# BIOPESTICIDES REGISTRATION

ACTION DOCUMENT

Paecilomyces fumosoroseus strain FE 9901

U.S. E NVIRONMENTAL 

## P ROTECTION 

## A GENCY



## O FFICE OF 

## P ESTICIDE 

## P ROGRAMS



## B IOPESTICIDES AND 

## P OLLUTION 

## P REVENTION 

## D IVISION



### May 8, 2011P. fumosoroseus strain FE 9901 page ii

Biopesticides Registration Action Document



## T ABLE OF 

## C ONTENTS

I. REGULATORY BACKGROUND ................................................................................ 1

A. Application for Pe sticide Regi stration ................................................................... 1

B. Petition to Establish an Exemption from Tolerance .............................................. 1

C. Public Pa rticipat ion ............................................................................................... 1

D. North American Free Trade Agreement Join t Review .......................................... 2

II. BIOLOGICAL CONSIDERATIONS ........................................................................... 2

III. ACTIVE INGREDIENT CHARACTERIZATION ........................................................ 2

A. Analysis and Cert ified Limi ts ................................................................................ 3

B. Physical and Chem ical Proper ties ........................................................................ 3

C. Identification and Characteri zation ....................................................................... 4

IV. HUMAN HEALTH RISK ASSESSMENT .................................................................. 4

A. Toxicity ................................................................................................................. 4

1. Acute To xicity .................................................................................................. 4

2. Hypersensitiv ity Inci dents ................................................................................ 5

3. Metabolites of Conc ern .................................................................................... 5

4. Effects on the Endocrine System ..................................................................... 5

B. Expos ure .............................................................................................................. 6

1. Occupationa l Exposu re ................................................................................... 6

2. Residential, School, and Day Care Ex posure .................................................. 6

C. Risk Charac terizati on ........................................................................................... 6

V. ENVIRONMEN TAL ASSESSMENT .......................................................................... 7

A. Risk Charac terizati on ........................................................................................... 7

B. Endangered Spec ies Assessm ent ........................................................................ 7

VI. RISK MANAGEMENT .............................................................................................. 7

A. Decision ................................................................................................................ 7

B. Terms of t he Registra tion ..................................................................................... 8

BIBLIOGRAPHY ............................................................................................................. 9

APPENDIX A: Data Requirements and How the Applican t Met Them........................ A-1

APPENDIX B: Biology and Taxonomy of P. fumosoroseus

........................................ B-1

APPENDIX C: Summary of Toxici ty Studies .............................................................. C-1



## Tables

Table 1. Select Physical and Chemical Properties of P. fumosoroseus strain FE 9901 . 3

Table 2. Summary of Toxicology Data for P. fumosoroseus strain FE 9901 .................. 4P. fumosoroseus strain FE 9901 page iii

Biopesticides Registration Action Document



## B IOPESTICIDES 

## R EGISTRATION 

## A CTION 

## D OCUMENT 

## T EAM



## Office of Pesticide Programs

Biopesticides and Pollution Prevention Division

Microbial Pesticides Branch

S CIENCE 

## R EVIEW



### Ibrahim Barsoum, Ph.D. Product Chem istry and Human Health Risk

John Kough, Ph.D. Secondary Review of Product Chemistry and Human Health Risk

Gail Tomimatsu, Ph.D. Environmental and Ecological Risk



## R EGULATORY



### Kathleen Martin, M.S.E.S. Regulatory Action Leader

Sheryl Reilly, Ph.D. Chief, Microbial Pesticides BranchP. fumosoroseus strain FE 9901 page iv

Biopesticides Registration Action Document



### ACRONYMS

Acronym Extension

ARS Agricultural Research Service

BRAD Biopesticides Registration Action Document

CFU Colony forming units

CFU/g Colony forming units per gram

DNA Deoxyribonucleic acid

EDSP Endocrine Disruptor Screening Program

EP End-use product

EPA U.S. Environmental Protection Agency or, the Agency

FFDCA Federal Food, Drug, and Cosmetic Act

FIFRA Federal Insecticide, Fungicide, and Rodenticide Act

g/cc Grams per cubic centimeter

LC 50 Median Lethal Concentration

LD 50 Median Lethal Dose

mg/kg bw Milligrams per kilogram of body weight

mg/L Milligrams per liter

MP Manufacturing-use product

MPCA Microbial pesticide control agent

MRID Master Record IDentification (number)

NAFTA North American Free Trade Agreement

NOF Notice of Filing

NOR Notice of Receipt

NR Not required

OECD Organization for Economic Cooperation and Development

OPP EPA’s Office of Pesticide Programs

OPPTS EPA's Office of Prevention, Pesticides and Toxic Substances;

now the Office of Chemical Safety and Pollution Prevention

P. fumosoroseus Paecilomyces fumosoroseus

PMRA Health Canada’s Pest Management Regulatory Agency

RAPD-PCR Random Amplification of Polymorphic DNA-Polymerase Chain Reaction

rDNA-ITS Ribosomal DNA-Internal Transcribed Spacer

TGAI Technical grade active ingredient

U.S. United States

UV Ultraviolet radiation

USDA U.S . Department of Agriculture

WP Wettable powderP. fumosoroseus strain FE 9901 page 1

Biopesticides Registration Action Document

In May 2009, Natural Industries Inc. (represented by Technology Sciences Group, Inc.) applied

to the U.S. Environmental Protection Agency (EPA or the Agency) for registration of two products

containing the new mycoinsecticide Paecilomyces fumosoroseus strain FE 9901:

Maunfacturing-Use End-Use

Product: NoFlyTM Technical NoFlyTM WP

EPA Reg. No: 73314-7 73314-6

% ai: 69.0% 18%

Docket: 1 EPA-HQ-OPP-2010-0093

Proposed

Uses: Nonfood (e.g., ornamentals) greenhouse and nursery crops to control

whiteflies, aphids, thrips, psyllid s, mealybugs, l eafhoppers, plant

bugs, weevils, grasshoppers, Mo rmon crickets, locust, and beetles

PC Code: 115003

The applicant also petitioned for an exemption from the requirement of tolerance for any

P.fumosoroseus strain FE 9901residues that may remain in or on food.

I. REGULATORY BACKGROUND

A. Application for Pesticide Registration

A Notice of Receipt (NOR), announcing EPA’s receipt of Natural Industries Incorporated’s

applications to register P.fumosoroseus strain FE 9901, was published in the Federal Register on

March 10, 2010 (75 FR 11175). You can find the NOR and supporting materials (e.g., draft label) at

regulations.gov; the docket number is EPA-HQ-OPP-2010-0093. EPA received no comments in

response to the NOR.

B. Petition to Establish an Exemption from the Requirement of Tolerance

Under section 408 of the Federal Food, Drug, and Cosmetic Act (FFDCA), the applicant

petitioned the Agency (PP 9F7587) to establish an exempt ion from the requirement of a tolerance for

residues of P.fumosoroseus strain FE 9901. A Notice of Filing (NOF) was published in the Federal

Register on March 10, 2010 (75 FR 11171); it can be found at regulations.gov under docket number

EPA-HQ-OPP-2010-0092. EPA received no comments in response to the NOF. In February 2011,

Natural Industries Inc. (as represented by Technology Sciences Group, Inc.) withdrew their petition

(TSG 2011).

