appendices described below, the primary report of the study should include 

these elements, as applicable.

(i) Study identification: Title, identifying study number(s), sponsor, study director, 

 investigators, name and location of the testing facility, and dates of the study. 

(A) Testing at locations outside the U.S. If tests are conducted outside the U.S., the 

relevance of the study for U.S. regulatory purposes should be justified in the study 

report.

(ii) Approved or proposed label directions for use; preferably as a copy of the proposed or 

 approved product label.

(iii) Study objective(s) 
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(iv) Test conditions: Temperature, relative humidity, ambient light and photoperiod, and air 

flow (where applicable).

(v) Test system, including but not limited to: 

• Test organisms including genus, species, and strains of susceptible, resistant and 

field bed bug populations; development stage, age and sex of bed bugs; and 

preparation of bed bugs for test (feeding/starving).

• Bed bug rearing, handling and maintenance

• Description of test substance, i.e., product or formulation to be tested

• Description of the experimental unit 

• Description of each product treatment and the untreated controls

• Treatment dose and method of application

• Number of product treatments

• Number of untreated control treatments

• Number of replicates per treatment 

• Number of bed bugs per replicate for each treatment.

• Length of bed bug exposure period to each treatment. 

• When appropriate, provide a description of a harborage for bed bugs with 

placement information. A harborage is typically provided in studies employing 

choice testing.

• When appropriate, the source of a bloodmeal. 

(2) Data reporting, Report the following information:

• Test results on all aspects of the research

• Copies of all raw data sheets. 

• Endpoints for each replicate of every treatment 

• Endpoint for each treatment

• Certification of the test substance’s identity and origin

• A copy of the protocol with all amendments and deviations

• Justification for selecting the approach to data analysis and statistical testing.

• A copy of the statistical analyses

(3) Study Conclusions. The report should include a discussion of the study results and conclusions 

based on treatment endpoints. Conclusions should state why and how the study results do or do 

not support the tested hypothesis.

(g) Retention of Records. The following record-keeping requirements apply to some or all records of 

research covered by this guideline. 

(1) The record-keeping requirements of 40 CFR §160.190 and §160.195 apply to records of any 

study conducted under the Good Laboratory Practices rule.
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(h) Recommended materials, procedures and environmental conditions for bed bug efficacy tests. The 

following materials, procedures and environmental conditions are recommended for the efficacy test 

methods described in Sections (i) to (n) unless otherwise specified: 

(1) Test substance: The test substance should be the pesticide product or formulation as proposed 

for use. 

(i) Liquids. For liquid concentrates, testing should be done with the lowest active ingredient 

concentration applied at the lowest application rate on the proposed label for bed bug 

control. Ready-to-Use products should be applied at the label rate without dilution. 

(ii) Dusts. The lowest label rate proposed for a dust formulation/product should be 

evaluated.

(2) Test organisms: Testing should be conducted with five strains of the common bed bug, Cimex 

lectularius – one susceptible strain, one resistant strain, and three field strains. Field strains 

should be collected from urban areas in three regions of the U.S. The preferred susceptible strain 

is the Harlan strain, which is known to have no history of pesticide exposure. For resistant and 

field strains, the level of resistance to a pesticide when compared to the susceptible strain should 

be characterized as stated in section “(i) Specific guidance for laboratory studies for 

resistance ratio determination, characterization of bed bug population strain susceptibility, 

and discriminating dose selection” of this guideline and reported. The bed bug life stage to be 

used in testing is specific to the target on the proposed label or test protocol as outlined below.

(i) Formulations that target bed bug adults. Testing should be conducted with unfed adult 

bed bugs. The ratio of males to females should be 1:1.

(ii) Formulations that target bed bug metamorphosis. Testing should be conducted with

mixed nymphal stages. Egg testing may be conducted but should be justified.

(iii) Formulations that target bed bug eggs (ovicidal product formulations). Testing 

should be conducted with the egg life stage. 

(3) Bed bug rearing, handling and maintenance. A description of the bed bug laboratory colony 

and rearing practices should be included. Maintenance procedures for field-collected strains 

should be described.

