Document ID: EPA-HQ-OPPT-2009-0150-0030
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2017-06-14T04:00Z

United States
Environmental Protection Agency
           Office of Chemical Safety and Pollution Prevention (7101)
                                                     EPA 712-C-17-001
June 2017
                                       
Product Performance
Test Guidelines

OCSPP 810.3900:

Laboratory Product Performance Testing Methods for Bed Bug Pesticide Products 

                                     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 (US EPA) 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 Act (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 https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances. 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 guideline.

OCSPP 810.3900: Laboratory Product Performance Testing Methods for Bed Bug Pesticide Products
 Introduction.
 Scope. This guideline provides recommendations for the design and execution of laboratory studies to evaluate the performance of pesticide products intended to repel, attract, and/or kill the common bed bug (Cimex lectularius) in connection with registration of pesticide products 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, if intended to be applied to have a pesticidal purpose such as to attract, repel, or kill bed bugs. It does not apply to repellent products applied to human skin, and does not apply to those products exempt from FIFRA Registration under 40 CFR 152.25.
 Purpose. This guideline provides appropriate laboratory study designs and methods for evaluating the product performance of pesticides against bed bugs and includes statistical analysis and reporting.
 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 (e) provide general guidance applicable to protocol development for bed bug product performance testing conducted in the laboratory. Sections (f) through (g) describe reporting of completed bed bug product performance studies. Sections (h) through (o) provide specific guidance on laboratory product performance testing of bed bug pesticide products. 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 product performance studies.
               (e)	Execution of bed bug product performance studies.
               (f)	Reporting of completed bed bug product performance studies to the Agency.
               (g)	Retention of records.
               (h)	Specific guidance for laboratory studies for resistance ratio determination and characterization of bed bug strain susceptibility.
               (i)	Specific guidance for laboratory studies for forced exposure (no-choice) residual surface treatments.
               (j)	General guidance for laboratory studies to determine the product performance of a repellent or attractant product.
               (k)	Specific guidance for laboratory studies for testing indoor pesticide total release aerosols, space sprays, and insecticide vapor strip products.
               (l)	Specific guidance for laboratory studies for direct application testing of pesticide products.
               (m)	Specific guidance for laboratory studies for testing ovicidal products.
               (n)	Specific guidance for laboratory studies for fumigant products against all bed bug life stages.
               (o)	Specific guidance for laboratory studies of insect growth regulators (IGRs).
               (p)	References.
 General Considerations. Any protocol and/or study developed using this guidance must meet the provisions 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.
            This guideline does not supersede or overrule the regulations governing research conducted with human subjects such as those contained in 40 CFR Part 26, 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.
 Good Laboratory Practice Standards. Good Laboratory Practice (GLP) Standards set forth in 40 CFR Part 160 apply to laboratory studies evaluating pesticide product performance. 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)]. However, 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) states that 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" must be submitted to aid in making that determination.
 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 in this guideline.
 Resistance Management Considerations for Registrants and Professional Applicators. Full doses of pesticide applications are often not delivered to bed bugs because of their use of obscure harborages, lack of tarsal pads, and other unique aspects of their biology. Resistance therefore builds quickly in bed bug populations making resistance management a vital part of combating these pests. Registrants are strongly encouraged to adhere to Insecticide Resistance Action Committee (IRAC) and EPA labeling guidance on resistance management when compiling final product packaging. Similarly, all professional applicators should be trained in and encouraged to alter modes of action as necessary when applying chemical control against bed bugs.
 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.
          An attractant is a substance that causes bed bugs to make oriented movements towards its source.
          Behavioral resistance refers to the avoidance by arthropods of a toxin through detection or recognition. (19)
          A product that controls bed bugs demonstrates residual product performance. See the definition for "residual efficacy."
          Crossing is the act of passage by a bed bug from an untreated surface to a treated surface or over a treated surface to another untreated surface.
          A fumigant is a pesticide that is applied in the gaseous state or that forms a gas. Fumigants may be used to kill bed bugs indoors or in containers.
          A harborage is a sheltered area or refuge for bed bugs.
          A host is a warm-blooded animal that bed bugs bite to obtain blood for their survival and reproduction.
          Host-seeking is the behavior of bed bugs actively seeking a host.
          An Insect Growth Regulator (IGR) is a pesticide that inhibits the maturation of a bed bug through its life cycle.
          Knockdown refers to a bed bug that is rendered incapable of coordinated movement or unable to right itself following exposure to a pesticide product.
