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: Kevin J. Sweeney, Senior Entomologist

Invertebrate

- Vertebrate Branch 1 Registration Division (7505P) Office of Pesticide Programs

E ric W. Bohnenblust, Ph.D., Entomologist

Invertebrate

- Vertebrate Branch 2 Registration Division (7505P) Office of Pesticide Programs

TO: Marietta Echeverria , Chief,

Invertebrate

- Vertebrate Branch 1 Registration Division (7505P)

Office of Pesticide Programs

REF: P alm, J. M. (2015) Field Testing of S.C. Johnson Personal Mosquito Repellent Products to Support Their Use of the EPA Repellency Awareness Graphic

– Project 864E1. Unpublished document prepared by S

. C. Johnson & Son, Inc., 1525 Howe Street Racine, WI 53 403, September 24 , 201 5. 154 p. (MRID 49750201) (D430001 )

ACTION REQUESTED

Conduct a science review of a completed field study testing the efficacy of a n insect repellent

formulated as a pressurized aerosol spray product containing diethyl toluamide (DEET) against mosquitoes

. 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.2 No. 4822

- 397

, Mark

- 2 OFF! Deep Woods Sportsmen I nsect Repellent II (Unscented Deep Woods Off! Insect Repellent) (30 % DEET ). The protocol used to conduct this study was

pre viously reviewed and accepted by EPA and the HSRB on April 23 , 201 5 . The protocol used in this study was amended to incorporate 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 EPA Human Studies Review

Board. The s tudy ( MRID 49750201 ) was conducted in accordance with Good Laboratory Practices as described in 40 CFR §160 ( with one minor GLP exception as described in Protocol

Deviation #7 ), 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 o f this study is to establish the c omplete protection time of

MARK

- 2 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 G uideline , 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 entification of the test system: In this study, landings of wild mosquitoes on r eplicate human subjects were used to evaluate the repellency of a pressurized aerosol insect repellent product (30 % DEET) applied to human skin. Mosquitoes were used because they are one of the insect pest groups targeted 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

, Culex

, Mansonia

, and Anopheles (Tables 3 and 4; p. 11 of 154)

.

Ta ble 3

. Wisconsin Site Mosquito Species C ollected

- August 3

, 2015

Species Number

C ollected % of Total

Coquillettidia perturbans 17 17.2 Psorophora ferox 1 1.0

Aedes vexans 1 1.0

Aedes trivittatus 80 80.8

Total 99 100

Table 4

. Florida Site Mosquito Species C ollected

- August 18

- 19

, 201 53 Species Number

Collected % of Total

Anopheles atropos 2 0.9 Anopheles crucians 1 0.4

Aedes atlanticus 76 32.6

Aedes infirmatus 116 49.8

Aedes taeniorhynchus 9 3.9

Culex erraticus 6 2.6

Mansonia dyari 3 1.3

Mansonia titillans 15 6.4
Psorophora ferox 5 2.1

Total 233 100.0

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 3 , 2015 , and one in Collier C ounty, Florida on August 18

- 19 , 2015

. At each site, t he experimental treatment groups consisted of 10 different treated su bjects and the untreated control group consisted of two untreated subjects. Subjects at each site were selected from a pool of trained participants (Table 7 )

. In Wisconsin , testing was conducted during the course of a single day while in Florida testing was conducted over the course of two consecutive days; each day with five treated subjects (§ 13.1, p. 20 of 154 )

.

Table 7

. Summary of Test Su bject Participation in GLP864E1

– EPA Reg. No. 4822

- 397 ( This

is the revised version of T able 7 appearing on p. 13 of 154 sent to EPA by S. C. Johnson & Son,

Inc. )

GLP 864E1

- 4822

- 397 Wisconsin Florida Total

Number of Subjects

Enrolled 24 30 54 (Note: S.C. Johnson corrected
the chart in the study which
indicated a total of 53, which

was a typographical error. )

Number of No

- Shows to

training and/or testing No

- shows for training: 3

No

- shows for testing: 0 No

- shows for training: 17

No

- shows for testing: 0 20 (20 no

- shows for training, 0

no

- shows for testing)

Number Assigned as Test
Subjects 12 12 24

Number Assigned as

Alternates or Extras 5 alternates

4 extras 0 alternates

0 extras 9

Number of Test Subjects

Withdrawn Voluntarily 0 1 on training day 1 on test day (post

treatment) 2 (1 on training day, 1 on test day)

Number of Test Subjects
Withdrawn Involuntarily 0 0 0

Number of Test Subjects
Completed Research 12 11 234 In Wisconsin: 12 test subjects (10 treated test subjects and 2 controls)

and 5 alternates were randomly selected as

described in the report

out of the pool of 21 trained participants

. In Florida: Only 1 2 trained
participants were available on this test day

. 10 treated test subj ects

and 2 controls were

selected as described in
the report out of the pool
of 12 trained participants

.

