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



### MEMORANDUM 

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SUBJECT:

FROM:

THROUGH:

TO: Jointreview ofdata for the registration ofa newTGAI, NoFlyTechnical, and a newend use product formulation, NoFlyWettable Powder, containingthe fungus 

### Paecilomycesfumosoroseus strainFE 9001. 

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## 1rrfo Ibrahim S. Barsoum, Ph.D., Microbiologist 

### ..JkW-- Microbial Pesticides Branch, Biopesticides and PollutionPrevention Division (751 lC) John Kough, Ph.D., Senior Scientist Microbial Pesticides Branch, Biope ·cid s and PollutionPreventionDivision (7511 Kathleen Martin, Regulatory Action Leader Microbial PesticidesBranch, Biopesticides and PollutionPreventionDivision (7511C) 

### ACTION REQUESTED: Ajointreview withPMRA, Canada, ofproduct chemistry, acute toxicity/pathogenicity studies, and waiverrequests theregistration ofa newTGAI, NoFlyTechnical, and a newend use productformulation, NoFly Wettable Powder, containing thefungus 

### Paecilomyces fumosoroseus strain FE 900lsubmittedbyNatural Industries Inc., Houston, TX



### CONCLUSION: UPGRADABLE to ACCEPTABLEpending on the response oftheregistrant to the deficienciesreported in the recommendations.



### CONTAINS CONFIDENTIAL BUSINESS INFORMATION

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DATA REVIEW·RECORD Active Ingredient: Product Name: CompanyName: Reg. No: Chemical Number: Decision Number: DP Barcode: MRIDNo:

BACKGROUND: Paecilomyces fumosoroseus strain FE 900I NoFly Technical, and NoFly Wettable Powder Natural Industries Inc., Houston, TX 73314-T l I5003 4I6429 38I252 47719-01, -02 Product Chemistry & Storage Stability ofNOFLY 

### WP 477I9-03

- 08 Acute Toxicity & Sensitization Studies ofNOFLY WP 477920-0I Product Chemistry ofNOFLY Technical 477920-02

- 07 Acute Toxicity & Waiver requests ofNOFLY Technical

Paecilomyces fumosoroseus is a naturally-occurring fungus in most countries ofthe world and occurs in various soil types at very low densities. The fungus has frequently been isolated from lepidopteran cadavers in Europe. Isolates occur in the Netherlands, Ireland, and Spain. The fungus has also been found invarious non-European countries such as China, Japan, Ghana, Pakistan, India, and the USA. Target Organisms: NoFly WP is intended to control all stages ofwhitefly (eggs, NI,N2, N3 and N4 larvae, and adults). The ability to infect whitefly eggs is unique among entomopathogenic fungi. The target organisms are the whiteflies Trialeurodes vaporariorum, Bemisisa tabaci, Bemisia argentifolii, and Lecanoideus floccissimus. In its natural environment, 

### P. fumosoroseus strain FE 990I is dispersed as conidia orblastospores, either by air orwater movement or by other insects and/or mites. The mode ofinfection ofthe various entomopathogenic fungi is basically similar where a typical infection cycle proceeds as such: conidial attachment, germination, penetration, vegetative growth, and then conidiogenesis. The infection cycle ofP. fumosoroseus strain FE 990I is particularly rapid under optimal conditions. Initially, conidia are attached to the cuticle orthe dorsum ofimmature or adult whiteflies. First symptoms ofinfection typically are apparent within one to two days after contact. Conidia and hyphae are present in the hosthemocoel within one day. Fungal multiplication takes place through formation ofhyphal bodies or blastospores. The mycelium is present on the dorsum ofthe insect body within two days. Sporulation occurs within three days and reaches a maximum within five to seven days

. Conidiospores generated from the mycelial growth on dead insects can infect additional live target pests. The ability ofP.fumosoroseus strain FE 990I to produce infections on eggs oftwo novel whitefly species was evaluated under laboratory conditions. The strain produced infections on the NI and N4 nymph stages and significantly reduced the hatching capacity ofeggs ofLecanoides jloccissimus. Italso produced infections onA/eurodicusdisperse NI and N4 nymph stages and reduced the percent ofhatching ofeggs by 50%

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DISCUSSION: With the notable exception ofbeauverolides, no impurities oftoxicological significance are associated with the TGAI orEP. NoFlyTechnical contains

Test substances in all toxicity tests included these components in additionto the MPCA. Therefore, the potential toxicity ofinert materials associated with the active ingredient in both TGAI andEP has been evaluated.

