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Kevin Sweeney

Kelly Sherman

Office of Pesticide Programs

EPA Review of 

USDA-ARS-CMAVE Protocol 

Proposal for a Laboratory Evaluation 

of Bite Protection from RepellentImpregnated Clothing for the 

United States Military
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Overview

 Protocol for a laboratory study of the 

repellent efficacy of military uniforms 

containing 1% etofenprox

 Submitted by Dr. Ulrich Bernier, United States 

Department of Agriculture, Center for 

Medical, Agricultural, and Veterinary 

Entomology

 Protocol is a special study, non-EPA guideline, 

that is not similar to previous mosquito field 

studies reviewed by the HSRB
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Overview 2

 Research is proposed to satisfy EPA 

registration requirements

 This protocol and subsequent study may be 

used to standardize the experimental 

approach to evaluating the efficacy of 

repellent treated textiles
Overview 3

 Sponsor will test the hypothesis that 

etofenprox treatment provides bite protection 

when mosquitoes are exposed to treated 

fabric compared to an untreated control

 Etofenprox is recommended by the World 

Health Organization for use in public health 

vector control programs as a direct spray to 

infested areas or indirectly by treating fabrics, 

such as mosquito nets 

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Comparisons to Skin-Applied Repellent 

Studies Reviewed by the HSRB

 Laboratory vs. Field

 Different repellent effect

 Different efficacy measures

 Subjects will receive mosquito bites

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Science Assessment: 

USDA-ARS Protocol for a Laboratory 

Evaluation of Bite Protection from 

Repellent-Impregnated Clothing for the 

United States Military

Kevin Sweeney

Registration Division

Office of Pesticide Programs
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Study Objectives

 This study is designed to determine the bite 

protection level of etofenprox-treated U.S. 

Military Fire Resistant Army Combat Uniforms 

(FRACUs) treated initially at an application 

rate of 1% wt/wt, and to assess the bite 

protection performance after 0x, 20x, and 

50x washes. 
Study Objectives 2

 The results of this research will allow for 

determination of whether etofenprox-treated 

FRACUs meet the Department of Defense’s 

specifications for minimum bite protection 

level. 

 The research has societal value because U.S. 

military personnel serving domestically and 

abroad are at risk of contracting insecttransmitted diseases. 

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Bite Protection Specifications (%)

Uniform 0x wash 

cycle

20x wash 

cycle

50x wash 

cycle

Army 

FRACUs 

(test material

in the 

proposed 

protocol) 

85% 80% 70%

Study Objectives 3
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Acute Toxicity of the Test Material

 Acute Dermal = LD50 >2,100 mg/kg body 

weight

 Acute oral = LD50 >5,000 mg/kg body 

weight

 Minimally irritating to the skin and eyes

 Not a skin sensitizer
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MOE Estimate

 Estimated maximum dose = based on the 

assumption of 100% etofenprox absorption 

from 6 treated sleeves = 635.4 mg/subject

 Assuming 70 kg subject, equivalent dose rate is 

635.4/70 = 9.08 mg/kg

 Margin of Exposure (MOE) = 231

 EPA’s Level of Concern > 100
Evaluating Skin Irritation 

 Dermal observations resembling skin 

irritation were observed in a previously 

conducted 28-day dermal toxicity study with 

technical etofenprox on rabbits

 The etofenprox registrant, Mitsui Chemicals, 

will soon be conducting a product-specific 

28-day dermal toxicity study in rabbits with 

etofenprox-treated fabric

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Subject Right Arm Subject Left Arm

Test

Set Treatment Condition Specimen Treatment Condition Specimen

1

Coat Untreated 

Unwashed Control Sleeve 1 Trouser Untreated Unwashed Control Sleeve 2

2

Coat Treated Washed 

50x Sleeve 3 Trouser Treated Washed 50x Sleeve 4

3

Coat Treated Washed 

20x Sleeve 5 Trouser Treated Washed 20x Sleeve 6

4

Coat Treated 

Unwashed (0x) Sleeve 7 Trouser Treated Unwashed (0x) Sleeve 8

Experimental Design 

Testing Paradigm for each Mosquito Species
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Experimental Design 2 

Fabric and Treatment Condition

Number of 

Fabric 

Specimens

Number

of 

Subjects

Number

of 

Species

Total 

Replicates 

per Fabric 

Type

Coat Untreated Unwashed Control 1 8 2 16

Coat Treated Washed 50x 1 8 2 16

Coat Treated Washed 20x 1 8 2 16

Coat Treated Unwashed (0x) 1 8 2 16

Trouser Untreated Unwashed Control 1 8 2 16

Trouser Treated Washed 50x 1 8 2 16

Trouser Treated Washed 20x 1 8 2 16

Trouser Treated Unwashed (0x) 1 8 2 16
Experimental Design 3

 The test cages are approximately 59,000 cm3

in volume and each will contain 175 to 225 

female mosquitoes (density of ~1 

mosquito/300 cm3)

