Page 1 of 26 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

O CSPP 810.3900: Laboratory Testing Methods for Bed Bug

Pesticide ProductsPage 2 of 26 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 de velop 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, FIF RA, 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. T hese guidelines are not binding on either EPA or any outside partie s, 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 harmoniz ed 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 h ttp://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: T his 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.Page 3 of 26 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 guida nce 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 appropria te 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 ar e 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 produc t la boratory 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 exposu re (no

- choice) residual surface treatments with concentrates and ready to use pr oducts. ( 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 guida nce 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 p esticides

.Page 4 of 26 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 t he 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. G ood 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 Sponsor s 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 r epellent 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 b ed bug from an untreated surface t o 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 b ed 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.Page 5 of 26 (5) LT 50 is the time required to kill 50% of a test bed bug population following exposure to an

insecticide treatment.

(6) LT 9 0 is the time required to kill 90% of a test bed bug population following exposure to an

insecticide treatment.

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

insecticide treatment.

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

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

(10) K n ock e d down refers to a bed bug that is unable to right itself following exposure to a pesticide product.

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

an insecticide treatment.

(12) KT 9 0 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 populatio n 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 d iscriminating 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 har borage is a sheltered area or refuge for bed bugs.

(19) An ovicid al product is a pesticide product that kills bed bug eggs.Page 6 of 26 (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 b ed bug studies include its scientific design, data collection, data analysis and reporting. Each of these topics is discu ssed in more detail in the sub

sections 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 LT 90

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

textiles , t he 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 ma terials. 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 e ndpoint 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 /cm 2 of test surface or volumetrically as

ml of test substance/cm 2 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. Th e 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.Page 7 of 26 (v) Test organisms. Testing should be conducted with the a dult life stage of the common bed bug, Cimex lectularius. The ratio of male s 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 di fferent 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, e xpressed 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 necessar y 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).

(vi i ) 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

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

insecticides.

(ix) Statistical analysis plan. P rotocols 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 regulat ions (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.Page 8 of 26 (i) Elements required by GLP regulations. Each proto col 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 r esearch 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 ad dition 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 stud y 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 pr oduct label.

(iii) Study objective(s)Page 9 of 26 (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 g enus, species, and strain s 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 treatmen t.

**•** Length of bed bug exposure period to each treatment.

**•** When appropriate, provide a description of a harborage for bed bugs with placement information. A ha rborage 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 re cords of any study conducted under the Good Laboratory Practices rule.Page 10 of 26 ( 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 propo se d

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

. T he lowest label rate proposed for a dust formulation/product should be

evaluated.

(2) Test organisms: Testi ng 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 i s 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 fo r 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 t hat target bed bug adults. Testing should be conducted with unfed adult bed bugs. The ratio of male s 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 ( o vicidal product formulations ). Testing

should be conducted with the egg life stage.

(3) Bed bug rearing, ha ndling 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.Page 11 of 26 (i) Specific guidance for laboratory studies for resistance ratio determination, characterization of bed bug population strain susceptibility, and discrim inating 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 d ose selection and ultimately, the discriminating dose . A large numbe r of bed bugs will be needed to complete this analysis and collection of field strains is necessary. U se 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 t est 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 sh ould 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 base d 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 combinat ion 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 treatment s

. 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 LD 50 and

LD 90 values for each bed bug strain tested. Concentration

- Response (dose

mortality) lines should be generated. If the data do not fit the probit model,Page 12 of 26 include an explanation for a lack of fit to the model, i.e., experimental err or or genetic heterogeneity in the target field population.

(G) Resistance ratios (RR) . 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

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

.

LD 50 (field strain)/LD 50 (susceptible strain) = RR 50 for deltamethrin

LD 90 (field strain)/ LD 90 (susceptible strain) = RR 90 for deltamethrin

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

be selected based on the LD 90 for the least susceptible field population tested. If the product contains more than one active ingredient, the LD 90 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 knoc ked 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.Page 13 of 26 (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 differen t surfaces in a forced exposure (no

- choice) test.