(4) Testing conditions. The temperature during the test should be kept at 25°C, with a relative 

humidity of 50-80%, and a photoperiod of 16:8. The temperature during the test should be kept 

as constant as possible because changes can affect the performance of the product treatments.

(5) Disposition of test bed bugs. Bed bugs should be used for only one test and should be 

destroyed immediately after the trial.
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(i) Specific guidance for laboratory studies for resistance ratio determination, characterization of bed 

bug population strain susceptibility, and discriminating dose selection. Response ratio 

determination, commonly known as a “Resistance Ratio”, is useful in characterizing the magnitude of 

tolerance or resistance to pesticides used in bed bug control. Documenting the susceptibility of bed bug 

population strains used in product testing is essential to determining test dose selection and ultimately, 

the discriminating dose. A large number of bed bugs will be needed to complete this analysis and 

collection of field strains is necessary. Use of field strains from at least three EPA Regions is 

recommended. 

(1) Study 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. 

(2) Materials and methods. 

(i) Identification of the test system

(A) Experimental units. One piece of white filter paper should be taped to the 

bottom of a 500 ml glass beaker. Paper should be taped in a manner that prevents 

bed bugs from crawling beneath. Each insecticide concentration should be 

applied to filter paper at a volume that saturates the paper, generally 50-100ul 

(100 ul = 0.1 ml). Papers should be allowed to dry for a minimum of three hours. 

An equal number of untreated control beakers with paper that was treated with the 

diluent should also be prepared. Papers should be weighed before and after 

treatment.

(B) Number of treatments. Five concentrations of the active ingredient should be 

prepared with the appropriate diluent. Active ingredient concentrations should be 

prepared based on a logarithmic scale, i.e., 0.0001%, 0.001%, 0.01%, 0.1%, and 

1.0%. Other concentrations may be used based on a previous knowledge of bed 

bug susceptibility to the insecticide being tested. The untreated control should be 

treated with the diluent for insecticide solution preparation. If a product contains 

a synergist, only the insecticide-synergist combination should be tested with 

solution concentration based on the active ingredient, not the synergist.

(C) Number of replicates and bed bug density. The test should be replicated ten 

times for each concentration with 20 bed bugs in each replicate.

(D) Bed bug exposure to treatments. Bed bugs should be exposed to the filter paper 

in each treatment for 24 hours. 

(E) Positive control. For the testing of pyrethroid insecticides, deltamethrin is 

recommended as a positive control. 

(F) Lethal dose values. A probit analysis is recommended to determine the LD50 and 

LD90 values for each bed bug strain tested. Concentration-Response (dosemortality) lines should be generated. If the data do not fit the probit model, 
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include an explanation for a lack of fit to the model, i.e., experimental error or

genetic heterogeneity in the target field population.

(G) Resistance ratios (RR). Resistance ratios should be calculated as follows: 

LD50 (field strain)/LD50 (susceptible strain) = RR50

LD90 (field strain)/LD90 (susceptible strain) = RR90

(H) Resistance ratio (RR) with deltamethrin when testing pyrethroid insecticides. 

LD50 (field strain)/LD50 (susceptible strain) = RR50 for deltamethrin

LD90 (field strain)/LD90 (susceptible strain) = RR90 for deltamethrin

(I) Discriminating dose selection. It is recommended that the discriminating dose 

be selected based on the LD90 for the least susceptible field population tested. If 

the product contains more than one active ingredient, the LD90 for the active 

ingredient that was least efficacious against bed bugs should be used.

(3) Reporting results. Study results should be reported in table form and be accompanied by a 

written summary.

(i) Protocol with amendments and deviations. A copy of the study protocol should be 

included with amendments and deviations. Protocol deviations should be described 

together with their impact on the validity of the study. 

(ii) Data and endpoints. Treatment data should be corrected for untreated control mortality 

with Abbott’s Formula or the equivalent. 

(A) Amount of product applied. The amount of product, expressed as weight, 

should be reported. 

(B) Knockdown and mortality. For each strain, report the percentage of bed bugs 

killed and knocked down, exclusively, for each treatment at each observation 

interval. 