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. This was true for all treated test subjects except for one treated subject (Subject ID #49) who drew an odd number but had her right arm treated. This was due to a prior injury to her

left arm.

Treated subjects were blinded to the identity of the test substance ( §13.3, p. 21)

. The t wo subjects most proficient at aspirating mosquitoes from the pool of subjects were used at each site

as untreated controls to determine mosquito landing rate. The untreated control subjects chose which arm to expose to mosquitoes so the dominant hand could be used to aspirate mosquitoes

.

Test preparation. At the testing sites, stu dy 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 (§ 13.4

- 13.5 , p

. 21 of 154 )

.

Measuring repellency

. The unit of measure for assessing repellent effects in this experiment

was mosquito landings, similar to previous skin applied repellent evaluations where the “Landings” measure is used and repellency is measured as CPT

. To determine CPT for each subject , su bjects were grouped into pairs. Pairs were separated from each other by at least twenty feet. Starting two hours after application of the repellent product ( Protocol Deviation #6

, Appendix B, p. 118 of 154) , subjects observed their limbs for five minutes for landing mosquitoes. Mosquitoes th at landed were aspirated by each subject or their partner , and if necessary, headlamps were used at night so subject s could see to aspirate mosquitoes. For this study, headlamps were required on August 18, 2015 only. After the f ive

- minute exposure period, subjects reported the number of mosquito landings on them to study staff who recorded

the number for each subject. A spirated mosquitoes were labeled and kept to be identified. The

five

- minute exposures were conducted every thirty minutes until repellent failur e occurred.

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 mosquitoe s for a five

- minute period every thirty minutes as described above for treated subjects. However, to reduce th e exposure 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 were5 considered the minimum necessary to ensure the mosquito population was large enough to determine repellency of the tested product. The time to reach five l andings on the untreated control subjects was recorded for ea ch five

- minute exposure period [ §13.7, p . 23 of 154 ].

Subject specific Complete Protection Time (CPT) was calculated as time from application of the test substance to a subject and the First Confirmed Landing on that subject. A landing occurred when a mosquito alighted on the treated skin of a subject. A Firs t Confirmed Landing is that which is followed by another landing within a 5 minute exposure period or, when one land occurs in such an exposure period and another landing occurs in the next exposure period (30 minutes later) ( § 3.1, p. 8 of 154). Subjects with repellent failures were removed from the field test when a First Confirmed Landing occurred. The test was terminated as determined by the study director.

The duration of repellency time to appear on the Repellency Awareness Graphic on the p roduct 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 t reatment : The target application rate was 1.0 g per 600

cm 2 + 10%

. 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 isopropa nol and 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 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

ont o the skin was determined. If the amount sprayed was below the target weight, more was sprayed onto the forearm and the can weighed again ( §14.6, p.24 of 154)

. The target amount and

the amount applied to each subject is found in Table 8 ( p. 25 of 154)

. The mean amount applied was the target amount (100% of target) with a range of 94

- 111% of the target amount

. Subject ID #51 had 11% more than the target amount applied but a p rotocol d eviation was not reported.

Formula 1. Weight of test substance to apply (g) = [Area of limb cm 2 /600 cm 2 ] * 1.0 g

