UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON , D. C. 20460

OFFICE OF CHEMICAL SAFETY ANO POLLUTION PREVENTION

MEMORANDUM

DATE: September 26, 2017

SUBJECT: Review of Product Characterization and Human Health Data for the microbial End­ Use product, Vectorite with CR-7, containing the active ingredient, Clonostachys rosea strain CR-7 (EPA File Symbol: 90641-E) in support for a Section 3 Registration submitted by Bee Vectoring Technology, Inc. [Decision No.: 520718; DP Barcode: 436549; Submission No: 9908.5~0] ~

FROM: Jennifer Wingeart, M.S., Biologist 1/2.-::;,/J 1- Pesticides Branch, Biopesticides and Pollution Prevention Division (751 lP)

THROUGH: John L. Kough, Ph.D., Senior Scientist Microbial Pesticides Branch, Biopestic des Pollution Prevention Division (751 lP)

TO: Nicola Steinmetz, Regulatory Action Leader Microbial Pesticides Branch, Biopesticides and Pollution Prevention Division (751 IP)

ACTION REQUESTED: To review product characterization and human health data submitted by Bee Vectoring Technology, Inc. (hereafter, "BVT") in support for a Sec. 3 Registration of an end-use (EP) fungicide Vectorite with CR-7 (hereafter, "Vectorite"), containing the active ingredient, Clonostachys rosea strain CR-7 (hereafter, "C. rosea'').

CONCLUSION:

The product characterization, and human health data submitted by BVT in support of Sec. 3 Registration for the new end-use product Vectorite with CR-7 is for use as a fungicide intended for agricultural food­ use purposes, with product delivery via bee vectoring is Acceptable. Vectorite contains the active ingredient C. rosea strain CR-7 which confers resistance to various Botrytis sp. and similar plant diseases within plant tissue.

CLASSIFICATION: ACCEPTABLE, provided BVT amends the label to account for the following:

*THIS REPORT DOES NOT CONTAIN FIFRA CONFIDENTI AL BUSINESS INFORMAT ION* 1. Acute Inhalation Toxicity Study (870.1300) - BVT submitted an incomplete study to support a Toxicity Category JV due to the rationale of the test material not being respirable at a concentration of 2.43mg/L with a Mass Median Aerodynamic Diameter (MMAD) of 9µm in rats. A lack of data to support a Tox. Cat. lV and lack of adequate rationale were provided. The Acute Inhalation Toxicity Category should be a category III and the label contain the Tox. Cat. III corresponding Worker Protection Standard (WPS) language. 2. BVT has requested an exemption from the Restricted Entry Interval (REI) requirement per 40 CFR § 156.208. EPA agrees that an REI in an open-system (field application via bee vectoring) is impractical. However, BVT intends on the same label for greenhouse (closed-system) agricultural food-uses as well, where adequate rationale on the toxicity of the microbe to humans in a closed-system was not provided. Given the low toxicity and lack of exposure, the zero-hour REI request for all applications is justified, provided standard WPS language that is consistent with a Toxicity Category III, is contained on the label and/or sub-labels for Acute Inhalation.

RECOMMENDATION: The submitted product characterization, and human health data are sufficient to support BVT's application for the Sec. 3 Registration of the new end-use product, Vectorite with CR-7, containing the active ingredient, Clonostachys rosea strain CR-7, pending the final printed label language as suggested above.

DATA REVIEW RECORD:

Active Ingredient: Clonastachys rosea strain CR-7 (5% TGAI) Product Name: CR-7 Vectorite Company Name: Bee Vectoring Technology ID No: 90641 Decision Number: 520718 DP Barcode: 436549 Submission Numbers: 990850

I. BACKGROUND

BVT is seeking a FIFRA Sec. 3(c)(5) Registration for the new end-use product, Vectorite with CR-7, containing the active ingredient C. rosea CR-7 for use as a fungicide intended for agricultural food-use purposes, with the product delivery via bee vectoring. C. rosea strain CR-7 confers resistance to various Botrytis sp. and simi lar plant diseases within plant tissue. Clonostachys rosea strain CR-7 is a non­ pathogenic fungal endophyte that controls Botrytis species and other similar diseases in plant tissues and induces plant natural resistance. C. rosea is formerly known in the literature as Gliocladium roseum (Schroers et al., 1999). It is proposed for application only via bee vectoring to labeled crops including strawberries, sun flowers, and canola. No foliar applications are proposed.