C. Public Participation

On October 1, 2009, EPA announced a new policy to provide a more meaningful opportunity for

the public to participate in major registration decisions before they occur. According to this new policy,

the Agency intends to provide a public comment period prior to making a registration decision for, at

minimum, the following types of applications: new ac tive ingredients; first food uses; first outdoor uses;

first residential uses; and any other registration decisions for which the Agency believes there may be

substantial public interest.

1 To access the public docket, go to www.regulations.gov; under “Enter Keyword or ID” enter the docket number, which is

‘EPA-HQ-OPP-2010-0093.’P. fumosoroseus strain FE 9901 page 2

Biopesticides Registration Action Document

Consistent with the new policy of making registration actions more transparent,

P. fumosoroseus strain FE 9901 was subject to a 30-day comment period as a “new active ingredient”

whose registration would result in a “first food use.” The public comment period ran from

October 6, 2010 to November 5, 2010; no comments were received.

D. North American Free Trade Agreement Joint Review

P. fumosoroseus strain FE 9901 was the subject of a North American Free Trade Agreement

(NAFTA) joint registration review conducted by EPA’ s Office of Pesticide Programs (OPP) and Health

Canada’s Pest Management Regulatory Agency (PMRA).

II. BIOLOGICAL CONSIDERATIONS (Altre and Vandenberg 2001; Bolckmans et aI.1995; Lacey

et. al. 1999; Osborne et. al. 1990; Osborne and Landa1992; Smith 1993; MRID 477920-01)

P. fumosoroseus is a naturally occurring fungus found in various soil types around the world,

including Europe (Netherlands, Ireland, Spain); Asia (China, Japan, Pakistan, India); Africa (Ghana);

and the United States. In the environment, P. fumosoroseus is dispersed as conidia, either by air or

water movement or by other insects and/or mites. Conidia are spores and they are supported on

specialized spore-producing “hyphae” (a filamentous stalk) known as conidiophores. As with other

entomopathogenic 2 fungi, P. fumosoroseus infects its target by conidial attachment followed by

germination, penetration into the host, vegetative gr owth within the organism, and then conidiogenesis

(i.e., production of more conidia).

In laboratory testing, investigators determined that P. fumosoroseus strain FE 9901 grows best

at (76.1o F; 24.5°C). At higher temperatures (79.7o F to 86.0o F; 26.5°C to 30°C), growth is significantly

reduced and at temperatures greater than 95.0o F (35°C), the fungus will no longer grow. At 98.6oF

(37oC), the organism is killed ( P. fumosoroseus strain FE 9901 was unable to resume growth at room

temperature after incubation at 98.6oF).

NoFlyTM WP’s target is the whitefly, and this microbial pesticide control agent (MPCA) is

intended to control all stages of whitefly development (i.e., eggs, N1, N2, N3 and N4 larvae, and

adults). The ability to infect whitefly eggs is unique among entomopathogenic fungi.

 The infection

cycle of P. fumosoroseus strain FE 9901 is particularly rapid under optimal conditions. Initially, conidia

attach to the cuticle or the dorsum of immature or adult whiteflies; the first symptoms of infection

typically are apparent within one to two days after contact. Conidia and hyphae are present in the host

hemocoel (body cavity) within one day. Fungal multiplication takes place through formation of hyphal

bodies or blastospores. The mycelium (a branching network of hyphae) is present on the dorsum of the

insect body within two days. Sporulation occurs within three days and reaches a maximum within five

to seven days.

P. fumosoroseus strain FE 9901 does not exhibit any atypical morphological, physiological,

biochemical, pesticidal, or resistance characteri stics. For more information on the biology and

taxonomy, see Appendix B.

III. ACTIVE INGREDIENT CHARACTERIZATION

Most of the data requirements under 40 CFR 158.2120 (Product Analysis) have been satisfied.

A sample of P. fumosoroseus strain FE 9901 has been deposited in the U.S. Department of

Agriculture’s Agricultural Research Service Collection of Entomopathogenic Fungal Cultures (USDA

ARS) under the accession number ARSEF 4490. Described below are the studies that are not

2 An entomopathogenic fungus is one that can act as a parasite of insects and kills or seriously disables them.P. fumosoroseus strain FE 9901 page 3

Biopesticides Registration Action Document

acceptable or need further information, as terms of registration (see Section VI.B: Terms of the

Registration, for a complete list). The physical and chemical properties of P. fumosoroseus strain FE

9901 are provided in Table 1.

A. Analysis and Certified Limits

Under Analysis of Samples, the registrant must submit a five-batch analysis of the EP (end-use

product) and MP (manufacturing-use product), from the proposed manufacturing site, as a term of

registration (OPPTS 885.1400).

B. Physical and Chemical Properties

Three Physical and Chemical Characteristics (as discussed below) must be completed as terms

of registration. Provided in Table 1 is a summary of the physical and chemical properties of NoFlyTM

WP and NoFlyTM Technical.

Stability to Normal and Elevated Temperatures, Metals and Metal Ions (OPPTS 830.6313) was

satisfied for NoFlyTM Technical (MRID 480514-01); however, it has not been satisfied for

NoFlyTM WP.

Storage Stability (OPPTS 830.6317) must be addressed for both NoFlyTM Technical and

NoFlyTM WP. For the Technical, Natural Industries Inc. has stated that they will conduct a

combined storage stability/corrosion characterist ic study as a term of registration (MRID

480514-01). Regarding NoFlyTM WP, a study was submitted (MRID 477919-02); however, it is

inadequate. First, the test guidelines for Storage Stability (OPPTS 830.6317; EPA 2002)

recommend a test period of one year, at a minimum; the duration of the petitioner’s study was

just six months. Further, one batch of the product was not produced at the proposed

manufacturing site and the relative viability after six months was mediocre. The existing data

support a three-month storage period, with the condition that a new study be conducted using

the proposed formulation, manufactured at the proposed site.

Corrosion Characteristics (OPPTS 830.6320) must be satisfied as a term of registration; Natural

Industries Inc. has indicated that they intend to complete these studies (MRID 480514-01).

Table 1 . Select Physical and Chemical Properties of Paecilomyces fumosoroseus

strain FE 9901 (MRIDs 477919-01, 477920-01, and 480514-01)

OPPTS

Guideline

Number Data

Requirement VALUE

EP (NoFlyTM WP) MP (NoFlyTM Technical)

830.6302 Color lilac (Pantone 435M/662C) lilac

830.6303 Physical state powder s lightly cohesive, dusty powder

830.6304 Odor yeast-like yeast-like

830.6313 Stability to normal and

elevated temperatures,

metals and metal ions Term of registration Above 104  ̊ F (40  ̊ C), this MPCA will

not survive for any length of time;

NoFlyTM Technical will not be

exposed to metal or metal ions.

830.6317 Storage stability Term of registration—submitted study

is inadequate Term of registration

830.6320 Corrosion characteristics Term of regist ration Term of registration; to be conducted

with storage stability

830.7000 pH (1% suspension

in water) 6.5 to 7.5 6.5 to 7.5

830.7300 Bulk density 0.65 to 0.75 g/cc 0.6 to 0.7 g/ccP. fumosoroseus strain FE 9901 page 4

Biopesticides Registration Action Document

C. Identification and Characterization

Identification and characterization of P. fumosoroseus strain FE 9901 has been made using

BioLog FF MicroPlateTM. Growth was observed on a variety of solid media agar formulations (e.g.,

Sabouraud dextrose, MacConkey, and chitin) over a wide range of pH values. Molecular fingerprints of

P. fumosoroseus strain FE 9901 were compared with those of three other P. fumosoroseus isolates;

two techniques were used: variable number tandem repeat, and inter-simple sequence repeat. Other

technologies available to identify and characterize the microbe include rDNA-ITS (Ribosomal DNAInternal Transcribed Spacer) sequencing and RAPD-PCR (Random Amplification of Polymorphic DNA

polymerase chain reaction).