 Female mosquitoes will be preselected from 

stock cages by using a specially designed 

draw box that uses odors from the hand of a 

laboratory staff person to attract mosquitoes 

upwind in to a trap

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Endpoints and Measures 

 Unit of measure for determination of the 

repellent effects is percent bite protection

 Presence of blood in the mosquito’s abdomen 

will confirm a ‘mosquito bite’

 For each test set, the treatment % bite 

values will be corrected to account for the 

bite through values in the untreated control 

using Abbott’s Formula
Endpoints and Measures 2

 Percent bloodfed in untreated control 

treatment after test interval

 Percent bloodfed in etofenprox treatment 

after the test interval

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Endpoints and Measures 3

 Percent Bite Protection =

[1 – (treatment rate) / (control rate) ] × 100%

 Treatment rate (or proportion) = 

(# bloodfed female mosquitoes after test interval) / 

(total # of female mosquitoes after test interval)

~~when subject used treated fabric~~

 Control rate (or proportion) =

(# bloodfed female mosquitoes after test interval) / 

(total # of female mosquitoes after test interval)

~~when subject used untreated fabric~~

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Statistical Analysis Plan 

The objective is to estimate the mean 

level of bite protection and associated 

95% confidence intervals for different 

‘treatments’ [i.e. different combinations 

of fabric types (coats and trousers), 

number of washes, and mosquito 

species].

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True bite-through rate 

for control fabric (θC)

50% 20%

True bite protection for 

treated fabric (βT)

80% 95% 80% 95%

Number of Subjects Expected half-width of a 95% confidence interval 

for % bite protection

3 5.2% 2.7% 8.8% 4.5%

4 4.5% 2.3% 7.5% 3.8%

5 4.0% 2.0% 6.7% 3.4%

6 3.7% 1.9% 6.0% 3.0%

7 3.4% 1.7% 5.6% 2.8%

8 3.2% 1.6% 5.2% 2.6%

9 3.0% 1.5% 4.9% 2.4%

10 2.8% 1.4% 4.7% 2.3%

15 2.3% 1.2% 3.8% 1.9%

20 2.0% 1.0% 3.3% 1.3%

Table 3.5.1 Impact of the Number of Replications on the Number of Subjects

Statistical Analysis Plan 2
Statistical Analysis Plan 3

 The proposed sample size of 8 subjects 

represents a reasonable compromise 

between decreasing confidence interval 

width and limiting unnecessary human 

experimentation.

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Statistical Analysis Plan 4

 Data Analysis 

The numbers of bloodfed and total 

female mosquitoes found with treated 

and control fabric for each subject 

will be analyzed as binomial 

distributed data in a generalized 

linear model (GLiM) using a log link. 

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Measures to Ensure Reliability

 Standard Operating Procedures (SOPs) will be in 

place that must meet Good Laboratory Practices 

requirements.

 Subjects’ attractiveness to mosquitoes will be 

determined prior to testing

 Laboratory technicians will assist subjects with 

placing the test sleeves on their arms and excluding 

all exposed skin from mosquito exposure. Laboratory 

technicians will assist subjects with insertion and 

removal of their arms in/from the cages.

 Counts of bloodfed mosquitoes and the total number 

of mosquitoes in the cage will be determined by a 

research technician.
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Compliance with Scientific Standards

The following elements are adequately 

addressed:

 Available toxicity studies with etofenprox 

• Adequately characterize toxicological profile of 

the formulation except for dermal irritation 

from intermediate exposures to treated fabric. 

• Support estimate of acceptable Margin of 

Exposure (MOE)
Compliance with Scientific Standards

The following elements are generally 

acceptable but require refinement 

and clarification: 

•Experimental design

•Statistical analysis

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Science Comments and 

Recommendations 

 Conduct a product-specific 28-day dermal 

toxicity study in rabbits with etofenproxtreated fabric

 EPA recommends that the proposed efficacy 

study not be conducted until the results of the 

product specific dermal toxicity study have 

been submitted to and reviewed by EPA
Science Comments and 

Recommendations 2

 Provide justification for testing two vector 

mosquito species instead of three

 Consider recruiting more than two alternates

 The statistical plan for analyzing the data will 

need to take into account how alternate 

subjects will be handled

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Science Comments and 

Recommendations 3

 Please add more details to the protocol about 

what will happen if a subject withdraws 

midway through the study and an alternate is 

brought into the study as a replacement. 

 Will an alternate who replaces an original 

subject complete all eight pairs of sleeves, or 

only the pairs of sleeves that were not 

completed by the original subject?