(C) Results of probit analyses. Provide a complete set of probit analyses for all of 

the strains tested. Goodness-of-Fit test statistics should be provided.

(D) Resistance ratios. The resistance ratios for each bed bug strain should be 

reported.

(iii) Discriminating dose. The selected dose should be reported.
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(4) Study conclusions. Describe the conclusions and justify the selection of the discriminating 

dose.

(j) Specific guidance for laboratory studies for forced exposure (no-choice) residual surface 

treatments with concentrates and ready to use products 

(1) Study objective: to determine the efficacy of a residual product formulations treatment made to 

five different surfaces in a forced exposure (no-choice) test.

(2) Materials and methods

(i) Identification of the test system

(A) Number treatments and experimental units. For each product, an application 

should be made to each of five surfaces. An equal number of untreated control 

treatments should be established on the same surfaces. The surfaces to be treated 

with the product should be: 1) unpainted 1⁄4" thick plywood; 2) commercial 

linoleum tile; 3) concrete board; 4) 100% cotton sheeting; and 5) medium pile 

100% nylon carpet. Surfaces should be pre-cut to 6" by 6" panels. Cotton sheet 

replicates should be affixed to the top of a glass panel to provide a flat, rigid 

surface for treatment. In order to determine the length of residual performance 

time, testing should be repeated every seven days with aged panels until failure. 

A panel should be tested only once. At the beginning of the study, prepare 25 

treated (5 surfaces x 5 replicates per surface) and 25 untreated panels for each test 

interval for each bed bug strain tested. A minimum of 300 treated and 300 

untreated panels should be prepared to test up to three months of residual control 

for each bed bug strain. 

(B) Application with a dilution from a concentrate. The lowest label dilution for 

bed bug control, equivalent to the lowest application rate, should be sprayed on 

each 36 square inch panel. A metered bench top sprayer is preferred as the 

delivery device to ensure consistent application volume and even distribution of 

spray particles. Generally, panel spraying is performed from a distance of one 

foot above the panel surface. Panels should be stored under ambient test site 

conditions and allowed to dry for a minimum of three hours before exposing bed 

bugs to them. Report the volume of spray applied and the weight of active 

ingredient delivered. 

(C) Ready-to-use application. The amount of product delivered by the ready to use 

spray container, expressed as amount of product sprayed per second, should be 

determined before treatments can be made. Five replicates of sprays lasting three 

seconds each should be made. The product container should be weighed before 

and after each replicate and the difference recorded. The mean value of the five 

replicates should be determined and the result divided by three to equal the 

average amount of product applied for each second of spraying. Panel treatments 

should be made with the amount of product to be sprayed per unit area as directed 
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by the label. Generally, panels are sprayed from a height of one foot above the 

surface. The same procedure should be conducted to evaluate dust product 

formulations except that application should be made from a height of six inches or 

less. Other modifications required to apply dusts should be described and 

justified.

(D) Number of replicates and bed bug density. A minimum of 5 replicates should 

be used for each product treatment and untreated control. Each replicate should 

have 10 bed bugs each. A total of 500 bed bugs should be used for each test 

interval for each bed bug strain tested. 

(E) Bed bug exposure to product treatments. For each replicate, bed bugs should 

be held in a glass Petri Dish with lubricant coated sides to prevent bed bug escape. 

The Petri dish should be inverted onto the test surface and tightly secured to 

prevent bed bug escape. Two types of timed exposures are commonly used to 

evaluate the efficacy of a product treatment for each evaluation interval.

1. Fixed exposure period. Bed bugs should be exposed to the product treated 

surface for a fixed time period, usually 24 hours. After 24 hours, transfer the 

bed bugs to a clean, untreated container for further observation and 

evaluation. Observations for knockdown and mortality should be made at 15 

and 30 minutes; and at 1, 2, 4, 24, 48, 72, and 96 hours, unless all bed bugs in 

the product treatment die or untreated control mortality exceeds 10%.

2. Continuous exposure period. Bed bugs should be kept in contact with the 

test surfaces until all bed bugs in the product treatment die or until 10% of the 

bed bugs in the untreated control die, whichever comes first. Bed bugs should 

not be transferred to clean, untreated containers.