          KT50 is the time needed to knockdown 50% of a test population following exposure to a specific dose of insecticide treatment.
          KT90 is the time needed to knockdown 90% of a test population following exposure to a specific dose of insecticide treatment.
          LD50 is a measure of lethality of a given toxicant calculated as the dose of toxicant needed to kill 50% of a test population.
          LD90 is a measure of lethality of a given toxicant calculated as the dose of toxicant needed to kill 90% of a test population.
          Moribund refers to bed bugs that are on their backs with only a single appendage twitching. Bed bugs exhibiting this behavior may not be considered dead.
          Mortality refers to bed bug death. A dead bed bug is a bed bug that does not move, even when poked or probed. Moribund bed bugs may not be considered dead.
          An ovicidal product is a pesticide product that kills bed bug eggs.
          Pesticide resistance is a heritable decrease in the susceptibility of a pest strain or population to a given pesticide. This change is revealed in the repeated failure of a product to achieve the expected level of mortality when used according to the label directions for that pest species. (19)
          Penetration resistance refers to a decrease in the absorption of a toxin through the cuticle of an arthropod. For instance, a barrier may develop in an arthropod's cuticle that slows the absorption of chemicals by the body. (19)
          Quick kill and/or kills on contact occur when bed bugs are exposed to a given pesticide and more than 90% of bed bugs exhibit knockdown within 30 seconds and, following transfer to clean containers within 4 hours following pesticide exposure, greater than 90% mortality is observed by 96 hours after initial pesticide exposure.
          A repellent is a substance intended to disrupt the host-seeking behavior of insects or other arthropods, including bed bugs, causing them to avoid a treated substrate in the presence of a host.
          Residual efficacy refers to a surface or space treated with a pesticide product continuing to provide the intended pesticidal effect at an acceptable level for an extended length of time after application. The product's residues should be effective for at least 24 hours post application.
          Resistance Ratio (RR) is a quantitative expression of the resistance of a bed bug strain to a specific active ingredient or product formulation. A resistance ratio (e.g., RR50) is calculated by dividing a quantitative measure of the lethality of a given insecticide (e.g., LD50 value) for a bed bug strain of unknown level of resistance by the corresponding measure of lethality for a strain known to be susceptible to the given insecticide.
          A resistant bed bug is a bed bug that survives a pesticide dose that is expected to kill or control bed bugs.
          Starts killing bed bugs is when the product begins killing bed bugs and there is a statistically significant difference in mortality and/or knockdown between the treatment and negative control at a specific point in time, usually expressed in seconds or minutes.
          A susceptible bed bug is a bed bug that exhibits mortality when exposed to an appropriate dose of a given pesticide for bed bugs. Ideally, susceptible bed bugs will not have a history of exposure to the insecticidal mode of action being tested and will not display behavioral or penetration resistance to insecticides.
 Development of protocols for bed bug studies. The first major stage of bed bug product testing is development of a study protocol.
General considerations in developing a study protocol for bed bug studies include scientific design of the study, data collection, data analysis, and reporting. Each of these topics is discussed in more detail in the sub-sections below with EPA recommendations included for additional guidance.
 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 (35). The design should include a detailed description of the experimental design, addressing topics (i) through (xi), given directly below.
 Objectives. In the case of claims that products kill and/or knockdown bed bugs, the objective of bed bug product performance testing is to determine that a product application made at the lowest proposed label rate kills or knocks down the bed bugs. For claims that products repel or attract bed bugs, the objective of product performance testing is to determine the ability of a product to encourage or deter bed bugs to or from a pre-determined locale. For "controls bed bugs" claims, the objective is to determine that the pesticide application has residual performance. In all cases the scientific objective should be stated clearly and all treated bed bugs should be compared to control bed bugs that have received no treatment (4, 11, 17, 24).
 Test materials and treatments. Product performance should be tested using the end-use formulation and application rates as registered or as proposed for use. Test materials should be stored at ambient temperature and humidity for at least one day before use.
 Product treatments for product performance tests.
 Products that target bed bug nymphs and adults. Testing should be conducted with adult bed bugs (unless nymphs are specifically targeted), preferably blood fed seven days before testing.
 Products that target bed bug development. Testing should be conducted with mixed nymphal stages or eggs as appropriate. A blood meal should be available to provide the nourishment needed for bed bugs to molt from one nymphal stage to the next. The blood meal should be identified.
 Products that target bed bug eggs (ovicidal products). Testing should be conducted with the egg life stage only.