C. rosea is a species of in the family . It colonizes living plants as an endophyte, utilizes dead fungal material in soil as a saprophyte and is also known as a parasite of other fungi and of nematodes. The ubiquitous fungus has been isolated from cultivated grassland, woodland/forest soil detritus, and marine/coastal soils; found particularly in soil with alkaline pH (Toledo, et al., 2005). This particular strain was isolated by BVT from the root of a wheat plant in Canada and is deposited in a

Page 2 of 6 Canadian collection bank. C. rosea is widely used as a biological control due to its history of safe use for food crops and wide geographical distribution in soil. As mentioned previously, the mode of action is a beneficial endophyte that is used to suppress spore production of Botrytis sp. and other similar plant diseases in plant ti ssues and is used to enhance the plant's natural resistance. Hyphae have been found to coil around, penetrate and grow inside the hyphae and conidia of Botrytis sp. (Zhang et al, 2008). The pest host range includes: Alternaria, Botrytis, Colletotrichum (Anthracnose), Fusarium, Pythium, Rhizoctoma, Rhizopus, Rhomopsis, Sclerotinia.

The Agency has previously registered a different strain, Clonostachys rosea Strain 321 U, for use to control blue stain and mold on freshly cut lumber and logs in hardwoods and softwoods (EPA, 2014). To support the Sec. 3 registration of the new product, Vectorite with CR-7, product characterization, and human health data have been submitted by BVT and are summarized in this memorandum.

II. Summary of Data Submitted:

STUDY TITLE STUDY SUMMARY MRTD N!!JI

Product Chemistry for The purpose of this study was to provide product chemistry to adequately 49949301 Vectorite with CR-7 characterize the new end-use product Vectorite with CR-7, including; (885.1100) Product Identity and Composition, (885 .1 200) Manufacturing Process, (885. 1300) Discussion on Impurities, (885.1400) Preliminary Analysis, (885.1500) Certified Limits, and (830.6302- 830.7300) Physical/Chemical Properties. The aforementioned data is considered as Confidential Business Information (CBI), and is contained within the associated MRlD listed.

CLASSIFICATION: ACCEPTABLE Vectorite with CR-7: Test substance CR-7 on Vectorite was evaluated for acute oral toxicity 49949302 Acute Oral Toxicity potential in female albino rats when administered as a gavage dose at 5000 mg/kg. The study was terminated following stopping rules of this procedure. No mortality occurred during the study. There were no clinical signs of toxicity during the study. Animals exhibited weekly weight gain during the study. Gross necropsy conducted at terminal sacrifice revealed no observable abnormalities. The test substance acute oral LD50, indicated by the data, was determined to be greater than 5000 mg/kg.

CLASSIFICATION: ACCEPTABLE. Toxicity category rv. Vectorite with CR-7: Test substance CR-7 on Vectorite was evaluated for dermal toxicity potential 49994303 Acute Dermal Toxicity and relative skin irritancy when a single dose at 5050 mg/kg, moistened with deionized (DI) water, was applied to the intact skin of albino rats. No mortality occurred during the study. There were no clinical signs of toxicity or signs of dermal irritation at any time throughout the study. Animals exhibited weekly weight gain during the study. Gross necropsy conducted at study termination revealed no observable abnormalities. The test substance LD50 was determined to be greater than 5050 mg/kg.

CLASSIFICATION: ACCEPTABLE. Toxicity Category IV.