IV. HUMAN HEALTH RISK ASSESSMENT (EPA 2010b)

In assessing the risk of a pesticide to human heal th, EPA considers the toxicity of the pesticide

in addition to exposure routes and levels. Provided in section A (below) is the toxicity of P.

fumosoroseus strain FE 9901 and in section B, the exposure. The Risk Characterization is provided in

section C.

A. Toxicity

1. Acute Toxicity

With one exception (additional information is needed for the acute injection

toxicity/pathogenicity study), all the data requirements under 40 CFR 158.2140 (Toxicology)

have been met. A summary of the toxicity data for NoFlyTM WP and NoFlyTM Technical is

provided in Table 2; for brief descriptions of the studies submitted, please see Appendix C.

A complete listing of the terms of registration is provided in section VI.B.

Table 2. Summary of Toxicology Data for Paecilomyces fumosoroseus strain FE 9901

OPPTS

Guideline

Number Data

Requirement TOXICITY AND CATEGORY 1

EP (NoFlyTM WP) MP (NoFlyTM Technical)

870.1100 Acute oral toxicity LD 50 >5,000 mg/kg bw

Category IV

(MRID 477919-03) LD 50 =600 mg/kg bw

Category III

870.1200 Acute dermal toxicity LD 50 >5,050 mg/kg bw

Category III

(MRID 477919-04) Data waived

(MRID 477920-06)

870.1300 Acute inhalation toxicity LC 50 >2.18 mg/L

Category III

(MRID 477919-05) Data waived

(MRID 477920-06)

870.2400 Acute eye irritation some redness

Category IV

(MRID 477919-06) Data waived

(MRID 477920-06)

870.2500 Primary dermal irritation slightly irritating

Category IV

(MRID 477919-07) Data waived

(MRID 477920-06)

885.3050 Acute oral toxicity/

pathogenicity TGAI 2 : Not infective or pathogenic to rats via the oral route.

885.3150 Acute pulmonary

toxicity/pathogenicity TGAI: Not infective or pathogenic to ferrets via the inhalation route.

885.3200 Acute injection

toxicity/pathogenicity

(intraperitoneal) TGAI: Not infective or pathogenic to rats via injection, based on the

available results (more information is needed). 3 (MRID 477920-05)P. fumosoroseus strain FE 9901 page 5

Biopesticides Registration Action Document

1 Category I has the highest toxicity while Category IV has the lo west. For a description of the Toxicity Categories along with their

limits, see 40 CFR 156.62.

2 TGAI=technical grade active ingredient.

3 Natural Industries Inc. must submit data showing microbial clear ance from matrices other than bl ood (e.g., organs, body fluids)

(EPA 2010b).

2. Hypersensitivity Incidents (OPPTS 885.3400; MRID 480514-02)

The applicant reported that they have seen no incidents of hypersensitivity throughout

the research, development, and testing of N oFlyTM Technical and WP; however, a literature

search of the PubMed database ( www.ncbi.nlm.nih.gov/pubmed ) using the keywords

“paecilomyces” and “allergy,” revealed 21 reports of hypersensitivity for this genus. In a

supplemental study (MRID 477919-08), study invest igators determined that NoFlyTM WP causes

sensitization when applied to the skin of female guinea pigs.

Based on the assumption that most microorganisms contain substances that could elicit

allergic reactions, EPA considers P. fumosoroseus strain FE 9901 to be a potential sensitizing

agent.

3. Metabolites of Concern

P. fumosoroseus strain FE 9901 may produce the metabolites Beauverolides M and I,

and 2,6-pyridinedicarboxylic acid during the fermentation process. EPA does not expect these

metabolites to be of concern because, overall, the acute toxicity of NoFlyTM Technical and WP is

low (see Table 1).

4. Effects on the Endocrine System

As required under FFDCA section 408(p) , EPA has developed the Endocrine Disruptor

Screening Program (EDSP) to determine whether certain substances (including pesticide active

and other ingredients) may have an effect in humans or wildlife similar to an effect produced by

a “naturally occurring estrogen, or other such endocrine effects as the Administrator may

designate.” The EDSP employs a two-tiered approach to making the statutorily required

determinations. Tier 1 consists of a battery of 11 screening assays to identify the potential of a

chemical substance to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal

systems. Chemicals that go through Tier 1 screening and are found to have the potential to

interact with E, A, or T hormonal systems will proceed to the next stage of the EDSP where EPA

will determine which, if any, of the Tier 2 tests are necessary based on the available data. Tier

2 testing is designed to identify any adverse endocrine related effects caused by the substance,

and establish a dose-response relationship between the dose and the E, A, or T effect.

Between October 2009 and February 2010, EPA issued test orders/data call-ins for the

first group of 67 chemicals, which contains 58 pesticide active ingredients and nine inert

ingredients. This list of chemicals was se lected based on the potential for human exposure

through pathways such as food and water, residential activity, and certain post-application

agricultural scenarios. This list should not be construed as a list of known or likely endocrine

disruptors.

P. fumosoroseus is not among the group of 58 pesticide active ingredients on the initial

list to be screened under the EDSP. Under FFDCA section 408(p) the Agency must screen all

pesticide chemicals. Accordingl y, EPA anticipates issuing future EDSP test orders/data call-ins

for all pesticide active ingredients.

For further information on the status of the EDSP, the policies and procedures, the list of

67 chemicals, the test guidelines and the Tier 1 screening battery, please visit our website:

http://www.epa.gov/endo/.P. fumosoroseus strain FE 9901 page 6

Biopesticides Registration Action Document

B. Exposure

1. Occupational Exposure

NoFlyTM WP, which is limited to greenhouse use (indoor), can be applied using

conventional spraying equipment, but use of manual sprayers is highly recommended.

Occupational exposure via the inhalation and derm al is expected for persons who mix and/or

applying this pesticide. Dermal exposure is expected for individuals who work in the

greenhouse following application.

2. Residential, School, and Day Care Exposure

Individuals in residential, school, and day-care settings may be exposed to residues of

P. fumosoroseus strain FE 9901 through the dermal route by touching treated ornamental

plants, but EPA expects that exposure will be low due to the nature of this fungus. At warm

temperatures, this MPCA does not grow well—at 80o F (26.7°C), growth is significantly reduced,

and at 95o F (35°C), it does not grow at all. Sunlight further inhibits the growth of P.

fumosoroseus (Fargues 1997; TSG 2008). Fargues et al. (1997) studied the effects of

ultraviolet (UV) radiation on the germinability, survival, and infectivity of P. fumosoroseus . They

found the UVB range of ultraviolet light (sunlight includes UVB light) to be the most important

factor “for the detrimental effects of sunlight on the persistence of conidia of entomopathogenic

fungi in insolated environments” (Fargues 1997). EPA expects that once the treated plants are

out of the greenhouse, residues of P. fumosoroseus strain FE 9901 would rapidly decline due to

temperature and sunlight.