Therefore, no side-products from chemical reactions are likely. The acute pulmonary infectivity and toxicity study was done in ferrets and not in rats. Although we usually preferthat this study be done in rats, the applicant has given enoughjustification for the use offerrets that is acceptable. Also, in the acute intraperitoneal Infectivity study in rats, the applicanthas studied microbial clearance only in blood samples and has not studied microbial clearance in other body organs. 

### RECOMMENDATION: The following data are required for the unconditional registration of NoFly Technical; andNoFly Wettable Powder:
1-Afive batch analysis ofEP and TGAI from the proposed manufacturing site must be submitted.
2- Microbial clearance from body organs in the acute intraperitoneal infectivity study in rats.



### SUMMARY OFDATA SUBMITTED: ProductChemistry & 

### StorageStability (MRID 47719-01, 47719-02, & 

### 47720-01) NoFly Technical is a wettable powder consisting of

. NoFly WP is a wettable powder consisting of

. NoFly Technical is a slightly cohesive, dusty powderwith a bulk density of0.6- 0.7 glee and 99% ofthe particles are less than 1 OOμm. Its coloris lilac and has a yeast-like odor. A 1 % suspension ofNoFly Technical in water has a pH of6.5

- 7.5. NoFly WP is a powder with a bulk density of0.65-0.75 glee. Its color 3

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is lilac (Pantone 435M/662C) and has a yeast-like odor. A suspension ofNoFly WP in water has a pH of6.5

- 7.5. The potency estimation data for Nofly Technical supports a nominal guarantee of 8.1 x l09 CFU/g. A lower limit of for the TGAI is also supported by the data. The potency estimation data for NoFly WP supports a nominal guarantee of2.0 x I09 CFU/g. A lower limit of for the EP is also supported by the data. Manufacturing Process: The TGAI, NoFly Technical, and the formulated EP, NoFly WP, are produced
process of

### P

.fumosoroseus strain FE 9901. Production ofNoFly Technical involves typical procedures for growth ofmicrobial products and is summarized in Figure 1

Figure 1. Flowchart for Manufacture of NoFfy•• T«lmlca/

Formation ofUnintentional Ingredients: NoFly Technical
Batch Number

Parameterl'v1easured Pf Pf Pf 0702 0706 0708 Coliform count Abs Abs Abs Gram (-) Enteric Abs Abs Abs bacteria Salmonella/Shigella Abs Abs Abs

4 Pf Pf 0712 0715 Abs Abs Abs Abs Abs Abs Quality l'v1ean Control Specification Abs Absent in 

### O.lg Abs Absent in O.lg Abs Absent in O.tg

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Lactobacilli 0 0 

### 0 0 0 0 < lOJ CF Other fungi 0 0 

### 0 0 0 0 < 1% 0 f'Umosoro

# !!j f P. seus
NoFly WP Batch Number Quality

ParameterMeasured FE07 FE07 FE07 FE07 FE08 Mean Control 0920 0927 1010 0207 0409 Specification Coliform count Abs Abs Abs Abs Abs Abs Absent m O.lg Gram (-) Enteric Abs Abs Abs Abs Abs Abs Absent m bacteria O.lg Salmonella/Shigella Abs Abs Abs Abs Abs Abs Absent m 0.lg Lactobacilli 0 0 0 

### 0 0 0 

### < lOj CFIJ/g Otherfungi 0 0 0 0 0 0 < 1% of P. fumosoroseus
Analysis for Other Unintentional Ingredients demonstrated thatknown mammalian toxins are not produced or are not present in final products. 

