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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON D.C., 20460

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION

December 15, 2015

MEMORANDUM

SUBJECT: Science Review of Field Testing of S.C. Johnson Personal Mosquito Repellent 

Products to Support Their Use of the EPA Repellency Awareness Graphic.

FROM: Eric W. Bohnenblust, Ph.D., Entomologist

Invertebrate-Vertebrate Branch 2

Registration Division (7505P) 

Office of Pesticide Programs

Kevin J. Sweeney, Senior Entomologist

Invertebrate-Vertebrate Branch 1

Registration Division (7505P) 

Office of Pesticide Programs

TO: Marietta Echeverria, Chief,

Invertebrate-Vertebrate Branch 1

Registration Division (7505P)

Office of Pesticide Programs

REF: Talbert, C. (2015) Field Testing of S.C. Johnson Personal Mosquito Repellent 

Products to Support Their Use of the EPA Repellency Awareness Graphic –

Unpublished document prepared by S. C. Johnson & Son, Inc., 1525 Howe Street 

Racine, WI 53403. October 21, 2015. 154 p. (MRID 49761601)(D430082)

ACTION REQUESTED 

Conduct a science review of a completed field study testing the efficacy against mosquitoes of an

insect repellent formulated as a pressurized aerosol product containing diethyl toluamide

(DEET). Determine the adequacy of the methods employed and the scientific validity of the 

reported data. These data are required to establish the median complete protection time (CPT) 

against mosquitoes for use in the EPA Repellency Awareness Graphic on the label of EPA Reg. 
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No. 4822-167, Mark-8 OFF! Insect Repellent Formula V (25% DEET). The protocol used to 

conduct this study was previously reviewed and accepted by EPA and the HSRB with comment

on April 23, 2015. The protocol used in this study was amended to address EPA and HSRB 

recommendations.

CONCLUSIONS

The EPA assessed the scientific aspects of the research in relation to the recommendations of the 

EPA §810.3700 product performance testing guideline and the Human Studies Review Board. 

The study (MRID 49761601) was conducted in accordance with Good Laboratory Practices as 

described in 40 CFR §160 (with one minor exception), and provides scientific data that are 

acceptable. The Human Studies Review Board will be asked to comment on this study.

SCIENCE REVIEW

Study objective: The objective of this study is to establish, for the EPA Repellency Awareness 

Graphic, the median complete protection time of MARK-8 in the field against populations of wild 

mosquitoes using human volunteer subjects. This is a guideline study designed to fulfill the 

requirements in OPPTS Series §810.3700 product performance guideline, Insect Repellents to be 

Applied to Human Skin. This study was conducted in accordance with EPA, FIFRA (Federal 

Insecticide, Fungicide and Rodenticide Act), Good Laboratory Practice Standards (GLP); 40 CFR, 

Part 160 (October 1989). (p. 3 of 154).

Identification of the test system: In this study, landings of wild mosquitoes on replicate human 

subjects were used to evaluate the repellency of an insect repellent product (25% DEET) applied 

to human skin. Mosquitoes were used because they are one of the insect pest groups repelled by 

the product and one of the insect groups represented on the EPA Repellency Awareness Graphic. 

The registrant conducted tests in two field locations, one in Florida and one in Wisconsin. The 

following mosquito genera were collected and identified, Coquillettidia, Psorophora, Aedes, 

Mansonia, and Wyeomyia (Tables 1, 2; §10.1, p. 11 of 154).