 Dose determination. The test dose in bed bug product performance studies is the lowest application rate from a proposed product label expressed as ounce(s) of product per square foot for surface area treatments or volumetrically as fluid ounces per cubic foot for space spray, total release aerosols, and fumigant treatments. A rate may also be reported using metric system measurements, as mg of test substance/cm[2] of test surface or volumetrically as ml of test substance/m[3]. The amount of active ingredient tested per unit area or time should also be given.
 Testing conditions. During bed bug product performance testing the temperature should be kept at 25 + 1 C, with a relative humidity of 50-80%, and a photoperiod ranging from 12 hours of light to 12 hours of darkness to 16 hours of light to 8 hours of darkness. The temperature during the test should be kept as constant as possible because changes can affect the performance of the product treatments.
 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. Generally, the endpoints tested will be bed bug knockdown, kill, repellency, or attraction tested at the lowest labeled application rate and will determine whether or not a product is efficacious. The endpoint selected should be claimed on the proposed label. Bed bugs should be removed and placed into a clean container within 4 hours after onset of exposure to pesticide application. For knockdown evaluation, observations should be made for up to 30 seconds post-treatment exposure. For mortality, evaluation observations should be reported at 24, 48, 72, and 96 hours post-treatment exposure unless all bed bugs die or negative control mortality exceeds 10%. Data will not be acceptable if control mortality exceeds 10%. Observations of mortality occurring after 96 hours should be justified based on the mode of action and application type. Survival of bed bugs beyond 96 hours in negative control replicates does not justify making observations after 96 hours. If a test recommends that a bed bug be contained during exposure to a pesticide, a bed bug should not be confined to a treated surface for more than 4 hours. The number of dead, knockdown and live bed bugs in each replicate should be recorded, and a count on mortality should be separate from a count on morbidity. The percentage of bed bugs killed and knockdown, exclusively, for each treatment at each test interval should be recorded. Confidence limits around the reported mean or median values should be reported. Evaluation of speed of kill or additional knockdown evaluations should include more observations on the first day of the test to record the data needed to support the desired claims.
 Test organisms. Testing should be conducted with one field-collected strain of the common bed bug, Cimex lectularius, and the laboratory-reared, susceptible Harlan strain (31, 42). Bed bugs should be blood fed and should be tested seven days after the blood meal. Field collected populations may be reared in the laboratory but testing should occur no later than the second lab-reared generation. If more generations are needed to produce sufficient numbers of bed bugs for testing, it should be indicated in the study report. Field populations should be collected from urban areas in the U.S.
 Representative sampling. The sampling scheme should target U.S. bed bug populations.
 Sample size. The sample should be large enough to likely 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.
 Replication. A minimum of five replicates of ten bed bugs each and balanced (equal number of treated and control replicates) experimental designs are recommended for most studies. Exceptions will be noted in the guidance that follows in this document.
                  Other factors that may affect sample size and replication 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). The protocol should fully describe how sample size and replication were determined.
 Bed bug rearing, handling, and maintenance. When applicable, a description of the bed bug laboratory colony rearing practices should be included. Collection details and maintenance procedures for field-collected strains should be described.
 Negative (untreated) control. A negative control should be included in all testing. The number of untreated control replicates should equal the number of replicates for each treatment. When appropriate, a negative control is typically treated with diluent only or receives no treatment at all.
 Positive controls. A positive control is recommended for determining a resistance ratio.
 Statistical analysis plan. Protocols should include a full description, explanation, and justification for the statistical methods proposed to analyze both resistance ratio determinations and product performance test results, taking into account the specific study objectives and variables. A statistician should be consulted when developing test protocols. Analysis of data is recommended to determine if the mean (arithmetic mean) mortality of the group treated with the product differs from the negative control mortality and if any within treatment effects were significant. Protocols should explicitly describe the model to be used and demonstrate whether or not assumptions underlying the model can be met for all proposed analyses. Restrictions on randomization of any testing components should be documented clearly and should be accounted for correctly in the statistical analyses. Generally, generalized linear models (GLMs) (21) are recommended to fit models directly to non-normal (e.g., binomial  -  which describe much of the collected product performance data sets) data using a probit link or logit link function. GLMs do not involve transforming the response variable, thereby allowing the data to remain on the original scale of measurement. Generalized linear mixed-models (GLMM)(12, 13, 16, 17) may also be appropriate. Software for analysis using GLMs or GLMMs is available in many widely sold statistical analysis packages. If survival analyses (23), such as the Kaplan-Meier Estimator, are used provide justification for use of the median value to characterize product performance and demonstrate that the underlying assumptions of these analyses have been met. Other analysis including assumption of the normal distribution should be described and justified (1, 8, 33, 36, 40).
