EPA Review of the

Carroll-Loye Biological Research 

Completed Study No Mas 003

Field Efficacy Test of a PMD1

Clara Fuentes

Kelly Sherman

Office of Pesticide Programs

Field Efficacy Test of a PMDand Lemongrass Oil-based Repellent

‘No Mas’ Against Mosquitoes
Background

 Completed report of a mosquito repellent field 

efficacy study 

 Test material is called ‘No Mas’

 ‘No Mas’ is a lotion formulation containing 16% 

2

PMD and 2% lemongrass oil

 Protocol reviewed by HSRB at October 2010 

meeting

 Research conducted in California, at two sites, 

in July 2011

 Final report submitted to EPA in August 2011
Background 2

 Sponsor is developing ‘No Mas’ as a low-cost 

repellent for distribution in developing countries 

with vector-borne disease

 Sponsor reports that the product has broad3

spectrum efficacy against more than 40 species of 

mosquitoes, including four of the most important 

malaria-vectoring anophelines

 The purpose of the present study was to test the 

product for efficacy against three mosquito 

genera – Culex, Anopheles, and Aedes
EPA Science Review of Carroll-Loye 

Efficacy Test of NO-MAS 003, MRID 48577201. 

Unpublished document prepared by Carroll-Loye Biological 

Research. 411 p. 

by Clara Fuentes, Ph.D.

EPA Science Review of 

Carroll-Loye Biological Research 

Completed Study ‘No Mas 003’

4

by Clara Fuentes, Ph.D.

Field Test of Mosquito Repellency for p-menthane-3,8-diol 

(PMD) and Lemongrass Oil-Based Repellent ‘No Mas’ 

Against Mosquitoes, 

Clara Fuentes, Ph.D.

Biopesticides and Pollution Prevention Division

Office of Pesticide Programs
Purpose of the Science Review

 Assess the validity of the efficacy data for 

evaluating the performance of ‘NO MAS’ 

repellent against mosquitoes in the field 

 Provide scientific review of the study and its 

consistency with approved protocol NO MAS003

5
EPA Scientific Assessment

The Carroll-Loye Efficacy Test, NO-MAS 003, 

MRID 48577201, was conducted in a manner 

substantially consistent with the study 

protocol reviewed by EPA and the HSRB

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Consistency Between Conduct and Protocol

 Protocol amendments were approved prior 

to study initiation

 Justification provided for choice of sample 

size

 Rationale provided for choice of statistical 

methods to analyze non-normally distributed 

data

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Deviation from the Protocol

 Data forms were reformatted to minimize 

data entry error and thus enhance accuracy. 

 The reported deviation is expected to 

improve data quality.

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Study Objectives

The test’s objectives are:

 to characterize the performance of ‘NO MAS’ 

against wild populations of mosquito species 

among the genera Culex, Anopheles and Aedes.

 to estimate the mean value of Complete Protection 

time (CPT) within 95% confidence interval

 to provide efficacy data required to support 

registration of NO MAS formulation

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Study Design

Dose determination:

 Standard consumer dose for arms and legs was determined from 

grand mean of 10 subject means (5 males and 5 females).

Efficacy testing:

 Sample size: 10 treated subjects (5 males and 5 females) per site.

 Test sites: 2 different mosquito habitats 

 Number of treatments: 1 lotion formulation

 Negative control: 2 untreated subjects/ site to monitor landing activity 

during testing.