Table 1. Wisconsin Site Mosquito Species collected - August 10,2015 (§10.1, p. 11 of 154)

Species Number 

Collected % of Total

Coquillettidia perturbans 4 3

Psorophora ferox 2 2

Aedes vexans 3 3

Aedes trivittatus 109 92

Total 118 100

Table 2. Florida Site Mosquito Species collected - August 25, 2015 (§10.1, p. 11 of 154)

Species

Number

Collected % of Total
3

Aedes atlanticus 51 28.7%

Aedes infirmatus 107 60.1%

Aedes taeniorhynchus 3 1.7%

Mansonia dyari 1 0.6%

Mansonia titillans 12 6.7%

Psorophora ferox 2 1.1%

Wyeomyia spp. 2 1.1%

Total 178 100.0%

Experimental design: This field study was conducted with human subjects at two

geographically and ecologically distinct field sites, one in Kenosha County, Wisconsin on 

August 10, 2015, and one in Collier County, Florida on August 25, 2015. At each site, the 

experimental treatment groups consisted of 10 different treated subjects and the untreated control 

group consisted of two untreated subjects. Subjects at each site were selected from a pool of 

trained participants (Table 3; §12.4, pp. 15-16 of 154). At each site, testing was conducted during 

the course of a single day (§13.1, p. 20 of 154).

Table 3. Summary of Test Subject Participation in GLP 873E1 (Revision of Table 7 in the study 

§12.4, pp. 15-16 of 154).

GLP 873E1 - 4822-167 Wisconsin Florida Total

Number of Subjects 

Enrolled 22 35 57

Number of No-Shows 

to training and/or 

testing

No-shows for training: 2

No-shows for testing: 0

No-shows for training: 18

No-shows for testing: 0 20

Number Assigned as 

Test Subjects 12 12 24

Number Assigned as 

Alternates or Extras

4 alternates

4 extras

4 alternates

0 extras

12

*(Note to HSRB: In the 

study, the table cited 11.)

Number of Test 

Subjects Withdrawn 

Voluntarily

1 on test day

(post-treatment) 1 on training day

2

**(Note to HSRB: In the 

study, the table cited 1*. 

However, it should be 2, 1 

on training day and 1 posttreatment on test day.)

Number of Test 

Subjects Withdrawn 

Involuntarily

0 0 0

Number of Test 

Subjects Completed 

Research

11 12

23 **(Note to HSRB: In 

the study, the table cited 

24**.) 

In Wisconsin: 12 test 

subjects (10 treated and 

2 controls) and 4 

alternates were 

randomly selected as 

described in the report

In Florida: Only 16 trained 

participants were available 

on this test day. 12 test 

subjects (10 treated and 2 

controls) and 4 alternates

were selected as described 
4

out of the pool of 20

trained participants.

in the report out of the pool 

of 16 trained participants.

*typo

**post-treatment withdrawal was previously considered as 'completing research' instead of 'withdrawn'

The experiment was partially randomized, the test subjects were randomly selected from a pool 

of potential subjects that met the inclusion criteria found in §12.3, pp. 14-15 of 154. Using an 

allocation table, treatments were assigned to the limbs of each subject. If the subject was 

assigned an odd number, the left limb was treated and if assigned an even number the right arm 

was treated. Treated subjects were blinded to the identity of the test substance (§13.3, p. 20 of 

154). Two untreated control subjects were used at each site to determine mosquito landing rate 

and aspirate mosquitoes. The control subjects chose which arm to expose to mosquitoes so the 

dominant hand could be used to aspirate mosquitoes (§13.7, p. 23 of 154).

At the testing sites, study staff prepared subjects for testing in a tent enclosure to protect them 

from mosquitoes during preparation. Subjects washed their limbs before dressing in bug suit 

pants and jackets, and gloves to protect untreated body parts from mosquito bites. To treat the 

forearm, the selected forearm of the bug suit was rolled up and the arm treated as discussed in 

the Test Substance Application Rate and Treatment section below (§13.2-13.5, pp. 20-21 of 

154).