 QA/QC plan. Protocols should provide for periodic quality assurance inspections that are adequate to ensure the integrity of the study and consistency with the provisions of EPA's Good Laboratory Practices regulations (40 CFR §160).
 Data Collection and Reporting. Study 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 (f) of this guideline.
 GLP regulations. Each study protocol should provide for collecting and reporting all elements provisioned by the GLP regulation at 40 CFR §160.120.
 Reporting results. Refer to (c)(1)(xi) of this guideline for guidance in reporting results and the data analysis.
 Review of protocols for bed bug product performance studies. EPA recommends protocol submission of studies that test repellents, attractants, and intentional exposure human studies covered by CFR part 26, and/or for studies to be conducted outside of the United States. Protocols proposing novel testing methods to support new use patterns for bed bug control should also be submitted to EPA for review before the study begins.
 Execution of bed bug product performance studies.
 Execution of protocol. In cases where a protocol has been submitted to EPA for review, it is recommended that testing be initiated when the EPA review is complete and if applicable, EPA comments are incorporated into the revised protocol.
 Quality Assurance (QA) oversight. Product performance testing is subject to the Good Laboratory Practices regulations at 40 CFR §160. The GLP regulation states that each testing facility should include an independent QA unit and that the QA unit monitors execution of each protocol and documents 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. Please see (a)(5)(i) above for the discussion of the use of GLP laboratory methods when conducting bed bug product performance studies.
 Protocol amendments. Amendments are planned changes to the protocol and should be made before the study is executed. All amendments to the protocol should be noted in the written report to the Agency.
 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).
 Reporting of completed bed bug product performance studies to the Agency.
 Study report. The study should include the following elements. Guidance for specific tests is provided in Sections (h)-(o).
  Study identification: Title, identifying study number(s), sponsor, study director, investigators, name and location of the testing facility, and dates of the study should be reported.
 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.
 Approved or proposed label directions for use: A copy of the proposed or approved product label should be included.
 Study objective(s): The purpose of the study should be stated.
 Testing conditions: Information on temperature, relative humidity, ambient light and photoperiod, and air flow (where applicable) should be reported.
 Testing system, including but not limited to:
 Bed bug species tested, including identification of strains of susceptible and field bed bug populations, where bed bugs stains were collected/obtained; development stage, age, and sex of bed bugs; and methods for preparation of bed bugs for test (feeding/starving), and when appropriate, the blood meal should be identified (18, 31, 38).
 Bed bug rearing, handling, and maintenance (31).
 Description of test substance (i.e., product, % active ingredient, and formulation to be tested). Negative control should also be described.
 Description of the experimental unit.
 Treatment application rate and method of application (rate should be consistent with label instructions).
 Number of product treatments.
 Number of negative control replicates.
 Number of replicates per treatment.
 Number of bed bugs per replicate for each treatment.
 Length of time for bed bug exposure period to each treatment.
 Data/Results reporting. Report the following information:
 Protocol with amendments and study deviations from the protocol. A copy of the study protocol should be included with amendments and deviations. Deviations should be justified, and described together with their impact on the validity of the study.
 Data and endpoints. Knockdown and/or mortality values should be corrected for untreated control knockdown and/or mortality with Abbott's Formula or the equivalent. Both endpoints should be reported as observed throughout the test, though percent mortality should be reported at the final evaluation.
 Amount of product applied. Report the amount of product, expressed as weight of product applied to each replicate. Report the quantity of active ingredient applied.
 Report the following:
                            Test results on all aspects of the research
                            Copies of all raw data
                            Certification of the test substance's identity and origin
                            Description of the experimental unit
                            Description of each product treatment and the untreated controls
                            Treatment application rate and method of application
                            Number of product treatments
                            Number of negative control replicates
                            Number of replicates per treatment
                            Number of bed bugs per replicate for each treatment
                            Length of bed bug exposure period to each treatment
 Data analysis. Provide a copy of the statistical analysis plan. Refer to Section (c)(1)(xi) for recommendations on data analyses.
 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.
 Retention of Records. The record-keeping provisions of 40 CFR §160.190 and §160.195 apply to records of any study conducted under the Good Laboratory Practices rule.
 Specific guidance for laboratory studies for resistance ratio determination and characterization of bed bug strain susceptibility. 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.
 Study objective: To estimate the susceptibility and magnitude of resistance of bed bug strains to pesticides used in product testing.
 Materials and methods.