(F) Positive control. A positive control is not recommended.

(3) Reporting results. Study results should be reported in table form for each testing interval and 

be accompanied by a written summary. Exposure time-response graphs from the probit analyses 

should be provided.

(i) Protocol with amendments and deviations. A copy of the study protocol should be 

included with amendments and deviations. Protocol deviations should be described 

together with their impact on the validity of the study. 

(ii) Data and endpoints for fixed exposure times for each bed bug strain. Treatment data 

corrected for untreated control mortality with Abbott’s Formula or the equivalent should 

be reported.

(A) Knockdown and mortality data. After bed bug exposure to the treated surfaces 

begins, observations should be reported at 15 and 30 minutes; and at 1, 2, 4, 24, 

48, 72 and 96 hours. Report results should include the number of dead, knocked 

down and live bed bugs in each replicate. The percentage of bed bugs killed and 
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knocked down, exclusively, for each treatment at each test interval should be

reported. Confidence limits around the reported mean values should be reported.

(B) Data analysis. A statistical analysis should examine the significance of within 

treatment surface differences, differences between surfaces, and the difference 

between treated and untreated surfaces. An analysis of variance (ANOVA) is 

recommended but other analysis may be considered and should be justified. KT50

and KT90 values should be reported based on a probit analysis for each bed bug 

strain tested.

(iii) Data and endpoints for continuous exposure times for each bed bug strain. 

Treatment data corrected for untreated control mortality with Abbott’s Formula or the 

equivalent should be reported.

(A) Knockdown and mortality data. After exposure to the treated surfaces begin, 

observations should be reported at 15 and 30 minutes; 1, 2, 4, 24, 48, 72, 96 

hours; and every 24 hours daily thereafter until 100% of the bed bugs in the 

product treatment replicates die or untreated control mortality exceeds 10%. 

Report results should include the number of dead, knocked down and live bed 

bugs in each replicate. The percentage of bed bugs killed and knocked down, 

exclusively, for each treatment at each test interval should be reported. The length 

of the test period should be reported in hours or days (as appropriate for the 

longevity of the test). 

(B) Data analysis. Data should be analyzed with a probit analysis to determine the 

LT50, LT90 and LT100 for each surface. Results of Goodness-of–Fit testing should 

be provided. KT50 and KT90 values should be reported based on a probit analysis 

for each bed bug strain tested.

(4) Study conclusions. Summarize study outcomes and discuss their implications for product 

labeling.

(k) Specific guidance for laboratory studies to determine if bed bugs are repelled by or attracted to 

pesticide product residue. For pesticide product treatments to be efficacious, bed bugs must contact 

pesticide residues. 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.

(1) Study objective: To determine if bed bugs are repelled by or attracted to pesticide product 

formulation residue in a choice-test. 

(2) Materials and methods

(i) Identification of the test system

(A) Treatments and experimental units. The treatments will evaluate group and 

individual bed bug responses. A container with sides and an area large enough to 

fit up to three harborages should be used as the experimental arena for each set of 
Page 16 of 26

replicates. The sides should be lubricated to prevent bed bug escape and the 

bottom covered with filter paper that has been secured with tape to prevent bed 

bugs from crawling under it.

1. Product treatments. The product treatments are: diluent treated control filter 

paper; product treated filter paper; diluent treatment to conditioned filter 

paper; and product treatment to conditioned filter paper. 

Filter papers should be weighed before and after treatment. 

2. Filter paper conditioning. For each bed bug strain tested, conditioning 

should consist of exposing at least ten bugs to the papers for at least 24 hours 

in order for bed bugs to deposit excretions typically found in harborages.

3. Experimental unit 1. Each replicate should have two pieces of filter paper, 

one treated with the pesticide product and the other with the product diluent or 

water if the product is not diluted. Once dry, each paper should be folded in 

half to form a “tent” that will serve as a harborage during the experiment. The 

tents should be placed on the bottom of the container. Another construction 

type may be used provided it is fully described and justified.

