# ROBERT I . ROSE, PH.D. Biotechnology Regulatory Consultant 8322 Sharon Drive Frederick, Maryland 21704 Phone & Fax 301-874-5593 Mobile 301-693-0659 rirose l @juno.com November 5, 2013 Document Processing Desk (AMEND) EPA Office of Pesticide Programs, (7504P) Rm. S-4910, 4th fl., 1 Potomac Yd. 2777 S. Crystal Dr. Arlington, VA 22202-4501 Attn: Ms. Kimberly Nesci, Branch Chief & Ms. Shanaz Bacchus, Regulatory Action Leader Microbial Pesticides Branch (PM-92) Biopesticides 

### & Pollution Prevention Div. (7511 P) Subject: Application to amend 89668-EUP-1 to add San Gabriel Valley Mosqu ito and Vector Control District, West Covina, CA with six experimental-use site-locations and request extension of time for EUP in NY up to September 30,2016 and in KY, CA, and FL for up to October 31, 2015. Product Name and Name of AI: Wolbachia pipientis, ZAP Strain Company: MosquitoMate, Inc., Lexington, KY Dear Ms. Bacchus, This cover letter is part of a MosquitoMate Inc. application to amend 89668-EUP-1 to add San Gabriel Valley Mosquito and Vector Control District, West Covina, CA with six experimental-use site-locations and request extensions of time for this EUP in NY for up to September 30, 2016 and in KY, CA, and FL for up to October 31,2015. San Gabriel Valley Mosquito and Vector Control District of West Covina, CA would like to add 5379 acres to the existing EUP's 2059 acres for a total of 7438 acres surveillance and monitoring for 89668-EUP-1. However, releases of male Aedes albopictus containing Wolbachia pipientis, ZAP Strain are to be done on only about 22 acres, total, of release-sites. Up to 100,000 male Ae. albopictus with W . pipientis, ZAP Strain for control of Ae. albopictus mosquitoes by sterile insect technique (SIT) are planned to be released per week by San Gabriel Valley Mosquito and Vector Control District as a collaborator to this EUP. The extension of time requested for NY for up to September 30,2016 and in KY, CA, and FL for up to October 31, 2015 is requested, because the extra time is needed to allow for a full-season of test-trials, so that control efficacy can be determined for the full-season of expected mosquito occurrence.

### Included with this letter are the following amendment application materials, all

submitted in triplicate via UPS: CONTENTS: 1) Cover/transmittal letter. 2) Application to amend Experimental Use Permit to Ship and Use a Pesticide for Experimental Purposes Only. Amendment, EPA Form 8570- 17. 3) Field Trial Information completed for the sites in San Gabriel Valley Mosquito and Vector Control District, West Covina, CA. The increased scope of 89668-EUP-1 is an improvement intended to better meet the public health pesticide performance data requirements for FIFRA Sec. 3 registration. The application for amendment of 89668-EUP-1 is not accompanied by a registration service fee payment (B621, $1158 with 75% waiver), because ofthe following Agency and FIFRA provisions: "(b) Registrant-initiated fast-track amendments are to be completed within the timelines specified in FIFRA Section 3(c)(3)(B) and are not subject to registration service fees." FIFRA 3(c)(3)(B) " IDENTICAL OR SUBSTANTIALLY SIMILAR ... (II) proposes an amendment to the registration of a registered pesticide that does not require scientific review of data ... if no review time is established, not later than 90 days after receiving a complete application, notify the registrant if the application has been granted or denied;" If you need additional information or have any questions regarding this application to amend 89668-EUP-1 to add one more mosquito control district and for minor time extensions for CA, FL, KY, and NY, please let me know, so that I may respond accordingly and promptly. Sincerely,

Robert I . Rose Regulatory Agent for MosquitoMate, Inc. cc. Drs. Stephen Dobson 

### & Jimmy Mains, MosquitoMate, Inc., 

### & Dr. J. Wakoli Wekesa, San Gabriel Valley Mosquito and Vector Control District



# Page 2 of2

# * U.S. GOVERNMENT PRINTING OFFICE: 1_ - 452-684/25008 Form A roved. OMS No. 2070-0040. 

# I

OPP Identifier Numb.r 

# &EPA United States 

## ENVIRONMENTAL PROTECTION AGENCY

Washington, DC 20460 Office of P.sticid •• Programs 17505C) 

## Application for Experimental Use Permit to Ship and Use a Pesticide for Experimental Purposes Only 1. Typ. of Application

New ~ Amendment (See No.2) 2. Briefly explain (attach a •• par at. sheet if necsssary) Application to amend 89668-EUP-1 to add San Gabriel Valley Mosquito & Vector Control District, West Covina, CA to Wolbachia EUP program. Total acres for monitoring/sampling is 5379 acres in six Covina, CA sites with about 6 acres total for release of up to 100,000 male mosquitoes per week. Also, extension of EUP time is requested to allow a full-mosquito-activity-season of test-trials in NY up to September 30,2016 and in KY, CA, and FL up to October 31 ,2015. 