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Science Comments and 

Recommendations 4 

 The subjects and alternates need to be 

randomly selected from a larger pool of 

qualified potential subjects.

 Please continue screening respondents to the 

advertisement until you have at least 20 

qualified potential subjects. Then, randomly 

select the 8 subjects and 2 or more alternates 

from the pool of qualified potential subjects. 

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Science Comments and 

Recommendations 5 

 Please address the distribution of male and 

female subjects and discuss if this will impact 

the results due to differences, if any, in 

attractiveness to mosquitoes. 

 Please revise the protocol to specify exactly 

what will happen if there is unequal 

distribution or if only one sex is represented. 

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Science Comments and 

Recommendations 6

 The statistical analysis used to analyze 

the study data should be justified in the 

final report. 

 Describe how the data will be analyzed 

if the number of test subjects at the 

end of the test is less than eight.

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Ethics Assessment 

USDA Protocol: Laboratory Evaluation 

of Bite Protection from Repellent-Impregnated 

Clothing for the United States Military

Kelly Sherman

Office of the Director

Office of Pesticide Programs
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Value to Society

 Proposed study would test the repellent 

efficacy of FRACU material that has been 

treated with etofenprox

 Would allow for a determination of whether 

etofenprox-treated FRACUs meet DoD’s

specifications for minimum bit protection level 

 Could allow for better protection of US forces 

serving abroad
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Subject Selection

 Subjects will be recruited through an 

advertisement placed in a newspaper and 

posted on university bulletin boards

 Callers will be informed about the study using 

an IRB-approved script

 Callers will be screened for eligibility and then 

scheduled for informed consent meetings
Subject Selection 2

 Inclusion/Exclusion criteria are complete 

and appropriate, except:

 Add exclusion for individuals known to be 

sensitive to pesticides or other chemical 

products

 Add exclusion for individuals with cuts, 

scrapes or skin conditions on their hands 

or forearms

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Subject Selection 3

 No potential subjects will be from a 

vulnerable population

 Subjects will be recruited through an 

advertisement printed in a local 

newspaper and posted on university 

bulletin boards

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Consent Process

 Study Director meets individually with each interested 

candidate

 Confirms eligibility criteria

 Provides detailed explanation of the procedures of the study

 Shows DVD further explaining testing process

 Informs candidate of how many mosquito bites they are 

likely to obtain 

 Reviews Informed Consent Document

 Answers questions

 Study Director confirms understanding and solicits 

consent to participate

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Risks and Risk Minimization

Four categories of risk; protocol provides 

appropriate measures to minimize each

 Exposure to biting mosquitoes

 Possible exposure to arthropod-borne disease

 Exposure to test material

 Breach of privacy (pregnancy testing)
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Benefits

 No direct benefit to subjects

 Primary direct beneficiary is sponsor

 If the treated materials are proven effective 

and superior to existing materials, indirect 

beneficiaries will include US military 

personnel who wear this etofenprox-treated 

FRACUs
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Risk:Benefit Balance 

 Risks have been effectively minimized

 Risks are reasonable in light of the expected 

societal benefits of the knowledge likely to 

be gained
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Respect for Subjects

 Effective methods for protecting 

subjects’ privacy

 Proposed level of compensation is 

appropriate

 Subjects will be free to withdraw at any 

time

 Medical care for research-related 

injuries will be provided at no cost to 

subjects 
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Independent Ethics Review

 The Western Institutional Review 

Board (WIRB) reviewed and approved 

the protocol and informed consent 

materials 
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Revisions Requested by EPA 

Before Research Proceeds

 Minor clarifications to protocol and consent 

form as detailed on pg. 4-5 of EPA Review

 Explain process for inspection of subjects hands and 

arms 

 Resolve inconsistency re what member of the research 

team will verify pregnancy test results

 Clarify that there are no benefits to subjects

 Add exclusion for people sensitive to pesticides or 

chemical products

 Add exclusion for cuts, scrapes, skin conditions on 

hands or forearms
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Compliance with Ethical Standards

 All requirements of §26.1111, §26.1116, and 

§26.1117 are met

 All requirements of §26.1125 are met

 Requirements of §26.1203 are met

 If EPA’s and HSRB’s requested corrections are 

made, research conducted according to this 

protocol will likely meet the applicable 

requirements of 40 CFR part 26, subparts K and L
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Charge Questions

If the proposed protocol is revised as suggested 

in EPA’s review and if the research is performed 

as described: 

 Is the protocol likely to generate scientifically 

reliable data, useful for estimating the level of 

mosquito bite protection provided by two different 

textiles treated with etofenprox?

 Is the research likely to meet the applicable 

requirements of 40 CFR part 26, subparts K and L?