C. Risk Characterization

Occupational exposure may occur through the dermal and inhalation routes. P. fumosoroseus

strain FE 9901 is not acutely toxic via these routes—the dermal LD 50 is 5,050 mg/kg bw (limit dose) and

inhalation LC 50 is >2.18 mg/L (limit dose). There is concern, however, regarding sensitization. In a

search of the open literature, EPA noted a number of hypersensitivity reports for the genus

Paecilomyces . And, a skin sensitization assay using P. fumosoroseus strain FE 9901 as the test

substance (MRID 477919-08) was positive—the test animals exhibited intense erythema and swelling

in areas where the test material was applied. To mitigate potential sensitization, the label requires

protective eyewear; chemical-resistant gloves ; long-sleeved shirts and long pants; and, for

mixer/loaders, a dust/mist-filtering respirator meeting NIOSH (National Institute for Occupational Safety

and Health) standards of at least N-95, R-95, or P-95.

EPA concludes that there is a reasonable certainty that no harm will result from the intended

use of P. fumosoroseus strain FE 9901 as a microbial pesticide. This is based on the available toxicity

and exposure information— P. fumosoroseus strain FE 9901 does not demonstrate toxic, pathogenic,

and/ or infective potential to mammals, and potential occupational and non-occupational exposure is

expected to be very low.P. fumosoroseus strain FE 9901 page 7

Biopesticides Registration Action Document

V. ENVIRONMENTAL ASSESSMENT

A. Risk Characterization

EPA did not require any environmental effects da ta, including those for non-target organisms,

because of the low toxicity of this MPCA and, low exposure from the intended use. P. fumosoroseus

strain FE 9901will only be used in greenhouses 3 ; therefore, the Agency does not expect that birds,

honeybees, and other nontarget organisms such as insects and fish, will be exposed. In a letter to the

applicant (EPA 2008), EPA pointed out that if “the intended use sites are restricted to indoors, then

neither a honey bee study nor other nontarget assessments are likely to be needed.” For more details,

including a complete list of the Nontarget Organisms and Environmental Fate Data requirements under

40 CFR 158.2150, see Table A-3 of Appendix A.

B. Endangered Species Assessment

EPA expects that nontarget organisms will not be exposed to P. fumosoroseus strain FE 9901.

Thus, the Agency has determined that it will have “No Effect” 4 on any currently listed threatened or

endangered species, or any designated critical habitat.

VI. RISK MANAGEMENT

A. Decision

The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) section 3(c)(5) provides for the

registration of new active ingredients if EPA determines that:

 its composition is such as to warrant the proposed claims for it;

 its labeling and other materials required to be submitted comply with the

requirements of FIFRA;

 it will perform its intended function without unreasonable adverse effects on

the environment; and

 when used in accordance with widespread and commonly recognized

practice, it will not generally cause unreasonable adverse effects on the

environment.

The four eligibility criteria for registration of new active ingredients are satisfied for NoFlyTM WP

(73314-A) and NoFlyTM Technical (73314-T). These products are not expected to cause unreasonable

adverse effects and are likely to provide protection as claimed when used according to label

instructions. The new active ingredient P. fumosoroseus strain FE 9901 is eligible for registration for

the labeled uses. Please note that certain terms of registration must be met.

3 In a letter to the Agency (TSG 2008), Technology Sciences Gr oup Inc. (the regulatory cons ultant) pointed out that

“Natural Industries has decided not to make NoFlyTM for outdoor use because Paecilomyces fumosoroseus

strain FE 9901is UV sensitive.”

4 The U.S. Fish and Wildlife Service defines the term “No Effect ” to mean that “its proposed action will not affect a listed

species or designated critical habitat” (www.f ws.gov/endangered/esa-library/pdf/CH1-3.PDF).P. fumosoroseus strain FE 9901 page 8

Biopesticides Registration Action Document

B. Terms of the Registration

As terms of registration, EPA requires that the following data requirements be completed within

the next several months; specific time frames (from the date of registration) are noted below:

40 CFR 158.2120 (Product Analysis)

Analysis and Certified Limits :

Analysis of samples

(OPPTS 885.1400)

EP and MP A five-batch analysis of the EP and MP from the proposed

manufacturing site must be submitted. DUE : 18 months

Physical and Chemical Characteristics :

Stability to normal and

elevated temperatures,

metals and metal ions

(OPPTS 830.6313)

EP This requirement has been satisfied for the MP (MRID

480514-01); however, it must be completed for the EP. DUE : 12 months

Storage stability

(OPPTS 830.6317)

EP and MP Regarding the MP, MRID 480514-01 reports that “a storage

stability/corrosion study will be conducted as a condition of

registration.”

For the EP, a storage stabilit y study (MRID 477919-02) was

submitted; however, it is inadequate and will only support a

three-month storage period. A storage stability study must be

conducted using the proposed formulation of NoFlyTM WP,

from the proposed manufacturing site. The study must

support a storage period of at least six months. DUE: 18 months

Corrosion

characteristics

(OPPTS 830.6320)

EP and MP MRID 480514-01 reports these will be completed as “a

condition of registration.” DUE : 18 months

40 CFR 158.2140 (Toxicology data)

Acute injection toxicity/

pathogenicity:

intraperitoneal

( OPPTS 885.3200)

TGAI This study was submitted (MRI D 477920-05); however, the

registrant must submit additional data showing microbial

clearance from matrices other than blood (e.g., organs, body

fluids) (EPA 2010b). DUE : six months

NOTE : EP =NoFlyTM WP and MP =NoFlyTM Technical.P. fumosoroseus strain FE 9901 page 9

Biopesticides Registration Action Document

BIBLIOGRAPHY

Studies, by MRID

477919-00. Natural Industries, Inc. 2009. Submission of Product Chemistry and Toxicity Data in Support of

the Application for Registration of NoFlyTM WP and the Petition for Tolerance of Paecilomyces

fumosoroseus strain FE 9901, blastospores. Transmittal of 8 Studies. May 7, 2009.

477919-01. Curry, K.; Brookman, D. 2009. Product Chemistry of NoFLYTM WP. Project Number:

NoFLYTM/PC/002. Unpublished study prepared by Technology Sciences Group, Inc. 64 p. April

29, 2009.

477919-02. Bell, A. 2006. Storage and Stability of NoFlyTM WP. Project Number: CEMS/2836.

Unpublished study prepared by MGS Laboratories, Lt d., and CEM Analytical Services, Ltd. 14 p.

August 22, 2006.

477919-03. Kuhn, J. 2009. Futureco NoFlyTM: Acute Or al Toxicity Study (UDP) in Rats. Final Report.

Project Number: 12412/08. Unpublished study prep ared by Stillmeadow, Inc. 12 p. January 13,

2009.

477919-04. Kuhn, J. 2009. Futureco NoFlyTM: Acute Derm al Toxicity Study in Rats. Final Report. Project

Number: 12413/08. Unpublished study prepared by Stillmeadow, Inc. 13 p. January 13, 2009.

477919-05. Younger, C. 2009. Futureco NoFlyTM: Acute Inhalation Toxicity Study in Rats. Final Report.

Project Number: 12414/08. Unpublished study prep ared by Stillmeadow, Inc. 19 p. January 29,

2009.

477919-06. Stahl, J. 2004. NoFlyTM WP Acute Eye I rritation Study (in Rabbits). Project Number:

04/831/005N. Unpublished study prepared by LAB Inte rnational Research Centre Hungary, Ltd.