(2) Materials and m ethods

(i) Identification of the t est 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. C otton sheet replicates should be affixed to the top of a glass panel to provide a flat, rigid

s urface 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, p repare 25

treated (5 sur faces 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) A pplication 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 d istribution 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 bug s 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 produc t 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 sprayin g. Panel treatments should be made with the amount of product to be sprayed per unit area as directedPage 14 of 26 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 produc t 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 us ed 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 treatment s. For each replic ate, 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 use d 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 shoul d

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 time s for each bed bug strain. Treatment data corrected for untreated control mortality with Abbott’s Formula or the equivalent should be reported.

(A) K nockdown 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 andPage 15 of 26 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 sh ould 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 b e justified. KT 50 and KT 90 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 thereaft er 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 kno cked 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 prob it analysis to determine the

LT 50 , LT 90 and LT 100 for each surface. Results of Goodness

- of

– Fit testing should be provided. KT 50 and KT 90 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 m ethods

(i) Identification of the t est system

(A) Treatments and e xperimental unit s

. 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 ofPage 16 of 26 replicat es. The sides should be lubricated to pr event 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. P roduct t reatments. The product treatments are: diluent treated control filter

paper; product treated filter paper; d iluent 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 exposi ng 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 conditione d 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 th is test for each str ain tested. Exposure time shoul d 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 eac h treatment for each strain tested. Exposure time shoul d

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

(C) Positive control . A positive control is not recommended.Page 17 of 26 (3) Reporting results

. Study results should be reported in table form and be accompanied by a written summary. Graphical summaries, such as histogram s , 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 c ontrol 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 t reated 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 unconditione d 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 m ethods

(i) Identification of the t est 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

supp orts 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 i s installed at the end of the tunnel or through a hole in the

bottom of the bait chamber. C arbon 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 replicate d 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 report ed. 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 report ed.

(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 produ ct

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 effica cy of pesticide fogging product formulations against bed bugs.

(2) Materials and methods.

(i) Identification of the t est 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 fogg er products should be delivered by an automatic dispenser calibrated for thePage 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 u nfed 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 P eet

- 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 a pplication 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

include d 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 eachPage 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. A ddress 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 t est 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 do es 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 s hould 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 allotte d 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 treatm ents. 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

shoul d 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 untr eated 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 untr eated 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. KT 50 and KT 90 data should be reported and may be based on a probit analysis for each bed bug strain tested. An analysis should be performed to de termine 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 laborat ory studies for testing ovicidal products

.

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

(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

re commended as the te st 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

par ticles. 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 mad e at the lowes t label rate from a height of six inches or less. Record the weight of

formulation applied and the weight of active ingredient delivered.

An u ntreated control should be established on the same type of plates.

2. C ontact wi th 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 cond itioned 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 ovip osit. 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 deposi ted 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 ovip osit.

(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 avail able for meaningful treatment comparisons.

1. D irect application

. The test should be replicated 10 times for each

treatment. Each treatment replicate should have a minimum of 2 0 bed bug eggs.

2. C ontact 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 dishe s . Each dish should be considered a replicate.

3. C ontact 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) E xposure time

. Plates or dishes should be stored in the laboratory

. Observations for mortality and hatching should be made every 24 hours fo r up to 30 days

. E ggs 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 desc ribed 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 r ecommendations.

Barile, J., R. Nauen, G. Nentwig, R. Pospischil and B. Reid. 2008. Laboratory and field e valuation of

deltamethrin and b endiocarb t o c ontrol Cimex l ectularius (Heteroptera: Cimicidae) . pp. 105

- 111. I n : W.

H . Robinson and D. Bajomi (eds )

. Proceedings of the Sixth International Conference on Urban Pests.

