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

Eric 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: Laznicka, E. (2015) Field Testing of S.C. Johnson Personal Mosquito Repellent Products to Support Their Use of the EPA Repellency Awareness Graphic

– Project Number 867E1. Unpublished document prepared by S

. C. Johnson & Son, Inc., 1525 Howe Street Racine, WI 53 403, October 21 , 201 5, 150 p. (MRID

49765901 ) (D43018 1 )

ACTION REQUESTED

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

formulated as a pump 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. No.2 4822

- 395

, Mark

- 5 ( Unscented OFF! Skintastic Spray Insect Repellent 7 % DEET ). The protocol used to conduct this study was previously reviewed and accepted by EPA and the HSRB on Apri l

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 49765901 ) was conducted in accordance with Good Laboratory Practices as described in 40 CFR §160 ( with one minor GLP exception as described in Protocol

Deviation #3 ), 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 complete protection time (CPT) of

MARK

- 5 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 150 )

.

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 pump

- spray insect repellent product (7 % 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 R epellency

A wareness 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: Psorophora

, Aedes

, Culex

, Mansonia

, and Anopheles (Tables 3 and 4; p. 11 of 150 )

.

Ta ble 3

. Wisconsin Site Mosquito Species C ollected

– July 28

, 2015

Species Number

C ollected % of Total

Aedes vexans 2 3 %

Aedes trivittatus 64 97 %

Total 66 100 %

Table 4

. Florida Site Mosquito Species C ollected

– September 29

, 201 5

Species Number

Collected % of Total

Anopheles atropos 3 5 % Anopheles crucians 1 2 %3 Aedes atlanticus 21 36 %

Aedes infirmatus 12 20 %

Aedes taeniorhynchus 9 15 %

Culex erraticus 3 5 %

Mansonia dyari 3 5 %
Psorophora ferox 7 12 %

Total 59 100 %

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 July

28 , 2015 , and one in Collier C ounty, Florida on September 29 , 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 different candidate pools (Table 7 )

. Testing at both sites was conducted in a single day (§13.1, p. 20 of

154 )

.

Table 7

. Summary of Test Su bject Participation in GLP867E1conducted with Mark

- 5. ( This is

the revised version of T able 7 appearing on p. 13 of 150 sent to EPA by S. C. Johnson & Son,

Inc. )

GLP 867E1

– Mark

- 5

- EPA Reg. No. 4822

- 395 Wisconsin Florida Total

Number of Subjects Enrolled 24 20 44 *( Note: SC Johnson
corrected the original figure of 45.)

Number of No

- Shows to

training and/or testing No

- shows for training: 1

No

- shows for testing: 2 No

- shows for
training: 7

No

- shows for

testing: 0 10

** ( Note: S.C. Johnson
corrected the original
figure of 8. It should be

10; 8 no

- shows for
training, 2 no

- shows for testing)
Number Assigned as Test
Subjects 12 12 24

Number Assigned as

Alternates or Extras 4 alternates (2 were used)

7 extras 1 alternate

0 extras ***( Note: S.C. Johnson
corrected original figure

of 5; it should be 12)
Number of Test Subjects

Withdrawn Voluntarily 0 0 0

Number of Test Subjects
Withdrawn Involuntarily 0 0 0

Number of Test Subjects
Completed Research 12 12 244 In Wisconsin: 12 test subjects (10 treated test subjects and 2 controls) and

4 alternates were randomly

selec ted as described in the

report out of the pool of 23 trained participants

. In Florida: Only 13

trained
participants were

available on this
test day. 12 test subjects (10

treated test
subjects and 2 controls) and 1

alternate were
selected as

described in the

report out of the pool of 13 trained
participants

. *typo graphical error

**no

- shows for testing were not previously included in the total

***7 extras were not previously included in the total

The experiment was partially randomized, the test subjects were randomly selected from a pool of potential subjects that met the incl usion criteria found in § 12.3, pp. 14

- 15 of 150

. 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 treat ed.

Treated subjects were blinded to the identity of the test substance ( §13.3, p. 20 of 150 )

. The two 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 han d could be used to aspirate mosquitoes

.

Test preparation. 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 pant s 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 p

. 20

- 21 of 150 )

.