Page 3 of 6 I STUDY TITLE I STUDY SUMMARY I MRID NO. I 49949304 Vectorite with CR-7: Test substance CR-7 on Vectorite was to be evaluated for acute inhalation Acute Inhalation toxicity potential in albino rats. A pre-study trial assay was conducted to Toxicity determine if aerosolizing the test substance into the exposure chamber would produce an acceptable concentration of at least 2 mg/L with mass median aerodynamic diameter (MMAD) between I and 4 µm. An aerosol was generated using a Venturi Aspirator connected to a 500 L exposure chamber. The attempt resulted in an exposure concentration of2.43 mg/L, but with MMAD of9 µm. This level would not provide meaningful acute inhalation toxicity data. BVT submitted rationale as to why further testing was not needed due to the determination of 'no toxicity' when the pulmonary test was conducted and has requested a Toxicity Category IV. The Agency has deemed the rationale acceptable with the stipulation of a Toxicity Category III assignment and has required the proper labeling to correspond with the updated category.

CLASSIFICATION: ACCEPT ABLE Toxicity category 111. Vectorite with CR-7: An acute eye irritation study was conducted on three albino rabbits using test 49949305 Acute Eye Irritation substance CR-7 on Vectorite. Test substance, 100 mg, was placed into the conjunctiva I sac of the right eye of each animal selected for testing. All treated eyes were washed with room temperature deionized (DI) water for one minute after recording the 24-hour observation. There were no positive effects exhibited in any eyes at 24 hours after treatment. The test substance is rated minimally irritating and assigned Toxicity Category IV.

CLASSIFICATION: ACCE PTABLE. Toxicity category TV. Vectorite with CR-7: A primary dermal irritation study was conducted on three albino rabbits using 49949306 Acute Dermal Irritation test substance CR-7 on Vectorite. There was one intact test site per animal. in Rabbits Each test site was treated with 500 mg oftest substance moistened with deionized (DI) water and covered with semi-permeable dressing. Test substance was maintained in contact with the skin for 4 hours. Observations for dermal irritation and defects were made at - 1, 24, 48 and 72 hours after unwrapping. Based on 0.0 PH {Primary Irritation Index), the test substance is rated non-irritating. Based on scores at only 72-hour observations, the test substance is assigned Toxicity Category IV.

CLASSIFfCATION: ACCEPTABLE. Toxicity category IV. Vectorite with CR-7: The purpose of this study was to determine the amount of active ingredient in 49949307 Pre and Post Toxicity Colony Forming Units (CFU) per gram of the test substance, CR-7 on Analysis of Active Vectorite, for pre- and post-dosing of toxicological studies, and to generate a Ingredient for a Certificate of Analysis from the results. Quantification on Potato Dextrose Microbial Pesticide Agar (PDA) plus streptomycin with incubation at 23°C was used to obtain the Control Agent CFU/g. The active ingredient content of CR-7 on Vectorite from plating on 19 (OCSPP 885.1400) Feb 20 16 was 7.3 x I 07 CFU/g and the active ingredient content from plating on 04 Apr 2016 was 5.9 x I 07 CFU/g. Based on these results, the active ingredient present in CR-7 on Vectorite was determined to be stable throughout the duration of the study.

CLASSIFICATION: ACCEPTABLE Supplemental Product BVT was issued a deficiency later by the Agency, indicating the original 50137801 Chemistry for CR-7 Acute Inhalation study was unacceptable and required to either repeat the Technical study, or submit adequate rationale as to why no further testing was needed. BVT submitted adequate rationale to satisfy this data requirement as a Toxivity Category I II, and not IV as requested by the registrant. In the original study, the test substance was not ground to a finer powder due to the potential

Page 4 of 6 I STUDY TITLE I STUDY SUMMARY I MRIDNO. I of deactivating the microbe (active ingredient). As a result, the final test substance was not respirable to the test subjects, as it was too large. BVT submitted adequate rationale and bridging study from the Acute Pulmonary/Pathogenicity Toxicity study that showed no abnormal clinical observations and was deemed not toxic to mammals. The bridging study is summarized as follows:

