United States 

Environmental Protection 

Agency 

Office of Chemical Safety and 

Pollution Prevention (7101) 

EPA 712-C-10-001 

July 7, 2010

 

Product Performance 

Test Guidelines 

OPPTS 810.3700: 

Insect Repellents to be 

Applied to Human Skin 
NOTICE 

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

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

Substances (OPPTS) prior to April 22, 2010),United States Environmental Protection Agency for 

use in testing pesticides and chemical substances to develop data for submission to the Agency under 

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

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

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

guidelines. 

 The harmonized test guidelines serve as a compendium of accepted scientific methods 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 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 and alternatives to the methods described 

in these guidelines, with your supporting rationale. The Agency will assess such proposals on a caseby-case basis. 

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

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

the navigation menu at the top of the screen. 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. 

 
OPPTS 810.3700: Insect Repellents to be Applied to Human Skin 

(a) Introduction. 

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

and execution of studies to evaluate the performance of pesticide products 

intended to repel insects and other arthropods in connection with the products’ 

registration under the Federal Insecticide, Fungicide, and Rodenticide Act 

(FIFRA) (7 U.S.C. 136, et seq.). This guidance applies to products in any 

formulation—such as lotion, liquid, or spray—intended to be applied directly to 

human skin. It does not apply to products applied to or impregnated into clothing 

or fabric, or used to repel insects from indoor or outdoor spaces. This guidance 

recommends appropriate study designs and methods for selecting subjects, 

statistical analysis, and reporting. 

(2) General Considerations. Any protocol 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 repellents, and EPA’s rules for 

the protection of human subjects of research, 40 CFR part 26, subparts K through 

Q. Because these studies would support a FIFRA registration and involve 

intentional exposure of human subjects to the test repellents, review of each 

protocol by an Institutional Review Board (IRB), by EPA, and by EPA’s Human 

Studies Review Board (HSRB) is required before the study is initiated. (See 40 

CFR §26.1109, §26.1125, and §26.1601.) 

This guideline does not supersede or overrule the regulations governing research 

conducted on human subjects contained in 40 CFR part 26, subparts K through Q, 

or any other Agency regulations. To the extent there are any unintended conflicts 

between this guideline and any EPA regulation, the regulation at issue governs. 

(3) Related Requirements. 

(i) FIFRA Informed Consent Requirement. Any research conducted under 

this guideline is subject to §12(a)(2)(P) of FIFRA, which defines it as an 

unlawful act for any person “to use any pesticide in tests on human beings 

unless such human beings (i) are fully informed of the nature and purposes 

of the test and of any physical and mental health consequences which are 

reasonably foreseeable therefrom, and (ii) freely volunteer to participate in 

the test.” Regulations implementing this statutory provision and defining 

associated record-keeping requirements can be found at 40 CFR §169.2(j) 

and (k). 

(ii) EPA’s Rule for the Protection of Human Subjects of Research. Any 

research conducted under this guideline is covered by the requirements of 

EPA regulations for the protection of human subjects of research set out at 

 1 
40 CFR part 26, subparts K, L, and M. Persons conducting and submitting 

topical repellent efficacy studies should ensure compliance with all 

applicable requirements of that rule; the following paragraphs highlight a 

few of them: 

(A) Applicability. Subparts K and L of 40 CFR part 26 apply to 

regulated third parties who conduct or sponsor research involving 

intentional exposure of human subjects which is intended for 

submission to EPA for consideration under the pesticide laws—

FIFRA and §408 of the Federal Food, Drug, and Cosmetic Act 

(FFDCA) (21 U.S.C. 346a). (40 CFR §26.1101) Efficacy testing 

of topically applied repellents typically meets the regulatory 

definitions of “research” with “human subjects” involving 

“intentional exposure,” and must therefore be conducted and 

submitted to EPA in compliance with the requirements of these 

subparts. (See definitions at 40 CFR §26.1102.) Subpart M 

applies to any person who submits data from research with human 

subjects for consideration in connection with any action that may 

be performed by EPA under FIFRA or section 408 of FFDCA. 

(B) Prohibition of research involving pregnant or nursing women 

or children. 40 CFR §26.1203 as amended effective August 22, 

2006, provides: 

“Under no circumstances shall a person conduct or sponsor 

[covered] research that involves intentional exposure of any human 

subject who is a pregnant woman (and therefore her fetus), a 

nursing woman, or a child.” 

(C) Required review by Institutional Review Board (IRB). 40 CFR 

§26.1109(a) provides: 

“An IRB shall review and have authority to approve, require 

modifications in (to secure approval), or disapprove all research 

activities covered by this subpart.” 

(D) Required Pre-testing submissions to EPA. 40 CFR §26.1125 

provides: 

“Any person or institution who intends to conduct or sponsor 

human research covered by §26.1101(a) shall, after receiving 

approval from all appropriate Institutional Review Boards (IRBs), 

submit to EPA prior to initiating such research all information 

relevant to the proposed research specified by §26.1115(a), and, to 

the extent not already included: 

 2 
(a) A discussion of: 

(1) The potential risks to human subjects; 

(2) The measures proposed to minimize risks to the 

human subjects; 

(3) The nature and magnitude of all expected benefits 

of such research, and to whom they would accrue; 

(4) Alternative means of obtaining information 

comparable to what would be collected through the 

proposed research; and 

(5) The balance of risks and benefits of the proposed 

research. 

(b) All information for subjects and written informed consent 

agreements as originally provided to the IRB, and as 

approved by the IRB. 

(c) Information about how subjects will be recruited, including 

any advertisements proposed to be used. 

(d) A description of the circumstances and methods proposed 

for presenting information to potential human subjects for 

the purpose of obtaining their informed consent. 

(e) All correspondence between the IRB and the investigators 

or sponsors. 

(f) Official notification to the sponsor or investigator, in 

accordance with the requirements of this subpart, that 

research involving human subjects has been reviewed and 

approved by an IRB.” 

(E) Required post-testing submissions to EPA. 40 CFR §26.1303 

provides: 

“Any person who submits to EPA data derived from human 

research covered by this subpart shall provide at the time of 

submission information concerning the ethical conduct of such 

research. To the extent available to the submitter and not 

previously provided to EPA, such information should include: 

(a) Copies of all of the records relevant to the research 

specified by §26.1115(a) to be prepared and maintained 

by an IRB. 

(b) Copies of all of the records relevant to the information 

identified in §26.1125(a) through (f). 

 3 
(c) Copies of sample records used to document informed 

consent as specified by §26.1117, but not identifying 

any subjects of the research.” 

(F) Prohibition of EPA reliance on unethical human research. 40 

CFR §26.1705 provides: 

“ . . . EPA shall not rely on data from any research initiated after 

April 7, 2006, unless EPA has adequate information to determine 

that the research was conducted in substantial compliance with 

subparts A through L of this part, or if conducted in a foreign 

country, under procedures at least as protective as those in subparts 

A through L of this part.” 

(iii) Good Laboratory Practice Standards. Good Laboratory Practice 

Standards (GLP) as defined in 40 CFR part 160 apply to both laboratory 

and field studies of repellent efficacy. According to 40 CFR §160.17: 

“EPA may refuse to consider reliable for purposes of supporting an 

application for a research or marketing permit any data from a study which 

was not conducted in accordance with this part.” 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, 40 CFR 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)). 

(iv) State Requirements. Investigators and Sponsors should ensure research 

is conducted in compliance with any applicable state laws or regulations, 

which are independent of and additional to those cited here. 

(4) Organization of the Guideline. This guideline begins with definitions of special 

importance in understanding this guideline (Section b). Sections (c) through (h) 

provide general guidance applicable to all topical repellent efficacy testing, 

whether conducted in the laboratory or in the field, and to all target species. Each 

of these six sections discusses one of the primary stages of repellent testing. 

(c) Development of protocols for repellent studies. 

(1) Scientific design of repellent studies. 

(2) Ethical justification for repellent studies. 

(3) Subject selection and informed consent. 

(4) Protection of subject privacy and confidentiality 

(d) Review of protocols for repellent studies. 

(e) Changes to IRB-approved research before execution. 

(f) Execution of repellent studies. 

 4 
(g) Reporting of completed repellent studies. 

(h) Retention of records. 

Sections (i) through (l) provide guidance specific to different kinds of testing of 

repellents. Section (i) addresses empirical estimation of a typical consumer dose, 

and sections (j) through (m) address testing of repellency in the laboratory or field 

to particular species of arthropods. 

(i) Specific guidance for dose-determination studies. 

(j) Specific guidance for laboratory studies of mosquito or biting fly 

repellency. 

(k) Specific guidance for field studies of mosquito or biting fly repellency. 

(l) Specific guidance for laboratory studies of tick or chigger repellency. 

The final section (m) lists references considered in the development of this 

guideline. 

Three appendices are attached as well: 

Appendix A: Checklist of Elements Required by 40 CFR §26.1125. 

Appendix B: Framework for Science and Ethics Reviews of Proposed 

Human Research. 

Appendix C: Checklist of Elements Required by 40 CFR §26.1303. 

(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) The following events may indicate a failure of repellent efficacy.

 (i) A landing is the act of a flying or jumping insect or other arthropod 

alighting on human skin without probing or biting. 

(ii) A probe is the act of penetrating human skin by the mouthparts of an 

insect or other arthropod without ingestion of blood. 

(iii) A bite is the act of penetrating human skin by the mouthparts of an insect 

or other arthropod with ingestion of blood, typically associated with 

abdominal swelling and color change. 

(iv) A crossing is the act of passage by a tick or chigger from an area of 

untreated skin to an area of treated skin. A crossing may be quantified 

either or both by the distance the tick or chigger moves onto treated skin 

or by how long the tick or chigger remains on treated skin. 

 5 
(2) An unconfirmed event is a landing, probe, bite, or crossing not followed by 

another similar event within 30 minutes. 

(3) A confirmed event is one landing, probe, bite, or crossing followed by another 

similar event within 30 minutes. The first event is confirmed by the second; the 

second event is the confirming event. 

(4) A human subject is a living individual about whom an investigator conducting 

research obtains either data through intervention or interaction with the individual 

or identifiable private information. By this definition, both untreated control 

subjects and treated subjects are considered human subjects of repellent efficacy 

testing. 

