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UNITED STATES ENVIRONMENTAL PROTECTIONAGENCY WASHINGTON, D.C. 20460
OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



### MEMORANDUM 

# MAR18 2013

SUBJECT: Review ofthe Registrant's response to the data deficiencies found bythe Agency in its review oftwo products: NoFly Technical and NoFly WP containingthe newAI

### Paecilomyces fumosoroseus strainFE 9901.


### FROM: Ibrahim S. Barsoum, Ph.D., Microbiologist,

# J1~.f-~**3{t~f ~D**  Microbial Pesticides Branch, Biopesticides and Pollution PreventionDivision


### THROUGH: John L. 

### Kough, Ph.D., Senior Scientist

# ct a

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### Microbial Pesticides Branch,



### TO: Biopesticides and Pollution Prevention · · ion A
Ann 

### Sibold, Regulatory Action Leader Microbial Pesticides Branch, Biopesticides and PollutionPreventionDivision (7511C)



### ACTION REQUESTED:

To reviewthe Registrant's response (letter dated 01/23/2013) to one ofthe deficiencies found bythe Agency (letter dated 05/12/2011) in its review ofproductchemistry, manufacturing process and toxicity studies oftwo products: NoFly Technical and NoFly WP containing the newAI 

### Paecilomycesfumosoroseus strain FE9901.



### CONCLUSION: ACCEPTABLE
The registrant has successfully responded to one ofthe deficiencies concerningthe demonstration ofmicrobial clearance from body organs in an acute toxicity/infectivity study ofthe product in experimental animals.



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DATA REVIEWRECORD
Active Ingredient: ProductName: CompanyName: Reg. No: ChemicalNumber: DecisionNumber: DP Barcode: MRIDNo:


### BACKGROUND: Paecilomycesfumosoroseus strainFE 9901 NoFly Technical, andNoFly Wettable Powder Natural Industries Inc., Houston, TX 73314-A, 733314-T 115003 475705 409640 490443-01



### Paecilomyces fumosoroseus is a naturally-occurring fungus inmost countries ofthe world and
occurs in various soil types atvery low densities. The Agencyhas reviewed a submissionfor registration ofNoFlyTechnical andNoFly WP containing

### Paeci/omycesfumosoroseus strain FE 9901byNatural Industries Inc. and found deficiencies intheir application in a letter dated 11/5/2010. The registrant has responded to one ofthe deficiencies in a letter dated 01/23/2013 concerningthe demonstration ofmicrobial clearance from body organs in an acute inhalation toxicity/pathogenicity study ofthe product inexperimental animals. 

### Response to MPCA Clearancefrom Toxicity/Pathogenicity Studies (MRID # 490443-01) ExperimentalMethods: In an acute pulmonary infectivity and toxicity study, groups ofyoung adult ferrets (14/sex) were exposed bythe 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 oflxl08 CFU (in 0.5 mL) per animal. Animals were then observed for up to 21 days. Another group offerrets ( 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: Thetest suspension was prepared by suspending 

### 3 g ofMPCA 

### Paecilomycesfumosoroseus strain FE 9901 into 15 mL of0.05% Tween 80 in sterile water.



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Solvent/vehicle: Thetest 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 NerLtd. Czech Republic).

Microbial enumeration: The dosing suspension was enumerated by plating an aliquot (0.1 mL) ofthe diluted dosing suspension (10-5) onto Sabouraud agar in triplicates. Colonies were counted afterthe samples were incubated at 22°C for 48 hours. Lungs from the test animals were examined for the presence ofMPCAto estimate clearance after dosing. Lung samples (O.lg) were homogenized in 1mL ofsaline solution andthe homogenate was diluted 10 times. An aliquot (0.1 mL) ofthis 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). Afterthe incubation period, the colonies were counted and the concentration ofMPCA in CFU/g was evaluated. The presence and/orthe enumeration of the MPCA from the kidney, brain, liver, spleen, blood, mediastinal lymph node and caecum contents were also made. Samples oftissues, organs and body fluids were subjected to screening ofsterility. Approximately 0.5 g ofthe 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 ofincubation at 22°C. All the samples used were also frozen (-20°C) immediately after necropsy. In case ofthe presence ofMPCA during screening sterility the exact quantification ofCFU/gcan be made. Samples ofsmall 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 bodyweights: The health oftestanimals 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 oftremors, convulsions, diarrhea, lethargy, salivation, sleep and coma. Body weights were measured priorto dosing and necropsy, as well as on a weekly basis duringthe study period. Necropsy and organ weights: Interim sacrifices (3/sex) were performed on Days 0, 3, 7, 10, 14 and 21. On the day ofscheduled sacrifice, test animals were weighed and sedated using an intramuscular injection ofKetamine (5 mg/kg) and Domitor (0.05 mg/kg) priorto blood collection/sacrifice by an intravenous injection ofKetamine (SO mg/kg) and Rometar (0.05 mg/kg). The necropsy included an examination for gross pathological changes. Microscopic examinations oforgans were only performed in cases where greater detail was required.

Duration ofstudy: 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. CLINICALOBSERVATIONS: The study author notedthat no symptoms ofintoxication
were observed after a single application ofthetest material. Cage side observations were partially affected by mild anesthetic that was administered tothetest animals. Some ofthe animals also reflexively licked their mouths and borders ofnostrils with residues ofthe test material. As a result, it is likely thattest animals were exposed viathe oral route. Within 1houroftreatment, 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 behaviorpattern were normal. No effect dependent on sex ofanimals was evaluated. Slight diarrhea was observed in the case of3 animals, but not longer than one day and without any relation withthe test material.
C. BODYWEIGHT: The study author noted thatthe body weight ofthe test animals oscillated or increased slightly duringthe 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 studyreport 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. Atthe 1hour interval, spores ofMPCAwere detected in the samples obtained from the middle partoflungs of each treated animal.The average concentration was 372 CFU/g ofwet lung weight. The MPCA, however, was not detected at any ofthe 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 oftreated animals.
Table 2. Clearance in Lungs



### Animal Time Dilution Count PerPlate 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 CountPerPlate 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 ofanimals after 1hr. 

### oftreatmentbutno spores were detected at subsequent intervals. No CFU was detected in any samples ofkidney, brain, liver, spleen, blood, mediational lymph node, caecum contents. MPCA proved no infectivity and minimal persistence (max. 3 days) inthe animals' organs.
Reviewers' remarks: 

### Plate countnumbers at 

### 102

### dilutions are small (0-9 CFUs). Plate counts shouldhave been done at 

### 10 1

### dilutions.
CONCLUSION: ACCEPTABLE

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