# D 

# o Extension (Gille Permit Number below) Permit Number 89668-EUP-1 3. Name and Address of Firm/Person to Whom the Experimental Use Parmit is to be Issued !include Zip Code' (Type or Print) MosquitoMate, Inc. 1122 Oak Hill Drive Lexington , KY 40505 4. Name and Addres. of Shipper only if shipment is intended or If different from applicant'. name and address !include Zip Codel I TIIJ» or Print) Same name/address as MosquitoMate, Inc. EPA Company Number 89668 6. Neme of Product Wolbachia pipientis, ZAP Strain 6. Is Product Registered with EPA7



# o lZl No

7. Total Quantity of Product Propo.ed for Shipment/Use 8. Acreege or Area to be Treated Pounds of formulated product _4 ~~_ 7438 acres total for all sampling areas. Male releases/

treatments on - 22 acres only. Pounds of active ingredient 4 Yes (Give Registration Number or File Symbol below) Registration Number 89668-EUP-1



# --------------------- File Symbol 89668-EUP-R

Until October 31 , 2015

This i. to certify that food or feed derived from the experimental program will not be used or offered for consumption or sale for consumption, except by laboratory or experimental animals, if illegal residue. lire pre.ent In or on such food or feed. 10. Places from which Shipped MosquitoMate, Inc.; 1122 Oak Hill Drive; Lexington , KY 40505 and/or Dimmock Bldg.; 1081 Veterans Drive; Lexington, KY 40546. 12. Spllclfy the nllme snd number of the contact person most familiar with thi. application. Robert I. Rose; (301) 874-5593; rirose1@juno.com 8322 Sharon Drive Frederick, MD 21704 9 . Propo •• d Period of Shipment/U.e

1 1. Crop/Site to be Treated Urban and local habitats of the Asian tiger mosquito, Aedes albopictus.

14. Title 15. Date Signed Biotechnology Regulatory Consultant/ Agent 

## I 11 103

/ 2013 

### Certification

I certify that the statements I have made on this form and all attachments thereto are true, accurllte, and complete. I acknowledge that any knowingly fllise or misleading statement may be punishable by fine or imprisonment, or both. under applicable law.

## Field Trial Information Requirements

Complete one form for each cooperator/participant field trial. The information

boxes will expand when completed in Microsoft Word. Participants and cooperators, i.e., persons who grant permission for an experimental use pesticide to be used on application sites they own or control : If participants change, the permit needs to be modified . Name of program participant or cooperator: Dr. J . Wakoli Wekesa Address: San Gabriel Valley Mosquito & Vector Control District 1145 N. Azusa Canyon Road West Covina, CA 91790 Telephone No., e-mail address: wwekesa@sgvmosquito.org Office: (626) 814-9466 Qualifications, Title, Position: J . Wakoli Wakesa, PhD Scientific Program Manager San Gabriel Valley Mosquito & Vector Control District Names of US state(s) or territory (ies) where the product is intended to be used: California, USA Names of state(s) or territory(ies) in which the product may be shipped for further distribution elsewhere: None Test material: What amount or quantity of active ingredient or test material to be used for testing? Approximately 100,000 ZAP strain Aedes albopictus males infected with the Wolbachia pipientis microbial pesticide per week for six months. What is the justification for the amount or quantity of active ingredient or test material proposed to be used, including how it was determined? Based on laboratory research , a 7: 1 ratio of Ae. albopictus males infected with the W. pipientis microbial pesticide to each wild-type Ae. albopictus male is needed to result in a population suppression effect (Fig . 1) . The number of target mosquitoes varies seasonally throughout the year (Fig. 2) . Based upon ongoing monitoring , we estimate a population size of approximately 1 ,500 mosquitoes at each of the field sites. To allow for mortality and potential underestimation of population size, we plan to release up to 100,000 males per week.



### Page 1 of 4~ 

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- Untreated Population

- Wolbachia Biopesticide Treated Population 1800 1 600 1400

. 1200 1 000 800 600 400 

# lli 200 

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~ 500 400 300 200 100 Figure 1. Replicate populations of Ae. albo pi ctus are monitored in a closed laboratory model system . (A) represents two control populations that did not receive W pipientis male releases and one treatment population received repeated releases of W pipi entis infected males. (8) represents one control population that did not receive W pipientis male releases and one treatment population received repeated releases of W pipientis infected males. Each incompatible male release is represented by a shaded vertical bar in (A) and (8) , with the height indicating the release size. All treatment populations were eliminated with repeated W oioientis infected male releases. - Wolbachia atcpesrtctce Treated Population

-===- Untreated Population o . . , ~ ., ~ ...........•.......... -- "':'-"'

--"--'-r-- "- ~""--':

Figure 2. Mean number of Ae. albopi ctus eggs deposited per month in 25 ovi-cups distributed throughout the infestation area in South Elmonte, CA, during the fi rst full year of surveillance after the detection of the invasive soecies. 10 so 100 1 1 0 120