(5) Questing is the behavior of ticks or chiggers actively seeking a host. 

(6) A repellent is a product intended to disrupt the host-seeking behavior of insects or 

other arthropods, driving or keeping them away from treated human skin. 

(7) Complete Protection Time (CPT) is the time from application of a repellent until 

efficacy failure as it is defined in each study—for example, the time from 

application until the first efficacy failure event confirmed within 30 minutes by a 

second similar event. 

(8) Dose determination is a testing procedure used to estimate a “typical consumer 

dose” of a topical repellent. 

(c) Development of protocols for repellent studies. The first major stage of repellent 

testing is development of a protocol. Under EPA’s amended rules for the protection of 

human subjects—40 CFR part 26—protocol development is the focus of far more 

attention than was the case before that rule took effect in April 2006. A detailed protocol 

must be approved by an Institutional Review Board (IRB) and, accompanied by 

supporting documentation meeting the requirements of 40 CFR §26.1125, must then be 

reviewed by EPA and the HSRB before research is initiated. 

It is critical to all later stages of repellent testing that protocols contain the elements 

required by EPA’s Good Laboratory Practices regulations at 40 CFR §160.120 and meet 

all other applicable scientific and ethical standards. The broad topics of scientific design, 

ethical justification, subject selection and informed consent, and protection of subject 

privacy and confidentiality are discussed in detail 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 such as to 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). 

 6 
(i) Objectives. The objective of most repellent efficacy testing to support 

registration is to estimate how long after treatment a repellent will 

continue to protect users from the target pest; this period of effective 

repellency is measured as CPT. Sometimes the objective of repellent 

efficacy testing is to compare the efficacy of one treatment to another 

treatment or to an untreated control; this ratio is expressed as Relative 

Protection (RP). In dose-determination testing associated with repellent 

testing the objective is typically to estimate a “typical consumer dose” for 

use in later repellent testing. In all cases the scientific objective should be 

stated clearly. 

(ii) Test materials. Repellent efficacy should be tested using the end-use 

formulation as registered or as proposed for registration. Test materials 

should be stored at ambient temperature and humidity before use. 

(iii) 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 while 

minimizing the risks to subjects. Considerations in endpoint selection for 

dose determination and repellency include: 

(A) Dose determination. To estimate a “typical consumer dose” the 

endpoint of concern is a rate of application, typically expressed 

either by weight as mg/cm2

 of treated skin surface or 

volumetrically as ml/cm2 of treated skin surface. Because each 

subject is likely to apply a different amount of repellent in 

uncontrolled trials, the “typical dose” should be calculated as the 

mean of multiple applications by each of many subjects. 

(B) Repellency. The endpoint of repellency testing should be selected 

to show a failure of repellent efficacy for subjects treated with a 

“typical consumer dose.” Efficacy failure in a test to determine 

Complete Protection Time (CPT) may be defined either as the first 

event—i.e., the first landing, bite, or crossing—or as the first 

confirmed event—i.e., the first landing, bite, or crossing confirmed 

within 30 minutes by another similar event. The Agency 

encourages the use of the first confirmed failure event to estimate 

complete protection time for individual subjects, and the median 

CPT across all subjects in each treatment arm as the summary 

measure of CPT. 

(1) In repellency testing with mosquitoes or biting flies the 

study design should choose either landings or bites as the 

event showing failure of repellent efficacy. The Agency 

encourages the choice of landings for field testing to reduce 

 7 
the risk of exposing subjects to vector-borne diseases. 

Even in laboratory testing using laboratory-reared, diseasefree insects, the choice of landings reduces the risk of an 

adverse reaction to bites. A proposal to use bites as an 

endpoint in either field or laboratory testing should be 

justified. 

(2) In repellency testing with ticks or chiggers the appropriate 

event to show failure of repellent efficacy is a “crossing” 

from untreated skin onto treated skin. 

(iv) Duration. Repellency testing should continue long enough to fairly 

assess the duration of protection provided by the repellent, and long 

enough that all or nearly all subjects experience efficacy failure. 

If testing ends before some subjects experience a failure of efficacy the 

resulting data-set is said to be “right-censored.” Right-censorship of data 

poses challenges for meaningful and accurate analysis. Right-censorship 

can be reduced through appropriate care in study design—for example, by 

planning a test of longer duration or, when appropriate, by pre-treating 

subjects well before their first exposure to target pests. Methods for 

analyzing right-censored datasets are discussed in paragraph (c)(1)(xi) 

below. 

(v) 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 in each protocol, taking into account the 

specific characteristics of the proposed research and the desired accuracy 

and precision of the results. Researchers are encouraged to consult a 

statistician to help determine appropriate sample size. 

Withdrawal of test subjects from the study before failure of efficacy 

decreases the sample size and may compromise the validity or utility of 

test results. The protocol should fully describe how the proposed sample 

size was determined, and how data for subjects who withdraw from the 

test prematurely will be treated. 

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

the experimental design, and the heterogeneity of the target population 

(e.g., by age, sex) and of the environment (different habitats, different 

conditions, different species and population densities). 

(vi) Allocation of subjects to treatments. Subjects should be allocated to 

treatments randomly and, to the extent possible, blindly. 

 8 
Multiple treatments per subject may be permissible if all treatments 

contain the same active ingredient at comparable concentrations, but care 

must be taken in the study design and execution to prevent crosscontamination or confounding interference. It is particularly important for 

subjects receiving different treatments on different limbs not to rub their 

limbs together or otherwise contaminate the repellent treatments, to keep 

results independent. The number of treatments per subject should be 

limited by the feeding behavior of the target species. For example, for 

mosquito species feeding close to the ground on lower limbs, treatments 

should be applied only to subjects’ lower legs, and only two treatments per 

subject will be possible. Treating the same subject more than once with 

the same treatment does not increase the sample size; a single subject can 

only be counted once in determining the sample size. 

When assessing relative protection provided by more than one product 

tested simultaneously, a Latin square design, in which each subject is 

tested with each treatment over the course of the study, is often 

appropriate. 

(vii) Untreated controls. To minimize risks to untreated control subjects, both 

the number of untreated subjects and the duration of their exposure should 

be minimized. Untreated controls should be exposed intermittently, and 

then only long enough to confirm adequate pest pressure throughout the 

study. Recent research (Barnard, et al. 2002) has shown that treated 

subjects should not serve as their own untreated controls. 

(viii) Positive controls. Positive controls are desirable to support comparison 

of results from different repellency studies or from testing on different 

days or at different locations. The recommended positive control material 

is 20% deet in ethanol, applied at a standard dose rate of 1 ml per 600 cm2

. 

Positive controls are particularly valuable when the objectives of the study 

include comparisons between formulations, and when testing spans 

different days. To minimize the likelihood of interference, subjects treated 

with the test material should not also serve as concurrent positive controls. 

(ix) Preparation of subjects. Before treatment with a test repellent or use as 

an untreated or positive control, subjects’ limbs should be washed with an 

unscented detergent and carefully rinsed and dried. Subjects should avoid 

alcohol, tobacco, and scented products (perfume, cologne, hair spray, 

lotion, soap, etc.) for at least twelve hours before and throughout the test. 

Subjects should avoid activities that increase perspiration, and avoid 

abrading, rubbing, touching, or wetting the treated area. 

(x) Treatment of subjects. Subjects in trials designed to estimate a typical 

consumer dose should self-treat with the repellent, which should be 

provided in the type of container and delivery system (e.g., pump spray, 

 9 
aerosol spray, towelette, or lotion) and with the directions for use intended 

for commercial distribution. Subjects in repellency trials should all be 

treated at the same standard dose rate, and treatment should be delivered 

so as to ensure consistent application and uniform coverage. 

(xi) Statistical analysis plan. Protocols should include a full description, 

explanation, and justification for the statistical methods proposed to 

analyze both dose determination and repellency test results, taking into 

account the specific study objectives and variables. 

The statistical analysis plan should provide for testing results for normal 

distribution. When results are normally distributed, it may be appropriate 

to report the mean CPT across all treated subjects with its standard error. 

When the data do not fit a normal distribution—more typical of repellency 

datasets—it may be possible to transform them to fit a distribution for 

which a parametric method of analysis can be employed. When the data 

do not fit and cannot be transformed to fit an underlying distribution, nonparametric analyses, such as Kaplan-Meier survival analysis1

, are 

suggested. 

If the study objective is to determine CPT across all subjects, any rightcensorship of repellency data will lead to underestimation of both the 

mean and the variance around it. Because right-censorship is common in 

repellency testing, EPA recommends use of the median CPT with its 95% 

confidence limits as the summary measure of CPT. 

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

inspections adequate to ensure the integrity of the study and consistent 

with the requirements of EPA’s Good Laboratory Practices regulations (40 

CFR part 160.) 

(2) Ethical Justification for Research. Because repellent efficacy testing is never 

directly beneficial to the subjects of the research, the risks to subjects must be 

minimized for the research to be ethically justified. The expected benefits to 

society from the knowledge likely to be gained in the research must also outweigh 

the minimized risks to subjects. (40 CFR §26.1111) 

(i) Alternatives to research with human subjects. If the research question 

can be answered without conducting new research with human subjects, 

human research is not justifiable. Investigators should consider possible 

alternatives to human research; protocols should discuss possible 

alternatives to human research and explain why they are infeasible or 

would not answer the research question. 

 

1

 For guidance see, for example, WHO (2009) Guidelines for Efficacy Testing of Mosquito Repellents for Human 

Skin: WHO/HTM/NTD/WHOPES/2009.A. Annex 3: Estimation of Median and Confidence Interval of Complete 

Protection Time Using the Kaplan-Meier Survivor Function. 

 10 
(ii) Prerequisite research. Before testing a repellent’s efficacy with human 

subjects, acute toxicity to animals of the active ingredients should be 

tested and reported in the protocol. Acute dermal toxicity, dermal 

irritation, eye irritation, and skin sensitization studies are needed to 

estimate the margin of exposure (MOE) for subjects participating in a 

repellent test. All additional components in the materials tested should be 

cleared by EPA for use in repellent formulations. 

(iii) Risks to subjects. Protocols should include a complete discussion of risks 

to subjects associated with their participation in the research, 

characterizing all risks and their likelihood, and describing steps proposed 

to minimize each of them. 

