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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY 

AND POLLUTION PREVENTION 

MEMORANDUM MAR18 2013 

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

Agency in its review of two products: NoFly Technical and NoFly WP 

containing the new AI Paecilomyces fumosoroseus strain FE 9901. 

FROM: Ibrahim S. Barsoum, Ph.D., Microbiologist, J1~.f-~ 3{t~f ~D Microbial Pesticides Branch, 

Biopesticides and Pollution Prevention Division 

THROUGH: John L. Kough, Ph.D., Senior Scientist ct a 1 

Microbial Pesticides Branch, 

TO: 

Biopesticides and Pollution Prevention · · ion A 

Ann Sibold, Regulatory Action Leader 

Microbial Pesticides Branch, Biopesticides and 

Pollution Prevention Division (7511 C) 

ACTION REQUESTED: 

To review the Registrant's response (letter dated 01/23/2013) to one of the deficiencies 

found by the Agency (letter dated 05/12/2011) in its review of product chemistry, 

manufacturing process and toxicity studies of two products: NoFly Technical and NoFly 

WP containing the new AI Paecilomycesfumosoroseus strain FE 9901. 

CONCLUSION: ACCEPTABLE 

The registrant has successfully responded to one of the deficiencies concerning the 

demonstration of microbial clearance from body organs in an acute toxicity/infectivity 

study of the product in experimental animals. 

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DATA REVIEW RECORD 

Active Ingredient: 

Product Name: 

Company Name: 

Reg. No: 

Chemical Number: 

Decision Number: 

DP Barcode: 

MRIDNo: 

BACKGROUND: 

Paecilomycesfumosoroseus strain FE 9901 

NoFly Technical, and NoFly Wettable Powder 

Natural Industries Inc., Houston, TX 

73314-A, 733314-T 

115003 

475705 

409640 

490443-01 

Paecilomyces fumosoroseus is a naturally-occurring fungus in most countries of the world and 

occurs in various soil types at very low densities. The Agency has reviewed a submission for 

registration ofNoFly Technical and NoFly WP containing Paeci/omycesfumosoroseus strain FE 

9901 by Natural Industries Inc. and found deficiencies in their application in a letter dated 

11/5/2010. The registrant has responded to one of the deficiencies in a letter dated 01/23/2013 

concerning the demonstration of microbial clearance from body organs in an acute inhalation 

toxicity/pathogenicity study of the product in experimental animals. 

Response to MPCA Clearance from Toxicity/Pathogenicity Studies (MRID # 490443-01) 

Experimental Methods: 

In an acute pulmonary infectivity and toxicity study, groups of young adult ferrets (14/sex) were exposed 

by the intranasal route to the microbial pest control agent (MPCA), Paecilomycesfumosoroseus strain FE 

9901 (100%; I.Oxl09 CFU/g), in 0.05% Tween 80 at a nominal dose of lxl08 CFU (in 0.5 mL) per 

animal. Animals were then observed for up to 21 days. Another group of ferrets ( 4 ferrets/sex) were 

untreated and served as a negative control. 

Table 1. Doses, mortality/animals treated 

Test Test Substance Dose Level Males Females Combined Group 

MPCA Paecilomyces 

1 fumosoroseus strain 1x108 CPU/animal 0/14 0/14 0/28 

FE 9901 

2 None Untreated 012 012 014 

Sample preparation: The test suspension was prepared by suspending 3 g of MPCA 

Paecilomycesfumosoroseus strain FE 9901 into 15 mL of 0.05% Tween 80 in sterile water. 

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Solvent/vehicle: The test material was suspended in sterile water (Batch no. 03B17 01; expired in 

February 2006; Bieffe Medital, Italy) containing 0.05% Tween 80 (Batch No. 306570404; 

expired on July 31, 2008; Lach Ner Ltd. Czech Republic). 

Microbial enumeration: The dosing suspension was enumerated by plating an aliquot (0.1 mL) 

of the diluted dosing suspension (10-5) onto Sabouraud agar in triplicates. Colonies were counted 

after the samples were incubated at 22°C for 48 hours. Lungs from the test animals were 

examined for the presence ofMPCA to estimate clearance after dosing. Lung samples (O.lg) were 

homogenized in 1 mL of saline solution and the homogenate was diluted 10 times. An aliquot 

(0.1 mL) of this suspension was then incorporated into Sabouraud agar in triplicates using 

standard sterile techniques. Spores germinated within 3 days following incubation at 22°C and 

colonies were white (1-3 mm in diameter). After the incubation period, the colonies were counted 

and the concentration of MPCA in CFU/g was evaluated. The presence and/or the enumeration of 

the MPCA from the kidney, brain, liver, spleen, blood, mediastinal lymph node and caecum 

contents were also made. Samples of tissues, organs and body fluids were subjected to screening 

of sterility. Approximately 0.5 g of the samples (kidney, brain, liver, spleen, mediastinal lymph 

node) was homogenized with scissors and inoculated into 5 mL Sabouraud broth then incubated 

at 22°C for 48 h. The definitive count ofMPCA in samples was determined on Sabouraud agar 

after 3 days of incubation at 22°C. All the samples used were also frozen (-20°C) immediately 

after necropsy. In case of the presence ofMPCA during screening sterility the exact 

quantification ofCFU/g can be made. Samples of small intestine contents (approximately 0.05 g) 

and heart blood (0.1 mL) were inoculated directly on Sabouraud agar plates and incubated 3 days 

at 22°C. 