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 30 minutes after application of the repellent product, 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 not require d because the study was

terminated during daylight hours. After the five

- minute exposure period, subjects reported the number of mosquito landings on them to study staff who recorded the number for each subject.5 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 population s 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 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 were considered the minimum necessary to ensure t he 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 ea ch five

- minute exposure period [ §13.7, p . 22

- 23 o f 150 ] except as described in Prot ocol Deviation 4 (Appendix B, p. 114 of 150) when the study director did not record the time for the control subjects to reach five lands on the first exposure period. Subsequent exposure periods reached 5 landings in less than 5 minutes and this deviation was unlikely to negatively impact the study.

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 150 ). 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 product

la bel 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 neare st whole number. ”( § 4.1, p. 8 of

150 ).

Test substance application rate and t reatment : The target application rate was 0.5 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 o nly exposed skin. The test substance was placed on

the balance and the balance tared. The target amount (by weight) of repellent was removed from the sample container with a pipette. Once the balance indicated that approximately the target

amount was wi thdrawn from the test substance container, the test substance was transferred from the pipette as droplets and distributed around the forearm treatment area ( §14.6, p. 23 of 150 )

.6 The target amount and the amount applied to each subject is found in Table 8 ( p. 24 of 150 )

. The mean amount applied was the target amount (10 0% of target) with a range of 100

- 109 % of the

target amount.

Formula 1. Weight of test substance to apply (g) = [Area of limb cm 2 /600 cm 2 ] * 0.5 g

Table 8

. Amount of Test Substance Applied

.

Site Test Subject

No. Date Limb

Treated Target

Amount 1

(g) Actual

Amount (g) % of
Target

Wisconsin 1 7/28 /15 Left Arm 0.36 0.36 100

Wisconsin 2 7/28 /15 Right Arm 0.39 0.4 103

Wisconsin 3 7/28 /15 Left Arm 0.46 0.46 100

Wisconsin 5 7/28 /15 Left Arm 0.37 0.38 103

Wisconsin 9 7/28 / 15 Left Arm 0.39 0.39 100

Wisconsin 13 7/28 /15 Left Arm 0.58 0.58 100

Wisconsin 14 7/28 /15 Right Arm 0.5 0.5 100

Wisconsin 15 7/28 /15 Left Arm 0.5 0.5 100

Wisconsin 16 7/28 /15 Right Arm 0.45 0.46 102

Wisconsin 17 7/28 /15 Left Arm 0.48 0.49 102

Florida 289 9/29 /15 Left Arm 0.67 0.67 100

Florida 292 9/29 /15 Right Arm 0.65 0.6 6 106

Florida 293 9/29 /15 Left Arm 0.58 0.59 102

Florida 295 9/29 /15 Left Arm 0.56 0.57 102

Florida 296 9/29 /15 Right Arm 0.6 0.61 102

Florida 301 9/29 /15 Left Arm 0.5 0.5 100

Florida 303 9/29 /15 Left Arm 0.46 0.46 100

Florida 304 9/29 /15 Right Arm 0.43 0.44 102

Florida 307 9/29 /15 Left Arm 0.33 0.36 109

Florida 310 9/29 /15 Right Arm 0.44 0.45 102

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

was one amendment to the pro tocol for the purpose of changing study directors ( Appendix A, p p

. 109

- 110 of 150 )

. F our protocol deviations occurred during the study (Appendix B, p. 110

- 114

of 150 ). The deviations did not have a negative impact on the outcome of the study.

Results and analyses :

Test s ystems: Test subjects collected mosquitoes representing six genera ( Aedes, Anopheles,
Culex, Mansonia, and Psorophora ) in this study across both field sites. One genus with two7 species was present on the day of testing in Wisconsin while five genera including eigh t species

were collected 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 o f human diseases, represented 100 % of the mo squitoes collected in Wisconsin

. M ajor disease vectors did not predominate the collections in Florida ( Tables 3 and 4 ; § 10.1

, p. 11 of 150 )

.