22 p. September 2, 2004.

477919-07. Stahl, J. 2004. NoFlyTM WP Primary Dermal Irritation Study (in Rabbits). Project Number:

04/831/006N. Unpublished study prepared by LAB Inte rnational Research Centre Hungary, Ltd.

20 p. September 2, 2004.

477919-08. Stahl, J. 2004. NoFlyTM WP Skin Sensitiz ation Study (in Guinea Pig). Project Number:

04/831/104T. Unpublished study prepared by LAB Inte rnational Research Centre Hungary, Ltd.

34 p. September 27, 2004.

477920-00. Natural Industries, Inc. 2009. Submission of Product Chemistry and Toxicity Data in Support of

the Application for Registration of NoFlyTM Technical and the Petition for Tolerance of

Paecilomyces fumosoroseus Strain FE 9901, Blasto spores. Transmittal of 7 Studies. May 7,

2009.

477920-01. Brookman, D.; Curry, K. 2009. Product Chem istry of NoFlyTM Technical. Project Number:

NoFLYTM/PC/001. Unpublished study prepared by Technology Sciences Group, Inc. 247 p. May

13, 2009.

477920-02. Cerna, Z. 2004a. NoFlyTM Technical Acute Oral Toxicity (in Rats). Project Number: 006/04/C.

Unpublished study prepared by BioT est, Ltd. 25 p. July 30, 2004.

477920-03. Mier, T.; Olivares-Redonda, G .; Navarro-Barranco, H.; et al. 2005. Acute Oral Intragastric

Pathogenicity and Toxicity in Mice of Paecilomyce s fumosoroseus Isolated from Whiteflies.

Antonie van Leeuwenhoek 88:103-111. February 9, 2005.

477920-04. Marhan, J. 2004. NoFlyTM Technical Acute Pulmonary Toxicity/Pathogenicity (in Ferrets).

Project Number: 004/04/C. Unpublished study prep ared by BioTest, Ltd. 26 p. July 30, 2004.

477920-05. Cerna, Z. 2004b. NoFlyTM Technical Acute Injection Toxicity/Pathogenici ty (Intraperitoneal) (in

Rats). Project Number: 005/04/C. Unpublished st udy prepared by BioTest, Ltd. 22 p. July 30,

2004.

477920-06. Even, M. 2009. Response to Tier 1 Microbial Pesticide Data Requirements for NoFlyTM

Technical. Unpublished study prepared by Technolog y Sciences Group, Inc. 41 p. May 7, 2009.

477920-07. Vertesr, A. 2004. NoFlyTM Technical Ba cterial Reverse Mutation Assay. Project Number:

04/831/007M. Unpublished study prepared by LAB Inte rnational Research Centre Hungary, Ltd.

41 p. September 2, 2004.P. fumosoroseus strain FE 9901 page 10

Biopesticides Registration Action Document

479705-00. Natural Industries, Inc. 2010. Submission of Efficacy Data in Support of the Application for

Registration of NoFlyTM WP. Transmittal of 1 Study. February 1, 2010.

479705-01. Corts, V.; Aucejo, S. 2007. Laboratory Trials to Test the Side Effect of NoFlyTM WP and Adults

of Apis Mellifera: One Acute Oral and One Cont act Toxicity Trial. Unpublished study prepared

by recerca agricola/SynTech Research Spain S.L. 49 p. July 12, 2007.

480514-00. Natural Industries, Inc. 2010. Submission of Product Chemistry and Toxicity Data in Support of

the Applications for Registration of NoFlyTM Tech nical and NoFlyTM WP. Transmittal of 3 Studies.

April 2, 2010.

480514-01. Bjornson, H.; Lara, J. 2010. NoFlyTM Technical and NoFlyTM WP Response to PMRA and EPA

Clarifications. Unpublished study prepared by Tech nology Sciences Group, Inc. 18 p. April 2,

2010.

480514-02. Bjornson, H. 2010. NoFlyTM Technical and NoFlyTM WP Response to Hypersensitivity Data

Requirement. Unpublished study prepared by Technology Sciences Group, Inc.

4 p. April 1, 2010.

480514-03. Luangsa, J.; Hyel-Jones, N.; Manoch, L.; et al. 2005. On the Relationships of Paecilomyces

sect. Isarioidea Species. Mycol. Res. 109(5):581-589. May 2005.

Reviews and other References

Altre, J.A. and Vandenberg, J. D. 2001. Penetration of cuticle and proliferation in hemolymph by Paecilomyces

fumosoroseus isolates that differ in virulence against lepidopt eran larvae. J. Invertebr. Pathol. 78:81-86.

Bolckmans et aI. 1995. PreFeRal, ( Paecilomyces fumosoroseus strain Apopka 97), a new microbial insecticide

for the biological control of whiteflies in greenhou ses. Med. Fac. Landbouww. Univ. Gent, 60/3a.

Brown, A.H.S. and Smith, G. 1957. The genus Paecilomyces Bainier and its perfect stage Byssochlamys

Westling. Transactions of the British Mycological Society. 40(1); 17-89.

Catalogue of Life. 2010. Bisby et al., editors. Catalogue of Life: 2010 Annual Checklist. Integrated Taxonomic

Information System (ITIS) and Species 2000. Reading, United Kingdom. Information retrieved on August 24,

2010. www.catalogueoflife.or g/annual-checklist/2010

.

Fargues J. et.al. 1997. Inactivation of Conidia of Paecilomyces fumosoroseus by Near-Ultraviolet (UVB and

UVA) and Visible Radiation. Journal of Invertebrate Pathology 69, 70-78 (1997).

Lacey, L.A. et. al. 1999. Ovicidal and larvici dal activity of conidia and blastospores of Paecilomyces

fumosoroseus (Deuteromycotina: Hyphomycetes) against Bemisia argentifolii (Homoptera: Aleyrodidae) with a

description of a Bioassay System allowing prolonged su rvival of control insects. Biocontrol Science and

Technology, 9:9-18.

MycoBank. 2010. Species Paecilomyces fumosorose us (Wize) A.H.S. Br. & G. Sm. 1957; MB302188.

MycoBank Fungal Databases. Information Retrieved on August 25, 2010 (searching with the term "Paecilomyces

fumosoroseus”). www.mycobank.org

.

Osborne, L.S. et. al. 1990. Pr ospects for biological control of Bemisia tabaci . International Organization for

Biological Control/West Paleartic Regional Section (IOBC/WPRS). Bulletin 13:153-160.

Osborne, L.S. and Landa, Z. 1992. Bi ological Control of whiteflies with entomopathogenic fungi. Florida Ent.,

75:457-471.

Padilla, M., Hernández, E., Carnero, A., Amador, S. 2004. Evaluation of the side e ffects of the Microbial Pest

Control Product FUTURECO NoFLYTM (B CA Paecilomyces fumosoroseus strain FE 9901) to whitefly natural

enemies in the Canary Islands. Company Report No NF-04001.

Riba, G. and Ravelojoana, M. 1984. The paras exual cycle in the entomopathogenic fungus Paecilomyces

fumosoroseus (Wize) Brown and Smith. Can. J. Microbiol. 30:922-926.

Smith, P. 1993. Control of Bemisia tabaci and the potential of Paecilomyces fumosoroseus as a Biopesticide.