 Experimental units. Place a piece of white filter paper on the bottom of a 6 or 10 cm glass Petri dish and secure a screen over the top of the Petri dish. An insecticide concentration should be applied to filter paper in each replicate at a volume that saturates the paper, generally at least 200 ul (200 ul = 0.2 ml). Allow paper to dry before exposing the bed bugs. Prepare an equal number of negative control dishes with paper treated with the diluent only.
 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 previous knowledge of bed bug susceptibility to the insecticide being tested but a justification should be provided (2, 6, 7, 15, 16, 26, 32, 39, 46, 48). If a product contains a synergist, use only the insecticide component with a solution concentration based on the active ingredient, not the synergist (28).
 Number of replicates. Ten replicates with ten bed bugs each are recommended.
 Bed bug exposure to treatments. Bed bugs should be exposed to the treated filter paper in each treatment for 24 hours.
 Negative control. The negative control should be treated with the diluent for insecticide solution preparation.
 Positive control. An appropriate positive control should be used. For the testing of pyrethroid insecticides, deltamethrin is recommended as a positive control. For testing neonicotinoid insecticides, imidacloprid is recommended as the positive control.
 Lethal dose (LD) values. An analysis using GLMs is recommended to determine the LD values for each bed bug strain tested. Use of a probit analysis should be justified (41).
 Resistance ratios (RR). Resistance ratios should be calculated and reported as follows:
                  LD (lab or field strain)/LD (susceptible strain) = RR
                  For example: LD50 (lab or field strain)/LD50 (susceptible strain) = RR50
                  LD90 (lab or field strain)/LD90 (susceptible strain) = RR90
                  A resistance ratio equal to or greater than 100 is characteristic of a resistant strain.
 Reporting results. Refer to (c)(1)(xi) of this guideline for guidance in reporting results and the data analysis. Report the resistance ratio values for each strain for each insecticide tested and the associated data analysis (25).
 Specific guidance for laboratory studies for forced exposure (no-choice) residual surface treatments.
 Study objective: To determine the residual product performance of an application made to three different surfaces in a forced exposure (no-choice) test.
 Materials and methods
 Experimental units. The surfaces to be treated with the product should be: 1) unpainted/unfinished (1/4)" thick plywood; 2) commercial linoleum tile; and 3) 100% cotton sheeting stretched over a cardboard panel. Surfaces should be pre-cut to 4" x 4" or larger panels. Cotton sheet replicates should be affixed to the top of the panel to provide a flat, rigid surface for treatment. An application should be made to each surface. An equal number of negative control replicates should be established with the same surfaces.
 Application of product dilutions. The lowest labeled application rate for bed bug control should be applied on each panel. A metered bench top sprayer is preferred as the delivery device to ensure consistent application volume and even distribution of spray particles. Generally, panels are sprayed from a distance of one foot above the panel surface. Use of other heights should be justified. Panels should be stored and exposed to ambient conditions at the test site to age residues. Panels should be fully dried before exposing bed bugs. Measure the volume of spray applied and calculate the weight of the active ingredient(s) delivered.
 Ready-to-use product application. Application to panel surfaces should be made at rates equivalent to the amount of product to be sprayed per unit area as directed by the label. In product performance testing, the amount of product delivered by a ready-to-use spray product (aerosol or pump-spray) is described as the amount of product sprayed per second, and should be determined before treatments can be made. To determine the quantity sprayed per second, spray five panels of each surface type for three seconds each for each treatment. The product container should be weighed before and after each spray and the difference recorded. The mean value of the five replicates should be determined and that result divided by three to determine the average amount of product applied per second of spraying. The same procedure should be conducted to evaluate dust product formulations except that application should be made from a height of six inches or as directed by the product label. Other modifications to the protocol needed to apply dusts should be described and justified.
 Replication. A replicate should consist of a treated or untreated panel, each with 10 bed bugs confined to the panel. A minimum of 5 replicates should be used for each product treatment and negative control. For each bed bug strain tested at each time point, prepare 15 treated (3 surfaces x 5 replicates per surface) and 15 untreated panels. Three hundred bed bugs are necessary for each time point tested per strain (including the untreated control specimens). After an initial test 24 hours post application, the pesticide residues should be tested regularly until the end of the test. One may consult with the Agency for a determination on an appropriate testing interval.
 Bed bug exposure to product treatments. Bed bugs should be exposed to treated panels for no more than 4 hours. After the exposure period, transfer the bed bugs to a clean, untreated container for further observation and evaluation. Treated panels should be retested though bed bugs should not be reused.