4. Experimental unit 2. The set-up is the same as described in experimental 

unit 1 except that only conditioned filter papers should be used. 

5. Experimental unit 3. This unit should include an unconditioned pesticide 

treated filter paper and a conditioned pesticide treated filter paper and a

diluent treated conditioned filter paper. 

 (B) Number of replicates, bed bug density and exposure to treatments. Two 

procedures are described. Other treatment and replication procedures may be 

considered provided they are fully described and justified. 

1. Individual responses. Individual adult bed bugs should be placed in each 

treatment arena. A minimum of 20 replicates with 1 bed bug in each replicate 

should be used for each treatment. A total of 60 bed bugs (1:1 sex ratio) 

should be used in this test for each strain tested. Exposure time should be a 

minimum of one hour but not more than 24 hours.

2. Group response. Each replicate should have ten bed bugs. The test should 

be replicated 5 times for each treatment. A minimum of 50 bed bugs are 

recommended for each treatment for each strain tested. Exposure time should 

be a minimum of one hour but not more than 24 hours.

(C) Positive control. A positive control is not recommended.
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(3) Reporting results. Study results should be reported in table form and be accompanied by a 

written summary. Graphical summaries, such as histograms, should be provided to describe the 

results.

(i) Protocol with amendments and deviations. A copy of the study protocol should be 

included with amendments and deviations. Protocol deviations should be described 

together with their impact on the validity of the study. 

(ii) Data and endpoints. Treatment data should be corrected for untreated control mortality 

with Abbott’s Formula or the equivalent. 

(A) Amount of product applied. Report the amount of product applied to each piece 

of filter paper.

(B) Bed bug location observations. Report the location of bed bugs at the end of 

every test. Report the number of dead, knocked down, and live bed bugs in each 

replicate. 

(C) Mortality and knockdown. Report the percentage of bed bugs killed and 

knocked down, exclusively, for each treatment at each test interval for group and 

individual response testing. 

(iii) Data analysis. An analysis should determine:

• whether there is a difference between the treated and untreated harborage without 

conditioning for individual responses

• whether there is a difference between the treated and untreated harborage with 

conditioning for individual responses

• whether there is a difference between the treated unconditioned and treated 

conditioned harborages for individual responses

The aforementioned analyses should be conducted for the group responses.

• Whether there is a difference between the individual and group responses

(4) Study conclusions. Based on the results, address whether or not bed bugs were repelled, 

attracted or indifferent to pesticide residue.

(l) Specific guidance for laboratory studies for testing pesticide impregnated material products. 

(1) Study objective. To test the efficacy of a pesticide impregnated material product formulations 

against the bed bug. This test should evaluate impregnated material repellency or attraction in the 

presence of a host and blood feeding inhibition.

(2) Materials and methods

(i) Identification of the test system

(A) Number of treatments and experimental units. 
Page 18 of 26

1. Experimental Unit. The tunnel assay is commonly used to evaluate the 

efficacy and persistence (mortality and blood-feeding inhibition) of 

insecticide treated or impregnated fabric or textile. Generally, a tunnel 

system is composed of glass or plastic cylinder surrounded by square 

supports at regular intervals that rest on a flat surface. The tunnel should 

be approximately 25 cm high, 21 cm wide and 120 cm long. A bait 

chamber containing a host animal (short-haired guinea pig or small rabbit) 

housed unconstrained in a metal cage (with a 1/2-3/4′′ hardware cloth) and 

provided with food and water is placed in the second section adjacent to 

the tunnel end. Three treatments are recommended. Instead of using an 

animal host, blood may be provided through an artificial membrane 

system that is installed at the end of the tunnel or through a hole in the 

bottom of the bait chamber. Carbon dioxide should be used as an 

attractant. 

a. Treatment 1. The tunnel chamber should be divided evenly into two 

sections. The first section should be lined with untreated (pesticide 

free) product fabric or textile, while the second section is lined with 

pesticide impregnated product fabric or textile.

b. Treatment 2. The tunnel should be lined only with pesticide 

impregnated product fabric or textile.

c. Treatment 3. The tunnel should be lined only with untreated product 

fabric or textile. 