The unit of measure for determining repellent effects (Complete Protection Time) in this 

experiment was mosquito landings, similar to previous skin applied repellent evaluations where 

the “Landings” measure is used and efficacy is measured as CPT. To assess CPT, subjects were 

grouped into pairs. Pairs were separated from each other by at least twenty feet. Starting two 

hours after application of the repellent product, subjects observed their limbs for five minutes for 

landing mosquitoes. Mosquitoes that landed were aspirated by the subjects or their partner, and if 

necessary, headlamps were used at night so subjects could see to aspirate mosquitoes. After the 

five-minute exposure period, subjects reported the number of mosquito landings to study staff 

who recorded the number for each subject, and aspirated mosquitoes were labeled and kept to be 

identified. The five-minute exposures were conducted every thirty minutes until repellent failure 

occurred. Repellent failure (i.e., first confirmed landing) is the exposure period in which two or 

more mosquito landings occurred, or when one land occurred in an exposure period and another 

landing occurred in the subsequent exposure period. A rain event occurred at the Florida site

causing exposure periods 6 and 7 to be skipped. The protocol did not address how to determine 

repellent failure in the event of a rain delay; therefore, the study staff treated landings in the first 

exposure period after the rain delay as the first confirmed landing and exposure period 6 was 

used for repellent failure (Deviation 3). If the repellent failed on a subject, the subject was 

removed from the test. However, the repellent did not fail on all subjects, thus at the discretion of 

the study direction the study at the site in Wisconsin was ended after 17 exposure periods (10 h 

post treatment), and the study in Florida was ended after 14 exposure periods (8.5 h posttreatment) [§13.6, pp. 21-23 of 154].
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Two untreated control subjects were paired together and used to confirm that mosquito 

populations were adequate to test the repellency of the product. Untreated control subjects were 

exposed to mosquitoes for a five-minute period every thirty minutes as described above for 

treated subjects. However, to reduce the exposure of untreated control subjects to mosquitoes, 

untreated subjects covered their exposed limb after five landings in the five-minute exposure 

period. Five landings on each untreated subject during the five-minute exposure period were

considered the minimum necessary to ensure the mosquito population was large enough to 

determine repellency of the tested product. The time to reach five landings on the untreated 

control subjects was recorded for each five-minute exposure period [§13.7, p. 23 of 154].

The duration of repellency to appear on the Repellency Awareness Graphic on the product label 

will be based on the EPA Repellency Awareness Guidance for Skin-Applied Insect Repellent 

Products, which states, “The mosquito claim should be calculated using the most conservative 

(i.e., lowest) CPT from all available studies (In this study - Florida compared to Wisconsin).” 

“And “The number of hours of protection appearing on the repellency awareness graphic should 

be expressed as a whole number. If the most conservative calculated CPT is not a whole 

number, EPA recommends rounding down to the nearest whole number.”(§4.1, p. 8 of 154).

Test Substance Application Rate and Treatment: The target application rate was 1.0 g per 

600 cm2

. Formula 1 (below) was used to calculate the amount of the test substance to apply to 

each subject. Prior to applying the test substance, each subject washed the limb to be treated with 

water and unscented soap, dried the limb, and then wiped the limb in a 70% solution of 

isopropanol and allowed the limb to dry. After drying, the area to be treated was marked with a 

felt-tipped pen. Adhesive surgical tape and adhesive bandage were wrapped around the area to 

be treated so the area to be treated was the only exposed skin. The test substance was placed on 

the balance and the balance tared. The test substance was applied directly from the aerosol can 

onto the skin. After spraying, the sample was returned to the balance and the amount sprayed 

onto the skin was determined. If the amount sprayed was below the target weight, more was 

sprayed onto the forearm and the can weighed again (§7.1.8.2, pp. 68-69 of 154). The amount 

applied to each subject is found in Table 4 (§13.7, p. 23 of 154).

Formula 1. Weight of test substance to apply (g) = [Area of limb cm2

/600 cm2] * 1.0 g

Table 4. Amount of Test Substance Applied (§13.7, p. 23 of 154).