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Dosimetry Endpoint and Standard Consumer Dose

Dosimetry Endpoint: Standard Consumer Dose 

Arms = 1.14 mg/cm2

Legs = 0.99 mg/cm2

Standard consumer dose used for testing efficacy;

-Arms = 1.14 mg/cm2 (0.9524 kg/L) =1.20 μl/cm2

-Legs = 0.99 mg/cm2 (0.9524 kg/L) =1.04 μl/cm2

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Margin of Exposure (MOE)

Margin of Exposure

Arms MOE > 583

Legs MOE > 287 

Based on the NOEL of dermal toxicity > 5,000 

mg/kg b.w. and standard dose 

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Efficacy Endpoint and Other Measurements 

 Complete Protection Time (CPT)

 Landing pressure (threshold=1 LIBe/minute)

 Exposure delay (min)-time between application 

and first exposure: 

- Site 1 = 3.2 hours

- Site 2 = 6 minutes

 Duration of exposure: 1 minute every 15 minutes

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Results from Field Test: 

Time Distribution of CPT by Site 

Sites Subjects CPT 

(hrs)

CLIBe

(Yes or

No?)

Number 

of LIBe

125 11.17 Y 2

106 10.85 Y 2

Sites Subjects CPT 

(hrs)

CLIBe

(Yes or

No?)

Numbe

r of 

LIBe

4 9.25 N 0

81 9.17 N 0

14

Site 1

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

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
Results from Field Test: Mosquito species by Site 

Mosquitoes collected at SITE 1

Mosquito 

species

Total 

collected

Total collected by 

subject

Aedes

melanimon

52

2 

Control 

subjects

10 

Treated 

subjects

45 7

Mosquitoes collected at SITE 2

Mosquito 

species

Total 

collected

Total collected by subject

Aedes

melanimon

76

2 

Control 

subjects

10

Treated 

subjects

68 7

Aedes vexans 5 5 0

15

45 7

Aedes

vexans

26 18 8

Total 

number of 

all species

78 63 15

Aedes vexans 5 5 0

Aedes

nigromaculis

1 0 1

Culex tarsalis 4 4 0

Anopheles 

freeborni

2 2 0

Total 

number of 

all species

88 79 8
Statistical Analysis

Dosimetry procedure

 Average testing dose was the grand mean (± SD) across 10 subjects’ 

means. 

Efficacy Testing

 Sample size: 10 subjects/test site.

 Statistical methods employed to calculate CPT within 95% CI:

 Weibull Mean 

 Kaplan-Meier median, and

 Normal Mean 

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Complete Protection Time Values by Site

(from Carroll-Loye Biological Research)

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

Site 1

CPT values within 95% CI by test site

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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 --
Statistical Questions for the HSRB

Which statistical method is appropriate to calculate 

the Complete Protection Time for the NO MAS 

repellent?

1. Parametric (with Weibull distribution) or 

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1. Parametric (with Weibull distribution) or 

2. Non-parametric (Kaplan-Meier)? 
Summary Assessment of Reliability

Accuracy measurements

 Test material was applied by laboratory technicians.

 Alternate subjects were enrolled to ensure 

adequate sample size.

 Alternate subjects were enrolled to ensure 

adequate sample size.

 All landings were verified and recorded by a 

research technician. 

 Pre-training of subjects on how to handle 

mosquitoes.

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Summary Assessment of Reliability (Cont.)

Measurement of uncertainty

Mean CPT was calculated across all 10 

subjects/site, and was presented within 95% 

confidence intervals, assuming a non-normal 

Weibull distribution.

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Compliance with Scientific Standards

The following elements are adequately addressed:

 Prerequisite acute toxicity research to characterize 

toxicological profile of the formulation and calculate 

margin of exposure (MOE)

 Dosimetry

 Experimental design 

 Verification of subject attractiveness to mosquitoes

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Conclusion

 The study is scientificallyacceptable

 The data provides scientifically reliable 

information because it satisfies the following 

scientific criteria:

 It produced important information that cannot be 

obtained except by research with human subjects

 It has a defined scientific objective

 The study design (subject selection, sample size, 

dosing, QA/QC, etc.) generated adequate data to 

meet the test objective

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EPA Ethics Review of 

Carroll-Loye Biological Research 

Completed Study ‘No Mas 003’