(B) Number of replicates and bed bug density. The test should be replicated five 

times for each treatment. Twenty unfed adult bed bugs (sex ratio 1:1) should be 

used for each replicate. The blood source should be replaced for every test. 

(C) Bed bug exposure to the treatments. Unfed adult bed bugs should be released 

through an opening in the tunnel located at the end opposite of the bait chamber 

and left in the dark for 24 hours. At 24 hours, the blood source should be 

removed. For each treatment, bed bugs should be recorded as live or dead and fed 

or unfed. For treatment 1, the location of each bed bug should also be recorded. 

Longer exposure and evaluation periods should be described and justified.

(D) Positive control. A positive control is not recommended.

(3) Reporting results. Study results for should be reported in table form and be accompanied by a 

written summary. 

(i) Protocol with amendments and deviations. A copy of the study protocol should be 

included with amendments and deviations. Protocol deviations should be described 

together with their impact on the validity of the study. 
Page 19 of 26

(ii) Data and endpoints. Treatment data should be corrected for untreated control mortality 

with Abbott’s Formula or the equivalent.

(A) Mortality data. The number of live and dead bed bugs should be reported for 

each replicate and treatment for each strain. The percentage of bed bugs killed in 

each treatment should be reported. Report confidence limits around the mean 

values.

(B) Bed bug locations in treatment 1. The location of each bedbug should be 

reported. 

(C) Blood feeding. The number of blood fed and unfed bed bugs in all replicates and 

treatments should be reported.

(D) Blood feeding inhibition. Percent reduction (inhibition) of bed bug blood 

feeding should be based on the following calculation:

Blood Feeding Inhibition (BFI) = 100 -

 Control

(Treated x 100)

(E) Data analysis. For each bed bug strain: 

1. Evaluate the effects of the product treatment on blood feeding 

inhibition.

2. Evaluate the effects of the product treatment on bed bug mortality.

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

treatment.

(4) Study conclusions. Discuss the efficacy (mortality and persistence) of the impregnated 

pesticide product.

(m) Specific guidance for laboratory studies for testing indoor pesticide fogger products. This guidance 

applies to testing total release foggers and space sprays including misters, and hand-held aerosol 

products used for indoor applications.

(1) Study objective: to determine the efficacy of pesticide fogging product formulations against bed 

bugs. 

(2) Materials and methods.

(i) Identification of the test system

(A) Experimental unit. Testing should be conducted in a Peet-Grady chamber with a 

volume of 216 cubic feet or greater. The chamber should have a window for 

observation. The wall, ceiling and floor of the room may be lined with plastic or 

other suitable materials to facilitate cleaning. Test doses for aerosols, misters and 

fogger products should be delivered by an automatic dispenser calibrated for the 
Page 20 of 26

proper droplet size and application rate. At the end of each replicate, the air in the 

chamber should be exhausted and any surface residues washed off. Surfaces 

should be clean and dry before the next test. Alternative product application 

methods may be considered but should be described and justified.

(B) Number of treatments. There should be one product treatment and one 

untreated control treatment for each bed bug strain.

(C) Replication and bed bug density. Bed bugs should be placed into cages for 

testing. Each cage should be a 6′′ long PVC pipe 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. Cage dimensions and 

design should be reported and other cage types should be justified as simulated 

refuges. Forty-eight cages should be prepared for each bed bug strain. Ten unfed 

adult bed bugs should be transferred to each cage. One half of the cages should 

be kept outside the chamber as untreated controls. In the Peet-Grady chamber 

two cages should be placed where the wall and floor meet; on the wall three feet 

from floor level; and where the wall and ceiling meet. This array should result in 

six cages arranged on each of four walls of the chamber before the fogger 

application is made.

(D) Bed bug exposure to the treatments. The chamber should be sealed and the 

product application made. After the application is made, the test cages should be 

left in place for two hours and removed after the chamber has been evacuated. 

Bed bugs should be left in the test cages. At 24 hours post-treatment, bed bugs 

from each cage should be transferred to a clean, untreated container. Replicates 

of the strains to be tested may be conducted in the chamber at the same time if 

space allows.