Site

Test 

Subject

No. Date

Limb 

Treated

Target

Amount 1

(g)

Actual

Amount

(g)

% of

Target

Wisconsin 121 8/10/15 Left Arm 0.72 0.73 101

Wisconsin 122 8/10/15 Right Arm 0.68 0.68 100

Wisconsin 123 8/10/15 Left Arm 0.72 0.73 101

Wisconsin 124 8/10/15 Right Arm 0.76 0.73 96

Wisconsin 125 8/10115 Left Arm 0.64 0.69 108
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Site

Test 

Subject

No. Date

Limb 

Treated

Target

Amount 1

(g)

Actual

Amount

(g)

% of

Target

Wisconsin 134 8/10/15 Right Arm 1.06 1.09 103

Wisconsin 135 8/10/15 Left Arm 1.02 1.08 106

Wisconsin 136 8/10/15 Right Arm 1.01 1.05 104

Wisconsin 139 8/10/15 Left Arm 0.91 0.9 99

Wisconsin 143 8/10/15 Left Arm 0.76 0.76 100

Florida 194 8/25/15 Right Arm 1.12 1.2 107

Florida 199 8/25/15 Left Arm 1.03 l.09 106

Florida 205 8/25/15 Left Arm 0.76 0.7 92

Florida 207 8/25/15 Left Arm 0.78 0.88 113

Florida 209 8/25/15 Left Arm 0.97 1.04 107

Florida 210 8/25/15 Right Arm 0.85 0.84 99

Florida 219 8/25/15 Left Arm 1.07 1.01 94

Florida 220 8/25/15 Right Arm 1.01 1.01 100

Florida 221 8/25/15 Left Arm 1.21 1.3 107

Florida 229 8/25/15 Left Arm 0.99 0.99 100

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Target amount was based upon forearm surface area calculation (Formula 1).

Protocol amendments and deviations: The approved protocol was dated June 26, 2015. There 

were no amendments to the protocol (§20.2 p. 30 of 154). 

Six protocol deviations occurred during the study (Appendix B, p. 112 – 117 of 154). Four of 

the six deviations did not affect the study. Deviation 4 (Appendix B, p. 115 of 154) documents 

that the test product was applied to subject 207 at a slightly higher volume; however, because 

lower doses of the repellent on other subjects also lasted the full duration of the study, this 

minimal increase in application volume did not affect the study. Deviation 3 (Appendix B, p. 114 

of 154), which did affect the study, resulted in the cancellation of two exposure periods because 

of heavy rain and lightning, the effect of this deviation on the study is discussed in the 

experimental design section above and the results section below. In future draft protocols for 

field studies testing insect repellents, EPA should ensure that the protocol addresses how to 

determine repellent breakdown points in the event of a weather delay.

Results and analyses:

Test Systems: Test subjects collected mosquitoes representing three genera (Coquillettidia, 

Psorophora, and Aedes) and four species in Wisconsin, and four genera (Mansonia, Psorophora, 

Wyeomyia, and Aedes) and seven species in Florida for a total of five genera and 10 species 

across the two sites. Aedes spp. mosquitoes, a mosquito genus containing numerous important

vectors of human diseases, represented 95% of the mosquitoes collected in Wisconsin, and 90% 

of the mosquitoes collected in Florida (Tables 1, 2; §10.1, p. 11 of 154).
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Duration of Repellency: At both the Florida and Wisconsin sites, at least 5 mosquitoes landed 

on the untreated control in all of the five-minute exposure periods. At the Florida site, the time to 

count five mosquito landings on control subjects ranged from 11 to 97 seconds. At the Wisconsin 

site, the time to count five mosquito landings on control subjects ranged from 15 seconds to 3 

minutes. One of the untreated control subjects withdrew after the fifth exposure period at the 

Wisconsin site and the subject that withdrew was not replaced with an alternate subject. 

However, landings on the remaining untreated control subject were adequate to confirm a 

mosquito population high enough to test for repellency throughout the study.

Median complete protection time and 95% confidence limits (lower confidence limit [LCL] and 

upper confidence limit [UCL]) were calculated by Kaplan Meier analysis using PROC 

LIFETEST in SAS, which employs a generalization of the Brookmeyer and Crowley (1982) 

method under a log-log transformation. The median duration of repellency for the test substance

was 8.25 hours in Wisconsin, and 8.0 hours in Florida (Tables 5, 6; §4.1, pp. 8-9 of 154). In 

Florida, because of a rain event the first confirmed landing on three treated subjects (Subject 

Nos. 199, 210, 229) occurred in the first exposure period immediately after two skipped 

exposure periods, the repellency duration for these three subjects is recorded as 4.5 hours

(Deviation 3).