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Kelly Sherman

Office of Pesticide Programs
Recruiting

• Recruiting process outlined in the 

protocol was followed

 32 subjects were selected randomly from a pool 

of 92 subjects

 10 subjects participated in the dosimetry phase

 22 subjects participated in the field testing at 

one or both sites

 6 subjects were enrolled as alternates

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Consent Process

 Per protocol, subjects were provided with the 

MSD sheet, study synopsis, consent form, 

California Experimental Bill of Rights, and 

other study-related information

 Subjects signed the consent form and the CA 

Experimental Bill of Rights prior to enrollment

 No reported or unreported deviations related 

to the consent process

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Subject Demographics

 Dosimetry phase:

 5 males, 5 females

 Field testing, at each site:

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 Treated subjects: 5 males, 5 females

 Untreated controls: 1 male, 1 female

 All subjects over the age of 18

 All female subjects were tested for pregnancy 

prior to participation
Monitoring

 Research was conducted without 

incident

 No subjects withdrew from the 

research

 No adverse events or incidents of 

concern were reported

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No Mas 003: Protocol Review

 Protocol was approved by IIRB, Inc. and submitted 

to EPA in July 2010

 EPA’s science and ethics review found the protocol 

acceptable with minor changes

Reviewed by the HSRB on 10/27/10

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

 HSRB concurred with EPA, recommended minor 

revisions

 Amendment 1 dated 11/15/10, addressed most EPA 

and HSRB comments; approved by IIRB 11/16/10

 Amended protocol was approved by CDPR on 

3/21/11
No Mas 003: Amendment 1

 Provided additional justification for the chosen 

sample size and discussion of data analysis 

approach

Adjusts wording in the protocol and consent 

29



form per recommendations from EPA, HSRB, 

and CDPR

 Excludes as permissible subjects employees of 

the sponsor or researchers
Responsiveness to Previous Ethics Reviews

 Most of EPA’s/HSRB’s comments from the protocol review 

were addressed in Amendment 1:

 Employees of the sponsor were excluded as permissible subjects

 Symptoms of heat stress and equine encephalitis were added to 

the consent form

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 Most drafting recommendations were incorporated

 Two HSRB comments were not addressed:

 The acronym PMD was not spelled out when first used

 “Child/Minor” was not added to the list of exclusion criteria, 

although the inclusion criteria specify that subjects must be 

between 18-55 years old

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Protocol Deviation

 One deviation: reformatting of dosimetry 

data form

 This deviation did not affect the rights or 

31

safety of the subjects, or compromise 

informed consent

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Completeness of Submission

 The primary study report, MRID 48577201, is 

complete

 All requirements of 40 CFR §26.1303 for 

documentation of ethical conduct are satisfied

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Substantive Acceptance Standards

 40 CFR §26.1703

 Prohibits reliance on data involving intentional exposure of 

pregnant or nursing women or of children

 40 CFR §26.1705

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 Prohibits reliance on data unless EPA has adequate 

information to determine substantial compliance with 

subparts A through L for 40 CFR 26

 FIFRA §12(a)(2)(P)

 Makes it unlawful to use a pesticide in human tests without 

fully informed, fully voluntary consent
Findings

 No Mas 003 did not involve intentional 

exposure of pregnant or nursing women or of 

children 

‘No Mas 003’ was conducted in substantial 

34



compliance with all applicable requirements of 

40 CFR part 26, subparts K and L

 Subjects were fully informed and participated 

voluntarily

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Conclusion

 If No Mas 003 is determined to be 

scientifically acceptable, I find no barrier in 

law or regulation to EPA’s reliance on it in 

actions under FIFRA

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No Mas 003 Completed Study: 

Charge Questions

1. Is the completed study No Mas 003 sufficiently 

sound, from a scientific perspective, to be used 

to estimate the duration of complete protection 

36

against mosquitoes provided by the tested 

repellent?

2. Does available information support a 

determination that the studies were conducted 

in substantial compliance with 40 CFR part 26, 

subparts K and L?