(A) Risk characterization. The nature of all risks to subjects should 

be described and the probabilities of occurrence of each type of 

risk should be estimated in the protocol. Potential risks in repellent 

testing typically include (but may not be limited to) reactions to the 

test substance or to arthropod bites, acquisition of vector-borne 

illness, physical risks (e.g., stresses imposed by the requirements 

or conditions of the test), or possible psychological risk associated 

with a breach of confidentiality in handling the results of 

pregnancy testing. 

(B) Risk minimization. As a condition of approval of proposed 

research, an IRB must determine that risks to subjects have been 

minimized by using procedures which are consistent with sound 

research design and which do not unnecessarily expose subjects to 

risk. (See 40 CFR §26.1111(a)(1).) Different actions are effective 

in minimizing different risks; protocols should identify specific 

steps taken to minimize each identified risk to subjects. 

Protocols should discuss specific steps proposed to prevent

research-induced harm—for example, by training subjects, using 

appropriate eligibility criteria, ensuring adequate supervision of 

subjects, and having a physician on call during testing. In addition, 

protocols should specify how any research-induced harm will be 

reversed—for example, by the investigator’s or sponsor’s 

committing to pay for uninsured costs of medical treatment of 

subjects for injuries or illnesses resulting from their participation in 

the research, and for longer-term treatment, if needed, of any 

arthropod-borne disease contracted during testing. 

Study protocols should include procedures for monitoring the 

safety of subjects, stopping rules specifying conditions under 

which a subject would be withdrawn from the research or the 

 11 
research would be terminated to protect subjects, and a medical 

management plan covering foreseeable contingencies. 

Methods of risk minimization are ultimately study-specific, but 

ensuring that risks to subjects are minimized in repellent testing 

may involve some or all of the following: 

• Monitoring of potential field testing sites for the presence 

of disease vectors at least weekly during the month before 

testing. Sites at which infected vectors are known to be 

present should not be used for field testing; 

• Serological or DNA-based assays of insects collected at the 

site of field testing to determine the presence or absence of 

disease organisms. Results of such testing should be 

reported to subjects after completion of the field test; 

• Using the minimum number of untreated controls 

consistent with statistical soundness; 

• Exposing untreated control subjects intermittently for only 

the minimum time required to confirm continued pest 

pressure; 

• Training subjects to use an aspirator to capture landing 

insects before they have time to bite; 

• Using only pathogen-free laboratory-reared insects in 

laboratory tests; 

• Excluding subjects known to be sensitive to repellents or to 

insect bites; 

• Using bites as the endpoint in either field or laboratory 

studies only when fully justified; 

• Ensuring that enough sub-investigators are present at all 

times so that the Principal Investigator can, if needed, 

attend to the safety of a subject without compromising the 

integrity of the research or endangering other subjects; 

• Initiating post-study contact with subjects to inquire about 

any signs of study-related injury or illness. 

(iv) Expected benefits of research. Participation as a subject in repellent 

testing has no direct benefit for individual subjects, yet is not risk-free, 

even after risks have been minimized. Thus the ethical justification for the 

research ultimately depends on the expected benefits to society of the 

information likely to be gained from the research. Protocols should 

characterize all anticipated benefits of the information to be gained from 

the research, and to whom or to what segment in society each identified 

benefit is likely to accrue. In addition, protocols should estimate the 

likelihood of achieving each identified societal benefit. 

 12 
 Societal benefits include only those directly resulting from the knowledge 

likely to be gained from the research. Benefits potentially resulting from 

use of effective repellents should not be attributed to the proposed 

research. Payments to subjects or other incentives for participation should 

not be treated as benefits, either to the subjects or to society. 

(v) Relation of benefits to risks. As a condition of approval of proposed 

research, an IRB must also determine that minimized “risks to subjects are 

reasonable in relation to anticipated benefits, if any, to subjects, and the 

importance of the knowledge that may reasonably be expected to result. In 

evaluating risks and benefits, the IRB should consider only those risks and 

benefits that may result from the research. . . . The IRB should not 

consider possible long-range effects of applying knowledge gained in the 

research . . . [to be] within the purview of its responsibility.” (See 40 CFR 

§26.1111(a)(2).) The protocol should discuss explicitly the balance of 

risks to subjects and anticipated societal benefits of the research. 

(3) Subject selection and informed consent. Subject selection is important both to 

the scientific merit of research and to its ethical acceptability. Selection of 

representative subjects is critical to the generalizability of the results of research, 

and to be ethically acceptable, subject selection must be equitable. (40 CFR 

§26.1111(a)(3)) Fully informed and fully voluntary consent of subjects is 

fundamental to ethical human research. (FIFRA §12(a)(2)(P) and 40 CFR 

§26.1116) 

(i) Representative sampling. The results of repellent testing should be as 

generalizable as possible to the target population of repellent users. 

Samples should include adults of various ages, of both sexes, and of a 

variety of races and ethnicities, and protocols should describe the 

demographic characteristics of the pool from which subjects will be 

recruited. Attractiveness of recruited candidates to the target species 

should be verified before they participate in repellency testing. 

Testing with a sample known to be unrepresentative is discouraged; if it is 

proposed, it should be fully justified. Arguments based on the 

convenience of the investigators or on the difficulties associated with 

recruiting a more representative sample do not justify testing with an 

unrepresentative sample. 

(ii) Inclusion/Exclusion factors. All exclusion/inclusion criteria proposed 

should be supported by a clear and explicit rationale. Because testing a 

topical repellent necessarily involves intentional exposure of subjects, 

children under 18 and pregnant or nursing women must be excluded. (See 

40 CFR §26.1203.) Because older adults are more susceptible to 

arthropod-borne diseases, candidates over 55 should be excluded as 

subjects in field testing, or their inclusion should be specifically justified. 

 13 
Members of certain vulnerable populations, including people of limited 

mental capacity, those not in good health or with compromised immune 

systems, those sensitive to chemicals, and students or employees of the 

investigators or sponsors should always be excluded. Other potential 

subjects who may be in a vulnerable position, including the educationally 

or economically disadvantaged, or those who communicate with difficulty 

because of language differences or disabilities, should not be excluded 

arbitrarily if appropriate specific provision can be made to ensure their 

safety and welfare. 

 

Subjects should generally be recruited from populations in the area where 

testing will be conducted. An offering of distant travel may unduly 

influence a candidate’s choice to enroll, and a subject who has accepted 

long-distance transportation may feel less than free to withdraw from a 

study. If it is proposed, transportation of subjects to distant locations 

should be justified, and specific mechanisms should be proposed to 

prevent any coercion or undue influence. 

(iii) Methods of recruiting. Protocols should describe in detail the proposed 

recruiting process, from the first contacts with potential candidates 

through all discussions of the research and the subjects’ enrollment. Any 

advertisements or flyers proposed for use in recruiting should be appended 

to the protocol, and should reflect IRB review and approval. If recruiting 

will be done through telephone calls, the script for recruiting calls should 

be appended to the protocol, and should reflect IRB review and approval. 

If any candidates may prefer to speak or read a language other than 

English, procedures for accommodating their needs in the recruiting 

process and in the conduct of the research itself should be described in 

detail. 

(iv) Compensation of subjects. It is reasonable to compensate participants in 

repellent efficacy research for their time and trouble. The level of 

compensation should not be so high as to constitute an undue influence in 

the choice to participate, nor should it be so low as to make participation 

in the research attractive only to the economically disadvantaged. 

Compensation should not be used or administered so as to compromise the 

freedom guaranteed to subjects to withdraw from participation at any time 

for any reason, without sacrificing benefits to which they are entitled. 

(v) Informed consent. It is a fundamental requirement for ethical research 

with human subjects that participation of subjects be both fully informed 

and fully voluntary. 

(A) Process. Informed consent is an extended process, involving 

much more than simply obtaining a subject’s signature on a form. 

 14 
It begins with recruiting, and continues after signature of the 

consent form throughout the conduct of the research. A single 

discussion in the protocol of the recruiting and consent processes 

as a continuous whole is recommended. 

As a condition of approval of proposed research, an Institutional 

Review Board (IRB) must determine that informed consent will be 

sought from each prospective subject in accordance with, and to 

the extent required by §26.1116, and that informed consent will be 

appropriately documented, in accordance with and to the extent 

required by §26.1117. (See 40 CFR §26.1111(a)(4) and (5).) 

The rule at §26.1116 requires that consent be sought “only under 

circumstances that provide the prospective subject . . . sufficient 

opportunity to consider whether or not to participate and that 

minimize the possibility of coercion or undue influence. The 

information that is given to the subject shall be in language 

understandable to the subject.” EPA recommends that consent 

discussions with individual candidates be conducted in private, and 

that all consent documents be written at or below an 8th grade 

reading level, as measured by the Flesch-Kincaid Grade Level 

score or another standard tool for assessing readability. (A tool to 

calculate the Flesch-Kincaid Grade Level score is a standard 

feature available to users of MS-Word.) If consent materials are 

translated into another language, it is important to ensure the 

translations are also at an appropriate reading level. 

The informed consent process should describe how the investigator 

will confirm each candidate’s understanding of the research and of 

what it will involve before he or she is enrolled. 

The rule at §26.1116 further defines the general requirements for 

informed consent, including at §26.1116(a) the basic elements 

always required, at §26.1116(b) other elements conditionally 

required, and at §26.116(e) a requirement to identify the pesticide 

and the nature of its pesticidal function. 

(B) Documentation of informed consent. The rule at §26.1117 

provides two options for documenting informed consent. The first 

option is a “written consent document that embodies the elements 

of informed consent required by § 26.1116.” The second is “a 

short form written consent document stating that the elements of 

informed consent required by § 26.1116 have been presented orally 

to the subject.” The short form requires that a witness be present 

in the consent interview. In repellent efficacy testing the short 

form is generally inappropriate; if use of a short form is proposed, 

 15 
it should be justified, and prior discussion with EPA is strongly 

encouraged. 

Ensure that any elements appearing in both the protocol and the 

consent form are in harmony. Elements common to both 

documents may include, for example, descriptions of the purpose 

of the research, subject inclusion/exclusion factors, risks to 

subjects and how they will be minimized, and what will happen in 

the course of the research. The protocol and the consent form 

should be consistent in substance, although the presentations 

should usually not be the same, because the readers and purposes 

differ. Consent forms should address candidates consistently in the 

second person—as “you”—and should present all information 

from the subject’s point of view. By contrast protocols should be 

written in the third person, and should present all information from 

the investigators’ point of view. 