Table 5. MARK-8 (25% DEET aerosol) Repellency Duration Results Summary, Hours, Sample 

size = 10 Wisconsin site, Sample size = 10 Florida Site (§4.1, p. 8 of 154).

Measure Wisconsin

Site

Florida

Site

Median 8.25 8.0

95% LCL 6.0 3.5

95% UCL 10.0 8.5

Range 6.0 - 10.0 3.5 - 8.5

Table 6. MARK-8 (25% DEET aerosol) Repellency Duration Results, Hours (§4.1, p. 9 of 154).

Wisconsin

Site

Florida

Site

Subject

No.

Repellency

Duration

Subject

No.

Repellency

Duration

121 10.0 194 8.5

122 8.0 199 4.5

123 10.0 205 8.5

124 8.5 207 8.5

125 9.0 209 7.5

134 7.5 210 4.5

135 6.5 219 8.5

136 8.0 220 8.5
8

139 6.0 221 3.5

143 8.5 229 4.5

Conclusions:

The methods used in this study were adequate to produce scientifically reliable results. The 

methods were based on the protocol reviewed and accepted by the EPA and HSRB on April 23, 

2015 as amended to incorporate EPA and HSRB recommendations before testing began. The 

data in the study are acceptable to support a median CPT of 8.0 hours against mosquitoes for the 

EPA Repellency Awareness Graphic on the label for EPA Reg. No. 4822-167, Mark-8 OFF! 

Insect Repellent Formula V (25% DEET).

Note:

The limb measurement forms should have been included with the raw data.

HSRB Comments and Science Recommendations on the Protocol and S.C. Johnson 

Responses from the April 2015 Meeting Report Dated June 23, 2015:

Product application rate: The Board understands that the data from this protocol will be used to 

calculate median CPT values across all subjects from each of two sites rounded down to the 

nearest integer and the lowest value will be used for the product graphic. (p. 11 of 39, Sweeney 

and Sherman). Given the stated use of the data, the Board agrees with the use of a standardized 

dose of 1 gram product per 600 cm2 treated skin. However, we strongly suggest changing the 

language from standardized dose to standardized application rate and reserve “dose” to describe 

how much active ingredient is applied.

The protocol specifies that the actual amount of product applied will be recorded. The percent 

active ingredient in each product is also available so the Agency can in fact assess the 

relationship between dose (active ingredient) and efficacy (or CPT) as a quality assurance check 

of the data. An assessment of the relationship between CPT and dose (mass active ingredient per 

treated area) can provide an indication of the quality of the data or point out data that might be 

suspect because it does not fall along the expected dose-response trend. The Board recommends 

that the data analysis include a dose-response comparison for all products where multiple 

concentrations are available (e.g., DEET content in the different products ranges from 5% to 

98.25%) to help assess data quality. In addition, the Agency might consider normalizing the CPT 

results to better represent the expected application rates derived from earlier dosimetry studies 

when calculating the final graphic number.

S.C. Johnson Response: The word “dose” was changed to “application rate” throughout. A 

dose-response comparison was not included in the data analysis because each study only tested a 

single product. 

Product application method: The protocol proposes to use a variety of application methods 

including using pipettes for transferring liquid contents from pump sprays and spatulas for 

lotions, while aerosol sprays are applied directly. In each case, the product is applied and spread 
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on the subject’s skin by a staff member. The Board was concerned that these application methods 

were not representative of actual application methods but ultimately concluded that the need for 

consistency outweighs the need for the protocol to be representative of consumer behavior. 

However, the Board stresses the importance of accurately reporting the application rate (mass of 

product per area of skin) for each subject.

A particular concern with the aerosol application method was that the iterative procedure leaves 

open the possibility to repeatedly apply more than the target 1 gram. Some means should be 

integrated within the protocol to limit or preclude the potential to bias the average dose upward. 