Biocontrol News and Information 14 (4):71N-78N.

TSG. 2008. Letter from Heather Bjornson, Technolog y Sciences Group Inc. (TSG) on behalf of Natural

Industries, Inc. to Jeannine Kaush, U.S. EPA. “NoFlyTM Pre-registrati on Meeting Minutes.” July 16, 2008.P. fumosoroseus strain FE 9901 page 11

Biopesticides Registration Action Document

TSG. 2009a. Letter from Heather Bjornson, Technol ogy Sciences Group Inc. (TSG) on behalf of Natural

Industries, Inc. to Sheryl Reilly, U.S. EPA. "NoFlyTM Technical—Application for new product registration...." May

7, 2009.

TSG. 2009b. Letter from Heather Bjornson, Technol ogy Sciences Group Inc. (TSG) on behalf of Natural

Industries, Inc. to Sheryl Reilly, U. S. EPA. "NoFlyTM WP—Application for new product registration...." May 7,

2009.

TSG. 2009c. Letter from Heather Bjornson, Technol ogy Sciences Group Inc. (TSG) on behalf of Natural

Industries, Inc. to Sheryl Reilly, U.S. EPA. “Petition for an exemption from the requirement of a tolerance for

residues of products containing the acti ve ingredient “Paecilomyces fumosoroseus strain FE 9901” in or on all

food commodities. May 7, 2009.

TSG. 2010a. Letter from Heather R. Bjornson, Regulatory Consultant to Natural Industries to Sheryl Reilly and

Kathleen Martin, U.S. EPA. “NoFlyTM Technical (EPA Reg. No. 73314-L). February 1, 2010.

TSG. 2010b. Letter from Heather R. Bjornson, Regulatory Consultant to Natural Industries to Sheryl Reilly, U.S.

EPA. Submission of Information to Support the App lication to Register NoFlyTM Technical (EPA File

Symbol:73314-L) and NoFlyTM WP (EPA File Symbol:73314-A). March 2, 2010.

TSG. 2011. Letter from Heather R. Bj ornson, Regulatory Consultant to Natu ral Industries to Sheryl Reilly, U.S.

EPA. Withdrawal of the Petition for an exemption from t he requirement of a tolerance for residues of products

containing the active ingredient “Paecilomyces fumosoro seus strain FE 9901” in or on all food commodities.

February 17, 2011.

U.S. EPA. 2002. Product Properties Test Guidelin es: OPPTS 830.6317—Storage Stability. U.S. EPA. EPA

712-C-02-026. June 2002.

U.S. EPA. 2008. Letter from Sheryl Reilly, U.S. EPA to Heather Bjornson, Tech nology Sciences Group Inc.

(TSG). Biopesticides and Pollution Prevention Division (BPPD) response to summary of the January 22, 2008

pre-registration meeting to discuss registration of a new microbial active ingredient, Paecilomyces fumosoroseus

strain FE 9901 blastospores. September 3, 2008.

U.S. EPA. 2010a. Letter from Letter from Sheryl Reilly , U.S. EPA to Heather Bjor nson, Technology Sciences

Group Inc. Data Deficiencies for Paecilomyces fumosoroseus strain FE 9901. April 21, 2010.

U.S. EPA. 2010b. Memorandum from Ibrahim Barsoum to Kathleen Martin. Joint Review of data for the

registration of a new TGAI, NoFly Technical, and a new end use product formulation, NoFly Wettable Powder,

containing the fungus Paecilomyces fumosorose us strain FE 9001. September 15, 2010.

U.S. EPA. 2011. Memorandum from Ib rahim Barsoum to Anna Gross. Review of the Registrant's response to

the data deficiencies found by the Agen cy in its review of product chemistry, manufacturing process of two

products: NoFly Technical and NoFly WP containing the new AI Paecilomyces fumosoroseus strain FE 9001.

January 28, 2011.A

‐ 1 

### APPENDIX A: Data Requirements and How the Applicant Met Them

In reviewing its data, EPA’s Office of Pesticide Programs classifies each study as either ‘Acceptable,’

Supplemental,’ ‘Supplemental: Upgradable,’ or ‘Unacceptable.’ An Acceptable study is one that the Agency

concludes is scientifically sound and is useful for risk assessment. A Supplemental study is one that may provide

some information that can be useful in risk assessment; often a study is classified as Supplemental because it

was not required under 40 CFR 158. A ‘Supplemental: Upgradable’ study is one that has the potential to be

upgraded to Acceptable, provided certain addition information is submitted. Finally, an Unacceptable study is

one that is not useful for risk assessment purposes.

Product Analysis. Provided in Table A

‐ 1 is a summary of the Product Analysis data requirements for

microbial pesticides (40 CFR 158.2120) with information regarding how the requirement was met. Note that

four requirements—Analysis of samples; Stability to normal and elevated temperatures, metals and metal ions;

Storage stability; and Corrosion characteristics—need to be completed as terms of registration.

Table A

‐ 1

. Product Analysis Data Requirements for Paecilomyces fumosoroseus strain FE 9901 (EPA 2010b)

OPPTS

Guideline

Number Data Requirement HOW THE REQUIREMENT WAS MET 3 (Conclusion, Review, Notes, MRID)

End

‐ Use Product (73314

‐ A) Manufacturing

‐ Use Product (73314

‐ T)

Product Chemistry and Composition

885.1100 Product Identity Acceptable study (477919

‐ 01) Acceptable study (477920

‐ 01)

885.1200 Manufacturing Process Acceptable study (477919

‐ 01) Acceptable study (477920

‐ 01)

Deposition of a sample in a

nationally recognized culture

collection A pure culture of P. fumosoroseus strain FE 9901 was deposited in the U.S. Department of

Agriculture’s Agricultural Research Service Collection of Entomopathogenic Fungal Cultures

(USDA ARS) under the accession number ARSEF 4490 (MRID 477920

‐ 01).

885.1300 Discussion of formation of

unintentional ingredients Acceptable study (477919

‐ 01) Acceptable study (477920

‐ 01)

Analysis and Certified Limits

885.1400 Analysis of samples Supplemental: Upgradable 1 (477919

‐ 01) Supplemental: Upgradable 1 (477920

‐ 01)

885.1500 Certification of limits Acceptable study (477919

‐ 01) Acceptable study (477920

‐ 01)

Physical and Chemical Characteristics

830.6302 Color Acceptable study (477919

‐ 01) Acceptable study (477920

‐ 01)

830.6303 Physical state Acceptable study (477919

‐ 01) Acceptable study (477920

‐ 01)

830.6304 Odor Acceptable study (477919

‐ 01) Acceptable study (477920

‐ 01)

830.6313 Stability to normal and

elevated temperatures,

metals and metal ions Term of registration Acceptable study (480514

‐ 01)

830.6317 Storage stability Unacceptable study 2 (477919

‐ 02) Term of registration

830.6319 Miscibility Only required for emulsifiable liquid forms of microbial pesticides. Because this product is a

solid, this requirement does not apply.

830.6320 Corrosion characteristics Term of registration

830.7000 pH Acceptable study (477919

‐ 01) Acceptable study (477920

‐ 01)

830.7100 Viscosity Only required for liquid forms of microbial pesticides. Because this product is a solid, this

requirement does not apply.

830.7300 Bulk density Acceptable study (477919

‐ 01) Acceptable study (477920

‐ 01)

1 A five

‐ batch analysis of EP and MP from the proposed manufacturing site must be submitted as a term of registration.