An Acute Pulmonary Toxicity / Pathogenicity study conducted to evaluate the potential effects of a single high dose pulmonary exposure to pure active ingredient Clonostachys rosea strain CR-7 indicated the test material is low in toxicity following acute exposure to the respiratory system (Doig, 2016). In this study, 36 male and 36 female rats were separated into 4 groups: Group I, untreated, housed in a separate room (6/sex), Group 11, shelf controls, housed with Group IV (4/sex), Group 111, inactive test material (8/sex), and Group IV, test material, treated ( 18/sex). Treated rats received by tracheal injection, a single dose of 0.3 ml Clonostachys rosea strain CR-7 with counts of 3.1 x I0 8 CFU Im I, or 9 .3 x I0 7 CFU/rat. Rats were observed often on the day of dosing and once daily thereafter for 21 days. Interim sacrifices of treated rats were conducted on days 0, 3, 7, 14, and 21 , and blood and tissue samples were collected from these subjects at each sacrifice and cultured to quantify the clearance pattern of the microbe. There were no abnormal health observations and no abnormalities identified during necropsy. The test organism was not detected in any tissues at any time point, and was considered cleared from all tissues and blood by day 21. The test substance Clonostachys rosea strain CR- 7 was determined to be non-toxic following tracheal injection exposure in a single dose of9.3 x 107 CFU/rat.

The inert ingredients in Vectorite with CR-7 are low in acute inhalation toxicity. Additionally, all inert ingredients in Vectorite with CR-7 are low in acute inhalation toxicity, and all are exempt from the requirement of a tolerance.

The aforementioned data fulfills the data requirement (870.1300) and includes the following: I) Clonostachys rosea strain CR-7 administered to the rat respiratory system by pulmonary injection caused no toxicity or abnormalities, 2) the inert ingredients are low in acute inhalation toxicity, 3) the mass median aerodynamic diameter (MMAD) of the aerosolized powder EP fomrnlation was larger than respirable size, 4) the EP is enclosed in trays and product labeling has inhalation personal protective equipment so inhalation exposure is not expected, and 5) a literature search resulted in no association between C/onostachys rosea and inhalation toxicity (NLM, 2016). Based on the above rationale and bridging study, the Agency has assigned Vectorite with CR-7 a Toxicity Category Ill due to an incomplete Acute Inhalation study and has required the registrant to apply the appropriate toxicity category language on the label to ensure worker/handler protection.

CLASSIFICATION: ACCEPTABLE

Page 5 of 6 REFERENCES

Doig, A 2016. Clonostachys rosea strain CR-7 Technical Acute Pulmonary Toxicity/Pathogenicity Study in Rats. Unpublished report by Stillmeadow, Inc., Laboratory ID 19441-15. June 9, 2016. MRID No. 499492-02.

Schroers H, Samuels G, Seifert K, & Garns W. (1999). Classification of the Mycoparasite Gliocladium roseum in Clonostachys as C. rosea, Its Relationship to ochroleuca, and Notes on Other Gliocladium-like Fungi. Mycologia, 91(2), 365-385.

Hartwell, TA, 2016. Clonostachys rosea strain CR-7 Technical Acute Oral Toxicity (UDP) in Rats. Unpublished report by Stillmeadow, Inc. Laboratory ID 19442-15. May 26, 2016. MRID No. 499492-03.

Toledo, A, Virla, E., Humber, R., Paradell, S. and Lastra, C. (2006). First record of Clonostachys rosea (: ) as an entomopathogenic fungus of Oncometopia tucumana and Sonesimia grossa (Hemiptera: Cicadellidae) in Argentina.Journal of Invertebrate Pathology, 92(1), pp.7-10.

Zhang, L., Yang, J., Niu, Q., Zhao, X., Ye, F., Liang, L. and Zhang, K. 2008. Investigation on the infection mechanism of the fungus Clonostachys rosea against nematodes using the green fluorescent protein. Applied Genetics and Molecular Biotechnology, April 2008, Volume 78, Issue 6, pp 983-990.

EPA, 2014. Biopesticides Registration Action Document for Clonostachys rosea strain 321U; PC Code: 2100. httl)s://www.regulations.gov/#!docketDetail:D=EPA-HQ-OPP-2012-0S07

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