UNITED ST A TES ENVIRONMENT AL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

JUL 0 3 2014 

OFFICE OF CHEMICAL SAFETY 

AND POLLUTION PREVENTION 

MEMORANDUM 

SUBJECT: Review of the Registrant's response to data deficiencies fo und by the 

Agency in its review of Product Chemistry, and Toxicity Studies for the 

registration of two products: NoFly Technical and NoFly WP containing 

the new AI Isaria fumosoroseus strain FE 9901. 

FROM: Ibrahim S. Barsoum, Ph.D., Microbiologist, 11~~~ b / 25 / l )l 

Microbial Pesticides Branch, 

THROUGH: 

Biopesticides and Pollution Prevention Division ~ 

John L. Kough, Ph.D., Senior Scienti s~ v 

TO: 

Microbial Pesticides Branch, 

Biopesticides and Pollution Prevention ision A 

Jeannine Kausch, Regulatory Action Leader 

Microbial Pesticides Branch, Biopesticides and 

Pollution Prevention Division (75 11 C) 

ACTION REQUESTED: 

To review the Registrant's response (letters dated 7/22/20 13 & 5/16/20 14) to the 

defici encies fo und by the Agency in its review of Product Chemistry, and Toxicity 

Studies oftwo products: NoFly Technical (EPA Reg. no. 733 14-7) and NoFly WP (EPA 

Reg. no. 73314-6) containing the new AI Jsaria fumosoroseus strain FE 990 1. 

CONCLUSION: ACCEPTABLE 

The registrant has successfully responded to the following deficiencies: 

1. Demonstrate that there is no significant toxic metabolites produced by the TGAI. 

2. Change the genus name of the active ingredient from Paecilomyces to Isaria. 

3. Submit Storage Stability & Corrosion Characteristics studies of the EP. 

CONTAINS FIFRA CONFIDENTIAL BUSINESS INFORMATION 
DAT A REVIEW RECORD 

Active Ingredient: 

Product Name: 

Company Name: 

Reg. No: 

Chemi cal Number: 

Decision Number: 

DP Barcode: 

MRJD No: 

BACKGROUND: 

lsaria fumosoroseus strain FE 9901 

NoFly Technical, and NoFly Wettable Powder 

Novozymes BioAg, Inc., Brookfield, WI 

733 14-6 7333 14-7 

11 5003 

481554, 48 1555, 481 557 

41761 2, 42040 1, 420406 

49118301 , 49378401 

lsariafim10soroseus is a naturally-occurring fungus in most countries of the world and occurs in 

various soil types at very low densities. Its target organisms are the whiteflies. In its natural 

environment, I. fumosoroseus strain FE 990 I is dispersed as conidia or blastospores, either by air 

or water movement or by other insects and/or mites. In an earlier study (MRID No. 4829 12-01 ), 

extracG of biomass and culture filtrate derived from still culture and stirred tank reactor (STR) 

fermentations of Jsaria fumosoroseus strain FE 990 I were analyzed to detect the presence of 

secondary metabolites and to eva luate their cytotoxicity on human lung cancer (Hep-2) and 

canine kidney (MOCK) cell lines. The analysis by HPLC combined with chromatography 

revealed the presence of beauverolides Mand I in the extracts from still cultures with no 

significant levels of these compounds detected in the STR extracts. The growth inhibition assay 

on cell lines revealed a dose dependent cytotoxicity in the extracts from still culture that is 6 -

!OX higher than in stirred cultures, and no antiproliferative activity was detected in any of the 

extracts. The authors deduced from these studies that fermentation of Jsariajitmosoroseus strain 

FE 990 I in stirred tank reactors does not produce unintentional compound of toxic concern. The 

Agency, however, concluded that there is a potential for the presence of toxic metabolites in the 

technical product, even though at a lower level than in still cultures; and as such, the Agency 

cannot suppo11 use of the product on food crops. To prove the absence of toxic metabolites, the 

registrant did fi ve batches analysis on the technical product, manufactured at the actual 

production site, and demonstrated that there are no significant toxic metabolites produced. Also, 

in the original data submitted, a viable count test (CFU/g) of the fo1mulation showed a 10-fold 

reduction over the storage period of six months set at 4°C (± 2 °C). There was no significant 

difference in the physical test results before and after storage. However, since the one batch of 

EP that was tested was not produced at the proposed manufacturing site, the Agency has asked 

that the registrant submit a study of storage stability and corrosion characteristics of the EP for 

six months at room temperature and at 4°C. 