2 As a term of registration, the applicant must conduct a storage stability study using the proposed formulation of NoFlyTM WP, from the

proposed manufacturing site. The study must support a storage period of at least six months

.A

‐ 2 Toxicology. Provided in Table A

‐ 2 is a summary of the Tier I Toxicology Data Requirements

(40 CFR 158.2140) along with an explanation regarding how the requirement was met. Toxicology testing was

limited to Tier I because the acute toxicity of P. fumosoroseus strain FE 9901 did not trigger the Tier II or III

studies.

Table A

‐ 2

. Tier I Toxicology Data Requirements for Paecilomyces fumosoroseus strain FE 9901 (EPA 2010b)

OPPTS

Guideline

Number Data

Requirement HOW THE REQUIREMENT WAS MET 3 (Conclusion, Review, Notes, MRID)

End

‐ Use Product (73314

‐ A) Manufacturing

‐ Use Product (73314

‐ T)

870.1100 Acute oral toxicity Acceptable study (477919

‐ 03) Acceptable study (477920

‐ 02)

870.1200 Acute dermal toxicity Acceptable study (477919

‐ 04) Acceptable data waiver (477920

‐ 06)

870.1300 Acute inhalation toxicity Acceptable study (477919

‐ 05) Acceptable data waiver (477920

‐ 06)

870.2400 Acute eye irritation Acceptable study (477919

‐ 06) Acceptable data waiver (477920

‐ 06)

870.2500 Primary dermal irritation Acceptable study ( 477919

‐ 07) Acceptable data waiver (477920

‐ 06)

885.3050 Acute oral toxicity/ pathogenicity TGAI: Acceptable study (477920

‐ 02; 477920

‐ 03; 480514

‐ 01)

885.3150 Acute pulmonary

toxicity/pathogenicity TGAI: Acceptable study (477920

‐ 04; 477920

‐ 06)

885.3200 Acute injection toxicity/pathogenicity TGAI: Supplemental study; additional information is needed. 1 (477920

‐ 05)

885.3400 Hypersensitivity incidents Acceptable statement (480514

‐ 02)

885.3500 Cell culture Data must be submitted only for products whose active ingredient is a virus;

P. fumosoroseus is a fungus, therefore this requirement does not apply.

1 As a term of registration, EPA requires that the applicant submit data showing microbial

clearance from matrices other than blood (e.g., organs, body fluids).

3 See EPA 2010b for the data review.

Environmental Fate and Ecotoxicity. Provided in Table A

‐ 3 are explanations regarding why

environmental fate and ecotoxicity data were not required (40 CFR 158.2150).

Table A

‐ 3

. Tier I Nontarget Organisms and Environmental Fate Data Requirements

for Paecilomyces fumosoroseus strain FE 9901

OPPTS

Guideline

Number Data Requirement How the Requirement Was Met (EPA 2010b)

885.4050 Avian oral toxicity Tests for pesticides intended solely for indoor application would be required on a

case

‐ by

‐ case basis... In a letter to the applicant, EPA pointed out that if “the

intended use sites are restricted to indoors, then neither a honey bee study nor

other non

‐ target assessments are likely to be needed” (EPA 2008). 885.4100 Avian inhalation toxicity/pathogenicity

885.4150 Wild mammal toxicity/pathogenicity NR 1 for greenhouse uses.

885.4200 Freshwater fish toxicity/ pathogenicity Tests for pesticides intended solely for indoor application would be required on a

case

‐ by

‐ case basis... In a letter to the applicant, EPA pointed out that if “the

intended use sites are restricted to indoors, then neither a honey bee study nor

other non

‐ target assessments are likely to be needed” (EPA 2008). 885.4240 Freshwater invertebrate toxicity/

pathogenicity

885.4280 Estuarine/Marine fish testing; estuarine

and marine invertebrate testing NR 1 for greenhouse uses.

885.4300 Nontarget plant testing NR 1 for greenhouse uses.

885.4340 Nontarget insect testing Tests for pesticides intended solely for indoor application would be required on a

case

‐ by

‐ case basis... In a letter to the applicant, EPA pointed out that if “the

intended use sites are restricted to indoors, then neither a honey bee study nor

other non

‐ target assessments are likely to be needed” (EPA 2008). 885.4380 Honey bee testing

1 NR=not required under 40 CFR 158B

‐ 1 

### APPENDIX B: Biology and Taxonomy of P. fumosoroseus and strain FE 9901

Provided below are additional biologic and taxonomic considerations. Unless otherwise noted, the

source of the information is from the information submitted to support the registration of this new active

ingredient.

Biology

. P. fumosoroseus is a naturally occurring fungus. It is found in most countries of the world and

occurs in various soil types, at very low densities. For example, it has frequently been isolated from

lepidopteran cadavers in Europe. Isolates occur in the Netherlands, Ireland, and Spain. And, it has been found

in various non

‐ European countries such as China, Japan, Ghana, Pakistan, India, and the United States. In the

environment, P. fumosoroseus is dispersed as conidia, which are supported on conidiophores. The

conidiophores are erect and can be about 100 μm in length and 1.5 to 2.0 μm in width (MycoBank 2010). The

conidial structures consist of single phialides or in whorls occurring irregularly along aerial hyphae.

Investigators have found that P. fumosoroseus strain FE 9901 grows best at 24.5oC (76.1oF). When

grown on potato dextrose agar, P. fumosoroseus strain FE 9901 is floccose (i.e., it is covered with tufts of soft

hair) with a circular margin. Colonies are white, often turning pale yellow from the margin out and changing to

brownish

‐ gray when forming conidia. Colonies reach a diameter of 25 to 30 mm after seven days and 50 to 65

mm after 14 days. (Brown and Smith 1957). P. fumosoroseus has an asexual life cycle. However, exposure to

ultraviolet light results in formation of auxotrophic mutants, suggesting that a parasexual life cycle also exists for

the organism (Riba and Ravelojoana 1984).

Taxonomy

. The taxonomic classification P. fumosoroseus is provided below (Catalog of Life 2010). For a

detailed explanation of the taxonomic designation, refer to the product characterization data review (EPA

2010b).

Kingdom : Fungi

Phylum : Ascomycota

Class : Eurotiomycetes

Order : Eurotiales Family : Trichocomaceae

Genus : Paecilomyces

Species : fumosoroseus

Strain : FE 9901

The strain FE 9901 is a wild type—it is neither a spontaneous nor an induced mutant and it has not been

genetically modified. P. fumosoroseus strain FE 9901 was first isolated from an adult of Bemisia tabaci

(a species of whitefly) in Padappai, India in 1992.

Synonyms

. P. fumosoroseus

, a mycoinsecticide, was first described by Wize in 1904 (Brown and

Smith 1957). Synonyms (basionym, obligate, and taxonomic) for this fungus include (MycoBank 2010,

EPA 2010b).