One (and perhaps not the best) way to preclude such a bias would be to place some upper bound 

on the highest level above 1 gram that would be allowed to proceed to the field testing phase. 

Such an upper bound (or similar restriction) could be applied to all application methods, but 

over-exposures seem most likely to occur with the aerosol application method.

S.C. Johnson Response: The test substance (aerosol formulation) was applied as directed by the 

revised protocol (Section 7.1.8.2, p. 68-69 of 154) and results reported in Table 8 (p. 23 of 154). 

Note the study incorrectly lists the application method as the method for pump sprays. The mean 

amount applied was equal to the target amount with a range of 92 - 113% of the target amount 

applied.

Use of sites outside the U.S: The protocol specifies the use of two established and ecologically 

distinct field sites in the United States for testing, and the Board agrees that this will provide 

sufficient representation for determining CPT, but the Board shares the concerns expressed by 

the Agency about using sites located outside of the U.S. The study sponsor has described that in 

addition to the two established sites in the U.S., there is at least one established site in Australia 

that could be used. The protocol needs to provide more information on what constitutes an 

“established site” either in the U.S. or another country (i.e., climate, mosquito species present, 

other hazards such as other mosquito borne diseases, presence of cell phone service, 

representativeness of local demographics) and more importantly should describe how the data 

collected at the alternate sites outside the U.S. will be related to the U.S. consumer demographics 

and the expected mosquito populations in the U.S.

S.C. Johnson Response: The protocol only states that sites will be qualified by confirming 

mosquito populations are adequate to achieve the minimum landing pressure (5 mosquitoes on 

an untreated control in five minutes) required to evaluate efficacy. In addition, the protocol was 

revised to include the requested information (e.g., mosquito species present, climate, etc.) for the 

proposed site in Australia. However, testing was not conducted outside of the U.S., so this 

information is not relevant to the submitted study.

Potential for cross contamination: There are a number of places in the protocol that provide 

opportunities for insecticide to be inadvertently either lost or gained from/to the treated area on 

subjects. Simple precautions can be taken to alleviate this issue, but the protocol should specify 

steps that will be taken to insure that the treated area on subjects is not impacted by activities that 
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take place before or during the experiment (i.e., rubbing sleeve or pant leg across the treated 

area).

S.C. Johnson Response: Subjects were not transported using a vehicle after the test substance 

was applied, and subjects were reminded not to touch or contact the treated skin in any manner. 

Any inadvertent contact with the treated area was reported to the study staff and documented in 

the raw data.

Potential for “carryover”: No justification was provided for the adequacy of separating 

multiple participations by any test subject by a minimum of one day (Science Response #6 in 

S.C. Johnson letter of 17 April 2015). It is important to verify that no carryover effect is present 

on subjects used on multiple days. The protocol suggests that a day between treatments will be 

sufficient when the same subject is used a second time, but justification or references are needed 

to support this. If a subject is treated with 98.25% DEET, is there any residual effect after 24 

hours that might affect a low dose treatment (application of 5.6% DEET wipe)?

S.C. Johnson Response: Subjects washed before and after each test, and a minimum of two 

days was required to pass before subjects could participate in another test. There is no indication 

that test subjects participated in multiple tests so we assume all subjects did not previously 

participate in other tests and therefore there was no “carryover” effect.

Landing pressure: The protocol includes untreated control subjects with each test to insure that 

there is sufficient landing pressure to provide valid results. However, the landing pressure is not 

measured in quantitative terms, only whether it is sufficient or not (five landings in five 

minutes). Discussions during the meeting seemed to imply that landing pressure will influence 

the measured CPT. If in fact the landing pressure can influence the resulting median CPT and 

products tested on different days are subjected to different landing pressures, then it would be 

important to collect quantitative information on landing pressure that could be used to correct, 

normalize, or at least interpret the resulting CPT values. The Board recommends that the Agency 

and S.C. Johnson consider how a quantitative estimate of landing pressure can be determined 

without increasing the likelihood of bites if landing pressure is excessive (e.g., recording the time 

of each landing, the time to reach 5 landings, or the total landings in 5 minutes) and how that 

information can be used to normalize or interpret CPTs measured under different landing 

pressure conditions.