### Storage Stability Testing
A storage stability study was conducted on FuturEco NoFly WP formulation; batch number FE 051216 before and after storage for 6 months at 4°C. The viable count test (CFU/g) ofthe formulation showed a I0-fold reduction over the storage period ofsix months set at4°C (±2 "C). There was no significant difference in the physical test results before and after storage. The reviewers agree that the details ofthe study support the conclusions drawn above. However, since the one batch ofEP that was tested was not produced at the proposed manufacturing site, differs in formulation and the relative viability ofEP after six months was mediocre, the proposed storage statement ofsix months at 4°C cannot be supported. A storage statement for a period of3 months at 4°C can be supported at this time with the condition that new databe produced usir

_ g the**propo~ed** formulation, manufactured at the proposed site

. Deficiencies: A storage stability study must be conductedusing the proposed formulation ofNoFly WP from the proposed manufacturing site. 

### Acute Oral Toxicity-Rat MRID # 47719-03 In an acute oral toxicity study, Futureco NoFly (containing 2x 109 colony forming units of 

### Paecilomycesfumosoroseus strain FE 9901 per gram) was administered to three female SpragueDawley rats as a gavage dose of5000 mg/kg bwfollowing the up and down procedme. The study

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was terminated following the stopping rules for this procedure. Each animal was then observed for a period ofup to 14 days. Oral LD50 is > 5000 mg/kg bw. None ofthe animals died or showed any symptoms ofintoxication following treatment. Thetest material had no negative effect upon the body weight and no gross pathological changes were noted. Based onthe results ofthis study, FuturecoNoFly is ofLOW Toxicity (EPA Toxicity Category IV) to the rat via the oral route. This acute oral study is classified acceptable. This study satisfies the guideline requirement for an acute oral study (OPPTS 870.1100; OECD 425) in the rat. 

### CLASSIFICATION: ACCEPTABLE Acute Dermal Toxicity - Rat MRID # 

### 47719-04 In an acute dermal toxicity study, a group of7 week-old Sprague-Dawley rats (5/sex) was dermally exposed to Futureco NoFly (containing 2xl09 colony forming units of

### Paecilomycesfumosoroseus strain FE 9901 pergram) at a limit dose of5050 mg/kg bodyweight for 24 hours to an area ofnot less than 10% ofthe total body surface. Following exposure, the animals were observed for a period of 14 days. Dermal LD5

### o Males Females Combined > 5050 mg/kg bw 

### >5050 mg/kg bw 

### > 5050 mg/kg bw There were no mortalities and all the test animals appeared normal for the duration ofthe study. All animals gained weight with the exception ofone female which failed to gain weight between Days 7 and 14. At necropsy, two male rats had pale lungs at study termination. Based onthese results, Futureco NoFly is ofLOW Toxicity to the rat via the dermal route and is minim'ally irritating (i.e., mean irritation score=O.1) following a 24-hour exposure. This acute dermal toxicity study is classified as acceptable. This study satisfies the guideline requirement for an acute dermal toxicity study (OPPTS 870.1200: OECD 402) in the rat. 

### CLASSIFICATION: ACCEPTABLE, Toxicity Category IV 

### Acute Inhalation Toxicity- Rat MRID # 

### 47719-0S In an acute inhalation toxicity study, a group of 10-week old Sprague-Dawley rats (5/sex) were exposed by the inhalation route to undiluted Futureco NoFly (containing 2x109 colony forming units of

### Paecilomycesfumosoroseus strain FE 9901 pergram) for 4 hours to nose only at a concentration of2.18 mg/L. Animals then were observed for 14 days. Inhalation LC50 Males 

### > 2.18 mg/L Females 

### > 2.18 mg/L Combined > 2.18 mg/L Limit test There were no mortalities noted throughout the study. Clinical observations included piloerection and decreases in activity which resolved by Day 3. All test animals gained body weight throughout the studyperiod and no observable abnormalities were observed at gross necropsy. Based on the results ofthis study, Futureco NoFly is ofLOW Toxicity in male and female SpragueDawley rats via the inhalation route. This acute inhalation study is classified as acceptable. It also satisfies the guideline requirement for an acute inhalation study (OPPTS 870.1300; OECD 403) in the rat.