 Positive control. A positive control is not recommended.
 Reporting results. Refer to Section (c)(1)(xi) of this guideline for guidance on reporting results and data analysis.
 Study conclusions. Summarize study outcomes for residual control of bed bugs and discuss their implications for product labeling.
 General guidance for laboratory studies to determine the product performance of a repellent or attractant product. The following guidance describes approaches that may be used to assess whether a pesticide product is a repellent or attractant (10, 20, 30, 34, 37, 44, 45). To make these determinations, no-choice and choice tests should be conducted. Protocols for testing should be submitted to EPA for review and approval before conducting the study.
 Study objective: To determine if a pesticide product repels or attracts bed bugs.
 Materials and methods.
 Repellents.
  No-choice test.
 Volatile repellents. To evaluate repellent vapors, a test should simultaneously expose a bed bug to a human host or mimic (e.g. carbon dioxide and heat), and the test substance to determine if the bed bug is repelled in the presence of a human host or mimic. Five replicates of ten bed bugs each with an equal number of untreated control replicates is recommended.
 Impregnated fabric. The test should include the testing of a treated piece of fabric and an untreated piece of fabric, separately, along with a human host or mimic (e.g. carbon dioxide and heat). The test should determine if a bed bug is repelled by the treated fabric in the presence of a human host or mimic.
 Choice test.
  Volatile repellents. A version of the still-air olfactometer (9) as modified by Weeks et al. 2013(43) may be considered. The still-air model should be adapted to provide a source such as carbon dioxide and heat to mimic a human host in the presence and absence of repellent as alternative choices in the same arena. The negative control should consist of the same arrangement in a separate arena provided with only carbon dioxide and heat and with no repellent. A Y-tube assay (14) may also be considered.
 Impregnated fabric. Untreated and impregnated fabric should be tested together. Bed bugs should be placed onto the untreated fabric, and their movement either towards or away from the treated fabric should be observed and measured. An untreated control arena with untreated fabric only should also be included in the study.
 Attractants. Choice tests are recommended for testing the product performance of attractant products (43, 44). Depending on its use pattern (whether the product is intended to attract bed bugs towards a harborage or "pull" them away from human hosts), the product performance of an attractant product may be determined by comparing its effect on bed bugs to that of bed bug aggregation cues or host cues such as carbon dioxide and heat. Therefore, in this experiment either host cues such as carbon dioxide and heat or bed bug aggregation cues deposited in a harborage should be presented as an alternative to the attractant formulation in the same arena. Observations of bed bug location should be recorded at the end of the exposure period. Testing in the dark under red light is recommended (44).
 Replication. Ten bed bugs per replicate for a total of five replicates per trial or 50 replications of one bed bug each per trial should be used. Justify the choice of individuals or groups of ten.
 Positive control. A positive control is not recommended.
 Reporting results. Refer to Section (c)(1)(xi) of this guideline for guidance on reporting results and data analysis. Analyzing data from replicates with one bed bug or replicates with ten bed bugs is likely to differ and should be justified.
 Percent repellency. For repellent testing report the number of bed bugs that avoided the host mimic and the ones that did not. Calculate percent repellency corrected for untreated control results.
 Percent attractancy. For attractant testing, report the number of bed bugs that were attracted to the attractant and the number that were not. Calculate the percentage of bed bugs found at each location at 15 minutes post exposure in treated choice and negative control arenas.
 Mortality and knockdown. Report the number of dead, knockdown, and live bed bugs in each replicate. All raw data should be reported.
 Study conclusions. Describe the product performance of the product treatment. Include a discussion on untreated control results, and the adequacy of the host or aggregation cues used in the study.
 Specific guidance for laboratory studies for testing indoor pesticide total release aerosols, space sprays, and insecticide vapor strip products. This guidance applies to testing total release aerosols and space sprays including misters, hand-held aerosol products, and vaporizing strips on surfaces used for indoor applications to control bed bugs.
 Study objective: To determine the performance of pesticide products intended for total release aerosols, space sprays, and vapor strip treatments against bed bugs.
 Materials and methods.
 Experimental unit. Testing should be conducted in a Peet-Grady chamber with a volume of 216 cubic feet or greater (47). 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 and mister products should be delivered by an automatic dispenser calibrated for the 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. Vaporizing strips should be hung from the ceiling in the center of the room or applied according to label directions.
 Number of treatments and replication. There should be one product treatment and one negative control treatment for each bed bug strain. One half of the replicates will be in the chamber treated with the pesticide product while the other half will be the negative control.