OOK

- Press Kft., H

- 8200 Veszprém, Pápai út 37/a, Hungary . Available at: http://www.icup.org.uk/reports/ICUP861.pdfPage 25 of 26 Busvine, J.R. 1958. Insecticide resistance in bed

- bugs. Bull. Wld. Hlth. Org. 19: 1041

- 1052. Available at: http://whqlibdoc.who.int/bulletin/1958/Vol19/Vol19

- No6/bulletin_1958_19(6)_1041

- 1052.pdf

Busvine, J.R . and J. Lien. 1961. Methods for measuring insecticide susceptibility levels in bed

- bugs, cone

nosed bugs fleas and Lice. Bull. Wld. Hlth. Org. 24: 502

- 517. Available at: http://whqlibdoc.who.int/bulletin/1961/Vol24/Vol24

- No4

- 5/bulletin_1961_24(4

- 5)_509

- 517.pdf

European Commission, Directorate

- General Environment. 2010. Technical Notes for Guidance (TNsG)

. Insecticides, acaricides and products to control oth er arthropods (PT 18) and repellents and attractants (only concerning arthropods) (PT19). Draft guidance document to replace part of appendices to chapter

7 (p age 187 to 200) of the TNsG on p roduct evaluation . Available at: http://ec.europa.eu/environment/biocides/pdf/guidance_efficacy_pt18_19.pdf

Gratz, N. 1959. A survey of bed

- bug insecticide resistance in Israel. Bull. Wld. Hlth. Org. 20: 835

- 840.

Available at: http://whqlibdoc.who.int/bulletin/1959/Vol20/Vol20

- No5/bulletin_1959_20(5)_835

- 840.pdf

Health and Safety Executive (HSE), Biocides and Pesticides Unit (BPU). 2004. Guidelines on the efficacy data for approval of non

- agricultural pesticide products: insecticides and acaricides. Merseyside, UK.

Available at: http://www.hse.gov.uk/bioci des/copr/pdfs/pestinsect.pdf

Harlan, H.J., M.K. Faulde, and G.J. Baumann. 2008. Bedbugs. p p . 131

- 151. In : X. Bonnefoy, H. Kampen, and K. Sweeney (eds.) Public Health Significance of Urban Pests. World Health Or ganization, Copenhagen,

Denmark. Available at: http://www.euro.who.int/__data/assets/pdf_file/0011/98426/E91435.pdf

Miller, D.M. and M.L. Fisher. 2008. Bed bug (Hemiptera: Cimicidae ) response to fumigation using sulfuryl fluoride. pp. 123

- 127. I n : W. H . Robinson and D. Bajomi (eds )

. Proceedings of the Sixth International

Conference on Urban Pests. OOK

- Press Kft., H

- 8200 Veszprém, Pápai út 37/a, Hungary . Available at: http://www.icup.org.uk/reports/ICUP864.pdf

Moore, D.J. and D.M. Miller. 2006. Laboratory e v aluations of insecticide product efficacy for c ontrol of Cimex

lectularius . J. Econ. Entomol. 99(6): 2080

- 20 86.

Myamba, J., C.A. Maxwell, A.Sidi and C.F. Curtis. 2002. Pyrethroid resistance in tropical bedbugs, Cimex hemipteru s, associated with use of treated bednets. Medical and Veterinary Entomology: 16:448

- 451.

Available at: http://pamverc.org/wp

- content/uploads/175921.pdf

Naylor, R. D. Bajomi and C. Boase. 2008. Efficacy of (s)

- Methoprene against Cimex lectularius (Hemiptera: Cimicidae) . pp. 115

- 121. I n : W. H . Robinson and D. Bajomi (eds )

. Proceedings of the Sixth

International Conference on Urban Pests. OOK

- Press Kft., H

- 8200 Veszprém, Pápai út 37/a, Hungary

. Available at: http://www.icup.org.uk/reports/ICUP863.pdf

Pennetier , C.