Registrant's Response to the Deficiencies found by the Agencv in its review of product 

chemistry, and acute toxicity studies of NoFlv Technical and NoFly WP containing the new 

AI Isaria {umosoroseus strain FE 9901 

2 
Deficiency 1: Discussion of formation of unintentional ingredients. 

The Agency is concerned that !saria fumosoroseus strain FE 9901 is capable of producing toxic 

metabolites in fermented cultures used in the manufacture of No Fly Technical and consequently 

cannot issue an exemption from the requirement of a tolerance for residues of Isaria 

fumosoroseus strain FE 9901 for food use. 

Registrant's Response: (MRID # 4911 8301) 

Five batches ofNoFLYTM Technical, containing !sariafumosoroseus strain FE 9901 , were 

evaluated for the presence of beauverolides, beauvericin, and 2, 6-pyridinedicarboxylic acid. 

Each of the metabolites was quantified in the pg/g and low μgig ranges. In this study, the most 

prevalent beauverolide peptide, beauverolide I, was identified along with 14 others in the class 

according to their chromatographic and mass spectral properties. Beauverolide I was determined 

to be present at a range of (Table 1 ). 

Table 1. Beauverolide Metabolite Formation 

No Fly 

Technical BVD-1 Standard Coeff. Of 

Sample (μg/g) Deviation (±) Variation(%) 

Beauverolide I was the predominant beauverolide peptide detected amongst 15 beauverolide 

peptide species. Beauverolide-1 was approximately of the overall peptides combined, and 

100% of the peptides combined are well below 0.1 % of the No FLYTM Technical TGAI; 

therefore, no metabolite formation rises to a level of toxicological concern~ '-

CONCLUSION: ACCEPTABLE 

Deficiency 2: Change the genus name of the active ingredient from Paecilomyces to Isaria. 

The name of the TGAI needs to be updated on the data matrix to reflect the current taxonomy. 

Also, the new CSF and label should reflect the most common taxonomy i.e. the genus name 

should be changed from Paecilomyces to /saria. 

Registrant's Response: 

The registrant has submitted revised data matrix, CSF and label with the genus name changed 

from Paecilomyces to Jsaria. 

CONCLUSION: ACCEPTABLE 

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*Manufacturing process information may be entitled to confidential treatment*
Deficiency 3: Submit Storage Stabilitv & Corrosion Characteristics studies of the EP 

A storage stability study must be conducted using the proposed formulation ofNoFly WP from 

the proposed manufacturing site. 

Registrant's Response: (MRID # 49378401) 

An initial dilution is perfo rmed by diluting 0.1 g of technical grade NoFly into 100 ml sterile 

phosphate buffer or sterile water. The fl ask contents are stir-mixed 300 rpm for 15 minutes in 

order to insure effective spore dispersal. A 1: 10 dilution is performed. A 10 μl aliquot from this 

dilution blank is transferred to a counting chamber (haemocytometer, improved Neubauer, 

Bright-lineTM, Buffalo, NY). Each dilution is examined at 400X (total magnification) with a light 

microscope. Dilution levels are adjusted if necessary to assure that the number of spores per fi eld 

is between 50 and 200. A total of I 0 squares are counted and the mean number of spores counted 

is calculated. The total number of spores per gram is calculated as fo llows: 

N = [M x (2.5 x 105) x dilution factor x reciprocal g/ml (mass/ml in original fl ask)]/number of 

squares counted. 

M = total number of cells in all squares counted (a square is comprised of 16 grids) 

2.5 x I 05 is the reciprocal volume in ml of each square 

Table 2. Viable spores (CFU/g) in NoFly WP up to 6 months at room t em perature and 8°C 

1 month 1 month 3 month 3 month 6 month 6 month 

Lot T=O 

@RT @BC @RT @BC @RT @BC 

538 1.4 x lOlU 5.4 x 10" 9.1 x 10" 1.2 x lOlU 1.7 x lOlU 4.3 x 10~ 1.4 X lQlU 

540 7.7 x 10" 3.B x 10" 1.1 x lOlU 7.3x 10" 1.6 x lOlU 0 1.0 x lOlU 

The data shows that NoFly WP (733 14-6) maintains the lower activity titer of 2 x 109, as noted 

on the approved CSF, for 3 months at room temperature and 6 months at 8°C. Based on the 

above results, the proposed wording on the NoFly WP (733 14-6) label in the Storage and 

Disposal section concerning stability is as follows: "Store for 6 months at 46°F (8°C). Storage at 

room temperature (76°F, 23°C) will reduce the shelf life to 3 months." Product was stored in 

commercial packaging for the duration of the study and was monitored for corrosion or other 

damage throughout the study by visual inspeclion. No corrosion was noted during the duration of 

the study. 

CONCLUSION: ACCEPTABLE 

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