## EPA Review of the

Carroll-Loye Biological Research Completed Study No Mas 003 Field Efficacy Test of a PMD1

Clara Fuentes Ke lly 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 broad 3 

### 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 AedesEP A Science R

eview of Carroll - Loye Efficacy T

est of NO- MAS 003, MRID 48577201. Unpublished document prepared by Carroll-Loye Biological Re search. 411 p. by Clara Fuentes, Ph.D .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 T est of Mosquito Repellency for p- menthane- 3,8-diol (PMD) and Lemongrass Oil - Based R epellent ‘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
7

### 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 9

### 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 . 10**Dosimetry Endpoint and Standard Consumer Dose** 

**Dosimetry Endpoint: Standard Consumer Dose** 

Arms = 1.14 mg/cm 2 Legs = 0.99 mg/cm 2 **Standard consumer dose used for testing efficacy;** 

-Arms = 1.14 mg/cm 2 (0.9524 kg/L) =1.20 μl/cm 2

- Legs = 0.99 mg/cm 2 (0.9524 kg/L) =1.04 μl/cm 2

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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 13**Results from Field Test:**

**Time Distribution of CPT by Site**  **Sites Subjects**  **CPT**

**(hrs) CLIBe**  **(Y es or**  **No?) Number**  **of LIBe**  125 11 .17 Y 2 106 10.85 Y 2 **Sites Subjects**  **CPT (hrs)**  **CLIBe (Y**  **es or No?)**  **Numbe**

**r o f**  **LIBe** 4 9.25 N 0 81 9.17 N 0

14 Site 1 106 10.85 Y 2

28 10.47 Y 2 11 8 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**  **To tal** 

**collected To** **tal collected**  **by**

**subject** Aedes melanimon 52 **2 Control**  **subjects 10** 

**Tr eated**  **subjects** 45 7 **Mosquitoes collected**  **at SITE 2**  **Mosquito species**  **To tal** 

**collected To** **tal collected**  **by subject** Aedes melanimon 76 **2 Control**  **subjects 10** 

**Tr eated**  **subjects** 68 7 Aedes ve xans 5 5 0

15 45 7 Aedes ve xans 26 18 8 **To tal**  **number of**  **all species** 78 63 15 Aedes ve xans 5 5 0 Aedes nigromaculis 1 0 1 Cule x tarsalis 4 4 0 Anopheles freeborni 2 2 0 **To tal**  **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**  **9 5%**  **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. Pa ra metric (with Weibull distribution) or

18 1. Pa ra metric (with Weibull distribution) or 2. Non - parametric (K

aplan - 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 

### . 19

## 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 22

## EPA Ethics Review of Carroll-Loye Biological Research Completed Study ‘No Mas 003’

23

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 24

## 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

28   

### 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 30

## 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 34

## 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

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### 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?