 Cage Placement. For each exposure period, twelve cages from each strain should be used. Stacked egg cartons should be added to each cage to serve as a harborage. Allot six cages of each strain to the product treatment and place in the chamber, while the other six should be kept outside the chamber as a negative control for each exposure period. Ten bed bugs should be transferred to every cage. In the chamber place one cage in each corner one foot above the floor. Place the last two cages where the wall and ceiling meet at the middle of two opposite walls.
 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 pesticide has been evacuated from the chamber. Knockdown and mortality should be recorded separately at two hours. Bed bugs from each cage should be transferred to a clean, untreated container after the assessments are made, but no later than 4 hours post treatment. Vaporizing strips should be assessed in a similar manner following 24 hours of exposure.
 Negative control. Negative control replicates should be placed in the lab under the same abiotic conditions as the treatments. Negative control replicates should be untreated because treating with diluent is impractical.
 Positive control. A positive control is not recommended.
 Reporting results. Refer to Section (c)(1)(xi) of this guideline for guidance on reporting results and data analysis. In addition, the following information should be reported.
 Data and endpoints. Mortality data from the treated group should be corrected for untreated control mortality with Abbott's Formula or the equivalent.
 Mortality and knockdown. Knockdown and mortality data should be reported from product and negative control treatments from observations made at 2, 24, 48, 72, and 96 hours post-treatment. For each strain, report the percentage of bed bugs killed and knockdown exclusively for each treatment replicate at each observation interval. Report mean mortality data for each strain in each treatment at each height level and all heights combined as corrected arithmetic mean values. Confidence limits around the mean values should be reported.
 Data analysis. The analysis should consider the effect of the treatment cage height and bed bug strain effects on product performance.
 Study conclusions. Report the percent mortality for each replicate and treatment.
 Specific guidance for laboratory studies for direct application testing of pesticide products.
 Study objective: To determine the product performance of direct application of pesticide product formulations against bed bugs.
 Materials and methods.
 Experimental unit. Testing should be conducted with caged bed bugs. Typically, a test cage unit is a 16-ounce 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 and should be made from one foot above the test cage. Report the weight applied and the weight of active ingredient delivered.
 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.
 Replication and bed bug density. The test should be replicated 5 times for each treatment. Each replicate should have ten bed bugs. An equal number of untreated control replicates and specimens should also be included.
 Bed bug exposure to the treatments. Bed bugs should be transferred to clean cups in less than 4 hours after product application. Cups should be stored under ambient test site conditions. Observations for knockdown and mortality should be made at a minimum of 24 and 96 hours, unless all bed bugs die or untreated control mortality exceeds 10%. Repeat this procedure for each strain to be tested.
 Positive control. A positive control is not recommended.
 Reporting results. See Section (c)(1)(xi) of this guideline for guidance on results reporting and data analysis.
 Study conclusions. Discuss knockdown and percent mortality of the product treatment.
  Specific guidance for laboratory studies for testing ovicidal products.
 Study objectives: To determine the product performance of pesticide products intended for use as ovicides.
 Materials and methods.
 Treatments.
 Direct application. Testing should be conducted with bed bug eggs laid on filter paper the night before the test. 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 pieces that fit into the depressions on the plate or in a Petri dish. Twenty eggs should be allotted to each depression or well. 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 and should be made from one foot above the test surface (or as directed on the label). For a dust formulation, application should be made at the lowest label rate from a height of six inches or less (or as directed on the label). Record the weight of formulation applied and the weight of active ingredient delivered. An equal number of untreated control eggs should be included in the study design on the same type of plates.
 Contact with residual surface application. Gravid female bed bugs should be separated from the colony, allowing them to lay eggs in a separate chamber on a filter paper surface. Females should be fed the same blood source, about the same age and the eggs used should be collected every day to ensure they are the same age. A 5-7 cm Petri dish is recommended as the test container. A piece of filter paper should be treated with the product formulation at the lowest label rate and the residues should be aged for the desired time. Another piece of filter paper should be left untreated to serve as the negative control. One piece of paper should be placed into each dish. Collect eggs from the gravid female chamber and transfer 20 eggs to each treated and untreated paper in the Petri dishes. Alternatively, egg-laden papers with 20 bed bug eggs can be turned upside down and laid on treated and untreated paper surfaces.
 Number of treatments. The test should include a product treatment against half of the eggs and an untreated control against the other half. The test may be repeated using residues aged for various amounts of time.
 Replication and bed bug density.