In repellent testing, the consent form should tell subjects how 

many bites, if any, they are likely to get, and what symptoms of 

arthropod-borne disease they should be alert for after participating 

in a field study. 

(4) Protection of subject privacy and confidentiality. It is important to protect the 

privacy and confidentiality of subjects in repellent efficacy research, and to 

inform candidates in the recruiting and consent processes of the extent, if any, to 

which confidentiality of records identifying them will be maintained. 

(i) Subject identification. Subjects should not be identified by name in data 

collection forms or study reports. Subject names unavoidably must appear 

on consent forms and on administrative documents, but subjects should be 

identified only by an arbitrary code on other study documents. The key 

linking identifying codes to subjects should be stored securely, away from 

other study records. 

(ii) Photographic images. If photographs or videos are made to document 

the research, care should be taken to minimize making recognizable 

images of subjects. If faces or identifying marks cannot be excluded from 

a photograph or video image, the image should be altered to protect the 

identity of the subject(s). 

(iii) Pregnancy testing. Testing of female candidates for pregnancy should be 

handled with care and discretion. The investigator is responsible for 

ensuring that pregnant or nursing women are excluded as subjects (See 40 

CFR §26.1203). It is not necessary to meet this responsibility by testing 

all female candidates for pregnancy. For example, it is neither respectful 

nor informative to require women who are post-menopausal or surgically 

 16 
incapable of pregnancy to take a pregnancy test. Investigators should 

consider how best to meet their responsibilities while fully respecting 

female subjects. 

When pregnancy testing is conducted, the circumstances of testing should 

protect the privacy of the candidate, both during the testing itself and in 

handling the results. Unexpected news of a pregnancy can cause 

significant psychological distress, which can be increased by any breach 

of discretion in handling the information. It is a good practice to recruit 

more candidates than are required by the study design, so that the design 

would not be compromised by withdrawal of one or more subjects. This 

approach permits a candidate with a positive pregnancy test to withdraw 

without stating a reason. No records of a positive pregnancy test should 

be retained. 

(d) Review of protocols for repellent studies. EPA’s Rule for the Protection of Human 

Subjects of Research requires extensive review of protocols before research is initiated. 

The rule requires that the complete protocol, consent form, and supporting materials be 

reviewed and approved by an IRB (See 40 CFR §26.1109), and having been approved by 

the IRB, that it be submitted, along with specified additional supporting documentation, 

to EPA for review by EPA and by the HSRB (See 40 CFR §26.1125 and §26.1601.) 

(1) Review by IRB. The membership, functions, general procedures, and decision 

criteria for IRBs are defined in the rule at 40 CFR §26.1107-§26.1111. Records 

to be maintained by IRBs are defined at 40 CFR §26.1115. Most IRBs review 

many other kinds of human research in addition to repellent efficacy studies. 

Extensive guidance to and about IRBs can be found on the website of the Office 

of Human Research Protections (OHRP), at www.hhs.gov/ohrp. 

(i) Required elements in submission to IRB. Each IRB has its own 

application forms and procedures. Typical requirements are for a 

complete protocol, a consent form, and all materials intended for use in the 

recruiting process or to be provided to candidates or subjects. A protocol 

responsive to section (c) of this guideline is likely to satisfactorily address 

most requirements of a reviewing IRB. 

(ii) Criteria for IRB approval. The criteria for IRB approval of proposed 

research are defined in the rule at 40 CFR §26.1111. In summary, this 

passage of the rule requires that the IRB determine that proposed research 

meets all these criteria: 

• Risks to subjects are minimized; 

• Risks to subjects are reasonable in relation to the importance of the 

knowledge that may be reasonably expected to result from the 

research; 

 17 
• Selection of subjects is equitable; 

• Informed consent will be sought from each prospective subject; 

• Informed consent will be appropriately documented; 

• When appropriate, adequate provision is made for monitoring 

collected data to ensure the safety of subjects; and 

• When appropriate, adequate provision is made to protect the 

privacy of subjects and to maintain the confidentiality of data. 

In addition, the IRB ensures that when any subjects are likely to be 

vulnerable to coercion or undue influence, additional safeguards are 

included in the study design to protect their rights and welfare. 

(iii) Documentation of IRB approval. IRBs have the authority to approve, 

require modifications in (to secure approval), or disapprove research 

proposals. (40 CFR §26.1109.) It is common for IRBs to grant 

conditional approval subject to the investigator’s making specified 

modifications in the proposal; once required modifications are made, the 

IRB will issue formal written approval of the research. This letter of 

approval, along with all other correspondence between the investigators or 

sponsors and the reviewing IRB, must be included in the subsequent 

submission to EPA. (See 40 CFR § 26.1125(e) and (f).) 

(2) Reviews by EPA and the HSRB. After receiving approval from an IRB, the 

protocol and supporting material must be submitted to EPA for review by EPA 

and by the HSRB. (See 40 CFR § 26.1125.) 

(i) Required elements in submission to EPA. The elements to be included 

in a protocol submission to EPA are defined in the rule at 40 CFR 

§26.1125 and summarized in Appendix A to this guideline. Records of 

two kinds are required. 

(A) Records describing the research proposal itself. 40 CFR 

sections 26.1125(a)–(d) call for a discussion of the risks and 

benefits of the proposed research, for the consent forms as 

submitted to and as approved by the IRB, for information about 

how subjects will be recruited, and for a description of the 

circumstances and methods proposed for presenting information to 

subjects and seeking their consent. A protocol responsive to the 

guidance in section (c) of this guideline will already include this 

range of information. 

In addition, 40 CFR §26.1125(e) and (f) call for submission of all 

correspondence between the IRB and the investigators or sponsors 

(§26.1125(e)) and official notification of IRB review and approval 

(§26.1125(f)). For purposes of §26.1125(e), “correspondence” 

does not include attachments transmitted by correspondence, such 

 18 
as the protocol or consent form, which are otherwise required to be 

submitted by other provisions of the rule. 

(B) Records of the IRB review. 40 CFR §26.1125 also requires 

submission of copies of records relevant to the proposed research 

which the IRB is required to maintain by 40 CFR §26.1108(a). 

There is some overlap between these records and those discussed 

above, but there are some important differences. 40 CFR 

§26.1108(a) calls for, among other records: 

• Minutes of IRB meetings, in sufficient detail to show 

attendance; actions taken; votes for, against, and 

abstaining; the basis for requiring changes, and 

summarizing the discussion of controverted issues; 

• A list of IRB members identified by name, earned degrees, 

representative capacity, indications of experience sufficient 

to describe each member’s chief anticipated contributions 

to IRB deliberations, and any employment or other 

relationship between each member and the research 

institution; and 

• Written procedures for the IRB in the same detail described 

in §26.1108(a) and §26.1108(b). 

These records can only be obtained from the IRB. By prior 

arrangement, however, an IRB may submit them directly to EPA. 

(ii) EPA Review. Timely EPA review of submitted proposals for research is required 

by the rule at 40 CFR §26.1601. EPA will first review submitted proposals for 

satisfaction of the requirements of 40 CFR §26.1125, and for substantiation of any 

claims of business confidentiality associated with the submission. If a submission 

is found to be incomplete, or to include unsubstantiated claims of business 

confidentiality, the submitter will be promptly notified; the submission will not be 

reviewed further until these deficiencies are corrected. EPA evaluates complete 

proposals in a single substantive review addressing both scientific and ethical 

aspects of the proposal, following the general outline in Appendix B to this 

guideline. 

EPA will provide its completed review to the submitter. EPA’s review may call 

for revision of the proposal before it is reviewed by the HSRB. If EPA 

determines that the proposal is ready for HSRB review, it will be scheduled for 

HSRB review at the earliest opportunity. The submitter may respond to EPA’s 

review in writing to the HSRB docket, and may also comment orally at the HSRB 

meeting. 

 19 
(iii) HSRB Review. As provided in 40 CFR §26.1601(d), “following initial 

evaluation of the protocol by Agency staff, EPA shall submit the protocol and all 

supporting materials, together with the staff evaluation, to the Human Studies 

Review Board.” The HSRB is an advisory committee, chartered under the 

Federal Advisory Committee Act, charged with advising EPA on scientific and 

ethical aspects of proposed and completed research with human subjects. The 

HSRB meets several times a year in public session, with opportunities for public 

participation. All materials they consider are kept in a public docket, except any 

which are subject to a substantiated claim of business confidentiality. HSRB 

recommendations are documented in a public report of each meeting, prepared as 

soon as possible after the meeting—typically within 60-90 days. As soon as it is 

available, EPA will provide a copy of the HSRB’s report to submitters of research 

proposals discussed by the HSRB. Additional information about the HSRB, 

including agendas and reports from past meetings and schedules of future 

meetings, can be found at the HSRB website: www.epa.gov/osa/hsrb. 

(e) Changes to IRB-approved research before execution. After EPA and the HSRB have 

reviewed and commented on proposed research, revisions to the protocol, consent form 

or other materials may be needed before the research is executed. When IRB-approved 

materials are revised prior to initiation of the research to address concerns raised in EPA 

and/or HSRB reviews or for any other reason, those revised materials must be resubmitted to the IRB for review and approval before the research is initiated. This 

requirement applies to all changes to an IRB-approved protocol, to all changes to an IRBapproved consent form, and to all changes to any other materials approved by an IRB. 

(40 CFR §26.1108(a)(4)). Although an IRB has the discretion to conclude that some 

changes do not require review by the full IRB, investigators and sponsors do not have this 

discretion. EPA will treat as a breach of the regulations any planned changes to proposed 

research which are implemented without prior approval by the IRB. 

(f) Execution of repellent studies 

(1) Execution of protocol. When EPA and HSRB reviews are completed and the 

IRB has approved any revisions resulting from those reviews, the research can be 

initiated. Subjects can be recruited and data can be collected as specified in the 

protocol. Care should be taken to ensure and document that these events occur in 

the proper sequence. Initiation of subject recruitment before obtaining IRB 

approval of the final protocol will be treated as a breach of the regulations. 

