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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON D.C., 20460 

OFFICE OF 

CHEMICAL SAFETY AND 

POLLUTION PREVENTION

 September 29, 2011 

MEMORANDUM

SUBJECT: Science Review of Completed Carroll-Loye Mosquito Repellent Field Efficacy 

Study No Mas 003 

FROM: Clara Fuentes, Ph.D., Entomologist, Biochemical Pesticides Branch 

Biopesticides & Pollution Prevention Division 

Office of Pesticide Programs 

TO: Linda Hollis, Chief, Biochemicals Pesticide Branch 

Biopesticides & Pollution Prevention Division 

 Office of Pesticide Programs 

REF: Carroll, S. (2011) Field Efficacy Test of PMD and Lemongrass Oil-Based 

Repellent, No Mas-003, against Mosquitoes. Efficacy Test Report NO MAS-003. 

 Unpublished document prepared by Carroll-Loye Biological Research. 411 pp.

ACTION REQUESTED 

Conduct a science review of the completed study, Field Efficacy Test of PMD and Lemongrass 

Oil-Based Repellent, No Mas 003, against Mosquitoes” (MRID 48577201) to determine the 

adequacy of the methods employed and the scientific validity of the reported data, and to assess 

the consistency with the approved protocol. 

The protocol No Mas 003 was initially submitted to EPA for review in August 2010. The 

protocol and EPA’s review dated October 1, 2010, were discussed by the HSRB on October 27, 

2010. The HSRB reviewed the protocol favorably, concluding in their December 13, 2010, final 

report of the October meeting that “the proposed field repellency study protocol No Mas-003, if 

revised as suggested in EPA’s review...and performed as described, is likely to generate 
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scientifically reliable data, useful for assessing the efficacy of the tested material in repelling 

mosquitoes.” 

CONCLUSIONS 

Scientific aspects of the research were assessed in terms of the recommendations of EPA 

Guideline §810.3700 and of the EPA Human Studies Review Board. Study MRID 48577201 is 

in compliance with Good Laboratory Practices (US EPA Pesticide Programs 40 CFR §160) (pg. 

3) and was inspected for Quality Assurance during various stages of the study (pg. 4). This 

study provides scientific data that are acceptable pending resolution of data analysis for 

characterization of NO MAS performance. The Human Studies Review Board will be asked to 

comment on the statistical analysis employed in this study. 

SCIENCE REVIEW 

Study Objectives: To determine the Complete Protection Time (CPT) of NO MAS repellent 

when applied at a typical consumer dose, against wild populations of mosquitoes, including but 

not limited to species of the genera Culex, Anopheles, and Aedes. To provide data required by the 

EPA as a condition of registration for NO MAS 003 mosquito repellent, containing 16% w/w of 

p-menthane-3,8-diol (PMD) and 2% w/w of lemongrass as active ingredients, and to estimate the 

mean and standard deviation for CPT, which is measured as the period of time between product 

application and the time of First Confirmed Landing with Intend to Bite (FCLIBe). 

Materials and Methods: 

Study Locations: Field test sites were located in the Central Valley of California. The sites 

represented different habitats for different mosquito species, and were chosen based on mosquito 

and virus surveillance data compiled weekly by the California State Department of Public 

Health. Site 1 was a tall floodplain with mature oak forest surrounding marshy areas and 

standing water, located in Glenn County. Site 2 was a relatively open irrigated landscape with 

hedgerows of willow trees growing along an active stream located in Butte County (Table 2 pg. 

11). West Nile Virus had not been reported in the test area in more than 5 years of weekly 

surveillance by Butte/Glenn County Mosquito and Vector Control District. Both sites differ in 

composition and distribution of mosquito species given in Table 7, pg. 20, in the study report. No 

mosquitoes collected at either site for 2 weeks prior to testing were positive for West Nile Virus, 

West Equine Encephalitis virus, or St. Louis Encephalitis virus. 

Study Dates: Dosimetry testing was conducted in Arthropod Behavior Laboratory at CarrollLoye Biological Research on July 5-7, 2011. Repellent product tests were conducted in the field 

at Sites 1 and 2 on July 23-24, 2011. 

Repellent Tested: NO MAS 003 mosquito repellent formulation, containing 16% w/w of pmenthane-3,8-diol (PMD) and 2% w/w of lemongrass as active ingredients. 
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Tested positive control/comparison repellent: There was no positive control in this study. 