 Direct application. The test should be replicated 10 times for each treatment. Each treated and untreated replicate should have a minimum of 20 bed bug eggs.
 Contact with residual surface application. Each dish is a replicate and should have 20 eggs. Ten dishes should be allotted to the product treatment and untreated control.
 Exposure time. Plates or dishes should be stored in the laboratory. Eggs should be exposed continuously for 14-30 days. 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, and the number of un-hatched and hatched eggs should be recorded from the treated and untreated control groups. Unhatched eggs should also be examined to determine if they are embryonated.
 Positive control. A positive control is not recommended.
 Reporting results. See Section (c)(1)(xi) of this guideline for guidance on results reporting and data analysis.
 Data and endpoints. Mortality data from the treated group should be corrected for untreated control mortality with Abbott's Formula or the equivalent.
 Egg mortality. Report observations every 24 hours 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.
 Study conclusions. Discuss ovicidal performance of the product formulation treatment.
 Specific guidance for laboratory studies for fumigant products against all bed bug life stages.
 Study objective: To determine the product performance of a fumigant in the laboratory against all bed bug life stages (5, 22).
 Materials and Methods.
 Experimental unit (29). Five, 3.8 liter sealed glass containers with tubing capable of delivering and evacuating fumigant in a closed system should be used as the fumigation chambers. Bed bugs should be placed in a separate ventilated glass vial that should be wrapped in mattress padding and placed in the chamber before fumigation.
 Product treatment. Treatment should be at the lowest rate directed by the product label. This rate should be monitored by chemical detection to ensure the target dose was achieved.
 Bed bug life stage. This test may be used to evaluate product performance against all bed bug life stages.
 Replication. Five replicates of 10 bed bugs from the same life stage of the same strain should be tested with the exception of eggs where 20 eggs should be included per each of 10 replicates.
 Exposure time. Bed bugs should be exposed for 24 hours. After fumigation, transfer the bed bugs to clean containers and record mortality through 96 hours. For experiments with eggs and nymphs, observe for egg hatch and survival of nymphs for 14-30 days.
 Environmental conditions. Replication and treatment should be repeated at 59° F (15° C) and 77° F (25° C).
 Negative control. Bed bugs in the control group should be held in untreated glass containers outside the fumigation chamber for the same period of time as the treatment group.
 Positive control. A positive control is not recommended.
 Reporting results. Refer to Section (c)(1)(xi) of this guideline for guidance on reporting results and data analysis. In addition, report the following information.
 Amount of product applied. The amount of product, expressed as weight of product per unit volume, should be reported for each replicate. A copy of the chemical analysis should be included.
 Mortality. Mortality should be reported as number of dead bed bugs and percent kill by 96 hours post-treatment.
 Egg hatch and survival of emerging nymphs. Report percent egg hatch and length of time the emerging nymphs survive.
 Study conclusions. Report the application rates at which product performance was achieved.
 Specific guidance for laboratory studies of insect growth regulators (IGRs). This guidance is based on approaches to evaluate juvenile hormone analogs against bed bugs (3, 27).
 Study objective: To determine the product performance of an insect growth regulator in the laboratory against bed bugs.
 Materials and Methods.
 Life stages. Life stages should be evaluated separately and individuals in the population should be the same age. These products may be tested against eggs, nymphs (all stages/instars), and adults.
 Replication. A minimum of five treated replicates and five untreated replicates each with 10 specimens should be tested for every life stage for every IGR product tested.
 Test substance. The insect growth regulator should be tested alone. Testing should not be performed with tank mixes (mixtures) containing IGRs or insecticide products formulated with IGRs in combination with other active ingredients.
 Direct spray testing. Testing should be conducted as described in (l)(2) of this guideline but evaluated at 30 days post-treatment.
 Confinement to treated surfaces. Testing should be conducted as described in (i)(2) of this guideline but evaluated at 30 days.
 Evaluation period. Bed bugs should be evaluated for 30 days.
 Blood meal. A blood meal is needed for a bed bug to molt from one instar to the next and from the final nymph instar to the adult stage. A source of blood should be available regularly throughout the testing period.
 Reporting results. See Section (c)(1)(xi) of this guideline for guidance on results reporting and data analysis. Additional data reporting is described below.
 Report any abnormalities in bed bug development including deformities.
 Report egg hatch success and development of hatching nymphs.
 Report survivorship of all life stages.
 If female bed bugs were tested, track egg production, hatching success of eggs, and developmental success and survivorship of nymphs.
 Study conclusions. Discuss results and describe whether or not IGR effects impacted bed bug survivorship and development.
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