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CLASSIFICATION: ACCEPTABLE, Toxicity Category IV 

### PrimaryEye Irritation

- Rabbit MRID # 47719-06 In a primary eye irritation study, 0.1 g ofFuturecoNoFly WP (containing 88%

### Paecilomyces fumosoroseus strain FE 9901) was instilled into the conjunctiva! sac ofthe left eye of3 adult male NewZealand white rabbits. Thetreated eyes were not washed following application. The untreated right eye ofeach test animal served as a negative control. Test animals then were observed for 72 hours. Irritation was scored by the method ofDraize. After I hour, some redness (grade 1) was observed in one rabbit, and slightto moderate increases in excretion discharge were noted in 3 rabbits. All test animals were free ofirritation at the 24-, 48- and 72-hour observation time points. In this study, Futureco NoFly WP 

### (P.fumosoroseus strain FE 990l) is SLIGHTLY IRRITATING to the eyes ofNew Zealand white rabbits. This study is classified as acceptable. This study satisfies the guideline requirement for a primary eye irritation study (OPPTS 870.2400; OECD 405) in the rabbit. 

### CLASSIFICATION: ACCEPTABLE, Toxicity Category IV 

### Dermal Irritation -Rabbit MRID# 47719-07 In a primary dermal irritation study, adult male New Zealand white rabbits (3) were dermally exposed to 0.5 g Futureco NoFly WP (containing 88%

### Paecilomycesfumosoroseus strain FE 9901 [ARSEF]) in water for 4 hours to an area ofskin ofapproximately 6 cm2. Animals then were observed for 72 hours. Irritation was scored by the method ofDraize. Very slight erythemawas noted in two test animals I hour after patch removal. All test animals were free ofirritation atthe 24-, 48- and 72-hourobservationtime points. Based on the calculated maximum irritation score and an irritation index of0.7/8 (1 h) and 0.17 (respectively), Futureco NoFly WP is slightly irritating to the skin ofmale New Zealand white rabbits. This study is classified as acceptable. This study satisfiesthe guideline requirement for a primary dermal irritation study (OPPTS 870.2500; OECD 404) in the rabbit. 

### CLASSIFICATION: ACCEPTABLE, Toxicity Category 

### IV Dermal Sensitization

- Guinea Pig 

### MRID # 47719-08 In a skin sensitization study with Futureco NoFly WP (containing 88% 

### Paecilomycesfumosoroseus strain FE9901), Dunkin Hartley guineapigs (10 females) were tested using Magnusson and Kligman method. Guineapigs were subjected to a two-stage induction procedure; a I% intradermal injection and a 25% topical application. Two weeks following the last induction exposure, a challenge dose was applied as a 25% topical application. Five additional female guinea pigs were exposedto the vehicle during induction then challenged with the test material. This group served as a negative control. The dermal reactions observed in test animals (left flank) following challenge includc:d confluent, intense erythema and swelling. No visible dermal reactions were observed to the vd1icles (rightflank) or control animals. 

### In this study, Futureco NoFly WP was a sensitizer in guinea pigs following the Magnusson and Kilgman procedure. This study satisfies the guideline**requirerni~nt** for a dermal sensitization study (OPPTS 870.2600; OECD 406) in the guinea pig. 