Table 8

. Amount of Test Substance Applied

.6 Site Test Subject

No. Date Limb

Treated Target

Amount 1

(g) Actual

Amount (g) % of
Target

Wisconsin 49 8/3 /15 Right Arm 0.85 0.85 100

Wisconsin 50 8/3 /15 Right Arm 0.79 0.85 108

Wisconsin 51 8/3 /15 Left Arm 0.94 1.04 111

Wisconsin 52 8/3 /15 Right Arm 0.92 0.91 99

Wisconsin 53 8/3/ 15 Left Arm 0.79 0.78 99

Wisconsin 62 8/3 /15 Right Arm 0.92 0.89 97

Wisconsin 63 8 / 3 /15 Left Arm 0.96 0.93 97

Wisconsin 66 8/3 /15 Right Arm 1.00 0.98 98

Wisconsin 68 8/3 /15 Right Arm 0.97 0.9 6 99

Wisconsin 69 8/3 /15 Left Arm 0.9 6 1.03 107

Florida 149 8/18 /15 Left Arm 0.65 0.67 103

Florida 151 8/18 /15 Left Arm 0.76 0.76 100

Florida 152 8/18 /15 Right Arm 0.77 0.7 9 103

Florida 156 8/18 /15 Right Arm 0.72 0.6 8 94

Florida 158 8/18 /15 Right Arm 0.97 0.99 102

Florida 162 8/18 /15 Right Arm 1.10 1.06 96

Florida 172 8/19 /15 Right Arm 0.60 0.58 97

Florida 174 8/19 /15 Right Arm 0.75 0.78 104

Florida 179 8/19 /15 Left Arm 0.68 0.64 94

Florida 182 8/19 /15 Right Arm 0.99 0.94 95

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

.

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

. There

were no amendments to the pro tocol ( Appendix A, p p

. 32

- 33 and 100

- 101 of 154 ) but s even protocol deviations occurred during the study (Appendix B, p. 112

- 119 of 154 ). The deviations did not have a negative impact on the study.

Results and analyses :

Test s ystems: Test subjects collected mosquitoes representing six genera ( Aedes, Anopheles, Coquillettidia, Culex, Mansonia, and Psorophora ) in this study across both field sites. Three

genera including f our species were present on the day of testing in Wisconsin while five genera including nine species were collected in Florida for a total of six genera and 12 species across the

two sites. Aedes spp. mosquitoes, a mosquito genus containing numerous important vectors o f human diseases, represented 81 % of the mo squitoes collected in Wisconsin with Coquillettidia peturbans , a vector of Eastern Equine Encephalomyelitis, accounting for almost the entire

remainder of the collection. Aedes spp. disease vectors were collected in Florida ( Tables 3 and 4 ; § 10.1

, p. 11 of 154)

.7 Landing frequency and duration of r epellency: 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 Wisconsin site, the t ime to count five mosquito landings on control subjects ranged from 12 to 146 seconds with only 6 of 17 exposure periods having untreated controls exceeding 60

seconds. At the Florida site, the time to count five mosquito land ing s on control subjects ranged

from 7

- 215 seconds on A ugust 18 with untreated control subjects in 6 of 17 exposure periods exceeding 60 seconds (Table 11, p. 28 of 154)

. A rain event occurred at the Florida site causing exposure period 4 to be skipped (P rotocol Deviation #5, p. 117 of 154) on August 18, 2015

. The protocol did not address how to determine repellent failure in the event of a rain delay but no subject received a landing in exposure period 4 or 6 . On August 19, 2015 at the Florida site 14 of the 15 untreated control exposure periods reached 5 landings in less than one minute.

The repellent failed on all subjects except: Subject ID #66 at the Wisconsin site due to the study

director ’s decision to stop testing at 9.5 hours post

- treatment; Subject ID #162 at the Florida site on August 18 because the study director stopped testing at 10 hours post

- treatment; and Subject

ID #158 , who withdrew from the study on August 19, 2015 in Florida after the end of exposure period 14 (8.5 hours after treatment without any landings) [ §13.6.4, pp. 22 of 154 ; Tables 12 and 13, pp. 29

- 30 of 154 ].

Median Complete Protection Time s. M edian complete protection times with 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 durat ion of repellency (CPT) for the test substance (Mark

- 2) was 7. 5 hours in Wisconsin and 8.5 hours in

Florida (Table s 1 and 2, pp. 8

- 9 of 154 ). However, the Florida site study was conducted over two days with 5 subjects each plus one untreated control. There was no discussion in the study on whether or not this affected the study outcome and data analysis.

Table 1

. MARK

- 2 (30% DEET aerosol) Re pellency Duration Results Summary, Hours, Sample

size = 10 per site (p. 8 of 154).

Measure Wisconsin

Site Florida

Site

Median 7.5 8.5

95% LCL 4.0 4.5

95% UCL 8.0 10

Range 4 .0 to 9.5 4 .5

- 10

Table 2

. MARK

- 2 (30 % DEET aerosol) Rep ellency Duration Results, Hours (p. 9 of 154)

Wisconsin

Site Florida

Site
Subject

No. Repellency

Duration Subject

No. Rep ell ency
Duration

49 4.0 149 6 .58 50 7.0 151 8.5

51 7.5 152 10.0

52 6.0 156 8.5

53 8 .0 158 8 .5 1

62 7.5 162 10.0

63 7 .5 172 5 .5

66 9.5 174 8.5

68 7.5 179 4 .5

69 8.0 182 9.0

1 Subject withdrew from study at 8.5 hrs. post

- treatment.