Untreated Control: Two experienced negative control subjects (female control subjects 4 and 84 

at Sites 1 and 2, respectively, and male control subjects 63 and 52 at Sites 1 and 2, respectively. 

pp. 57-58) exposed untreated limbs for 1 minute every 15 minutes at the same time as treated 

subjects to monitor the ambient Landing with Intent to Bite (LIBe) pressure throughout the test. 

There were no statistical comparisons to the untreated controls. 

Mosquito species and life stage: NO MAS repellent was evaluated against natural populations of 

adult female mosquitoes. The species, number and distribution of collected mosquitoes are listed 

in Table 7 on page 20 of the study report. The most common species identified in Site 1 and Site 

2 was Aedes melaniomon, followed by Ae. vexan, more common at Site 1 than at site 2. At Site 

2, Ae. vexan, was equally common to Culex tarsalis, followed by Anopheles freeborni and Aedes 

nigromaculis. Anopheles freeborni and Ae. nigromaculis were the least common species only 

collected at Site 2. 

Number of Test Subjects/Treatment Regime: A total of 32 healthy subjects, between the ages of 

18 and 55 years old, were randomly selected from a pool of 92 subjects to participate in the 

study. A total of 10 subjects (female subjects 4, 23, 84, 105, 116 and male subjects 13, 14, 15, 

51, 64), were employed for determination of repellency dose. Four of the 10 dosimetry subjects 

also participated in the repellency test. Dosimetry subjects 64 (male) and 105 (female) 

participated in the efficacy test at Site 1, and dosimetry subjects 4 (female) and 14 (male) 

participated in the efficacy test at Site 2. 

For field testing, repellent-treated female subjects 28, 92, 105, 118, 125, and repellent-treated 

male subjects 29, 41, 64, 106, 123 participated in repellency testing at Site 1 (Randomized 

Treatment Allocation Table, pg. 55, and pg. 16 of study report). Repellent-treated female 

subjects 4, 39, 76, 81, 85, and repellent-treated male subjects 14, 63, 88, 120, 121 participated in 

repellency testing at Site 2 (Randomized Treatment Allocation Table, pg. 56 and pg. 16 of study 

report). Treatment was replicated 10 times (on 10 subjects) per site. 

Protocol used including amendments: CLBR Protocol NO MAS 003 (pp. 309-322) obtained 

prior review from EPA and the HSRB. EPA’s science and ethics review was dated 10/01/10 and 

the HSRB reviewed the protocol at its October 2010 meeting. The HSRB’s report dated 

12/13/10 concluded that “t”he proposed field repellency study protocol No Mas-003, if revised as 

suggested in EPA’s review...and performed as described, is likely to generate scientifically reliable 

data, useful for assessing the efficacy of the tested material in repelling mosquitoes.” Following 

HSRB review, the protocol and consent form were modified through Amendment 1 (dated 

11/15/10). This amendment incorporated changes responsive to the comments of EPA, HSRB, 

and California Department of Pesticide Regulation (CDPR), as well as additional corrections 

initiated by the investigators and/or sponsor. The Independent Investigational Review Board, Inc. 

granted approval to Amendment 1 on 11/16/10. 

 
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Protocol Deviations: One protocol deviation was reported. Data forms were reformatted to 

minimize data entry error and thus enhance accuracy. Therefore, the reported deviation is 

expected to improve data quality. 

Consistency of study with approved protocol: The study was conducted in accordance with the 

IIRB approved protocol as augmented by protocol amendment 1 (pp. 309-322). One protocol 

deviation was reported by the investigators (discussed above). I did not note any unreported 

deviations. 

Responsiveness to Prior EPA and HSRB Comments 

Table 1: 

EPA and/or HSRB Science-Related Comments 

on the No Mas 003 Protocol 

Addressed prior to 

initiation of the study? 

1. Amend the protocol to remove erroneous reference to spray 

repellents (e.g. the first sentence on page 79, under 

“Rationale”; Carroll 2010, 79). 

Yes. 

2. Consider whether the proposed sample size is large enough to 

be likely to yield a definitive answer to the research question 

and its size justified statistically in the protocol.

Yes. 

3. In the statistical analysis plan, discuss how non-normally 

distributed data points will be treated. That is, how the 

researcher plans to analyze and interpret results from nonnormally distributed data points that may follow an 

unknown distribution. 

Yes 

4. On page 28, line 41, delete the phrase “for each test 

material” because this protocol is for testing one single 

formulation. 

Yes 

5. Update the sequential exposure table to include 80 

sequential exposure intervals. 