(2) Quality Assurance (QA) oversight. Repellent 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 to monitor execution of each protocol and 

document its conduct in accordance with the GLP regulations. (40 CFR §160.35) 

 20 
(3) Deviations from protocol. In executing even the best designed and most 

comprehensive protocols, unanticipated deviations from the protocol may occur. 

All such deviations from the protocol should be promptly and fully reported to the 

IRB, and both the deviations and their impact on the research should be discussed 

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. 

Planned changes must be treated as amendments to the protocol, and must be 

approved by the IRB before they are implemented. (40 CFR §26.1108(a)(4)) 

(4) Changes in IRB-approved research after initiation. Any amendment to the 

protocol, consent form, or other materials approved by the IRB must be submitted 

to the IRB for review, and must be approved by the IRB before it is implemented, 

unless immediate implementation is required to avert an imminent hazard to 

subjects. (40 CFR §26.1108(a)(4)) 

(5) Reports of adverse events. Any adverse events affecting the subjects of a 

repellent efficacy test must be promptly reported to both the overseeing IRB and 

to EPA, including adverse events not established to be related to product exposure 

or study participation. (40 CFR §26.1108(b)(1)) 

(g) Reporting of completed repellent studies 

(1) Study report. In addition to the standard elements required by the GLP 

regulations at 40 CFR §160.185 and the appendices described below, the primary 

report of the study should include these elements, as applicable. 

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

director, investigators, name and location of the testing facility, and dates 

of the study. 

(ii) Approved or intended label directions for use. 

(iii) Recruiting and enrollment statistics, including the following: 

• Demographic characteristics of the pool from which subjects were 

recruited and of the subject sample selected; 

• Numbers of candidates contacted, interviewed, and screened; and 

• Numbers of subjects enrolled, withdrawn voluntarily, withdrawn 

involuntarily, and completing the research. 

(iv) Test arthropods: Genus, species, subspecies or strain (if information is 

available) of target pests; and pest pressure in each cage or field site. For 

laboratory studies, the source of test arthropods, methods used to establish 

 21 
and maintain their disease-free status, rearing techniques and conditions, 

developmental stage, age and sex of target arthropods and methods used to 

sex them; preparation of arthropods before testing, and density per cage or 

container. 

(v) Test conditions: For field studies, the location and type of habitat, species 

distribution, climate and environmental conditions. For laboratory studies, 

a complete description of cages or containers, temperature, relative 

humidity, ambient light, and air flow. 

(vi) Test procedures, including but not limited to: 

• Preparation of subjects: Training, demonstrating attractiveness, 

clothing and protective equipment worn, calculation of skin area 

and individual dose, washing, rinsing, and drying of limbs; 

• Allocation of subjects to treatments; number of treatments per 

subject; blinding key, if used; 

• Method of dose determination; 

• Method of application of repellent to each subject and individual 

dose applied; 

• Time of application of repellent to each subject; 

• Time of start and end of exposure of each subject. 

(vii) Complete accounting for all events during the testing period. 

(viii) Results for each subject. 

(ix) Reports of all statistical analyses 

(x) Conclusions 

(xi) Discussion 

(2) Appendices. 

(i) Complete protocol as approved by the IRB. 

(ii) Consent form and any additional recruiting materials as approved by the 

IRB. 

 22 
(iii) All amendments to the protocol, consent form, or other materials as 

approved by the IRB. 

(iv) Reports of all deviations from the protocol and assessment of their impact 

on the integrity of the research. 

(v) Raw data and data collection sheets. 

(vi) Documentation of ethical conduct of the research, as required by 40 CFR 

§26.1303, including all correspondence with IRB not previously submitted 

to EPA. The requirements of this section of the rule are summarized in 

Appendix C to this guideline. 

(h) 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 in 40 CFR §26.1115 apply to Institutional 

Review Boards (IRBs) that review human research conducted under EPA’s 

Human Research Rule. 

(2) The record-keeping requirements of 40 CFR §169.2(j) apply to investigators who 

conduct pesticide research with humans subject to FIFRA §12(a)(2)(P). 

(3) The record-keeping requirements of 40 CFR §169.2(k) apply to any person who 

submits the results of research to EPA in support of a petition for a tolerance or 

tolerance exemption or in support of registration or an application for registration. 

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

records of any study conducted under the Good Laboratory Practices rule. 

(i) Specific guidance for dose-determination studies. All subjects participating in 

repellency testing should be treated with the test material at a standard dose rate, 

expressed by weight of repellent per unit area, mg/cm2

 of treated skin, or volumetrically 

as ml/cm2

 of treated skin. Recommended methods for choosing the standard dose depend 

in part on the active ingredient(s) and formulation(s) of the test material. For testing of 

lotion formulations containing DEET (N,N-diethyl-meta-toluamide) eet, a standard dose 

rate of 1 g per 600cm2

 (equivalent to 1.67 mg per cm2) has been used in many tests of 

repellency. Repellents in lotion form containing DEET may be tested for repellency at 

this standard dose rate, or at a “typical consumer dose” rate determined empirically 

through a dose-determination study. For other ingredients and formulations no 

comparable standard dose has been identified, and it is recommended that a “typical 

consumer dose” be estimated through empirical dose-determination testing as described 

below. The rationale for the dose rate actually used in repellent testing should be 

documented in all study reports. 

 23 
(1) Test material. The formulated product should be used as it is or will be 

marketed, in the same type of container and bearing the same directions for use. 

(2) Subjects. Subjects used for dose determination should be recruited and informed 

as described above, and must provide written informed consent for their 

participation. The same or very similar eligibility criteria would apply as for 

repellency testing, although subjects participating only in dose-determination 

testing would not be exposed to any insects or to any of the risks associated with 

exposure to insects. The number of subjects used for dose determination should 

be justified statistically. The sample should include adults of both sexes and 

varied race, and should be as representative as possible of the repellent user 

population, consistent with ethical and feasibility constraints. 

(3) Measuring subject’s skin area. The surface area of subject’s limb in cm2

 can be 

estimated by measuring the circumference of the forearm in centimeters at the 

wrist and elbow, or of the leg at the ankle and knee, and in either case at one or 

two equally spaced intermediate points; then multiplying the average 

circumference by the length of the limb in centimeters from the wrist to elbow or 

from the ankle to the knee. 

(4) Methods. Each subject should be instructed to apply the test repellent to his or 

her own limbs—to forearms or lower legs, or both—as they normally would 

apply a repellent to achieve complete coverage. Once the quantity applied has 

been measured and recorded, the applied repellent should be washed completely 

off the limb. This process should be repeated at least 3 times for each limb 

treated by each subject. 

The applied quantity of a lotion or towelette formulation can be determined by 

weighing the container or towelette before and after use. Because less than all the 

spray released from a pump-spray or aerosol container will reach the target limb, 

a different method is needed to estimate the applied quantity of a spray 

formulation. The target limb should be wrapped in gauze “bracelets” of known 

area so that the bracelets cover only part of the skin. So long as the area of the 

bracelets is known, the quantity of a spray formulation applied to the skin per cm2

can be estimated by weighing the bracelets before and after application. Skin 

should be left exposed between bracelets to help subjects determine when they 

achieve complete coverage. Dose determination with spray formulations should 

be conducted out-of-doors. 

(5) Calculating standard dose for use in repellency trials. The mean dose applied 

by each subject to each limb and the grand mean across all subjects of mean doses 

applied to forearms and to lower legs should be calculated. The specific gravity 

of the test material should be used to convert the dose expressed by weight as mg 

per cm2

 to a volumetric dose expressed as ml per cm2; this calculation should be 

included in the study report. The volumetric dose should be used as the standard 

dose for repellency testing, scaling it to the treated surface area of each subject’s 

 24 
limb or limbs. The protocol should include the specific gravity of all test 

materials and should specify this calculation, and should specify doses both by 

weight and by volume. 

(j) Specific guidance for laboratory studies of mosquito or biting fly repellency. 

(1) Species. Mosquito tests should be conducted with Aedes aegypti, an Anopheles

species such as An. albimanus, or a Culex species, preferably Cx. 

quinquefasciatus. Stable fly tests should be conducted with Stomoxys calcitrans. 

The source of test insects should be reported, and if possible, they should be 

identified by subspecies or strain. 

(2) Stage, age, and sex. Mosquito testing should be conducted with adult females 5-

10 days old; methods of sexing should be reported. Stable flies should be male or 

female adults 3-10 days old. The age range of test insects should be reported. 

(3) Rearing techniques. Mosquito and biting fly larvae should be reared under 

optimal conditions for the species—typically at 27° ± 3°C, relative humidity 80% 

± 10%, and photoperiod 16:8 hours (light:dark). Alternative rearing methods for 

mosquitoes are discussed by Gerberg et al. (1998). Use of alternative rearing 

techniques for biting flies and/or mosquitoes should be fully documented and 

justified. 

(4) Preparation of insects. Test mosquitoes should be fed 10% sucrose and no 

blood meal before use in a test, and starved for 12 to 24 hours immediately before 

the test. Test stable flies may be fed dry sugar cubes with a separate water source; 

they should also receive no blood meal before use in a test, and should be starved 

for 24 hours immediately before the test. Test insects should be established to be 

free of disease; the source of insects and the methods used to ensure they are 

disease-free should be reported. 

(5) Disposition of insects. Test insects should be used only once and destroyed 

immediately after the trial. 

(6) Test cages. Cage size may vary, but is typically 2’ x 2’ x 2’ (8 cubic feet in 

volume, roughly equivalent to 232,000 cm3

), square or rectangular in plan, with 

one or more sleeved openings for the subject’s arms. A mirror on the bottom of 

the cage helps observers see insects landing on the underside of subjects’ arms. 

Larger cages may be used for two subjects at a time, so long as they are treated 

with the same repellent. Cage dimensions and design should be reported in detail. 

(7) Insect density. There should be at least 200 mosquitoes in each 2’x2’x2’ cage 

(equivalent to one for each 1,160 cm3

) or at least 50 stable flies per cage (one for 

each 4,640 cm3

.) After each exposure period all stable flies should be removed by 

vacuuming from each cage, and 50 fresh stable flies should be added to each cage. 