Clinical observations and body weights: The health oftest animals was monitored daily 

throughout the study period. Observations included viability/vitality as well as changes in the skin 

and fur, eyes and mucous membranes, respiratory, circulatory, and autonomic and central nervous 

system, somatomotor activity, and behavioral pattern. Special attention was directed to 

observations of tremors, convulsions, diarrhea, lethargy, salivation, sleep and coma. Body 

weights were measured prior to dosing and necropsy, as well as on a weekly basis during the 

study period. 

Necropsy and organ weights: Interim sacrifices (3/sex) were performed on Days 0, 3, 7, 10, 14 

and 21. On the day of scheduled sacrifice, test animals were weighed and sedated using an 

intramuscular injection of Ketamine (5 mg/kg) and Domitor (0.05 mg/kg) prior to blood 

collection/sacrifice by an intravenous injection of Ketamine (SO mg/kg) and Rometar (0.05 

mg/kg). The necropsy included an examination for gross pathological changes. Microscopic 

examinations of organs were only performed in cases where greater detail was required. 

Duration of study: 21 days. 

RESULTS 

A. MORTALITY There were no mortalities noted during the entire observation period. 

The pulmonary LD50 for males is >9.0 x 107 CFU/animal; females is >9.0 x 107 CFU/animal; 

combined is >9.0 x 107 CFO/animal 

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B. CLINICAL OBSERVATIONS: The study author noted that no symptoms of intoxication 

were observed after a single application of the test material. Cage side observations were partially 

affected by mild anesthetic that was administered to the test animals. Some of the animals also 

reflexively licked their mouths and borders of nostrils with residues of the test material. As a 

result, it is likely that test animals were exposed via the oral route. Within 1 hour of treatment, all 

the animals woke from the mild anesthetic and exhibited normal activities. No changes in the skin 

and fur, eyes and mucous membranes were observed. Respiratory, circulatory, autonomic and 

central nervous systems, somatomotor activity and behavior pattern were normal. No effect 

dependent on sex of animals was evaluated. Slight diarrhea was observed in the case of 3 animals, 

but not longer than one day and without any relation with the test material. 

C. BODY WEIGHT: The study author noted that the body weight of the test animals oscillated or 

increased slightly during the entire observation period. According to the study author, the test 

material had no negative effect on body weight (see Table 2). Please note that the study report did 

not include any bodyweight data for June 7, 2004 (Day 14) and June 14, 2004 (Day 21). 

D. MICROBIAL ENUMERATION: Clearance data are summarized in Table 2. At the 1 hour 

interval, spores ofMPCA were detected in the samples obtained from the middle part of lungs of 

each treated animal.The average concentration was 372 CFU/g of wet lung weight. The MPCA, 

however, was not detected at any of the subsequent intervals. The study author concluded that 

complete clearance occurred after several hours, but definitely less than 3 days. The presence of 

MPCA in tissues, organs, and body fluids was determined at the same intervals as the lungs 

examinations. The MPCA was not detected in the kidneys, brains, livers, spleens, blood, 

mediastinal lymph nodes, or caecum contents of treated animals. 

Table 2. Clearance in Lungs 

Animal Time Dilution Count Per Plate Mean Standard Mean 

No. point 1 2 3 Deviation CFU/g 

1 2 3 5 3.3 1.5 330 

2 3 1 4 2.7 1.5 270 

3 1 0 2 1.0 1.0 100 

4 2 2 3 2.3 0.6 230 

5 Afterl 10-2 6 9 5 6.7 2.1 670 

6 hour 4 8 7 6.3 2.1 630 

29 0 0 0 0 0 0 (control) 

30 0 0 0 0 0 0 (control) 

7 0 0 0 0 0 0 

8 0 0 0 0 0 0 

9 After 3 10-2 0 0 0 0 0 0 

10 days 0 0 0 0 0 0 

11 0 0 0 0 0 0 

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Animal Time Dilution Count Per Plate Mean Standard Mean 

No. point 1 2 3 Deviation CFU/g 

12 0 0 0 0 0 0 

13 0 0 0 0 0 0 

14 0 0 0 0 0 0 

15 After 10 10·2 0 0 0 0 0 0 

16 days 0 0 0 0 0 0 

17 0 0 0 0 0 0 

18 0 0 0 0 0 0 

19 0 0 0 0 0 0 

20 0 0 0 0 0 0 

21 0 0 0 0 0 0 

22 0 0 0 0 0 0 

23 0 0 0 0 0 0 

24 0 0 0 0 0 0 

25 After 21 10·2 0 0 0 0 0 0 

26 days 0 0 0 0 0 0 

27 0 0 0 0 0 0 

28 0 0 0 0 0 0 

31 0 0 0 0 0 0 (control) 

32 0 0 0 0 0 0 (control) 

Spores ofMPCA (average 372 CFU/g) were detected in the lung samples of animals after 1 hr. 

of treatment but no spores were detected at subsequent intervals. No CFU was detected in any 

samples of kidney, brain, liver, spleen, blood, mediational lymph node, caecum contents. MPCA 

proved no infectivity and minimal persistence (max. 3 days) in the animals' organs. 

Reviewers' remarks: Plate count numbers at 102 dilutions are small (0-9 CFUs). Plate counts 

should have been done at 101 dilutions. 

CONCLUSION: ACCEPTABLE 

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