What are the test area acres, hectares, square feet, or square meters? The total combined areas for Monitoring/Sampling is 5379 acres. The total combined areas for release/application is 6 acres. The test areas are located in neighborhoods of EI Monte and South EI Monte, CA. Specifically, -1 acre plots will be identified (with landowner permission) within these neighborhoods. • Residential area Corner of Lambert Ave & Cypress Ave, (1 , 114 acres for Monitoring/Sampling (M/S) . Release/Application (R/A) at 1 acre site) • Residential area Corner of Lambert Ave & La Madera Ave (1 ,574 acres for M/S; 1 acre of R/A) • Residential area Burkett Rd & Elliott Ave. (557 acres for M/S; 1 acre of R/A) • Residential area Maxon Rd & Elliott Ave. (640 acres for M/S; 1 acre of R/A) • Residential area Gage Ave. & Garvey Ave. (501 acres for MIS; 1 acre of RIA) 80 20 30 40 60 70 90 Ti me (Weeks)

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### '-r- '-r- '-rPage 2 of 4• Residential area Sastre Ave. & Whitmore St. (993 acres for MIS; 1 acre of RIA)

Information on the proposed stud : What types of pests or organisms are targeted? Ae. albopictus mosquitoes (the Asian tiger mosquito, an invasive alien species established in the USA) What are the crop(s), animal(s), surface(s), or site(s) to be treated? Male ZAP strain Ae. albopictus mosquitoes infected with the W pipientis microbial pesticide will be released. The male mosquitoes will mate with indigenous wild-type females, causing conditional sterility and resultinq in population decline and potential elimination. The purpose of study is to determine pesticidal value, toxicity, or other properties. What are the objectives and values of this study, including what type(s) of data is/are to be collected? The objectives and values are to determine the pesticidal value of a W pipientis microbial pesticide for the suppression and elimination of an Ae. albopictus mosquito populations. While the results of contained trials are encouraging (Fig. 1), the competitiveness and suppressive effect of the released males must be assessed in the field. The experiment will test for a hypothesized increased frequency of Ae. albopictus eggs failing to hatch (i.e., due to W pipientis induced conditional sterility) and a significant reduction in the Ae. albopictus population size compared to a control site, which is not treated . What is/are the site location(s) where the test material is to be used, i.e. GPS coordinates and/or other details to be able to find the site(s)? The potential sites in EI Monte and South EI Monte, California: • Residential area Corner of Lambert Ave & Cypress Ave, (1,114 acres for MonitoringlSampling (MIS). ReleaselApplication (RIA) at 1 acre site); (34 0 5'1.50" ; 118 0 1'35.81"W) • Residential area Corner of Lambert Ave & La Madera Ave (1,574 acres for MIS; 1 acre of RIA); (340 4'49.89"N; 1180 0'49.42"W) • Residential area Burkett Rd & Elliott Ave. (557 acres for MIS; 1 acre of RIA); (340 3'0.47"N; 1180 1'0.05"W) • Residential area Maxon Rd & Elliott Ave. (640 acres for MIS; 1 acre of RIA); (340 3'17.08"N; 1180 1 '19.43") • Residential area Gage Ave. & Garvey Ave. (501 acres for MIS; 1 acre of RIA); (34 0 3'46. 17"N; 118 0 2'25.28"W) • Residential area Sastre Ave. & Whitmore St. (993 acres for MIS; 1 acre of RIA); (34 0 4'6.50"N, 118°3'12.40"W) What are the intended plot sizes, number of plots, number of replicates, and other relevant test parameters? Releases and mosquito sampling will be performed weekly at the identified -1 acre plots. Ideally, two to three plots will be established for each of the listed neighborhoods. Pending the results and permission, the approach is to be replicated at additional sites. How will the test material be used? What is/are the mode(s) of application and rate(s) of use or application? Up to 100,000 W pipientis-infected male mosquitoes will be released weekly at selected sites. Mosquito populations will be sampled using BG (mosquito) Trap sampling of the adult population and ovicups to sample eggs. BG trap adult collection and ovicup egg collection data from the experimental site will be compared to the control site to examine for an effect of the release of the product on the indigenous mosquito population. 

### Page 3 of 4How will the test be supervised?

J. Wakoli Wakesa will oversee the distribution and monitorin a ulations. What are the proposed time dates and duration for the experiment? Male releases will start during the Summer of 2014. The male releases will continue for approximately six months from the start date. If delayed, these dates may be delayed accordingly. However, a later start will be an experimental complication, due to a potential increase in the tarqeted population size. How will the test material be stored and how will unused test materials and their containers be disposed of at the end of the experiment: Male ZAP mosquitoes will be reared in Lexington, KY within a contained facility and shipped to San Gabriel Valley Mosquito & Vector Control District headquarters in West Covina, CA. Male mosquitoes will be shipped in sealed, insulated shipping containers. San Gabriel Valley Mosquito 

### & Vector Control staff will transport the male mosquitoes to field sites where they will be released immediately. At the conclusion of the experiment all equipment used in the rearing process (e.g., cages and larval pans) will be frozen. For disposal, if males are not to be applied or used, they should be frozen to kill them, then heated to 212 F/100 C, cooled, put in a Ziploc-type PE bag and disposed of with trash and/or garbage.



### Page 4 of 4