Yes 

Dose determination: A dosimetry determination phase was conducted prior to efficacy testing to 

estimate typical consumer dose. The amount of lotion applied to limbs (forearms and lower legs) 

was averaged over 3 applications per subject. The overall average grams of lotion applied on 

forearms and lower legs across 10 dosimetry subjects was 0.57 ± 0.18 and 1.13 ± 0.40 grams, 

respectively. Overall average dose rates for arms and legs are 0.00114 and 0.00099 g/sq. cm, 

respectively, which are calculated from each subject individual limbs’ surface area value. The 

dose rates in g/sq. cm were modified to convert grams to mL based on specific gravity of the 

repellent (0.9524 kg/L) (Calculations are shown in raw data summary table on pp. 59 – 60). 

Dose rates: In the efficacy testing phase, test material applications were made volumetrically 

(mL) based on specific gravity of the formulation and adjusted to surface areas of repellent 

treated subjects’ limbs so that for efficacy testing at Site 1 and Site 2, all subjects’ arms and legs 

were treated at a volumetric dose rate of 1.20 μl/cm2 and 1.04 μl/cm2

, respectively, based on the 
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grand mean of arm and leg dosimetry. The efficacy testing dose rates of 1.20 μl/cm2

 (forearms) 

and 1.04 μl/cm2 (lower legs) were used to estimate Margin of Exposure (MOE) values > 583 and 

>287 for arms and legs, respectively, relative to NO MAS acute dermal toxicity limit dose > 

5,000 mg/kg in males and female rats. The resulting values are close to those predicted in the 

study protocol, presented in Section 4.6, Margin of Exposure and Dosimetry, on page 113 of the 

study protocol, included in study report MRID 48577201. (Toxicological profile of NO MAS 

repellent in summarized Appendix 1 of this review). 

Experimental design: There was no blinding in the study since the test was designed to evaluate 

one formulation at 2 different field sites, using 2 different groups of 10 treated subjects per site, 

and 2 experienced control subjects per site to monitor mosquito landing pressure throughout the 

test. Subjects were trained in the laboratory to use mechanical aspirators and handle mosquitoes 

prior to field testing. The pre-test training process is described in the Informed Consent 

Authorization as a Research Study Subject (no repellent applied) on pg. 129, and in Informed 

Consent Authorization as a Research Study Subject (repellent applied) on pg. 140. Subject 

preparation for testing was done consistently with the test guideline for Product Performance of 

Skin-applied Insect Repellents of Insect and Other Arthropods Test Guidelines (OPPTS Test 

Guideline No. 810.3700). 

The NO MAS formulation was tested at each site on 2 consecutive dates. The first trial was 

conducted on Site 1 on July 23, 2011, and the second trial was conducted at Site 2 on July 24, 

2011. For Site 1, the subjects were treated in the CLBR laboratory early in the morning, prior to 

travel to the field. For Site 2, the subjects were treated upon arrival at the field later in the day. 

Time between application and first exposure varied among individual subjects approximately 3.2 

hours at Site 1, and approximately 6 minutes at Site 2 (pg. 16). Repellent treatments were 

applied by Carroll-Loye Biological Research (CLBR) technicians and staff, using 1 mL syringes 

(0.01 ml increments). The material was spread evenly on subjects’ skin using one fingertip in a 

surgical glove. Repellent performance was evaluated on two different groups of ten subjects, 

five males and five females. To ensure adequate landing pressure throughout the test, two 

experienced subjects, one male and one female, monitored landings coincidentally with treated 

subjects exposure periods. 

During exposure in the field, treated subjects were arranged in pairs to monitor landings on their 

own exposed limbs and the back of the exposed limb of their partner. They were equipped with 

mechanical aspirators to remove landing mosquitoes before they would bite. Exposure to 

mosquitoes lasted 1 minute every 15 minutes until repellency failure or cessation of the test. The 

results of each exposure were monitored by technicians, who were equipped with mechanical 

aspirators to remove mosquitoes landing on subjects. Technicians reported all landings to a 

scientist in charge of recording each landing by subject and time of occurrence (during exposure 

interval). A stopping rule was invoked and the subject was withdrawn from further exposure 

when he or she experienced a confirmed landing; that is, a landing following another within 30 

minutes. At Site 1, testing stopped once all subjects had experienced a First Confirmed Landing 

with Intent to Bite (FCLIBe). At Site 2, the test ended at the onset of darkness when landing 

activity ceased (Table 9, pg. 23, and raw data tables, pp. 71-72). At that point, only four of the 

ten subjects had experienced a Confirmed Landing with Intent to Bite. 
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The study was conducted under suitable environmental conditions summarized in Table 6, pg. 

19, of the study report. Mosquito landing pressure throughout the test was adequate in that it was 

at or above the minimum of one mosquito landing per minute. 