S.C. Johnson Response: Study staff recorded the time to reach five landings if less than five 

minutes. Because all studies were performed at the same two sites, landing pressure appears to 

be fairly consistent across sites, therefore normalization is not required.

Delayed start: The Board recognizes the advantages of delaying the exposure to mosquitoes for 

subjects treated with products that are known from previous experience to last for a long time. 

However, the protocol needs to provide more information about the criteria used to determine 

how long to wait before starting the test cycles (5 minute exposure at 30 minute intervals). 
11

Regardless of how long the subject’s exposure is delayed, the protocol should require a 

minimum number of completed cycles to insure valid results. For example, following a delayed 

exposure, the subject should complete at least three exposure cycles before getting a confirmed 

landing.

S.C. Johnson Response: Exposures were delayed until two hours after application (Deviation 

6). The test substance contained 25% DEET as the active ingredient and according to the 

protocol exposures were to be delayed by three hours for products containing more than 16% 

DEET. This deviation did not affect the study, and all subjects were exposed to mosquitoes for at 

least three exposure cycles before a confirmed landing was recorded. In addition, delaying 

exposure periods reduced the exposure of the subjects to bites from mosquitoes.

Experimental design: The design as presented tests all ten subjects assigned to a product on a 

single day. The downside of this design is that it does not allow results to be easily generalized to 

a range of environmental conditions that may affect the attractiveness of a subject (e.g., sweating 

due to temperature and humidity levels). An alternative would be to test each product on several 

days; e.g., five subjects on each of two days or three subjects on each of two days and the 

remaining four subjects on a third day. Each day would form a block for the analysis of that 

product’s data. Such a design would allow testing of multiple products on a given day. If it were 

of interest to compare product formulations and/or application methods, the combined data could 

be analyzed as a block design with multiple replications of each product within each block (day). 

These alternative designs that utilize blocking (e.g., by individual test subject) could be 

considered to account for known sources of variation (e.g., individual effects).

S.C. Johnson Response: The two different sites in different areas of the country should allow 

for generalizing results to a greater range of environmental conditions than a single site. Also, 

the main goal of the study is to determine the CPT for an individual product, not to compare 

multiple products.

Randomization: The randomization mechanism should be described in more detail and rationale 

should be given for any given choice of randomization within the protocol. For example, it is not 

clear whether/how cross-substance relations are to be evaluated in the data analysis and why 

randomization among test substances is needed. An explanation of this would be helpful. In 

addition, when the conditions support use of arm rather than leg for exposure, then it may be 

more important to consider handedness when selecting what arm to treat, rather than randomly 

assigning to left or right hand, so the subject can have their dominant hand to remove landing 

mosquitoes before they bite. 

S.C. Johnson Response: One test substance was tested on each day, therefore randomizing the 

treatment was not necessary. The mechanism for randomizing the arm was not provided in the 

study; however, after discussions with S.C. Johnson, they indicated that the mechanism for 

randomizing the arm to be treated was based on the random selection of test ID numbers. 

Subjects assigned odd numbers had their left arm treated, and subjects with even numbers had 
12

their right arms treated. In addition, the protocol notes that aspirating mosquitoes is not difficult 

even with a non-dominant hand. The untreated control subjects were allowed to choose which 

arm to expose.

Sample size determination: A sample size calculation would be useful here to inform the power 

of testing and the width of confidence intervals. Power and sample size calculation can be 

implemented using existing SAS procedures. Information about appropriate sample size 

calculations is included in the EPA document “Product performance Test Guidelines 

OPPTS810.3700: Insect Repellents to be Applied to Human Skin.”