Isaria fumosorosea (basionym; first described by Wize in 1904),

Spicaria fumosorosea (obligate synonym; Wize),

Spicaria aphodii (Vuillemin in 1910),

Penicillium aphodii (Vuillemin),

Monilia aquatilis (Malguth, in 1928),

Paecilomyces hibernicus (Kennelly & Grimes, in 1930), and

Paecilomyces isarioides (Inagaki, in 1962).C

‐ 1 

### APPENDIX C: Summary of Submitted Toxicity Studies

Oral (OPPTS 870.1100; MRID 477919

‐ 03). Following the Up

‐ and

‐ Down Method (OECD Guideline 425),

three female Sprague

‐ Dawley rats were gavaged with P. fumosoroseus strain FE 9901(containing 2×10 9 colony

forming units per gram, or CFU/g) at a dose of 5,000 mg/kg bw. Investigators terminated the study following

the stopping rules for this procedure. Each animal was then observed for a period of up to 14 days. None of the

animals died or showed any symptoms of intoxication following treatment. The test material had no negative

effect upon the body weight, and no gross pathological changes were noted. Based on the results of this study,

the oral LD 50 is >5,000 mg/kg bw (which is the limit dose). According to the regulations at 40 CFR 156.62, the

Acute Toxicity Category is IV.

Dermal (OPPTS 870.1200; MRID 477919

‐ 04). A group of seven

‐ week

‐ old Sprague

‐ Dawley rats (five/sex)

was exposed dermally to P. fumosoroseus strain FE 9901(containing 2×10 9 CFU/g) at a limit dose of 5,050 mg/kg

bw for 24 hours to an area of not less than 10% of the total body surface. Following exposure, the animals were

observed for a period of 14 days. No mortalities were observed, and all the test animals appeared normal for

the duration of the study. Very slight erythema and discoloration was observed in one female. At necropsy, two

male rats had pale lungs at study termination. Based on these results, the dermal LD 50 is 5,050 mg/kg bw; the

Acute Toxicity

Category is III.

Inhalation (OPPTS 870.1300; MRID 477919

‐ 05). A group of 10

‐ week

‐ old Sprague

‐ Dawley rats (five/sex)

was exposed via the inhalation route to P. fumosoroseus strain FE 9901 (containing 2×10 9 CFU/g) for four hours

to nose only at a concentration of 2.18 mg/L (the limit dose is 2 mg/L). Animals then were observed for 14 days.

No mortalities were observed; clinical observations included piloerection and decreases in activity which both

resolved by Day 3. All test animals gained body weight throughout the study period, and no observable

abnormalities were observed at gross necropsy. Based on the findings of this study, the LC 50 is >2.18 mg/L; the

Acute Toxicity Category is III.

Eye Irritation (OPPTS 870.2400; MRID 477919

‐ 06). In a primary eye irritation study, 0.1g of NoFlyTM WP

(containing 88% P. fumosoroseus strain FE 9901) was instilled into the conjunctival sac of the left eye of three

adult male New Zealand White rabbits. The treated eyes were not washed following application. The untreated

right eye of each test animal served as a negative control. Test animals then were observed for 72 hours.

Irritation was scored by the Draize Method. After one hour, some redness was observed in one rabbit, and

slight to moderate increases in excretion discharge were noted in three rabbits. All test animals were free of

irritation at the 24

‐ , 48

‐ and 72

‐ hour observation timepoints. Based on the results of this study, NoFlyTM WP is

slightly irritating to the eyes of New Zealand White rabbits. EPA determined that the Acute Toxicity Category is

IV.

Primary Dermal Irritation (OPPTS 870.2500; MRID 477919

‐ 07). In a primary dermal irritation study,

three adult male New Zealand White rabbits were dermally exposed to 0.5g NoFlyTM WP (containing 88% P.

fumosoroseus strain FE 9901) in water for four hours to an area of skin of approximately six cm 2

. Animals then

were observed for 72 hours. Irritation was scored by the method of Draize. Very slight erythema was noted in

two test animals one hour after patch removal. All test animals were free of irritation at the 24

‐ , 48

‐ and 72

‐

hour observation timepoints. Based on the results of this study, NoFlyTM WP is slightly irritating to the skin of

male New Zealand White rabbits. EPA determined that the Acute Toxicity Category is IV.C

‐ 2 Oral toxicity/pathogenicity (OPPTS 885.3050; MRID 47720

‐ 02). In an acute oral toxicity/pathogenicity

study, groups of fasted, young Wistar rats (15/sex) were given NoFlyTM Technical (1.0×10 9 CFU/g) in a single dose

of 100 mg of test material per animal, each dose contained 10 8 CFU. The animals were then observed for a

period of up to 21 days with scheduled sacrifices 24 hours after application and on Days 3, 7, 14 and 21. None of

the 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 findings of this study, P. fumosoroseus strain FE

9901 is not considered infective or pathogenic in the rat. In terms of an LD 50 , the animals showed no signs of

toxicity at a dose of 600 mg/kg bw. 5

Pulmonary toxicity/pathogenicity (OPPTS 885.3150; MRID 47720

‐ 04). In an acute pulmonary infectivity

and toxicity study, groups of young adult ferrets (14/sex) were exposed by the intranasal route to NoFlyTM

Technical at a nominal dose of 1×10 8 CFU (in 0.5 mL) per animal. Animals were then observed for up to 21 days.

None of the animals died or showed any symptoms of intoxication following treatment. No gross pathological

changes were noted. Microbiological examination of lungs, other prescribed organs, and body fluids proved

minimal persistence and no infectivity of the test material. Based on the results of this study, P. fumosoroseus

strain FE 9901 is not considered infective or pathogenic in the ferret. Please note that the rat is the preferred

species for acute pulmonary toxicity.pathogenicity testing. For this MPCA, the applicant provided acceptable

justification for using ferrets instead of rats.

Acute injection toxicity/pathogenicity —intraperitoneal (OPPTS 885.3200; MIRD 47720

‐ 05). In an acute

intraperitoneal infectivity study, a group of young adult Wistar rats (three/sex) were injected with NoFlyTM

Technical (1.0×10 9 CFU/g) in a single dose of 107 CFU per animal. Animals were then observed for up to 21 days.

None of the animals died or showed any symptoms of intoxication after treatment. The test item had no

negative effect upon the body weight, and no gross pathological changes were noted. Blood samples were

collected during necropsy to evaluate clearance. Based on the available results from the study, P. fumosoroseus

strain FE 9901 is not considered infective or pathogenic in the rat via the intraperitoneal route of exposure. As a

term of registration, EPA is requiring that the applicant submit data showing microbial clearance from matrices

other than blood (e.g., organs, body fluids).

Hypersensitivity incidents (OPPTS 885.3400; MRID 480514

‐ 02). The applicant stated reported that no

hypersensitivity incidents have been reported through the research, development, and testing of NoFlyTM

Technical or WP. However, the EP has been found to be a dermal sensitizer (EPA 2010b). In addition, a

literature search of the PubMed database www.ncbi.nlm.nih.gov/pubmed ) using the keywords “paecilomyces”

and “allergy” revealed 21 reports of hypersensitivity. Based on the assumption that most microorganisms

contain substances that would elicit allergic reactions,

P. fumosoroseus strain FE 9901 is considered a potential sensitizing agent.

Supplemental Toxicity Test

Skin Sensitization

. In a skin sensitization test (477919

‐ 08) using the Magnusson and Kligman Method,

female guinea pigs were exposed to NoFlyTM WP that was administered as a 1% intradermal injection and a 25%

topical application. Later, a challenge dose was applied (25% topical application). Study investigators observed

a localized area of intense erythema and swelling in treated animals. EPA concluded that NoFlyTM WP is a skin

sensitizer.

5 Technical NoFlyTM mg 600

bw kg 0.163 Technical

NoFlyTM mg 100