(E) Positive control. A positive control is not recommended.

(3) Reporting results. Study results should be reported in table form and be accompanied by a 

written summary. Graphical descriptions of the results, such as histograms, should be included.

(i) Protocol with amendments and deviations. A copy of the study protocol should be 

included with amendments and deviations. Protocol deviations should be described 

together with their impact on the validity of the study. 

(ii) Data and endpoints. Treatment data should be corrected for untreated control mortality 

with Abbott’s Formula or the equivalent.

(A) Mortality and knockdown. Mortality data should be reported from product and 

untreated control treatments from observations made at 24, 48, 72 and 96 hours 

post-treatment. For each strain, report the percentage of bed bugs killed and 

knocked down, exclusively, for each treatment replicate at each observation 

interval. Report strain specific pooled product treatment mortality data for each 
Page 21 of 26

height level and entire treatments as corrected mean values. Confidence limits 

around the mean values should be reported.

(B) Amount of product applied. The amount of product applied to each replicate 

should be reported.

(iii) Data analysis. The analysis should consider treatment, cage height, and bed bug 

strain effects on product efficacy. 

(4) Study conclusions. Address whether or not the fogger treatment was efficacious.

(n) Specific guidance for laboratory studies for direct application testing of pesticide products.

(1) Study objective: to determine the direct application efficacy of pesticide product formulations 

against bed bugs. 

(2) Materials and methods.

(i) Identification of the test system

(A) Experimental unit. Testing should be conducted with caged bed bugs. 

Typically, a test cage unit is a 16 oz squat plastic cup with a screened bottom that 

has the inside lined with a lubricant to prevent bed bug escape. Other cage 

designs are acceptable provided the spray does not pool in the cage after spraying. 

A metered bench top sprayer is preferred as the delivery device to ensure 

consistent application volume and even distribution of spray particles. 

Applications should be made at the lowest label rate. Application should be made 

from one foot above the test cage. Report the weight applied and the weight of 

active ingredient delivered.

(B) Number of treatments. The test should include a product treatment and 

untreated control. One half of the cages should be allotted to product treatment 

while the other half should be for the untreated control. 

(C) Replication and bed bug density. The test should be replicated 5 times for each 

treatment. Each replicate should have ten bed bugs.

(D) Bed bug exposure to the treatments. Bed bugs should be transferred to clean 

cups in less than 10 minutes after product application. Cups should be stored 

under ambient test site conditions. Observations for knockdown and mortality 

should be made at 30 minutes, 1, 2, 4, 24, 48, 72, and 96 hours; unless until all 

bed bugs die or untreated control mortality exceeds 10%. Repeat this procedure 

for each strain to be tested. 

(E) Positive control. A positive control is not recommended.
Page 22 of 26

(3) Reporting results. Study results should be reported in table form and be accompanied by a 

written summary. Probit analysis should be included.

(i) Protocol with amendments and deviations. A copy of the study protocol should be 

included with amendments and deviations. Protocol deviations should be described 

together with their impact on the validity of the study. 

(ii) Data and endpoints. Product treatment data should be corrected for untreated control 

mortality with Abbott’s Formula or the equivalent.

(A) Amount of product applied. The amount of product, expressed as weight of 

product, applied to each replicate should be reported. 

(B) Knockdown and mortality. Results should report observations made at 30 

minutes; 1, 2, 4, 24, 48, 72, and 96 hours, unless all bed bugs die or untreated 

control mortality exceeds 10%. The percentage of bed bugs killed and knocked 

down, exclusively, for each treatment at each observation interval should be 

reported. Report the confidence limits around the mean values for each treatment.

(iii) Data analysis. KT50 and KT90 data should be reported and may be based on a probit 

analysis for each bed bug strain tested. An analysis should be performed to determine if 

the mean product treatment mortality differed from the untreated control mortality and if 

any within treatment effects were significant. 

(4) Study conclusions. Discuss knockdown and killing efficacy of the product treatment.

(o) Specific guidance for laboratory studies for testing ovicidal products. 

(1) Study objectives: to determine the efficacy of ovicidal products formulations.

(2) Materials and methods. 

