1 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON D.C., 2046 0

OFFICE OF CHEMICAL SAFETY AND

POLLUTION PREVENTION

December 1 5 , 2015

MEMORANDUM

S UBJECT: 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– Unpublished document prepared by S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 , 201 5. 154 p. (MRID 49761601)(D43008 2)

ACTION REQUESTED

Conduct a science review of a completed field study testing the efficacy against mosquitoes of a n i nsect 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 mosquit oes for use in the EPA Repellency Awareness Graphic on the label of EPA Reg.2 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 , 201 5

. The protocol used in this study was amended to address EPA and HSRB

recommendations.

CONCLUSIONS

The EPA assessed the s cientific 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 s tudy ( MRID 49761601 ) was conducted in accordance with Good Laboratory Practices as described in 40 CFR §160 ( with one minor exception ), and provides scientific data tha t are acceptable. The Human Studies Review Board will be asked to comment on this study.

SCIENCE REVIEW

Study objective : The objective o f this study is to establish

, for the EPA Repellency Awareness

G raphic, th e median complete protection time of MARK

- 8 in the field against populations of wild mosquitoes using human volunteer subjects

. This is a guideline study designe d 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 )

.

Id en tification of the test system: In this study, landings of wild mosquitoes on r eplicate 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 on e of the i nsect groups represented on the EPA R epellency Awareness G raphic

. 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 )

.

Ta ble 1

. Wisconsin Site Mosquito Species collected

- August 10, 2015 ( §10.1, p. 11 of 154 )

Species Number

C ollected % 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, 201 5 ( §10.1, p. 11 of 154 )

Species Number

Collected % of Total3 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 d esign : This field study was conducted with human subjects at two

g eographically and ecologically distinct field sites, one in Kenosha C ounty, Wisconsin on August 10, 2015 , and one in Collier C ounty, Florida on August 25, 2015

. At each site, t he experimental treatment groups consisted of 10 different treated su bjects and the untreated control

group consis ted 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 post

treatment 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 selec ted 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 described4 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 s ubject

. 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 i dentity 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 f rom 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 , p p . 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 , su bjects were

gro uped 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 subject s could see to aspirate mosquitoes. After the

five

- minute exposure period, subjects reported the number of mosquito landings t o study staff

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

identified. The five

- minute exposures were conducted every thirty minutes until repellent failure

occu rred. Repellent failure (i.e., first confirme d landing) is the exposure period in which two or more mosquito landings occurred, or when one land occurred in an exposure period and anoth er landing occurred in the subsequent exposure period. A ra in 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 ex posure period 6 was used for repellent failure (D eviation 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 s ite 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 post

treatment) [ §13.6, pp. 21

- 23 of 154 ]

.5 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 descri bed above for treated subjects. However, to reduce th e expos ure of untreated control subjects to mosquitoes, untreated subjects covered their exposed limb after five landings in the five

- minute ex posure period. Five landings on each untreated subject during the five

- minute exposure period were

considered the minimu m necessary to ensure the mosquito population was large enough to

determine re pellency of the tested product. The time to reach five landings on the untreated control subjects was recorded for ea ch 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 mo st 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 conse rvative 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 cm 2

. Formula 1 (below) was used to calculate the amount of the test subst ance 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 isopropa nol an d allowed the limb to dr y. 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 substanc e 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 spray ed 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 cm 2 /600 cm 2 ] * 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 8/10 115 Left Arm 0.64 0.69 1086 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

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

.

Pro tocol amendments and deviations: The approved protocol was dated June 26 , 2015

. There

were no amendments to the pro tocol ( §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 dos es 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. Deviatio n 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 r esults section below

. In futur e draft protocols for

field studies testing insect repellents , EPA should ensure that the protocol addresses h ow to determine repellent break down 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)

.7 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 t ime to count five mosquito landings on control subject s ranged from 11 to 97 seconds. At the Wisconsin site, the time to count five mosquito land ing s on control subjects ranged from 15 seconds to 3

minutes. One of the untreated control subjects withdrew after the fifth exposu re peri od at the Wisconsin site and the subject that withdrew was not repla ced with an alternate subject.

H owever, 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 Cro wley (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 (Table s 5 , 6 ; § 4.1, p p

. 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 i s 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. Rep ell ency
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 8.58 139 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 recomme ndations 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 Rec ommendations 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 cm 2 treated skin. However, we strongly suggest changing the language from standardized dose to standardized application rate and reserve “dose” to describe

how muc h 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 qua lity 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 t he expected dose

- response trend. The Board recommends that the data analysis inc lude 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 ass ess 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

i ncluding 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 spread9 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 impor tance 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 fo r 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 Uni ted 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 minim um landing pressure (5 mosquitoes on an untreated control in five minutes) required to evaluate efficacy. In addition, t he protocol was revised to include the requested information (e.g., mosquito species present, climate, etc.) for the proposed site in Au stralia. However, t esting 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 activ ities that10 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 t he 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 multipl e 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 be tween 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 w ith 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 coul d participate in another test. There is no indication that test subjects par ti cipated in multiple tes ts 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 pre ssure 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 increasi ng 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 diff erent landing pressure conditions.

S.C. Johnson Response: Study staff recorded t he 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 sit e s , 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 protoc ol 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

m inimum 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 ho urs 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 exposur e 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 wo uld 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 blo ck 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 c ountry 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, no t 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 whet her/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, the n 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: O n e test substance was tested on each day, therefore randomizing t he

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

randomi zing 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 had12 the ir right arms treated. In addition, the protocol notes that aspirating mosquito es is not difficult

even with a non

- dominant hand. T he untreated control subjects w ere 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 Repellent s 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 summa rizes 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 me eting 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 acknow ledged 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 differe nces 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 Rec ommendations on the Protocol and S.C. Johnson

Re sponses 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 cou nted 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 c ontrol 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 jus tified.

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 delive r 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. John son 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 dat a sheet.

S.C. Johnson R esponse: 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: M edian 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.