 25 
(8) Testing conditions. Ambient temperature during the test should be maintained at 

21° ± 8°C, and relative humidity at 55-85%. Ambient light appropriate to the 

preferred feeding time for the test species should be provided—e.g., more light 

for day feeders like Aedes aegypti, and less for night feeders like Anopheles spp.

and Culex spp. When testing in subdued light, care should be taken not to 

compromise the investigators’ ability to observe insect activity. Investigators and 

subjects should avoid exhaling into the test cage; introduction of CO2 could bias 

insects towards biting. 

(9) Treated area size and preparation. The recommended treatment area is the 

subject’s forearm (wrist to elbow). This area should be washed with unscented 

soap, rinsed with a solution of ethanol or isopropyl alcohol in water, and dried 

with a clean towel. The surface area of each test subject’s treated forearm (in 

cm

2

) and the volumetric dose administered to each subject (in ml) should be 

reported. Skin adjacent to the treated area should be covered with a light-colored 

material that test insects cannot bite through. Hands should be covered with 

impenetrable (e.g., latex) gloves. 

(10) Untreated controls. One or two untreated control subjects should verify 

continued landing pressure in tests of CPT. Results for these untreated subjects 

should not be compared to those for treated subjects; multiple untreated controls 

should be used as a standard of comparison only in studies of relative 

protection/percent repellency. To minimize potential interference, untreated 

subjects should not be treated on their other forearm. Forearms of untreated 

subjects should be washed, rinsed, and dried exactly like treated forearms. 

(11) Positive controls. A positive control group treated at a rate of 1 ml per 600 cm2

with 20% DEET in ethanol is recommended to calibrate the test system. 

(12) Establishing subject attractiveness to test insects. Before the test, subjects 

should expose their untreated forearms to the target insects in a test cage to 

establish their attractiveness. Five mosquito landings in one minute or less or 2 

stable fly landings in two minutes or less are sufficient to establish attractiveness. 

(13) Establishing and confirming landing pressure. Before exposing any treated 

subjects during each exposure period, an untreated subject’s forearm should be 

inserted into each cage to confirm landing pressure. Landing insects should be 

removed from the subject’s arm, by shaking it or by other means, before they 

have time to bite. The untreated subject should remove his or her forearm from 

the cage as soon as it has received the requisite number of landings. If at any time 

fewer than 5 mosquitoes land on the untreated control forearm within one minute, 

all mosquitoes should be removed from all cages in the study and fresh insects 

should be added to each cage. If at any time fewer than 2 stable flies land on the 

untreated control forearm within two minutes, all stable flies should be removed 

 26 
from that cage and 50 fresh flies should be added. The aggressiveness of the fresh 

insects should be confirmed before repellency testing continues. 

(11) Exposure period. Approximately 30 minutes after treatment with the repellent, 

test subjects should insert their treated forearms into the cage for the first time. 

Exposures of pre-defined duration should be repeated at regular intervals—e.g., 5-

minute exposures at 30-minute intervals—until efficacy failure for each subject or 

the end of the study, whichever occurs first. Subjects should avoid rubbing their 

arms when inserting them into or removing them from the cage and between 

exposure periods.

(k) Specific Guidance for field studies of mosquito or biting fly repellency. 

(1) Pre-test subject preparation. Subjects in field studies of repellency should be 

trained in the laboratory to identify landings, probes, and bites of the target 

species and to use aspirators to collect landing insects before they have time to 

bite. 

(2) Choice of field testing sites. Field tests for mosquito repellency should be 

conducted in at least two distinct habitats (e.g., forest, grassland, salt marsh, 

wetland, beach, barns, or urban environments) where the predominant mosquito 

species differ. So long as the environments and species distribution differ, it is 

not important for test sites to be widely separated geographically. Repellency 

against black flies, stable flies, or other biting flies typically occurring in only one 

habitat may be tested in only one habitat. 

Potential sites for testing mosquito repellency should be monitored at least weekly 

for a month before testing is scheduled. To minimize risks to subjects, field 

testing should not be conducted where West Nile virus (WNV) or other mosquitovectored diseases have been detected within the previous two weeks. Because 

biting flies are not known to vector human diseases in the USA, no comparable 

site monitoring is required for studies of repellency to biting flies. 

(3) Species identification. Mosquito tests should be conducted where more than one 

species is present. 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. 

Landing insects should be aspirated or trapped before and during the test, and 

labeled with the time of collection. After the field study, collected insects should 

be identified by genus and species, and if possible, by subspecies or strain. The 

number in each taxon collected in each time period should be reported. 

After identification, mosquitoes should be subjected to serological or other 

analysis to determine the presence or absence of WNV or other disease 

organisms. The results of these analyses should be reported to subjects and 

included in the study report. 

 27 
(4) Untreated controls. Untreated control subjects are necessary in all study 

designs. To minimize the potential for interference, untreated controls should be 

subjects who are not treated with a repellent on any limb. 

When the objective is to estimate CPT, two untreated control subjects are 

sufficient. They should expose an untreated arm or leg briefly at regular intervals 

during the test to confirm continued acceptable landing pressure. Results for the 

untreated controls should not be compared to results for treated subjects. When 

the objective is to evaluate relative protection, more untreated control subjects are 

needed to provide a standard of statistical comparison. 

 

Untreated limbs used as controls should be washed, rinsed, and dried exactly like 

treated limbs. 

(5) Positive controls. A positive control group treated at a rate of 1 ml per 600 cm2

with 20% deet in ethanol is recommended to calibrate the test system. 

(6) Minimum landing pressure to initiate or continue test. Landing pressure 

should be measured before treatment and intermittently throughout the course of 

the test by untreated control subjects. Testing should not be conducted or 

continued unless landing pressure of the target species is at least one mosquito 

landing within one minute, or at least one stable fly, black fly, ceratopogonid or 

tabanid landing within five minutes. Insects landing on untreated subjects should 

be collected for later identification, and labeled with the time of collection. 

(7) Treated area size and preparation. The recommended treatment area is the 

subject’s forearm (wrist to elbow) or lower leg (ankle to knee), depending on the 

feeding behavior of the predominant species at the selected test site. A smaller 

treated area on the appropriate limb of at least 250 cm2

 is also acceptable. The 

treated area should be washed with unscented soap, rinsed with a solution of 

ethanol or isopropyl alcohol in water, and dried with a clean towel. The surface 

area treated for each test subject limb and the specific dose administered (ml) to 

each subject should be recorded. With the exception of the treated area, subjects’ 

heads, trunks, and limbs should all be covered with light-colored material through 

which insects cannot bite. 

(7) Exposure period. Exposure of treated subjects may be intermittent—for 

example, for 1 or 2 minutes at 15-minute intervals, or for 5 minutes at 30-minute 

intervals—to reduce risks to subjects and allow them to rest between exposures. 

When testing a repellent with a long period of effectiveness, it is critical to the 

reliability of the data to minimize “right-censoring” of the data—i.e., the number 

of subjects who do not experience a failure of efficacy before the end of the test. 

Reliable results may be obtained for extended periods of protection by treating 

subjects up to several hours before the first field exposure, with subsequent 

 28 
exposures timed to coincide with periods of target insect activity. This approach 

reduces prolonged exposure of subjects to insects in the field, helps to reduce 

early withdrawal of subjects attributable to excessively long trials, and increases 

the likelihood that most or all subjects will experience a failure of efficacy. 

Another acceptable design for assessing long-term repellency involves treating 

different subjects at different times (e.g., 2, 4, 6, 8, or 10 hours before exposure) 

and then exposing all subjects at the same time when target insects are active. 

(8) Minimizing variation. Many factors contributing to variability in repellent field 

studies cannot be controlled, but minimizing variation when possible and 

designing studies with insect feeding patterns in mind can make a study design 

more efficient. Standardization is possible for such factors as time of testing, 

allocation to treatment, subject attractiveness to target insects, etc. Other factors 

which may be managed to minimize variation include targeting a predominant 

species in its habitat, synchronizing exposure periods with peak feeding activity 

of target insects, and treating limbs consistent with the preference of the target 

species to feed on legs, arms, or both. 

(9) Environmental conditions. The time of day at which subjects are treated and at 

which exposure to target insects begins and ends should be recorded and reported. 

Weather conditions during testing (including temperature, relative humidity, 

cloud cover, precipitation, light intensity, and wind speed) should be monitored 

periodically throughout the study and reported. Testing should be not be 

conducted or continued if wind speed exceeds 16 kph/10 mph. 

(10) Test subject placement and behavior. Subjects may work in pairs to assist each 

other to identify landings and collect insects. Because clustering of subjects may 

confound results, at least 3 m/10 ft should be maintained between pairs. Subjects 

should avoid strenuous exercise and sweating, since these could affect test results. 

(11) Data collection and reporting. Under the supervision of the investigator, an 

associate or another subject should record the number and timing of each landing 

or bite during each exposure period for each subject. If possible, all landing 

insects should be collected for identification and labeled with the time of 

collection. 

(l) Specific guidance for laboratory studies of tick and chigger repellency. Because 

reliable field tests have not been developed, EPA recommends testing for repellency 

against ticks and chiggers in the laboratory. If field tests are conducted, proposed label 

directions for re-application frequency should reflect field study results. 

(1) Summary of recommended method. Each subject should place the fingers of 

one hand on a flat surface, with the elbow above the wrist and the forearm held at 

an angle of 30° or more to the surface. With a suitable instrument (such as an 

 29 
artist’s paintbrush, forceps, or a cotton swab) the investigator should place a tick 

or chigger, one at a time, on the subject’s wrist, at a release point marked 3 cm 

below the boundary of the treated area of the forearm. The tick or chigger should 

be oriented gently (e.g., with paint brush, forceps, or cotton swab) toward the 

treated area. After its first movement up the arm toward the margin of the treated 

area, each tick or chigger should be allowed 3 minutes to move across the 

boundary onto the treated area. A tick or chigger that crosses at least 3 cm into 

the treated area (toward the elbow) is reported as ‘not repelled’. One that does not 

cross into the treated area, or that crawls into the treated area but immediately 

turns back or falls off, is reported as ‘repelled.’ Fresh ticks or chiggers are 

exposed to the treated area one at a time, at regular intervals for the duration of 

the test. 

While the general definition of a “crossing” is constant, the details of the method 

of scoring a crossing must be operationalized in each protocol in a way 

appropriate to the species and life stage of ticks to be tested. A release point 3 cm 

distant from the treated area and scoring of a crossing when a tick moves at least 3 

cm into the treated area have worked effectively in tests with nymphal 

Dermicentor variabilis and Ixodes scapularis, and are provided here as examples.