, V

. Corbel

, P. Boko , A. Odjo

, R. N'Guessan

, B. Lapied and J

- M Hougard . 2007. Synergy between repellents and non

- pyrethroid insecticides strongly extends the efficacy of treated nets against Anopheles gambiae

. Malaria Journal 6:38

. doi:10.1186/1475

- 2875

- 6

- 38 . Available at: http://www.malariajournal.com/content/6/1/38Page 26 of 26 Randall, J.R. and D.O. Brower. 1986. A new method to determine repellent, neutral, or aggregative properties

of chemicals on Blatttella germanica (Dictyoptera: Blattellidae). J. Med. Entomol. 23(3) : 251

- 255

.

Robertson, J.L., R.M. Russel l, H.K. Preisler and N.E. Savin (eds). 2007. Bioassays with Arthropods. Second edition. CRC Press, Boca Raton, Florida, USA.

Romero, A.

, M.F. Potter, D.A. Potter, and K .F. Haynes. 2007. Insecticide r esistance in t he bed b ug: A factor in the pest’s sudden r esurgence? J. Med. Entomol. 44(2): 175

- 178.

Romero, A.

, M.F. Potter, and K.F. Haynes. 2009. Behavioral r esponse of the bed bug to insecticide r esidues. J. Med. Entomol. 46(1): 51

- 57.

Romero, A. 2011. Moving from the old to the new: insecticide research on bed bugs since the resurgence.

Insects 2:210

- 217. doi : 10.3390/insects202010. Available at: http://www.mdpi.com/journal/insects/

Steelman, C.D., A. L. Szalanski, R. Trout, J. A. McKern, C. Solorzano, and J. W. Austin. 2008. Susceptibility

of the bed b ug Cimex lectularius L. (Heteroptera: Cimicidae) collected in poultry p roduction facilities to

selected i nsecticides . J. Agric. Urban Entomol. 25(1): 41

– 51

. Available at: http://comp.uark.edu/~aszalan/pdf/steelman_et_al_bed_bugs_jaue_2008.pdf

White, G.B. 2007. Chapter 2: Terminology of Insect Repellents. pp. 31

- 47. In: M. Debboun, S.P. Frances, and D. Strickman (eds). Insect Repellents: Principles, Methods, and Uses. CRC Press, Boca Rat on, Florida,

USA.

World Health Organizat ion Pesticide Evaluation Scheme (WHOPES). 1998. Test procedures for insecticide resistance monitoring in malaria vectors, bio

- efficacy and persistence of insecticide on treated surfaces, World Health Organization, Geneva, Switzerland. Available at: http://whqli bdoc.who.int/hq/1998/WHO_CDS_CPC_MAL_98.12.pdf

WHOPES . 2005. Guidelines for laboratory and field testing of long

- lasting insecticidal mosquito nets. World

H ealth O rganization, Geneva, Switzerland. Available at: http://whqlibdoc.who.int/hq/2005/WHO_CDS_WHOPES_GCDPP_2005.11.pdf

WHOPES . 2009. Guidelines for efficacy testing of household insecticide products

- Mosquito coils, vaporizer mats, liquid vaporizers, ambient emanato rs and aerosols. World Health Organization, Geneva,

Switzerland. Available at: http://whqlibdoc.who.int/hq/2009/WHO_HTM_NTD_WHOPES_2009.3_eng.pdf

WHOPES and Global Malaria Program Vector Control Unit. 2011. Guidelines for monitoring the durability of long

- lasting insecticidal mosquito nets under operational conditions

. World Health Organization, Geneva, Switzerland. Available at: http://whqlibdoc.who.int/publications/2011/9789241501705_eng.pdf

WHO Vector Control and Biology Division. 1982. VI. Bed bugs. WHO/VBC/82.857 , 9 pp. World Health Organization, Geneva, Switz erland. Available at: http://www.scent

- tek.com/bed_bugs/continued_learning/world_health_organization_paper.pdf