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 but the study director did not record untreated control landings in exposure period 1 in Wisconsin on July 28, 2015 as described in Protocol Deviation # 4

. At the Wisconsin site, the t ime to count five mosquito landings on control subjects ranged from 46 to 195 seconds

. At the Florida site, the time to count five mosquito land ing s on control subjects ranged from 30 seconds to just under 5

minutes (Table 1 1, p. 26 of 150 )

. The repellent failed on all subjects in Wisconsin. In Florida, eight subjects reported failures but Subjects ID #295 and ID #310 did not have a first confirmed landing through 3 .0 hours post

- treatment in Florida when the study director terminated the study due to weather (Appendix F2, p.138 and 149 of 154) ( [ §18.5, pp. 26 of 150; Tables 12 and 13, p.

27 of 150 ].

Median Complete Protection Time s. M edian complete protection times with 95% confidence

limits [Lower Confidence Limit (LCL) a nd 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 (CPT) for the test substance (Mark

- 5 ) wa s 2

. 0 hours in Wisconsin and 2 .5 hours in

Florida (Table s 1 and 2, pp. 8

- 9 of 150 ).

Table 1

. MARK

- 5 (7 % DEET aerosol) Repellency Duration Results Summary, Hours, Sample

size = 10 per site (p. 8 of 150 ).

Measure Wisconsin

Site Florida

Site

Median 2.0 2 .5

95% LCL 1.5 1.0

95% UCL 2.0 3.5

Range 1.5 to 2 .5 1.0

- 3.5

Table 2

. MARK

- 5 (7 % 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

1 2 .0 289 2.5

2 2.0 292 1 .5

3 2.0 293 2.5

5 2.5 295 3.58 9 1.5 296 3.5

13 2.0 301 2.5

14 1.5 303 1.0

15 2.0 304 2.0

16 2.0 307 3 .5

17 2.5 310 3.5

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 address EPA and HSRB recommendations before testing began. The data in the study are accepta ble to support a median CPT of 2 .0 hours against mosquitoes for the EPA Repellency Awareness Graphic on the label of EPA Reg. No. 4822

- 395, Mark

- 5 (Unscented OFF! Skintastic Spray Insect Repellent 7% DEET).

HSRB Comments and Science Rec ommendations from the April 2015 Me eting 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 va lue 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 langu age 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 ac tive 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 inc lude a dose

- respo nse 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 re present 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 t he data analysis because each study only tested a single product.

Product application method:9 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 concerned that these application methods were not representative of actual application methods but ul timately 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

e ach 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 t he 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. J ohnson Response: The test substance ( pump

- spray formulation) was applied as directed by the revised protocol ( study Section 14.6, p. 23 of 150 ) and re sults reported in Table 8 (p. 24 of

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

- 109 % 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 i nformation 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 demographi cs) 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 tha t 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, t he protocol was

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

information is not relevant to the submitted study

.10 Potential for cross contamin ation : 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 take place before or during the experiment ( i.e. , rubbing sleeve or pant leg across the treated area).

S.C. Johnson Response: Subjects were n ot 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

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 partic i pate in another test. There was no indication that test subjects par ticipated in multiple test days

.

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 c orrect, 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 pressu re 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. Jo hnson Response: Study staff recorded t he time to reach five landings if they occurred in

less than five minutes and these results were reporte d in the study (Table 11 , p. 26 of 150 ). 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.11 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). Regardless of how l ong 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 confirme d landing.

S. C. Johnson Response: This product has less than 16% DEET, therefore, exposure to mosquitoes began at 30 minutes post

- treatment.

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 subj ect (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 da y 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 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 one day with 10 subjects at the Florida site

.

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 analy sis 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 a rm 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

trea tment was not necessary. The mechanism for randomizing the arm was not provided in the12 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 I D numbers. Subjects assigned odd numbers had their left arm treated, and subjects with even numbers had their right arms treated. In addition, the protocol notes that aspirating mosquitoes is not difficult

even with a non

- dominant hand. T he untreated con trol 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 th e 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 pr otocol 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 betw een 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 subjec ts, 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?13 S.C. Johnson Response: None of the subjects withdrew.

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 Respon se: 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 numbe r 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. 23 of 150 ).

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.14 S.C. Johnson Response: Median CPT and 95% confidence limits were calcul ated 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.