S.C. Johnson Response: After discussions with S.C. Johnson, they indicated that they did not 

conduct a sample size calculation or power analysis but the table which summarizes the effect of 

sample size on the width of the confidence interval for median CPT presented in the protocol for 

testing repellents against ticks to the Human Studies Review board at the October meeting would 

apply to these studies because both studies use 10 subjects.

Sources of variation: Multiple sources of variation including, for example, site selection, 

treatment dosage, application rate, mosquito type/age/condition, and landing pressure can impact 

the results. For the most part, however, they are not accounted for explicitly within the study, and 

when the source of variation is not controlled (e.g. as it is using a standard application rate) then 

the contribution to variance should be acknowledged or discussed. The protocol does not 

currently specify the conditions that might cause the CPT data from the two sites to differ; 

however, the researchers should consider collecting information to explain any large and 

potentially significant differences in the CPT values between otherwise matched studies 

conducted at two different sites.

S.C. Johnson Response: The researchers identified mosquitoes to species, recorded habitat 

characteristics, climatic conditions, and the time to five landings on the untreated control 

subjects, information which could explain large and significant differences in CPT. Note the 

CPTs at both sites were similar.

EPA Comments and Science Recommendations on the Protocol and S.C. Johnson 

Responses from the April 2015 Meeting Review Dated March 31, 2015:

EPA Comment: Change “mosquito biting pressure” to “mosquito landing rate” as subject bites are 

not counted or recorded in this study.

S.C. Johnson Response: The term “mosquito biting pressure” was changed to “mosquito landing 

rate” throughout the protocol.

EPA Comment: Describe how the data will be analyzed if the number of test subjects at the end of 

the test is less than ten. In other words, what if subjects withdraw? If alternates replace them, how 

will Johnson account for this change of subjects in the data analysis?
13

S.C. Johnson Response: None of the treated subjects withdrew from the study at either location. 

One of the untreated control subjects withdrew from the study, but the untreated control subjects 

were not used to calculate CPT.

EPA Comment: The protocol states that up to 10% of the exposure periods in a test may have less 

than the minimum landing (biting in the protocol) pressure of five mosquitoes landing in five minutes 

or less. Will treatment exposures occur during periods of insufficient landing pressure? If treatment 

data are collected during these periods, how will they be used in CPT calculation? If they are not 

used, how will the lack of data points be considered in the K-M analysis and calculation of Median 

CPT? 

S.C. Johnson Response: Landing pressure at both locations reached the minimum biting 

pressure for all exposure periods therefore this comment does not apply to this study.

EPA Comment: State/justify why no positive control substance is to be used.

S.C. Johnson Response: A positive control was not used because the Agency did not provide 

information on how positive control data would be used to normalize the data across sites. 

Therefore exposing additional subjects to repellent products and mosquitoes was not justified.

EPA Comment: Product application is not fully described. After weighing the set dose, how is the 

product applied to the limb for pump sprays and lotions? For instance, is the required amount left in 

the container and the pump used to spray it on the limb? For lotions, the amount to be applied is 

removed with a spatula instead of a larger syringe so transfer to the subject might be easier? For 

aerosols, Johnson could estimate the delivery of the prescribed amount of product by counting the 

number seconds needed to deliver the dose to the limb and determine the amount applied per second 

of spraying to more closely estimate the application amount? How does this compare to the product’s 

label directions? Will study staff be spreading the lotion with a gloved hand?

S.C. Johnson Response: The exact method of determining the amount applied to each subject in 

this study is described in §7.1.8.2 (pp. 68-69 of 154).

EPA Comment: Appendix III – Land Data Form. Identification of which limb was treated needs to 

be added to this data sheet. 

S.C. Johnson Response: A line was added to the data form to identify the treated limb.

EPA Comment: Data compilation and processing. Little detail is provided in the protocol on how 

the data from these sheets will be compiled and processed before entry into Excel, JMP, or SAS, etc. 
14

S.C. Johnson Response: Median confidence limits were calculated by Kaplan Meier analysis

using PROC LIFETEST in SAS, which employs a generalization of the Brookmeyer and 

Crowley (1982) method under a log-log transformation.