(2) Species. Tick tests should be conducted using laboratory colonies of the tick 

species the label claims to repel. Common tick species in the United States 

include the blacklegged tick (deer tick, Ixodes scapularis), western blacklegged 

tick (deer tick, Ixodes pacificus), lone star tick (Amblyomma americanum), 

American dog tick (Dermacentor variabilis), and relapsing fever tick (softbacked 

tick, Ornithodoros turicata). Chigger tests should be conducted using laboratory 

colonies of chiggers in the Trombiculidae family; Eutrombicula splendens; or E. 

cinnabarrs. Test arthropods should be identified by genus and species, and if 

possible by subspecies or strain. It is permissible to test two species concurrently, 

alternating so that each subject qualifies and tests a tick or chigger from each 

species within each exposure period. 

(3) Stage, age, and sex. When testing with blacklegged (deer tick), lone star, or softbacked ticks, either adult or nymphal life stages are appropriate for testing. Only 

the adult American dog tick is recommended, since nymphs of this species do not 

feed on humans. Tests with chiggers should use immature chiggers. The age or 

age range of all test arthropods should be reported. 

(4) Rearing techniques. Ticks and chiggers should be reared at 25° ± 3°C, at high 

relative humidity (> 90%), and photoperiod of approximately 16:8 hours 

(light:dark). Any alternative rearing techniques should be fully described and 

justified. 

(5) Preparation of test arthropods. Ticks and chiggers should be disease-free. The 

source of test animals and the methods used to ensure they are disease-free should 

be reported. 

 30 
(6) Disposition of test arthropods. Each tick or chigger should be used only once 

and should be destroyed immediately after use. 

(7) Number of ticks or chiggers. Ticks or chiggers are tested for questing behavior 

and for repellency one at a time, according to the study design. Some studies 

using a single species of tick test 5 ticks in each 30-minute exposure period; other 

studies test one tick of each of two species in each 15-minute exposure period. 

Either pattern is acceptable. 

(8) Testing conditions. Temperature should be maintained during the test at 20° to 

25°C, with relative humidity at ≥ 35% and indirect ambient light 50 to 80%. The 

lights should be kept on. 

(9) Subject preparation. Before treatment, both forearms of each subject are 

washed with unscented detergent and carefully rinsed and dried. One forearm 

from wrist to elbow is treated with the test material, and two lines are drawn on 

the subject’s wrist: a ‘boundary line’ at the edge of the treated area, and another 

line—the ‘release line’—3 cm distant from the boundary line, outside the treated 

area toward the fingers. Similar lines are drawn in the same positions on the wrist 

of the subject’s other, untreated arm—one line at the wrist, and another 3 cm 

away toward the fingers. 

(10) Untreated control. The untreated forearm of each treated subject is used to 

screen ticks or chiggers for questing behavior; only actively questing ticks or 

chiggers should be selected for repellency testing. With an appropriate 

instrument, such as an artist’s paint brush, forceps, or a cotton swab, each tick or 

chigger should be picked up carefully to prevent damaging its body or forelegs 

and placed on the release line on the wrist of the subject’s untreated arm. A tick 

or chigger that moves steadily from the release line across the boundary line and 

upward along the subject’s untreated forearm is actively questing, and appropriate 

for use in repellency testing. A tick or chigger failing this test should be 

immediately destroyed. 

(11) Positive controls. A positive control group treated at a rate of 1 ml per 600 cm2

with 20% deet in ethanol is recommended to calibrate the test system.

(12) Test procedure. With the treated forearm held upright above the table, one fresh 

tick or chigger at a time, immediately after demonstrating active questing on the 

subject’s untreated arm, is placed at the release line on the treated arm. A 

“crossing” is recorded if the test organism crosses the boundary line at least 3 cm 

into the treated area within 3 minutes, and remains in the treated area for at least 

one minute. 

(13) Exposure period. At intervals of 15 to 30 minutes according to the study design, 

fresh ticks or chiggers are placed one at a time at the release point on the 

 31 
untreated arm and tested for active questing. Qualified ticks or chiggers are then 

placed at the release point on the treated arm and exposed to the treated area. The 

cycle continues until efficacy failure or the end of the study, whichever occurs 

first. 

(14) Data collection and reporting. Recording of crossings and repelled test ticks or 

chiggers arthropods during the exposure period should be supervised by 

investigators. The investigator, an associate, or another subject should record the 

number and timing of all events occurring during in each exposure period for each 

subject. 

(m) References. The following publications were consulted in developing this guideline. 

(1) American Society for Testing and Materials. (2006) E 939-94 Standard Test 

Method of Field Testing Topical Applications of Compounds as Repellents for 

Medically Important and Pest Arthropods (Including Insects, Ticks and Mites): I 

Mosquitoes. 

(2) American Society for Testing and Materials. (2003) E 1488-02 Standard Guide 

for Statistical procedures to use in Developing and Applying test methods. 

(3) Barnard, D.R.; Ulrich, R.B.; Xue, R.; and Debboun, M. (2007) Chapter 5: 

Standard methods for testing mosquito repellents. In Insect Repellents: Principles, 

Methods and Uses. Debboun, Frances, and Strickman, eds. CRC Press. (495 p.) 

(4) Bernard, D. R., Bernier, U. R., Posey, K. H. and Xue, R-D (2002). Repellency of 

IR 3535, KBR 3023, para-menthane-3,8,-diol, and deet to Black Salt Marsh 

Mosquitoes (Diptera:Culicidea) in Everglades National Park. J. Med. 

Entomology39(6): 895-899 

(5) Barnard, D.R. (1998) Mediation of Deet repellence in mosquitoes (Diptera: 

Culicidae) by species, age, and parity. J. Med. Entomol. 35(3): 340-343. 

(6) Barnard, D.R.; Posey, K.H.; Smith, D.; and Shreck, C.E. (1998) Mosquito 

density, biting rate and cage size effects on repellent tests. Med. Vet. Entomol. 

12:39-45. 

(7) Carroll, S. P. (2007) Chapter 12: Evaluation of topical insect repellents and 

factors that affect their performance. In Insect Repellents: Principles, Methods 

and Uses. Debboun, Frances, and Strickman, eds. CRC Press. (495 p.) 

(8) Frances, S.P. (1994) Response of a chigger, Eutrombicula hirsti (Acari: 

Trombiculidae) to repellent and toxicant compounds in the laboratory. J. Med. 

Entomol. 31(4): 628-630. 

 32 
 33 

(9) Frances, S.P.; Eikarat, N.; Sripongsai, B.; and Eamsila, C. (1993) Response of 

Anopheles dirus and Aedes albopictus to repellents in the laboratory. J. Am. Mos. 

Con. Assoc. 9(4): 474-476. 

(10) Gerberg, E. J. ; Barnard, D.R.; and Ward, R.A. (1998) Manual for Mosquito 

Rearing and Experimental Techniques. AMCA Bull. 5. 

(11) Govere, J.M.; and Durrheim, D. N. (2007) Chapter 8: Techniques for evaluating 

repellents. In Insect Repellents: Principles, Methods and Uses. Debboun, Frances, 

and Strickman, eds. CRC Press. (495 p.) 

(12) Huntsberger, D. and Billingsley, P. (1981) Elements of Statistical Inference. 5th

edition. Allyn and Bacon. Inc., Boston. 

(13) Klun, J.A. and Debboun, M. (2000) A new module for quantitative evaluation of 

repellent efficacy using human subjects. J. Med. Entomol. 37(1): 177-181. 

(14) Schofield, S.; Tepper, M.; and Gadawski, R. (2007) Field evaluation against 

mosquitoes of regular and polymer-based deet formulations in Manitoba, Canada, 

with comment on methodological issues. J. Med. Entomol. 44: 457-62. 

(15) Smith, C.N. (1955) Insect repellents. Quarterly Report, Entomological Research. 

Entomology Research Branch, U.S. Department of Agriculture. 8 p. 

(16) Verwey, R.E. (1996) Laboratory method for testing insect repellents on human 

test subjects against chiggers in the laboratory. Unpublished document prepared 

by S.C. Johnson & Sons, Inc., Racine, WI. 3 p. 

(17) WHO (2009) Guidelines for Efficacy Testing of Mosquito repellents for Human 

Skin: CDS/NTD/WHOPES/2009.4. 

(18) WHO (1996) Report of the WHO Informal Consultation on the Evaluation and 

Testing of Insecticides: CTD/WHOPES/1C/96.1. 
Appendix A: Checklist of Elements Required by 40 CFR §26.1125

 34 

40 CFR 26.1125 Prior submission of proposed human research for EPA review

Any person or institution who intends to conduct or sponsor human research covered by §26.1101(a) shall, after receiving 

approval from all appropriate IRBs, submit to EPA prior to initiating such research all information relevant to the proposed 

research specified by §26.1115(a), and the following additional information, to the extent not already included: 

Requirement Y/N Comments/Page Refs 

(1) Copies of 

• all research proposals reviewed by the IRB, 

• scientific evaluations, if any, that accompanied the proposals 

reviewed by the IRB, 

• approved sample consent documents, 

• progress reports submitted by investigators, and reports of injuries 

to subjects. 

(2) Minutes of IRB meetings . . . in sufficient detail to show 

• attendance at the meetings; 

• actions taken by the IRB; 

• the vote on these actions including the number of members voting 

for, against, and abstaining; 

• the basis for requiring changes in or disapproving research; 

• a written summary of the discussion of controverted issues and 

their resolution. 

(3) Records of continuing review activities. 

(4) Copies of all correspondence between the IRB and the investigators. 

(5) 

• A list of IRB members identified by name; earned degrees; 

representative capacity; indications of experience such as board 

certifications, licenses, etc., sufficient to describe each member’s 

chief anticipated contributions to IRB deliberations; 

• any employment or other relationship between each member and 

the institution, for example, full-time employee, a member of 

governing panel or board, stockholder, paid or unpaid consultant. 

(6) Written procedures for the IRB in the same detail as described in 

§26.1108(a) and §26.1108(b). 

All information relevant to the proposed research specified by § 26.1115(a) 

(7) Statements of significant new findings provided to subjects, as required 

by §26.1116(b)(5). 