### CLASSIFICATION: ACCEPTABLE, Toxicity Category IV 

### Acute Oral lnfectivity and 

### Toxicity- Rat MRID # 47720-02 In an acute oral toxicity study, groups offasted, young adult (6-7 weeks) Wistar rats (15 rats/sex)
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were given a single oral dose ofthe MPCA, Paecilomycesfumosoroseus strain FE 9901 (100%; I.Ox 109 CFU/g), in 0.05% Tween 80 at a single dose of 108 CFU peranimal. The animals were then observed for a period ofup to 21 days with scheduled sacrifices 24 hours after application and on Days 3, 7, 14 and 21. Another group ofrats (4 rats/sex) were untreated and served as a negative control. Oral LOSO Males Females Combined 

### > 108 CFU/anirnal 

### > I08 CFU/animal 

### > 108 CFU/animal None ofthe animals died and no clinical observations were noted during the study period. No gross pathological changes were noted in sacrificed test animals. Based on the results ofthis study, 

### P. fumosoroseus strain FE 9901 is ofLOW Toxicity and is not infective orpathogenic in the rat. 

### CLASSIFICATION: ACCEPTABLE, Toxicity CategoryIV 

### Acute Pulmonary Infectivity and Toxicity

- Ferret MRID # 

### 47720-04 In an acutepulmonary infectivity andtoxicity study, groups ofyoung adult ferrets (14/sex) were exposed by the intranasal route to the microbial pest control agent (MPCA), Paecilomyces fumosoroseus strain FE 9901 (100%; I.OxI09 CFU/g), in 0.05% Tween 80 at a nominal dose of

Ix 108 CFU (in 0.5 mL)peranimal. Animals were then observed for up to 21 days. Another group offerrets (4 ferrets/sex) were untreated and served as a negative control. Pulmonary LD50 Males 

### > 0.9x 108 CFU/animal Females > 0.9x 108 CFU/anirnal Combined 

### > 0.9xI08 CFU/animal None ofthe animals died or showed any symptoms ofintoxication following treatment. Some= animals lost weight during the first week ofthe study and it is not known ifthese effects were reversed by the end ofthe study since these data were not provided in the study report. No gross pathological changes were noted. Microbiological examination oflungs, otherprescribed organs and body fluids proved minimal persistence and no infectivity ofMPCA. Based on the results ofthis study, P.fumosoroseus strain FE 9901 is ofSLIGHT Toxicity and the MPCA is not infective orpathogenic in the ferret. This acute pulmonary infectivity and toxicity study is classified as ACCEPTABLE. Ferrets are usually not used for acute pulmonary toxicity/pathogenicity testing, but is acceptable because ofthejustification given for theiruse 

### CLASSIFICATION: ACCEPTABLE, Toxicity Category IV 

### Acute Intraperitoneallnfectivity MRID # 

### 47720-05 Jn an acute intraperitoneal infectivity study, a group ofyoung adult Wistar rats (3/sex) were**ir~ected** with the MPCA, Paecilomycesfumosoroseus strain FE 9901 (100%; t.Oxl09 CFUig), in 0.05% Tween 80 at a dose of 107 CFU per animal. Animals were then observed for up to 21 days. Another group ofrats ( 4 rats/sex) were untreated and served as a negative control. Based on the**resul~** of this study, P.fumosoroseus strain FE 9901 is not infective or pathogenic in therat viathe intraperitoneal route ofexposure. None ofthe animals died or showed any symptoms ofintoxication after treatment. The test item had no negative effect upon the body weight and no gross pathological changes were noted. Microbial clearance was shown from blood samples but not from body organs. This intraperitoneal infectivity study is classified as supplemental. 

### CLASSIFICATION: SUPPLEMENTAL
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Hypersensitivity Incidence MRID # 

### 47720-06 The registrant submitted a statement indicating that there were no reports ofhypersensitivity reactions with the microbial pest control agent (MPCA), Paecilomyces fumosoroseus strain FE 9901, among researchers, product developers and manufacturers. An end-useproduct containing P. fumosoroseus strafo. FE 990l was determined to be a sensitizer in a skin sensitization study and 21 reports ofhypersensitivity reactions were found in the PubMed database using the keywords ''paecilomyces" and "allergy". Based onthe assumption that mostmicroorganisms contain substances that would elicit allergic reactions, thetechnical grade ofthe active ingredient, NoFly Technical, is considered a potential sensitizing agent. 

### CLASSIFICATION: ACCEPTABLE

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