(i) Identification of the test systems 

(A) Treatments

1. Direct application. 

a. Experimental test unit. Testing should be conducted with 

viable bed bug eggs laid on filter paper. A tissue culture plate is 

recommended as the test container but small Petri dishes may be 

used. Pieces of egg-laden filter paper should be cut into piece that 

should fit into the depressions on the plate or in a Petri dish. Up to 

six eggs should be allotted to each well or depression. 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 label rate. 
Page 23 of 26

Application should be made from one foot above the test surface.

For a dust formulation, application should be made at the lowest 

label rate from a height of six inches or less. Record the weight of 

formulation applied and the weight of active ingredient delivered. 

An untreated control should be established on the same type of 

plates. 

2. Contact with residual surface application. 

a. Experimental unit 1. A 5-7cm Petri dish is recommended as the 

test container. A piece of conditioned filter paper should be treated 

with the product formulation at the lowest label rate. Another piece 

of conditioned filter paper should be left untreated to serve as the 

control. One piece of paper should be placed into each dish. One 

newly molted female bed bug, which is gravid, should be placed 

into each dish and left in the dish for up to seven days to oviposit. 

After oviposition is complete, the bed bugs should be removed and 

the dish covered. 

b. Experimental unit 2. The set-up is the same as for unit 1 except 

that bed bug eggs are transferred to treated and untreated paper 

instead of being deposited by female bed bugs. Alternatively, egg

laden papers can be turned upside down and laid on treated and 

untreated surfaces. Use of this unit may be necessary when 

insecticide residue on the paper kills female bed bugs used in unit 

1 before they can oviposit.

(B) Number of treatments. The test should include a product treatment and 

untreated control. One half of the eggs should be allotted to product treatment 

while the other half should be for the untreated control. 

(C) Replication and bed bug density. Not all eggs laid by bed bugs in the untreated 

control group will hatch during testing. In order to account for this condition, at 

least 20 bed bug eggs should be included in every replicate to ensure adequate 

quantities of bed bug eggs are available for meaningful treatment comparisons. 

1. Direct application. The test should be replicated 10 times for each 

treatment. Each treatment replicate should have a minimum of 20 bed 

bug eggs.

2. Contact with residual surface application with experimental 

unit 1. The number of eggs laid by each female bed bug will be variable. 

A treatment should have 25 dishes. Each dish should be considered a 

replicate. 

3. Contact with residual surface application with experimental unit 2. 

Each dish is a replicate and should have 10 eggs. Twenty dishes should 

be allotted to each treatment. 
Page 24 of 26

(D) Exposure time. Plates or dishes should be stored in the laboratory. Observations 

for mortality and hatching should be made every 24 hours for up to 30 days. Eggs 

should be examined microscopically to determine if egg hatch has taken place. 

Eggs should be observed every day and the number of un-hatched and hatched 

eggs should be recorded for the product formulation treatment and untreated 

control. Eggs should be examined to determine if they are embryonated.

(E) Positive control. A positive control is not recommended.

(3) Reporting results. Study results should be reported in table form and be accompanied by a 

written summary. 

(i) Protocol with amendments and deviations. A copy of the study protocol should be 

included with amendments and deviations. Protocol deviations should be described 

together with their impact on the validity of the study. 

(ii) Data and endpoints. Product treatment data should be corrected for untreated control 

mortality with Abbott’s Formula or the equivalent.

(A) Amount of product applied. The amount of product, expressed as weight of 

product, applied to each replicate should be reported. 

(B) Egg mortality. Results should report observations made at 24 hours and daily 

thereafter for a minimum of 14 but no more than 30 days post-treatment. The 

percentage of bed bug eggs killed for each treatment at each observation interval 

should be reported. Report the confidence limits around the mean values for each 

treatment.

(iii) Data analysis. An analysis should be performed to determine if the mean product 

treatment mortality differed from the untreated control mortality.

(4) Study conclusions. Discuss ovicidal efficacy of the product formulation treatment.

(p) References. The following publications were consulted for supporting guideline recommendations.

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http://www.icup.org.uk/reports/ICUP861.pdf
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