(1) The potential risks to human subjects 

(2) The measures proposed to minimize risks to the human 

subjects; 

(3) The nature and magnitude of all expected benefits of such 

research, and to whom they would accrue 

(4) Alternative means of obtaining information comparable to what 

would be collected through the proposed research; and 

§1125(a) 

a discussion of: 

(5) The balance of risks and benefits of the proposed research. 

§1125(b): All information for subjects and written informed consent 

agreements as originally provided to the IRB, and as approved by the IRB. 

§1125(c): Information about how subjects will be recruited, including any 

advertisements proposed to be used. 

§1125(d): A description of the circumstances and methods proposed for 

presenting information to potential human subjects for the purpose of 

obtaining their informed consent. 

§1125(e): All correspondence between the IRB and the investigators or 

sponsors. 

The following Information, to the 

extent not already included: 

§1125(f): Official notification to the sponsor or investigator . . . that 

research involving human subjects has been reviewed and approved by 

an IRB. 
Appendix B: Framework for Science and Ethics Reviews of Proposed Human Research

 35 

Questions to be Addressed in EPA Protocol Review 

Protocol Identification 

(a) Title 

(b) Date 

(c) Principal Investigator and any sub-investigators 

(d) Participating Laboratories 

(e) Sponsor 

(f) Reviewing IRB 

1. Societal Value of Proposed Research 

(a) What is the stated purpose of the proposed research? 

(b) What research question does it address? Why is this question important? Would the 

research fill an important gap in understanding? 

(c) How would the study be used by EPA? 

(d) Could the research question be answered with existing data? If so, how? 

(e) Could the question be answered without newly exposing human subjects? If so, how? 

2. Study Design 

(a) What is the scientific objective of the study? If there is an explicit hypothesis, what is it? 

(b) Can the study as proposed achieve that objective or test this hypothesis? 

2.1 Statistical Design 

(a) What is the rationale for the choice of sample size? 

(b) What negative and positive controls are proposed? Are proposed controls appropriate 

for the study design and statistical analysis plan? 

(c) How is the study blinded? 

(d) What is the plan for allocating individuals to treatment or control groups? 

(e) Can the data be statistically analyzed? 

(f) What is the plan for statistical analysis of the data? 

(g) Are proposed statistical methods appropriate to answer the research question? 

(h) Does the proposed design have adequate statistical power to definitively answer the 

research question? 

2.2 How and to what will human subjects be exposed? 

(a) To what will subjects be exposed? 

(b) What is the rationale for the choice of test material and formulation? 

(c) What is the rationale for the choice of dose/exposure levels and the staging of dose 

administration? 

(d) What duration of exposure is proposed? 
Appendix B: Framework for Science and Ethics Reviews of Proposed Human Research 

 36 

2.3 Endpoints and Measures 

(a) What endpoints will be measured? Are they appropriate to the question(s) being 

asked? 

(b) What steps are proposed to ensure measurements are accurate and reliable? 

(c) What QA methods are proposed? 

(d) How will uncertainty be addressed? Will reported point values be accompanied by 

measures of uncertainty? 

 

3. Subject Selection 

3.1 Representativeness of Sample 

(a) What is the population of concern? How was it identified? 

(b) From what populations will subjects be recruited? 

(c) Are expected participants representative of the population of concern? If not, why 

not? 

(d) Can the findings from the proposed study be generalized beyond the study sample? 

3.2 Equitable Selection of Subjects 

(a) What are the inclusion/exclusion criteria? Are they complete and appropriate? 

(b) What, if any, is the relationship between the investigator and the subjects? 

(c) If any potential subjects are from a vulnerable population, what is the justification for 

including them? 

(d) What process is proposed for recruiting and informing potential subjects? 

(e) If any subjects are potentially subject to coercion or undue influence, what specific 

safeguards are proposed to protect their rights and welfare? 

3.3 Remuneration of Subjects 

(a) What remuneration, if any, is proposed for the subjects? 

(b) Is proposed remuneration so high as to be an undue inducement? 

(c) Is proposed remuneration so low that it will only be attractive to economically 

disadvantaged subjects? 

(d) How and when would subjects be paid? 

4. Risks to Subjects 

4.1 Risk characterization 

(a) Have all appropriate prerequisite studies been performed? What do they show about 

the hazards of the test materials? 

(b) What is the nature of the risks to subjects of the proposed research? 

(c) What is the probability of each risk associated with the research? How was this 

probability estimated? 
Appendix B: Framework for Science and Ethics Reviews of Proposed Human Research 

 37 

4.2 Risk minimization 

(a) What specific steps are proposed to minimize risks to subjects? 

(b) How do proposed dose/exposure levels compare to established NOELs/NOAELs for 

the test materials? 

(c) What stopping rules are proposed in the protocol? 

(d) How does the protocol provide for medical management of potential illness or injury 

to subjects? 

(e) How does the protocol provide for safety monitoring? 

(f) How does the protocol provide for post-exposure monitoring or follow-up? Is it of 

long enough duration to discover adverse events which might occur? 

(g) How and by whom will medical care for research-related injuries to subjects be paid 

for? 

5. Benefits 

(a) What benefits of the proposed research, if any, would accrue to individual subjects? 

(b) What benefits to society are anticipated from the information likely to be gained through 

the research? 

(c) How would societal benefits be distributed? Who would benefit from the proposed 

research? 

(d) What is the likelihood that each identified societal benefits would be realized? 

6. Risk/Benefit Balance 

(a) How do the risks to subjects weigh against the anticipated benefits of the research, to 

subjects or to society? 

7. Independent Ethics Review 

(a) What Institutional Review Board (IRB) reviewed the proposed research? 

(b) Is this IRB independent of the investigators and sponsors of the research? 

(c) Is this IRB registered with OHRP? 

(d) Is this IRB accredited? If so, by whom? 

(e) Does this IRB hold a Federal-Wide Assurance from OHRP? 

(f) Are complete records of the IRB review as required by 40 CFR §26.1125 provided? 

(g) What standard(s) of ethical conduct would govern the work? 

8. Informed Consent 

(a) Will informed consent be obtained from each prospective subject? 

(b) Will informed consent be appropriately documented, consistent with the requirements of 

40 CFR §26.1117? 

(c) Do the informed consent materials meet the requirements of 40 CFR §26.1116, including 

adequate characterization of the risks and discomforts to subjects from participation in 

the research, the potential benefits to the subject or others, and the right to withdraw from 

the research? 
Appendix B: Framework for Science and Ethics Reviews of Proposed Human Research 

 38 

(d) What is the literacy rate in English or other languages among the intended research 

subjects? 

(e) What measures are proposed to overcome language differences, if any, between 

investigators and subjects? 

(f) What measures are proposed to ensure subject comprehension of risks and discomforts? 

(g) What specific procedure will be followed to inform prospective subjects and to seek and 

obtain their consent? 

(h) What measures are proposed to ensure fully voluntary participation and to avoid coercion 

or undue influence? 

9. Respect for Subjects 

(a) How will information about prospective and enrolled subjects be managed to ensure their 

privacy? 

(b) How will subjects be informed of their freedom to withdraw from the research at any 

time without penalty? 

(c) How will subjects who decline to participate or who withdraw from the research be dealt 

with? 
Appendix C: Checklist of Elements Required by 40 CFR §26.1303 

 39 

§ 26.1303 Submission of Completed Human Research for EPA Review 

Any person who submits to EPA data derived from human research covered by this subpart shall provide at the time of submission 

information concerning the ethical conduct of such research. To the extent available to the submitter and not previously provided to 

EPA, such information should include: 

Requirement Y/N Comments/Page References 

§1115(a)(1): Copies of 

• all research proposals reviewed, 

• scientific evaluations, if any, that accompany the proposals, 

• approved sample consent documents, 

• progress reports submitted by investigators, and reports of injuries to 

subjects. 

§1115(a)(2): Minutes of IRB meetings which shall be in sufficient detail to show 

• attendance at the meetings; 

• actions taken by the IRB; 

• the vote on these actions including the number of 

• members voting for, against, and abstaining; 

• the basis for requiring changes in or disapproving research; 

• a written summary of the discussion of controverted issues and their 

resolution. 

§1115(a)(3): Records of continuing review activities. 

§1115(a)(4): Copies of all correspondence between the IRB and the investigators. 

§1115(a)(5): 

• A list of IRB members identified by name; earned degrees; representative 

capacity; indications of experience such as board certifications, licenses, 

etc., sufficient to describe each member’s chief anticipated contributions 

to IRB deliberations; 

• any employment or other relationship between each member and the 

institution, for example, full-time employee, a member of governing panel 

or board, stockholder, paid or unpaid consultant. 

§1115(a)(6): Written procedures for the IRB in the same detail as described in § 

26.1108(a) and § 26.1108(b). 

(a) Copies of all of the records relevant to the research specified by 

§26.1115(a) to be prepared and maintained by an IRB 

§1115(a)(7): Statements of significant new findings provided to subjects, as 

required by § 26.1116(b)(5). 

(1) The potential risks to human subjects; 

(2) The measures proposed to minimize risks to the human subjects; 

(3): The nature and magnitude of all expected benefits of such research, 

and to whom they would accrue; 

(4) Alternative means of obtaining information comparable to what would 

be collected through the proposed research; and 

§1125(a) 

A discussion of: 

(5) The balance of risks and benefits of the proposed research. 

§1125(b): All information for subjects and written informed consent agreements as 

originally provided to the IRB, and as approved by the IRB. 

§1125(c): Information about how subjects will be recruited, including any 

advertisements proposed to be used. 

§1125(d): A description of the circumstances and methods proposed for 

presenting information to potential human subjects for the purpose of obtaining 

their informed consent. 

§1125(e): All correspondence between the IRB and the investigators or sponsors. 

(b) Copies of all of the records relevant to 

the information identified in §26.1125(a)-(f) 

§1125(f): Official notification to the sponsor or investigator, in accordance with the 

requirements of this subpart, that research involving human subjects has been 

reviewed and approved by an IRB. 

(c) Copies of sample records used to document informed consent as specified by 

§26.1117, but not identifying any subjects of the research 

(d) If any of the information listed in paragraphs (a) through (c) of this section is not 

provided, the person shall describe the efforts made to obtain the information.