Mosquito disease pathogen detection: No collected mosquitoes were found positive for viral 

pathogens. 

Data analysis: Results from each site were analyzed independently using descriptive statistics 

without making comparisons between sites. The means and medians of CPT, with their 

associated confidence levels, are tabulated by site in Table 8, pg. 22, of the study report. 

Survival data were analyzed using both non-parametric (Kaplan-Meier) and parametric (Weibull, 

Normal) methods. Kaplan-Meier is a non-parametric method which does not assume any 

parametric distribution of the data. On the other hand, parametric methods such as Weibull and 

Normal methods make distributional assumptions about the data. The Weibull probability 

density distribution, which is an exponential function commonly used to model the probabilities 

of times to an event when the event is inevitable, was used in survival analysis to estimate mean 

CPT within 95% confidence intervals from a non-normally distributed set of values. The 

researcher explains that mean and variances modeled by the Weibull distribution better 

characterize the mean and 95% confidence intervals of complete protection times even in the 

event of substantial right censoring. 

Results: 

Table 2. Results as presented in Table 8, pg. 22, of the Study Report, MRID 48577201. 

Site/Parameter CPT (hours) Lower 95% Upper 95% 

Site 1

Weibull mean 9.8 9.0 10.6 

Normal mean 9.2 8.1 10.2 

Kaplan-Meier median 9.6 6.4 10.5 

Site 2

Weibull mean 10.1 8.2 12.5 

Normal mean 8.5 7.8 9.2 

Kaplan-Meier median -- 6.8 -- 

Table 2 above summarizes the results of the field test by site. The Weibull mean values have a 

smaller confidence interval when compared to the Kaplan-Meier survival analysis; thus, the 

investigator asserts that use of the Weibull distribution provides a better estimate of CPT with a 

higher level of confidence. The Weibull mean CPT values for both sites analyzed independently 

are nearly the same, 9.8 and 10.1 hours. Individual subject data are tabulated in Table 9, page 

23, of the study report. 

 
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Table 3. Individual Subject Data as presented in Table 9, pg. 23, of the Study Report, MRID 

48577201. 

Sites Subjects CPT (hrs) CLIBe 

(Yes or No?) Number of LIBe

Site 1 

125 11.17 Y 2 

106 10.85 Y 2 

28 10.47 Y 2 

118 9.60 Y 3 

123 9.60 Y 2 

41 8.95 Y 4 

105 8.80 Y 2 

92 8.42 Y 2 

29 7.72 Y 2 

64 6.40 Y 3 

Site 2 

4 9.25 N 0 

81 9.17 N 0 

39 9.12 N 0 

76 9.08 N 1 

85 9.05 N 0 

88 9.02 N 0 

14 8.38 Y 2 

120 8.08 Y 5 

63 6.77 Y 2 

121 6.77 Y 2 

At Site 1, all subjects experienced first confirmed landings ranging from a CPT of 6.40 to 11.17 

hours. At Site 2, the normal mean of CPT is generated from a sample of 10 subjects, of which 

only 4 experienced confirmed landings at around 8 and 6.77 hours. The test ended after 9:25 

hours of exposure periods, when mosquito landing pressure declined below acceptable levels. 

The time when the test ended was assigned as the time of product failure (Table 9 pg. 23). The 

mean and median protection time for Site 2 were calculated from 4 actual and 6 estimated CPT. 

The average number of landings experienced by individual subjects was 2.4 at Site 1, and 1.2 at 

Site 2 (pg.22). Figure 10 on page 24, illustrates Kaplan-Meier survival plots for lasting 

repellency of NO MAS formulation against mosquitoes tested at both sites. 

Question to the HSRB regarding statistical analysis of the data: 

Which statistical method is appropriate to calculate the Complete Protection Time for the NO 

MAS repellent? 

1. Parametric (with Weibull distribution or normal distribution); or 

2. Non-parametric (Kaplan Meier) 
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Conclusion: 

The methods employed in this study were adequate to produce scientifically reliable data. They 

were based on study protocol No Mas 003 as amended in accordance with EPA and HSRB 

recommendations before testing began. No Mas 003 repellent provided a high degree of 

repellency against against several mosquito species (Aedes melanimon, A. vexans, A. nigromalis, 

Culex tarsalis, and Anopheles freeborni) encountered in the field. The Weibull mean CPT was 

9.8 hours at Site 1, and 10.1 hours at Site 2. The reported protocol deviation was nonsubstantive in nature and did not affect the design or conduct of the research, or the resulting 

data. 

Recommendation: The study is scientifically sound and acceptable.