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 address EPA and HSRB recommendations before testing began. The data in the study are accepta ble to support a median CPT of 7 .0 hours against mosquitoes for the EPA Repellency Awareness Graphic on the label of EPA Reg. No. 4822

- 397, Mark

- 2 OFF! Deep Woods Sportsmen Insect Repellent II (Unscented Deep Woods Off! Insect Repellent) (30% DEET)

.

Note: One subject (Subject ID # 51) received 11% o f the target dose but no protocol deviation was reported.

HSRB Comments and Science Rec ommendations 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 wit h 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 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 alo ng the 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 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.9 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 on the subject ’s skin by a staff

member. The Board was c oncerned 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 mor e 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 l evel 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. J ohnson Response: The test substance ( aerosol formulation) was applied as directed by the revised protocol (study Section 14.6, p. 24 of 154) and results reported in Table 8 (p. 25 of 154). The mean amount applied was equal to the target amount with a ra nge of 94

- 111% 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 est ablished 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 bor ne 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 mo squito 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) requ ired to evaluate efficacy. In addition, t he protocol was10 revised to include the requested information (e.g., mosquito species present, climate, etc.) for the proposed site in Australia. However, t esting was not conducted outside of the U.S., so this

inform ation 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. Si mple 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 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 w ith 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

w ith 98.25% DEET, is th ere 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 partici pate in another test. There is no indication that test subjects par ticipated in multiple tes ts except as described in Protocol Deviation #3 where an untreated control subject on August 18, 2015 was asked to participate as an alternate on August 19, 2015. Since the subject had been an untreated control, the deviation was justified because an alternate was needed on August 19, 2015 and participation of an untreated control subject from the previous day eliminated any possibility of a carry

- over effect.

L anding 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 la ndings 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 dif ferent 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

con sider how a quantitative estimate of landing pressure can be determined without increasing11 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 t he time to reach five landings if they occurred in

less than five minutes and these re sults were reporte d in the study (Table 11 , p. 28 of 154). Because all studies were performed at the same two sites, landing pressure appeared to be fairly

consistent across site s , therefore normalization was not required.

Delayed start : The Board rec ognizes 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 h ow long to wait before starting the test cycles (5 minute exposure at 30 minute intervals). 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, fol lowing 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 ( Protocol

Deviation # 6)

. The test substance contained 30 % 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. There was no negative impact on the study by having two additional expo sure periods to the beginning of the study. Subjects completed at least three exposure cycles before getting a confirmed landing.

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 pro duct 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 mu ltiple 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 greater generalization of results to a 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. That being stated, this study was conducted in one day with 10 subjects at the Wisconsin site and on two consecutive days with 5 subjects each at the Florida12 site. However, this approach was not planned Protocol Deviation s #2, #3 and #4 report why this took place and corrective actions taken (p p

. 11 5

- 116 of 154). The data were not analyzed differently.

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

w hether/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: O ne 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

ran domizing 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 their right arms treated. This was true for all treated test subjects except for one treated subject (Subject ID #49) who drew an odd number but had her right arm treated. This was due to a prior injury to her left arm. In addition, the protocol notes that aspirating mosquitoes is not difficult

even with a non

- dominant hand. T he u ntreated 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 include d in the EPA

document “Product P erformance 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 regarding sample size presented in the protocol for testing repellents against tic ks to the Human Studies Review B oard at the October meeting would apply to these studies.

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

valu es between otherwise matched studies conducted at two different sites.13 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 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?

S. C. Johnson Response: One of the treated subjects (Subject ID # 158) withdrew f rom the study at the Florida site on August 19, 2015 after completing 14 exposure periods (8.5 hours) without a landing. There was one subject left after this exposure period that had a FCL at 9.0 hrs. The data from Subject ID #158 was included the analysis with a reported CPT of 8.5 hrs. (Table 2, p. 9 of

154 )

.

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 Respon se: 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 Respons e: 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 lim b? For lotions, the amount to be14 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 §14.6 (p. 24 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 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.

S.C. Johnson Response: Median complete protection time and 95% confiden ce 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.